Jianxin Huo

Phospholipase Cγ2 Is Critical for Dectin-1-mediated Ca2+ Flux and Cytokine Production in Dendritic Cells

Dectin-1 is a C-type lectin that recognizes β-glucan in the cell walls of fungi and plays an important role in anti-fungal immunity. It signals via tyrosine kinase Syk and adaptor protein Card9 to activate NF-κB leading to proinflammatory cytokine production in dendritic cells (DCs). Other than this, not much else is known of the mechanism of Dectin-1 signaling. We demonstrate here that stimulation of DCs with zymosan triggers an intracellular Ca2+ flux that can be attenuated by a blocking anti-Dectin-1 antibody or by pre-treatment of cells with the phospholipase C (PLC) γ-inhibitor U73122, suggesting that Dectin-1 signals via a PLCγ pathway to induce Ca2+ flux in DCs. Interestingly, treatment of DCs with particulate curdlan, which specifically engages Dectin-1, results in the phosphorylation of both PLCγ1 and PLCγ2. However, we show that PLCγ2 is the critical enzyme for Dectin-1 signaling in DCs. PLCγ2-deficient DCs have drastic impairment of Ca2+ signaling and are defective in their secretion of interleukin 2 (IL-2), IL-6, IL-10, IL-12, IL-23, and tumor necrosis factor α. PLCγ2-deficient DCs also exhibit impaired activation of ERK and JNK MAPKs and AP-1 and NFAT transcription factors in response to Dectin-1 stimulation. In addition, PLCγ2-deficient DCs are also impaired in their activation of NF-κB upon Dectin-1 engagement due to defective assembly of the Card9-Bcl10-Malt1 complex and impaired IKKα/β activation and IκBα degradation. Thus, our data indicate that pattern recognition receptors such as Dectin-1 could elicit Ca2+ signaling and that PLCγ2 is a critical player in the Dectin-1 signal transduction pathway.

Bruton's tyrosine kinase phosphorylates Toll-like receptor 3 to initiate antiviral response

Toll-like receptor 3 (TLR3) mediates antiviral response by recognizing double-stranded RNA. Its cytoplasmic domain is tyrosine phosphorylated upon ligand binding and initiates downstream signaling via the adapter TIR-containing adaptor inducing interferon–β (TRIF). However, the kinase responsible for TLR3 phosphorylation remains unknown. We show here that Brutons tyrosine kinase (BTK)-deficient macrophages failed to secrete inflammatory cytokines and IFN-β upon TLR3 stimulation and were impaired in clearing intracellular dengue virus infection. Mutant mice were also less susceptible to d-galactosamine/p(I:C)-induced sepsis. In the absence of BTK, TLR3-induced phosphoinositide 3-kinase (PI3K), AKT and MAPK signaling and activation of NFκB, IRF3, and AP-1 transcription factors were all defective. We demonstrate that BTK directly phosphorylates TLR3 and in particular the critical Tyr759 residue. BTK point mutations that abrogate or led to constitutive kinase activity have opposite effects on TLR3 phosphorylation. Loss of BTK also compromises the formation of the downstream TRIF/receptor-interacting protein 1 (RIP1)/TBK1 complex. Thus, BTK plays a critical role in initiating TLR3 signaling.

BAFF costimulation of Toll-like receptor-activatedB-1 cells

B cell‐activating factor belonging to the TNF family (BAFF) and its receptor BAFF‐R play critical roles in the maturation and survival of conventional peripheral B cells. However, they appeared to be dispensable for the generation and maintenance of CD5+ B‐1 cells as BAFF–/– and BAFF‐R–/– mice have normal B‐1 cell populations. Hence, it is presently unclear if B‐1 cells are responsive to BAFF and if BAFF regulates some aspects of B‐1 cell function. We show here that BAFF‐R and transmembrane activator and CAML interactor (TACI) are the major receptors expressed by B‐1 cells. Specifically, we show that BAFF treatment of B‐1 cells leads to increased NF‐κB p100 processing and CD21/CD35 expression. Interestingly, toll‐like receptor (TLR) engagement of B‐1 cells augmented the surface expression of BAFF receptors and rendered them responsive to BAFF costimulation, as evidenced by their increased proliferation, expression of cell surface activation markers and secretion of the pro‐inflammatory cytokine IL‐6 and the anti‐inflammatory cytokine IL‐10. This costimulatory effect is achieved primarily through BAFF‐R as BAFF failed to costimulate B‐1 cells obtained from A/WySnJ mice which have defective BAFF‐R signaling. Thus, as TLR are innate immune receptors and B‐1 cells are innate‐like lymphocytes, our data provide evidence that BAFF plays a role in innate immunity.

The RNase III enzyme Dicer is essential for germinal center B-cell formation.

MicroRNAs (miRNAs) are short noncoding RNAs that regulate gene expression and are important for pre-B and follicular B lymphopoiesis as demonstrated, respectively, by mb-1-Cre- and cd19-Cre-mediated deletion of Dicer, the RNase III enzyme critical for generating mature miRNAs. To explore the role of miRNAs in B-cell terminal differentiation, we use Aicda-Cre to specifically delete Dicer in activated B cells where activation-induced cytidine deaminase is highly expressed. We demonstrate that mutant mice fail to produce high-affinity class-switched antibodies and generate memory B and long-lived plasma cells on immunization with a T cell-dependent antigen. More importantly, germinal center (GC) B-cell formation is drastically compromised in the absence of Dicer, as a result of defects in cell proliferation and survival. Dicer-deficient GC B cells express higher levels of cell cycle inhibitor genes and proapoptotic protein Bim. Ablation of Bim could partially rescue the defect in GC B-cell formation in Dicer-deficient mice. Taken together, our data suggest that Dicer and probably miRNAs are critical for GC B-cell formation during B-cell terminal differentiation.

Cbp Deficiency Alters Csk Localization in Lipid Rafts but Does Not Affect T-Cell Development

ABSTRACT The ubiquitously expressed transmembrane adaptor Csk-binding protein (Cbp) recruits Csk to lipid rafts, where the latter exerts its negative regulatory effect on the Src family of protein tyrosine kinases. We have inactivated Cbp in the mouse germ line. In contrast to Csk gene inactivation, which leads to embryonic lethality and impaired T-cell development, Cbp-deficient mice were viable and exhibited normal T-cell development but with an increased thymocyte population. In the absence of Cbp, the amount of Csk that localizes to the lipid rafts was greatly reduced. Interestingly, this altered lipid raft localization of Csk did not lead to any detectable biochemical or functional defect in T cells. The T-cell receptor-induced intracellular calcium flux, cell proliferation, and cytokine secretion were not affected by the absence of Cbp. Peripheral T-cell tolerance to superantigen SEB was also largely intact in Cbp-deficient mice. Thus, Cbp is dispensable for T-cell development and activation.

Activated dectin-1 localizes to lipid raft microdomains for signaling and activation of phagocytosis and cytokine production in dendritic cells.

Lipid rafts are plasma membrane microdomains that are enriched in cholesterol, glycosphingolipids, and glycosylphosphatidylinositol-anchored proteins and play an important role in the signaling of ITAM-bearing lymphocyte antigen receptors. Dectin-1 is a C-type lectin receptor (CLR) that recognizes β-glucan in the cell walls of fungi and triggers signal transduction via its cytoplasmic hemi-ITAM. However, it is not known if similar to antigen receptors, Dectin-1 would also signal via lipid rafts and if the integrity of lipid raft microdomains is important for the physiological functions mediated by Dectin-1. We demonstrate here using sucrose gradient ultracentrifugation and confocal microscopy that Dectin-1 translocates to lipid rafts upon stimulation of dendritic cells (DCs) with the yeast derivative zymosan or β-glucan. In addition, two key signaling molecules, Syk and PLCγ2 are also recruited to lipid rafts upon the activation of Dectin-1, suggesting that lipid raft microdomains facilitate Dectin-1 signaling. Disruption of lipid raft integrity with the synthetic drug, methyl-β-cyclodextrin (βmD) leads to reduced intracellular Ca2+ flux and defective Syk and ERK phosphorylation in Dectin-1-activated DCs. Furthermore, βmD-treated DCs have significantly attenuated production of IL-2, IL-10, and TNFα upon Dectin-1 engagement, and they also exhibit impaired phagocytosis of zymosan particles. Taken together, the data indicate that Dectin-1 and perhaps also other CLRs are recruited to lipid rafts upon activation and that the integrity of lipid rafts is important for the signaling and cellular functions initiated by this class of innate receptors.

Fas apoptosis inhibitory molecule regulates T cell receptor-mediated apoptosis of thymocytes by modulating Akt activation and Nur77 expression.

Fas apoptosis inhibitory molecule (FAIM) has been demonstrated to confer resistance to Fas-induced apoptosis of lymphocytes and hepatocytes in vitro and in vivo. Here, we show that FAIM is up-regulated in thymocytes upon T cell receptor (TCR) engagement and that faim−/− thymocytes are highly susceptible to TCR-mediated apoptosis with increased activation of caspase-8 and -9. Furthermore, injection of anti-CD3 antibodies leads to augmented depletion of CD4+CD8+ T cells in the thymus of faim−/− mice compared with wild-type control, suggesting that FAIM plays a role in thymocyte apoptosis. Cross-linking of the TCR on faim−/− thymocytes leads to an elevated protein level of the orphan nuclear receptor Nur77, which plays a role in thymocyte apoptosis. Interestingly, in the absence of FAIM, there are reduced ubiquitination and degradation of the Nur77 protein. Faim−/− thymocytes also exhibit a defective TCR-induced activation of Akt whose activity we now show is required for Nur77 ubiquitination. Further analyses utilizing FAIM-deficient primary thymocytes and FAIM-overexpressing DO-11.10 T cells indicate that FAIM acts upstream of Akt during TCR signaling and influences the localization of Akt to lipid rafts, hence affecting its activation. Taken together, our study defined a TCR-induced FAIM/Akt/Nur77 signaling axis that is critical for modulating the apoptosis of developing thymocytes.

Mir-17-92 regulates bone marrow homing of plasma cells and production of immunoglobulin G2c.

The polycistronic mir-17-92 cluster, also known as oncomir-1, was previously shown to be essential for early B lymphopoiesis. However, its role in late-stage B-cell differentiation and function remains unexplored. Here we ablate mir-17-92 in mature B cells and demonstrate that mir-17-92 is dispensable for conventional B-cell development in the periphery. Interestingly, mir-17-92-deficiency in B cells leads to enhanced homing of plasma cells to the bone marrow during T-cell-dependent immune response and selectively impairs IgG2c production. Mechanistically, mir-17-92 directly represses the expression of Sphingosine 1-phosphate receptor 1 and transcription factor IKAROS, which are, respectively, important for plasma cell homing and IgG2c production. We further show that deletion of mir-17-92 could reduce IgG2c anti-DNA autoantibody production and hence mitigate immune complex glomerulonephritis in Shp1-deficient mice prone to autoimmunity. Our results identify important roles for mir-17-92 in the regulation of peripheral B-cell function.

Phospholipase Cγ2 Dosage Is Critical for B Cell Development in the Absence of Adaptor Protein BLNK

B cell linker (BLNK) protein and phospholipase Cγ2 (PLCγ2) are components of the BCR signalosome that activate calcium signaling in B cells. Mice lacking either molecule have a severe but incomplete block in B lymphopoiesis. In this study, we generated BLNK−/−PLCγ2−/− mice to examine the effect of simultaneous disruption of both molecules on B cell development. We showed that BLNK−/−PLCγ2−/− mice had compounded defects in B cell maturation compared with either single mutant, suggesting that these two molecules cooperatively or synergistically signaled B lymphopoiesis. However, Ig H chain allelic exclusion was maintained in single and double mutants, indicating that signals propagated by BLNK and PLCγ2 were not involved in this process. Interestingly, in the absence of BLNK, B cell development was dependent on plcγ2 gene dosage. This was evidenced by the proportionate decrease in splenic B cell population and increase in bone marrow surface pre-BCR+ cells in PLCγ2-diploid, -haploid, and -null animals. Intracellular calcium signaling and ERK activation in response to BCR engagement were also proportionately decreased and delayed, respectively, with stepwise reduction of plcγ2 dosage in a BLNKnull background. Thus, these data indicate the importance of BLNK not only as a conduit to specifically channel BCR-signaling pathways and as a scaffold for the assembling of macromolecular complex, but also as an efficient aggregator or concentrator of PLCγ2 molecules to effect optimal signaling for B cell generation and activation.

Loss of Fas apoptosis inhibitory molecule leads to spontaneous obesity and hepatosteatosis

Altered hepatic lipogenesis is associated with metabolic diseases such as obesity and hepatosteatosis. Insulin resistance and compensatory hyperinsulinaemia are key drivers of these metabolic imbalances. Fas apoptosis inhibitory molecule (FAIM), a ubiquitously expressed antiapoptotic protein, functions as a mediator of Akt signalling. Since Akt acts at a nodal point in insulin signalling, we hypothesize that FAIM may be involved in energy metabolism. In the current study, C57BL/6 wild-type (WT) and FAIM-knockout (FAIM-KO) male mice were fed with normal chow diet and body weight changes were monitored. Energy expenditure, substrate utilization and physical activities were analysed using a metabolic cage. Liver, pancreas and adipose tissue were subjected to histological examination. Serum glucose and insulin levels and lipid profiles were determined by biochemical assays. Changes in components of the insulin signalling pathway in FAIM-KO mice were examined by immunoblots. We found that FAIM-KO mice developed spontaneous non-hyperphagic obesity accompanied by hepatosteatosis, adipocyte hypertrophy, dyslipidaemia, hyperglycaemia and hyperinsulinaemia. In FAIM-KO liver, lipogenesis was elevated as indicated by increased fatty acid synthesis and SREBP-1 and SREBP-2 activation. Notably, protein expression of insulin receptor beta was markedly reduced in insulin target organs of FAIM-KO mice. Akt phosphorylation was also lower in FAIM-KO liver and adipose tissue as compared with WT controls. In addition, phosphorylation of insulin receptor substrate-1 and Akt2 in response to insulin treatment in isolated FAIM-KO hepatocytes was also markedly attenuated. Altogether, our data indicate that FAIM is a novel regulator of insulin signalling and plays an essential role in energy homoeostasis. These findings may shed light on the pathogenesis of obesity and hepatosteatosis.