Xin Ming Jia

C-type Lectin Receptor Dectin-3 Mediates Trehalose 6,6′-Dimycolate (TDM)-induced Mincle Expression through CARD9/Bcl10/MALT1-dependent Nuclear Factor (NF)-κB Activation

Background: Recent studies suggest that Mincle expression is induced by Dectin-3-mediated signaling in response to TDM stimulation. Results: Deficiency in Dectin-3 and the CARD9-Bcl10-Malt1 complex are defective for TDM-induced NF-κB activation and Mincle. Conclusion: Dectin-3- and CARD9/Bcl10/Malt1-dependent NF-κB activation plays an essential role for TDM-induced Mincle expression. Significance: This study provides the molecular insight for designing adjuvants that stimulate the immune system. Previous studies indicate that both Dectin-3 (also called MCL or Clec4d) and Mincle (also called Clec4e), two C-type lectin receptors, can recognize trehalose 6,6′-dimycolate (TDM), a cell wall component from mycobacteria, and induce potent innate immune responses. Interestingly, stimulation of Dectin-3 by TDM can also induce Mincle expression, which may enhance the host innate immune system to sense Mycobacterium infection. However, the mechanism by which Dectin-3 induces Mincle expression is not fully defined. Here, we show that TDM-induced Mincle expression is dependent on Dectin-3-mediated NF-κB, but not nuclear factor of activated T-cells (NFAT), activation, and Dectin-3 induces NF-κB activation through the CARD9-BCL10-MALT1 complex. We found that bone marrow-derived macrophages from Dectin-3-deficient mice were severely defective in the induction of Mincle expression in response to TDM stimulation. This defect is correlated with the failure of TDM-induced NF-κB activation in Dectin-3-deficient bone marrow-derived macrophages. Consistently, inhibition of NF-κB, but not NFAT, impaired TDM-induced Mincle expression, whereas NF-κB, but not NFAT, binds to the Mincle promoter. Dectin-3-mediated NF-κB activation is dependent on the CARD9-Bcl10-MALT1 complex. Finally, mice deficient for Dectin-3 or CARD9 produced much less proinflammatory cytokines and keyhole limpet hemocyanin (KLH)-specific antibodies after immunization with an adjuvant containing TDM. Overall, this study provides the mechanism by which Dectin-3 induces Mincle expression in response to Mycobacterium infection, which will have significant impact to improve adjuvant and design vaccine for antimicrobial infection.

CARD9 mediates Dectin-1–induced ERK activation by linking Ras-GRF1 to H-Ras for antifungal immunity

CARD9 is dispensable for NF-κB activation induced by Dectin-1 ligands in mice. However, Dectin-1–induced H-Ras activation is mediated by a complex with CARD9, which leads to ERK activation for host innate immune responses to Candida albicans infection.

Tyrosine phosphatase SHP-2 mediates C-type lectin receptor–induced activation of the kinase Syk and anti-fungal T H 17 responses

SUMMARY Fungal infection stimulates the canonical C-type lectin receptors (CLRs) signaling pathway via Syk activation. Here we show that SHP-2 plays a crucial role in mediating CLRs-induced Syk activation. Genetic ablation of Shp-2 (Ptpn11) in dendritic cells (DCs) and macrophages impaired Syk-mediated signaling and abrogated pro-inflammatory gene expression following fungal stimulation. Mechanistically, SHP-2 operates as a scaffold facilitating the recruitment of Syk to dectin-1 or FcRγ, through its N-SH2 domain and a previously unrecognized C-terminal ITAM motif. We demonstrate that DC-derived SHP-2 is crucial for the induction of IL-1β, IL-6 and IL-23, and anti-fungal TH17 cell responses to control Candida albicans infection. Together, these data reveal a mechanism by which SHP-2 mediates Syk activation in response to fungal infections

JNK1 negatively controls antifungal innate immunity by suppressing CD23 expression

Opportunistic fungal infections are a leading cause of death among immune-compromised patients, and there is a pressing need to develop new antifungal therapeutic agents because of toxicity and resistance to the antifungal drugs currently in use. Although C-type lectin receptor– and Toll-like receptor–induced signaling pathways are key activators of host antifungal immunity, little is known about the mechanisms that negatively regulate host immune responses to a fungal infection. Here we found that JNK1 activation suppresses antifungal immunity in mice. We showed that JNK1-deficient mice had a significantly higher survival rate than wild-type control mice in response to Candida albicans infection, and the expression of JNK1 in hematopoietic innate immune cells was critical for this effect. JNK1 deficiency leads to significantly higher induction of CD23, a novel C-type lectin receptor, through NFATc1-mediated regulation of the CD23 gene promoter. Blocking either CD23 upregulation or CD23-dependent nitric oxide production eliminated the enhanced antifungal response found in JNK1-deficient mice. Notably, JNK inhibitors exerted potent antifungal therapeutic effects in both mouse and human cells infected with C. albicans, indicating that JNK1 may be a therapeutic target for treating fungal infection.

E3 ubiquitin ligase Cbl-b negatively regulates C-type lectin receptor–mediated antifungal innate immunity

Innate immune responses mediated by C-type lectin receptors Dectin-2 and Dectin-3 against fungal infections are negatively regulated by Cbl-b ubiquitination.

CARD9S12N facilitates the production of IL-5 by alveolar macrophages for the induction of type 2 immune responses

The adaptor CARD9 functions downstream of C-type lectin receptors (CLRs) for the sensing of microbial infection, which leads to responses by the TH1 and TH17 subsets of helper T cells. The single-nucleotide polymorphism rs4077515 at CARD9 in the human genome, which results in the substitution S12N (CARD9S12N), is associated with several autoimmune diseases. However, the function of CARD9S12N has remained unknown. Here we generated CARD9S12N knock-in mice and found that CARD9S12N facilitated the induction of type 2 immune responses after engagement of CLRs. Mechanistically, CARD9S12N mediated CLR-induced activation of the non-canonical transcription factor NF-κB subunit RelB, which initiated production of the cytokine IL-5 in alveolar macrophages for the recruitment of eosinophils to drive TH2 cell–mediated allergic responses. We identified the homozygous CARD9 mutation encoding S12N in patients with allergic bronchopulmonary aspergillosis and revealed activation of RelB and production of IL-5 in peripheral blood mononuclear cells from these patients. Our study provides genetic and functional evidence demonstrating that CARD9S12N can turn alveolar macrophages into IL-5-producing cells and facilitates TH2 cell–mediated pathologic responses.CARD9 serves as an adaptor for C-type lectin receptor signaling. Xin Lin and colleagues show that CARD9 inhibits RelB-mediated IL-5 expression. The CARD9S12N mutant, prevalent in humans, cannot interact with RelB and promotes enhanced allergic responses to fungal pathogens.

The CARMA3-BCL10-MALT1 (CBM) complex contributes to DNA damage-induced NF-κB activation and cell survival

Chemotherapy is one of major means for cancer treatments, and many of chemotherapeutic drugs are DNA damaging agents that reduce tumor growth through triggering cancer cell apoptosis or necrosis. Following DNA damage, ataxia telangiectasia mutated (ATM), a protein kinase, was activated and a cytosolic complex containing ATM, NEMO, RIP1 were formed (Biton and Ashkenazi, 2011). This ATM/NEMO/ RIP1 complex cooperates to activate cytoplasmic adaptor protein TRAF6 (Hinz et al., 2010) and Ser/Thr kinase, TGFβ-activated kinase 1 (TAK1) and IKK complex to induce NFκB activation through multiple signal transduction mechanisms depending on the severity of genotoxic stress and cell type (Wu et al., 2010). It has been shown that irradiation or chemotherapeutic drugs induce NF-κB activation through the functional IKK complex, but some reports suggest that UV irradiation activates NF-κB signal through IKK complexindependent but CK2-dependent manner (Kato et al., 2003). Of note, many DNA damaging agents can also activate NFκB family of transcription factors that induce the expression of anti-apoptotic genes, thereby protecting cancer cells from apoptosis and resulting in the resistance of cancer cells to the chemotherapy. Although there are many studies on the mechanism of DNA damage-induced drug resistance, the molecular mechanism by which DNA damage induces NFκB activation is not fully defined. The caspase recruitment domain (CARD) and membrane-associated guanylate kinase-like domain protein (CARMA) family of proteins has three members, CARMA1, CARMA2, and CARMA3 that are encoded by three different genes (Blonska and Lin, 2011). CARMA proteins share the same set of structural domains, but a different pattern of tissue expression profile. Upon different stimuli, all CARMA proteins can form a complex with BCL10 and MALT1, and the CARMA-BCL10-MALT1 (CBM) complex functions to activate NF-κB signaling (Pomerantz et al., 2002; Grabiner et al., 2007; Jiang et al., 2011). Besides in NF-κB signaling, CBM complex is also involved in antiviral innate immune response by suppressing IRF3-type I IFN expression through inhibiting the formation of MAVS oligomerization in mitochondrion (Jiang et al., 2016). CARMA3 was reported to be relatively overexpressed in many tumor cells and associated with the malignant behavior of cancer cells (Li et al., 2012; Pan et al., 2016). It has been shown that CARMA3 and TRAF6 form a complex to mediating GPCRand EGFRinduced NF-κB activation (Grabiner et al., 2007). Therefore, we hypothesized that the CBM complex contributes to DNA damage-induced NF-κB activation to protect genotoxic agent-induced cell death. To determine the role of CBM complex in DNA damageinduced NF-κB activation, MEF cells isolated from Malt1 Het (+/−) or knockout (−/−) mice were stimulated with doxorubicin, a genotoxic reagent inhibiting the activity of topoisomerase II. Consistent with our hypothesis, NF-κB was potently activated by doxorubicin in control cells, but completely impaired in Malt1-deficient cells (Fig. 1A). To confirm that Malt1 is responsible to DNA damage-induced NF-κB activation, MEFs (Malt1) and MALT-deficient MEFs (Malt1) were treated with two other genotoxic reagent VP16 (etoposide) (10 μmol/L) and CPT (2 μmol/L), respectively. Consistently, we found that VP16and CPT-induced NF-κB activation were defective in MALT1-deficient cells compared with controls (Fig. 1B), indicating that MALT1 is required for DNA damage-induced NFκB activation. Since MALT1 forms a complex with CARMA1 and Bcl10 in hematopoietic cells, and CARMA3 and Bcl10 in non-hematopoietic cells to activate NF-κB, we examined the impact of CARMA3 or Bcl10 on DNA damage-induced signaling (Fig. 1C). CARMA3-deficient (Carma3) and BCL10deficient (Bcl10) MEFs together with wild-type controls were treated with doxorubicin. Consistent with the result in Malt1 KO MEF cells, both CARMA3-deficient and BCL10deficient cells were completely defective for NF-κB activation upondoxorubicin treatment. Similarly, we found that genotoxic reagents-induced NF-κB activation was significantly reduced in JPM50.6 cells (Fig. S1), in which the expression of CARMA1 is defective (Wang et al., 2002). Together, these results indicate that CBM complex mediates DNA damageinduced NF-κB activation in both hematopoietic and non-hematopoietic cells. PKC family of kinases has been shown to play important roles in CBM complex-mediated NF-κB activation upon

Dectin-3 recognizes cryptococcal glucuronoxylomannan to initiate host defense against cryptococcosis

Cryptococcus neoformans and Cryptococcus gattii cause life-threatening meningoencephalitis and pneumonia in immunosuppressed and immunocompetent individuals. Given the structural differences of major polysaccharide glucuronoxylomannan (GXM) between C. neoformans and C. gattii, it remains unclear that how innate immune system recognizes GXM. Here, we report that C-type lectin receptor Dectin-3 (MCL encoded by Clec4d) is a direct receptor for GXMs from C. neoformans serotype AD (C.n-AD) and C. gattii serotype B (C.g-B). GXMs from C.n-AD and C.g-B activated both NF-κB and ERK pathways to induce the pro-inflammatory cytokine production, whereas it was completely abolished due to deficiency of Dectin-3 or its downstream adaptor protein CARD9. Upon pulmonary C.n-AD and C.g-B infection, Dectin-3- and CARD9-deficient mice were highly susceptible and showed augmented lung injury due to impairment of alveolar macrophage accumulation and killing activities. These results demonstrate that Dectin-3 contributes to host immunity against Cryptococcus infection through selectively recognizingGXM.

C-Type Lectin Receptor CD23 Is Required for Host Defense against Candida albicans and Aspergillus fumigatus Infection

Infection by invasive fungi, such as Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans, is one of the leading death causes for the increasing population of immunocompromised and immunodeficient patients. Several C-type lectin receptors (CLRs), including Dectin-1, -2, and -3 and Mincle can recognize fungal surface components and initiate the host antifungal immune responses. Nevertheless, it remains to be determined whether other CLRs are involved in antifungal immunity. Our recent study suggests that CD23 (CLEC4J), a CLR and also a well-known B cell surface marker, may function to sense C. albicans components in antifungal immunity. However, it is not clear how CD23 functions as a fungal pattern recognition receptor and whether the antifungal role of CD23 is specific to C. albicans or not. In this study, we show that CD23 can recognize both α-mannan and β-glucan from the cell wall of C. albicans or A. fumigatus but cannot recognize glucuronoxylomannan from Cryptococcus. Through forming a complex with FcRγ, CD23 can induce NF-κB activation. Consistently, CD23-deficient mice were highly susceptible to C. albicans and A. fumigatus but not to C. neoformans infection. The expression of CD23 in activated macrophages is critical for the activation of NF-κB. CD23 deficiency results in impaired expression of NF-κB–dependent genes, especially iNOS, which induces NO production to suppress fungal infection. Together, our studies reveal the CD23-induced signaling pathways and their roles in antifungal immunity, specifically for C. albicans and A. fumigatus, which provides the molecular basis for designing potential therapeutic agents against fungal infection.

Dectin-3 Recognizes Glucuronoxylomannan of Cryptococcus neoformans Serotype AD and Cryptococcus gattii Serotype B to Initiate Host Defense Against Cryptococcosis

Cryptococcus neoformans and Cryptococcus gattii cause life-threatening meningoencephalitis or lung diseases in immunocompetent individuals or immunocompromised ones. C. neoformans and C. gattii are subdivided into five serotypes based on their capsular glucuronoxylomannan (GXM). C. neoformans consists of serotypes A, D, and AD hybrid, and C. gattii consists of serotypes B and C. Given structural differences of GXM between C. neoformans and C. gattii, it remains unclear that how innate immune system recognizes GXM. Here, we report that C-type lectin receptor Dectin-3 (MCL encoded by Clec4d) is a direct receptor for GXMs from C. neoformans serotype AD (C.n-AD) and C. gattii serotype B (C.g-B). GXMs from C.n-AD and C.g-B activated NF-κB and ERK pathways to induce pro-inflammatory cytokine production, whereas it was completely abolished due to deficiency of Dectin-3 or caspase recruitment domain family member 9 (CARD9). Upon pulmonary C.n-AD and C.g-B infection, Dectin-3- and CARD9-deficient mice were highly susceptible and showed augmented lung injury due to impairment of alveolar macrophage accumulation and killing activities. Our study provides the first biological and genetic evidence demonstrating that Dectin-3 recognizes GXM of C.n-AD and C.g-B to initiate host defense against cryptococcosis.