Caspase recruitment domain (CARD) family (CARD9, CARD10, CARD11, CARD14 and CARD15) are increased during active inflammation in patients with inflammatory bowel disease

Ulcerative Colitis (UC) is one of the two forms of Inflammatory Bowel Disease (IBD). Epidemiologic data from developing countries reveals that the incidence and prevalence of IBD are increasing in different regions around the world [1]. Family-based studies have identified a number of familial IBD loci using nonparametric linkage analyses, where IBD1 locus contains NOD2 gene [2, 3]. The NOD2 or CARD15, the first gene associated to Crohn’s disease (CD), is an intracellular receptor of the Nod Like Receptor (NLR) family that binds to muramyl-dipeptide (MDP), the minimal bioactive component of bacterial peptidoglycan [4], and through Nuclear Factor κB (NF-κB) pathway, it regulates the expression of tumor necrosis factor (TNF) and other pro-inflammatory cytokines [5].

The CARD-containing adaptors and sensors represent an important family of 15 molecules involved in innate host defense against gastrointestinal pathogens and in the regulation of inflammatory responses. CARD15 has been found to be a key orchestrator of the intestinal mucosal barrier homeostasis through the regulation of intestinal microbiota as well as innate and adaptive immune responses [6‐8]. It belongs to CARD family, which also includes CARD9, CARD10, CARD11 and CARD14, among others.

The CARD9 is an adaptor molecule found mainly in lymphoid tissues, it plays a role in the innate immune response to fungi, bacteria, virus and mycobacteria, linking innate immunity with T-cell differentiation and type 17 T helper (Th17)-cell development [9‐12]; it is known that the presence of certain genetic variants of CARD9 implies an increased risk of IBD [13]. It participates in the activation of NF-κB under dectin-1 (C-type lectin domain family 7-member A, CLEC7A) stimulation and interactions between commensal fungi and the c-type lectin receptor dectin-1 influence colitis [11], which haplotype rs2078178–rs16910631 was found to be associated with CD and UC [13]. It physically associates with CARD15 in response to MDP and regulates the activation of the kinases p38 and JNK, required for the expression of inflammatory cytokines [12‐14]. With the appearance of double-stranded DNA (dsDNA) in the cytoplasm, it forms part of the signaling complex dsDNA-Rad50-CARD9 and culminates with activation of the transcription factor NF-kB and the generation of pro-IL-1β [15]. CARD10, CARD11 and CARD14 are members of the membrane-associated guanylate kinase (MAGUK) superfamily, and function as molecular scaffolds by using multiple discreet protein interaction domains to cluster receptors and cytosolic signaling molecules at the cell membrane [13‐15]. They contain three defining interaction domains: the PSD-95/Dlg/Z0–1 homologous (PDZ) domain, the Src- homology (SH3) domain and the guanylate kinase (GUK)-like domain. Their CARD domain, interacts specifically with the CARD of BCL-10, and under different stimuli, they activate NF-κB [16]. These three molecules have shown distinct tissue distributions but share 50–60% sequence identity in the CARD and coiled-coil domains, and 20–30% identity in the PDZ, SH3 and GUK domains [17‐19]. CARD10 is expressed in a variety of adult tissues like heart, kidney, liver and multiple cancer cell lines including colorectal adenocarcinoma SW480 cells [20]. It interacts with Bcl10 and MALT1 in the platelet-activating factor (PAF)-induced inflammatory pathway in intestinal epithelial cells, and the pro-inflammatory effects of PAF play prominent roles in the pathogenesis of IBD [21]. CARD11 is expressed in the spleen, thymus and peripheral blood leukocytes, it is an important component of the antigen-induced NF-κB signaling pathway in T cells [16], and interestingly, certain genetic variants were associated with an incremented risk of UC [3]. CARD14 gene expression has been found in placenta, skin and mucosa, and it is the only member of the family so far shown to be expressed in different alternatively spliced isoforms [22‐25]; its role in the context of IBD is unknown. The aim of this study was to determine the gene expression and to enumerate the protein-expressing cells of some members of the CARD family (CARD9, CARD10, CARD11, CARD14 and CARD15) in patients with IBD and normal controls without colonic inflammation.

CARDs family is composed by 15 molecules that are involved in innate host defense against gastrointestinal pathogens and in the regulation of inflammatory responses, suggesting that further insights into their physiological functions may yield new pharmacological strategies for treating intestinal inflammatory conditions [30].

The great interest to study the CARD family emerged from the impact that CARD15 has in the CD pathophysiology, regarding its implication in avoiding bacterial mucosal inflammation [7]. No previous studies have been evaluated in UC patients.

The findings of the present study showed that there is a differential gene and protein expression of some CARD family members due to the up-regulated expression of CARD9, CARD14 and CARD15, contrasting with the lack of significant expression in CARD 10 and 11 members in patients with UC.

Our findings suggest that CARD14 might be involved in the pathophysiology of UC, suggesting that CARD14 might play an important role concerning intestinal inflammation. Its relevance had previously been focused in patients with psoriasis, for instance, a Single Nucleotide Polymorphism (SNP) of this gene, named PSORS2 has been considered one of the major genetic risk factors for psoriasis. It has been proposed that CARD14 mutations lead to enhanced NF-kB activation and up-regulation of a subset of psoriasis-associated genes in keratinocytes, which up-regulates an acute inflammatory response throughout excessive activation of NF-kB responsive genes and initiating the recruitment of the inflammatory infiltrate where CARD14 seems to be a regulator of skin inflammation [31]. Similar findings have been made regarding generalized pustular psoriasis [32] and pitiriasis rubra pilaris [33].

Our results are relevant regarding the study of CARD14 as part of the large list of molecules implicated in the puzzling study of IBD etiology, besides suggesting the importance of studying other members of the CARD family. Therefore, this is a fertile ground to other studies regarding the further events that this study was not possible to address.

For instance, in the context of skin immunology, CARD14 protein expression has been found to be reduced in the basal layer, and more diffusely up regulated in the suprabasal layers of the epidermis, unlike the normal CARD14 expression localized mainly in the basal and suprabasal layers of healthy skin epidermis [31].

On the other hand, CARD9 is an important adaptor of innate immune signaling pathways and has not been explored in UC patients. CARD9 is predominantly expressed in immune cells such as: myeloid dendritic cells, macrophages, T lymphocytes and B lymphocytes. Previously, Bertin J et al., showed that CARD9 expression in the small intestine and colon was low under normal conditions, but it is probably that increases during mucosal inflammation due to an influx of myeloid cells [17]. It has been demonstrated that CARD9 promotes recovery from colitis through activation of the IL-22 pathway. Moreover, Card9^−/− mice has an altered microbiota with increased load of gut-resident fungi, and reduced IL-22 production, and the transfer of microbiota from knockout mice to wild-type germ-free mice increases their susceptibility to colitis. In Card9^−/− administration of Lactobacillus murinos, L. reuteri and L. taiwanensis strains attenuates intestinal inflammation through catabolites generated by metabolism of tryptophan. These metabolites act as ligands for aryl hydrocarbon receptor (AHR) that can modulate IL-22 production. Interestingly, reduced AHR ligands production was also observed in patients with IBD, mainly in those with CARD9 risk alleles associated with IBD [34‐36]. We found a protein overexpression of CARD9 in inflamed colonic tissue of UC groups compared to controls without inflammation. These results can be explained due to CARD9 is required for signaling from TLRs in dendritic cells and Triggering Receptor Expressed on Myeloid Cells (TREM) family of receptors in myeloid cells, since activation of nuclear factor-κB and/ or Mitogen-Activated Protein Kinases (MAPK), and induction of the proinflammatory cytokines TNF and IL-6. Probably, the increase of CARD9 observed in our patients could be a compensatory mechanism of inflammation due to dysbiosis. However, we cannot rule out the possible lack of functionality or poor function of the protein.

In genetic studies from other populations, the presence of certain genetic variants of CARD9 has shown to imply an increased risk of UC, and the haplotype rs2078178–rs16910631 was found to be associated with refractory UC [11, 13]. Zhernakova A et al., observed that the haplotype including the rs2287037*Grs1035130*G-rs2241116*C and rs6706002*A alleles occurred more frequently in IBD cases (26.1%) than in controls (21.4%) (P = 0.006) [11, 13].

In the same vein, CARD10 had not been explored in IBD patients. Our results showed that there is no significantly different gene expression of CARD10 in patients with active and remission UC compared to the non-inflamed control group.

Regarding the CARD11 gene expression no significant difference was found between UC patients and normal controls. The role of CARD11 mutations has been demonstrated in other types of diseases such as diffuse large B cell lymphoma [37], rheumatoid arthritis [38] and severe combined immunodeficiency [39]. In contrast, no previous studies have been performed in IBD patients.

We propose that further studies should be considered with a larger number of patients in order to find a statistically significant difference regarding CARD9 and CARD11 gene expression. This study entails the potential opportunity of finding one more possible explanation for the intense inflammation that characterizes IBD, waiting to be confirmed or rejected. The clinical relevance of these findings lies in the better understanding of UC pathophysiology.

Nevertheless, it has been suggested that not only the importance of the coiled-coil domains of CARD14, but also the ones of its related family members, such as CARD11, are important in regulating inflammation [40]. For this reason, attention needs to be further focused on the whole family role in intestinal mucosa immunity and the interaction between its members.

Although no risk alleles of NOD2/CARD15 have been associated in patients with UC, the NOD2/CARD15 gene expression was significantly higher in our cohort of patients with active UC compared to normal controls. No significant associations were found in the CARD15 gene expression regarding to clinical characteristics of UC. In this sense, Csillag et al., [41] previously demonstrated that gene expression profiling of mucosal biopsies from the descending colon of patients with CD could not be correlated with CARD15 status or with familial disposition for IBD. The over expression of CARD15 in CD immune cells was associated with expression of TNF and other pro-inflammatory cytokines, through NF-kB pathway [5].

Rosentiel P, et al. [42] demonstrated that TNF and IFN-γ up-regulate the expression of CARD15 gene in human intestinal epithelial cells and subsequently increased their LPS susceptibility, proposing that CARD15 could be part of the complex pathophysiology of barrier disruption, as it is observed in IBD. These results are of interest because they suggest an up-regulation of CARD15 gene expression in inflammatory conditions.

The findings related to gene and protein CARDs expression are based on the increased cellular infiltrates, activation of the cells and treatment, besides the mRNA presence not always is related to protein expression (correlation between the RNA and protein profile is ∼33 and 40%). It depends on half-life of different proteins (minutes to days) and/or posttranscriptional modification, RNA transport mechanisms, mRNA degradation (2–7 h), complex gene regulatory mechanisms, RNA processing, alternative splicing, RNA stability, and so forth [43].

Besides, samples used to determine mRNA expression are only from colonic mucosa, due to the fact that tissue was obtained by colonoscopy. Nonetheless for immunohistochemistry surgical specimens were employed.

This study has the strength of being the first depiction about the role of the CARD family in UC. This is the first time that other members of the family were studied and also the differential expression of CARD14 and CARD15 between UC and non-inflamed controls.

The role of CARD9 and CARD14 in the immunobiology of IBD is important in order to carry out studies in animal models by eliminating these receptors in specific cell lineages such as intestinal epithelial cells or cells from innate immunity.

The novel findings of the present study about gene expression of CARD9 and CARD14 can differently distinguish active and remission UC suggesting that blocking these receptors with an antagonist might be used as new therapeutic target in UC patients.

CARD14 gene expression was increased in patients with active UC, and its low gene expression was associated with a benign clinical course characterized by long-term remission, suggesting that an up-regulation is a defense mechanism in the colonic epithelia in response to decreased bacterial invasion and inflammatory activity.

The CARD family might be involved in UC pathophysiology, due to the fact that CARD9, 14 and 15 gene expression was increased in patients with active UC.