Abstract
CARD9 is a caspase recruitment domain-containing signaling protein that plays a critical role in innate and adaptive immunity. It has been widely demonstrated that CARD9 adaptor allows pattern recognition receptors to induce NF-κB and MAPK activation, which initiates a “downstream” inflammation cytokine cascade and provides effective protection against microbial invasion, especially fungal infection. Here our aim is to update existing paradigms and summarize the most recent findings on the CARD9 signaling pathway, revealing significant mechanistic insights into the pathogenesis of CARD9 deficiency. We also discuss the effect of CARD9 genetic mutations on the in vivo immune response, and highlight clinical advances in non-infection inflammation.
Similar content being viewed by others
Facts
CARD9 is a critical adaptor protein that operates downstream of these PRRs in myeloid cells.
CARD9 initiates inflammation cytokine cascade in myeloid cells.
CARD9 exhibits a critical role in human infectious diseases.
Open questions
What are the underlying mechanisms of CARD9 in microbial infection?
What is the relationship between CARD9 genetic mutations and clinical diseases?
What is the clinical significance of CARD9 in non-infection inflammation diseases?
Introduction
Innate immune cells are equipped with germ-line-encoded receptors, called pattern recognition receptors (PRRs), which sense the pathogen-associated molecular patterns of foreign pathogens and initiate the host’s immune defenses against invading microbes. Of note, caspase-associated recruitment domain 9 (CARD9) is a critical adaptor protein that operates downstream of these PRRs in myeloid cells. Following receptor engagement, CARD9 selectively interacts with the CARD domain of B-cell CLL/lymphoma 10 (BCL10) and mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1), which triggers an immune response against fungi, bacteria, and viruses1,2,3,4,5.
Although considerable progress has been made in determining its functions, CARD9 full characterization remains still unclear. Recently, Roth et al.6 confirmed the formation of dsDNA-Rad50-CARD9 signaling complexes in dendritic cells (DCs), revealing a distinct recognition pathway that links DNA sensors to viral infection in a CARD9-dependent manner. Some research groups had found that CARD9 knockdown in neutrophils impaired cytokine release in response to fungal invasion7,8. Our group explored the clinical role of CARD9 in aseptic pancreatitis, indicative of its function in non-infectious inflammation9,10. Thereby, this review mainly discusses recent advances in determining the role of CARD9 in activating inflammatory reactions.
CARD9 signaling pathway
CARD9 as a central adaptor molecule
There is evidence to suggest that PRR signaling pathways, C-type lectin receptors (CLRs), nucleotide-oligomerization domain (NOD), and toll-like receptors (TLRs), all converge on CARD9 when a microbial infection is encountered (Fig. 1).
CLRs are a class of transmembrane PRRs including Dectin-1, Dectin-2, and Mincle. After CLR recognition, spleen tyrosine kinase (SYK) activates protein kinase C (PKC), the signal-induced assembly of the CARD9/BCL10/MALT1 (CBM) complex. Furthermore, the CBM complex can trigger the activation of NF-κB1. Recent studies have indicated that Vav proteins are essential regulators of CLR-induced CARD9-NF-κB in mice and humans. The phosphorylation and activation of Vav proteins may bring CARD9 into the vicinity of key upstream regulators such as PKC and potentially engage CBM complexes for NF-κB control against fungal infection11.
Numerous studies have reported that CARD9 is required for NOD2-mediated activation of MAPK signaling components, such as p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun-NH2-terminal kinase (JNK), but not the activation of NF-κB4. After treatment with muramyl dipeptide, CARD9-deficient macrophages display normal NF-κB induction but selective defects in p38 and JNK signaling12. Thus, CARD9 signaling is involved in the innate immune response against certain intracellular bacteria4,13.
TLR3 ligand polyinosinic-polycytidylic acid and TLR7 ligand loxoribine were found to severely inhibit the generation of IL-6, IL-12, and TNF-α in CARD9−/− macrophages, whereas inflammatory cytokines induced by LPS (TLR4 ligand), diacylated lipopeptide FSL-1(TLR2 ligand), flagellin (TLR5 ligand), and CpG DNA (TLR9 ligand) were not defective3,4. These results indicated that CARD9 was essential for cytokine secretions through TLR3 and TLR7 but not TLR4, TLR2, TLR5, and TLR9. In addition, the ability of TLRs to recruit CARD9 may be partly explained by other adaptors or enzymes that interact with CARD9 being differentially expressed. In this study, receptor-interacting protein 2 was also found to be essential for CARD9-dependent MAPK activation ‘downstream’ of TLRs2. Furthermore, CARD9 signaling was shown to cooperate with TLR/MyD88 signaling to enhance immune cell activation14.
CARD9 proteins are modified by phosphorylation
Protein kinase Cδ (PKCδ), a PKC isoform, has recently been identified as the molecular link between SYK and CARD9 in bone marrow-derived macrophages (BMDMs) from mice1. Upon Dectin-1 ligation activation, SYK directly induced PKCδ phosphorylation at position Tyr311. Next, reconstitution experiments using WT-CARD9, CARD9 (T231A), and CARD9 (S303A) mutants demonstrated that PKCδ phosphorylates CARD9 at position Thr231 and Thr30315 (Fig. 1). Because global proteomic profiling revealed CARD9 phosphorylation at Thr95 in vivo16, it could be speculated that another PKCδ target site could be Thr95. However, due to unsuccessful attempts to express the CARD9 (T95A) mutant in mammalian cells, it was not possible to establish the physiological function of Thr95 phosphorylation.
Casein kinase 2 (CK2)-mediated phosphorylation of CARD9 was also found in renal carcinoma cells. In this study, the VHL tumor suppressor protein (pVHL) was found to serve as an adaptor that promoted the phosphorylation of the CARD9 C-terminus by CK2. pVHL was reported to be a CK2 substrate17, bringing CK2 into proximity with CARD9. The CARD9 C-terminus was composed of multiple threonine and serine residues that resembled CK2 phosphorylation sites. CK2 phosphorylated the CARD9 peptide at T531 and T53318. Phosphorylation of the CARD9 C-terminus by CK2 was responsible for CARD9-BCL10 complex assembly (Fig. 1).
CARD9 proteins are modified by ubiquitination
The N-terminal portion of CARD9 has been identified as very important in the recruitment of BCL10 and MALT1 to form a CBM complex. However, CARD9 has no clear domain within its C terminus composed of an oligomerization domain and its mode of regulation is not fully defined1,12 (Fig. 2). A recent study indicated a rare mechanism that involved TRIM62-mediated K27-linked ubiquitination of the CARD9 C-terminus19. TRIM62 was identified as a novel binding partner with the CARD9 C-terminus (aa 416–536), and facilitated K27-linked polyubiquitination of CARD9. TRIM62 ubiquitinated CARD9 at K125 and, in turn, a CARD9 mutation at this residue (K125R) abrogated CARD9-induced NF-κB signaling. Furthermore, Trim62−/−mice showed significant impairment in CLR-CARD9-dependent cytokine production. In parallel, the CARD9 Δ11 variant acted in a dominant-negative manner, disrupting the CARD9-TRIM62 interaction and abolishing TRIM62-mediated ubiquitination (Fig. 1)19.
DsDNA/Rad50/CARD9 signaling complexes for NF-κB activity
The presence of double-stranded DNA (dsDNA) in the cytoplasm, which is normally a DNA-free microenvironment, triggers the production of IL-1β against viral and bacterial invasion. Rad50 is a DNA-binding protein located in the nucleus that is involved in the response to eukaryotic DNA damage.
Roth et al. defined a distinct DNA recognition pathway through the formation of dsDNA-Rad50-CARD9 signaling complexes in DCs6,20. After the transfection of DCs with virus DNA, Rad50 translocated rapidly from the nucleus to the cytoplasm, forming a DNA-Rad50 complex. Following the cytosolic localization of Rad50 and DNA, CARD9 was recruited and interacted directly with Rad50 via its Zn-hook region. The formation of viral dsDNA-Rad50-CARD9 complexes mediated NF-κB activation for IL-1β generation. Crucially, Rad50 or CARD9-deficient DCs significantly impaired IL-1β production in response to pathogen DNA. Thus, the Rad50/CARD9 complexes, as viral cytoplasmic DNA sensors, were critical for the host immune response (Fig. 3).
TRAF6/BCL10/CARD9 signaling complexes for JNK activity
This study showed that CARD9 induced a multiprotein complex with BCL10 proteins, which recruited TRAFs in pVHL-deficient renal cell carcinomas (RCC). After the mitigation of inhibitory phosphorylation of CARD9 by CK2, CARD9-TRAF6 complexes form. Furthermore, CARD9 expression mediated TRAF6 polyubiquitination through K63 linkages. In turn, CARD9 knockdown directly led to an obvious reduction in TRAF6 K63 polyubiquitination in pVHL-deficient cells. As TRAF6 was known to function upstream of TAK121, the formation of a CARD9/BCL10/TRAF6 complex as a proximal signal triggered the sequential activation of TAK1, MKK4, and JNK. This finding indicated that CARD9-dependent TRAF6 drove JNK activity in RCC (Fig. 3)22.
CARD9-mediated inflammasome-induced IL-1β production
CARD9 plays the positive roles in fighting fungal and viral infections through NLRP3 inflammasome. The mycobacterial synthetic adjuvant analog trehalose-6,6′-dibehenate triggers SYK and CARD9 signaling molecules and relies on the C-type lectin Mincle. Trehalose-6,6′-dibehenate could induce NLRP3 inflammasome-dependent IL-1β secretion in BMDMs, in which CARD9 had been identified as an essential mediator23,24. Microsporum canis (a pathogenic fungus) was found to mediate IL-1β production in the human monocytic cell line THP-1, which was also dependent on the NLRP3 inflammasome. The pathways involved required Dectin-1, SYK, and CARD9, and CARD9 in particular exerted a critical role in the upregulation of pro-IL-1β25. Agrocybe aegerita lectin isolated from the edible mushroom A. aegerita promoted inflammatory cytokine secretion by regulating macrophage activation. A. aegerita lectin likely targeted macrophages through Mincle receptor, resulting in SYK/CARD9 signaling activation, which then coupled to NLRP3 and upregulated IL-1β synthesis26. Oxidized low-density lipoprotein-mediated IL-1β production also converged on CARD9 (Fig. 3)27.
In contrast to the above report, Milton et al. identified a negative regulatory role for CARD9 on IL-1β production. CARD9 markedly down-regulate NLRP3-induced IL-1β in response to Salmonella infection in BMDMs. The potential mechanism involved reducing NLRP3 activation via modulation of SYK and Caspase-8 activity28.
In addition, other CARD9-independent inflammasomes, Caspase-8 and caspase-1, have been detected. First, Dectin-1 receptors induced the activation of a noncanonical Caspase-8 inflammasome in response to fungal and mycobacterial infection29, following the formation of CARD9-BCL-10-MALT1 and the recruitment of MALT1-Caspase-8, in which ASC was required in this scaffold for noncanonical Caspase-8-induced IL-1β30. Second, RIG-I served as an RNA virus sensor that could also trigger CARD9 and Caspase-1inflammasome signaling for IL-1β production (Fig. 3)31.
Dectin-1 induces the CARD9-ERK pathway
It established that Dectin-1induces CARD9-ERK activation against fungal infection. CARD9 regulated Dectin-1-mediated ERK activation by linking Ras-GRF1 to H-Ras, which was dispensable for NF-κB activation induced by curdlan or Candida albicans yeast. Specifically, Dectin-1 initiated SYK-dependent Ras-GRF1 phosphorylation, and then the phosphorylated Ras-GRF1 induced H-Ras recruitment and activation by forming a complex with CARD9, which finally led to ERK phosphorylation32. In addition, the Dectin-1-induced SYK-CARD9-ERK pathway was identified during the process of conversion from tumor-associated macrophages into an M1-like phenotype33.
Rubicon-dependent feedback inhibition
The Rubicon protein binds to the Beclin-1complex, and localizes to late endosomes/lysosomesin phagocytic cells. Rubicon serves as a negative regulator of the maturation step of autophagy or the endocytic pathway, depending on the environmental stimulus34, and contains a RUN domain, a coiled-coil domain, an N-terminal serine-rich region, a helix-coil-rich region, a C-terminal serine-rich region, and a cysteine-rich region. Yang et al. reported that Rubicon negatively regulated inflammatory cytokine production in response to Rig-I-dependent sensing of RNA viruses and Dectin-1-dependent fungal ligands by displacing CARD9 from the CBM complex, thereby terminating the CARD9-mediated signaling pathway35,36. This displacement was dependent on the serine phosphorylation status of Rubicon, which bound to 14-3-3β, known as a phospho-serine/threonine binding protein. Indeed, S248-phosphorylation of Rubicon within an N-terminal serine-rich domain completely abolished its interaction with 14-3-3β, which led Rubicon to associate constitutively with CARD9. Furthermore, Rubicon robustly interacted with CARD9 through its C-terminal helix-coil-rich region, disassembling the CARD9-BCL10 signaling complex. Thus, Rubicon acted as a specific feedback inhibitor of CARD9-mediated PRR signal transduction, and markedly suppresses IL-6, IL-1β, and TNF-α production. It remains unclear whether molecular mechanisms specifically alter the binding partner of Rubicon from 14-3-3β to CARD9. It is possible that the interaction of Rubicon with 14-3-3β during the initial period of microbial stimulation, in which Rubicon sterically hinders a CARD9 interaction, and permits CBM signal transduction, triggers NF-κB activation. At the late stage, Rubicon dephosphorylates serine 248, dissociating from the 14-3-3β complex to allow an interaction with CARD9, thereby disrupting CBM signaling. Other unknown proteins and/or alterations in subcellular localization may be attributed to the change of interactions of Rubicon with 14-3-3β and CARD935.
Troglitazone-dependent negative feedback
The PPAR-γ ligand troglitazone impaired Dectin-1-mediated NF-κB and MAPK activation in human monocyte-derived DCs through the inhibition of CARD9 expression. CARD9 protein expression in the cytosol was dramatically downregulated in troglitazone-treated monocyte-derived DCs. The reduction in CARD9 protein was not due to proteasomal degradation and transcript levels. Thereby, troglitazone, as a negative feedback regulator, helps to alleviate the incoming inflammatory signaling pathways37.
Functional role of CARD9 in neutrophils
Patients with CARD9-deficient polymorphonuclear neutrophils (PMNs) have been found to be highly susceptible to systemic candidiasis that specifically targets the central nervous system (CNS). A novel CARD9 missense mutation (p.R57H; c.170 G > A) was reported in an11-year-old girl who was suffering from Candida meningoencephalitis infection in the CNS7. In this case, the c.170 G > A CARD9 mutation did not prevent the generation of full-length CARD9 proteins, but imparted CARD9 function. As a result, CARD9 mutations dramatically decreased IL-6, IL-1β, and TNF-α production upon fungal stimulation. Furthermore, CARD9-deficient patients had been found to be significantly impaired in the induction of neutrophil-recruiting CXC chemokines in the cerebrospinal fluid, leading to an absence of neutrophil accumulation in the infected CNS, and the impaired killing of unopsonized Candida albicans. A 13-year-old, CARD9-deficient, Asian girl suffering from chronic invasive Candida infection of the brain was reported in 201338. Molecular analysis revealed two previously undescribed mutations, c.214 G > A and c.1118 G > C, in CARD9. CARD9-deficient PMNs exhibited selective killing of unopsonized C. albicans conidia, which was independent of NAPDH oxidase-derived reactive oxygen species generation.
Moraxella catarrhalis as an important causal agent of exacerbations in chronic obstructive pulmonary disease could induce CARD9-dependent NF-κB activation in human granulocytes39. CARD9-deficient PMNs isolated from patients exhibited defects in proinflammatory cytokine production and Phialophora verrucosa killing40. Furthermore, CARD9-deficient patients with spontaneous CNS candidiasis had been successfully treated by granulocyte-macrophage colony-stimulating factor treatment of CARD9-dependent pathways41. Finally, Roel et al. demonstrated that human PMNs were primarily responsible for phagolysosomal killing of C. albicans through two independent mechanisms, complement receptor 3-mediated uptake of unopsonized C. albicans and FcγR-mediated uptake of serum-opsonized C. albicans42. The first mechanism was found to be dependent on complement receptor 3 via PI3K, SYK, and CARD9, but completely independent of phagocyte NADPH oxidase activity. The second mechanism strictly depended on Fcγ receptors and reactive oxygen species formation by the NADPH oxidase system, in which SYK played a positive role but PI3K does not.
Investigation of mice harboring CARD9-deficient PMNs further confirmed the high susceptibility to invasive fungal infection7. Subsequently, research groups provided direct evidence that CARD9 signaling induced CXCL chemokine and initiated neutrophil recruitment in response to fungal infection in CARD9 knockout mice7,43. In addition, CARD9−/− neutrophils also played a key role in non-infectious inflammation by suppressing autoantibody-induced arthritis and dermatitis in mice8,44.
CARD9 genetic mutations
Human CARD9 genetic mutations may result in protein structure modifications, lower expression, or function loss. CARD9 genetic mutations are associated with inflammatory responses, a susceptibility to fungal infection45,46, Crohn’s disease47,48, ulcerative colitis49,50, intestinal failure51, ankylosing spondylitis52,53, as well as the severity of pulmonary tuberculosis54.
A novel homozygous R101L mutation in CARD9 was identified in a Brazilian patient with deep dermatophytosis, resulting in impaired fungal killing55. Some patients have been reported to harbor a homozygous Q295X point mutation in CARD9, which led to increased susceptibility to dermatophytes56 and invasive chronic Candida57. One particular variant in the CARD9 gene, c.191-192InsTGCT, p.L64fsX59, was associated with Corynespora cassiicola infection in a Chinese patient58. Patients with the R70W CARD9 mutation were associated with impaired NF-кB activation, which resulted in a defective antifungal response and susceptibility to chronic, invasive fungal infections59. The CARD9c.3 G > C mutation predisposed patients to extrapulmonary Aspergillus infection in the abdomen and brain, along with reduced neutrophil recruitment to these fungal-infected tissues60. Another CARD9 mutation, Ser12Asn, rs4077515, was found to increase susceptibility to idiopathic recurrent vulvovaginal candidiasis, a common symptom of Candida infection61. The CARD9 S12N (c.35 G.A, rs4077515) mutations, an amino acid substitution from a serine to an asparagine residue, was strongly linked to the development of candidemia46. A homozygous R18W CARD9 mutation in patients who had functional CARD9 deficiency was identified as a risk factor for invasive Exophiala infection62.
Recent studies have demonstrated that CARD9 mutations are closely associated with Crohn’s disease and ulcerative colitis development. The genetic mutations c.IVS11 + 1 G > C could cause the loss of function of CARD9, which resulted in diminished immune responses and significant protection against IBD. However, a high-risk variant S12N may induce overexpression of CARD9, which led to a hyper-reactive immune state and high susceptibility to IBD49,63. CARD9 mutations (rs10870077 and rs10781499) were significantly associated with both Crohn’s disease and ulcerative colitis64, but showed no significant association with IBD in the Chinese Han population50. Interestingly, a novel rare non-synonymous variant rs200735402 in CARD9 was shown to play a functionally protective role in Crohn’s disease47. CARD9 variant rs4077515 may contribute to ileal Crohn’s disease susceptibility, positive regulation of TNF-α and IL-6 production, and positive regulation of the innate immune response65.
The CARD9 mutations, rs4077515, were found to be protective against intestinal failure. One possibility was that this genetic mutations decreased the level of sustained conjugated hyperbilirubinemia and inhibited NOD2-dependent signal pathways51. The CARD9 mutations rs4077515 significantly decreased the ankylosing spondylitis risk in a HLA-B27-negative Iranian population, possibly by contributing to the CARD9-IL23 axis for the pathogenesis of inflammatory disorders52. CARD9 genetic variants, rs4077515, rs10781499, and rs10870077, were found to increase susceptibility to tuberculosis and the severity of pulmonary tuberculosis in a Caucasian cohort of Romanian ethnicity13,54.
Importance of CARD9 in non-infection-related inflammation
Emerging studies indicated that CARD9 was also associated with sterile inflammation disease in the absence of pathogen infections (Table 1).
Cardiovascular disease
Early cardiovascular pathologies are characterized by inflammatory cell infiltration into the tissues66. During this process, the circulating immune cells including neutrophils and monocytes/macrophages follow a cascade of tethering, rolling, arrest, and adhesion, and display a close interaction with endothelial cells in the inflamed tissues67,68. Several studies showed that macrophages played a critical role in initiating inflammation in grafted veins. Depletion of macrophages and inhibition of proinflammatory cytokine expression could attenuate neointima formation in rat vein grafts69. In this study, CARD9 was highly expressed in the macrophages of grafted veins. Furthermore, necrotic smooth muscle cells induced an inflammatory reaction in macrophages via the CARD9-dependent NF-κB signaling pathway. Finally, CARD9−/− mice significantly inhibited neointima formation of vein grafts by suppressing the migrtion and proliferation of smooth muscle cells. These data suggested that CARD9 mediated necrotic smooth muscle cell-induced inflammation and contributed to neointima formation in vein grafts70. Another study showed that angiotensin II systems contributed to all stages of the inflammatory response, leading to cardiac fibrosis, hypertensive cardiac remodeling, and heart failure. Furthermore, CARD9 markedly inhibited the cardiac inflammation and fibrosis induced by angiotensin II infusion. CARD9−/− mice markedly inhibited the expression of collagen I and TGF-β, indicating that CARD9 attenuated cardiac fibrosis by inhibiting myofibroblast formation. CARD9 was also involved in angiotensin II-mediated cardiac inflammation through the regulation of NF-κB and MAPK activity in peritoneal macrophages71. Recently, CARD9 was found to suppress obesity-related cardiac hypertrophy through decreasing CBM formation and p38 MAPK production72.
Autoimmune disease
Crohn’s disease and ulcerative colitisare thought to be chronic relapsing immune-mediated diseases arising from dysregulated intestinal immune responses to the gutflora in genetically susceptible genetic backgrounds, which are characterized by inflammation and ulceration of the gut mucosa. Crohn’s disease and ulcerative colitis patients usually show dysregulated microbiomes that may contribute to the dysregulation of the immune system. The CARD9 locus encodes a key pattern recognition receptor of the innate immune system, and specific variants of the CARD9 gene are involved in Crohn’s disease and ulcerative colitis pathogenesis. CARD9-null mice showed increased vascular leakage and epithelial permeability at the site of injury, delayed intestinal injury recovery, and a defect in epithelial responses due to the impaired expression of IL-22, IL-17A, IL-6, and RegIIIγ. As a result, CARD9 played a pivotal role in intestinal homeostasis, mediating intestinal epithelial cell restitution, innate lymphoid cell and T-helper 17 cell responses, maintenance of the intestinal mucosal barrier, and control of intestinal bacterial infection73,74,75,76. The gut microbiota in CARD9−/− mice failed to activate the tryptophan metabolites that served as aryl hydrocarbon receptor ligands, which was responsible for the hypersusceptibility of mice to colitis. Patients with crohn’s disease and ulcerative colitis were observed to reduce the production of aryl hydrocarbon receptor ligands and tryptophan metabolites. These results suggested that CARD9 gene disorder altered the composition of the gut microbiota, affected the production of microbial metabolites, and increased sensitivity to colitis73,74,75,76.
IgA nephropathy is the most common form of primary glomerulonephritis, which is characterized by deposition of IgA-containing immune complexes in the glomerular mesangium. One study reported a genome-wide association study of IgA nephropathy, and follow-up in 20,612 individuals of European and East Asian ancestry. A significant association was detected between the CARD9 gene and the IgA nephropathy risk. A high-risk allele, rs4077515, was discovered, resulting in a p.Ser12Asn substitution in CARD9. This substitution induced the increased expression of CARD9 in monocytes, which were involved in activation of canonical pathways77. In addition, the CARD9 gene has also been associated with other immune-related diseases, such as autoimmune polyendocrinopathy candidiasis ectodermal dystrophy78, autoimmune uveitis79, ankylosing spondylitis53, and primary sclerosing cholangitis80.
Cancer
Interestingly, CARD9 as an inflammation-related gene also promote the proliferation and migration of tumor cells, induce cancer, and affect tumor severity and prognosis81.
The APCmin (multiple intestinal neoplasia, min) mouse harbors a point mutation in the murine homolog of the adenomatous polyposis coli (APC) gene and is an animal model for studies of human familial adenomatous polyposis, precancerous lesions associated with small-intestinal and colonic cancer82. CARD9 had been shown to play a key role in APCmin tumor incidence and progression, markedly reducing viability and promoting colonic tumorigenesis in male mice. The positive effect of CARD9 on tumor multiplicity and burden was due to decreased plasma IL6, G-CSF, and RANTES levels, and decreased macrophage and T-cell infiltration into the tumor83.
Gastric mucosa-associated lymphoid tissue (MALT) lymphomas frequently occur in translocation-negative tumors. Studies had shown that aberrant CARD9 upregulation may contribute to the development or progression of MALT lymphomas mediated by activation of the NF-isB signaling pathway, especially among patients who were not infected with Helicobacter pylori84,85. RCC is a common form of kidney tumor. Several lines of evidence suggested that CARD9 linked the VHL tumor suppressor protein (pVHL) to trigger tumor-related signaling molecule, thereby controlling RCC cell growth. pVHL, which was expressed upstream or parallel to CARD9, and was bound to CK2, promoted the phosphorylation of CARD9. Consequently, VHL-deficient cancer cells failed to phosphorylate CARD9, resulting in constitutive activation of CARD9, an upstream trigger for NF-κB activation to induce tumor occurrence18. Another study reported that pVHL+/+ RCCs sequentially stimulate CARD9 and TRAF and the JNK signaling pathway in a hypoxia-inducible factor alpha (HIFα)-independent manner. In the context of pVHL loss, CARD9 silencing led to a sharp reduction in TRAF6, which was known to function upstream of JNK, retarding tumor growth22.
Macrophages are critical immune effector cells that respond to the tumor microenvironment. Substantial experimental and clinical evidence indicates that tumor cell metastasisis associated with the presence of a high number of macrophages in various cancers and dynamic changes in the specific phenotypes of macrophage subpopulations86. High expression of CARD9 was reported in tumor-infiltrating macrophages and clinicopathologic analysis of colon cancer patients suggested that CARD9 expression was strongly correlated with tumor progression. Furthermore, CARD9-null mice provided evidence that CARD9 facilitated liver metastasis of colon carcinoma cells. Mechanistic studies revealed that CARD9 contributes to tumor metastasis through enhancing metastasis-associated macrophage polarization, independent of the number of infiltrating macrophages. CARD9 polarized macrophages toward a M2 phenotype in the tumor microenvironment by NF-κB activation87.
Pancreatitis
Severe acute pancreatitis (SAP) is characterized by a progressive inflammatory response with a high mortality rate88. To the best of our knowledge, the early stages of SAP manifest a sterile inflammation in response to endogenous substances. It is currently accepted that the activation of mononuclear cells is an early event in SAP patients, which induces an inflammatory cascade that leads to systemic organ failure. In our studies, mononuclear cells isolated from SAP patients were found to overexpress CARD9 that may serve as an upstream molecule of NF-κB and p38. What was more, CARD9 overexpression was positively correlated with the outcome and severity of pancreatic injury in SAP patients10. In another study, siRNA silencing of the CARD9 gene in SAP rats was used to investigate the therapeutic effects and potential mechanisms of CARD9. Interestingly, our findings provided strong evidence that blocking CARD9 expression failed to trigger NF-κB and p38 activity, which could effectively alleviate pancreatitis severity, as well as liver and lung injury9.
Hypersensitivity
Allergic contact dermatitis is caused by the reaction of T cells to various allergens. Allergens can effectively penetrate the skin and bind covalently to skin proteins to form hapten. Skin-resident DCs can recognize haptenated proteins, transfer to the skin-draining lymph nodes, and then prime hapten-specific T cells. This process is called sensitization, and indicates that DCs are essential for allergic contact sensitization to haptens. However, the mechanism by which haptens stimulate DCs to sensitize T cells remains unclear. In this study, CARD9 overexpression in DCs increased their ability to prime T cells. The potential signaling pathway was found to be the coupling of ITAM-SYK-CARD9 signaling to IL-1 secretion in DCs, involving the sequential activation of ITAM, SYK, CARD9, BCL10, the NLRP3 inflammasome, and IL-1 signaling. DC-specific deletion of CARD9 was sufficient to abolish hapten-induced IL-1 secretion and hinder allergic contact sensitization to haptens89.
Obesity
Obese patients are associated with low grade chronic inflammation, known as “metabolic inflammation”, the heightened infiltration of macrophages, which has detrimental effects on metabolism and cardiovascular dysfunction. A recent study reported that CARD9−/− mice had significantly decreased numbers of infiltrating macrophages in the heart, which prevented myocardial dysfunction and ameliorated high fat diet-induced insulin resistance and glucose intolerance, leading to alleviation of high fat diet-induced obesity potentially through CARD9-dependent p38 suppression90.
Conclusion
In summary, evidence is emerging of the molecular and cellular mechanisms of CARD9 activation of inflammatory reactions, particularly regarding cytosolic DNA-recognition signaling, the ubiquitination pathway, negative feedback regulation, genetic mutations, and aseptic inflammation. However, important questions still remain, such as (1) how CARD9 is translocated from the cytoplasm to the nucleus, (2) the role of CARD9 in the early and late inflammatory phases, and (3) the nature and mechanism by which CARD9 is involved in non-infection diseases, such as tumor development and cardiac fibrosis.
Change history
18 February 2019
The original version of this Article contained errors in the author affiliations.
References
Roth, S. & Ruland, J. Caspase recruitment domain-containing protein 9 signaling in innate immunity and inflammation. Trends Immunol. 34, 243–250 (2013).
Colonna, M. All roads lead to CARD9. Nat. Immunol. 8, 554–555 (2007).
Hara, H. et al. The adaptor protein CARD9 is essential for the activation of myeloid cells through ITAM-associated and toll-like receptors. Nat. Immunol. 8, 619–629 (2007).
Hsu, Y. M. et al. The adaptor protein CARD9 is required for innate immune responses to intracellular pathogens. Nat. Immunol. 8, 198–205 (2007).
Gross, O. et al. Card9 controls a non-TLR signalling pathway for innate anti-fungal immunity. Nature 442, 651–656 (2006).
Roth, S. et al. Rad50-CARD9 interactions link cytosolic DNA sensing to IL-1beta production. Nat. Immunol. 15, 538–545 (2014).
Drummond, R. A. et al. CARD9-dependent neutrophil recruitment protects against fungal invasion of the central nervous system. PLoS Pathog. 11, e1005293 (2015).
Nemeth, T., Futosi, K., Sitaru, C., Ruland, J. & Mocsai, A. Neutrophil-specific deletion of the CARD9 gene expression regulator suppresses autoantibody-induced inflammation in vivo. Nat. Commun. 7, 11004 (2016).
Yang, Z. W., Meng, X. X., Zhang, C. & Xu, P. CARD9 gene silencing with siRNA protects rats against severe acute pancreatitis: CARD9-dependent NF-kappaB and P38MAPKs pathway. J. Cell Mol. Med. 21, 1085–1093 (2017).
Yang, Z. W., Weng, C. Z., Wang, J. & Xu, P. The role of Card9 overexpression in peripheral blood mononuclear cells from patients with aseptic acute pancreatitis. J. Cell Mol. Med. 20, 441–449 (2016).
Roth, S. et al. Vav proteins are key regulators of CARD9 signaling for innate antifungal immunity. Cell Rep. 17, 2572–2583 (2016).
Hara, H. & Saito, T. CARD9 versus CARMA1 in innate and adaptive immunity. Trends Immunol. 30, 234–242 (2009).
Dorhoi, A. et al. The adaptor molecule CARD9 is essential for tuberculosis control. J. Exp. Med. 207, 777–792 (2010).
Ruland, J. CARD9 signaling in the innate immune response. Ann. N. Y. Acad. Sci. 1143, 35–44 (2008).
Strasser, D. et al. Syk kinase-coupled C-type lectin receptors engage protein kinase C-sigma to elicit Card9 adaptor-mediated innate immunity. Immunity 36, 32–42 (2012).
Choudhary, C. et al. Mislocalized activation of oncogenic RTKs switches downstream signaling outcomes. Mol. Cell 36, 326–339 (2009).
Lolkema, M. P. et al. Tumor suppression by the von Hippel-Lindau protein requires phosphorylation of the acidic domain. J. Biol. Chem. 280, 22205–22211 (2005).
Yang, H. et al. pVHL acts as an adaptor to promote the inhibitory phosphorylation of the NF-kappaB agonist Card9 by CK2. Mol. Cell 28, 15–27 (2007).
Cao, Z. et al. Ubiquitin ligase TRIM62 regulates CARD9-mediated anti-fungal immunity and intestinal inflammation. Immunity 43, 715–726 (2015).
Bowie, A. G. Rad50 and CARD9, missing links in cytosolic DNA-stimulated inflammation. Nat. Immunol. 15, 534–536 (2014).
Adhikari, A., Xu, M. & Chen, Z. J. Ubiquitin-mediated activation of TAK1 and IKK. Oncogene 26, 3214–3226 (2007).
An, J. et al. Hyperactivated JNK is a therapeutic target in pVHL-deficient renal cell carcinoma. Cancer Res. 73, 1374–1385 (2013).
Schweneker, K. et al. The mycobacterial cord factor adjuvant analogue trehalose-6,6′-dibehenate (TDB) activates the Nlrp3 inflammasome. Immunobiology 218, 664–673 (2013).
Shenderov, K. et al. Cord factor and peptidoglycan recapitulate the Th17-promoting adjuvant activity of mycobacteria through mincle/CARD9 signaling and the inflammasome. J. Immunol. 190, 5722–5730 (2013).
Mao, L. et al. Pathogenic fungus Microsporum canis activates the NLRP3 inflammasome. Infect. Immun. 82, 882–892 (2014).
Zhang, Z. et al. AAL exacerbates pro-inflammatory response in macrophages by regulating Mincle/Syk/Card9 signaling along with the Nlrp3 inflammasome assembly. Am. J. Transl. Res. 7, 1812–1825 (2015).
Rhoads, J. P. et al. Oxidized low-density lipoprotein immune complex priming of the Nlrp3 inflammasome involves TLR and FcgammaR cooperation and is dependent on CARD9. J. Immunol. 198, 2105–2114 (2017).
Pereira, M., Tourlomousis, P., Wright, J., Monie, T. P. & Bryant, C. E. CARD9 negatively regulates NLRP3-induced IL-1beta production on Salmonella infection of macrophages. Nat. Commun. 7, 12874 (2016).
Rieber, N. et al. Pathogenic fungi regulate immunity by inducing neutrophilic myeloid-derived suppressor cells. Cell Host Microbe. 17, 507–514 (2015).
Gringhuis, S. I. et al. Dectin-1 is an extracellular pathogen sensor for the induction and processing of IL-1beta via a noncanonical caspase-8 inflammasome. Nat. Immunol. 13, 246–254 (2012).
Poeck, H. et al. Recognition of RNA virus by RIG-I results in activation of CARD9 and inflammasome signaling for interleukin 1 beta production. Nat. Immunol. 11, 63–69 (2010).
Jia, X. M. et al. CARD9 mediates Dectin-1-induced ERK activation by linking Ras-GRF1 to H-Ras for antifungal immunity. J. Exp. Med. 211, 2307–2321 (2014).
Liu, M. et al. Dectin-1 activation by a natural product beta-glucan converts immunosuppressive macrophages into an M1-like phenotype. J. Immunol. 195, 5055–5065 (2015).
Matsunaga, K. et al. Two Beclin 1-binding proteins, Atg14L and Rubicon, reciprocally regulate autophagy at different stages. Nat. Cell Biol. 11, 385–396 (2009).
Yang, C. S. et al. The autophagy regulator Rubicon is a feedback inhibitor of CARD9-mediated host innate immunity. Cell Host Microbe. 11, 277–289 (2012).
Bradfield, C. J., Kim, B. H. & MacMicking, J. D. Crossing the Rubicon: new roads lead to host defense. Cell Host Microbe. 11, 221–223 (2012).
Kock, G. et al. Regulation of dectin-1-mediated dendritic cell activation by peroxisome proliferator-activated receptor-gamma ligand troglitazone. Blood 117, 3569–3574 (2011).
Drewniak, A. et al. Invasive fungal infection and impaired neutrophil killing in human CARD9 deficiency. Blood 121, 2385–2392 (2013).
Heinrich, A. et al. Moraxella catarrhalis induces CEACAM3-Syk-CARD9-dependent activation of human granulocytes. Cell Microbiol. 18, 1570–1582 (2016).
Liang, P. et al. CARD9 deficiencies linked to impaired neutrophil functions against Phialophora verrucosa. Mycopathologia 179, 347–357 (2015).
Gavino, C. et al. CARD9 deficiency and spontaneous central nervous system candidiasis: complete clinical remission with GM-CSF therapy. Clin. Infect. Dis. 59, 81–84 (2014).
Gazendam, R. P. et al. Two independent killing mechanisms of Candida albicans by human neutrophils: evidence from innate immunity defects. Blood 124, 590–597 (2014).
Jhingran, A. et al. Compartment-specific and sequential role of MyD88 and CARD9 in chemokine induction and innate defense during respiratory fungal infection. PLoS Pathog. 11, e1004589 (2015).
Futosi, K. & Mocsai, A. Tyrosine kinase signaling pathways in neutrophils. Immunol. Rev. 273, 121–139 (2016).
Firinu, D. et al. Genetic susceptibility to Candida infection: a new look at an old entity. Chin. Med. J. 126, 378–381 (2013).
Rosentul, D. C. et al. Genetic variation in the dectin-1/CARD9 recognition pathway and susceptibility to candidemia. J. Infect. Dis. 204, 1138–1145 (2011).
Hong, S. N. et al. Deep resequencing of 131 Crohn’s disease associated genes in pooled DNA confirmed three reported variants and identified eight novel variants. Gut 65, 788–796 (2016).
Fairfax, B. P. et al. Innate immune activity conditions the effect of regulatory variants upon monocyte gene expression. Science 343, 1246949 (2014).
Beaudoin, M. et al. Deep resequencing of GWAS loci identifies rare variants in CARD9, IL23R and RNF186 that are associated with ulcerative colitis. PLoS Genet. 9, e1003723 (2013).
Wang, Z. et al. Genetic association between CARD9 variants and inflammatory bowel disease was not replicated in a Chinese Han population. Int. J. Clin. Exp. Pathol. 8, 13465–13470 (2015).
Burghardt, K. M. et al. A CARD9 polymorphism is associated with decreased likelihood of persistent conjugated hyperbilirubinemia in intestinal failure. PLoS ONE 9, e85915 (2014).
Momenzadeh, P. et al. Determination of IL1 R2, ANTXR2, CARD9, and SNAPC4 single nucleotide polymorphisms in Iranian patients with ankylosing spondylitis. Rheumatol. Int. 36, 429–435 (2016).
Pointon, J. J. et al. Elucidating the chromosome 9 association with AS; CARD9 is a candidate gene. Genes Immun. 11, 490–496 (2010).
Streata, I. et al. The CARD9 Polymorphismsrs4077515, rs10870077 and rs10781499 are uncoupled from susceptibility to and severity of pulmonary tuberculosis. PLoS ONE 11, e0163662 (2016).
Grumach, A. S. et al. A homozygous CARD9 mutation in a Brazilian patient with deep dermatophytosis. J. Clin. Immunol. 35, 486–490 (2015).
Glocker, E. O. et al. A homozygous CARD9 mutation in a family with susceptibility to fungal infections. N. Engl. J. Med. 361, 1727–1735 (2009).
Herbst, M. et al. Chronic Candida albicans Meningitis in a 4-Year-Old Girl with a homozygous mutation in the CARD9 gene (Q295X). Pediatr. Infect. Dis. J. 34, 999–1002 (2015).
Yan, X. X. et al. CARD9 mutation linked to Corynespora cassiicola infection in a Chinese patient. Br. J. Dermatol. 174, 176–179 (2016).
Alves de Medeiros, A. K. et al. Chronic and invasive fungal infections in a family with CARD9 deficiency. J. Clin. Immunol. 36, 204–209 (2016).
Rieber, N. et al. Extrapulmonary Aspergillus infection in patients with CARD9 deficiency. JCI Insight 1, e89890 (2016).
Rosentul, D. C. et al. Gene polymorphisms in pattern recognition receptors and susceptibility to idiopathic recurrent vulvovaginal candidiasis. Front. Microbiol. 5, 483 (2014).
Lanternier, F. et al. Inherited CARD9 deficiency in 2 unrelated patients with invasive Exophiala infection. J. Infect. Dis. 211, 1241–1250 (2015).
Uniken Venema, W. T., Voskuil, M. D., Dijkstra, G., Weersma, R. K. & Festen, E. A. The genetic background of inflammatory bowel disease: from correlation to causality. J. Pathol. 241, 146–158 (2017).
Zhernakova, A. et al. Genetic analysis of innate immunity in Crohn’s disease and ulcerative colitis identifies two susceptibility loci harboring CARD9 and IL18RAP. Am. J. Hum. Genet. 82, 1202–1210 (2008).
Lee, Y. H. & Song, G. G. Pathway analysis of a genome-wide association study of ileal Crohn’s disease. DNA Cell Biol. 31, 1549–1554 (2012).
Galkina, E. & Ley, K. Immune and inflammatory mechanisms of atherosclerosis. Annu. Rev. Immunol. 27, 165–197 (2009).
Kelly, M., Hwang, J. M. & Kubes, P. Modulating leukocyte recruitment in inflammation. J. Allergy Clin. Immunol. 120, 3–10 (2007).
Peterson, M. R., Haller, S. E., Ren, J., Nair, S. & He, G. CARD9 as a potential target in cardiovascular disease. Drug Des. Devel. Ther. 10, 3799–3804 (2016).
Dai, X., Ding, Y., Liu, Z., Zhang, W. & Zou, M. H. Phosphorylation of CHOP (C/EBP Homologous Protein) by the AMP-activated protein kinase Alpha 1 in macrophages promotes CHOP degradation and reduces injury-induced neointimal disruption in vivo. Circ. Res. 119, 1089–1100 (2016).
Liu, Y. et al. CARD9 mediates necrotic smooth muscle cell-induced inflammation in macrophages contributing to neointima formation of vein grafts. Cardiovasc. Res. 108, 148–158 (2015).
Ren, J. et al. Proinflammatory protein CARD9 is essential for infiltration of monocytic fibroblast precursors and cardiac fibrosis caused by Angiotensin II infusion. Am. J. Hypertens. 24, 701–707 (2011).
Wang, S. et al. Zinc rescues obesity-induced cardiac hypertrophy via stimulating metallothionein to suppress oxidative stress-activated BCL10/CARD9/p38 MAPK pathway. J. Cell Mol. Med. 21, 1182–1192 (2017).
Sokol, H. et al. Card9 mediates intestinal epithelial cell restitution, T-helper 17 responses, and control of bacterial infection in mice. Gastroenterology 145, 591–601 (2013).
Imhann F. et al. Interplay of host genetics and gut microbiota underlying the onset and clinical presentation of inflammatory bowel disease. Gut 2016. https://doi.org/10.1136/gutjnl-2016-312135.
Lamas, B. et al. CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands. Nat. Med. 22, 598–605 (2016).
Lamas, B., Richard, M. L. & Sokol, H. CARD9 is involved in the recovery of colitis by promoting the production of AhR ligands by the intestinal microbiota. Med. Sci. 32, 933–936 (2016).
Kiryluk, K. et al. Discovery of new risk loci for IgA nephropathy implicates genes involved in immunity against intestinal pathogens. Nat. Genet. 46, 1187–1196 (2014).
Pedroza, L. A. et al. Autoimmune regulator (AIRE) contributes to Dectin-1-induced TNF-alpha production and complexes with caspase recruitment domain-containing protein 9 (CARD9), spleen tyrosine kinase (Syk), and Dectin-1. J. Allergy Clin. Immunol. 129, 464–472 (2012).
Lee, E. J. et al. Mincle activation and the Syk/Card9 signaling axis are central to the development of autoimmune disease of the eye. J. Immunol. 196, 3148–3158 (2016).
Janse, M. et al. Three ulcerative colitis susceptibility loci are associated with primary sclerosing cholangitis and indicate a role for IL2, REL, and CARD9. Hepatology 53, 1977–1985 (2011).
Tobias, H. et al. Card9 controls Dectin-1-induced T-cell cytotoxicity and tumor growth in mice. Eur. J. Immunol. 47, 872–879 (2017).
Sur, I. K. et al. Mice lacking a Myc enhancer that includes human SNP rs6983267 are resistant to intestinal tumors. Science 338, 1360–1363 (2012).
Leo, V. I. et al. CARD9 promotes sex-biased colon tumors in the APCmin mouse model. Cancer Immunol. Res. 3, 721–726 (2015).
Nakamura, S. et al. Overexpression of caspase recruitment domain (CARD) membrane-associated guanylate kinase 1 (CARMA1) and CARD9 in primary gastric B-cell lymphoma. Cancer 104, 1885–1893 (2005).
Zhou, Y. et al. Distinct comparative genomic hybridisation profiles in gastric mucosa-associated lymphoid tissue lymphomas with and without t(11;18)(q21;q21). Br. J. Haematol. 133, 35–42 (2006).
Afik, R. et al. Tumor macrophages are pivotal constructors of tumor collagenous matrix. J. Exp. Med. 213, 2315–2331 (2016).
Yang, M. et al. Tumor cell-activated CARD9 signaling contributes to metastasis-associated macrophage polarization. Cell Death Differ. 21, 1290–1302 (2014).
Yang, Z. W., Meng, X. X. & Xu, P. Central role of neutrophil in the pathogenesis of severe acute pancreatitis. J. Cell Mol. Med. 19, 2513–2520 (2015).
Yasukawa, S. et al. An ITAM-Syk-CARD9 signalling axis triggers contact hypersensitivity by stimulating IL-1 production in dendritic cells. Nat. Commun. 5, 3755 (2014).
Cao, L. et al. CARD9 knockout ameliorates myocardial dysfunction associated with high fat diet-induced obesity. J. Mol. Cell Cardiol. 92, 185–195 (2016).
Acknowledgements
This work was supported by the National Basic Research Program of China (No 81370569).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The authors declare that they have no financial interests.
Additional information
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Edited by H.-U. Simon
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Zhong, X., Chen, B., Yang, L. et al. Molecular and physiological roles of the adaptor protein CARD9 in immunity. Cell Death Dis 9, 52 (2018). https://doi.org/10.1038/s41419-017-0084-6
Received:
Revised:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41419-017-0084-6
This article is cited by
-
CARD9 contributes to ovarian cancer cell proliferation, cycle arrest, and cisplatin sensitivity
BMC Molecular and Cell Biology (2022)
-
CARD9 Expression Pattern, Gene Dosage, and Immunodeficiency Phenotype Revisited
Journal of Clinical Immunology (2022)
-
Primary Cutaneous Aspergillosis in a Patient with CARD9 Deficiency and Aspergillus Susceptibility of Card9 Knockout Mice
Journal of Clinical Immunology (2021)
-
Myeloid-derived suppressor cells—new and exciting players in lung cancer
Journal of Hematology & Oncology (2020)
-
CARD9 promotes autophagy in cardiomyocytes in myocardial ischemia/reperfusion injury via interacting with Rubicon directly
Basic Research in Cardiology (2020)