CN114286684B - Prevention and/or treatment of STAT 3-related diseases - Google Patents
Prevention and/or treatment of STAT 3-related diseases Download PDFInfo
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- CN114286684B CN114286684B CN202180003577.6A CN202180003577A CN114286684B CN 114286684 B CN114286684 B CN 114286684B CN 202180003577 A CN202180003577 A CN 202180003577A CN 114286684 B CN114286684 B CN 114286684B
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Abstract
The present invention relates to the prevention and/or treatment of STAT 3-related diseases by modulation of 14-3-3σ.
Description
Technical Field
The present invention relates to the prevention and/or treatment of STAT 3-related diseases by modulation of 14-3-3σ.
Background
Signal transduction and transcription activator protein 3 (STAT 3) is a member of the STAT protein family, and is composed of a number of transcription factors that regulate genes involved in many cellular functions. STAT proteins are activated by JAK kinases (JAKs). Upon stimulation by cytokines or growth factors, STAT3 envelope receptor-associated JAKs phosphorylate, forming homo-or heterodimers, and are transported to the nucleus for transcription. Since STAT3 controls the expression of a variety of genes, and in many cellular processes (including cell growth and apoptosis 1 ) Plays a key role in the mouse model, and thus STAT3-KO has embryonic lethality 2 。
The function of STAT3 in immune cells has been widely studied. STAT3 is essential for differentiation of TH17 cells 3-6 . During viral infection, STAT 3-deficient mice in T cells completely lost the production of follicular helper T cells (Tfh ) And antibody Capacity 7 . Significant STAT3 mutations in humans are loss of function (LOF) mutations, which can lead to High Immunoglobulin E Syndrome (HIES). Patients suffering from this disease may develop recurrent infections and skeletal and dental dysplasia 8，9 . On the other hand, a function-acquired mutation (GOF) of STAT3 gene causes autoimmune diseases 10 . It has been reported that mutations in other genes such as Dock8, PGM3, SPINK5 and TYK2 can also induce HIES 11-14 . We bred DOCK8 mutant mice to mimic mutations in DOCK8 patients and found that early activation of memory B cells was disrupted. Furthermore, DOCK8 deficiency results in reduced CD19 and WASP expression 15 . Previous studies have shown that in TLR 9-mediated B cell activation, STAT3 is downstream of DOCK8, and DOCK8 inhibits IgE production by activating STAT3 16 . However, the correlation between STAT3 and DOCK8 in the presence of modulation of IgE production is not clear.
Recently, a correlation has been found between micrornas (mirnas) and high IgE. The use of miRNA146A transgenic mice can demonstrate that miRNA146A promotes IgE class switching by up-regulating 14-3-3σ,14-3-3σ being a key factor in immunoglobulin class switching DNA recombination (CSR) in B cells 17-19 . Ectopic expression of miRNA146A in Jurkat T cells results in moderate downregulation of STAT3 expression 20 . Furthermore, when activation of STAT3 is blocked in hepatocellular carcinoma (HCC) cells, expression of miRNA146A is reduced 21 . In addition, miRNA146A controls the expression of SOCS1/STAT3 and cytokines in monocytes during hepatitis c infection 22 . However, the underlying mechanism of HIES by STAT3 mutation is still unclear, especially in B cells.
B Cell Receptor (BCR) signaling is critical for regulating B cell processes and functions. BCR signaling determines the fate of B cells and the extent of their antibody responses. When the antigen binds to BCR, conformational changes of the receptor may be initiated and trigger signaling cascades such as CD19, lyn, syk, btk, PLC gamma and phosphorylation of negative regulators (including SHIP and PTEN) in BCR signaling 23-25 . BCR signaling can also induce actin through several actin modulators such as WASP, abp1 and N-WASPRecombination, whereas actin recombination provides feedback for BCR signaling by modulating BCR movement 26-29 . It is not clear how STAT3 affects BCR signaling in STAT3 LOF and GOF patient B cells.
In one study, STAT3 deficiency prevented early development of bone marrow B cells, but STAT3 deficiency was due to Mx1 not characteristic of B cells Cre Driven by a motor 30 . In contrast, CD19 Cre Stat3 fl/fl No developmental defects were observed in mice, so that stat3 loss occurred at the later stages of B cell development and only the T cell dependent (TD) IgG responses were severely compromised, whereas the TD-IgM, igE, and IgA responses or non-T cell dependent (TI) IgM and IgG3 responses were not compromised 30,31 . STAT3 signaling is critical to maintaining Germinal Centers (GC) and accounts for the pathogenesis of Systemic Lupus Erythematosus (SLE) by modulating GC activity 32 . Memory B cell (including converted and unconverted immunoglobulins) depletion in STAT3 patients 33-35 . To accurately investigate the potential mechanism of STAT3 mutation to trigger HIES only in B cells using a mouse model, we incubated Mb1 with STAT3 deleted early in B cell development Cre stat3 flox/flox Mice (STAT 3 KO).
Disclosure of Invention
In a first aspect, the present invention provides a method of preventing and/or treating STAT 3-related diseases in a patient in need of such prevention and/or treatment, the method comprising modulating 14-3-3σ in said patient.
In one embodiment of the first aspect, the STAT3 is a loss-of-function STAT3 or a gain-of-function STAT3. In another embodiment of the first aspect, the STAT3 is a loss of function STAT3.
In another embodiment of the first aspect, the STAT 3-related disease is a hyper IgE syndrome.
In another embodiment of the first aspect, modulating 14-3-3σ is down-modulating 14-3-3σ or up-modulating 14-3-3σ. In another embodiment of the first aspect, the modulation is down-regulation of 14-3-3σ. In another embodiment of the first aspect, the down-regulating 14-3-3σ is performed with a 14-3-3σ inhibitor. At the position ofIn another embodiment of the first aspect, the 14-3-3σ inhibitor is a peptide. In another embodiment of the first aspect, the peptide comprises the amino acid sequence NH 2 -YGRKKKRQRRRPHCVPRDLSWLDLEANMCLP-COOH。
In a second aspect, the invention provides a 14-3-3 sigma modulator, a pharmaceutical composition comprising said 14-3-3 sigma modulator or a kit comprising said 14-3-3 sigma modulator for preventing and/or treating STAT3 related diseases in a patient in need of such prevention and/or treatment.
In one embodiment of the second aspect, the 14-3-3σ modulator is a 14-3-3σ down-regulator or a 14-3-3σ up-regulator. In another embodiment of the second aspect, the 14-3-3σ modulator is a 14-3-3σ down-regulator. In another embodiment of the second aspect, the 14-3-3σ down-regulator is a 14-3-3σ inhibitor. In another embodiment of the second aspect, the 14-3-3σ inhibitor is a peptide. In another embodiment of the second aspect, the peptide comprises the amino acid sequence NH 2 -YGRKKKRQRRRPHCVPRDLSWLDLEANMCLP-COOH。
In another embodiment of the second aspect, the STAT3 is a loss-of-function STAT3 or a gain-of-function STAT3. In another embodiment of the second aspect, the STAT3 is a loss of function STAT3.
In another embodiment of the second aspect, the STAT 3-related disease is hyper IgE syndrome.
In another embodiment of the second aspect, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.
In another embodiment of the second aspect, the 14-3-3σ modulator included in the kit is in the form of a pharmaceutical composition. In another embodiment of the second aspect, the kit further comprises instructions for using the 14-3-3σ modulator.
In a third aspect, the invention provides the use of a 14-3-3 sigma modulator for the prevention and/or treatment of STAT 3-related diseases in a patient in need of such prevention and/or treatment.
In the third aspectIn one embodiment, the 14-3-3σ modulator is a 14-3-3σ down-regulator or a 14-3-3σ up-regulator. In another embodiment of the third aspect, the 14-3-3σ modulator is a 14-3-3σ down-regulator. In another embodiment of the third aspect, the 14-3-3σ down-regulator is a 14-3-3σ inhibitor. In another embodiment of the third aspect, the 14-3-3σ inhibitor is a peptide. In another embodiment of the third aspect, the peptide comprises the amino acid sequence NH 2 -YGRKKKRQRRRPHCVPRDLSWLDLEANMCLP-COOH。
In another embodiment of the third aspect, the STAT3 is a loss-of-function STAT3 or a gain-of-function STAT3. In another embodiment of the third aspect, the STAT3 is a loss of function STAT3.
In another embodiment of the third aspect, the STAT 3-related disease is a hyper IgE syndrome.
In a fourth aspect, the present invention provides the use of a 14-3-3 sigma modulator in the manufacture of a medicament for the prevention and/or treatment of STAT 3-related diseases in a patient in need of such prevention and/or treatment.
In one embodiment of the fourth aspect, the 14-3-3σ modulator is a 14-3-3σ down-regulator or a 14-3-3σ up-regulator. In another embodiment of the fourth aspect, the 14-3-3σ modulator is a 14-3-3σ down-regulator. In another embodiment of the fourth aspect, the 14-3-3σ down-regulator is a 14-3-3σ inhibitor. In another embodiment of the fourth aspect, the 14-3-3σ inhibitor is a peptide. In another embodiment of the fourth aspect, the peptide comprises the amino acid sequence NH 2 -YGRKKKRQRRRPHCVPRDLSWLDLEANMCLP-COOH。
In another embodiment of the fourth aspect, the STAT3 is a loss-of-function STAT3 or a gain-of-function STAT3. In another embodiment of the fourth aspect, the STAT3 is a loss of function STAT3.
In another embodiment of the fourth aspect, the STAT 3-related disease is hyper IgE syndrome.
Drawings
Fig. 1: STAT3 is critical to maintaining peripheral blood B cell homeostasis, but not bone marrow B cells.
(a-C) flow cytometry data from WT and STAT3KO mice (n=8) bone marrow cells of pre-progenitor B cells (a), progenitor B cells (B), early pre-B cells (C), late pre-B cells (D), immature B cells (E) and recycled B cells (F), as well as the percentage and absolute number of each of these cells. (D) MFI of CD127 from different populations of a and B gating. (E-H) flow cytometry data from T1, T2, FO cell populations of WT and STAT3KO mouse spleen cells (n=8) and the percentage and absolute number of each of these cells. (I and J) flow cytometry data of MZ B cells from mouse spleen cells (n=8) and the percentage and absolute number of these cells. (K and L) flow cytometry data from GC cells of mouse spleen cells (n=8) and the percentage and absolute number of these cells. (M-O) flow cytometry analysis of B1a and B1B cells from WT and STAT3KO mice (n=6) and the percentage and absolute number of each of these cells. (P-R) IgE from WT and STAT3KO mice (n=4) with or without 5 days of anti-CD 40 and IL-4 stimulation + Flow cytometry data of spleen B cells, percentage and absolute number of these cells. (S) titers of WT and STAT3 KO mouse serum (n=6) IgE by ELISA. Representative results of three independent experiments are shown. * P (P)<0.05 and P<0.01。
Fig. 2: STAT3 positively regulates proximal BCR signaling.
(A and B) spleen B cells from WT and STAT3 KO mice were labeled and stimulated with AF 546-mono-biotinylated Fab' -anti-IgG plus streptavidin via soluble antigen (sAg) for various lengths of time at 37 ℃. After fixation and permeabilization, cells were stained for pCD19, imaged by confocal microscopy, and the correlation coefficient between BCR and pCD19 was quantified. (C) pCD19/CD19 levels in spleen B cells of sAg stimulated WT and STAT3 KO mice analyzed by western blot. (D and E) confocal microscopy images of pY and pBtk in spleen B cells of mice stimulated with sAg, and the correlation coefficients between BCR and pY/pBtk were quantified. (F) pY and pBtk/Btk levels in spleen B cells of mice stimulated with sAg by Western blot analysis. (G and H) pSHIP in spleen B cells of sAg-stimulated mice analyzed by confocal microscopy, and correlation coefficients between BCR and pSHIP were measured. (I) Western blot of pSHIP/SHIP levels in spleen B cells of sAg stimulated mice. Representative results of three independent experiments are shown. Scale bar, 2.5 μm. Correlation coefficients were quantified for more than 50 cells using NIS Elements AR 3.2 software. Mannheim U test (FIG. 2B, FIG. 2E, FIG. 2H). * P <0.05; * P <0.01.
Fig. 3: the absence of STAT3 reduces F-actin accumulation mediated by WASP and WIP, BCR clustering and forward signaling complex recruitment.
(A) Confocal microscopy analysis of pwsp and actin in spleen B cells of WT and STAT3KO mice. B cells were stimulated with sAg at 37 ℃ for different lengths, then fixed, permeabilized and stained for pwascp and actin (using phalloidin). (B) Spleen B cells from WT and STAT3KO mice were labeled with BV 510-anti B220, stimulated with sAg for different lengths of time at 37 ℃, then fixed, permeabilized and stained as described in a. MFI of pwsp and actin was measured by flow cytometry. (C) pWASP/WASP levels in spleen B cells of sAg stimulated WT and STAT3KO mice analyzed by Western blotting. (D) Expression of WIP and DOCK8 in spleen B cells from WT and STAT3KO mice (left panel) and in PBMC (right panel) of Healthy Control (HC) and loss of function (LOF) STAT3 patients. GAPDH was used as a loading control. (E-J) spleen B cells from WT and STAT3KO mice were stimulated with AF 546-mono-biotinylated Fab' -anti-IgG over different time periods on membrane-tethered antigens (mAg), then fixed, permeabilized and stained for pWASP and actin. Cells were analyzed using TIRFm and MFI of BCR, pwassp and actin were measured, and B cell contact area was measured using Interference Reflection Microscopy (IRM). Representative images and mean (+ -SD) are shown, wherein more than 50 cells were analyzed using NIS Elements AR 3.2 software. Scale bar, 2.5mm. * P <0.05; * P <0.01. Mannheim U test (FIGS. 2G-2J).
Fig. 4: STAT3 deficiency resulted in reduced recruitment of pCD19, pBtk and pY, but increased recruitment of pSHIP.
(A and D) spleen B cells from WT and STAT3 KO mice were stimulated with mAg at 37℃for various lengths of time, then fixed, permeabilized and stained for pCD 19. Images were taken using TIRFm and analyzed for MFI of pCD19 in the B cell contact zone. (B, E, F) mice spleen B cells stimulated with mAg and stained for pBtk and pY were imaged using TIRFm and the MFI of pBtk and pY within the contact zone was measured. (C and G) mAg stimulated and stained mouse spleen B cells for pSHIP were imaged using TIRFm and the MFI of pSHIP within the contact area was measured. Representative images and mean (+ -SD) are shown, wherein more than 50 cells were analyzed from 3 independent experiments using NIS Elements AR 3.2 software. Scale bar, 2.5mm. * P <0.05; * P <0.01. Mannheim U test (FIG. 2D, FIG. 2E, FIG. 2F, FIG. 2G).
Fig. 5: STAT3 lacks a pathway that reduces PI3K-Akt-mTORC 1-mediated metabolic signaling.
(A) Spleen B cells from WT and STAT3 KO mice were stimulated with sAg at different times, lysed, and probed for pPI3k/PI3k, pAkt/Akt, pFOXO1/Foxo1, pMtor/Mtor, and pS6/S6 using Western blotting. (B) Spleen B cells from WT and STAT3 KO mice respond to basal oxygen rate (OCR) of the inhibitors oligomycin, FCCP (carbonyl cyanide p-trifluoromethoxybenzohydrazone) and rotenone treatment. (C and D) spleen B cells (1 x 10) from WT and STAT3 KO mice (n=4) 6 ) Labeling with Cell Trace Violet and stimulation with LPS and CpG for 72 hours. Detection of B220 by flow cytometry + Proliferation of cells, and the percentage of proliferating cells is quantified. (E and F) spleen B220 of mice after 72 hours of stimulation with LPS and CpG + Expression of annexin V in cells. Annexin V + The percentage of cells (n=4) was quantified. Representative results of three independent experiments are shown. * P<0.05；***P<0.01。
Fig. 6: memory B cells from STAT3 LOF patients disrupt early BCR activation.
(A, D-F) B cells sorted from peripheral blood (P1-P4) of HC and STAT3 LOF patients were stimulated with mAg for different lengths of time, fixed, permeabilized and stained for CD27 (to label memory B cells) and actin (using phalloidin). Images were taken using TIRFm, B cell contact areas were analyzed using IRM, and MFI of BCR and actin in the contact areas was measured. (B and G) HC and STAT3 LOF patient B cells stained for CD27 and pCD19 and stimulated with mAg were imaged using TIRFm and MFI of pCD19 in the contact zone was measured. (C and H) HC and STAT3 LOF patient B cells stained for CD27 and pY and stimulated with mAg were imaged using TIRFm and MFI of pY in the contact zone was measured. Representative images and mean (+ -SD) are shown, wherein more than 50 cells were analyzed from 3 independent experiments using NIS Elements AR 3.2 software. Scale bar, 2.5mm. * P <0.05; * P <0.01. Mannheim U test (FIGS. 2D-2H). (I and J) HC and STAT3 LOF (P1-P6) and GOF (P7) patient PBMC (n=6), as well as quantitative percentages of primary B cells, memory B cells, transitional B cells and plasmablasts. (K and L) B cells sorted from HC, STAT3 LOF (P1-P4) and GOF (P7) patient PBMC were sAg stimulated for different lengths of time and analyzed for percentage of primary and memory B cells (n=4) using flow cytometry. Representative results of three independent experiments are shown. * P <0.05; * P <0.01.
Fig. 7: STAT3 deficiency results in high IgE by enhancing the miRNA146A-14-3-3σ axis.
(A) Analysis of miRNA146A and 14-3-3σmrna in spleen B cells from WT and STAT3-KO mice (n=6). (B) Analysis of miRNA146A and 14-3-3σmrna in B cells from PBMC (n=6) (P1-P7) of HC and LOF patients. (C) Expression of 14-3-3σ in B cells from spleen (left panel) of WT and STAT3 KO mice and PBMC (right panel) of HC and LOF patients (P4-P6) using Western blotting detection. GAPDH was used as a loading control. (D) PCR analysis of STAT3 binding to the 14-3-3σ promoter in B cells from WT mice. IgG was used as a negative control. (E) Spleen B cells from WT and STAT3 KO mice were treated with or without 10 μ M R18 for 1 hour, stimulated with sAg, then lysed and probed for pAkt and pfox 1 using western blotting. Beta-actin was used as a loading control. (F) WT and STAT3 KO mice were injected intraperitoneally for 14 days with or without R18 (0.2 μg/kg body weight) and thenSpleen B cells were isolated and stimulated with sAg, lysed, and pAkt and pfox 1 were probed using western blotting. Beta-actin was used as a loading control. (G-J) WT and STAT3 KO mice (n=3) were continuously injected intraperitoneally with or without R18 (0.2 μg/kg body weight) for 14 days and analyzed for the percentage of FO and GC B cells by flow cytometry. (K and L) IgE was measured by flow cytometry on WT and STAT3 KO mice with or without R18 treatment and with or without stimulation + Percentage of B cells and IgE-converted B cells. (M) IgE titers in serum of WT and STAT3 KO mice (n=5) with or without R18 treatment were measured by ELISA. Representative results of three independent experiments are shown. * P (P)<0.05 and P<0.01。
FIG. 8
(A) Analysis of STAT3 mRNA in spleen B cells from WT and STAT3-KO mice (n=4). (B and C) quantitative percentages of annexin V expression in spleen B220, MZ, FO, T1 and T2 cells from WT and STAT3 KO mice, and cell populations (n=8). (D and E) quantitative percentages of Ki67 expression in spleen B220, MZ, FO, T1 and T2 cells from WT and STAT3 KO mice, and cell populations (n=8). Representative results of three independent experiments are shown. (F and G) WT and STAT3 KO mice were injected intraperitoneally for 14 days with or without R18 (0.2. Mu.g/kg body weight). The data show flow cytometry analysis of MZ cells and their percentages (n=3). Representative results of three independent experiments are shown. * P <0.05 and P <0.01.
Examples
The present application will be explained in detail as follows.
In this study, we found that in STAT3 KO B cells, proximal positive BCR signaling was reduced, but negative modulator SHIP was enhanced. In STAT3 KO B cells, activation of WASP and thus actin recombination is reduced, and early activation of B cells (including BCR clustering, B cell spreading, and signal complex recruitment) is also impaired. By using Peripheral Blood Mononuclear Cells (PBMCs) from STAT3 LOF patients, we found that early activation of memory B cells and accumulation of actin was also reduced. Notably, we found that memory B was thin Cells and plasmablasts were decreased, but the initial B cells were increased in both STAT3 LOF and GOF cells. In addition, primary B cells from STAT3 LOF readily differentiate into memory B cells. Mechanistically, STAT3 KO B cells have more and are prone to IgE formation in vitro + B cells. Finally, we found that the expression of both miRNA146A and 14-3-3σ was enhanced in both STAT3 KO B cells and STAT3 LOF patients.
Transcription factors play a critical role in maintaining a properly functioning immune system; thus, transcription factor mutations can lead to immune disorders. The loss of STAT3 or Dock8 function mutations lead to immunodeficiency and IgE overproduction, known as hyper IgE syndrome (HIES), the underlying mechanisms of this disease are largely unknown. In this study, we studied the mechanism of HIES using samples from STAT3 loss of function (LOF) and gain of function (GOF) patients and samples from our cultured STAT 3B cell specific deletion mice (STAT 3 KO). Interestingly, we found that peripheral blood B cell homeostasis in STAT3 KO mice was similar to the phenotype of STAT3 LOF patients, with reduced Follicular (FO) and Germinal Center (GC) B cells, but increased Marginal Zone (MZ) B cells, resting B cells, and activated ige+b cells. In addition, STAT3 KO B cells have reduced B Cell Receptor (BCR) signaling under antigen stimulation due to reduced BCR clustering and accumulation of WASP and F-actin. It is exciting that a central junction protein 14-3-3 sigma is increased in B cells in STAT3 KO mice and STAT3 LOF patients, and that 14-3-3 sigma is essential for increasing IgE production. An increase in 14-3-3σ was associated with an increase in expression of the upstream mediator miRNA 146A. Inhibition of 14-3-3σ with the R18 peptide in STAT3 KO mice rescued BCR signaling and FO, GC and IgE+B cell differentiation to the extent of Wild Type (WT) mice. In summary, our study established a novel regulatory pathway for STAT3-miRNA146A-14-3-3σ to regulate BCR signaling, peripheral blood B cell differentiation, and IgE production, which could provide targets for HIES treatment.
Materials and methods
A mouse
By combining Mb1 Cre Mice (Jackson Laboratory) and stat3 flox/flox A mouse 36 Hybridization to obtain STAT3 conditional knockout mice with C57/BL6 background (referred to herein as STAT3 KO mice), STAT3 with the same genetic background flox/flox Mice were used as wild-type controls (WTs). All mice were kept in separate ventilated cages according to the protocol of the national institutional children hospital animal center of Chongqing medical university. Unless otherwise indicated, mice were analyzed at 8-12 weeks of age. Isolation of bone marrow, spleen Single cell suspension and B cells as described previously 26 . For in vivo treatment with R18, WT and STAT3 KO mice were continuously injected with R18 (0.2 μg/kg body weight) Intraperitoneally (IP) for 14 days. All animal experiments were performed according to protocols approved by the institutional animal care and use committee of the child hospital at Chongqing university.
Patient(s)
From 2017 to 2018, a total of 7 chinese patients participated in the study, including 6 STAT3 loss of function (LOF) patients and 1 STAT3 gain of function (GOF) patient (table 1).
Table 1 patient information including gender, age and STAT3 mutation type.
Diagnosis of STAT3 LOF and GOF patients is based on clinical signs, symptoms and STAT3 mutations. Healthy control subjects consisted of 7 age-matched subjects. Human Peripheral Blood Mononuclear Cells (PBMC) were collected and were used with EasySep TM Human B cell isolation kit (17954; stemcell) B cells were isolated from human PBMC. The signing agreement of all children parents is obtained after approval by the ethical committee of the children hospital at Chongqing medical university.
R18 peptide synthesis
Synthesis of R18 amino acid sequence NH by GL Biochem 2 -YGRKKKRQRRRPHCVPRDLSWLDLEANMCLP-COOH。
Cell culture
Obtaining WT and STAT3 KO mouse spleen B cells (1X 10 5 ) 10. Mu.g/ml of anti-mouse in 5ml of RPMI 1640 containing 10% FCS (Hyclone)CD40 (BE 0016-2, bioXcell) and 8ng/ml IL-4 (R)&D Systems) for 5 days, and then the cells were analyzed for IgE expression by flow cytometry. To detect B cell proliferation, mouse spleen B cells (1 x10 5 ) Was isolated and labeled with Cell Trace Violet, then incubated with 5mg/mL LPS (Sigma-Aldrich) or 10. Mu.g/mL B class CpG oligonucleotide (ODN 1826, invivogen) in a 96 well round bottom plate at a volume of 200. Mu.l/well for 5 days in RPMI 1640 containing 10% FCS (Hyclone), 10mmol/L beta-mercaptoethanol, and 1% streptomycin-penicillin, and then analyzed by cytometry.
Flow cytometry and antibodies
For flow cytometry analysis, single cell suspensions were isolated from bone marrow and spleen and incubated with fcγ receptor (fcγr) blocking antibodies (anti-mouse CD16/CD32; BD Bioscience) on ice, followed by staining with the following antibodies (abs) in PBS with 2% fbs. For surface markers, anti-mouse antibodies used included FITC-anti-CD 127 (135008; bioLegend), APC-anti-CD 43 (143208), PE-anti-BP-1 (108307; bioLegend), bright violet 510-anti-B220 (103247; bioLegend), PE/Cy 7-anti-CD 24 (101822; bioLegend) and Bright violet 421-anti-IgM (406518; bioLegend), FITC-anti-CD 19 (101506; bioLegend), FITC-anti-CD 95 (554257;BD Biosciences), FITC-anti-annexin V (640906; bioLegend), PE-anti-CD 23 (101608; bioLegend), perCP/Cy5.5-anti-IgD (405710; bioLegend), APC-anti-CD 21 (123412; bioLegend), alexa Fluor 647-anti-GL 7 (144606; bioLegend), bright 421-anti-IgM (406518; bioLed), anti-CD (PE) and anti-CD 11 (BioLegend). For intracellular staining, cells were fixed and permeabilized using Foxp3 staining buffer kit (eBioscience) according to the manufacturer's instructions, and stained with Ki67 (25-56698-82, ebioscience) and PE-anti-IgE (406907; bioLegend). Anti-human antibodies and reagents for staining human B cell surface markers include FITC-anti-CD 19 (302206; bioLegend), pacific blue-anti-CD 38 (356628; bioLegend), BV 510-anti-IgD (348220; bioLegend), PE-anti-CD 24 (311106; bioLegend), and AF 647-anti-CD 27 (302812; bioLegend).
Phosphorylation signalling has been previously describedDetection of protein levels 37 . Briefly, spleen B cells were isolated and stained with leucinia 510-anti-B220 (103247; bioLegend) and then conjugated with biotin F (ab) 2 Soluble antigen (sAg) against mouse IgG was incubated for 30 min and then with streptavidin for 10 min, followed by BCR signaling induction at 37 ℃ for different lengths of time. Immediately after BCR activation, cells were fixed with phosphorylated flow cytometry (Phosflow) lysis/fixation buffer (BD Biosciences) and permeabilized with phosphorylated flow cytometry permeabilization buffer III (BD Biosciences). Cells were stained with antibodies to pWASP (S483/S484) (A300-205A; bethy) and F-actin (R37110; thermo Fisher) using Alexa Fluor 405-goat anti-rabbit IgG antibody (A-31556;Thermo Fisher). Flow cytometry data were collected on FACS Canto (BD Biosciences) and analyzed using FlowJo software.
Total internal reflection fluorescence microscopy and confocal microscopy
As previously described, total internal reflection fluorescence microscopy (TIRFm) and confocal fluorescence microscopy images were obtained 26 . Briefly, B cells from human PBMCs and mouse spleens were incubated at 37 ℃ on membrane tethered antigens (mAg) at different time points. After fixation and permeabilization, cells were stained for pCD19 (ab 203615; abcam), pBtk (5082S;Signaling Technology, denver, massachusetts), pY (05-321; merck-Millipore), pSHIP (3941S;Cell Signaling Technology, denver, massachusetts) and pWASP (A300-205A;Bethyl Laboratories). TIRFm analysis was performed using NIS Elements AR 3.2 software. B cell contact area was determined using Interference Reflection Microscopy (IRM) images. For Mean Fluorescence Intensity (MFI) analysis, background fluorescence was subtracted and MFI was measured in the B cell contact zone. For each set of data, for samples from 2 or 3 independent experiments >20 individual cells were analyzed.
For confocal microscopy analysis spleen B cells from WT and STAT3 KO mice were isolated and sAg stimulated by incubating the cells with AF 546-mono biotinylated Fab' -anti-IgG and streptavidin at 4 ℃, followed by washing and warming the cells to 37 ℃ for different time points. After fixation and permeabilization, cells were stained for pCD19 (ab 203615; abcam), pBtk (5082S;Signaling Technology), pY (05-321; merck-Millipore), pSHIP (3941S;Cell Signaling Technology, denver, massachusetts) and pWASP (A300-205A;Bethyl Laboratories), then imaged by confocal microscopy and analyzed using NIS Elements AR3.2 software.
Immunoblotting
Immunoblot analysis was performed as described previously 37 . For detection of phosphorylated signaling proteins, the cells from spleen B cells from WT and STAT3 KO mice were isolated from biotinylated anti-mouse IgG F (ab) 2 The cells were subjected to sAg stimulation by incubation on ice for 30 minutes and then 10 minutes with streptavidin, then washed and warmed to 37 ℃ for the indicated time and lysed. Cell lysates were run on SDS-PAGE gels and immunoblots were performed against pAkt (Ser 473; cell Signaling Technology), pBtk (5082S;Cell Signaling Technology), pCD19 (3571;Cell Signaling Technology), pY (05321; merck-Millipore), pSHIP (3941S;Cell Signaling Technology), pWASP (ab 5278; abacm), p-mTOR (5536S;Cell Signaling Technology), pS6 (4856S;Cell Signaling Technology), pFaox 1 (9461S;Cell Signaling Technology) and pPI3K (17366S;Cell Signaling Technology). To detect total protein expression, human or mouse B cells were lysed and immunoblotted against Btk (8547S;Cell Signaling Technology), SHIP (2728S;Cell Signaling Technology), WASP (sc-13139;Santa Cruz Biotechnology), akt (9272S;Cell Signaling Technology), S6 (2217S;Cell Signaling Technology), foxo1 (2880S;Cell Signaling Technology), mTOR (7C10;Cell Signaling Technology), PI3K (4292;Cell Signaling Technology), CD19 (3574;Cell Signaling Technology), btk (8547;Cell Signaling Technology), DOCK8 (sc-292124;Santa Cruz Biotechnology), WIP (sc-271113;Santa Cruz Biotechnology), human/mouse/rat 14-3-3 sigma antibodies (AF 4424; bio-Techne) and anti-mouse GAPDH and beta-actin were used as loading controls. For the treatment with R18 in vitro, Spleen B cells from WT and KO mice were incubated with vehicle or 10 μ M R for 1h prior to incubation with sAg.
Seahorse assay
Using biotinylated anti-mouse IgG F (ab) 2 Spleen B cells isolated from WT and STAT3 KO mice were incubated with sAg for 1 hour at 37 ℃ and then cells were incubated in XF medium (buffer-free DMEM containing 5mM glucose, 2mM L-glutamine and 1mM sodium pyruvate). Oxygen Consumption Rate (OCR) in response to 1M oligomycin, 2M fluorocarbonylphenylhydrazone cyanide (FCCP) and 1M rotenone was measured using an XF-24 extracellular flow analyzer (Seahorse Bioscience).
ELISA
Serum was collected from WT and STAT3 KO mice and IgE levels were measured using ELISA kit (eBioscience) according to the manufacturer's instructions.
Quantitative RT-PCR
To detect differences in STAT3, miRNA146A and 14-3-3 delta mRNA expression in WT and STAT3 KO mice as well as Healthy Control (HC) and LOF patients, B cells were isolated from mouse spleen and patient PBMC, then RNA was isolated by RNAPURE kit (RP 1202; bioTeke) and reverse transcribed using PrimeScript RT kit (RR 037A; takara). The transcribed cDNA was used to analyze the expression of the different genes with Advanced SYBR Green Supermix (Bio-Rad) on a CFX96 Touch Real-Time System (Bio-Rad). STAT3 (mouse) F5'-TGTCAGATCACATGGGCTAAAT-3'; STAT3 (mouse) R5'-GGTCGATGATATTGTCTAGCCA-3'; 14-3-3σ (mouse) σF:5'-AGAACCCAGCGTTACTCTCGA-3';14-3-3σ (mouse) σR 5'-CCA CCACGTTCTTGTAAGCT-3';14-3-3σ (human) σF:5'-GAAGTTGCAGCTGATTAAGGAC-3';14-3-3σ (human) σR 5'-TCTGGATTAGTTGCATTGGCTA-3'. Expression of miRNA146A was analyzed using Taqman microrna transcription and microrna assay kits according to the manufacturer's protocol.
Chromatin immunoprecipitation (ChIP) assay
According to the manufacturer's scheme, useChIP studies were performed using enzymatic chromatin IP kit (agarose beads) (9002S;Cell Signaling Technology). At the position ofB cells from WT C57BL/6 mice were treated with formaldehyde for 10 min at room temperature. The cell lysate is sonicated to produce sheared cross-linked chromatin having a length of 100-1000 base pairs. Soluble chromatin was immunoprecipitated with antibodies to STAT3 (9139S;Cell Signaling Technology) and PCR detected on a Bio-Rad PCR instrument. The primers used were as follows: mouse 14-3-3σ promoter forward 5'-CACACCCACACTACCTCACA-3' and reverse 5' -GTGGTAGTGCTGTCCAGGTG-3.
Statistical analysis
Statistical significance was assessed by a two-tailed unpaired student t-test using Prism 7 software (p <0.05; p <0.01; p < 0.001), unless otherwise indicated. Data are expressed as mean +/-Standard Deviation (SD).
Results
STAT3 is critical to maintaining peripheral blood B cell homeostasis, but not bone marrow B cells.
To investigate whether STAT3 is required during bone marrow B cell development we performed by combining Mb1 Cre Mouse and stat3 flox /flox Mouse hybridization to generate STAT 3B cell specific knockout mouse Mb1 Cre stat3 flox/flox (STAT 3 KO). The expression of STAT3 mRNA was significantly reduced in STAT3 KO B cells, indicating an effective deletion (fig. 8A). The bone marrow B cell subclass of STAT3 KO mice was stained with BP-1 and CD24 antibodies to determine pre-progenitor B cells, and early pre-B cells, while late pre-B cells, immature B cells, and recycled B cells were identified using B220 IgM antibodies. No abnormalities were observed in all sub-populations examined except for the decreasing frequency of recycle B cells (fig. 1A, 1B, 1C). IL-7 signaling was critical for BM B cell development, however, there was no difference in IL-7 receptor expression (CD 127) between WT and STAT3 KO mice (FIG. 1D). This indicates that STAT3 is essential for bone marrow B cell development. We further determined how STAT3 affects peripheral blood B cells by staining T1, T2 and Follicular (FO) B cells with IgM IgD antibodies, defining edge zone (MZ) B cells with CD23 and CD21 antibodies, and identifying Germinal Center (GC) B cells with GL7 and CD95 antibodies. At STAT3 KO SmallIn mice, the percentage and number of T1B cells were normal, but the percentage and number of T2B cells were reduced (fig. 1E, 1F, 1G). Notably, in STAT3 KO mice, the percentage and number of FO was significantly reduced (fig. 1E, 1H), but the percentage and number of MZ B cells was increased (fig. 1I, 1J). In addition, STAT3 KO mice also had reduced ratios and numbers of spontaneous GC B cells (fig. 1K, 1L). We examined the proliferation and apoptosis of resting B cells by staining with Ki67 and annexin V and found no difference in the expression of Ki67 and annexin V in FO, MZ, GC, T and T2 cells between WT and KO mice (fig. 8B-8E). Second, we found that the percentage and number of B1a cells were normal, but the ratio of B1B cells was significantly reduced (fig. 1M, fig. 1N, fig. 1O). Finally, we examined IgE staining of resting and activated B cells stimulated with IL-4 and anti-CD 40 for 5 days. Interestingly, we found that STAT3 KO B cells had significantly increased IgE percentages in both resting and activated states (fig. 1P-1R). We also examined IgE levels in serum of WT and STAT3 KO mice and found that IgE titers in STAT3 KO mice were also increased (fig. 1S). These results indicate that STAT3 is critical to maintaining peripheral blood B cell homeostasis.
STAT3 positively regulates proximal BCR signaling.
Since STAT3 is critical for maintaining the homeostasis of peripheral blood B cells, which is highly correlated with antigen BCR signaling 38-40 We therefore studied how STAT3 modulates BCR signaling expansion. We examined the effect of STAT3 deletion on CD19 spatiotemporal organization using confocal microscopy, CD19 being a co-receptor involved in amplifying upstream BCR signaling. Co-localization between pCD19 and BCR was significantly reduced in STAT3 KO B cells at 5 and 10 minutes after sAg stimulation (FIGS. 2A, 2B). Furthermore, by immunoblotting lysates of spleen B cells stimulated with soluble antigen (sAg) over different time periods, a decrease in pCD19 levels in STAT3 KO B cells was determined (fig. 2C). In summary, this means that STAT3 affects the spatiotemporal organization and activation of CD 19. Next, we examined tyrosine phosphorylated proteins by confocal microscopy and immunoblotting using conditions similar to those above(pY) and pBtk (direct downstream kinase of CD 19). In STAT3 KO B cells, the correlation coefficient between pY/pBtk and BCR was significantly reduced at 10min (fig. 2D, fig. 2E). In addition, pY and pBtk levels were significantly reduced in STAT3 KO B cells (fig. 2F). Finally, we determined the effect of STAT3 deficiency on co-localization between pSHIP and BCR and SHIP (which is a proximal negative regulator of BCR signaling) activation using the same conditions as above. Co-localization between BCR and pSHIP in STAT3 KO B cells was significantly reduced at 5min (FIG. 2G, FIG. 2H), and SHIP phosphorylation was enhanced in STAT3 KO B cells (FIG. 2I). Taken together, these results indicate that STAT3 deficiency disrupts the balance of positive and negative BCR signaling.
STAT3 deficiency reduced F-actin accumulation mediated by WASP and WIP, BCR clustering and forward signaling complex recruitment.
Previous reports confirm the correlation between STAT3 and actin in macrophage lineage 41 . Thus, we attempted to determine if aberrant BCR signaling in STAT3KO B cells would lead to actin destruction. To check this, sAg stimulated B cells were fixed, permeabilized, stained for actin modulators pwascp and F-actin using specific antibodies and phalloidin, respectively, and then analyzed by confocal microscopy, immunoblotting and phosphoflow cytometry. In immunoblots and phosphorylated flow cytometry of STAT3KO B cells, only the expression of pwsp was reduced, whereas the expression of total WASP was not reduced (fig. 3A-3C), which correlates with reduced actin accumulation. To further investigate how STAT3 promotes the potential mechanism of expression of pwsp, WIP expression was examined by immunoblotting, which stabilized the phosphorylation of WASP and prevented WASP degradation. Without being expected, WIP expression was reduced in STAT3KO murine B cells and PBMCs of STAT3 LOF patients (fig. 3D). This suggests that the decrease in pWASP levels may be due to reduced WIP expression. DOCK8 upstream of WASP 15 And it has been reported that DOCK8 deficient patients also suffer from HIES 42 . Therefore, we examined the expression of DOCK8 in STAT3 KO mice and LOF patients. Interestingly, DOCK8 expression in STAT3 KO murine B cells and PBMC of STAT3 LOF patients was significantReduction (fig. 3D). Our previous studies indicate that defective F-actin reorganization on plasma membranes affects the rate of BCR and subsequent BCR clustering and BCR signaling 43 . To investigate the effect of STAT3 depletion on early B cell activation, including BCR clustering and signal complex accumulation, we observed membrane tethered antigen (mAg) -stimulated B cells using total internal reflection fluorescence microscopy (TIRFm). Spleen B cells were stained with phalloidin, pWASP, pCD19, pBtk, pY and pSHIP after activation with mAg for various lengths of time. In STAT3 KO B cells, B cell spreading and BCR clustering as measured by Mean Fluorescence Intensity (MFI) within the contact zone was significantly reduced (fig. 3E, 3G, 3H). The recruitment of F-actin and pWASP was also significantly reduced in the contact zone of STAT3 KO B cells (FIGS. 3F, 3I, 3J). In addition, within the B cell contact region, recruitment of pCD19 (fig. 4A, 4D), pBtk and pY (fig. 4B, 4E, 4F) signal complexes was significantly reduced, but in STAT3 KO B cells, recruitment of pSHIP was enhanced (fig. 4C, 4G). Taken together, these results indicate that STAT3 deficiency reduces F-actin recruitment mediated by WASP on the plasma membrane, resulting in BCR clustering and a reduction in BCR signaling, which may be due to a lack of driving force to sequester BCR into clusters.
STAT3 lacks a pathway that reduces PI3K-Akt-mTORC 1-mediated metabolic signaling.
PI3K is involved in metabolic signaling pathways and is a direct downstream effector of Btk activation 44 . Thus, we examined mTORC1 and mTORC2 signaling mediated by PI 3K. First, STAT3KO B cells stimulated with sAg were lysed and probed with a pPI 3K-specific antibody and a decrease in pPI3K levels was found (fig. 5A). Next, we examined mTORC2 signaling molecules, including pAkt and pfox 1, using similar conditions. pAkt and pfox 1 levels were also reduced in STAT3KO B cells after stimulation (fig. 5A). In addition, the levels of phosphorylation of mTORC1 and mTORC2 common subunits in STAT3KO B cells after stimulation were also reduced (fig. 5A). Finally, activation of the downstream kinase of Akt-mTORC1 was determined by examining pS6 levels. We found that pS6 levels were also reduced (FIG. 5A). Since mTORC activity is highly correlated with cellular metabolism, we utilized SeahorThe se XF technique analyzes oxidative phosphorylation in real time. Oxygen Consumption Rate (OCR) was used as a measure of oxidative phosphorylation. Cells were first treated with oligomycin to block mitochondrial ATP production, then treated with carbonyl cyanide 4- (trifluoromethoxy) phenylhydrazone (FCCP) to induce maximum oxygen consumption, and then treated with rotenone plus antimycin to inhibit the electron transfer chain. After stimulation of cells with sAg, both basal and maximum respiration rates of STAT3KO B cells were lower than WT B cells (fig. 5B). We also examined proliferation and apoptosis of LPS or CpG pulsed B cells by flow cytometry using annexin V and Cell Trace Violet staining and found an increase in STAT3KO B cell proliferation (fig. 5C, 5D), but no difference in annexin V expression (fig. 5E, 5F). Thus, STAT3KO B cells are not proliferation defective when stimulated, nor are they debilitating and die from apoptosis. Taken together, these results suggest that STAT3 deficiency inhibits B cell metabolism in response to BCR stimulation.
Memory B cells from STAT3 LOF patients disrupt early activation.
Activation of CD19 is critical for early activation of memory B cells 45 And to determine the effect of STAT3 deficiency on early activation of human memory B cells, we examined BCR clustering and B cell spreading of B cells from HC and STAT3 LOF patients upon mAg stimulation by TIRFm. Compared with memory B cells of HC, memory B cells of STAT3 LOF patients (identified as CD27 + ) Is significantly reduced (fig. 6A, 6D). BCR clustering in the memory B cell contact zone was also significantly reduced in STAT3 LOF patients compared to HC (fig. 6A, 6E). Next, we examined actin reorganization in memory B cells by phalloidin staining and found a significant decrease in actin accumulation in memory B cell contact areas from STAT3 LOF patients (fig. 6A, 6F). To further determine the effect of STAT3 deficiency on signal complex recruitment in memory B cells, we examined pY and pCD19 levels within the contact zone. pY and pCD19 levels were significantly reduced in memory B cell contact regions of STAT3 LOF patients compared to HC (FIG. 6B, FIG. 6C, FIG. 6G, FIG. 6H). These results indicate that STAT3 deficiency blocks early activation of memory B cells. We analyzed further by different antibody staining combinations Immunophenotype of STAT3LOF and GOF patients were examined. Interestingly, we found that STAT3LOF and GOF patients had significantly reduced percentages of memory B cells, while STAT3LOF and GOF patients had increased percentages of initial B cells (fig. 6I, 6J). Notably, the frequency of transitional B cells was significantly increased in STAT3LOF patients, while the frequency of transitional B cells was significantly decreased in STAT3 GOF patients. Finally, STAT3LOF and GOF patients had a reduced percentage of plasmablasts (fig. 6I, 6J). Next, we studied the effect of STAT3 deficiency on the transition of naive B cells to memory B cells in vitro. PBMCs from STAT3LOF patients were stimulated with sAg for 5min and stained for IgD and CD 27. We found that in STAT3LOF patients, the primary B cells (IgD + CD27 - ) Is significantly reduced in frequency, while memory B cells (IgD - CD27 - ) Is significantly increased (fig. 6K, 6L). These results indicate that initial B cells lacking STAT3 are prone to forming memory B cells, which may be in line with the high IgE model of STAT3LOF patients.
STAT3 deficiency results in high IgE by enhancing the miRNA146A-14-3-3σ axis.
Previous studies have shown that miRNA146A promotes IgE conversion by up-regulating 14-3-3σ expression 18 . To determine whether STAT3 modulates the miRNA146A-14-3-3 sigma axis, we examined the expression of miRNA146A and 14-3-3 sigma in B cells of STAT3 KO mice or STAT3LOF patients using RT-PCR and immunoblotting. Interestingly, mRNA levels of miRNA146a and 14-3-3σ were increased in STAT3 KO B cells (FIG. 7A), and expression of 14-3-3σ protein was also significantly increased in STAT3 KO mouse B cells (FIG. 7C). The phenotype found in STAT3 KO mice was reproduced in STAT3LOF patients (fig. 7B, 7C). Mechanistically, we found STAT3 to regulate 14-3-3σ expression by binding to the 14-3-3σ promoter using ChIP (fig. 7D). To further confirm that STAT3 modulates BCR signaling, igE production, and B cell differentiation through central hub 14-3-3 sigma, STAT3 KO B cells or mice were treated with 14-3-3 sigma inhibitor peptide R18, respectively, in vitro or in vivo. We found that in STAT3 KO B cells, the levels of BCR signaling molecules such as pAkt and pDOXO 1 were rescued to the extent of WT B cells (FIGS. 7E, 7F). In addition, we analyzed 14-3-3 sigma inhibition Agent-treated STAT3 KO mice peripheral blood B cell subclass, igE + Percentages of B cell and IgE serum, and found FO (fig. 7G, fig. 7H), GC (fig. 7I, fig. 7J) and IgE in addition to MZ B cells (fig. 8F, fig. 8G) + The percentage of B cells (fig. 7K, fig. 7L) was saved to the extent of WT mice in both resting and activated states. In addition, serum IgE titers of STAT3 KO mice injected with 14-3-3σ inhibitor (fig. 7M) were also saved. These results indicate that STAT3-miRNA146A-14-3-3σ axis is important for BCR signaling, igE production, and FO and GC B cell differentiation.
Discussion of the invention
STAT3 deficiency results in HIES, but its underlying molecular mechanism is not yet clear. By studying the early stages of B cell development using a mouse model of STAT3 deletion and patient samples, we found that the differentiation of FO, MZ and GC B cells was disrupted. It is exciting that IgE in vitro in STAT3 KO mice + Both B cell production and IgE isotype switching were enhanced, demonstrating that this model is the best model for studying the molecular mechanism of high IgE caused by STAT3 deficiency. In addition, we found STAT3 to disrupt the balance of positive and negative BCR signaling and reduce BCR signaling. Mechanically, we found that BCR clustering, B cell spreading, and BCR signaling complex recruitment were reduced in the naive B cells, probably due to the lack of driving force from actin. Furthermore, in STAT3 deficient cells, early activation of memory B cells (including B cell spreading, BCR clustering, and BCR signaling complex recruitment) is also reduced. Mechanistically, we found that a key factor in determining IgE isotype switching is the enhancement of 14-3-3σ expression in STAT3 KO B cells, and its upstream mediator miRNA146A. Interestingly, we found that inhibition of 14-3-3σ in vitro or in vivo can rescue STAT3 KO B cells or BCR signaling and IgE production in STAT3 KO mice to normal levels. This is the first report of enhanced 14-3-3σ expression in STAT3 KO B cells.
The role of 14-3-3σ in B cells has been studied previously. 14-3-3 sigma deficient mice have reduced peripheral blood B cells (e.g., FO, MZ and recycle B cells) and T cell independent antigen responses 46 . In the case of the STAT3 KO mouse,although 14-3-3σ expression was enhanced, MZ B cells were reduced, indicating that optimal expression of 14-3-3σ was critical for homeostasis of peripheral blood B cells. 14-3-3 sigma deficient mice have reduced pAkt and pErk1/2 signaling upon anti-IgM stimulation 46 . Our STAT3 KO mice also have reduced proximal and distal BCR signaling under antigen stimulation. This also suggests that optimal expression of 14-3-3σ is important for normal BCR signaling.
The relationship between BCR signaling and IgE production remains to be elucidated. DOCK8 patients suffer from HIES, and we found that BCR signaling levels were reduced in DOCK8 deficient B cells 15 . Similarly, in STAT3 patients we also found reduced DOCK8 expression and reduced BCR signaling levels. These results suggest that a decrease in BCR signaling can lead to IgE isotype switching. Previous studies have shown that BCR cross-linking blocks class switching of IgG1 and IgE by addition of LPS with IL-4 47 。
In our study, inhibition of BCR signaling in STAT3 KO B cells by the 14-3-3σ inhibitor R18 reduced IgE production. Thus, we determined that low BCR signaling favors IgE isotype switching. We have also found that STAT3 expression is critical for early activation of memory B cells. Notably, both STAT3 LOF and GOF patients had a decrease in memory B cells and an increase in primary B cells, indicating that optimal expression of STAT3 is critical for maintaining a memory response in B cells.
One problem remaining is the detailed molecular mechanism by which STAT3 regulates BCR signaling. The transcript levels of CD19, BTK and SHIP in STAT3KO B cells were not altered at all, suggesting that STAT3 does not regulate transcription of the proximal BCR signaling gene. The key is to find regulatory regions of the target gene using ChIP assay and STAT3 antibody as decoys. STAT3 has been shown to interact with several proteins such as NF-kB, relA, HIF1a, jun, STAT1, mTOR and ICOS 26、48-53 。
One contradiction is that STAT3 patients have reduced memory B cells, but STAT3KO initial B cells are anyway susceptible to memory B cell formation, probably due to a compensatory mechanism. Previous studies have shown memoryB cells may require less STAT3 activity to respond to cytokines, allowing plasmablast differentiation 35 . Our results demonstrate that STAT3-KO naive B cells are prone to become memory B cells and IgE-switching plasmablasts; this is probably due to the higher expression of miRNA146A and 14-3-3σ proteins. We need to further determine the expression of activation-induced deaminase (AID) in STAT3KO B cells. However, it is not clear why memory B cells of STAT3 patients are reduced. A recent study showed that mTORC1 deficiency resulted in Ag-specific memory B cell and plasma and GC B cell depletion 54 . Our results indicate that both mTORC1 and mTORC2 activity in STAT3 KO B cells are reduced and that STAT3-mTORC axis modulates memory B cell formation. Furthermore, STAT3 KO has a low metabolic activity of the primary B cells, which may be responsible for the conversion of the primary B cells to memory B cells.
In general, our study reveals the underlying mechanisms of STAT3 patient hyperige syndrome by using optimized mouse models and patient samples. Enhancement of 14-3-3σ expression in STAT3 KO mice results in increased IgE + B cell production and lower BCR signaling.
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Claims (3)
- Use of a 14-3-3 sigma inhibitor in the manufacture of a medicament for the prevention and/or treatment of a STAT3 related disease in a patient in need of such prevention and/or treatment,wherein the 14-3-3 sigma inhibitor is a peptide, andthe peptide consists of an amino acid sequenceNH 2 -YGRKKKRQRRRPHCVPRDLSWLDLEANMCLP-COOH of the composition,wherein the STAT 3-related disease is a nonfunctional STAT3, and the nonfunctional STAT3 is a hyper IgE syndrome.
- 2. Use of a pharmaceutical composition comprising a 14-3-3 sigma inhibitor for the manufacture of a medicament for the prevention and/or treatment of STAT 3-related diseases in a patient in need of such prevention and/or treatment,Wherein the 14-3-3 sigma inhibitor is a peptide consisting of the amino acid sequenceNH 2 -YGRKKKRQRRRPHCVPRDLSWLDLEANMCLP-COOH of the composition,wherein the STAT 3-related disease is a nonfunctional STAT3, and the nonfunctional STAT3 is a hyper IgE syndrome.
- 3. Use of a kit comprising a 14-3-3 sigma inhibitor for the manufacture of a medicament for the prevention and/or treatment of a STAT3 related disease in a patient in need of such prevention and/or treatment,wherein the 14-3-3 sigma inhibitor is a peptide consisting of the amino acid sequenceNH 2 -YGRKKKRQRRRPHCVPRDLSWLDLEANMCLP-COOH of the composition,wherein the STAT 3-related disease is a nonfunctional STAT3, and the nonfunctional STAT3 is a hyper IgE syndrome.
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| CN101854930A (en) * | 2007-09-10 | 2010-10-06 | 波士顿生物医药公司 | A novel group of STAT3 pathway inhibitors and cancer stem cell pathway inhibitors |
| CN101925364A (en) * | 2007-11-27 | 2010-12-22 | 不列颠哥伦比亚大学 | Be used to prevent 14-3-3 antagonist with treatment of arthritis |
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| JP7411315B2 (en) * | 2018-04-19 | 2024-01-11 | トゥヴァルディ セラピューティクス，インク． | STAT3 inhibitor |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101854930A (en) * | 2007-09-10 | 2010-10-06 | 波士顿生物医药公司 | A novel group of STAT3 pathway inhibitors and cancer stem cell pathway inhibitors |
| CN101925364A (en) * | 2007-11-27 | 2010-12-22 | 不列颠哥伦比亚大学 | Be used to prevent 14-3-3 antagonist with treatment of arthritis |
Non-Patent Citations (3)
| Title |
|---|
| 14-3-3 Proteins Mediate an Essential Anti-apoptotic Signal;Shane C. Masters等;THE JOURNAL OF BIOLOGICAL CHEMISTRY;第276卷(第48期);摘要，第45194页，左栏第二段 * |
| 14-3-3蛋白的新认识;潘伟男等;北京生物医学工程;第34卷(第3期);第324页，左栏，第二-第三段 * |
| Isolation of High-Affinity Peptide Antagonists of 14-3-3 Proteins by Phage Display;Bingcheng Wang等;Biochemistry;第38卷;摘要，第12501页，表1 * |
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