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

Balancing intestinal and systemic inflammation through cell type-specific expression of the aryl hydrocarbon receptor repressor

As a sensor of polyaromatic chemicals the aryl hydrocarbon receptor (AhR) exerts an important role in immune regulation besides its requirement for xenobiotic metabolism. Transcriptional activation of AhR target genes is counterregulated by the AhR repressor (AhRR) but the exact function of the AhRR in vivo is currently unknown. We here show that the AhRR is predominantly expressed in immune cells of the skin and intestine, different from other AhR target genes. Whereas AhRR antagonizes the anti-inflammatory function of the AhR in the context of systemic endotoxin shock, AhR and AhRR act in concert to dampen intestinal inflammation. Specifically, AhRR contributes to the maintenance of colonic intraepithelial lymphocytes and prevents excessive IL- 1β production and Th17/Tc17 differentiation. In contrast, the AhRR enhances IFN-γ-production by effector T cells in the inflamed gut. Our findings highlight the physiologic importance of cell-type specific balancing of AhR/AhRR expression in response to microbial, nutritional and other environmental stimuli

Complex morphology and functional dynamics of vital murine intestinal mucosa revealed by autofluorescence 2-photon microscopy

The mucosa of the gastrointestinal tract is a dynamic tissue composed of numerous cell types with complex cellular functions. Study of the vital intestinal mucosa has been hampered by lack of suitable model systems. We here present a novel animal model that enables highly resolved three-dimensional imaging of the vital murine intestine in anaesthetized mice. Using intravital autofluorescence 2-photon (A2P) microscopy we studied the choreographed interactions of enterocytes, goblet cells, enteroendocrine cells and brush cells with other cellular constituents of the small intestinal mucosa over several hours at a subcellular resolution and in three dimensions. Vigorously moving lymphoid cells and their interaction with constituent parts of the lamina propria were examined and quantitatively analyzed. Nuclear and lectin staining permitted simultaneous characterization of autofluorescence and admitted dyes and yielded additional spectral information that is crucial to the interpretation of the complex intestinal mucosa. This novel intravital approach provides detailed insights into the physiology of the small intestine and especially opens a new window for investigating cellular dynamics under nearly physiological conditions

The CD40-CD40L Pathway Contributes to the Proinflammatory Function of Intestinal Epithelial Cells in Inflammatory Bowel Disease

In inflammatory bowel diseases (IBD), intestinal epithelial cells (IECs) are involved in the outbalanced immune responses toward luminal antigens. However, the signals responsible for this proinflammatory capacity of IECs in IBD remain unclear. The CD40/CD40L interaction activates various pathways in immune and nonimmune cells related to inflammation and was shown to be critical for the development of IBD. Here we demonstrate CD40 expression within IECs during active IBD. Endoscopically obtained biopsies taken from Crohn’s disease (n = 112) and ulcerative colitis patients (n = 67) consistently showed immunofluorescence staining for CD40 in IECs of inflamed ileal or colonic mucosa. In noninvolved mucosa during active disease, tissue obtained during Crohn’s disease or ulcerative colitis in remission and biopsies from healthy controls (n = 38) IECs almost entirely lacked CD40 staining. Flow cytometry and RT-PCR analysis using different intestinal epithelial cell lines (HT29, SW480, and T84) showed IFN-γ to effectively induce CD40 in IECs. Cells were virtually unresponsive to LPS or whole E. coli regarding CD40 expression. In addition, a moderate induction of CD40 was found in response to TNF-α, which exerted synergistical effects with IFN-γ. CD40 ligation by CD40L-transfected murine fibroblasts or soluble CD40L increased the secretion of IL-8 in IFN-γ pretreated HT29 cells. Our findings provide evidence for the epithelial expression and modulation of CD40 in IBD-affected mucosa and indicate its involvement in the proinflammatory function of IECs

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 the histological evaluation of intestinal lesions.

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

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 assessment of the disease activity index.

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