The C-type lectin receptor SIGNR3 binds to fungi present in commensal microbiota and influences immune regulation in experimental colitis

Inflammatory bowel disease is a condition of acute and chronic inflammation of the gut. An important factor contributing to pathogenesis is a dysregulated mucosal immunity against commensal bacteria and fungi. Host pattern- recognition receptors (PRRs) sense commensals in the gut and are involved in maintaining the balance between controlled responses to pathogens and overwhelming innate immune activation. C-type lectin receptors (CLRs) are PRRs recognizing glycan structures on pathogens and self-antigens. Here we examined the role of the murine CLR specific intracellular adhesion molecule-3 grabbing non-integrin homolog-related 3 (SIGNR3) in the recognition of commensals and its involvement in intestinal immunity. SIGNR3 is the closest murine homolog of the human dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) receptor recognizing similar carbohydrate ligands such as terminal fucose or high-mannose glycans. We discovered that SIGNR3 recognizes fungi present in the commensal microbiota. To analyze whether this interaction impacts the intestinal immunity against microbiota, the dextran sulfate sodium-induced colitis model was employed. SIGNR3(-/-) mice exhibited an increased weight loss associated with more severe colitis symptoms compared to wild-type control mice. The increased inflammation in SIGNR3(-/-) mice was accompanied by a higher level of TNF-α in colon. Our findings demonstrate for the first time that SIGNR3 recognizes intestinal fungi and has an immune regulatory role in colitis

Group A Streptococcal M1 Protein Sequesters Cathelicidin to Evade Innate Immune Killing.

The antimicrobial peptide LL-37 is generated upon proteolytic cleavage of cathelicidin and limits invading pathogens by directly targeting microbial membranes as well as stimulating innate immune cell function. However, some microbes evade LL-37-mediated defense. Notably, group A Streptococcus (GAS) strains belonging to the hypervirulent M1T1 serogroup are more resistant to human LL-37 than other GAS serogroups. We show that the GAS surface-associated M1 protein sequesters and neutralizes LL-37 antimicrobial activity through its N-terminal domain. M1 protein also binds the cathelicidin precursor hCAP-18, preventing its proteolytic maturation into antimicrobial forms. Exogenous M1 protein rescues M1-deficient GAS from killing by neutrophils and within neutrophil extracellular traps and neutralizes LL-37 chemotactic properties. M1 also binds murine cathelicidin, and its virulence contribution in a murine model of necrotizing skin infection is largely driven by its ability to neutralize this host defense peptide. Thus, cathelicidin resistance is essential for the pathogenesis of hyperinvasive M1T1 GAS

Group A Streptococcal M1 Protein Sequesters Cathelicidin to Evade Innate Immune Killing.

The antimicrobial peptide LL-37 is generated upon proteolytic cleavage of cathelicidin and limits invading pathogens by directly targeting microbial membranes as well as stimulating innate immune cell function. However, some microbes evade LL-37-mediated defense. Notably, group A Streptococcus (GAS) strains belonging to the hypervirulent M1T1 serogroup are more resistant to human LL-37 than other GAS serogroups. We show that the GAS surface-associated M1 protein sequesters and neutralizes LL-37 antimicrobial activity through its N-terminal domain. M1 protein also binds the cathelicidin precursor hCAP-18, preventing its proteolytic maturation into antimicrobial forms. Exogenous M1 protein rescues M1-deficient GAS from killing by neutrophils and within neutrophil extracellular traps and neutralizes LL-37 chemotactic properties. M1 also binds murine cathelicidin, and its virulence contribution in a murine model of necrotizing skin infection is largely driven by its ability to neutralize this host defense peptide. Thus, cathelicidin resistance is essential for the pathogenesis of hyperinvasive M1T1 GAS

Histological analysis of colon sections from wild-type and DCIR^−/− mice.

<p>Paraffin sections of the colon from untreated or 3% DSS-treated wild-type and DCIR^−/− mice were prepared at day seven and were stained with hematoxylin and eosin (H&E) for histological evaluation in a blinded manner. (<b>A</b>) Representative images of paraffin-embedded sections of the rectal part of the colon are shown (40x magnification). Arrows indicate a severe ulcer in the colon from DCIR^−/− mice. Each colon was divided into three segments of identical length (oral, middle, rectal) which were separately analyzed. The degree of leukocyte infiltration (<b>B</b>) and mucosal erosion/ulceration (<b>C</b>) was graded from none (score 0) to mild (score 1), moderate (score 3), or severe (score 4). The scores for both, cell infiltration as well as mucosal ulceration in the rectal part of the colon from DCIR^−/− mice were significantly increased compared to wild-type mice. Data are expressed as mean + SEM (n = 5). The <i>p</i>-values were determined using Mann-Whitney’s U test (*<i>p</i><0.05, **<i>p</i><0.01). Significance is indicated by asterisks (*), ns = no significance.</p

Scoring for the histological evaluation of intestinal lesions.

<p>Scoring for the histological evaluation of intestinal lesions.</p

Local cytokine concentrations in the colon of wild-type, MCL^−/−, and DCIR^−/− mice.

<p>Colons from untreated wild-type mice or from wild-type and MCL^−/− mice (n = 6) (<b>A</b>), or wild-type and DCIR^−/− mice (n = 7 for wild-type and n = 8 for DCIR^−/− mice) (<b>B</b>) treated with 3% DSS for seven consecutive days were homogenized and used for cytokine determination by cytometric bead array. Data are expressed as mean + SEM. Significance is indicated by asterisks (*), ns = no significance.</p

MCL and DCIR recognize commensal intestinal microbiota and modulate APC and T cell cytokine production.

<p>Binding of MCL- and DCIR-hFc fusion proteins to stained gut microbes was analyzed by flow cytometry. (<b>A</b>) Representative dot plots of one binding experiment with MCL- and DCIR-hFc, with hFc as negative control, and with MGL1-hFc as positive control. Gating and frequencies indicate binding events of CLR-hFc fusion proteins to commensal microbiota. For analysis, it was first gated on Syto 61 positive events ( = stained microbiota) followed by gating on PE positive events ( = CLR-Fc fusion proteins). Data are representative of three independent experiments (triplicates each). (<b>B</b>) MCL^−/− and wild-type BMMs or (<b>C</b>) DCIR^−/− and wild-type BMDCs were stimulated with various concentrations of heat-killed gut microbiota, LPS or coated zymosan for 18 h (triplicates each). TNF-α levels in the culture supernatants were determined by ELISA. TNF-α production was significantly increased for MCL^−/− BMMs and DCIR^−/− BMDCs compared to wild-type APCs. Data are representative of three independent experiments. For analysis of T cell activation, purified OT-II transgenic T cells were co-cultivated with BMMs or BMDCs in the presence of heat-killed gut microbiota and 30 µg/mL OVA for 72 h. (<b>D</b>) IL-2 and (<b>E</b>) IFN-γ levels were determined in the culture supernatants of stimulated MCL^−/− and wild-type BMMs. Similarly, (<b>F</b>) IL-2 and (<b>G</b>) IFN-γ levels were analyzed in the culture supernatants of stimulated DCIR^−/− and wild-type BMDCs. Data are representative of three independent experiments (triplicates each) and are expressed as mean + SEM. The <i>p</i>-values were determined with unpaired Student’s t-test (*<i>p</i><0.05, **<i>p</i><0.01). Significance is indicated by asterisks (*), ns = no significance.</p

Scoring for assessment of the disease activity index.

<p>Scoring for assessment of the disease activity index.</p

Histological analysis of colon sections from wild-type and MCL^−/− mice.

<p>Paraffin sections of the colon from untreated or 3% DSS-treated wild-type and MCL^−/− mice were prepared at day seven and were stained with hematoxylin and eosin (H&E) for histological evaluation in a blinded manner. Each colon was divided into three segments of identical length (oral, middle, rectal) which were separately analyzed. (<b>A</b>) Representative images of paraffin-embedded sections of the rectal part of the colon are shown (40x magnification). The degree of leukocyte infiltration (<b>B</b>) and mucosal erosion/ulceration (<b>C</b>) was graded from none (score 0) to mild (score 1), moderate (score 3), or severe (score 4). Data are expressed as mean + SEM (n = 8 for wild-type and n = 5 for MCL^−/− mice). The <i>p</i>-values were determined using Mann-Whitney’s U test. Significance is indicated by asterisks (*), ns = no significance.</p