The multifaceted role of CARD9 in inflammatory bowel disease

Abstract Inflammatory bowel disease (IBD) involves a dysregulated immune response to the gut microbiota. Emerging evidence has demonstrated that dysfunctions in caspase recruitment domain‐containing protein 9 (CARD9) may contribute to the pathogenesis of IBD. Interestingly, an allelic series of Card9 variants have both a common predisposing and rare protective function in IBD patients. In this review, we provide mechanistic insights into the role of the CARD9 adaptor molecule in intestinal inflammation and determine a potential CARD9‐targeting therapeutic approach against IBD.


| INTRODUC TI ON
These PRRs on the surface of innate cells effectively sense distinct microbial components and can trigger an inflammatory cascade in a CARD9-dependent manner. CARD9 is required for TLR pathways to activate mitogen-activated protein kinase (MAPK) and CLRs pathways to activate nuclear factor-kappaB (NF-κB). As a result, CARD9, a signalling adaptor known to regulate innate immune activation, plays a major role in the sensing of pathogenic microorganisms. 2 Emerging evidence has demonstrated that CARD9 dysfunction may contribute to the pathogenesis of IBD. 3,4 Unlike other IBD risk genes, Card9 alleles in IBD patients have both a common predisposing and rare protective function. In this review, we summarize recent progress on the molecular mechanisms of CARD9 in IBD and discuss the feasibility of small-molecule inhibitors targeting CARD9 for the treatment of IBD.

| THE DUAL ROLE OF C ARD9 G E N E TI C MUTATI ON S IN IB D PATIENTS
In 2008, a functional candidate gene analysis of the innate immune pathway was used to investigate Card9 rs10870077, the intronic substitution of base C (cytosine) for G (guanine) in the Card9 genetic locus on chromosome 9. The variant rs10870077 in the Card9 locus was found to be a high-risk factor for phase III IBD patients, showing a strong association with UC and a moderate association with CD. 5 This was the first time that the Card9 gene was identified as a risk factor for IBD patients. In 2012, genome-wide association studies were conducted to analyse Card9 single-nucleotide polymorphisms from IBD patients. In this study, Card9 variant rs4077515 known to create substitution S12N was established as a risk factor in the development of ileal CD, which initiated an innate immune response to peptidoglycan, a bacterial cell wall macromolecule. 6 In 2018, another variant Card9 rs10781499 was confirmed to be a high IBD genetic risk factor, which altered the composition of the gut microbiota in patients with IBD. 7 In contrast to the above mentioned studies, some rare variants of Card9 have also been identified as protective against IBD. A rare splicesite variant, c.IVS11 + 1G>C with the substitution of G for C at position 1 of exon 11 of the Card9 gene, suggested a potentially strong protective effect against disease development. Of note, c.IVS11 + 1G>C (rs141992399) as a protective variant actually occurs on a haplotype carrying the risk allele at S12N, indicating that not only were the two associations independent, but that the splice variant also completely eliminated the risk normally associated with the common haplotype. As the CD risk allele at S12N may lead to higher expression of CARD9, a consistent allelic series existed if the splice variant was substantially low or non-functional and therefore highly protective. 8,9 Similarly, a rare nonsynonymous variant, Card9 rs200735402 with the substitution of C for T at position 139265120, was also shown to have a functionally protective role in CD patients. 10 Some Card9 variants exhibited an increased risk while others were shown to have a protective effect on IBD ( Figure 2). This can be attributed to the Card9 variants having different mechanisms of pathogenesis, and thus different disease susceptibilities. CARD9 F I G U R E 1 Topology of inflammation in IBD. A, Health status. The intestinal immune system protects the largest mucosal surface against infection and injury; B, IBDs. Abnormal intestinal immunity is thought to lead to the development of IBD F I G U R E 2 The dual role of Card9 genetic mutations in IBD patients. Card9 alleles in IBD patients have both a common predisposing and rare protective function. The rs10870077, rs4077515 and rs10781499 variants in Card9 were confirmed to be high genetic risk factors, but rs141992399 and rs200735402 were shown to have a functional protective role. Of note, c.IVS11 + 1G>C (rs141992399) is a protective variant and occurs on a haplotype carrying the risk allele at S12N contains a protein interaction domain known to participate in the activation or suppression of other CARD-containing proteins. 10 The Card9 gene variant is likely to change the protein function or structure level in a positive and negative manner. Therefore, the predisposing variants (rs10870077, rs10781499 and rs4077515) were associated with increased expression of CARD9 mRNA and activation of the immune response to pathogens. 5,11 The protective variants, c.IVS11 + 1G>C and rs200735402, may have lost the biological function of CARD9, inhibiting the immune response to the pathogens. 5,11 Different from the above study, a recent study reported that Card9 predisposing variants, rs10870077 and rs10781499, did not increase susceptibility to IBD in the Chinese Han population. These discrepancies may be partly explained by the different incidences, epidemiologies and phenotypes among patients from the Chinese Han population and Western countries. 12

| C ARD9 DEFICIEN C Y IMPAIRED IMMUNE RE S P ONS E S
Dextran sulphate sodium (DSS) is often used to induce an UC model in mice, which exhibits several clinical and histological features similar to human UC. 13 Following DSS administration, Card9null mice had an impaired immune response, defective expression of interleukin (IL)-6, IL-17A, IL-22 and regenerating islet-derived 3 gamma (RegIIIγ). 3 IL-6 protected against intestinal epithelium injury by secreting intestinal trefoil factor and antimicrobial peptides. 14,15 IL-17A increased the expression of colonic β-defensin. 16 IL-22-mediated STAT3 signalling was involved in mucosal wound healing of intestinal epithelial cells. 17 RegIIIγ in epithelial cells acts as an antibacterial protein facilitating intestinal epithelial wound healing by preventing wound infection and inflammation. 18 In addition, Card9-null mice were also found to have fewer colonic T-helper (Th) 17 cells and exhibited impaired Th17 responses. As predicted, DSS-induced colitis in Card9 −/−mice inhibited intestinal epithelial restoration and impaired gut recovery by significantly increasing apoptosis and reducing proliferation of intestinal epithelial cells. 4 Given the impaired immune responses and injured intestinal epithelial cells, Card9-null mice were less efficient in controlling fungal colonization, increasing their susceptibility to intestinal inflammation. 3,4 Citrobacter rodentium infection in mice can induce acute, self-limited colitis. C rodentium is a Gram-negative and murine-specific bacterial pathogen that is equivalent to the human enteropathogenic Escherichia coli. 19 During C rodentium infection, Card9-null mice were found to have an impaired immune response along with defective expression of colonic IL-6, IL-17A, IL-22 and RegIIIγ. To further investigate the gut immune response elicited by C rodentium, antigen-specific T cell cytokine production in mesenteric lymph nodes (MLNs) was assessed, which suggested that the inappropriate or ongoing inflammation in Card9 knockout mice was characterized by decreased levels of IFNγ and Th17 cytokine responses. As reported previously, innate lymphoid cells (ILCs) in the gut also secrete IL-17A and IL-22 cytokines, particularly in response to C rodentium infection. 20,21 The Th17-dependent C rodentium colitis model showed that Card9 deletion leads to significantly fewer CD3 + CD4 + IL − 17A + Th17 cells and CD3 − CD4 − NKP46 + ILC populations. Card9 is thus required for both Th17 cells and ILC-mediated intestinal immune responses. As a result, Card9-null mice were unable to control bacterial propagation and were more susceptible to intestinal inflammation during C rodentium infection ( Figure 3A). 3

| C ARD9 CONTROLLED G UT MICROB IOTA
Card9, as an IBD susceptibility gene, is required to shape the normal gut microbiota. 3 However, it also raises a question about the specific F I G U R E 3 The potential functions of Card9 in IBD. A, CARD9 deficiency impaired immune responses; B, CARD9 controlled gut microbiota; C, CARD9 altered gut microbiota metabolism; D, the positive therapeutic effect of CARD9targeting small-molecule inhibitor role of the gut microbiota in susceptibility to colitis. To address this question, C rodentium-induced and DSS-induced colitis mouse models were used in the following studies ( Figure 3B).
In C rodentium-induced colitis (Table 1), CARD9 effectively controlled the virulence of C rodentium to promote pathogen eradication through both gut microbiota-independent and microbiota-dependent mechanisms. 22 Previous studies reported that the specific intestinal IgG response was required to eliminate C rodentium infection. 23 However, Card9 −/− (Card9 −/− →germ free) mice in this study failed to stimulate the C rodentium-specific IgG response. As a result, Card9 −/− mice were associated with a defective intestinal humoral immune response and exhibited a higher susceptibility to C rodentium in the late-phase of infection. This finding indicated that CARD9 controlled pathogen virulence by supporting a specific humoral response in a microbiota-independent manner. Several studies have shown that the gut microbiota also played a key role in the clearance of C rodentium. 24,25 Resistance to C rodentium infection was associated with a decrease in Firmicutes, 26 and an increase in Clostridiaceae. 27 Interestingly, CARD9 in C rodentium-induced colitis mice decreased In DDS-induced colitis (Table 1), CARD9 also had a key role in shaping the ecosystem of bacteria and fungi in the gut. The faecal fungal composition in Card9 −/− mice was changed and was dominated by members of the phyla Ascomycota, Basidiomycota and Zygomycota. In parallel, composition of the faecal bacterial microbiota in Card9 −/− mice was altered, but the alterations were less than those for fungi. These findings indicate that the susceptibility gene Card9 controls fungal microbiota expansion for gut homeostasis in DDS-induced colitis. 4

| C ARD9 ALTERED G UT MICROB I OTA ME TABOLIS M
Tryptophan catabolites from gut microbiota play a key role in intestinal mucosal immune responses, in which they act as aryl hydrocarbon receptor (AHR) ligands by modulating the production of IL- 22. 28 In this study, the gut bacteria of Card9 −/− mice failed to metabolize tryptophan into AHR ligands, leading to defective AHR activation and aggravation of intestinal inflammation. Administration of an AHR agonist can rescue the susceptibility of Card9 −/− →germ free mice to colitis. Moreover, AHR ligand production was normalization in the presence of exogenous IL-22, which effectively relieved colitis susceptibility in Card9 −/− mice. Following analysis of faecal samples, reduced AHR activity and tryptophan metabolites were observed in IBD patients, particularly in those with Card9 rs10781499 as a risk allele associated with IBD. Consequently, these results provided strong evidence that CARD9 was required for intestinal microbiota metabolism and production of AHR agonists, altering the gut immune response that resulted in the loss of intestinal homeostasis ( Figure 3C). 4

| C ARD9 AC TIVATI ON WA S REQU IRED FOR TRIM62-MED IATED UB I QU ITINATI ON
CARD9 is structurally similar to the CARMA family members, but lacks the C-terminal MAGUK motif, and contains an amino-terminal CARD and a carboxy-terminal coiled-coil domain. 29 The N-terminal CARD region of CARD9, comprising 7-98 amino acids, is involved in the association of CARD motifs, with an increased susceptibility to microbial infection. 2,30 However, the biological functions of the Cterminus of CARD9 are unknown. CARD9 S12NΔ11, located in the C-terminal portion of the protein, was defined as a rare splice variant in which exon 11 of CARD9 is deleted. It provided strong protection against IBD in two previous studies. 8,9 In order to determine its potential mechanism, CARD9 S12NΔ11, as a protective variant, was used to investigate the biological functions of CARD9 in IBD. Interestingly, CARD9 S12NΔ11 was responsible for a dominant negative effect on CARD9 function, exhibiting impaired CARD9-mediated cytokine production via direct hetero-oligomerization interacting with full-length CARD9.

TA B L E 1 The role of Card9 in Citrobacter rodentium and DSS-induced colitis mouse models
As CARD9 is a scaffold protein and coordinates the assembly of a multiprotein complex, it was determined whether loss of specific protein-protein interactions by the CARD9 C-terminal portion might alter its function. It should be noted that TRIM62 is a novel CARD9 interactor and can directly bind the C-terminus of CARD9.
Furthermore, the presence of CARD9 S12NΔ11 could effectively disrupt the CARD9-TRIM62 interaction. As a result, CARD9-dependent signalling pathways were blocked. TRIM62 generally modified their targeted proteins via several types of ubiquitin linkages, mainly K48-and K63-based linkages that contributed to degradation and stabilization of protein complexes. 31 The findings suggest that  Figure 3D). 33 Patients with complete CARD9 functional deficiency via missense or early stop codon mutations are susceptible to life-threatening microbial infections. 33 In the current work, these lead compounds provide precision immunomodulatory intervention rather than broad immunosuppression. Thus, targeting CARD9 with small molecules that mimicked the protective CARD9Δ11 mechanism, compared favourably with complete ablation of CARD9 activity, and may be a safe and effective targeted treatment.

| CON CLUS IONS
CARD9 is a central integrator in innate immune cell activation, which triggers the inflammatory signalling pathway in response to microbial infection. Along with an in-depth understanding of pathological CARD9 signalling, CARD9 was confirmed to play an essential role in the pathogenesis of inflammatory bowel diseases. CARD9, as a susceptibility factor for colitis, mainly induces the intestinal mucosal immune response and mediates gut microbiota composition and metabolism. Interestingly, an allelic series of Card9 variants have both a common predisposing and rare protective function in IBD patients. These rare protective variants may provide crucial clues in order to achieve targeted treatments and improve current clinical approaches. Some lead compounds were confirmed to mimic the protective CARD9Δ11 mechanism that lost the key C-terminal functions. These findings provide a novel CARD9-targeting approach towards improved IBD therapeutics.
However, some important questions in this field still remain. The gut commensal microbiota contains a complex population of microorganisms, and CARD9 deficiency in IBD patient results in their being highly predisposed to fungal infections. It is unclear how CARD9 can reestablish intestinal epithelial homeostasis and restore beneficial bacterial colonization after inflammatory injury. Considering the close connection between protective genetic variants and therapeutic advances, this connection may be used as a clinical guide for the rational design of IBD therapeutics. The present work also showed a positive therapeutic effect of CARD9-targeting small-molecule inhibitor in the in vitro cellular model. However, it is unknown whether these lead compounds could progress to clinical practice as further data on human and experimental colitis are required.