WO2016172219A1

WO2016172219A1 - Methods and compositions for inhibition of toll-like receptors (tlrs)-mediated inflammation

Info

Publication number: WO2016172219A1
Application number: PCT/US2016/028451
Authority: WO
Inventors: Kwang Chul Kim
Original assignee: Temple Univ School of Medicine; University of Arizona; Arizona's Public Universities
Current assignee: Temple Univ School of Medicine; University of Arizona; Arizona's Public Universities
Priority date: 2015-04-24
Filing date: 2016-04-20
Publication date: 2016-10-27
Anticipated expiration: 2017-10-24

Abstract

The invention is directed to a compound that binds to a Toll-like Receptor (TLR) and prevents TLR-mediated inflammation. The present invention is further directed to methods for blocking an interaction between TLR and MyD88 and/or TRIF, methods for inhibiting TLR-mediated inflammation in a mammalian cell, methods for preventing and/or treating a mammal suffering or at risk for suffering from TLR-mediated inflammation, and methods for preventing and/or treating a mammal suffering or at risk for suffering from a disorder related to TLR-mediated inflammation (e.g., chronic inflammatory disorders).

Description

METHODS AND COMPOSITIONS FOR INHIBITION OF TOLL-LIKE RECEPTORS (TLRS)-MEDIATED INFLAMMATION

CROSS REFERENCE TO RELATED APPLICATIONS

The present invention claims priority to U.S. Provisional Patent Application

62/152,296, filed April 24, 2015, which is incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No. R01 HL047125 awarded by NIH. The government has certain rights in the invention.

FIELD OF THE INVENTION

INTRODUCTION

Toll-like receptors (TLRs) are a class of proteins that play a key role in the innate immune system. They are single, membrane-spanning, non-catalytic receptors usually expressed in various cell types associated with host defense, that recognize structurally conserved molecules called pathogen-associated molecular patterns (PAMPs) derived from microbes. Once PAMPs are in contact with the cells they are recognized by TLRs, which activate immune cell responses through well-defined signaling pathways.

Indeed, TLRs participate in the first line of defense against invading pathogens and play a significant role in inflammation, immune cell regulation, survival, and proliferation. To date, 11 members of the TLR family have been identified, of which TLRl, TLR2, TLR4, TLR5, TLR6, and TLRl 1 are located on the cell surface and TLR3, TLR7, TLR8, and TLR9 are localized to the endosomal/lysosomal compartment. The activation of the TLR signaling pathway originates from the cytoplasmic Toll/IL-1 receptor (TIR) domain that associates with a TIR domain-containing adaptor, MyD88 and/or TRIF. Upon stimulation with ligands, MyD88 recruits IL-1 receptor-associated kinase-4 (IRAK-4) to TLRs through interaction of the death domains of both molecules. IRAK-1 is activated by phosphorylation and associates with TRAF6, thereby activating the IKK complex and leading to activation of MAP kinases (JNK, p38, ERK) and NF-κΒ. Tollip and IRAK-M interact with IRAK-1 and negatively regulate the TLR-mediated signaling pathways. Additional modes of regulation for these pathways include TRIF-dependent induction of TRAF6 signaling by RIP1 and negative regulation of TIRAP-mediated downstream signaling by ST2L, TRIAD3A, and SOCS1. Activation of MyD88- independent pathways occurs via TRIF and TRAF3, leading to recruitment of ΙΚΚε/ΤΒΚΙ, phosphorylation of IRF3, and expression of interferon-β. TIR domain containing adaptors such as TIRAP, TRIF, and TRAM regulate TLR-mediated signaling pathways by providing specificity for individual TLR signaling cascades. TRAF3 plays a critical role in the regulation of both MyD88-dependent and TRIF-dependent signaling via TRAF3 degradation, which activates MyD88-dependent signaling and suppresses TRIF-dependent signaling (and vice versa).

Improved methods for treating disorders related to TLR-mediated inflammation are needed. SUMMARY OF THE INVENTION

Mucin 1(MUC1) is a membrane-tethered glycoprotein expressed in various mucosal epithelial cells as well as hematopoietic cells and plays an anti-inflammatory role during the resolution phase of airway bacterial infection. Recently, it was shown that the antiinflammatory effect of MUC1 is attributable to its cytoplasmic tail, specifically the presence of the EGFR phosphorylation site (YEKV (SEQ ID NO: 1)). In experiments conducted during the course of developing embodiments for the present invention, a compound which consists of an 11-mer peptide of MUC1 cytoplasmic tail containing YEKV (SEQ ID NO: 1) and two covalently linked carrier molecules - TAT peptide and palmitic acid - was synthesized in order to determine whether this compound exhibits an anti-inflammatory effect in an in vitro inflammation model. Such experiments demonstrated that this compound can inhibit

Pseudomonas aeruginosa-induced IL-8 release in a dose-dependent, sequence specific manner without cytotoxicity in an identical mechanism as endogenous MUC1 : by

interference of MyD88 recruitment to TLR5→ NF-kB activation→ IL-8 release. In addition, it was shown that mutations on YEKV (SEQ ID NO: 1) of the 11 mer abolished the antiinflammatory activity, indicating that tyrosine phosphorylation by EGFR is required.

Furthermore, this MUCl- like synthetic compound may provide an alternative therapy for chronic inflammatory disorders such as COPD.

Accordingly, the invention is directed to a compound that binds to a Toll-like

Receptor (TLR) and prevents TLR-mediated inflammation. The present invention is further directed to methods for blocking an interaction between TLR and MyD88 and/or TRIF (see, e.g., Kato, et al, Am J Respir Cell Mol Biol 2014, Vol. 51(3):446-454; Ueno, et al, Am J Respir Cell Mol Biol 2008 Vol. 38:263-268), methods for inhibiting TLR-mediated inflammation in a mammalian cell, methods for preventing and/or treating a mammal suffering or at risk for suffering from TLR-mediated inflammation, and methods for preventing and/or treating a mammal suffering or at risk for suffering from a disorder related to TLR-mediated inflammation (e.g., chronic inflammatory disorders).

In certain embodiments, the present invention provides a composition comprising a compound that binds to a Toll-like Receptor (TLR) polypeptide and inhibits TLR-mediated inflammation, wherein the compound comprises a MUCl peptide or MUCl mimetic.

In some embodiments, the MUCl peptide or MUCl mimetic comprises at least a portion of the wild type amino acid sequence of MUCl. In some embodiments, the MUC-1 peptide or MUCl mimetic comprises at least a portion of the wild type MUCl amino acid sequence which permits the resulting compound to inhibit interaction between a TLR polypeptide (e.g., TLR5) and MyD88 and/or TRIF. In some embodiments, the MUCl peptide or MUCl mimetic comprises at least a portion of the wild type MUCl amino acid sequence which permits the resulting compound to inhibit TLR-mediated inflammation (e.g., TLR- mediated inflammation resulting from interaction between a TLR polypeptide (e.g., TLR5) and MyD88 and/or TRIF). In some embodiments, the MUCl peptide or MUCl mimetic comprises at least a portion of the wild type MUCl amino acid sequence which permits the resulting compound to inhibit NF-kB activation resulting from interaction between a TLR polypeptide (e.g., TLR5) and MyD88). In some embodiments, the MUCl peptide or MUCl mimetic comprises at least a portion of the wild type MUCl amino acid sequence which permits the resulting compound to inhibit IL-8 release resulting from NF-kB activation (e.g., resulting from interaction between a TLR polypeptide (e.g., TLR5) and MyD88 and/or TRIF). In some embodiments, such a MUCl peptide or MUCl mimetic comprises the following amino acid sequence: YEKV (SEQ ID NO: l). In some embodiments, such a MUCl peptide or MUCl mimetic comprises the following amino acid sequence:

TDRSPYEKVSA (SEQ ID NO:2). The wild type MUCl nucleic acid sequence and amino acid sequence is provided at Figs. 9A (NM_013605.2) and 9B.

In some embodiments, the MUCl peptide or MUCl mimetic further comprises a cell- penetrating peptide. Such embodiments are not limited to a particular type of a cell- penetrating peptide. For example, in some embodiments, the cell-penetrating peptide is selected from the group consisting of HIV-derived TAT peptide, penetratins, transportans, SS peptides, and hCT derived cell-penetrating peptides. In some embodiments, the cell- penetrating peptide is a Trans-Activator of Transcription (TAT) sequence. For example, in some embodiments, the TAT sequence has the amino acid sequence RRRQRRKKRGY (SEQ ID NO: 3).

In some embodiments, the MUCl peptide or MUCl mimetic further comprises at least one biocompatible carrier. In some embodiments, the biocompatible carrier is selected from the group consisting of poly-lactic acid, poly-gly colic acid, and copolymers of poly- lactic acid and poly-gly colic acid. In some embodiments, the biocompatible carrier comprises at least one biodegradable fatty acid or a metal salt thereof. In some embodiments, the biodegradable fatty acid is selected from the group consisting of palmitic acid, stearic acid, oleic acid, myristic acid, and metal salts thereof. In some embodiments, the biocompatible carrier comprises a salt selected from the group consisting of porous or non-porous calcium phosphates, porous or non-porous hydroxyapatites, porous or non-porous tricalcium phosphates, porous or non-porous tetracalcium phosphates, porous or non-porous calcium sulfates, and combinations thereof.

In some embodiments, the MUCl peptide or MUCl mimetic is directly linked to the cell-penetrating peptide. In some embodiments, the cell-penetrating peptide is directly linked to the MUCl peptide or MUCl mimetic, and the MUCl peptide or MUCl mimetic is directly linked to the biocompatible carrier. In some embodiments, the cell-penetrating peptide is directly linked to the biocompatible carrier, and the cell-penetrating peptide is directly linked to the MUCl peptide or MUCl mimetic.

In some embodiments, the compound is within a composition comprising a pharmaceutically acceptable excipient.

In some embodiments, the compound inhibits TLR-mediated inflammation through inhibiting an interaction between MyD88 and/or TRIF and a TLR polypeptide. In some embodiments, the TLR polypeptide is selected from TLRl , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11 , TLRl 2, and TLRl 3.

In certain embodiments, the present invention provides methods for inhibiting interaction between MyD88 and/or TRIF and a TLR polypeptide in a mammalian cell, the method comprising contacting the TLR polypeptide with a compound as described herein, wherein the mammalian cell expresses MyD88 and/or TRIF and a TLR polypeptide. In some embodiments, the mammalian cell is experiencing or is at risk for experiencing TLR- mediated inflammation. In some embodiments, the mammalian cell is a mucosal epithelial cell. In some embodiments, the mammalian cell is in a living human. In some embodiments, the TLR polypeptide is selected from TLRl , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR1 1, TLR12, and TLRl 3.

In certain embodiments, the present invention provides methods of inhibiting TLR- inflammation in a mammalian cell, the method comprising treating the cell with a compound as described herein, wherein the mammalian cell is experiencing or is at risk for experiencing TLR-mediated inflammation. In some embodiments, the mammalian cell is a mucosal epithelial cell. In some embodiments, the mammalian cell is in a living human. In some embodiments, the compound inhibits TLR-mediated inflammation through inhibiting an interaction between MyD88 and/or TRIF and a TLR polypeptide. In some embodiments, the TLR polypeptide is selected from TLRl , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLRl 1, TLRl 2, and TLR13.

In certain embodiments, the present invention provides methods for preventing and/or treating a mammal experiencing or at risk for TLR-mediated inflammation, the method comprising administering the composition of claim 1 to the mammal. In some embodiments, the mammal is suffering from or is at risk for developing a disorder related to TLR-mediated inflammation. In some embodiments, the disorder is selected from pulmonary fibrosis,

COPD, asthma, cardiovascular disorder, diabetes, obesity, metabolic syndrome, autoimmune disorders, neuroinflammatory disorders, schizophrenia, bipolar disorder, autism, clinical depression, chronic fatigue syndrome, alcohol abuse, and toluene inhalation. In some embodiments, the mammal is a human. In some embodiments, the compound inhibits TLR- mediated inflammation through inhibiting an interaction between MyD88 and/or TRIF and a TLR polypeptide. In some embodiments, the TLR polypeptide is selected from TLRl, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR1 1, TLR12, and TLRl 3. In some embodiments, the present invention provides an isolated polypeptide comprising the amino acid sequence TDRS P YEKVS ARRRQRRKKRGYK (SEQ ID NO:4).

In some embodiments, the present invention provides a peptide or mimetic comprising the amino acid sequence NH2-TDRSPYEKVSARRRQRRKKRGYK-Palmitic Acid (SEQ ID NO: 4).

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 A-B. Structure of Peptide 1 (A) The sequence of 72 amino acids consisting of the MUC1 cytoplasmic tail (CT). (B) The structure of Peptide 1 which consists of three components - 11 mer, TATp, and CI 6 palmitic acid, which are covalently linked through an extra lysine (K) moiety.

FIG. 2A-C. Effect of Peptide 1 on PAK-induced IL-8 release and LDH release 293- hTLR5-HA cells were incubated in the presence of varying concentrations of Peptide 1 for 1 h prior to stimulation with PAK (10⁶ CFU/ml) for 2 h. Spent media were collected and analyzed for IL-8 contents by ELISA (A) and LDH activity using a LDH assay kit (B). (C) Cells were pretreated with 10 μΜ of Peptide 1 for 1 or 3 h prior to PAK stimulation. Each bar represents the mean ± SEM (n=4). *, p<0.05; **, pO.01; NS, not significant (p>0.05). The results are representative of at least two independent experiments.

FIG. 3. Effect of Peptide 1 mutants on PAK-induced IL-8 release 293-hTLR5-HA cells were pretreated with 10 μΜ of Peptide 1 or its derivatives for 1 h prior to stimulation with PAK (10⁶ CFU/ml) for 2 h. While Peptide 1 has YEKV (SEQ ID NO: 1) sequence, Peptide 2 and 3 have FEKV (SEQ ID NO: 10) and YKEV (SEQ ID NO: 11) sequence, respectively (see Figure 1 A and Methods). TATp-C16 refers to the carrier molecule used for delivering the peptides into the cell. The amounts of IL-8 in the spent media were measured by ELISA. Each bar represents the mean ± SEM (n=4). **, pO.01. The results are representative of at least two independent experiments.

FIG 4A-B. Effect of Peptide 1 on PAK-induced NF-κΒ activation (A) 293-hTLR5- HA cells were transiently transfected with both aNF-κΒ dependent luciferase plasmid (reporter gene) and a Renilla luciferase plasmid (reference gene) as described in Methods. After transfection, cells were incubated for 1 h with Peptide 1 or PBS prior to stimulation with PAK (10⁶ CFU/ml) for 2 h. Relative luciferase activities of cell lysates were measured by a luminometer using the Dual-luciferase reporter system. (B) 293-hTLR5 NF- κΒ/SEAPorter cells were treated with Peptide 1 and PAK for 4 h under the same condition as above. Aliquots of spent media were subjected to the SEAP assay as described in Methods.

Each bar represents the mean ± SEM (n=4). **, p<0.01. The results are representative of at least two independent experiments.

FIG. 5. Effect of Peptide 1 on PAK-induced ΙκΒ-α degradation and p65

phosphorylation 293-hTLR5-HA cells were pretreated with Peptide 1 (10 μΜ) for 1 h prior to treatment with PAK (10⁶ CFU/ml) for the indicated time periods. Equal protein amounts of cell ly sates were subjected to Western blotting with indicated antibodies. The results are representative of three independent experiments.

FIG. 6. Effect of Peptide 1 on PAK-induced nuclear translocation of p65 293-hTLR5- HA cells were pretreated with Peptide 1 (10 μΜ) for 1 h prior to treatment with PAK (10⁶

CFU/ml) for 30 min. Cells were subjected to immunohistochemistry for localizing NF-KB p65 as described in Methods. DAPI was used to counterstain nuclei. Scale bars, 200 μηι. The results are representative of two independent experiments.

FIG. 7. Effect of Peptide 1 mutants on PAK-induced NF-κΒ activation All the experimental conditions were identical with those in Figure 4B except for using various

Peptide 1 mutants as in Figure 3. Each bar represents the mean ± SEM (n=4). **, p<0.01. The results are representative of at least two independent experiments.

FIG. 8A-B. Effect of Peptide 1 on PAK-induced TLR5/MyD88 interaction 293- hTLR5-HA cells were pretreated with Peptide 1 (10 μΜ) for 1 h followed by treatment with PAK (10⁶ CFU/ml) for 30 min. Equal protein amounts of cell lysates were used for immunoprecipitation with anti-MyD88 Ab or isotype-matched normal rabbit IgG, and the immunoprecipitated proteins were subjected to Western blotting with the indicated Abs (A).

Protein expression levels of TLR5 and MyD88 were verified in the same lysates used for immunoprecipitation (B). The results are representative of three independent experiments.

FIG. 9A and 9B show the wild type MUC1 nucleic acid sequence and amino acid sequence.

DETAILED DESCRIPTION OF THE INVENTION

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death in the United States. Although its symptoms may be ameliorated with proper treatments, it still remains incurable due to the nature of the progressive, irreversible structural remodeling of the lung. Given that chronic inflammation results from the failure to control inflammation, one possible therapeutic approach toward COPD would be to control the initial stage of pulmonary inflammation in order to prevent it from developing into the chronic phase of inflammation.

MUC1 (MUC in human and Muc in animals) is a membrane-tethered mucin-like glycoprotein expressed in mucosal epithelial cells as well as some hematopoietic cell types (13, 14). Studies with Mucl knockout (KO) mice revealed that these animals are much more inflammatory compared to their wild type (WT) littermates in a mouse model of acute infection with Pseudomonas aeruginosa (Pa) (5, 17) suggesting that Mucl is an antiinflammatory molecule in the lung during bacterial infection. The anti-inflammatory role of Mucl was also demonstrated in a chronic Pa infection model in which Mucl KO mice showed much greater airspace enlargement - which is a major phenotype of emphysema - compared to their WT (21). Based on the results of these animal experiments, experiments conducted during the course of developing embodiments for the present invention hypothesized that MUCl/Mucl may play a crucial role in controlling pulmonary

inflammation and also dysfunctional MUCl/Mucl may result in the development of chronic inflammatory disease such as COPD.

Using various genetic and molecular biological methods, it has been shown that the anti-inflammatory activity of MUCl/Mucl is mediated through its inhibition of Toll-like receptor (TLR) signaling (12, 17, 20). The detailed mechanistic studies revealed that MUCl/Mucl binds to TLRs and thus preventing the interaction between TLRs and MyD88 (10) or TRIF (9), the two adaptor proteins required for TLR signaling following activation of TLRs by their agonists (1). Furthermore, the interaction between MUC1 and TLR5 was greatly enhanced by tyrosine phosphorylation of MUC1 on Y46 by EGFR (10). Since it has also been shown that the anti-inflammatory activity of MUC 1 requires only the cytoplasmic tail (CT) domain and not the extracellular domain (10, 17, 20), it was presumed that it would be reasonable to predict that a "small" peptide segment containing Y46 of the MUC1 CT domain may be able to mimic the anti-inflammatory activity of MUC 1.

Cell-penetrating peptides (CPPs) have been used to overcome the lipophilic barrier of the cellular membranes and deliver a large variety of cargoes such as proteins, DNA, antibodies, imaging agents, toxins, and nanoparticular drug carriers including liposomes (7) into the cells. The TAT peptide (TATp) is derived from the transactivator of transcription

(TAT) of human immunodeficiency virus (3) and has been successfully used as a CPP (18). In the present experiment, a TAT sequence (YGRKKRRQRRR; amino acids 47 to 57 of TAT)(4) and palmitic acid was used to introduce an 11-mer MUC1 CT domain containing Y46 in the center in order to determine whether this peptide can mimic the anti -inflammatory activity of MUC1 without cytotoxicity. Such experiments demonstrated that this compound can inhibit Pseudomonas aeruginosa-induced IL-8 release in a dose-dependent, sequence specific manner without cytotoxicity in an identical mechanism as endogenous MUC1 : by

interference of MyD88 recruitment to TLR5→ NF-kB activation→ IL-8 release. In addition, it was shown that mutations on YEKV (SEQ ID NO: 1) of the 11 mer abolished the antiinflammatory activity, indicating that EGFR binding is required. Furthermore, this MUC1- like synthetic compound may provide an alternative therapy for chronic inflammatory disorders such as COPD.

Accordingly, the present invention is based on the discovery that peptides having a portion of the sequence of MUC1 inhibits interaction between TLR and MyD88, which thereby inhibits TLR-mediated inflammation.

Thus, in some embodiments, the invention is directed to compounds that bind to a TLR polypeptide and prevent binding between the TLR polypeptide and MyD88 and/or TRIF. The compounds are not limited to binding a particular TLR polypeptide. In some embodiments, the TLR polypeptide is selected from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLRl l, TLR12, and TLR13. In some embodiments, the invention is directed to compounds that bind to TLR5 and prevent binding between the TLR5 polypeptide and MyD88.

The invention is not limited to a particular type or kind of compound that binds to a

TLR polypeptide. In some embodiments, the compound is a peptide or mimetic of MUC1. For example, in some embodiments, such peptides or mimetics comprise a portion of the amino acid sequence of MUC1 which permits the resulting peptide or mimetic to bind to a TLR polypeptide and thereby prevent TLR-mediated inflammation. In some embodiments, the MUC1 peptide or MUC1 mimetic comprises the amino acid sequence YEKV (SEQ ID NO: l). In some embodiments, the peptides or mimetics comprise the amino acid sequence TDRSPYEKVSA (SEQ ID NO:2).

The MUC1 peptide or MUC1 mimetic can also comprise one or more functional groups, such as a moiety that facilitates purification, e.g., a (His)6 moiety or an antibody - binding epitope.

In some embodiments, the MUC1 peptide or MUC1 mimetic further comprises at least one biocompatible carrier (e.g., poly-lactic acid, poly-gly colic acid, and copolymers of poly-lactic acid and poly-gly colic acid). In some embodiments, the MUC1 peptide or MUC1 mimetic further comprises at least one biocompatible carrier selected from the group consisting of porous or non-porous calcium phosphates, porous or non-porous hydroxyapatites, porous or non-porous tricalcium phosphates, porous or non-porous tetracalcium phosphates, and porous or non-porous calcium sulfates, or a combination thereof.

In some embodiments, the biocompatible carrier is palmitic acid.

In some embodiments, the MUC1 peptide or MUC1 mimetic further comprises a cell- penetrating moiety that facilitates delivery of the peptides to an intracellular space, e.g., HIV- derived TAT peptide, penetratins, transportans, SS peptides (alternating aromatic residues and basic amino acids (aromatic-cationic peptides)), SA, SM, or SNL peptides, or hCT derived cell-penetrating peptides (see, e.g., Caron et al, (2001) Mol. Ther. 3(3):310-8; Langel, Cell- Penetrating Peptides: Processes and Applications (CRC Press, Boca Raton Fla. 2002); El- Andaloussi et al, (2005) Curr Pharm Des. 11(28):3597-611; Lindgren et al, Trends

Pharmacol Sci. 21(3):99-103 (2000); Zhao et al, J Biol Chem 279:34682-34690 (2004); Szeto, AAPS Journal 2006; 8 (2) Article 32; Deshayes et al, (2005) Cell Mol Life Sci.

62(16): 1839-49; Horn et al, J. Med. Chem., 46: 1799 (2003); Bonny et al, Diabetes, 50:77-82 (2001), and U.S. Patent Nos. 6,841,535 and 7,576,058). In some embodiments the cell- penetrating moiety is linked to the MUC1 peptide or MUC1 mimetic, e.g., as a single fusion protein; thus, the invention includes fusion proteins comprising a MUC1 peptide as described herein and a cell-penetrating peptide, e.g., TAT, penetratins, transportans, or hCT derived cell-penetrating peptides. In some embodiments, the cell-penetrating peptide is attached to the N-terminus of the MUC1 peptide; in some embodiments, the cell-penetrating peptide is attached to the C-terminus of the MUC1 peptide. In some embodiments, the fusion protein further comprises a cleavable moiety as known in the art between the cell-penetrating peptide and the MUC1, that cleaves off the cell-penetrating peptide, leaving the MUC1 peptide intact.

An additional useful functional group here is a moiety that facilitates detection of the MUC1 peptide or MUC1 mimetic, such as a fluorescent moiety, a radioactive moiety, or an antigen.

In some embodiments, the compound comprises a TAT sequence and a palmitic acid sequence. For example, the MUC1 peptide or MUC1 mimetic may be linked directly to the TAT sequence, and the TAT sequence is directly linked to the palmitic acid sequence. In another embodiment, the TAT sequence is directly linked to the MUC1 peptide or MUC1 mimetic, and the MUC1 peptide or MUC1 mimetic is directly linked to the palmitic acid sequence. In yet another embodiment, the TAT sequence is directly linked to the palmitic acid sequence, and the palmitic acid sequence is directly linked to the MUC1 peptide or MUC1 mimetic.

For therapeutic uses, the MUC1 peptide or MUC1 mimetic is preferably in a pharmaceutically acceptable excipient. Such compositions can be formulated without undue experimentation for administration to a mammal, including humans, as appropriate for the particular application. Additionally, proper dosages of the compositions can be determined without undue experimentation using standard dose-response protocols.

Accordingly, the MUC1 peptide or MUC1 mimetic compositions designed for oral, lingual, sublingual, buccal and intrabuccal administration can be made without undue experimentation by means well known in the art, for example with an inert diluent or with an edible carrier. The compositions may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the pharmaceutical compositions of the present invention may be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums and the like.

Tablets, pills, capsules, troches and the like may also contain binders, recipients, disintegrating agent, lubricants, sweetening agents, and flavoring agents. Some examples of binders include microcrystalline cellulose, gum tragacanth or gelatin. Examples of excipients include starch or lactose. Some examples of disintegrating agents include alginic acid, com starch and the like. Examples of lubricants include magnesium stearate or potassium stearate. An example of a glidant is colloidal silicon dioxide. Some examples of sweetening agents include sucrose, saccharin and the like. Examples of flavoring agents include peppermint, methyl salicylate, orange flavoring and the like. Materials used in preparing these various compositions should be pharmaceutically pure and nontoxic in the amounts used.

The MUC1 peptide or MUC1 mimetic compositions of the present invention can easily be administered parenterally such as for example, by intravenous, intramuscular, intrathecal or subcutaneous injection. Parenteral administration can be accomplished by incorporating the compositions of the present invention into a solution or suspension. Such solutions or suspensions may also include sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents. Parenteral formulations may also include antibacterial agents such as for example, benzyl alcohol or methyl parabens, antioxidants such as for example, ascorbic acid or sodium bisulfite and chelating agents such as EDTA. Buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose may also be added. The parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic.

Rectal administration includes administering the pharmaceutical peptide or mimetic compositions into the rectum or large intestine. This can be accomplished using suppositories or enemas. Suppository formulations can easily be made by methods known in the art. For example, suppository formulations can be prepared by heating glycerin to about 120° C, dissolving the composition in the glycerin, mixing the heated glycerin after which purified water may be added, and pouring the hot mixture into a suppository mold.

Transdermal administration includes percutaneous absorption of the composition through the skin. Transdermal formulations include patches (such as the well-known nicotine patch), ointments, creams, gels, salves and the like.

The present invention includes nasally administering to the mammal a therapeutically effective amount of the composition. As used herein, nasally administering or nasal administration includes administering the composition to the mucous membranes of the nasal passage or nasal cavity of the patient. As used herein, pharmaceutical compositions for nasal administration of a composition include therapeutically effective amounts of the composition prepared by well-known methods to be administered, for example, as a nasal spray, nasal drop, suspension, gel, ointment, cream or powder. Administration of the MUC 1 peptide or MUC1 mimetic composition may also take place using a nasal tampon or nasal sponge.

In some embodiments, the invention is directed to methods of inhibiting interaction between a TLR polypeptide (e.g., TLR5) and MyD88 and/or TRIF. Such methods comprise contacting the cells expressing a TLR polypeptide (e.g., TLR5) and MyD88 and/or TRIF with any of the compounds described above. In some embodiments of these methods, the TLR polypeptide (e.g., TLR5) and MyD88 and/or TRIF are is in a mammalian cell. Addition of the peptides and mimetics as described herein inhibits such binding between the TLR polypeptide (e.g., TLR5) and MyD88 and/or TRIF. In some embodiments, such mammalian cells are expressed in a living mammal. The invention methods would be expected to work in any mammal, however, in the most preferred embodiments, the mammal is a human.

In some embodiments, the invention is directed to methods of inhibiting TLR- mediated inflammation in a mammal. In some embodiments, methods of inhibiting TLR- mediated inflammation in a mammal is accomplished through inhibiting interaction between a TLR polypeptide (e.g., TLR5) and MyD88 and/or TRIF. Such methods comprise contacting the cells expressing a TLR polypeptide (e.g., TLR5) and MyD88 and/or TRIF with any of the compounds described above. In some embodiments, such mammalian cells are expressed in a living mammal. The invention methods would be expected to work in any mammal, however, in the most preferred embodiments, the mammal is a human.

In some embodiments, the invention is directed to methods of preventing and/or treating an individual suffering from or at risk for suffering from TLR-mediated

inflammation. In some embodiments, methods of inhibiting TLR-mediated inflammation in a mammal is accomplished through inhibiting interaction between a TLR polypeptide (e.g., TLR5) and MyD88 and/or TRIF. Such methods comprise administering to the individual a composition comprising any of the compounds described above. In some embodiments, the individual is a living mammal (e.g., a living human).

In some embodiments, the invention is directed to methods of treating an individual suffering from or at risk for suffering from a disorder related to TLR-mediated inflammation. In some embodiments, methods of treating a disorder related to TLR-mediated inflammation is accomplished through inhibiting interaction between a TLR polypeptide (e.g., TLR5) and MyD88 and/or TRIF. Such methods comprise administering to the individual a composition comprising any of the compounds described above. In some embodiments, the individual is a living mammal (e.g., a living human). Examples of disorders related to TLR-mediated inflammation include, but are not limited to, pulmonary fibrosis, COPD, asthma,

cardiovascular disorder, diabetes, obesity, metabolic syndrome, autoimmune disorders, neuroinflammatory disorders, schizophrenia, bipolar disorder, autism, clinical depression, chronic fatigue syndrome, alcohol abuse, and toluene inhalation.

In some embodiments, the compound comprises a peptide or mimetic that comprises the sequence of SEQ ID NO: l or SEQ ID NO:2.

In some embodiments, the present invention provides a polypeptide comprising SEQ

ID NO: l or SEQ ID NO:2. Thus, the invention is further directed to polypeptides comprising SEQ ID NO: l or SEQ ID NO:2. Also useful are polynucleotides encoding this polypeptide, and vectors comprising this polynucleotide.

One of ordinary skill in the art will readily recognize that the foregoing represents merely a detailed description of certain preferred embodiments of the present invention.

Various modifications and alterations of the compositions and methods described above can readily be achieved using expertise available in the art and are within the scope of the invention. EXAMPLES

The following examples are illustrative, but not limiting, of the compounds, compositions, and methods of the present invention. Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered in clinical therapy and which are obvious to those skilled in the art are within the spirit and scope of the invention.

Example I.

This example demonstrates that a MUC1 CT 11-mer has an ability to suppress PAK- induced IL-8 release without cytotoxicity. It has been shown that the anti-inflammatory effect of MUC1 resides in its CT and requires tyrosine phosphorylation of the CT by EGFR on Y46 (10, 17, 20). To determine whether a small peptide containing Y46 can mimic the antiinflammatory effect of MUC1, we chose an 11-mer from MUC1 CT that contains Y46 in its center (Fig. 1A). To introduce the 11-mer into the cell, it was covalently linked to both TATp sequence (4) and palmitic acid (Fig. IB). The resulting peptide is referred to as Peptide 1 throughout this study. Confluent 293-hTLR5-HA cells were treated with various

concentrations of Peptide 1 for 1 h followed by stimulation with PAK for 2 h, and culture spent media were analyzed for the amounts of IL-8 using ELISA. Fig. 2A shows that treatment with PAK drastically increased IL-8 release from the cells, which was suppressed by pretreatment with Peptide 1 in a dose-dependent manner except for 100 μΜ which showed an increase, suspecting a possible cytotoxicity. To test for the possible cytotoxicity of Peptide 1, the spent media were analyzed for LDH activity. Fig. 2B shows that treatment with Peptide 1 didn't cause a significant release of LDH until the concentration reached 100 μΜ - suggesting that a significant increase in IL-8 release by 100 μΜ of Peptide 1 was most likely due to the plasma membrane damage at this concentration. Cytotoxicity was also observed under light microscopy (data not shown). Based on the efficacy and cytotoxicity data, we decided to use 10 μΜ for the remaining experiments of this study. This concentration suppressed PAK-induced IL-8 release from these cells by 40-60% in a consistent manner. Next, we tried to determine whether the 1 h pretreatment with Peptide 1 is sufficient to see a maximum effect of IL-8 release. Fig. 2C shows that there is no difference in the level of IL-8 release between the 1 h and 3 h treatment period. Together, the results of these initial experiments demonstrated that Peptide 1 has an ability to suppress PAK-induced IL-8 release in an in vitro inflammation model and served to establish the experimental condition for the remainder of this study.

Example II.

This example demonstrates that the anti -inflammatory effect of Peptide 1 is sequence specific. Next, we determined whether the anti-inflammatory effect of Peptide 1 was due to the presence of the carrier (i.e., TATp-C16) or the cargo peptide sequence (i.e., 11-mer) or both. We tested the carrier and several derivatives of 11-mer for their possible antiinflammatory effect. Fig. 3 shows that, while the presence of the 11-mer (Peptide 1) exhibits the anti -inflammatory effect, neither the carrier alone (TATp-C16) nor its two mutants

(Peptide 2: a mutation of Peptide 1 on Y46; Peptide 3: a mutant of Peptide 1 by substitution of YKEV (SEQ ID NO: 11) for YEKV (SEQ ID NO: l)) (see Methods and Fig. 1A) succeeded in displaying any anti-inflammatory effect under exactly the same treatment conditions. Since Y46EKV is the consensus sequence motif for EGFR tyrosine phosphorylation on MUC1 CT, these results seem to confirm the requirement of EGFR interaction with MUC1 CT for the anti-inflammatory effect of MUC1 (10). In summary, the results clearly indicate that the antiinflammatory effect of Peptide 1 is sequence specific and requires YEKV (SEQ ID NO: l), the EGFR phosphorylation consensus sequence. Example III

This example demonstrates that the anti-inflammatory effect of Peptide 1 is mediated through activation of NF-κΒ. TLR5 regulates IL-8 gene expression mainly through the NF- KB signaling pathway (12, 20). To determine whether the suppression of PAK-induced IL-8 release was mediated through inhibition of NF-κΒ activation, we measured the activation of PAK-induced NF-κΒ following pretreatment with Peptide 1 using two different methods - the NF-κΒ dependent ELAM-1 promoter assay and the SEAP assay. 293-hTLR5-HA cells transiently transfected with pELAMl-luc and 293-hTLR5 NF-KB/SEAPorter cells were pretreated with Peptide 1 prior to treatment with PAK and, at the end of the treatment, cell lysates were assayed for NF-κΒ activity using luciferase assay and the spent media using SEAP assay, respectively. Our results show that Peptide 1 suppressed the PAK-induced NF- KB activity by 66% with luciferase assay (Fig. 4A) and by 50% with SEAP assay (Fig. 4B). The inhibitory effect of Peptide 1 was confirmed in two additional experiments by measuring the levels of pp65 and ΙκΒ-α by Western blot analysis and monitoring nuclear translocation of p65 by immunohistochemistry. NF-κΒ activation involves the phosphorylation and degradation of ΙκΒ-α with a concomitant phosphorylation and nuclear translocation of p65, a subunit of NF-KB (2, 19). Our results show that pretreatment with Peptide 1 inhibits PAK- induced degradation of ΙκΒ-α with a concomitant increase in the levels of p65 compared with its control (PBS treatment) (Fig. 5) and nuclear translocation of p65 (Fig. 6). Finally, we determined whether the NF-κΒ inhibition by Peptide 1 is also sequence specific using the SEAP assay. Fig. 7 shows that only 11-mer exhibited the inhibitory activity among all the derivatives tested. Together, these results clearly indicate that Peptide 1 has an ability to suppress PAK-induced NF-κΒ activation, which is most likely responsible for its suppression of PAK-induced IL-8 release.

Example IV.

This example demonstrates that Peptide 1 inhibits PAK-induced TLR5/MyD88 association. We have recently demonstrated that the anti-inflammatory effect of MUC1 during PAK infection was mediated through suppression of TLR5 signaling, specifically at the level of TLR5 and MyD88 interaction (10). To determine whether Peptide 1 possesses the same ability as MUC1, we measured the interaction between TLR5 and MyD88 following treatment of 293-hTLR5-HA cells with PAK in the presence or absence of Peptide 1. Fig. 8 shows that pretreatment with Peptide 1 blocked PAK-induced increase in the binding between MyD88 and TLR5 (Panel A) suggesting that the anti-inflammatory effect of Peptide 1 is mediated through inhibition of MyD88 recruitment to TLR5, the very first step of the TLR5 signaling pathway.

Example V.

This example describes the materials and methods for Examples I-IV.

Reagents

All chemicals and reagents were from Sigma (St. Louis, MO) unless otherwise indicated. TLR5 mouse monoclonal Ab (IMG-664A) was purchased from Imgenex (San Diego, CA) and the following Abs were obtained from Cell Signaling Technology (Beverly, MA): MyD88 rabbit monoclonal Ab (D80F5), Phosphor-NF-κΒ p65(Ser536) rabbit monoclonal Ab (93H1), ΙκΒ-α rabbit monoclonal Ab (44D4), NF-κΒ p65 rabbit monoclonal Ab (D14E12). β-Actin rabbit polyclonal antibody (Thermo Fisher Scientific Inc., Waltham, MA) was used for controlling the amount of protein loading. Pseudomonas aeruginosa K strain (PAK) and heat-inactivated PAK were prepared as described (11).

Synthesis of Peptides

All chemicals were used without further purification. Fmoc-protected amino acids, O- benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) and Rink amide MBHA resins were purchased from Novabiochem. Preparative HPLC purification was performed on a Gilson Preparative HPLC system with a CI 8 reversed phase column (waters XTerra Prep RP8 column, 10 μπι, 19 mm x 250 mm) using a linear gradient from 3% B to 10% B by changing solvent composition over 35 minutes (solvent A: H20, 0.01% TFA; B: ACN, 0.01% TFA). MALDI-TOF MS was performed on ABI 4800 mass spectrometers (Applied Biosystems) using a-cyano-4-hydroxycinnamic acid as a matrix.

Peptide 1 : TATp-C16-l lmer [NH2-TDRSPYEKVSA(RRRQRRKKRGY)K-Pal] (SEQ ID NO: 4)

Peptide 2: TATp-C 16-11 mer with a mutation (YEKV (SEQ ID NO. : l) to FEKV (SEQ ID NO: 10))

Peptide 3: TATp-C16-l l-mer with a mutation (YEKV (SEQ ID NO.: l) to YKEV (SEQ ID NO: 11))

These three peptides were synthesized using exactly the same procedure except for the replacement of the underlined amino acids. A brief summary of the procedure is as follows: Rink amide MBHA resins (100 mg, 75 μπιοΐ) were swollen with DMF (2 ml) in a 5 ml fritted syringe for 2 h. The Fmoc protecting group was removed by treating with 20% piperidine in DMF (2 x 10 min). The resins were treated with Fmoc-amino acid (4.4 equiv) in the presence of HBTU (4 equiv) and DIPEA (4 equiv) in DMF (1 ml) at room temperature. After shaking for 2 h, the reaction mixture was drained and the resins were washed with

DMF (3 x), CH2C12 (2x), MeOH (2x), and DMF (3 x). This process was repeated respectively with the amino acid residues and palmitic acid using the same reaction conditions. The Alloc protecting group was removed by treatment with Pd(PPh3)4 (0.2 equiv) and PhSiH3 (10 equiv) in anhydrous CH2C12 (1 ml). After shaking for 2 h, the reaction mixture was drained and the resins were washed with DMF (3 x), CH2C12 (2x), MeOH (2x), and DMF (3 x). The resins were treated with Fmoc-amino acid (4.4 equiv) in the presence of HBTU (4 equiv) and DIPEA (4 equiv) in DMF (1 ml) at room temperature. After shaking for 2 h, the reaction mixture was drained and the resins were washed with DMF (3 x), CH2C12 (2x), MeOH (2x), and DMF (3 x). This process was repeated respectively with the amino acid residues. The products were cleaved from the resins using a cleavage cocktail (95% trifluoroacetic acid (TFA), 2.5% triisopropylsilane, and 2.5% water) for 2 h at room temperature. The mixture was purified by Prep-HPLC.

Cells

293-hTLR5-HA cells were obtained by stable transfection of HEK293 cells with the pUNO-hTLR5-HA plasmid, which expresses the human TLR5 gene fused at the 3'end to the influenza hemagglutinine (HA) (InvivoGen, San Diego, CA). 293-hTLR5 NF-kB/SEAPorter cells were obtained by co-transfection of HEK293 cells with the human TLR5 gene and an inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene. The SEAP is under the control of the promoter that contains NF-κΒ binding sites (InvivoGen). 293/hTLR5-HA cells and 293-hTLR5 NF-kB/SEAPorter cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 50 U/ml penicillin, 50 μg/ml streptomycin and 10% FBS.

IL-8 ELISA assay

IL-8 ELISA assay was performed as described (16). LDH activity assay

Cytotoxicity was monitored by measuring LDH release using a LDH assay kit as previously described (15).

NF-KB dependent ELAM-1 luciferase reporter assay

An NF-KB-dependent ELAM-1 -luciferase reporter plasmid (pELAMl-luc) (6). was transfected into 293-hTLR5-HA cells and ELAM-1 -luciferase reporter assay was performed as we previously described (11).

SEAP Reporter Assay

HEK 293-hTLR5 NF-KB/SEAPorter cells (InvivoGen), a stably co-transfected cell line which expresses full-length human TLR5 and the SEAP reporter gene under the transcriptional control of an NF-κΒ response element, were seeded in 48-well plates. When cells reached 90% confluence, the cells were pretreated with peptides (10 μΜ) for 1 h and then treated with PAK for 4 h, and cell culture media were assayed using SEAP Assay Kit (Novus Biologicals, Littleton, CO) following the manufacture's protocol.

Western blot and immunoprecipitation analyses

Western blot analysis was performed as described (20).

NF-KB p65 nuclear translocation

Image analysis of p65 nuclear translocation was performed as described with a slight modifications (8). Briefly, 293-hTLR5-HA cells were grown in 8-well chamber slides, fixed in cold 95% ethanol for 20 min at -20°C, washed once using washing buffer (PBS [pH7.0], 0.1% Triton 100) and incubated in permeabilizing buffer (PBS [pH7.0], 0.2% Triton 100) for 20 min at RT and blocked in PBS containing 5% bovine serum albumin for 1 h at RT. The cells were incubated overnight with anti-NF-κΒ p65 rabbit monoclonal Ab followed by incubation for 1 h with Alexa-Fluor-488 -conjugated goat anti -rabbit IgG Ab (Invitrogen) at RT. Nuclei were counterstained with 1 μg/ml of 4', 6-diamidino-2-phenylindole (DAPI).

Coverslips were applied in Antifade solution (Invitrogen) and samples were visualized using a fluorescent microscope.

Statistical analysis

Differences between groups were assessed by comparing mean ± SEM values using the Student t-test and were considered significant at p<0.05.

Having now fully described the invention, it will be understood by those of skill in the art that the same can be performed within a wide and equivalent range of conditions, formulations, and other parameters without affecting the scope of the invention or any embodiment thereof. All patents, patent applications and publications cited herein are fully incorporated by reference herein in their entirety.

INCORPORATION BY REFERENCE

The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.

The following reference and related reference numbers pertain to the instant application: 1. Akira S, and Takeda K. Toll-like receptor signalling. Nature reviews Immunology 4: 499-511, 2004.

2 Brockman JA, Scherer DC, Mckinsey TA, Hall SM, Qi XX, Lee WY, and Ballard DW. Coupling of a Signal Response Domain in I-Kappa-B-Alpha to Multiple Pathways for Nf-Kappa-B Activation. Molecular and cellular biology 15: 2809-2818, 1995.

3. Chauhan A, Tikoo A, Kapur AK, and Singh M. The taming of the cell penetrating domain of the HIV Tat: myths and realities. Journal of controlled release : official journal of the Controlled Release Society 117: 148-162, 2007.

4 Chen L, Hahn H, Wu G, Chen CH, Liron T, Schechtman D, Cavallaro G, Band L, Guo Y, Bolli R, Dorn GW, 2nd, and Mochly-Rosen D. Opposing cardioprotective actions and parallel hypertrophic effects of delta PKC and epsilon PKC. Proceedings of the National Academy of Sciences of the United States of America 98: 11114-11119, 2001.

5. Choi S, Park YS, Koga T, Treloar A, and Kim KC. TNF-alpha is a key regulator of MUC1, an anti -inflammatory molecule, during airway Pseudomonas aeruginosa infection.

American journal of respiratory cell and molecular biology 44: 255-260, 2011.

6 Chow JC, Young DW, Golenbock DT, Christ WJ, and Gusovsky F Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction. The Journal of biological chemistry 274: 10689-10692, 1999.

7. Foged C, Franzyk H, Bahrami S, Frokjaer S, Jaroszewski JW, Nielsen HM, and Olsen CA. Cellular uptake and membrane-destabilising properties of alpha-peptide/beta- peptoid chimeras: lessons for the design of new cell-penetrating peptides. Biochimica et biophysica acta 1778: 2487-2495, 2008.

8. Kato K, Lillehoj EP, Kai H, and Kim KC. MUC1 expression by human airway epithelial cells mediates Pseudomonas aeruginosa adhesion. Frontiers in bioscience 2: 68-77, 2010.

9. Kato K, Lillehoj EP, and Kim KC. MUC1 regulates epithelial inflammation and apoptosis by PolyLC through inhibition of Toll/IL-1 receptor-domain-containing adapter- inducing IFN-beta (TRIF) recruitment to Toll-like receptor 3. American journal of respiratory cell and molecular biology 51: 446-454, 2014.

10 Kato K, Lillehoj EP, Park YS, Umehara T, Hoffman NE, Madesh M, and Kim KC. Membrane-Tethered MUC1 Mucin Is Phosphorylated by Epidermal Growth Factor Receptor in Airway Epithelial Cells and Associates with TLR5 To Inhibit Recruitment of MyD88. Journal of immunology 188: 2014-2022, 2012.

11 Kato K, Lillehoj EP, Park YS, Umehara T, Hoffman NE, Madesh M, and Kim KC. Membrane-Tethered MUC1 Mucin Is Phosphorylated by Epidermal Growth Factor Receptor in Airway Epithelial Cells and Associates with TLR5 To Inhibit Recruitment of MyD88. J Immunol 188: 2014-2022, 2012.

12. Kato K, Lu W, Kai H, and Kim C. Phosphoinositide 3-kinase is activated by MUC1 but not responsible for MUC1 -induced suppression of Toll-like receptor 5 signaling. Am J Physiol-Lung C 293: L686-L692, 2007.

13. Kim KC. Role of epithelial mucins during airway infection. Pulmonary

pharmacology & therapeutics 25: 415-419, 2012.

14. Kim KC, and Lillehoj EP. MUC1 mucin: a peacemaker in the lung. American journal of

respiratory cell and molecular biology 39: 644-647, 2008.

15. Kuwahara I, Lillehoj EP, Hisatsune A, Lu W, Isohama Y, Miyata T, and Kim KC.

Neutrophil elastase stimulates MUC1 gene expression through increased Spl binding to the MUC1 promoter. American journal of physiology Lung cellular and molecular physiology 289: L355-362, 2005.

16 Kuwahara I, Lillehoj EP, Lu W, Singh IS, Isohama Y, Miyata T, and Kim KC

Neutrophil elastase induces IL-8 gene transcription and protein release through p38/NF- {kappa}B activation via EGFR transactivation in a lung epithelial cell line. American journal of physiology Lung cellular and molecular physiology 291 : L407-416, 2006.

17 Lu W, Hisatsune A, Koga T, Kato K, Kuwahara I, Lillehoj EP, Chen W, Cross AS, Gendler S J, Gewirtz AT, and Kim KC. Cutting edge: enhanced pulmonary clearance of Pseudomonas aeruginosa by Mucl knockout mice. Journal of immunology 176: 3890-3894, 2006.

18 Mondrinos M J, Zhang T, Sun S, Kennedy PA, King D J, Wolfson MR, Knight LC, Scalia R, and Kilpatrick LE. Pulmonary endothelial protein kinase C-delta (PKCdelta) regulates neutrophil migration in acute lung inflammation. The American journal of pathology 184: 200-213, 2014.

19 Scherer DC, Brockman JA, Chen Z J, Maniatis T, and Ballard DW Signal- Induced Degradation of I-Kappa-B-Alpha Requires Site-Specific Ubiquitination. Proceedings of the National Academy of Sciences of the United States of America 92: 11259-11263, 1995. 20 Ueno K, Koga T, Kato K, Golenbock DT, Gendler S J, Kai H, and Kim KC

MUC1 mucin is a negative regulator of Toll-like receptor signaling. American journal of respiratory cell and molecular biology 38: 263-268, 2008.

21 Umehara T, Kato K, Park YS, Lillehoj EP, Kawauchi H, and Kim KC Prevention of lung injury by Mucl mucin in a mouse model of repetitive Pseudomonas aeruginosa infection. Inflammation research : official journal of the European Histamine Research Society fet al] 61 : 1013-1020, 2012.

EQUIVALENTS

The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

What Is Claimed Is:

1. A composition comprising a compound that binds to a Toll-like Receptor (TLR) polypeptide, wherein the compound comprises a MUCl peptide or MUCl mimetic comprising at least a portion of the wild type amino acid sequence of MUCl.

2. The composition of claim 1, wherein the MUCl peptide or MUCl mimetic comprises at least a portion of the wild type MUCl amino acid sequence which permits the resulting compound to inhibit interaction between a TLR polypeptide and MyD88 and/or TRIF.

3. The composition of claim 1, wherein the MUCl peptide or MUCl mimetic comprises at least a portion of the wild type MUCl amino acid sequence which permits the resulting compound to inhibit TLR-mediated inflammation.

4. The composition of claim 3, wherein the compound is capable of inhibiting TLR- mediated inflammation through inhibiting interaction between a TLR polypeptide and MyD88 and/or TRIF.

5. The composition of claim 1, wherein the MUCl peptide or MUCl mimetic comprises at least a portion of the wild type MUCl amino acid sequence which permits the resulting compound to inhibit NF-kB activation resulting from interaction between a TLR polypeptide and MyD88 and/or TRIF.

6. The composition of claim 1, wherein the MUCl peptide or MUCl mimetic comprises at least a portion of the wild type MUCl amino acid sequence which permits the resulting compound to inhibit IL-8 release resulting from NF-kB activation.

7. The composition of claims 1-6, wherein the MUCl peptide or MUCl mimetic comprises the following amino acid sequence: YEKV (SEQ ID NO: 1).

8. The composition of claims 1-6, wherein the MUCl peptide or MUCl mimetic comprises the following amino acid sequence: TDRSPYEKVSA (SEQ ID NO:2).

9. The composition of claim 1, wherein the MUC1 peptide or MUC1 mimetic further comprises a cell-penetrating peptide selected from the group consisting of HIV-derived TAT peptide, penetratins, transportans, SS peptides, and hCT derived cell-penetrating peptides.

10. The composition of claim 9, wherein the cell-penetrating peptide is a Trans-Activator of Transcription (TAT) sequence.

11. The composition of claim 10, wherein the cell-penetrating peptide is a TAT sequence having the amino acid sequence RRRQRRKKRGY (SEQ ID NO: 3).

12. The composition of claim 1, wherein the composition comprises at least one biocompatible carrier.

13. The composition of claim 12, wherein the biocompatible carrier is selected from the group consisting of poly -lactic acid, poly-gly colic acid, and copolymers of poly-lactic acid and poly-gly colic acid.

14. The composition of claim 12, wherein the biocompatible carrier comprises at least one biodegradable fatty acid or a metal salt thereof.

15. The composition of claim 14, wherein the biodegradable fatty acid is selected from the group consisting of palmitic acid, stearic acid, oleic acid, myristic acid, and metal salts thereof.

16. The composition of claim 12, wherein said biocompatible carrier comprises a salt selected from the group consisting of porous or non-porous calcium phosphates, porous or non-porous hydroxyapatites, porous or non-porous tricalcium phosphates, porous or non- porous tetracalcium phosphates, porous or non-porous calcium sulfates, and combinations thereof.

17. The composition of claim 12, wherein the MUC1 peptide or MUC1 mimetic is directly linked to the cell-penetrating peptide.

18. The composition of claim 12, wherein the cell-penetrating peptide is directly linked to the MUCl peptide or MUCl mimetic, and the MUCl peptide or MUCl mimetic is directly linked to the biocompatible carrier.

19. The composition of claim 12, wherein the cell-penetrating peptide is directly linked to the biocompatible carrier, and the cell-penetrating peptide is directly linked to the MUCl peptide or MUCl mimetic.

20. The composition of claim 1 in a pharmaceutically acceptable excipient.

21. The composition of claims 1-6, wherein the TLR polypeptide is selected from TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR1 1, TLR12, and TLR13.

22. The composition of claims 1 -6, wherein the TLR polypeptide is TLR5.

23. A method for inhibiting interaction between MyD88 and a TLR polypeptide in a mammalian cell, the method comprising contacting the TLR polypeptide with the composition of claim 1 , wherein the mammalian cell expresses MyD88 and/or TRIF and a TLR polypeptide.

24. The method of claim 23, wherein the mammalian cell is experiencing or is at risk for experiencing TLR-mediated inflammation.

25. The method of claim 23, wherein the mammalian cell is a mucosal epithelial cell.

26. The method of claim 23, wherein the mammalian cell is in a living human.

27. The composition of claim 23, wherein the TLR polypeptide is selected from TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR1 1, TLR12, and TLR13.

28. The composition of claim 23, wherein the TLR polypeptide is TLR5.

29. A method of inhibiting TLR-inflammation in a mammalian cell, the method comprising treating the cell with the composition of claim 1 , wherein the mammalian cell is experiencing or is at risk for experiencing TLR-mediated inflammation.

30. The method of claim 29, wherein the mammalian cell is a mucosal epithelial cell.

31. The method of claim 29, wherein the mammalian cell is in a living human.

32. The method of claim 29, wherein the compound inhibits TLR-mediated inflammation through inhibiting an interaction between MyD88 and/or TRIF and a TLR polypeptide.

33. The method of claim 32, wherein the TLR polypeptide is selected from TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR1 1, TLR12, and TLR13.

34. The method of claim 32, wherein the TLR polypeptide is TLR5.

35. A method for treating a mammal experiencing or at risk for TLR-mediated inflammation, the method comprising administering the composition of claim 1 to the mammal.

36. The method of claim 35, wherein the mammal is suffering from or is at risk for developing a disorder related to TLR-mediated inflammation.

37. The method of claim 36, wherein the disorder is selected from pulmonary fibrosis, COPD, asthma, cardiovascular disorder, diabetes, obesity, metabolic syndrome, autoimmune disorders, neuroinflammatory disorders, schizophrenia, bipolar disorder, autism, clinical depression, chronic fatigue syndrome, alcohol abuse, and toluene inhalation.

38. The method of claim 35, wherein the mammal is a human.

39. The method of claim 35, wherein the compound inhibits TLR-mediated inflammation through inhibiting an interaction between MyD88 and/or TRIF and a TLR polypeptide.

40. The method of claim 39, wherein the TLR polypeptide is selected from TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR1 1, TLR12, and TLR13.

41. The method of claim 39, wherein the TLR polypeptide is TLR5.

42. An isolated polypeptide comprising the amino acid sequence

TDRSPYEKVSARRRQRRKKRGYK (SEQ ID NO:4).

43. A peptide or mimetic comprising the amino acid sequence NH2- TDRSPYEKVSARRRQRRKKRGYK-Palmitic Acid (SEQ ID NO:4).

44. A method for preventing a mammal from experiencing TLR-mediated inflammation, the method comprising administering the composition of claim 1 to the mammal.

45. The method of claim 44, wherein the mammal is suffering from or is at risk for developing a disorder related to TLR-mediated inflammation.

46. The method of claim 45, wherein the disorder is selected from pulmonary fibrosis, COPD, asthma, cardiovascular disorder, diabetes, obesity, metabolic syndrome, autoimmune disorders, neuroinflammatory disorders, schizophrenia, bipolar disorder, autism, clinical depression, chronic fatigue syndrome, alcohol abuse, and toluene inhalation.

47. The method of claim 44, wherein the mammal is a human.

48. The method of claim 44, wherein the compound inhibits TLR-mediated inflammation through inhibiting an interaction between MyD88 and/or TRIF and a TLR polypeptide.

49. The method of claim 48, wherein the TLR polypeptide is selected from TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLRl l, TLR12, and TLR13. The method of claim 48, wherein the TLR polypeptide is TLR5.