The Role of CARD9 in Metabolic Diseases*

Caspase recruitment domain containing protein 9 (CARD9) is an adaptor protein that plays a critical role in pattern recognition receptors (PRRs)-mediated activation of NF-κB and mitogen-activated protein kinase (MAPK). This elicits initiation of the pro-inflammatory cytokines and leads to inflammatory responses, which has been recognized as a critical contributor to chronic inflammation. Current researches demonstrate that CARD9 is strongly associated with metabolic diseases, such as obesity, insulin resistance, atherosclerosis and so on. In this review, we summarize CARD9 signaling pathway and the role of CARD9 in metabolic diseases.

Metabolic diseases are recognized as a group of diseases characterized by metabolic disorder, such as obesity, insulin resistance (IR), atherosclerosis and so on. In the modern era, metabolic diseases have become the leading risk factor affecting disability-adjusted life-years in many countries, with a skyrocketing incidence [1] . Metabolic diseases have been validated to be strongly associated with inflammation [2] . For example, elevated level of pro-inflammatory cytokines is detected in IR mice, and these cytokines drive macrophage polarization into M1 type. Besides, nuclear factor-κB (NF-κB) signaling pathway in macrophages is activated in mice with IR or atherosclerosis, which contributes to activation of macrophages and propagation of pro-inflammatory signals. Currently, extensive studies in terms of the potential involvement of caspase recruitment domain containing protein 9 (CARD9), an upstream molecule of NF-κB, in inflammation have been conducted [3,4] . As a result, CARD9 may be involved in metabolic diseases, which has been proved by some researches [5,6] .
CARD9, possessing a coiled-coil region in C terminus and a caspase recruitment domain (CARD) in N terminus, was originally identified via Millennium Pharmaceuticals proprietary database search for CARD-containing proteins [7] . CARD9, a critical integrator of innate immunity, is highly expressed in myeloid cells, especially in macrophages and dendritic cells (DCs) [4] . In addition, CARD9 has been identified as a vital adaptor protein that integrates signals from pattern recognition receptors (PRRs) in myeloid cells [8,9] . Following PRRs engagement, CARD9 can activate NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways by forming CARD9-B cell lymphoma 10 (BCL10)-mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) (CBM) complex, and then triggers an inflammatory immune response [10,11] .
Recently, accumulating evidence has proved that CARD9 plays an indispensable role in multiple metabolic diseases. In this review, we summarize CARD9 signaling pathway and the role of CARD9 in metabolic diseases, which is conducive to exploring the pathogenesis of metabolic diseases.

CARD9 Integrates Signals from Transmembrane PRRs
Common transmembrane PRRs include C-type lectin receptors (CLRs) and Toll-like receptors (TLRs). Dectin-1, one of CLRs, initiates NF-κB and MAPK signaling in CARD9-dependent manners [15] . Ligation of Dectin-1 leads to the recruitment and activation of spleen tyrosine kinase (SYK), which subsequently phosphorylates protein kinase C δ (PKCδ) at position Tyr311. Activated PKCδ induces CARD9 phosphorylation at position Thr231 and the assembly of CBM complex [15,16] . Then, CBM complex triggers the production of pro-inflammatory cytokines, including IL-6, TNF-α and IL-1β, via activating extracellular regulated protein kinases (ERK) and NF-κB, the former of which happens through a RasGrf1-H-Ras pathway and the latter of which occurs via a classic NF-κB signaling pathway [17,18] . There are numerous regulators of CARD9-dependent signaling pathways induced by CLRs, such as Vav proteins, tripartite motif-containing 62 (TRIM62), Rubicon and Casein kinase 2 (CK2) [19][20][21][22] . Both Vav proteins and TRIM62 are indispensable to the activation of CARD9-mediated NF-κB signaling pathway, while both Rubicon and CK2 are crucial in down-regulation of CARD9-mediated inflammatory responses. The phosphorylation of Vav proteins at position Tyr174 promotes the formation of CBM complex and subsequent NF-κB activation. And TRIM62, facilitating K27-linked polyubiquitination of the CARD9 C-terminus, ubiquitinates CARD9 at position Lys125 and promotes CARD9-mediated NF-κB activation, but is dispensable to the assembly of CBM complex. Rubicon, a critical autophagy regulator, competitively binds to CARD9 and leads to the disassembly of the CBM complex, thus turning off inflammatory signaling and preventing excessive inflammatory responses. In addition, CK2-mediated phosphorylation of CARD9 at Thr531 and Thr533 inhibits the formation of CBM complex, and the process is promoted by the VHL tumor suppressor protein (pVHL).
TLRs were observed to trigger CARD9mediated MAPK activation [23] . Hara et al reported that CARD9 -/-DCs could not be activated by Zymosan, the ligand of TLRs and Dectin-1. Mechanically, CARD9 was required for TLR-induced DCs activation through transducing MyD88-mediated signals and activating MAPK. Further researches revealed that CARD9 was required for innate immune responses to selective ligands [13,23] . For example, the generation of IL-6 and TNF-α in CARD9 -/macrophage was induced by the ligands of TLR3 or TLR7, but not by the ligands of TLR2, TLR4, TLR5, and TLR9. In addition to inducing innate immune responses, CARD9 activates adaptive immunity to pathogens [11] . For instance, Flagellin-specific T-cell responses were shown to require CARD9-expressing DCs.
It has been validated that CARD9 is essential for NOD2-mediated activation of MAPK [13] . Hsu et al reported that CARD9 -/macrophages showed defective activation of p38 and c-Jun-NH2-terminal kinase (JNK) following stimulation with muramyl-dipeptide (MDP), a ligand of intracellular NOD2 receptor. However, CARD9 -/macrophages displayed normal NF-κB activation after MDP treatment. Further evidence suggested that CARD9 could form trimolecular complex with NOD2 and receptor interacting protein 2 (RIP2), a CARD kinase, which further induced NOD2mediated activation of p38 and JNK in innate immune responses.
RLRs, such as RIG-1, sense viral double-strand (ds) RNA in the cytoplasm and trigger the production of IL-1β [24] . Loo et al showed that RLRs-mediated signals were relayed to interferon (IFN)-regulatory factor (IRF) and NF-κB by interacting with the adaptor mitochondrial antiviral-signaling protein (MAVS). After selective RIG-I ligand dsRNA treatment, CARD9-BCL10 module was induced to activate NF-κB to promote the synthesis of pro-IL-1β, while RIG-I triggered caspase-1-dependent inflammasome activation by binding to apoptosis-associated specklike protein containing CARD (ASC). Subsequently, caspase-1 processed pro-IL-1β into mature and bioactive IL-1β [14] .

The Promotive Role of CARD9 in Obesity
Obesity is regarded as low grade chronic inflammation, with increased infiltration of immune cells into adipose tissue and heightened levels of circulating and localized pro-inflammatory cytokines [27,28] . Troglitazone (TGZ), a synthetic agonist of peroxisome proliferator-activated receptor-gamma (PPARγ), improves insulin sensitivity in obese objects [29] . Kock et al showed that TGZ inhibited Dectin-1-mediated activation of MAPK and NF-κB signaling pathways in DCs through interfering with CARD9 [30] , which suggests that TGZ may partially ameliorate obesityrelated symptoms by suppressing CARD9-associated signaling pathways. And the activation of Dectin-1, inducing innate immune responses through activating CARD9-mediated NF-κB signaling pathway [17] , aggravated adiposis and IR in MyD88 -/mice [31] . Furthermore, monocyte chemoattractant protein-1, the serum level of which was closely correlated with SNPs in CARD9, was increased in obese human [32] . These studies provide a possibility that CARD9 contributes to the development of obesity.
A recent study conducted by Yang et al indicated that CARD9 deficiency mitigated the high fat diet (HFD)-induced metabolic disorders and inflammation by suppressing p38 MAPK and NF-κB signaling pathways [5] . In HFD-treated mice, CARD9 deficiency ameliorated adiposis, diminished the excessive lipid accumulation, and alleviated adiposity-associated symptoms including glucose tolerance impairment, inflammation and hepatopathy. Compared to HFDfed wild type (WT) mice, HFD-fed CARD9 -/mice exhibited inhibited p38 and NF-κB signaling pathways. In another study, CARD9 -/mice preserved autophagy, suppressed p38 phosphorylation and reduced macrophages infiltration into heart, thus ameliorating myocardial dysfunction related to HFDinduced obesity [6] . Similarly, zinc deficiency and HFD synergistically increased the activation of p38 signaling pathways through increasing the expression of BCL10 and CARD9, giving rise to exacerbating obesity-related cardiac hypertrophy [33] . As a result, these data suggest that CARD9 is a potential target for attenuating obesity and obesity-associated symptoms.

The Promotive Role of CARD9 in Insulin Resistance
A large body of evidence suggests that high level of pro-inflammatory mediators in obese mice adversely affects insulin sensitivity and contributes to IR [34,35] . For example, TNF-α levels in bloodstream and peripheral tissues are increased in IR mice, and the neutralization of TNF-α prevents IR induced by HFD [36] . CARD9 mediates the activation of NF-κB signaling pathway, further promoting the production of pro-inflammatory cytokines [37,38] . In addition, increased infiltration of immune cells into adipose tissue is associated with IR [28] . For instance, activation of DCs infiltrating into adipose tissue of obese mice, which is induced via SYK-CARD9 pathway, promotes inflammation and IR through inducing Th17 cell responses [39,40] . These results reveal the potential correlation between CARD9 and IR.
Yang et al revealed that CARD9 accelerated the development of IR and increased the activation of p38 and NF-κB in HFD-fed mice [5] . Besides, Cao et al found that CARD9 deficiency retarded the development of IR and decreased p38 activation [6] . And TGZ, a drug for ameliorating IR, inhibited activation of DCs by suppressing CARD9-mediated activation of p38 and NF-κB [30] . Collectively, CARD9-mediated activation of p38 and NF-κB plays a promotive role in IR.

The Protective Role of CARD9 in Atherosclerosis
Inflammation, clearly implicated in CARD9, has been known as a crucial contributor to atherosclerosis [41] . A recent research reported that deletion of hematopoietic Mincle, a member of CLRs correlated with CARD9, reduced atherosclerotic lesion formation and lesion severity [42] . Furthermore, atherosclerosis is associated with increased titers of oxidized low-density lipoprotein (LDL) immune complex [43] . And oxidized LDL immune complex priming Nlrp3 inflammasome relies on CARD9 and involves TCR and FcγR synergy [44] . And CARD9 has been recognized as a potential target in cardiovascular diseases [45] . As a result, CARD9 may promote the development of atherosclerosis. However, a recent study conducted by Thiem et al indicated that hematopoietic CARD9 deletion increased atherosclerotic plaque formation in hyperlipidemic LDL receptor (LDLR)-knockout mice [46] . In this study, deletion of hematopoietic CARD9 or Dectin-2 in LDLR -/mice did not affect plasma lipids, circulating immune cell composition and peripheral cytokines. But deletion of hematopoietic CARD9, not Dectin-2, promoted atherosclerotic plaque formation and increased severity. This research provides an intriguing possibility that CARD9 protects against atherosclerosis with an uncertain mechanism. But it is also surprising because hematopoietic deletion of several CLRs, such as Mincle and Dectin-2, has a protective or void effect on atherosclerosis in LDLR -/mice [42,46] , and these CLRs could initiate signals by CARD9-dependent manners. We speculate that multiple signaling pathways are involved in pathogenesis of atherosclerosis, and deletion of hematopoietic CARD9 compensatorily activates or inhibits other signaling pathways, ultimately promoting atherosclerosis initiation. The accurate mechanism remains to be elucidated.

The Dual Role of CARD9 in Heart Diseases
Some studies demonstrate that heart diseases are associated with obesity and inflammation [47][48][49] . For instance, mice fed on HFD were more likely to develop myocardial dysfunction than normal mice [6] . And CARD9 deficiency alleviated obesityinduced myocardial dysfunction and inflammation through suppressing CARD9-dependent MAPK phosphorylation. Another research showed that CARD9-dependent MAPK signaling pathway was involved in the development of obesity-related cardiac hypertrophy (ORCH) [33] . Zinc supplement alleviated ORCH, induced by HFD and zinc deficiency, via stimulating metallothionein to inhibit oxidative stress-activated CARD9-dependent MAPK signaling pathway. In addition, zinc modulates the assembly of CARD9-BCL10 complex by binding with CARD9 [50] . In summary, zinc supplement ameliorates CARD9mediated inflammation by activating metallothionein to repress CARD9 activation and directly binding with CARD9, and further attenuates cardiac diseases. Furthermore, Ren et al demonstrated that CARD9 played a critical role in regulating angiotensin II (Ang II)-induced cardiac remodeling [51] . CARD9 deficiency alleviated Ang II-induced cardiac fibrosis and inflammation by inhibiting the activation of NF-κB, JNK and p38 in macrophages.
In addition to mediating inflammation, CARD9 inhibits mitochondria-dependent apoptosis of cardiomyocytes under oxidative stress [52] . During myocardial ischemia-reperfusion (I/R) injury, excessive ROS stimulates the release of cytochrome C from mitochondria into the cytoplasm. Then, cytochrome C, Apaf-1, and procaspase-9 assemble into an apoptosome which stimulates the activation of caspase-9. Activated caspase-9 subsequently activates caspase-3 inducing the apoptosis of cardiomyocytes. However, CARD9 competitively binds with Apaf-1 and disrupts apoptosomes formation through a CARD domain-dependent mechanism, which subsequently inhibits the activation of caspase-9 and caspase-3 and thus suppresses cardiomyocytes apoptosis induced by I/R injury.
These findings suggest that CARD9 has a dual role in heart diseases. On one hand, CARD9 deficiency in macrophages alleviates myocardial dysfunction, cardiac hypertrophy and cardiac remodeling via inhibiting the activation of CARD9-mediated inflammatory signals. On the other hand, CARD9 represses cardiomyocytes apoptosis induced by oxidative stress through interacting with Apaf-1.

The Promotive Role of CARD9 in Neointima Formation of Vein Grafts
CARD9 deficiency not only alleviates the obesity-associated heart diseases through suppressing CARD9-dependent MAPK phosphorylation, but ameliorates the intimal hyperplasia of vein grafts by inactivating CARD9-dependent NF-κB signaling pathway [6,33,53] . Vein grafts are generally used for vascular reconstruction. However, the graft patency is limited by neointima formation [54] . Inflammation plays a crucial role in intimal hyperplasia of vein grafts. Increased production of pro-inflammatory cytokines and infiltration of inflammatory immunocytes in grafted vein precede neointima formation and are throughout intimal hyperplasia of vein grafts [53,55,56] . Liu et al found that CARD9-dependent NF-κB activation in macrophages contributed to necrotic smooth muscle cells (SMCs)-induced inflammation and facilitated intimal hyperplasia of vein grafts [53] . Necrosis of SMCs was induced in early vein grafts and induced production of IL-1β, IL-6 and MCP-1 in macrophages. These pro-inflammatory cytokines ulteriorly recruited macrophages and exacerbated inflammation. Further study found that CARD9 was highly expressed in infiltrated macrophages of grafted veins. Furthermore, depletion of CARD9 alleviated SMC-induced inflammation by inactivating NF-κB and finally ameliorated neointima formation of vein grafts. Therefore, CARD9 contributes to neointima formation of vein grafts.

CONCLUSIONS AND PERSPECTIVES
A surge of interest in the role of innate immune in metabolic regulation has revealed the close relation between innate immune and metabolic disease. As a key adaptor transducing PRRs-mediated signals in innate immune, CARD9 is involved in the pathogenesis of multiple metabolic diseases via activating p38 and NF-κB signal pathways and inducing cascaded inflammatory reaction. TGZ was reported to interfere with CARD9 and inhibited Dectin-1-mediated secretion of cytokines and chemokines via p38 and NF-κB pathway in DCs [30] . Pharmacological inhibition of SYK in inflammatory macrophage, which may indirectly inhibit CARD9, ameliorated fibrosis, inflammation and steatosis in mice with non-alcoholic steatohepatitis [57] . Also, CARD9 participates in the regulation of cell apoptosis. Li et al found that CARD9 protects against myocardial I/R injury by inhibiting cardiomyocytes apoptosis [52] . Interestingly, the researchers found that CARD9 induced cell apoptosis in human leukemic cells [58,59] , such as NB4 and HL60 cells, but inhibited that in oral squamous cell carcinoma (OSCC) and cardiomyocytes. Collectively, CARD9 is not only a critical adaptor of innate immunity, but an apoptosisregulating protein ( fig. 1).
In conclusion, this review summarizes the role of CARD9 in immunity-based metabolic diseases. It not only facilitates the further study of pathogenesis of metabolic diseases, but provides a theoretical As an adaptor of innate immunity, CARD9 accelerates inflammation by activating MAPK and NF-κB signaling pathway in innate immune cells, which promotes multiple metabolic diseases, including obesity, IR, cardiac fibrosis, neointima formation of vein grafts, and inhibits atherosclerosis via the unknown mechanism.
As an apoptosis-regulating protein, CARD9 suppresses caspase-9 activation by combining with Apaf-1, which inhibits apoptosis of cardiomyocytes and further relieves I/R injury.
foundation for the study of drugs preventing or treating metabolic diseases. As a signal transducer in innate immune, CARD9 regulates immune-based metabolic diseases through delivering inflammatory signals to p38 and NF-κB pathways, but some blind spots still exist. Upon the role in metabolic disease, it is unclear whether CARD9 directly regulates metabolic pathways such as mTOR and AMPK pathways. As an apoptosis-regulating protein, how CARD9 affects the apoptosis of immune cells and whether it affects the crosstalk between cascaded inflammatory reaction and the programmed cell apoptosis remain unclear.