WO2017197243A1

WO2017197243A1 - Cblb inhibition for treating fungal infections

Info

Publication number: WO2017197243A1
Application number: PCT/US2017/032370
Authority: WO
Inventors: Jian Zhang; Chad RAPPLEYE
Original assignee: Ohio State Innovation Foundation
Current assignee: Ohio State Innovation Foundation
Priority date: 2016-05-13
Filing date: 2017-05-12
Publication date: 2017-11-16
Anticipated expiration: 2018-11-13

Abstract

Disclosed herein is a method for treating a fungal infection in a subject that involves administering to the subject a composition comprising a therapeutically effective amount of a casitas B lymphoma-b (CBLB) inhibitor. Specifically, wherein the CBLB inhibitor is an siRNA. Further disclosed are specific nucleic acid sequences of siRNA CBLB inhibitors.

Description

CBLB INHIBITION FOR TREATING FUNGAL INFECTIONS

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application Serial No. 62/335,901 filed May 13, 2016 and U.S. Provisional Patent Application Serial No. 62/384,526 filed September 7, 2016, the disclosures of which are expressly incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

This invention was made with Government Support under Grant Nos. R01AI090901, ROl AI123253, and R21 All 17547 awarded by the National Institutes of Health. The Government has certain rights in the invention.

BACKGROUND

C. albicans is the most common cause of fungal infections in humans and has become one of the leading causes of hospital-acquired blood stream infections. Despite the availability of several antifungal drugs, invasive candidiasis still has a high mortality rate ranging from 45 to 75% (Brown, G.D. et al. Sci. Transl. Med. 4, 165rvl3 (2012)). The high morbidity and mortality associated with disseminated candidiasis are mainly due to the lack of early and accurate diagnostic tools, limited antifungal drugs, and the emergence of drug resistance. These factors highlight the need to further understand host-pathogen interactions and the mechanisms of immune resistance to fungal spread, and to develop immune-based strategies to combat candidemia.

SUMMARY

Disclosed herein is a method for treating a fungal infection in a subject that involves administering to the subject a composition comprising a therapeutically effective amount of a casitas B lymphoma-b (CBLB) inhibitor.

The disclosed methods can in some embodiments be used to treat any fungal infection. In particular embodiments, the disclosed methods can be used to treat a Candida spp. infection. In some embodiments, the fungal infection comprises Candida auris, aspergillosis, Pneumocystis carinii pneumonia (PCP), coccidioidomycosis (valley fever), cryptococcosis, histoplasmosis, or a combination thereof. In some embodiments, the CBLB inhibitor comprises functional nucleic acid that knocks down expression of the Cbl-b gene in the subject. For example, the functional nucleic acid comprises an siRNA.

In particular embodiments, the siRNA comprises the nucleic acid sequence 5'- AAAUUCUCGAAGUAUGCUCUU-3 ' (SEQ ID NO : 1 ), or a variant thereof having at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1.

In some embodiments, the fungal infection comprises a Candida albicans infection. In some embodiments, the fungal infection comprises aspergillosis, Pneumocystis carinii pneumonia (PCP), coccidioidomycosis (valley fever), cryptococcosis, histoplasmosis, or a combination thereof. In some embodiments, the fungal infection comprises aspergillosis, Pneumocystis carinii pneumonia (PCP), coccidioidomycosis (valley fever), or a combination thereof.

In some embodiments, the CBLB inhibitor is an siRNA, a miRNA, a shRNA, a small molecule, an antisense molecule, a peptide, or a protein.

In some embodiments, the CBLB inhibitor comprises a functional nucleic acid that knocks down expression of the Cbl-b gene in the subject. In some embodiments, the functional nucleic acid comprises an siRNA. In some embodiments, the functional nucleic acid is from about 15 to about 25 nucleotides.

In some embodiments, the siRNA comprises the nucleic acid sequence 5'- UUUGCUAACGGACCAGUACUU-3 ' (SEQ ID NO:2) or 5'- UAAUACCCAAAAUUCGACCUU-3 ' (SEQ ID NO:3).

In particular embodiments, the siRNA comprises the nucleic acid sequence 5'- UUUGCUAACGGACCAGUACUU-3 ' (SEQ ID NO:2), or a variant thereof having at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:2.

In particular embodiments, the siRNA comprises the nucleic acid sequence 5'- UAAUACCCAAAAUUCGACCUU-3 ' (SEQ ID NO:3), or a variant thereof having at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:3. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

In another aspect, disclosed herein is a method for treating or preventing a fungal infection in a subject, comprising administering to the subject a composition comprising a therapeutically effective amount of a casitas B lymphoma-b (CBLB) inhibitor, wherein the casitas B lymphoma-b (CBLB) inhibitor reduces the activity of a casitas B lymphoma-b (CBLB) protein in the subject.

In some embodiments, the casitas B lymphoma-b (CBLB) inhibitor reduces the activity of a casitas B lymphoma-b (CBLB) protein in the subject comprises reducing the expression of the gene encoding the casitas B lymphoma-b (CBLB) protein. In some embodiments, the reducing the expression of the gene comprises RNA interference using a functional nucleic acid that knocks down expression of the gene in the subject.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.

FIGS. 1 A-1C. CBLB inhibits pro-inflammatory cytokine production by macrophages after infection with C. albicans yeast cells or hyphae, and A. fumigatus conidia. (a) TNF-a (left), IL-6 (middle), and IL-Ιβ (right) production, as determined by ELISA, in the culture supernatants of WT and Cblb^_/~ BMDMs that were infected with C. albicans capl mutant yeast cell (top) and hyphal (bottom) forms (WT strain SC5314) (MOI: 1 : 1) for 1 h and 3 h. (b) TNF-a (left) and IL-6 (right) production, as assessed by ELISA, in the culture supernatants of WT and Cblb^_/~ BMDMs that were infected with swollen A. fumigatus conidia (AF293) (MOI = 1 : 1) for 2 h and 4 h. (c) IL- IRA production, as measured by ELISA, in culture supernatants of WT and Cblb^_/~ BMDMs that were infected with C. albicans yeast (left) and hyphal (right) forms. For all ELISA experiments data are representative of three independent experiments (biological replicates); n = 3 mice per group, each with three repeated wells. Error bars are mean ± s.d. *P < 0.05, **P < 0.01; by unpaired two-tailed Student's t-test.

FIGS. 2A-2G. CBLB associates with dectin-1 and dectin-2 in macrophages after infection with C. albicans yeast cells or hyphae. (a,b) Immunoblot analysis of dectin-1 (a) or dectin-2 (b), SYK, and CARD9 after immunoprecipitation (IP) with CBLB-specific antibodies, using lysates of BMDMs that were uninfected or infected with C. albicans yeast cells (a) or hyphae (b). Images are representative of three independent experiments (biological replicates), and each IP was analyzed separately. (c,d) Immunoblot analysis of dectin-1 (top) or dectin-2

(bottom), SYK, and CARD9 after IP with CBLB-specific antibodies, from lysates either of WT

BMDMs ± Syk gene silencing (c) or of WT and Card9^~!~ BMDMs (d) that were uninfected or infected with C. albicans yeast cells (top) or hyphae (bottom). The numbers below the SYK (c) and CARD9 (d) blots indicate the extent of SYK expression in BMDMs of C57BL/6 mice that were treated with the control siRNA or the Syk-specific siRNA (c) or of CARD9 expression in

WT and Card9^~h BMDMs (d). Images are representative of two independent experiments

(biological replicates), and each IP was analyzed separately, (e) Immunoblot analysis of dectin-1 after CBLB immunoprecipitation from lysates of Clec7a^~ BMDMs that were reconstituted with constructs expressing Flag-tagged dectin-1 or CLEC7A^Y15F and that were infected with C.

albicans yeast. The numbers below the Flag blots in e,f indicate the extent of dectin-1 expression in Clec7a^_/~ BMDMs reconstituted with constructs expressing WT dectin-1 or CLEC7A^Y15F (e), or of dectin-2 expression in Fcerlg^_/" BMDMs reconstituted with constructs expressing WT

FcR-γ or FcR-y^Y65F'^Y76F (f). Images are representative of three independent experiments

(biological replicates), and each IP was analyzed separately, (f) Immunoblot analysis of dectin-2 after CBLB immunoprecipitation from lysates of Fcerlg^~h BMDMs that were reconstituted with constructs expressing Flag-tagged FcR-γ or FcR-yY65F,Y76F and that were infected with C. albicans hyphae. Images are representative of two independent experiments (biological replicates), and each IP was analyzed separately, (g) Immunoblot analysis of FcR-γ after CBLB immunoprecipitation from lysates of WT and Fcerlg^_/" BMDMs that were infected with C. albicans hyphae. The numbers below the FcR-γ blot indicate that the extent of FcR-γ expression in WT BMDMs relative to that in Card9^~ BMDMs. Images are representative of three independent experiments (biological replicates), and each IP was analyzed separately.

Throughout, actin was used as a loading control. CL, cell lysates.

FIGS. 3A-3H. CBLB targets dectin-1 and dectin-2 for polyubiquitination and subsequent degradation in the lysosome. (a) Immunoblot analysis for the indicated proteins, using lysates from WT and Cblb- BMDMs that were infected with C. albicans yeast or hyphal forms (MOI

= 1 : 1) for the indicated amounts of time. Images are representative of five independent experiments (biological replicates), (b) Immunoblot analysis of WT BMDMs that were pretreated with E-64 (10 μΜ), MG-132 (5 μΜ), or both for 30 min, and were then infected with

C. albicans yeast cells or hyphae (MOI = 1 : 1). Images are representative of three independent experiments (biological replicates). (c,d) Immunoblot analysis for ubiquitination of dectin-1 (c) or dectin-2 (d) immunoprecipitates that were isolated from WT and CblbC373A BMDMs that were infected with C. albicans yeast (c) and hyphae (d). Images are representative of four independent experiments (biological replicates), and each IP was analyzed separately, (e) Immunoblot analysis for ubiquitination of dectin-1 in Clec7a^~ BMDMs reconstituted with constructs expressing WT dectin-1 (CLEC7A^WT), CLEC7A^K2R, CLEC7A^K27R, CLEC7A^K34R, or CLEC7A^K2R' ^¹¹^' ^K34R (CLEC7^3KR) and infected with C. albicans yeast. Images are

representative of three independent experiments (biological replicates), and each IP was analyzed separately, (f) Immunoblot analysis for ubiquitination of dectin-2 in Clec4n^_/" BMDMs reconstituted with constructs expressing WT dectin-2 (CLEC4N^WT) or CLEC4N^K10R after infection with C. albicans hyphae. Images are representative of three independent experiments (biological replicates), and each IP was analyzed separately. (g,h) T F-α (left) and IL-6 (right) production, as determined by ELISA, in Clec7a^_/~ BMDMs reconstituted with constructs expressing WT dectin-1 or CLEC7A^{K2R K27R K34R} after infection with C. albicans yeast (g) or in Clec4n BMDMs reconstituted with constructs expressing WT dectin-2 or CLEC4N^K10R after infection with C. albicans hyphae (h). Data are representative of three independent experiments (biological replicates). In g,h, n = 3 mice per group, each with three repeated wells. Error bars are mean ± s.d. *P < 0.05, **P < 0.01; by unpaired two-tailed Student's t-test.

FIGS. 4A-4D. Loss of CBLB impairs dectin-1 and dectin-2 internalization and their downregulation at the cell surface. (a,b) Cell surface (left) and intracellular (right) expression of dectin-1 (a) and dectin-2 (b) in WT and Cblb^~ BMDMs infected with C. albicans yeast cells (a) or hyphae (b) (MOI = 1 : 1) for the indicated times, as determined by flow cytometry (n = 3 mice per group; each with three repeated wells). For internalization of dectin-1 and dectin-2, WT and Cblb^~h BMDMs were treated with acid buffer to strip the antibodies remaining at the cell surface after infection at each time point. Data are representative of three independent experiments (biological replicates). Error bars are mean ± s.d. *P < 0.05; by unpaired two-tailed Student's t- test. (c,d) Representative confocal images of dectin-1 (c) and dectin-2 (d) internalization and lysosome sorting in WT and Cblb^~ BMDMs that were uninfected or infected with either C. albicans yeast (c) or hyphae (d) (MOI = 1 : 1) for 30 min (n = 3 mice per group; each with three repeated wells.). DIC, differential interference contrast; LAMP, lysosomal-associated membrane protein; DAPI, 4,6-diamidino-2-phenylindole dihydrochloride, was used to stain nuclei. Images are representative of five independent experiments (biological replicates). Scale bars, 5 μπι.

FIGS. 5A-5F. Introducing a dectin-1 or dectin-2 deficiency, or a double deficiency, into Cblb^~ mice renders Cblb^~ mice susceptible to systemic C. albicans infection, (a) Kaplan- Meier survival curve of WT, Cblb^~ , and Cblb^C313A mice (n = 10 per group) after infection with 5 ^χ 10⁵ c.f.u. of C. albicans (SC5314). Data are representative of three independent experiments (biological replicates). *P < 0.05; by log-rank test, (b) Fungal burden in paired kidneys of WT, Cblb^_/~ and Cblb^C373A mice (n = 10 per group) at day 2 after infection with 1 ^χ 10⁵ c.f.u. of C. albicans. Data are representative of three independent experiments (biological replicates). **P < 0.01; by unpaired two-tailed Student's t-test. (c) Kidney histopathology analysis by H&E staining (top) and PAS staining to visualize fungal burden (hyphae) (bottom) (n = 10 mice per group). Images are representative of two independent experiments (biological replicates). Scale bars, 200 μιη. (d) Serum TNF-a (left), IL-6 (middle), and IL-Ιβ (right) levels in WT and Cblb^~ mice infected with 1 x 10⁵ c.f.u. of C. albicans at 2, 6, 12, and 24 h after infection (n = 10 mice per group; each with three repeated wells). Data are representative of three independent experiments (biological replicates). Error bars are mean ± s.d. #P > 0.05, **P < 0.01; by unpaired two-tailed Student's t-test. (e) Survival rate of Ragl^~ and Ragl^~h Cblb^~ mice infected with 1 x 10⁵ c.f.u. of C. albicans (n = 8 mice per group). Data are representative of three independent experiments (biological replicates). *P < 0.05; by log-rank test, (f) Survival analysis of WT, Cblb^{" "} Clec7a-^/- Clec4_n-^/- Cblb-^/ lec7_a-^/- Cblb^{" "} Clec4_n-^/- and

Cblb^~/~Clec7a^~/~Clec4n^~/~ mice infected with C. albicans (3.5 x 10⁵ c.f.u.) by i.v. injection (n = 5 mice per group). Data are representative of three independent experiments (biological replicates). #P < 0.01, Cblb^_/" versus all other groups; *P < 0.05, Cblb^"/"Clec7a^"/" versus Clec7a^_/", or Cblb^"/"Clec4n-^/- versus Clec4n^_/-; and §P < 0.05, Cblb^"/"Clec7a^"/" Clec4n^_/- versus

Clec7a^~/~Clec4n^"/"; by log-rank test.

FIGS. 6A-6C. Systemic in vivo delivery of Cblb-specific siRNA into C57BL/6 mice protects them from lethal disseminated candidiasis, (a) Survival of C57BL/6 mice treated with in vivo-grade 0>/¾-specific siRNA (5'-AAAUUCUCGAAGUAUGCUCUU-3', SEQ ID NO: 1) or a nonsense (Ctr) siRNA (2 mg per kg body weight per mouse) via tail vein injection 24 h after infection with C. albicans (5 x 10⁵ c.f.u.) (n = 9 mice per group). Data are representative of three independent experiments (biological replicates). P < 0.05 by log-rank test, (b) Fungal burden in the kidneys on day 2 after infection (n = 9 mice per group). Data are representative of three independent experiments (biological replicates). Error bars are mean ± s.d. *P < 0.05; by unpaired two-tailed Student's t-test. (c) Immunoblot analysis for CBLB in spleen cells from C57BL/6 mice that were treated with the control siRNA or the Cblb-specific siRNA (n = 3 mice per group). Actin was used as a loading control. The numbers below the blots in c indicate the extent of CBLB expression in C57BL/6 mice that were treated with the control siRNA or the Cblb-specific siRNA. Data are representative of four independent experiments (biological replicates). FIGS. 7A-7B. CBLB inhibits signaling via the dectin-1. (a,b) ELIS A of TNF-a and IL-6 production by WT and Cblb^''- BMDMs and BMDCs were stimulated with TLR ligands 1 to 9, zymosan, and curdlan for 48 h. For all ELISA experiments data are representative of three independent experiments (biological replicates). Error bars are mean ± s.d. *P < 0.05, **P < 0.01; unpaired two-tailed Student's t test, n = 3 per group, each with three repeated wells.

FIGS. 8A-8B. CBLB inhibits TNF-a and IL-6 production by dendritic cells but has a limited role in the production of these cytokines by neutrophils upon infection with C. albicans yeast or hyphae. (a) ELISA of TNF-a and IL-6 production in the supernatants collected from BMDCs of WT and Cblb-/- mice infected with C. albicans yeast and hyphae forms (MOI = 1 : 1) for 1, 3, and 6 h. (b) ELISA of TNF-a and IL-6, production in the supernatants collected from BM neutrophils of WT and Cblb-/- mice infected with C. albicans yeast and hyphae forms (MOI = 1 : 1) for 1, 3, and 6 h. The data are representative of three independent expendents (biological replicates). Error bars are mean ± s.d. *P < 0.05, unpaired two-tailed Student's t test, n = 3 per group, each with three repeated wells.

FIGS. 9A-9D. Human macrophages lacking CBLB fail to down-regulate dectin-1 and dectin-2 expression and produce more pro-inflammatory cytokines upon infection with C.

albicans yeast and hyphae. (a,b) ELISA of TNF-a and IL-6 production by MDMs with or without Cblb gene silencing, infected with yeast (a) or hyphae (b) of C. albicans (MOI = 1 : 1), or left uninfected. For all ELISA experiments data are representative of two independent experiments (biological replicates). Error bars are mean ± s.d. *P < 0.05, **P < 0.01; unpaired two-tailed Student's t test, n = 3 per group, each with three repeated wells. (c,d) Immunoblot analysis of CBLB, dectin-1 and dectin-2, and actin expression in MDM lysates. Data are representative of two independent experiments.

FIGS. 10A-10E. CBLB deficiency impairs ubiquitination of dectin-1, dectin-2, and SYK in macrophages induced by C. albicans yeast and hyphal infection. (a,b) Immunoblot analysis of dectin-1 and dectin-2 ubiquitination of dectin-1 or dectin-2 immunoprecipitates isolated from BMDMs from WT and Cblb^_/" mice infected with C. albicans yeast and hyphae, respectively, by anti-ubiquitin and anti-K48 ubiquitin antibodies. (c,d) Immunoblot analysis of SYK

ubiquitination of SYK immunoprecipitates isolated from BMDMs from WT and Cblb^C373A mice infected with C. albicans yeast and hyphae, respectively, by anti-K48-specific ubiquitin antibody, (e) Immunoblot analysis of phospho-SYK and NF-κΒ p65 in BMDMs from WT and Cblb^_/" mice infected with C. albicans yeast and hyphae, respectively, by anti-phospho-SYK (Y525,Y526) and anti-phospho-p65 (S536). Data are representative of three independent experiments (biological replicates). FIGS. 11A-11D. Loss of CBLB or CBLB C373 A mutation enhances ROS production and fungal killing but does not affect phagocytosis, (a) ROS production by WT, Cblb^_/" or Cblb^C373A BMDMs co-cultured with C. albicans at MOI = 5: 1 and 2: 1, and monitored in real time over the indicated time period using the luminol assay. Values are expressed as relative light units (RLU) per 1,000 cells and represent mean values, (b) Killing capacity of WT or Cblb^_/~ BMDMs as assessed by 24 h co-culture with C. albicans. Assays were performed in triplicate. Data are shown as means ± s.d. (c) ROS production by WT, Cblb^_/" , or Cblb^C373A BM neutrophils co- cultured with C. albicans at 2: 1, and monitored in real time over the indicated time period using the luminol assay. Values are expressed as relative light units (RLU) per 1,000 cells and represent mean values (upper panel). Killing capacity of WT, Cblb^~!~ or Cblb^C313A BM neutrophils as assessed by 24 h co-culture with C. albicans (lower panel), (d) Phagocytosis of C. albicans. WT or Cblb^_/" BMDMs co-cultured with Alexa Fluor 488-labeled C. albicans. After 45 min of co- culture, WT or Cblb^~!~ BMDMs were analyzed for C. albicans uptake by flow cytometry. * P <0.05, ** P <0.01, Student's t test.

FIGS. 12A-12B. CBLB C373 A mutation significantly reduces fungal burden in kidneys, livers, lungs, and spleens, and leads to less myeloid cell recruitment to the kidneys, (a) Fungal burden in depicted organs at 48 h after systemic C. albicans infection, shown as colony forming units (CFU)/gram organ weight. Each dot represents an individual mouse, (b) Percentages and absolute numbers of hematopoietic cells in the kidneys from WT and Cblb^C313A mice at 48 h after infection with C. albicans (1 x 10⁶ CFU). n = 5 for WT or Cblb^{C 1 A} mice. Data are shown as means ± s.d. and analyzed using the Student's t test. *P < 0.05, **P < 0.01.

FIGS. 13A-13E. CBLB C373 A mutation facilitates fungal killing by enhancing ROS activity in the blood and within spleens and kidneys by monocytes/macrophages, (a) Fungal burden of WT and Cblb^C373A in the blood at 2 and 6 h after systemic C. albicans infection, and killing capacity of WT or Cblb^C373A mice as assessed by 24 h co-culture with C. albicans, (b) Killing capacity of splenic monocytes and neutrophils of WT or Cblb^C313A mice as assessed by 24 h co-culture with C. albicans, (c) ROS staining in splenic monocytes, macrophages and neutrophils infected with 1 x 10⁶ CFU of C. albicans performed at day 2 after infection, (d) ROS staining in monocytes, macrophages and neutrophils in the kidneys of WT and Cblb^C313A infected with 1 x 10⁶ CFU of performed at day 2 after infection. Killing capacity of CD45- enriched cells isolated from C. albicans, (e) ELISA of TNF-a, IL-6, and IL-Ιβ levels of kidney homogenates of WT or Cblb^{C 1 A} mice infected with 1 x 10⁶ CFU of C. albicans at 48 h after infection, n = 5 mice per group and are representative data from three independent experiments. Error bars, mean ± s.e.m. Data were analyzed using the Student's t test. *P < 0.05, **P < 0.01. FIGS. 14A-14B. Introducing dectin-1 or dectin-2 deficiency, or both into Cblb^~!~ mice significantly reduces serum T F-α and IL-6 which correlates with increased fungal burden in the kidneys, (a) ELISA of serum TNF-a and IL-6 at 24 and 48 h from WT, Cblb^{" "}, Clec7a-^/" Clec4n-^/- Cblb-^/-Clec7_a-^/- Cblb-^/-Clec4_n-^/- Clec7a^"/- Clec4n^"/- and Cblb-^/-Clec7_a-^/-Clec4_n-^/- mice infected with C. albicans (3.5 x 10⁵ CFU) by i.v. injection, (b) Fungal burden in the above mice measured at 48 h after infection. Data are representative of two independent experiments.

FIGS. 15A-15D. Autoantibody, IgG, and IgE titers in the sera and IL-17/IFN-Y in the sera and kidneys of WT, Cblb^_/", and Cblb^C373A mice before and after C. albicans infection, (a-c) Serum titers of anti-dsDNA and anti-ssDNA antibodies, IgG, IgE, IL-17, and IFN-γ in WT, Cblb^" ^/_, and Cblb^C373A mice before or 48 h after C. albicans infection (1 x 10⁶ CFU). (d) IL-17 and IFN-γ in the kidneys of WT, Cblb^~!~, and Cblb^C373A mice before or 48 h after C. albicans infection, n = 5 mice per group and are representative data from three independent experiments. Error bars, mean ± s.e.m. Data were analyzed using the Student's t test.

FIG. 16. Loss of CBLB stabilizes dectin-3, but not MR, Mincle and DC-SIGN.

Immunoblot analysis of lysates of WT and Cblb^~!~ BMDMs infected with C. albicans hyphae for the times indicated with antibodies against dectin-3, MR, Mincle, and DC-SIGN. Results are representative data from three independent experiments (n = 3).

DETAILED DESCRIPTION

Disseminated C. albicans infection in patients who have a weakened immune system is life-threatening. In hospitals 40% of bloodstream infections (candidemia) are caused by Candida spp. Despite the availability of several antifungal drugs, invasive candidiasis still has a high mortality rate ranging from 45 to 75%. The high morbidity and mortality associated with disseminated candidiasis are mainly due to the lack of early and accurate diagnostic tools, the limited antifungal drugs, and the emergence of drug resistance, thus highlighting the need to further understand host-pathogen interactions and the mechanisms of immune resistance to fungal spread, and to develop alternative immune-based strategies to combat candidemia. In normal hosts, C. albicans is controlled after activation of innate immune cells via cell surface pattern recognition receptors (PRRs) such as TLR2 and C-type lectin receptors (CLRs) that detect the infecting fungi. The CLRs Dectinl and Dectin2/3 recognize C. albicans yeast cells and hyphae by binding to the surface β-glucans and a-mannans of the two fungal forms, respectively. Recognition of these molecules results in release of inflammatory cytokines from dendritic cells and macrophages, which is critical for antifungal immunity. The mechanisms that control this CLR-mediated pro-inflammatory response to fungal infection are completely unknown. As disclosed herein, E3 ubiquitin ligase Cbl-b targets K48-linked poly-ubiquitination of Dectinl and 2, two key pattern recognition receptors for sensing C albicans, leading to Dectin internalization and degradation. Loss of Cbl-b function protects mice from systemic infection with a lethal dose of C albicans and deficiency of Dectin- 1, -2, or both in Cbl-b^"/_mice negates this protection. Importantly, silencing Cbl-b gene in vivo protects mice from lethal systemic C albicans infection. In addition, knocking down Cbl-b in human macrophages significantly enhances TNF-alpha and IL6 production upon infection with C albicans yeast and hyphae. Therefore, the disclosed data reveals that Cbl-b's negative regulation of Dectinl and 2 is crucial for homeostatic control of innate immune responses against C albicans infection. The disclosed data also indicate that Cbl-b is a host-directed therapeutic drug target for protection from disseminated Candidiasis.

Terminology

The term "subject" refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human or veterinary patient. The term "patient" refers to a subject under the treatment of a clinician, e.g., physician.

The term "therapeutically effective" refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.

By the term "effective amount" of a therapeutic agent is meant a nontoxic but sufficient amount of a beneficial agent to provide the desired effect. The amount of beneficial agent that is "effective" will vary from subject to subject, depending on the age and general condition of the subject, the particular beneficial agent or agents, and the like. Thus, it is not always possible to specify an exact "effective amount." However, an appropriate "effective" amount in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an "effective amount" of a beneficial can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts.

An "effective amount" of a drug necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. As used herein, a "therapeutically effective amount" of a therapeutic agent refers to an amount that is effective to achieve a desired therapeutic result, and a "prophylactically effective amount" of a therapeutic agent refers to an amount that is effective to prevent an unwanted physiological condition. Therapeutically effective and prophylactically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject.

The term "therapeutically effective amount" can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the drug and/or drug formulation to be administered (e.g., the potency of the therapeutic agent (drug), the concentration of drug in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art.

The term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.

The term "carrier" means a compound, composition, substance, or structure that, when in combination with a compound or composition, aids or facilitates preparation, storage, administration, delivery, effectiveness, selectivity, or any other feature of the compound or composition for its intended use or purpose. For example, a carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.

The term "treatment" refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

As used herein, the terms "treating" or "treatment" of a subject includes the administration of a drug to a subject with the purpose of preventing, curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving, stabilizing or affecting a disease or disorder, or a symptom of a disease or disorder. The terms "treating" and "treatment" can also refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, prevention of the occurrence of symptoms and/or their underlying cause, and improvement or remediation of damage.

As used herein, the term "preventing" a disorder or unwanted physiological event in a subject refers specifically to the prevention of the occurrence of symptoms and/or their underlying cause, wherein the subject may or may not exhibit heightened susceptibility to the disorder or event.

The phrase "small interfering RNA" or "siRNA" as used herein, refers to a double stranded RNA duplex of any length, with or without single strand overhangs, wherein at least one strand is homologous to the target mRNA to be degraded. The difference between antisense and double stranded small interfering molecules is that an antisense molecule is a single stranded oligonucleotide which is complementary to a section of the target RNA and must hybridize or bind to it in a 1 : 1 ratio in order to cause its degradation. In contrast, siRNA provides a substrate for the RNA-induced silencing complex (RISC), and unlike antisense, is inactive until incorporated into this macromolecular complex. This RISC complex is then guided by the unwound siRNA to its target gene. Once the target gene is located, it is destroyed by cleaving the target gene into small pieces, and thereby preventing its expression. Reference to an siRNA can also refer to just one strand of the siRNA duplex, where the opposing strand is inferred by perfect complementarity (with or without additional nucleotide overhangs). In a preferred embodiment, the siRNA comprises a double-stranded RNA duplex of at least about 15, or at least about 19, nucleotides with no overhanging nucleotides. In another embodiment, the siRNA has nucleotide overhangs. For example, the siRNA may have two nucleotide overhangs, thus the siRNA can comprise a 21 nucleotide sense strand and a 21 nucleotide antisense strand paired so as to have a 19 nucleotide duplex region. The number of nucleotides in the overhang can be in the range of about 1 to about 6, about 2 to about 4, or about 3 homologous nucleotide overhangs at each of the 5' and 3' ends. The nucleotide overhang can be modified, for example to increase nuclease resistance. The terms "about" and "approximately" are defined as being "close to" as understood by one of ordinary skill in the art. In one non-limiting embodiment the terms are defined to be within 10%. In another non-limiting embodiment, the terms are defined to be within 5%. In still another non-limiting embodiment, the terms are defined to be within 1%.

Methods

In one aspect, disclosed herein is a method for treating a fungal infection in a subject that involves administering to the subject a composition comprising a therapeutically effective amount of a casitas B lymphoma-b (CBLB) inhibitor.

The disclosed methods can in some embodiments be used to treat any fungal infection. In particular embodiments, the disclosed methods can be used to treat a Candida spp. infection. In some embodiments, the fungal infection comprises Candida auris, aspergillosis, Pneumocystis carinii pneumonia (PCP), coccidioidomycosis (valley fever), cryptococcosis, histoplasmosis, or a combination thereof.

In some embodiments, the CBLB inhibitor comprises functional nucleic acid that knocks down expression of the Cbl-b gene in the subject. For example, the functional nucleic acid comprises an siRNA.

In particular embodiments, the siRNA comprises the nucleic acid sequence 5'- AAAUUCUCGAAGUAUGCUCUU-3 ' (SEQ ID NO: 1), or a variant thereof having at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1.

In some embodiments, the CBLB inhibitor comprises a functional nucleic acid that knocks down expression of the Cbl-b gene in the subject. In some embodiments, the functional nucleic acid comprises an siRNA. In some embodiments, the functional nucleic acid is from about 15 to about 25 nucleotides. In some embodiments, the siRNA comprises the nucleic acid sequence 5'- UUUGCUAACGGACCAGUACUU-3 ' (SEQ ID NO:2) or 5'- UAAUACCCAAAAUUCGACCUU-3 ' (SEQ ID NO:3).

In particular embodiments, the siRNA comprises the nucleic acid sequence 5'- UAAUACCCAAAAUUCGACCUU-3 ' (SEQ ID NO:3), or a variant thereof having at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:3.

In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

In particular embodiments, the siRNA comprises the nucleic acid sequence 5'- AAAUUCUCGAAGUAUGCUCUU-3 ' (SEQ ID NO : 1 ), or a variant thereof having at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1. In some embodiments, the fungal infection comprises a Candida albicans infection. In some embodiments, the fungal infection comprises aspergillosis, Pneumocystis carinii pneumonia (PCP), coccidioidomycosis (valley fever), cryptococcosis, histoplasmosis, or a combination thereof. In some embodiments, the fungal infection comprises aspergillosis, Pneumocystis carinii pneumonia (PCP), coccidioidomycosis (valley fever), or a combination thereof.

In particular embodiments, the siRNA comprises the nucleic acid sequence 5'- UAAUACCCAAAAUUCGACCUU-3 ' (SEQ ID NO:3), or a variant thereof having at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 3.

In some embodiments, the Cbl-b gene sequence is the human Cbl-b gene sequence is SEQ ID NO:4 (Homo sapiens Cbl proto-oncogene B (CBLB), transcript variant 1, mRNA; NCBI Reference Sequence: NM 001321786.1). In some embodiments, the Cbl-b gene sequence is the mouse Cbl-b gene sequence Accession Number NM_001033238.1 (Mus musculus Casitas B- lineage lymphoma b (Cblb), mRNA; NCBI Reference Sequence: NM_001033238.1). In some embodiments, the siRNA comprises a nucleic acid sequence from about 15 to about 25 nucleotides targeting the human Cbl-b gene sequence. In some embodiments, the siRNA comprises a nucleic acid sequence from about 15 to about 25 nucleotides targeting SEQ ID NO:4.

In some embodiments, the shRNA comprises a nucleic acid sequence from about 15 to about 25 nucleotides (length of the duplex region is from 15 to 25 nucleotides) targeting the human Cbl-b gene sequence. In some embodiments, the shRNA comprises the nucleic acid sequence from about 15 to about 25 nucleotides targeting SEQ ID NO:4.

In some embodiments, disclosed herein is a method for treating a Candida albicans fungal infection in a subject that involves administering to the subject a composition comprising a therapeutically effective amount of an siRNA, wherein the siRNA comprises a nucleic acid sequence from about 15 to about 25 nucleotides targeting SEQ ID NO:4.

In some embodiments, disclosed herein is a method for treating a Candida albicans fungal infection in a subject that involves administering to the subject a composition comprising a therapeutically effective amount of an siRNA, wherein the siRNA is selected from the SEQ ID NO: l, SEQ ID NO:2, or SEQ ID NO:3.

CBLB inhibitors are well known in the prior art. For example, siRNA against cbl-b for treating cancer are disclosed in U.S. Patent No. 9,186,373, which is incorporated by reference in its entirety for the teaching of these siRNA. CBLB inhibitors have also been described for example in Loeser et al. (JEM (2007) doi: 10.1084/iem.20061699; Chiang et al. (Journal of Clinical Investigation 117 (4) (2007): 1033-1034); Lametschwandtner et al. (Journal of

Immunotherapy 31 (9) (2008): 943); Paolini et al. (J. Immunol. 2011 Feb. 15; 186 (4): 2138-47). US 2007/0087988 relates to a method for regulating HPKl, whose expression may be enhanced by increasing the expression of Cbl-b, and vice versa (e. g. by Cbl-b siRNA inhibition).

Additional CBLB inhibitors are disclosed, for example, in US20070054355; WO 2005007141; US 9334522; which are incorporated by reference in their entirety.

In some embodiments, the CBLB inhibitor reduces or inhibits the function of CBLB by reducing or inhibiting the expression of CBLB or Cbl-b. The terms "reduce/reduction" or "inhibit/inhibition" relate to a reduction or inhibition of the function (or expression) of CBLB or Cbl-b as compared to the unmodified natural function, optionally including the complete inhibition of said function. Preferably, the function (or expression) is reduced by at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%. In some embodiments, the reduction or inhibition of the function of CBLB or Cbl-b is transient, i. e. the function is only temporarily reduced as described in the above and can therefore recover again, e. g. by consumption or degradation of inhibitors, such as Cbl-b siRNA, or by restructuring or non-CW- -impaired cells in vivo. The transient reduction of Cbl-b in immune cells can also be performed in a repetitive manner, e. g. until a therapeutic success has been achieved.

In some embodiments, the expression of CBLB or Cbl-b is reduced or inhibited by the use of Cbl-b antisense RNA or siRNA. For this purpose, short DNA and/or RNA sequences that are complementary to one of the regions of the target {Cbl-b) mRNA sequence are employed, so that hybridization and inactivation of the corresponding sequences will occur. These sequences preferably have a length of at least 15, 18, 20, 22, 25, 28, 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180 or up to 200 bases until the length of the complete target sequence is reached, preferably up to 2500, 2000, 1500, 1000, 500 or 300 bases. In some embodiments, the sequence of SEQ ID No. 1 is used.

The CBLB inhibitor of the provided method can be a functional nucleic acid. Functional nucleic acids are nucleic acid molecules that have a specific function, such as binding a target molecule or catalyzing a specific reaction. Functional nucleic acid molecules can be divided into the following categories, which are not meant to be limiting. For example, functional nucleic acids include antisense molecules, aptamers, ribozymes, triplex forming molecules, RNAi, and external guide sequences. The functional nucleic acid molecules can act as affectors, inhibitors, modulators, and stimulators of a specific activity possessed by a target molecule, or the functional nucleic acid molecules can possess a de novo activity independent of any other molecules.

Functional nucleic acid molecules can interact with any macromolecule, such as DNA, RNA, polypeptides, or carbohydrate chains. Thus, functional nucleic acids can interact with the mRNA of Cbl-b or the genomic DNA of Cbl-b or they can interact with the polypeptide CBLB.

Often functional nucleic acids are designed to interact with other nucleic acids based on sequence homology between the target molecule and the functional nucleic acid molecule. In other situations, the specific recognition between the functional nucleic acid molecule and the target molecule is not based on sequence homology between the functional nucleic acid molecule and the target molecule, but rather is based on the formation of tertiary structure that allows specific recognition to take place.

Antisense molecules are designed to interact with a target nucleic acid molecule through either canonical or non-canonical base pairing. The interaction of the antisense molecule and the target molecule is designed to promote the destruction of the target molecule through, for example, RNAseH mediated RNA-DNA hybrid degradation. Alternatively the antisense molecule is designed to interrupt a processing function that normally would take place on the target molecule, such as transcription or replication. Antisense molecules can be designed based on the sequence of the target molecule. Numerous methods for optimization of antisense efficiency by finding the most accessible regions of the target molecule exist. Exemplary methods would be in vitro selection experiments and DNA modification studies using DMS and DEPC. It is preferred that antisense molecules bind the target molecule with a dissociation constant (Kd)less than or equal to 10^"6, 10^"8, 10^"10, or 10^"12. A representative sample of methods and techniques which aid in the design and use of antisense molecules can be found in U.S. Patent Nos. 5, 135,917, 5,294,533, 5,627, 158, 5,641,754, 5,691,317, 5,780,607, 5,786, 138, 5,849,903, 5,856, 103, 5,919,772, 5,955,590, 5,990,088, 5,994,320, 5,998,602, 6,005,095, 6,007,995, 6,013,522, 6,017,898, 6,018,042, 6,025, 198, 6,033,910, 6,040,296, 6,046,004, 6,046,319, and 6,057,437.

Aptamers are molecules that interact with a target molecule, preferably in a specific way.

Typically aptamers are small nucleic acids ranging from 15-50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets. Aptamers can bind small molecules, such as ATP (U. S. Patent No. 5,631, 146) and theophiline (U.S. Patent No. 5,580,737), as well as large molecules, such as reverse transcriptase (U. S. Patent No. 5,786,462) and thrombin (United States patent 5,543,293). Aptamers can bind very tightly with K_d's from the target molecule of less than 10-12 M. It is preferred that the aptamers bind the target molecule with a Kd less than 10^"6, 10^"8, 10^"10, or 10^"12. Aptamers can bind the target molecule with a very high degree of specificity. For example, aptamers have been isolated that have greater than a 10,000 fold difference in binding affinities between the target molecule and another molecule that differ at only a single position on the molecule (U. S. Patent No.

5,543,293). It is preferred that the aptamer have a Kd with the target molecule at least 10, 100, 1000, 10,000, or 100,000 fold lower than the Kd with a background binding molecule. It is preferred when doing the comparison for a polypeptide for example, that the background molecule be a different polypeptide. Representative examples of how to make and use aptamers to bind a variety of different target molecules can be found in U.S. Patent Nos. 5,476,766, 5,503,978, 5,631, 146, 5,731,424 , 5,780,228, 5,792,613, 5,795,721, 5,846,713, 5,858,660 , 5,861,254, 5,864,026, 5,869,641, 5,958,691, 6,001,988, 6,01 1,020, 6,013,443, 6,020, 130, 6,028, 186, 6,030,776, and 6,051,698. Ribozymes are nucleic acid molecules that are capable of catalyzing a chemical reaction, either intramolecularly or intermolecularly. Ribozymes are thus catalytic nucleic acid. It is preferred that the ribozymes catalyze intermolecular reactions. There are a number of different types of ribozymes that catalyze nuclease or nucleic acid polymerase type reactions which are based on ribozymes found in natural systems, such as hammerhead ribozymes, (U.S. Patent Nos. 5,334,711, 5,436,330, 5,616,466, 5,633,133, 5,646,020, 5,652,094, 5,712,384, 5,770,715, 5,856,463, 5,861,288, 5,891,683, 5,891,684, 5,985,621, 5,989,908, 5,998, 193, 5,998,203;

International Patent Application Nos. WO 9858058 by Ludwig and Sproat, WO 9858057 by Ludwig and Sproat, and WO 9718312 by Ludwig and Sproat) hairpin ribozymes (for example, U.S. Patent Nos. 5,631,115, 5,646,031, 5,683,902, 5,712,384, 5,856,188, 5,866,701, 5,869,339, and 6,022,962), and tetrahymena ribozymes (for example, U.S. Patent Nos. 5,595,873 and 5,652, 107). There are also a number of ribozymes that are not found in natural systems, but which have been engineered to catalyze specific reactions de novo (for example, U.S. Patent Nos. 5,580,967, 5,688,670, 5,807,718, and 5,910,408). Preferred ribozymes cleave RNA or DNA substrates, and more preferably cleave RNA substrates. Ribozymes typically cleave nucleic acid substrates through recognition and binding of the target substrate with subsequent cleavage. This recognition is often based mostly on canonical or non-canonical base pair interactions. This property makes ribozymes particularly good candidates for target specific cleavage of nucleic acids because recognition of the target substrate is based on the target substrates sequence. Representative examples of how to make and use ribozymes to catalyze a variety of different reactions can be found in U.S. Patent Nos. 5,646,042, 5,693,535, 5,731,295, 5,811,300, 5,837,855, 5,869,253, 5,877,021, 5,877,022, 5,972,699, 5,972,704, 5,989,906, and 6,017,756.

Triplex forming functional nucleic acid molecules are molecules that can interact with either double-stranded or single- stranded nucleic acid. When triplex molecules interact with a target region, a structure called a triplex is formed, in which there are three strands of DNA forming a complex dependant on both Watson-Crick and Hoogsteen base-pairing. Triplex molecules are preferred because they can bind target regions with high affinity and specificity. It is preferred that the triplex forming molecules bind the target molecule with a Kd less than 10-6, 10-8, 10-10, or 10-12. Representative examples of how to make and use triplex forming molecules to bind a variety of different target molecules can be found in U.S. Patent Nos.

5, 176,996, 5,645,985, 5,650,316, 5,683,874, 5,693,773, 5,834,185, 5,869,246, 5,874,566, and 5,962,426. External guide sequences (EGSs) are molecules that bind a target nucleic acid molecule forming a complex, and this complex is recognized by RNase P, which cleaves the target molecule. EGSs can be designed to specifically target a RNA molecule of choice. RNAse P aids in processing transfer RNA (tRNA) within a cell. Bacterial RNAse P can be recruited to cleave virtually any RNA sequence by using an EGS that causes the target RNA:EGS complex to mimic the natural tRNA substrate. (WO 92/03566 by Yale, and Forster and Altman, Science 238:407-409 (1990)).

Similarly, eukaryotic EGS/RNAse P-directed cleavage of RNA can be utilized to cleave desired targets within eukarotic cells. (Yuan et al., Proc. Natl. Acad. Sci. USA 89:8006-8010 (1992); WO 93/22434 by Yale; WO 95/24489 by Yale; Yuan and Altman, EMBO J 14: 159-168 (1995), and Carrara et al., Proc. Natl. Acad. Sci. (USA) 92:2627-2631 (1995)). Representative examples of how to make and use EGS molecules to facilitate cleavage of a variety of different target molecules be found in U.S. Patent Nos. 5, 168,053, 5,624,824, 5,683,873, 5,728,521, 5,869,248, and 5,877,162.

Gene expression can also be effectively silenced in a highly specific manner through RNA interference (RNAi). This silencing was originally observed with the addition of double stranded RNA (dsRNA) (Fire,A., et al. (1998) Nature, 391 :806-11; Napoli, C, et al. (1990) Plant Cell 2:279-89; Hannon, G.J. (2002) Nature, 418:244-51). Once dsRNA enters a cell, it is cleaved by an RNase III -like enzyme, Dicer, into double stranded small interfering RNAs (siRNA) 21- 23 nucleotides in length that contains 2 nucleotide overhangs on the 3 ' ends (Elbashir, S.M., et al. (2001) Genes Dev., 15: 188-200; Bernstein, E., et al. (2001) Nature, 409:363-6; Hammond, S.M., et al. (2000) Nature, 404:293-6). In an ATP dependent step, the siRNAs become integrated into a multi-subunit protein complex, commonly known as the RNAi induced silencing complex (RISC), which guides the siRNAs to the target RNA sequence (Nykanen, A., et al. (2001) Cell, 107:309-21). At some point the siRNA duplex unwinds, and it appears that the antisense strand remains bound to RISC and directs degradation of the complementary mRNA sequence by a combination of endo and exonucleases (Martinez, J., et al. (2002) Cell, 110:563-74). However, the effect of iRNA or siRNA or their use is not limited to any type of mechanism.

Short Interfering RNA (siRNA) is a double-stranded RNA that can induce sequence- specific post-transcriptional gene silencing, thereby decreasing or even inhibiting gene expression. In one example, an siRNA triggers the specific degradation of homologous RNA molecules, such as mRNAs, within the region of sequence identity between both the siRNA and the target RNA. For example, WO 02/44321 discloses siRNAs capable of sequence-specific degradation of target mRNAs when base-paired with 3' overhanging ends, herein incorporated by reference for the method of making these siRNAs. Sequence specific gene silencing can be achieved in mammalian cells using synthetic, short double-stranded RNAs that mimic the siRNAs produced by the enzyme dicer (Elbashir, S.M., et al. (2001) Nature, 411 :494 498) (Ui- Tei, K., et al. (2000) FEBS Lett 479:79-82). siRNA can be chemically or in vitro-synthesized or can be the result of short double-stranded hairpin-like RNAs (shRNAs) that are processed into siRNAs inside the cell. Synthetic siRNAs are generally designed using algorithms and a conventional DNA/RNA synthesizer. Suppliers include Ambion (Austin, Texas), ChemGenes (Ashland, Massachusetts), Dharmacon (Lafayette, Colorado), Glen Research (Sterling, Virginia), MWB Biotech (Esbersberg, Germany), Proligo (Boulder, Colorado), and Qiagen (Vento, The Netherlands). siRNA can also be synthesized in vitro using kits such as Ambion's SILENCER® siRNA Construction Kit. Disclosed herein are any siRNA designed as described above based on the sequences for Cbl-b.

The production of siRNA from a vector is more commonly done through the transcription of a short hairpin RNAs (shRNAs). Kits for the production of vectors comprising shRNA are available, such as, for example, Imgenex's GENESUPPRESSOR™ Construction Kits and Invitrogen's BLOCK-IT™ inducible RNAi plasmid and lentivirus vectors. Disclosed herein are any shRNA designed as described above based on the sequences for the herein disclosed inflammatory mediators.

Also disclosed is pharmaceutical composition comprising a CBLB inhibitor in a pharmaceutically acceptable carrier. In some embodiments, the composition comprises a pharmaceutically acceptable carrier that is suitable for the intracellular administration in a patient. In particular, the composition comprises vehicles such as liposomal or microsomal formulations which are particularly preferred for the administration of nucleic acids.

Pharmaceutical compositions may comprise pharmaceutically suitable salts as well as additional buffers, tonicity components or pharmaceutically acceptable carriers. In particular, inhibitory nucleic acids, such as antisense nucleic acids, siRNA and shRNA, may be provided in suitable therapeutic vector systems. Pharmaceutical carrier substances are provided to improve the tolerability of the composition and to improve the solubility and bioavailability of the active ingredients. Examples include emulsifying agents, thickening agents, redox components, starch, alcohol solutions, polyethylene glycol or lipids. The selection of a suitable pharmaceutical carrier strongly depends on the mode of administration. For oral administration, liquid or solid carriers can be used, whereas final compositions in liquid form are advantageous for injections.

In some embodiments, the pharmaceutical composition comprises buffer substances or tonic substances. By using a buffer, it is possible to adjust the pH value of the pharmaceutical composition to physiological conditions and to attenuate or buffer pH variations. An example for such a substance is a phosphate buffer. Tonicity agents are used to adjust the osmolarity and may comprise ionic substances, such as inorganic salts, e. g. NaCl, or non-ionic substances, e. g. glycerol, or carbohydrates.

In some embodiments, the composition is provided to be suitable for a systemic, topical, oral or intranasal administration. The pharmaceutical composition can be suitable for intravenous, intraarterial, intramuscular, intravascular, intraperitoneal or subcutaneous administration. For instance, injection or transfusions are suitable for this purpose. Administering the pharmaceutical composition directly into the bloodstream will have the advantage that the active ingredients of the pharmaceutical composition are distributed throughout the body and are thus capable of reaching their target tissues quickly. In addition, topical applications are provided. The administration either directly to or in the vicinity of a site at which an immune response is to be induced or enhanced, e. g. the site of an infection.

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

EXAMPLES

Example 1: Targeting CBLB as a therapeutic approach for disseminated candidiasis.

The fungi-responsive C-type lectin receptors (CLRs) have a central role in the detection of Candida during bloodstream infection. In normal hosts, Candida albicans is controlled by activation of innate immune cells via cell surface pattern-recognition receptors (PRRs) such as toll-like receptor 2 (TLR2) and CLRs that detect the infecting fungus. The CLRs dectin-1 (encoded by Clec7a in mice) and dectin-2 (encoded by Clec4n in mice) recognize C. albicans yeast cells and hyphae by binding to surface β-glucans and a-mannans on the two fungal forms, respectively (Taylor, P.R. et al. Nat. Immunol. 8, 31-38 (2007); Saijo, S. et al. Immunity 32, 681-691 (2010); Zhu, L.L. et al. Immunity 39, 324-334 (2013)). Recognition of these molecules results in the release of inflammatory cytokines from innate immune cells, which is critical for antifungal immunity (Hernandez-Santos, N. et al. Cell Host Microbe 11, 425-435 (2012)).

However, the regulation of dectin-mediated signaling pathways, including those involving spleen tyrosine kinase (SYK), that control the pro-inflammatory response to fungal infection, is completely unknown.

Casitas B lymphoma-b (CBLB), a member of the RING-finger-type E3 ubiquitin ligases, directs the ubiquitination of an array of signaling proteins. A crucial role has been shown for

CBLB in T cell activation, tolerance induction, and TH2 and TH9 cell differentiation (Bachmaier, K. et al. Nature 403, 211-216 (2000); Jeon, M.S. et al. Immunity 21, 167-177 (2004); Qiao, G. et al. Mol. Cell. Biol. 28, 2470-2480 (2008); Li, D. et al. J. Immunol. 173, 7135-7139 (2004); Zhang, J. et al. J. Immunol. 169, 2236-2240 (2002); Guo, H. et al. Cell Rep. 1, 472-482 (2012); Qiao, G. et al. J. Immunol. 191, 632-639 (2013); Qiao, G. et al. Cell Rep. 6, 709-723 (2014)), but its role in innate immune responses is unclear. Here it is disclosed that CBLB functions as a negative regulator of fungal recognition during systemic C albicans infection by targeting dectin-1, dectin -2, and SYK for Lys48 (K48)-linked polyubiquitination. Negative regulation of dectin-1- and dectin-2-mediated signaling by CBLB is crucial for restraining the magnitude of the innate immune responses against C albicans infection, but it leads to suboptimal protection of the host. Systemic in vivo delivery of Cblb-speciiic siRNA protects C57BL/6 mice from systemic C albicans infection. Therefore, the disclosed data show that CBLB is a drug target for systemic candidiasis.

Results

CBLB inhibits signaling via dectin receptors

To determine the role of CBLB in innate immune responses, wild-type (WT) and Cblb^~h bone marrow (BM)-derived macrophages (BMDMs) and BM-derived dendritic cells (BMDCs) were stimulated with ligands for TLRs 1-9 or with zymosan (a ligand for TLR2 and dectin-1).

Whereas TLR ligand-induced production of tumor necrosis factor (TNF)-a and interleukin (IL)-6 was comparable between WT and Cblb^~h BMDMs and BMDCs, zymosan-induced TNF-a and

IL-6 production was substantially higher in Cblb^~h BMDMs and BMDCs than in WT cells (Fig.

7a,b). Given that zymosan activates both TLR2 and dectin-1 (Brown, G.D. et al. Nat. Rev.

Immunol. 6, 33^13 (2006)), this result suggests that CBLB could regulate the dectin-1 signaling pathway. To directly test this WT and Cblb^~h BMDMs and BMDCs were stimulated with curdlan, a purified β-glucan that specifically activates dectin-1 (Yoshitomi, H. et al. J. Exp. Med.

201, 949-960 (2005)). Curdlan stimulation induced a markedly higher level of TNF-a and IL-6 in Cblb^~'^~ BMDMs and BMDCs than in WT cells (Fig. 7a,b).

To confirm this observation and to determine whether CBLB regulates other dectin family members BMDMs, BMDCs, and BM neutrophils from WT and Cblb^~ mice were infected with a C albicans yeast-only mutant (capl; hereafter referred to as yeast), in which the adenylate-cyclase-associated protein-1 (Capl) gene was disrupted, causing the failure of yeast- hypha transition due to the lack of cAMP (Bahn, Y.S. et al. J. Bacterid. 183, 3211-3223

(2001)). Dectin-1 and dectin-2 recognize the yeast and hyphal forms of C albicans, respectively, by binding to the surface β-glucans (dectin-1) and a-mannans (dectin-2) of the two fungal forms (Taylor, P R. et al. Nat. Immunol. 8, 31-38 (2007); Saijo, S. et al. Immunity 32, 681-691 (2010); Zhu, L.L. et al. Immunity 39, 324-334 (2013)). CBLB deficiency resulted in increased production of TNF-a and IL-6 by BMDMs and BMDCs in response to signaling via both the yeast and hyphal forms of C. albicans after infection (Fig. la, Fig. 8a). In contrast, Cblb^~h neutrophils produced comparable amounts of TNF-a and IL-6 to those produced in WT neutrophils, except at the 3-h time point after infection (Fig. 8b), suggesting that CBLB may have a limited role in affecting the inflammatory response of neutrophils to C. albicans infection. Cblb^~h BMDMs also produced more TNF-a and IL-6 than WT BMDMs infected with

Aspergillus fumigatus conidia (Fig. lb), a prevalent fungus that causes potentially lethal infections in immunosuppressed patients (Hohl, T.M. et al. Eukaryot. Cell 6, 1953-1963 (2007)). This finding is notable, as dectin-1 is a major PRR recognizing A. fumigatus (Steele, C. et al. PLoS Pathog. 1, e42 (2005); Gersuk, G.M. et al. J. Immunol. 176, 3717-3724 (2006); Rivera, A. et al. J. Exp. Med. 208, 369-381 (2011)). Therefore, CBLB has the potential to regulate the dectin family of CLRs in response to some fungal pathogens. Because several studies have indicated that either the NLRP3 inflammasome or a noncanonical, caspase-8-mediated inflammasome participates in host defense to C. albicans infection (Hise, A.G. et al. Cell Host Microbe 5, 487-497 (2009); Gringhuis, S.I. et al. Nat. Immunol. 13, 246-254 (2012)), IL-Ιβ production by WT and Cblb^_/~ BMDMs after infection with C. albicans yeast and hyphae was measured. Both WT and Cblb^_/~ BMDMs produced comparable levels of IL-Ιβ (Fig. la), suggesting that CBLB does not regulate the inflammasome activation that is mediated by dectin- 1 or dectin-2.

A recent report showed that β-glucan of C. albicans induces a strong IL-IRA response in human peripheral blood mononuclear cells (PBMCs), which is independent of dectin-1 and complement receptor 3 (CR3) (Smeekens, S.P. et al. Cytokine 71, 215-222 (2015)). To test whether CBLB affects the release of anti-inflammatory stimuli such as IL-IRA, the production of IL-IRA in BMDMs from WT and Cblb^~ mice after infection with live C. albicans yeast and hyphae was measured. Data showed that there was no significant difference in IL-IRA release between WT and Cblb^~ BMDMs infected with both forms of C. albicans (Fig. lc). These data suggest that CBLB does not modulate the release of IL-IRA.

To determine whether CBLB has a similar effect on human macrophages infected with C. albicans, human monocyte-derived macrophages (hMDMs) were generated (Kang, P.B. et al. J.

Exp. Med. 202, 987-999 (2005); Rajaram, M.V. et al. Proc. Natl. Acad. Sci. USA 108, 17408-

17413 (2011)) and transfected with a Cblb-speciiic siRNA or a control scrambled siRNA.

Consistent with the mouse results, silencing Cblb in hMDMs resulted in markedly increased production of TNF-a and IL-6 after infection with C. albicans yeast and hyphae, with IL-6 production being more profound (Fig. 9a,b). These results also correlated with impaired down- modulation of dectin-1 and dectin-2 expression (Fig. 9d), thus indicating that our observations in mouse macrophages can be recapitulated in human macrophages.

CBLB binds to dectin-1 and dectin-2 in macrophages after infection with C. albicans Dectin family CLRs have a major role in fungal recognition and host innate responses against fungal infection (Brown, G.D. et al. Nat. Rev. Immunol. 6, 33-43 (2006); Brown, G.D. et al. Annu. Rev. Immunol. 29, 1-21 (2011); Hardison, S.E. et al. Nat. Immunol. 13, 817-822 (2012)). Dectin-l 's cytoplasmic tail contains an immunoreceptor tyrosine-based activation motif (IT AM) that can be phosphorylated by Src family kinases. Phosphorylated dectin-1 in turn, recruits and activates SYK, thereby initiating downstream signaling via the CARD9-BCL10- MALT1 complex (Brown, G.D. et al. Nat. Rev. Immunol. 6, 33-43 (2006); Hardison, S.E. et al. Nat. Immunol. 13, 817-822 (2012)). Because dectin-2 lacks this ITAM-like motif, it binds FcR-γ (Saijo, S. et al. Immunity 32, 681-691 (2010)), which contains ITAMs (Osorio, F. et al.

Immunity 34, 651-664 (2011)) that recruit SYK and transduce dectin-2 signaling (Kerscher, B. et al. Int. Immunol. 25, 271-277 (2013); Miyake, Y. et al. Immunity 38, 1050-1062 (2013); Sato, K. et al. J. Biol. Chem. 281, 38854-38866 (2006)). Experiments were conducted to determine whether and how CBLB regulates signaling via dectin-1 and dectin-2 during C.

albicans infection. First, it was determined whether CBLB physically interacts with dectin receptors or their signaling intermediates, and if so, how this occurs. To this end, WT BMDMs were infected with C. albicans yeast cells or hyphae for different amounts of time. Using co- immunoprecipitation (co-IP) analyses, that CBLB was inducibly associated with dectin-1, dectin- 2, SYK, and CARD9 after infection with C. albicans yeast cells or hyphae (Fig. 2a,b).

It has previously been shown that CBLB binds to SYK in B cells after B cell receptor (BCR) stimulation (Sohn, H.W. et al. J. Exp. Med. 197, 1511-1524 (2003)) or to CARD11 (also known as CARMA1), a homolog of CARD9, in NKT cells (Kojo, S. et al. Proc. Natl. Acad. Sci. USA 106, 17847-17851 (2009)). To determine whether SYK and CARD9 are potential binding partners of CBLB in the signaling pathways downstream of dectin-1 and dectin-2, Syk gene expression was silenced in WT BMDMs by using a -S ^-specific siRNA. Knocking down Syk expression did not affect the association of CBLB with either dectin-1 or dectin-2 (Fig. 2c). Similarly, CARD9 deficiency also did not affect CBLB-dectin-1 or CBLB-dectin-2 association (Fig. 2d). Experiments were next conducted to determine whether phosphorylation of the IT AM within dectin-1 and the ITAMs within the IgE high-affinity receptor FcR-γ (encoded by Fcergl in mice) is required for CBLB association in macrophages after C. albicans infection (with yeast cells and hyphae). To accomplish this, the tyrosine of the hemi-ITAM was mutated to phenylalanine in dectin-l 's cytoplasmic tail (CLEC7A^Y15F) and the tyrosines within the ITAMs of FcR-γ were mutated to phenylalanine (FCER1G^{Y65F Y76F}), then Clec7a^~'^~ BMDMs and Fcergl^~h BMDMs with these mutated alleles were reconstituted and infected with C. albicans yeast and hyphae, respectively. Mutating dectin-1 at Tyrl5, or FcR-γ at Tyr65 and Tyr76, completely abrogated the binding of CBLB to dectin-1 or dectin-2 (Fig. 2e,f), indicating that phospho-Tyrl5 of dectin-1 or phospho-Tyr65 and phospho-Tyr76 of FcR-γ is critical for their binding to CBLB. Indeed, CBLB bound to FcR-γ in WT BMDMs after infection with C.

albicans hyphae (Fig. 2g).

Dectin-1, dectin-2, and SYK are targets of CBLB

To determine whether dectin-1 and dectin-2, or the downstream signaling molecules, are the targets of CBLB, protein stability of dectin-1, dectin-2, SYK, and CARD9 was first examined in macrophages infected with C albicans yeast cells or hyphae. Notably, dectin-1 and dectin-2, but not SYK or CARD9, underwent degradation in WT BMDMs after infection with C. albicans yeast cells and hyphae, but not in BMDMs lacking CBLB (Fig. 3a). These findings suggest that dectin receptors are the likely targets of CBLB. Furthermore, dectin-1 and dectin-2 degradation was completely abrogated by pretreatment with E-64, a lysosome inhibitor, but not with MG-132, a proteasome inhibitor (Fig. 3b), suggesting that dectin-1 and dectin-2 undergo lysosome-mediated degradation.

To further determine whether CBLB is the E3 ubiquitin ligase for dectin-1 or dectin-2,

BMDMs generated from WT mice, Cblb^~ mice, or mice expressing an E3-ligase-dead mutant of CBLB (Cblb^C373A) (Oksvold, M.P. et al. Mol. Immunol. 45, 925-936 (2008)) were infected with C albicans yeast cells or hyphae. Expression of the CBLB^C373A mutant abrogated ubiquitination of dectin-1 and dectin-2 (Fig. 3c,d, upper panel, Fig. 1 la,b). To determine whether ubiquitination of dectin-1 or dectin-2 is K48- or K63-linked, K48-ubiquitin- or K63-ubiquitin-specific antibodies were used. It was confirmed that both dectin-1 and dectin-2 underwent K48-linked polyubiquitination, and that this K48-linked polyubiquitination of dectin-1- and dectin-2 was abrogated in BMDMs lacking CBLB or expressing the CBLB^C373A mutant (Fig. 3c,d, bottom, Fig. l la,b).

It was previously shown that CBLB targets SYK for polyubiquitination but not for degradation in B cells (Sohn, H.W. et al. J. Exp. Med. 197, 1511-1524 (2003)). To determine whether SYK is also a potential target of CBLB in macrophages triggered by dectin-1 or dectin-2 receptor-ligand interactions, SYK ubiquitination was examined in WT and C£/£^C373A-expressing

BMDMs after infection with C. albicans yeast cells or hyphae. Indeed, SYK underwent K48- linked polyubiquitination after infection with both C. albicans yeast and hyphae, but this ubiquitination was greatly reduced in BMDMs expressing CBLB^C373A (Fig. 1 lc,d). Therefore, data suggest that dectin-1, dectin-2, and SYK are targets of CBLB and that CBLB keeps the expression of these CLRs in check. Consistent with these data, SYK and the transcription factor F-KB were highly activated in BMDMs lacking CBLB after infections with C. albicans yeast and hyphae (Fig. l ie).

To examine the functional relevance of CBLB-mediated ubiquitination of dectin-1 and dectin-2, site-directed mutagenesis was used to generate single and triple lysine-to-arginine mutations in the genes encoding dectin-1 and dectin-2 (to yield CLEC7A^K2R, CLEC7A^K27R, CLEC7A^K34R, CLEC7A^K2R'^K27R'^K34R, and CLEC4N^K10R). BMDMs lacking dectin-1 (from

Clec7a^~ mice) were reconstituted with constructs expressing either WT dectin-1 or the dectin-1 lysine-to arginine mutants and BMDMs lacking dectin-2 (from Clec4n^~ mice) with a construct expressing either WT dectin-2 or the CLEC4N^K10R mutant. These reconstituted BMDMs were then infected with C. albicans yeast cells or hyphae. Reconstituting Clec7a^~ BMDMs with WT dectin-1 or the single mutants completely or partially restored dectin-1 ubiquitination; however, expression of the dectin-1 triple mutant did not result in dectin-1 ubiquitination (Fig. 3e). As expected, Clec4n^~h BMDMs expressing WT dectin-2, but not the dectin-2 mutant

(CLEC4N^K10R), restored ubiquitination of dectin-2 (Fig. 3f). These data indicate that Lys2, Lys7, and Lys34 in dectin-1 and LyslO in dectin-2 are the sites of ubiquitination in dectin-1 and dectin- 2, respectively. Consistent with these data, Clec7a^~ BMDMs expressing the dectin-1 triple mutant (CLEC7A^{K2R K27R K34R}) or Clec4n '^~ BMDMs reconstituted with CLEC4N^K10R produced significantly higher amounts of TNF-a and IL-6 after infection with C. albicans yeast cells or hyphae (Fig. 3g,h).

CBLB regulates the internalization of dectin-1 and dectin-2, and their trafficking to the lysosome

Cell surface receptor internalization can occur when receptors are mono- or

polyubiquitinated following ligand-induced activation, and these receptors are subsequently sorted into endocytic vesicles for delivery to the lysosome for degradation (Sorkin, A. et al. Nat.

Rev. Mol. Cell Biol. 3, 600-614 (2002); Haglund, K. et al. Nat. Cell Biol. 5, 461-466 (2003); Lin, Q. et al. Mol. Cell. Biol. 30, 1541-1554 (2010)). Internalization of dectin-1 has been shown to terminate inflammatory responses to keep inflammation in check (Hernanz-Falcon, P. et al.

Eur. J. Immunol. 39, 507-513 (2009)). Thus, impaired down-modulation of dectin-1 and dectin-

2 could be due to a lack of internalization or a block in intracellular vesicle sorting to the lysosome. To determine whether CBLB is critical for this process, the levels of cell-surface- expressed and internally expressed dectin-1 and dectin-2 in BMDMs from WT and Cblb^~h mice were investigated. There was a minimal level of intracellular dectin-1 or dectin-2 in Cblb^~h BMDMs (Fig. 4a,b), suggesting that CBLB promotes internalization of dectin-1 or dectin-2 after infection with C. albicans yeast cells or hyphae.

Next to be investigated was whether retention of ligand-engaged dectin-1 or dectin-2 in Cblb^~h BMDMs is due to impaired sorting of endosomal vesicles to lysosomes. The subcellular localization of ligand-engaged dectin-1 or dectin-2 was compared in WT and Cblb^~h BMDMs by confocal microscopy. In support of the idea of impaired lysosomal degradation of dectin-1 and dectin-2 in BMDMs lacking CBLB, intracellular trafficking of internalized dectin-1 or dectin-2 to the lysosome was markedly reduced in the absence of CBLB (Fig. 4c,d).

CBLB negatively regulates ROS production and fungal killing but not phagocytosis of C. albicans

Neutrophils and macrophages are professional phagocytes of the innate immune system that are essential in controlling bacterial and fungal infections by phagocytosis and killing mechanisms (Nicola, A.M. et al. Curr. Opin. Microbiol. 11, 313-317 (2008)). The production of highly reactive oxygen species (ROS) is one of the primary effector mechanisms used by phagocytes to control or clear microbial infections. ROS have an important role in the initial step of fungal killing in phagosomes (Brown, A.J. et al. Curr. Opin. Microbiol. 12, 384-391 (2009)) and can be potentiated by dectin signaling. ROS was measured production by co-culturing the C. albicans capl mutant yeast or hyphae with WT or Cblb^~ BMDMs. Cblb^~ and Cblb^C373A- expressing BMDMs produced more ROS than WT controls at a multiplicity of infection (MOI) of 5: 1 or 2: 1 (Fig. 12a). Enhanced ROS activity in Cblb^~ BMDMs correlated with an increase in their fungal-killing potency (Fig. 12b). Consistent with a limited role of CBLB in proinflammatory cytokine production by neutrophils, there was no substantial increase in ROS activity and fungal killing in neutrophils that were isolated from the BM of Cblb^~h or Cblb^C373A- expressing mice, as compared to those in the WT controls (Fig. 12c). However, phagocytosis of C albicans by Cblb^_/~ BMDMs was not increased, as compared to that by WT BMDMs (Fig. 12d).

CBLB inhibits dectin-mediated innate immune responses to systemic C. albicans infection

The recognition of β-glucans and a-mannans by dectin-1 and dectin-2, respectively, is thought to trigger immune responses that are primarily designed for the control of fungal pathogens (Taylor, P.R. et al. Nat. Immunol. 8, 31-38 (2007); Saijo, S. et al. Immunity 32, 681-

691 (2010); Zhu, L.L. et al. Immunity 39, 324-334 (2013)). To assess the role of CBLB in antifungal immunity, WT, Cblb^~ , and C£/£^C373A-expressing mice were infected with a lethal dose of C. albicans to monitor survival and with a sublethal dose to measure serum cytokines and fungal burden. Most Cblb^~h and C£/£^C373A-expressing mice were protected from lethal systemic infection with C. albicans (Fig. 5a), which correlated with heightened levels of TNF-a and IL-6 in the sera of Cblb^~ and C£/£^C373A-expressing mice, lower fungal burden in the kidney, lung, spleen, and liver, and decreased amounts of C. albicans hyphae in the kidney on day 2 as assessed by PAS staining (Fig. 5b-d, Fig. 12a). Multifocal tubulointerstitial nephritis was observed in WT mice infected with C. albicans, which was ameliorated in mice lacking CBLB or expressing the CBLB^C373A mutant (Fig. 5c). This observation is consistent with the fact that more immune cells— including macrophages, DCs, and neutrophils— traffic to the kidneys in WT than in CblbC373A-expressing mice (Fig. 12b). An improved survival rate was also observed m Rag Cblb^~h mice that lack functional adaptive immune cells (Fig. 5e), supporting a critical role of CBLB in downregulating innate immune responses.

To further determine whether monocytes, macrophages, and neutrophils have a greater capacity to kill C. albicans during systemic infection, fungal burdens were monitored in the blood of WT and Cblb^C373A-expressing mice at 2 h and 6 h after infection (Fig. 13a). Fungal burden in the blood of Cblb^C373A-expressing mice was substantially lower than that of WT mice at 2 h and 6 h after infection. The lower fungal burden in the blood of CblbC373A-expressing mice correlated with enhanced fungal killing activity by PBMCs but not by neutrophils in these mice (Fig. 13a). Increased fungal killing was also observed in monocytes from the spleen of Cblb^C373A-expressing mice (Fig. 13b). ROS activity was also monitored in monocytes, macrophages and neutrophils from WT and CblbC373A spleens and kidneys by CellRox dye (Fig. 13c). Monocytes and macrophages, but not neutrophils, showed augmented ROS expression in C. albicans-miected Cblb^C373A-expressing mice after they were infected in vitro with C. albicans. Consistent with the lower fungal burden and decreased inflammation in Cblb^C373A kidneys, trafficking of CD45.2+ leukocytes, including macrophages, DCs, and neutrophils, to Cblb^C373A kidneys was significantly reduced (Fig. 12b; P < 0.01). Even with decreased numbers of myeloid cells in Cblb^C373A kidneys after infection with C. albicans, there was an increase in ROS expression in monocytes and macrophages, increased fungal killing in vitro using CD45+ cells isolated from CblbC373A kidneys (Fig. 13d), and increased amounts of TNF-a and IL-6 in the kidney homogenates of CblbC373A-expressing mice (Fig. 13e).

To further determine whether heightened inflammatory responses caused by CBLB deficiency are mediated by dectin-1 and dectin-2, Cblb^~ Clec7a^~ , Cblb^~ Clec4n^~ ,

Clec7a^~l~Clec4n^~ , and Cblb^~ Clec7a^~ Clec4n^~ mice were generated. WT, Cblb^~ , Clec7a^~ ,

Cblb- Clec7a-'-, Clec4n , Cblb- Clec4n , Clec7a-'-Clec4n , and Cblb- Clec7a- Clec4n ^l- mice were infected with C. albicans. Dectin-1 or dectin-2 single deficiency rendered Cblb^_/~ mice susceptible to C. albicans infection, and a deficiency in both dectin-1 and dectin-2 greatly increased the sensitivity of Cblb^~ mice to systemic C. albicans infection. All of the triple- knockout mice died within 4 d after infection at a dose at which all of the Cblb^~h mice survived (Fig. 5f), which correlated with markedly lower levels of TNF-a and IL-6 in their sera and a lower fungal burden in the kidneys of the triple-knockout mice (Fig. 14a,b). Therefore, results suggest that CBLB negatively regulates both dectin-1 and dectin-2, and that CBLB dampens inflammatory responses mediated by dectin-1 and dectin-2 during systemic fungal infection. Notably, Cblb^~h or Cblb^C313A mice at 8-12 weeks of age did not show signs of autoimmunity, as revealed by comparable amounts of autoantibody titers to double-stranded (ds) DNA and single- stranded (ss) DNA, and of IL-17 and IFN-γ levels, in the sera of WT and Cblb^~ or Cblb^C313A mice, as well as no elevated IL-17 and IFN-γ in the kidneys of Cblb^~ or Cblb^C313A mice, as compared to that of WT mice (Fig. 15a-d). These data suggest that a pre-existing autoimmunity in Cblb^~ or Cblb^{C 1 A} mice does not account for strong differences in antifungal immune responses, relative to those in WT mice, after fungal infection.

Clec7a^~ and Clec4n^~ mice die at a similar rate after systemic C. albicans infection, suggesting that both dectin-1 and dectin-2 are equally important for fungal recognition (Fig. 5f). Because Cblb^~ Clec7a^~ , Cblb^~ Clec4n^~ , and Cblb^~ Clec7a^~ Clec4n^~ mice did not die at the same rate after infection as did Clec7a^~ , Clec4n^~ , or Clec7a^~ Clec4n^~l~ mice (Fig. 5f), these results suggest that CBLB may regulate an additional CLR(s), such as the mannose receptor (MR), dectin-3, or Mincle, which have been shown to be involved in host defense against C. albicans infection (Zhu, L.L. et al. Immunity 39, 324-334 (2013); Wells, C.A. et al. J. Immunol. 180, 7404-7413 (2008); van de Veerdonk, F.L. et al. Cell Host Microbe 5, 329-340 (2009); Cambi, A. et al. Eur. J. Immunol. 33, 532-538 (2003)). Indeed, loss of CBLB appeared to stabilize the protein expression of dectin-3, but not MR, Mincle and dendritic-cell-specific intercellular-adhesion-molecule-3 -grabbing non-integrin (DC-SIGN) (Fig. 16).

CBLB is a therapeutic target for antifungal infection

Because CBLB downregulates dectin family CLR signaling and host innate immune responses, decreasing CBLB expression may enhance phagocyte antifungal responses, providing evidence for a new therapeutic approach. Experiments were performed using in vivo delivery of

Cblb-specific siRNA to knock down Cblb. WT mice were first infected with C. albicans by intravenous (i.v.) injection, and 24 h later the Cblb-specific siRNA or a nonsense siRNA was injected via the tail vein. Mortality of the mice was monitored for 7 d. Although all of the WT mice that were treated with the nonsense siRNA died within 7 d after infection, seven of nine WT mice that were treated with the siRNA to Cblb survived. There was a significantly higher fungal burden in the kidneys of WT mice that received the nonsense siRNA than those that received the Cblb-specific siRNA (Fig. 6). These data indicate that CBLB can serve as a potent therapeutic target for enhancing host defense against fungal infections.

Methods

Mice. C57BL/6 mice and Rag^~ mice were purchased from the Jackson Laboratory. Fcerlg^~h mice were purchased from Taconic (Hudson, NY). Cblb^~h mice7 were provided. Cblb^C373A mice and Clec7a^~h were described previously (Taylor, P.R. et al. Nat. Immunol. 8, 31-38 (2007); Oksvold, M.P. et al. Mol. Immunol. 45, 925-936 (2008)). Clec4rf'^~ mice were described previously (Saijo, S. et al. Immunity 32, 681-691 (2010)). Cblb^~h mice on a C57BL/6 background were crossed with Clec7a^~ or Clec4n^_/" mice to generate C£/£^~/~Clec7a^~/~ and Cblb^~ Clec4n^~ mice, or Cblb^~ Clec7a^~ Clec4n^~ mice. Cblb^~ mice were also crossed with Ragl^~h mice to generate Cblb^~hRag mice. The mice were used at 8-12 weeks of age, and both male and female mice were used in this study. All animal experimentation involving systemic C. albicans infection and in vivo delivery of control and Cblb-specific siRNA was approved by the Institutional Animal Care and Use Committees (IACUCs) of the Ohio State University and the Xiangya School of Medicine, Central South University.

Reagents. Antibodies against CBLB (G-l), SYK (N-19), CARD9 (H-90), DC-SIGN (T- 13), CD206 (H-300), and ubiquitin (P4D1) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-dectin-1 (GE2; Ab82888) was obtained from Abeam (Cambridge, MA). Anti-dectin-2 (217611) and mouse IL-1RA/IL-1F3 Quantikine ELISA Kit (MRA00) was purchased from R&D Systems (Minneapolis, MN). The following items were purchased from BioLegend (San Diego, CA): PE-conjugated anti-dectin-1 (144204), FITC-conjugated anti- LAMP-1 (1D4B), anti-mouse CD45.2 antibody (104), anti-mouse CD 8 (53-6.7), anti-mouse CDl lb (Ml/70), anti-mouse F4/80 (BM8), anti-mouse CDl lc (N418), anti-mouse I-A/I-E (M5/114.15.2), anti-mouse Ly6C (HK1.4), anti-mouse Ly6G (1A8), and ELISA kits for mouse IL-17A (432504), IFN-γ (430805), IL-6 (431304) and TNF-a (430904). PE-conjugated anti- dectin-2 (MCA2415PE) was obtained from AbD Serotec (Raleigh, NC). Anti-Mincle (D292-3) was purchased from MBL Life Science (Woburn, MA). ELISA kits for mouse IgG (88-50400) and IgE (88-50460) were purchased from eBioscience (San Diego, CA). ELISA kits for anti- ssDNA (5310) and anti-dsDNA (total (A + G + M) (5110) were purchased from Alpha

Diagnostic International Inc. (San Antonio, TX). The plasmids encoding Clec7a (dectin-1) and

Clec4n (dectin-2) (pCMV2-Flag) were purchased from Sino Biologicals, Inc. (Beijing, P.R. China). Anti-K48-linkage-specific polyubiquitin (4289), anti-K63 -linkage-specific polyubiquitin (D7A1 1), anti-phospho-SYK (Y525/526; #271 1), and anti-phospho-NF-κΒ p65 (S536; #3031), and anti-phospho-ΙκΒα (Ser32/36) (5A5; #9246) were purchased from Cell Signaling

Technology, Inc. (Danvers, MA). Anti-dectin-3 was provided. Mouse neutrophil isolation kit, monocyte isolation kit, and CD45 microbeads (mouse) were purchased from Miltenyi Biotec (San Diego, CA). Histopaque 1 1 19 (Sigma 1 1 191), Histopaque 1077 (Sigma 10771), and anti- Flag (M2) were obtained from Sigma-Aldrich (St. Louis, MO). Collagenase type IV (021951 10) was purchased from MP Biomedicals (Santa Ana, CA). CellRox Deep Red (C I 0422) was purchased from ThermoFisher Scientific (Waltham, MA). The validation of the antibodies used is provided on the manufacturers' websites.

Site-directed mutagenesis. Single and triple lysine-to-arginine (K-to-R)-encoding mutations of dectin-1 (CLEC7A^K2R, CLECTA¹"™, CLEC7A^K34R, and CLEC7A^{K2R K27R K34R}) and dectin-2 (CLEC4N^K10R), CLEC7A^Y15F, and FCR-Y^{Y65F Y76F} were generated by site-directed mutagenesis at Mutagenex Inc. (Piscataway, NJ).

Generation ofBMDMs and BMDCs, and isolation of mouse BM neutrophils. BM cells were harvested from the femurs and tibias of mice. Cells were cultured in Dulbecco' s modified Eagle' s medium (DMEM) (Sigma-Aldrich, St. Louis, MO) containing 10% FBS and 30% conditioned medium from L929 cells expressing macrophage colony stimulating factor (M- CSF). After 1 week of culture, non-adherent cells were removed, and adherent cells were 80- 90%) F4/80⁺CDl lb⁺, as determined by flow cytometric analysis. Mouse BMDCs were generated using granulocyte-macrophage colony stimulating factor (GM-CSF) and purified from bulk cultures by magnetic selection with anti-CD 1 lc microbeads. This routinely gave purities of >98%>. For isolation of BM neutrophils, total BM cells were recovered from the femurs and tibias by flushing with RPMI medium (Sigma-Aldrich) with an 18-gauge needle; erythrocytes were lysed with red blood cell (RBC) lysis buffer (eBioscience) and BM neutrophils were isolated by neutrophil isolation kit (Milteny); neutrophil purity (>98%>) was confirmed by flow cytometry.

Isolation of mouse PBMCs and neutrophils from blood, and splenic monocytes, neutrophils, and kidney CD 45+ cells. WT and Cblb^C313A mice were anesthetized, and blood was collected from the tail vein. The RBC were lysed using RBC lysis buffer (eBioscience). PBMCs and neutrophils were isolated by gradient centrifugation over Histopaque 1 1 19 (density, 1.1 19 g/ml) and Histopaque 1077 (density, 1.077 g/ml), according to the manufacturer's instructions, at

400g for 30 min at 25 °C (Swamydas, M., et al. Curr. Protoc. Immunol. 1 10, 3.20 (2015).).

PBMCs were collected from the interface between the plasma and Histopaque 1077. Neutrophils were recovered at the interface of the interface of the Histopaque 1 1 19 and Histopaque 1077 layers, and they were 80-90% pure and >95% viable, as determined by flow cytometry. PBMCs and neutrophils were washed twice and resuspended in RPMI 1640 medium supplemented with 10%) FBS. Splenic monocytes and neutrophils of WT and Cblb^C313A mice were isolated by monocyte isolation and neutrophil isolation kits (Milteny). Monocyte and neutrophil purities (>98%) were confirmed by flow cytometry.

WT and CblbC373 A mice were killed at 48 h after infection with C. albicans (by tail vein injection) at a dose of 1 χ 10⁶ c.f.u. Kidneys were perfused, minced, and placed in 2 ml of Hank' s balanced salt solution (HBSS) (50 mM HEPES, 12 mM Dextrose, 280 mM NaCl, 10 mM KC1, 1.5 mM Na2HP04, pH to 7.05) containing 2 mg/ml collagenase IV, and incubated at 37 °C for 30 min with gentle agitation. Digested kidney tissues were passed through a 40-μπι

Falcon cell strainer, by using the rubber end of a 1-ml syringe plunger, and a cell suspension was obtained via centrifugation at 1,200 r.p.m. for 10 min. The CD45⁺ cells were purified with CD45 MicroBeads (Milteny). The purity (-90%) was determined by flow cytometry.

In vitro infection of macrophages, dendritic cells, and neutrophils with C. albicans yeast and hyphal forms. A single colony of C. albicans strain SC5314 was grown overnight at 30 °C in yeast peptone dextrose (YPD) medium. The cells were washed twice with PBS before use as live yeasts. The capl yeast-only mutant described previously (Bahn, Y. S. et al. J. Bacterid. 183, 321 1-3223 (2001)) was obtained. For the hyphal forms, the washed yeasts were resuspended at 10⁷ cells/ml in RPMI 1640 with 10% FCS and grown for 3 h at 37 °C. After washing in PBS, the hyphae were used for live stimulations. For analysis of cytokine production, 10⁵ BMDMs, BMDCs, or neutrophils were cultured overnight in a 96-well U-bottom plate with live C.

albicans capl mutant or hyphae at an MOI of 1 for the times indicated; cytokine levels in the supernatant were measured by sandwich ELISA.

BMDM reconstitution. Clec7a^~ BMDMs were transfected with constructs expressing Flag-tagged dectin-1, CLEC7A^K2R, CLEC7A^K27R, CLEC7A^K34R, CLEC7A^{K2R K27R K34R}, or

CLEC7A^Y15F by Lipofectamine 2000. Clec4n '^~ or Fcergl^~'^~ BMDMs were transfected with Flag-tagged dectin-2, CLEC4N^K10R, FcR-γ, and FCR-Y^{Y65F Y76F}, respectively.

ROS assay, phagocytosis of C. albicans and fungal killing assay. For the ROS production assay, 2 10⁵ WT, Cblb^~ , or Cblb^C313A BMDMs were washed with PBS twice and replated in PBS containing 100 mM luminol and 5 units of horseradish peroxidase. The cells were incubated at 37 °C for 30 min and were then infected with C. albicans at an MOI = 5 : 1 and 2: 1, respectively. The relative amount of ROS generated by neutrophils was detected at regular intervals over 75 min by measuring the luminescence. Relative light units (RLU) were plotted as a function of time to evaluate the chemiluminescence (CL) rate. To measure ROS expression in monocytes, macrophages, and neutrophils in spleens and kidneys, WT and Cblb^~h mice were infected with C. albicans by tail vein injection at a dose of 1 x 10⁶ c.f.u. 48 h later, mice were killed, and leukocytes from spleens and kidneys were infected with C. albicans for 30 min, and stained with CellRox and cell surface markers to determine ROS expression in monocytes (kidney: CD45.2⁺CDl lb⁺ Lye^I^G^"; spleen: CD1 lb⁺Ly6C⁺Ly6G-), macrophages (kidney: CD45.2⁺ CDl lb⁺F4/80⁺Ly6C^loCDl lc^"; spleen: CD1 lb⁺F4/80⁺Ly6C^loLy6G^"), and neutrophils (kidney: CD45.2⁺CDl lb⁺ Ly6C^l0 Ly6G⁺; spleen: CD1 lb⁺Ly6G⁺Ly6C^").

For phagocytosis of C. albicans, C. albicans yeast were labeled with Alexa Fluor 488 (Invitrogen) in 100 mM HEPES buffer (pH 7.5) (diluted to 1 :500) and then co-cultured with WT or Cblb^~ BMDMs for 45 min at 37 °C. Adherent fungal cells were quenched with trypan blue, and the rate of phagocytosis was determined by flow cytometry (Wirnsberger, G. et al. Nat. Genet. 46, 1028-1033 (2014)).

For in vitro fungal killing assay, WT or Cblb^~ BMDMs (1 x 10⁵/well) were incubated with C. albicans at an MOI of 1 :500 for 24 h. To determine the fungal killing capacity of PBMCs, blood neutrophils, splenic monocytes, and neutrophils and of kidney CD45⁺ cells, WT and Cblb^C373A mice were infected with C. albicans by tail vein injection (1 x 10⁶ c.f.u.). PBMCs, blood neutrophils, and splenic monocytes, neutrophils and kidney CD45⁺ cells were co-cultured with a C. albicans form at an MOI of 1 : 10 for 24 h. After co-culture, a 100-μ1 suspension was spread (1 : 10⁴ dilution) on YPD plates. After incubation at 37 °C for 36 h, killing was determined by counting the Candida colonies, with and without the indicated cells (Wirnsberger, G. et al. Nat. Genet. 46, 1028-1033 (2014)).

Immunoprecipitation and western blotting. For co-immunoprecipitation, WT BMDMs were infected with C. albicans yeast cells or hyphae (MOI = 1 : 1) for various times and lysed in

0.5% NP40 lysis buffer. The cell lysates were immunoprecipitated with anti-CBLB (1 : 100) and blotted with anti-dectin-1 (1 : 1,000) or anti-dectin-2 (1 :5,000), anti-SYK (1 : 1,000), and anti-

CARD9 (1 : 1,000). For detection of dectin-1 or dectin-2 ubiquitination, BMDMs from WT and either Cblb^~ or Cblb^C373A mice were infected with C. albicans yeast capl mutant or hyphae

(MOI = 1 : 1) for various times and lysed in RIPA buffer containing 2% SDS, which were then diluted to 0.5% of SDS. The cell lysates were immunoprecipitated with anti-dectin-1 (1 : 100) or anti-dectin-2 (1 : 100), and blotted with anti-ubiquitin (1 : 1,000), or with anti-K48- or anti-K63- specific ubiquitin antibodies (1 : 1,000). To assess the protein stability of dectin-1, dectin-2, dectin-3, MR, Mincle, DC-SIGN, SYK and CARD9, BMDMs from WT and Cblb^~'^~ mice were infected with C. albicans yeast cells or hyphae (MOI = 1 : 1) at the indicated times and lysed for immunoblotting with antibodies against dectin-1 (1 : 1,000), dectin-2 (1 : 1,000), dectin-3 (1 : 1,000), MR (1 : 1,000), Mincle (1 : 1,000), DC-SIGN (1 : 1,000), SYK, and CARD9, respectively. To determine whether dectin-1 and dectin-2 undergo proteasome- or lysosome- mediated degradation, WT BMDMs were pretreated with MG-132 (5 μΜ) or E64 (10 μΜ) for 30 min, and were then infected with C. albicans yeast capl mutant or hyphae (MOI = 1 : 1) for various times and lysed. The cell lysates were blotted with anti-dectin-1 or anti-dectin-2.

Detection of serum and kidney cytokines, serum IgG and IgE, and autoantibodies by ELISA. For detection of TNF-a, IL-6, IL-Ιβ, and IL-IRA in macrophage culture supernatants, 10⁵ BMDMs from WT, Cblb^~ or Cblb^C313A mice were infected with live C. albicans capl mutant cells or hyphae at MOI 1 : 1 for the times indicated, and cytokine production in the supernatant was measured by ELISA. WT and Cblb^~ BMDMs were also infected with fumigatus conidia (MOI = 1 : 1) for the times indicated, and TNF-a and IL-6 levels in the supernatant were measured by ELISA.

For detection of serum IL-17, IFN-γ, IL-6, TNF-a and IL-Ιβ, WT, Cblb^~'^~ or Cblb^C373A mice were infected with C. albicans (5 x 10⁴, or 1 x 10⁶ c.f.u. for some experiments), sera were collected at different time points and subjected for ELISA analysis. The kidneys harvested at 48 h after infection were homogenized, and the supernatant was recovered following centrifugation at 15,000g for 20 min at 4 °C. The cytokines, including IL-17, IFN-γ, and IL-6, in the kidney homogenates were determined by using ELISA kits according to the manufacturer's instructions. The ELISA results were expressed as 'pg per g of kidney' . For detection of serum IgG, IgE, anti- ssDNA, and anti-dsDNA, sera were collected from WT, Cblb^~h or Cblb^C313A mice before C. albicans infection and at 48 h after infection and were subjected to ELISA analysis.

Internalization of dectin-1 and dectin-2 in macrophages after infection with C. albicans yeast and hyphae. WT and Cblb^~ BMDMs were infected with C. albicans yeast capl mutant (MOI: 1 : 1) for the times indicated. Flow cytometry was then used to determine the surface expression of dectin-1 and dectin-2. For dectin-1 internalization, BMDMs from WT and Cblb^~ mice were labeled with PE-conjugated anti-dectin-1 (1 :200) or anti-dectin-2 (1 :200). Cells were then incubated at 37 °C for 5, 15, and 30 min. To remove uninternalized dectin-1- or dectin-2- coupled antibodies from the cell surface, half the cells from each time point were treated briefly with ice-cold acidic buffer (1% BSA at pH 3.0) and immediately neutralized in PBS containing 1% BSA and 0.5% NaN3. Both treated and untreated cells were stained with anti-F4/80 and anti- CD1 lb. Dectin-1 internalization was calculated with gated F4/80 and CD 1 lb-positive cells using the formula: % of dectin-1 or dectin-2 internalization = 100 x ((MFI of acid-resistant PE fluorescence (at time t) - MFI of acid-resistant PE fluorescence (at time 0))/MFI of total PE fluorescence of untreated cells). Confocal microscopy. WT and Cblb^~ BMDMs were attached to poly(l-lysine)-coated coverslips and surface-labeled with PE-conjugated anti-dectin-1 or anti-dectin-2 on ice. Labeled cells were infected with C. albicans yeast capl mutant or hyphae for 30 min at 37 °C to allow dectin-1 or dectin-2 internalization to occur. The cells were fixed in 1% paraformaldehyde, permeabilized in 0.05% saponin and stained with FITC-conjugated anti-LAMP-1. Imaging was performed on a Leica TCS-SP2 confocal microscope (1 : 100). Imaging was performed on a laser- scanning confocal microscope (Flowview 1000, Olympus).

Systemic C. albicans dissemination. For survival analysis, mice were infected with C. albicans i.v. at 1-5 x 10⁵ c.f.u. and monitored daily. After infection, mice were weighed and monitored daily. Mice were euthanized if they lost >20% of their body weight. In a separate group, the kidneys were harvested 2 d after infection. The left kidneys were photographed and homogenized for enumeration of fungal burden. The right kidneys were fixed for histological analysis. The fungal burden in the kidneys, spleens, livers, and lungs was determined by c.f.u. in kidney, spleen, liver, and lung homogenates. The fungal burden in the blood at 2 and 6 h after infection was also determined. Mice were allocated to experimental groups based upon their genotypes and randomized within their sex- and age-matched groups. No blinding was done in this study.

Generation of human monocyte-derived macrophages (MDM) and silencing of Cblb gene expression. Human MDMs were generated as previously described (Kang, P.B. et al. J. Exp. Med. 202, 987-999 (2005); Rajaram, M.V. et al. Proc. Natl. Acad. Sci. USA 108, 17408-17413 (2011)). In brief, peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated from heparinized blood on Ficoll-sodium diatrizoate gradients and then cultured for 5 d in RPMI containing 20% autologous serum (2.0 x 10⁶ mononuclear cells/ml) at 37 °C. On day 5, human MDMs were transfected with control siRNA or Cblb-specific siRNA (100 or 200 nM; Dharmacon RNA Technologies) by using Lonza nucleofector reagent, and they were then plated in RPMI 1640 containing 20% autologous serum. After 36 h, the MDMs were washed and infected with yeast cells or hyphae of C. albicans. The protocol was approved by The Ohio State University Institutional Review Board.

In vivo delivery of Cblb-specific siRNA. WT mice were injected with C. albicans i.v. at 5 x 10⁵ c.f.u., and 24 h later they were treated with in vivo-grade Cblb-specific siRNA (5'-

AAAUUCUCGAAGUAUGCUCUU-3 ' (SEQ ID NO: 1) or a nonsense siRNA (2 mg/kg/mouse) (Dharmacon RNA Technologies) in In vivo-j etPEI-FluoF (Polyplus-transfection, Inc.; New York, NY) via tail vein injection. Three days later, the spleen cells were collected and lysed in RIPA buffer. The cell lysates were subjected to SDS-PAGE, transferred and blotted with anti- CBLB and anti-actin, respectively.

Data analysis and statistical analysis. Differences in concentrations of cytokines and fungal burden were analyzed by using the Student' s t-test. Survival data were analyzed by using the Kaplan-Meier log-rank test. Differences were considered significant at P < 0.05. No animals were excluded from the analysis. Mice were allocated to experimental groups based on their genotypes and were randomized within their sex- and age-matched groups. No statistical method was used to predetermine sample size. It was assumed that normal variance occurs between the experimental groups.

Discussion

The fungal cell wall consists mainly of carbohydrates, including mannose-based structures (the mannoproteins), β-glucan, and chitin. Recognition of β-glucans and a-mannans by dectin-1 and dectin-2 is essential for antifungal immunity (Brown, G.D. et al. Annu. Rev.

Immunol. 29, 1-21 (201 1)). However, the regulation of dectin family receptors is unknown. As disclosed herein, CBLB functions as a negative regulator of the dectin-1 and dectin-2 CLRs, which initiate innate immune responses to fungal pathogens in human and mouse macrophages. CBLB targets dectin-1 and dectin-2, and SYK for K48-linked polyubiquitination, which inhibits dectin-1- or dectin-2-mediated signaling pathways. CBLB deficiency or inactivation leads to increased pro-inflammatory responses that decrease dissemination of C. albicans and bolster host defense.

CLEC7A^K2R' ^K34R and CLEC4N^K10R mutants, in which ubiquitination is abrogated, result in increased production of TNF-a and IL-6 by macrophages infected with C. albicans yeast cells or hyphae (Fig. 3g,h), thus mirroring the data obtained from Cblb^~ and Cblb^C313A mice. The disclosed data therefore provide evidence that ubiquitination of dectin-1 and dectin-2 is a key mechanism for terminating innate immune responses during fungal infection, thereby avoiding excessive inflammation and subsequent tissue damage, while at the same time dampening optimal host-defense properties.

Phagocytosis is a key cellular process, both during homeostasis and after infection or tissue damage, and dectin-1 has been shown to be a phagocytic receptor (Goodridge, H.S. et al.

Traffic 13, 1062-1071 (2012)). ROS production by phagocytes is associated with pathogen killing (Dupre-Crochet, S. et al. J. Leukoc. Biol. 94, 657-670 (2013)), and it was reported that dectin-1 activates SYK in macrophages and is important for dectin-1 -stimulated ROS

production, but not for phagocytosis (Underhill, D M. et al. Blood 106, 2543-2550 (2005)). Consistent with this report, the disclosed data show that CBLB regulates both dectin-1 and dectin-2 expression, and ROS production, by macrophages but does not affect fungal phagocytosis. The disclosed data suggest that additional receptor(s), such as those belonging to the Fc-γ receptor family or DC-SIGN (Cambi, A. et al. Eur. J. Immunol. 33, 532-538 (2003); Goodridge, H.S. et al. Traffic 13, 1062-1071 (2012)), may be involved in controlling fungal phagocytosis independently of regulation by CBLB.

Because CBLB is critical for T cell activation, tolerance induction, and TH2 and TH9 cell differentiation (Liu, Q. et al. Cell Cycle 13, 1875-1884 (2014)), it is possible that the enhanced antifungal immune response in the absence of CBLB may result in heightened adaptive T cell responses. However, this possibility was excluded by the observation that the phenotype of Cblb^~hRag mice, which do not have T and B cells, phenocopies that of Cblb^~h mice after C. albicans infection (Fig. 5e), supporting the notion that CBLB is crucial for controlling innate immune responses against systemic C. albicans infection. The heightened innate immune responses observed during systemic C. albicans infection is mediated by dectin-1 and dectin-2, because introducing mutants of dectin-1, dectin-2, or both into Cblb^~h mice abrogates these heightened responses and renders Cblb^~h mice susceptible to C. albicans infection (Fig. 5f). More notably, systemic in vivo delivery of a Cblb-speciiic siRNA to C57BL/6 mice protects them from lethal systemic C. albicans infection (Fig. 6). These data show that CBLB is a therapeutic target for controlling disseminated candidiasis. Of note, inhibition of CBLB may have detrimental effects due to unchecked inflammation, particularly on patients in intensive care. However, inhibition of Cblb by using an siRNA in vivo may be a more viable approach because the siRNA would have a limited half-life and dosages could be modulated to minimize the degree of inflammation.

In summary, the disclosed data provide the first evidence that CBLB has an essential role in regulating dectin-mediated innate immune responses to fungal pathogens following inflammatory responses to fungi in immunocompetent hosts. One consequence of this dampening of inflammatory responses is the creation of a less-than-optimal host defense program. Targeting CBLB therefore serves as a new and important therapeutic strategy in fighting fungal infections.

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Human Cbl-b gene sequence (SEQ ID NO:4)

(Homo sapiens Cbl proto-oncogene B (CBLB), transcript variant 1, mRNA; NCBI Reference Sequence: NM_001321786.1)

1 ggggccggga aggggcctga gcagggaagg gacacaggcg tcctgaaaca aagctttggg

61 ttgagggcag aggcgaggag ccagagaagt gaactcctcc tttccgtggc cgctcttgtc

121 atttttctct tttctctttt caaatgggct atttgtgtgt taatttcatt tggttcttgg

181 gaataacgac tcaccgcgtt gatttaaaga aagaactaaa attccagatg gcaaactcaa

241 tgaatggcag aaaccctggt ggtcgaggag gaaatccccg aaaaggtcga attttgggta

301 ttattgatgc tattcaggat gcagttggac cccctaagca agctgccgca gatcgcagga

361 ccgtggagaa gacttggaag ctcatggaca aagtggtaag actgtgccaa aatcccaaac

421 ttcagttgaa aaatagccca ccatatatac ttgatatttt gcctgataca tatcagcatt

481 tacgacttat attgagtaaa tatgatgaca accagaaact tgcccaactc agtgagaatg

541 agtactttaa aatctacatt gatagcctta tgaaaaagtc aaaacgggca ataagactct

601 ttaaagaagg caaggagaga atgtatgaag aacagtcaca ggacagacga aatctcacaa

661 aactgtccct tatcttcagt cacatgctgg cagaaatcaa agcaatcttt cccaatggtc

721 aattccaggg agataacttt cgtatcacaa aagcagatgc tgctgaattc tggagaaagt

781 tttttggaga caaaactatc gtaccatgga aagtattcag acagtgcctt catgaggtcc

841 accagattag ctctggcctg gaagcaatgg ctctaaaatc aacaattgat ttaacttgca

901 atgattacat ttcagttttt gaatttgata tttttaccag gctgtttcag ccttggggct

961 ctattttgcg gaattggaat ttcttagctg tgacacatcc aggttacatg gcatttctca

1021 catatgatga agttaaagca cgactacaga aatatagcac caaacccgga agctatattt

1081 tccggttaag ttgcactcga ttgggacagt gggccattgg ctatgtgact ggggatggga

1141 atatcttaca gaccatacct cataacaagc ccttatttca agccctgatt gatggcagca

1201 gggaaggatt ttatctttat cctgatggga ggagttataa tcctgattta actggattat

1261 gtgaacctac acctcatgac catataaaag ttacacagga acaatatgaa ttatattgtg

1321 aaatgggctc cacttttcag ctctgtaaga tttgtgcaga gaatgacaaa gatgtcaaga

1381 ttgagccttg tgggcatttg atgtgcacct cttgccttac ggcatggcag gagtcggatg

1441 gtcagggctg ccctttctgt cgttgtgaaa taaaaggaac tgagcccata atcgtggacc

1501 cctttgatcc aagagatgaa ggctccaggt gttgcagcat cattgacccc tttggcatgc

1561 cgatgctaga cttggacgac gatgatgatc gtgaggagtc cttgatgatg aatcggttgg

1621 caaacgtccg aaagtgcact gacaggcaga actcaccagt cacatcacca ggatcctctc

1681 cccttgccca gagaagaaag ccacagcctg acccactcca gatcccacat ctaagcctgc

1741 cacccgtgcc tcctcgcctg gatctaattc agaaaggcat agttagatct ccctgtggca

1801 gcccaacggg ttcaccaaag tcttctcctt gcatggtgag aaaacaagat aaaccactcc 1861 cagcaccacc tcctccctta agagatcctc ctccaccgcc acctgaaaga cctccaccaa

1921 tcccaccaga caatagactg agtagacaca tccatcatgt ggaaagcgtg ccttccagag

1981 acccgccaat gcctcttgaa gcatggtgcc ctcgggatgt gtttgggact aatcagcttg

2041 tgggatgtcg actcctaggg gagggctctc caaaacctgg aatcacagcg agttcaaatg 2101 tcaatggaag gcacagtaga gtgggctctg acccagtgct tatgcggaaa cacagacgcc

2161 atgatttgcc tttagaagga gctaaggtct tttccaatgg tcaccttgga agtgaagaat

2221 atgatgttcc tccccggctt tctcctcctc ctccagttac caccctcctc cctagcataa

2281 agtgtactgg tccgttagca aattctcttt cagagaaaac aagagaccca gtagaggaag

2341 atgatgatga atacaagatt ccttcatccc accctgtttc cctgaattca caaccatctc 2401 attgtcataa tgtaaaacct cctgttcggt cttgtgataa tggtcactgt atgctgaatg

2461 gaacacatgg tccatcttca gagaagaaat caaacatccc tgacttaagc atatatttaa

2521 agggagatgt ttttgattca gcctctgatc ccgtgccatt accacctgcc aggcctccaa

2581 ctcgggacaa tccaaagcat ggttcttcac tcaacaggac gccctctgat tatgatcttc

2641 tcatccctcc attaggtgaa gatgcttttg atgccctccc tccatctctc ccacctcccc 2701 cacctcctgc aaggcatagt ctcattgaac attcaaaacc tcctggctcc agtagccggc

2761 catcctcagg acaggatctt tttcttcttc cttcagatcc ctttgttgat ctagcaagtg

2821 gccaagttcc tttgcctcct gctagaaggt taccaggtga aaatgtcaaa actaacagaa

2881 catcacagga ctatgatcag cttccttcat gttcagatgg ttcacaggca ccagccagac

2941 cccctaaacc acgaccgcgc aggactgcac cagaaattca ccacagaaaa ccccatgggc 3001 ctgaggcggc attggaaaat gtcgatgcaa aaattgcaaa actcatggga gagggttatg

3061 cctttgaaga ggtgaagaga gccttagaga tagcccagaa taatgtcgaa gttgcccgga

3121 gcatcctccg agaatttgcc ttccctcctc cagtatcccc acgtctaaat ctatagcagc

3181 cagaactgta gacaccaaaa tggaaagcaa tcgatgtatt ccaagagtgt ggaaataaag

3241 agaactgaga tggaattcaa gagagaagtg tctcctcctc gtgtagcagc ttgagaagag 3301 gcttgggagt gcagcttctc aaaggagacc gatgcttgct caggatgtcg acagctgtgg

3361 cttccttgtt tttgctagcc atatttttaa atcagggttg aactgacaaa aataatttaa

3421 agacgtttac ttcccttgaa ctttgaacct gtgaaatgct ttaccttgtt tacagtttgg

3481 caaagttgca gtttgttctt gtttttagtt tagttttgtt ttggtgtttt gatacctgta

3541 ctgtgttctt cacagaccct ttgtagcgtg gtcaggtctg ctgtaacatt tcccaccaac 3601 tctcttgctg tccacatcaa cagctaaatc atttattcat atggatctct accatcccca

3661 tgccttgccc aggtccagtt ccatttctct cattcacaag atgctttgaa ggttctgatt

3721 ttcaactgat caaactaatg caaaaaaaaa aaagtatgta ttcttcacta ctgagtttct

3781 tctttggaaa ccatcactat tgagagatgg gaaaaacctg aatgtataaa gcatttattt

3841 gtcaataaac tgccttttgt aaggggtttt cacataacat agaggagctt cccttttttg 3901 tttaagtttt gtaaccttta atcctccata ttctcatgtc tgtcatccca ggggtgtcac

3961 aactgtaaaa atctacaata ttagaaagca gctactacag atgtggaaga gagaacactt

4021 gtataagaca atgctgtact gaagttatat aacaaggtcc cctgatactt atgcctgcat

4081 tactttgagg gatgctgaat gagaggacca tctccctgaa atatattaat aattttcaga

4141 taaaattatg aacataacat accttttctc aagtggaatt agagttcatt tcccatttaa 4201 gtgattacac tctatctcct aagagctgtg ggaaccagtt catattgcag tggaaacatt

4261 aactcttcgt tgcactgttg agtgtcaagg ctactgtggc aatgtttttt gcaaagttct

4321 cagtaatttt ctctctgcaa tcaaattaga gttcatattt aatttgcaga ttctctcatt

4381 cattgctcca cactgcttta tcaaatcagg tgaaagaaat taatgtagtt ttgcctattt

4441 tacaaaatgg tggttggttc tttaaacacc atgttattac tcttaataaa tctacaaata 4501 tttattgagc atctatgata tgcttggtgc tgttaggcac tgtgttttta aagcacaaac

4561 tctcaaaagc tgattccagc gtaatgtttt atttttgttt ttctgtgttt tttatgacaa

4621 agtctgtctg atagacaaag cctggaaggt ttcccccagg gctttagaat ttagaattag

4681 ctggttttca tttcaaatgg gaaattaatg gagaaatgag ataaaatcat ctctgtgtaa 4741 atgcttgaac ccacagaaag ttttctaaat atggactaat tgtgggtttg tgcatatttc

4801 tgtgtctcct cttttcactt cacacccacc atgatataaa aagatgcatc agagagtgag

4861 cctgccttca ggacctgaaa ggacatgact gaacatctag tagatctggt ggaaattaat

4921 gttatacaaa agacacgtag actaagttga ccctgggaga ggagttaata cattcctttg

4981 caaagtgaca tgtgcccttt ttttgacagt ctggctttca tggtatgaga tgaggactca 5041 caggccttgg gtaatttctg cttttgtatg aattttttca gatggttctg agctggttat

5101 gtccctggag gggaattttt catttgcctt agatgttctt cccatgagta ggtacatgag

5161 tgaagtttct tacactttta aaattgcatt ggcaaatttt cttttgcttt atgaacccct

5221 gttttgtcat tatgattttt cacttgtttt aacagtttca gatgtcacct tggaatagca

5281 ctgtttattt tggccttttt atgattgctg atgataggta cagtctaaag gccaaaagtt 5341 gccacttcat tgtgataaca ttgttgcaac attcctaata tattagagaa tgatgtaaaa

5401 taagaatgtt agagaacata gcacattctc attcagttta taaacaatat ttgaaaattt

5461 ccagtgagaa ttaaaaaagg actagtgatg atatagcaaa cctggacctg aaatttgggg

5521 gtggggggtg gggggatatc tgttttgcag agaaaattta ccagcctcca gcaaaatctc

5581 tgcatcttgt actcaatagc aaccattagt attgtcattt aaaaagaatg aatcaaaaat 5641 tcatacgtaa tggaatgaaa agaggaaaac tttctattaa acactgtcaa gaaagaccca

5701 ttgctatcta tcacctatga aattaaacct aaaaaaattt ttttttattg taaaagcgac

5761 tgctcgttga tgtctggaag tgatccagac tttgtttcac tgtgatccaa aaagttagac

5821 attccttcca tttctcaaga tttggtaaca attttttttt aagtcagcat ttcatataat

5881 acataaaatt aagacaaatt aacttactga tatgtggcca atatcaaccc ttctcaccca 5941 acccaaacat gttctggaga tagaaggact tatcctcaat tatattccag taaatttatc

6001 tccataatct gttttatccc ctaatgtatg atatatctca ccaaattttt ctagaattga

6061 ataaaagcag tttttgctta acttgaattt ccttccacaa atacaacctt cagctcttcc

6121 ctctctgttt cccttttgcc cacattgcaa atagtcttca taatagaacc ctcactgcaa

6181 cttaaaaggg tcccatttct taactcagca tttcatatac caagaacact tgcaaagatt 6241 tcttcataat gtgatttttt aatttttcag ttccagaagc atttcctaca gttcgaacgt

6301 tattttttct ctcagatttt caacttgaag ctttgatttg cactgaatta acttggataa

6361 aattttagtt tctctcttca gctaacagat ttggaagcaa attacagtat acttgaataa

6421 ttcaggtact acctgttgac ctggaatttt agtgattctg ggaaagtttt ccagacaaag

6481 acaccaatag agtgaagggt tttttcccca attactattt tattgttttt taaaatgcct 6541 ttttaaaact tgtgtgatgt ggcaaaatag tcatatgttc aattaattta tattttattc

6601 attccttttc aattctggtt attatgtaac ctcaagtact ttatctgttc ttttaaagta

6661 ttttataaaa tgaacgttaa ctaaa (SEQ ID NO : 4 )

Claims

WHAT IS CLAIMED IS:

1. A method for treating a fungal infection in a subject, comprising administering to the subject a composition comprising a therapeutically effective amount of a casitas B lymphoma-b (CBLB) inhibitor.

2. The method of claim 1, wherein the fungal infection comprises a Candida spp. infection.

3. The method of claim 2, wherein the fungal infection comprises a Candida albicans infection.

4. The method of claim 1, wherein the fungal infection comprises aspergillosis,

Pneumocystis carinii pneumonia (PCP), coccidioidomycosis (valley fever), cryptococcosis, histoplasmosis, or a combination thereof.

5. The method of claim 1, wherein the fungal infection comprises aspergillosis,

Pneumocystis carinii pneumonia (PCP), coccidioidomycosis (valley fever), or a combination thereof.

6. The method of any one of claims 1 to 5, wherein the CBLB inhibitor is an siRNA, a miRNA, a shRNA, a small molecule, an antisense molecule, a peptide, or a protein.

7. The method of any one of claims 1 to 6, wherein the CBLB inhibitor comprises a functional nucleic acid that knocks down expression of the Cbl-b gene in the subject.

8. The method of claim 7, wherein the functional nucleic acid comprises an siRNA.

9. The method of claim 7, wherein the functional nucleic acid is from about 15 to about 25 nucleotides.

10. The method of any one of claims 6 to 9, wherein the siRNA comprises the nucleic acid sequence 5'-AAAUUCUCGAAGUAUGCUCUU-3' (SEQ ID NO: l).

11. The method of any one of claims 6 to 9, wherein the siRNA comprises the nucleic acid sequence 5'-UUUGCUAACGGACCAGUACUU-3' (SEQ ID NO:2) or 5'- UAAUACCCAAAAUUCGACCUU-3 ' (SEQ ID NO:3).

12. The method of any one of claims 1 to 11, wherein the subject is a mammal.

13. The method of claim 12, wherein the subject is a human.

14. A method for treating or preventing a fungal infection in a subject, comprising administering to the subject a composition comprising a therapeutically effective amount of a casitas B lymphoma-b (CBLB) inhibitor, wherein the casitas B lymphoma-b (CBLB) inhibitor reduces the activity of a casitas B lymphoma-b (CBLB) protein in the subject.

15. The method of claim 14, wherein the fungal infection comprises a Candida spp.

infection.

16. The method of claim 15, wherein the fungal infection comprises a Candida albicans infection.

17. The method of claim 14, wherein the fungal infection comprises aspergillosis,

18. The method of claim 14, wherein the fungal infection comprises aspergillosis,

19. The method of any one of claims 14 to 18, wherein the CBLB inhibitor is an siRNA molecule, a miRNA molecule, a shRNA molecule, a small molecule, an antisense molecule, a peptide, or a protein.

20. The method of any one of claims 14 to 19, wherein the CBLB inhibitor comprises a functional nucleic acid that knocks down expression of the Cbl-b gene in the subject.

21. The method of claim 20, wherein the functional nucleic acid comprises an siRNA.

22. The method of claim 20, wherein the functional nucleic acid is from about 15 to about 25 nucleotides.

23. The method of any one of claims 19 to 22, wherein the siRNA comprises the nucleic acid sequence 5'-AAAUUCUCGAAGUAUGCUCUU-3' (SEQ ID NO: l).

24. The method of any one of claims 19 to 22, wherein the siRNA comprises the nucleic acid sequence 5'-UUUGCUAACGGACCAGUACUU-3' (SEQ ID NO:2) or 5'- UAAUACCCAAAAUUCGACCUU-3 ' (SEQ ID NO:3).

25. The method of any one of claims 14 to 24, wherein the subject is a mammal.

26. The method of claim 25, wherein the subject is a human.

27. The method of any one of claims 14 to 26, wherein the casitas B lymphoma-b (CBLB) inhibitor reduces the activity of a casitas B lymphoma-b (CBLB) protein in the subject comprises reducing the expression of the gene encoding the casitas B lymphoma-b (CBLB) protein.

28. The method of claim 27, wherein reducing the expression of the gene comprises RNA interference using a functional nucleic acid that knocks down expression of the gene in the subject.