Gastroenterology

Gastroenterology

Volume 145, Issue 3, September 2013, Pages 591-601.e3
Gastroenterology

Original Research
Full Report: Basic and Translational—Alimentary Tract
Card9 Mediates Intestinal Epithelial Cell Restitution, T-Helper 17 Responses, and Control of Bacterial Infection in Mice

https://doi.org/10.1053/j.gastro.2013.05.047Get rights and content

Background & Aims

Caspase recruitment domain 9 (CARD9) is an adaptor protein that integrates signals downstream of pattern recognition receptors. CARD9 has been associated with autoinflammatory disorders, and loss-of-function mutations have been associated with chronic mucocutaneous candidiasis, but the role of CARD9 in intestinal inflammation is unknown. We characterized the role of Card9 in mucosal immune responses to intestinal epithelial injury and infection.

Methods

We induced intestinal inflammation in Card9-null mice by administration of dextran sulfate sodium (DSS) or Citrobacter rodentium. We analyzed body weight, assessed inflammation by histology, and measured levels of cytokines and chemokines using quantitative reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay. Cell populations were compared between wild-type and Card9-null mice by flow cytometry analysis.

Results

Colon tissues and mesenteric lymph nodes of Card9-null mice had reduced levels of interleukin (IL)-6, interferon-γ, and T-helper (Th)17 cytokines after administration of DSS, compared with wild-type mice. IL-17A and IL-22 expression were reduced in the recovery phase after DSS administration, coincident with decreased expression of antimicrobial peptides and the chemokine (C-C motif) ligand 20 (Ccl20). Although Card9-null mice had more intestinal fungi based on 18S analysis, their Th17 responses remained defective even when an antifungal agent was administered throughout DSS exposure. Moreover, Card9-null mice had impaired immune responses to C rodentium, characterized by decreased levels of colonic IL-6, IL-17A, IL-22, and regenerating islet-derived 3 gamma (RegIIIγ), as well as fewer IL-22−producing innate lymphoid cells (ILCs) in colon lamina propria.

Conclusions

The adaptor protein CARD9 coordinates Th17- and innate lymphoid cell–mediated intestinal immune responses after epithelial injury in mice.

Section snippets

DSS

Mice were fed 3% (wt/vol) DSS (molecular weight, 36,000–50,000; MP Biomedicals, Solon, OH) dissolved in sterile drinking water ad libitum. Animals were monitored daily for weight loss.

C rodentium Infection

Mice were inoculated orally with C rodentium (strain DBS100; American Type Culture Collection, Manassas, VA) as previously described.13, 14 Bacteria were grown overnight in Luria broth and resuspended in phosphate-buffered saline (PBS) before infection (0.5 mL/mouse, 1 × 109 colony forming units [CFU] unless

Card9-Null Mice Show Impaired Recovery After DSS-Induced Injury

To determine the role of Card9 in mucosal immune responses, we worked with 2 mouse models of epithelial injury. Previous studies have shown that Card9-null mice have normal B-cell, T-cell, and myeloid cell development and function, suggesting that the immune system is intact.1 Upon histologic examination, intestinal epithelial cell distribution appeared normal, including Paneth and goblet cells, and we did not observe spontaneous colitis (data not shown). We therefore decided to induce

Discussion

CARD9 is a key adapter molecule that regulates responses to various microbes as well as responses to tissue injury associated with high levels of apoptosis.33 Although the role of CARD9 in systemic immunity against fungi and other microbes has been explored,1 its function in the gastrointestinal tract was unknown before this report. Here, we present evidence that Card9 is important in epithelial injury, ILC, and Th17 responses. Card9-null mice were more susceptible to DSS colitis than wild-type

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    • Cited by (0)

    Author names in bold designate shared co-first authorship.

    Conflicts of interest The authors disclose no conflicts.

    Funding Supported by grants from the French Society of Gastroenterology, the Bettencourt Schueller Foundation, the Ferring Fellowship, and the Arthur Sachs Scholarship (H.S.); The Netherlands Organization for Scientific Research (C.W.); by grants from the National Institutes of Health (DK083756, DK043351, DK086502, and DK060049), the Crohn's and Colitis Foundation of America, and The Leona M. and Harry B. Helmsley Charitable Trust (R.J.X.).

    Editorial assistance was provided by Natalia Nedelsky, as supported by National Institutes of Health grant DK043351 (R.J.X.).

    ∗

    Authors share co-first authorship.

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