Caspase Recruitment Domain Family Member 9 Research Articles

Abstract 15726: Large-Scale Plasma Proteomics and Mendelian Randomization of Cardiovascular Performance and Peak Arterio-Venous Oxygen Difference Identify Novel Biology Related to VO 2 max

Introduction: VO 2 max is the product of maximal cardiac output (Q) (maximal stroke volume (SVmax) x heart rate) and peak arterio-venous oxygen content difference (a-vO 2 diff). SVmax and peak a-vO 2 diff have important implications across the spectrum of human health and performance. However, the molecular mechanisms underlying SVmax and peak a-vO 2 diff remain largely unknown. We characterize SVmax and peak a-vO 2 diff using large-scale plasma proteomics and genotyping to identify circulating proteins and putative mediators of these traits. Methods: 763 physically inactive adults without overt cardiovascular disease underwent cardiopulmonary exercise testing with rigorous measurements of Q. High-throughput plasma proteomic profiling was performed to identify proteins associated with SVmax, a-vO 2 diff, and VO 2 max. cis-Mendelian Randomization (MR) analysis was conducted to assess causal relationships between protein markers and cardiovascular performance traits. Results: Proteomic profiling revealed associations between SVmax and several adiposity-related proteins, as well as secretory muscle factors (cathepsin B, α-actinin 2, and troponin I) (Figure). Proteins associated with peak a-vO2diff reflected oxygen-carrying capacity, muscle biology, and glucose metabolism (erythropoietin, myoglobin, fructose-1,6-bisphosphatase). Significant overlap was observed between proteins associated with SVmax, a-vO 2 diff, and VO 2 max. Cis-MR identified myocilin (MYOC) and caspase recruitment domain family member 9 (CARD9) as potential mediators of cardiovascular performance and VO 2 max. Conclusions: This study provides novel insights into the molecular influences of cardiovascular performance and VO 2 max. The identified protein markers associated with SVmax and a-vO 2 diff, along with the possible mediators MYOC and CARD9, merit further investigation into their role in endurance exercise capacity and cardiometabolic health.

Macrophage CARD9 mediates cardiac injury following myocardial infarction through regulation of lipocalin 2 expression

Immune cell infiltration in response to myocyte death regulates extracellular matrix remodeling and scar formation after myocardial infarction (MI). Caspase-recruitment domain family member 9 (CARD9) acts as an adapter that mediates the transduction of pro-inflammatory signaling cascades in innate immunity; however, its role in cardiac injury and repair post-MI remains unclear. We found that Card9 was one of the most upregulated Card genes in the ischemic myocardium of mice. CARD9 expression increased considerably 1 day post-MI and declined by day 7 post-MI. Moreover, CARD9 was mainly expressed in F4/80-positive macrophages. Card9 knockout (KO) led to left ventricular function improvement and infarct scar size reduction in mice 28 days post-MI. Additionally, Card9 KO suppressed cardiomyocyte apoptosis in the border region and attenuated matrix metalloproteinase (MMP) expression. RNA sequencing revealed that Card9 KO significantly suppressed lipocalin 2 (Lcn2) expression post-MI. Both LCN2 and the receptor solute carrier family 22 member 17 (SL22A17) were detected in macrophages. Subsequently, we demonstrated that Card9 overexpression increased LCN2 expression, while Card9 KO inhibited necrotic cell-induced LCN2 upregulation in macrophages, likely through NF-κB. Lcn2 KO showed beneficial effects post-MI, and recombinant LCN2 diminished the protective effects of Card9 KO in vivo. Lcn2 KO reduced MMP9 post-MI, and Lcn2 overexpression increased Mmp9 expression in macrophages. Slc22a17 knockdown in macrophages reduced MMP9 release with recombinant LCN2 treatment. In conclusion, our results demonstrate that macrophage CARD9 mediates the deterioration of cardiac function and adverse remodeling post-MI via LCN2.

CARD9 deficiency aggravated nonalcoholic steatohepatitis in mice through increasing inflammatory response

Nonalcoholic steatohepatitis (NASH), a subtype of nonalcoholic fatty liver disease (NAFLD), is the leading cause of liver-related morbidity worldwide. Caspase recruitment domain family member 9 (CARD9), a myeloid cell-specific signaling protein, belongs to the CARD protein family. However, its role in NASH is unknown. Therefore, this study aimed to investigate the role of CARD9 in the development of NASH. NASH models were established using CARD9-knockout and wild-type mice. They were either fed a methionine/choline deficient (MCD) diet for 6 weeks or a high-fat high-cholesterol (HFHC) diet for 16 weeks. Liver fibrosis model was also developed using CCl4. CARD9 deficiency accelerated steatohepatitis development in MCD or HFHC diet-fed mice, accompanied by an upregulation of fibrosis, adipogenesis, and proinflammatory genes. CARD9 deficiency was found to exacerbate CCl4-induced liver fibrosis. In vitro studies demonstrated that CARD9 deficiency induced the expression of S100a8/a9 through Toll-like receptor in Kupffer cells treated with palmitate. This led to an increased expression of proinflammatory, fibrosis, and lipid metabolism-related genes in NASH progression. These results highlight the role of CARD9 in the development of NASH and provide new insights into the therapeutic strategies for NASH.

Heterozygous CARD9 mutation favors the development of allergic bronchopulmonary aspergillosis.

Previous research demonstrated that a homozygous mutation of g.136372044G>A (S12N) in caspase recruitment domain family member 9 ( CARD9 ) is critical for producing Aspergillus fumigatus -induced ( Af -induced) T helper 2 (T H 2)-mediated responses in allergic bronchopulmonary aspergillosis (ABPA). However, it remains unclear whether the CARD9S12N mutation, especially the heterozygous occurrence, predisposes the host to ABPA. A total of 61 ABPA patients and 264 controls (including 156 healthy controls and 108 asthma patients) were recruited for sequencing the CARD9 locus to clarify whether patients with this heterozygous single-nucleotide polymorphisms are predisposed to the development of ABPA. A series of in vivo and in vitro experiments, such as quantitative real-time polymerase chain reaction, flow cytometry, and RNA isolation and quantification, were used to illuminate the involved mechanism of the disease. The presence of the p.S12N mutation was associated with a significant risk of ABPA in ABPA patients when compared with healthy controls and asthma patients, regardless of Aspergillus sensitivity. Relative to healthy controls without relevant allergies, the mutation of p.S12N was associated with a significant risk of ABPA (OR: 2.69 and 4.17 for GA and AA genotypes, P = 0.003 and 0.029, respectively). Compared with patients with asthma, ABPA patients had a significantly higher heterozygous mutation (GA genotype), indicating that p.S12N might be a significant ABPA-susceptibility locus ( aspergillus sensitized asthma: OR: 3.02, P = 0.009; aspergillus unsensitized asthma: OR: 2.94, P = 0.005). The mutant allele was preferentially expressed in ABPA patients with heterozygous CARD9S12N , which contributes to its functional alterations to facilitate Af -induced T H 2-mediated ABPA development. In terms of mechanism, Card9 wild-type ( Card9WT ) expression levels decreased significantly due to Af -induced decay of its messenger RNA compared to the heterozygous Card9S12N . In addition, ABPA patients with heterozygous CARD9S12N had increased Af -induced interleukin-5 production. Our study provides the genetic evidence showing that the heterozygous mutation of CARD9S12N , followed by allele expression imbalance of CARD9S12N , facilitates the development of ABPA.

Dimethyl Fumarate Suppresses the Proliferation of HTLV-1-infected T Cells by Inhibiting CBM Complex-triggered NF-B Signaling.

Adult T-cell leukemia (ATL) is a peripheral T-lymphocytic malignancy influenced by human T-cell leukemia virus type 1 (HTLV-1) infection. Aggressive ATL has a poor prognosis, therefore newer agents are desperately needed. We revealed that dimethyl fumarate (DMF) causes ATL cell death via inhibition of nuclear factor-kappa B (NF-B) and signal transducer and activator of transcription 3 signaling. Here, we evaluated the specific mechanism of DMF effects on NF-B signaling in MT-2 HTLV-1-infected T-cells. We examined the effects of DMF on the caspase recruitment domain family member 11 (CARD11)-BCL10 immune signaling adaptor (BCL10)-mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) (CBM) complex and upstream signaling molecules which are critical for NF-B signaling in MT-2 cells by immunoblotting. We also explored its effects on cell-cycle distribution. Furthermore, we assessed whether the BCL2 apoptosis regulator (BCL2)/BCL2-like 1 (BCL-xL) inhibitor navitoclax promoted the inhibitory effect of DMF on cell proliferation and apoptosis-associated proteins by trypan blue exclusion test and immunoblotting, respectively. DMF inhibited constitutive phosphorylation of CARD11 followed by suppression of inhibitory-B kinase α/β phosphorylation at serine in a dose-dependent fashion in MT-2 cells. Furthermore, DMF inhibited MALT1 and BCL10 expression in the same fashion. However, DMF did not prevent the phosphorylation of protein kinase C-β, an upstream signaling molecule of CARD11. Cell-cycle analysis highlighted that DMF treatment at 75 μM resulted in the accumulation of cells at the sub-G1 and G2/M phases. Navitoclax modestly promoted DMF-induced suppression of MT-2 cells via inhibition of cellular inhibitor of apoptosis protein-2 expression and c-JUN N-terminal kinase phosphorylation. The suppression of MT-2 cell proliferation by DMF makes its further evaluation as an innovative agent for therapy of ATL worthwhile.

Genetic association of PRKCD and CARD9 polymorphisms with Vogt–Koyanagi–Harada disease in the Chinese Han population

BackgroundProtein kinase C delta (PRKCD) and caspase recruitment domain family member 9 (CARD9) are genes involved in B and T cell activation, and cytokine production, which are vital mechanisms underlying autoimmune disease development. This study aimed to explore the association of the PRKCD and CARD9 genes with Vogt–Koyanagi–Harada disease (VKH) disease. The case–control study was performed to in 912 patients with VKH and 878 normal controls. MassARRAY system, SHEsis online platform, real-time PCR, and enzyme-linked immunosorbent assay were used to detect genotyping, haplotyping, mRNA expression, and cytokine levels, respectively.ResultsWe found that rs74437127 C allele of PRKCD, rs3812555 CC genotype, and C allele of CARD9 were associated with increased susceptibility of VKH (Pc = 0.020, OR = 1.624; Pc = 2.04 × 10–5, OR = 1.810; Pc = 2.76 × 10–5, OR = 1.698, respectively). However, the rs74437127 T allele, and rs3812555 TC genotype and T allele were linked with decreased susceptibility to VKH (Pc = 0.020, OR = 0.616; Pc = 7.85 × 10–5, OR = 0.559; Pc = 2.76 × 10–5, OR = 0.589, respectively). PRKCD ATG and CARD9 GCTTA haplotypes decreased susceptibility to VKH (Pc = 3.11 × 10–3, OR = 0.594; Pc = 5.00 × 10–3, OR = 0.639, respectively). Functional studies on rs3812555 genotyped individuals revealed that CC carriers had significantly higher CARD9 mRNA expression and tumour necrosis factor-α production than TC/TT carriers (P = 1.00 × 10–4; P = 2.00 × 10–3, respectively).ConclusionsWe found an association between PRKCD rs74437127 and CARD9 rs3812555 polymorphisms and VKH susceptibility and revealed that the increased susceptibility of rs3812555 for VKH may be mediated by regulating CARD9 gene expression and the production of pro-inflammatory cytokines, such as TNF-α.

Polymorphisms of CARD9 Gene Predict Disease Progression and Renal Survival of Immunoglobulin A Nephropathy

Introduction: A previous genome-wide association study has identified CARD9 (caspase recruitment domain family member 9) as a susceptibility gene for immunoglobulin A nephropathy (IgAN), which encodes an adapter protein and is related to mucosal immunity. This study aimed to investigate the association of CARD9 variants with the clinicopathological phenotypes and prognosis of IgAN. Methods: Eight single nucleotide polymorphisms within CARD9 were genotyped using Sequenom MassARRAY iPLEX for 986 IgAN patients in this study. Logistic and linear regression analyses adjusted for age and gender were performed to evaluate the effects of CARD9 gene polymorphisms on clinicopathological phenotypes. The Kaplan-Meier method and Cox proportional hazard models were applied to analyze the associations between genetic variants and renal survival. Results: The T allele of rs10747047 was strongly associated with higher levels of serum creatinine (p = 0.005) and lower levels of estimated glomerular filtration rate (p = 0.005). The rs10870149-G and rs10870077-C alleles were associated with elevated 24-h urine protein excretion (p = 0.041 and 0.022, respectively) and more serious segmental glomerulosclerosis lesions (p = 0.005 and 0.041, respectively) in IgAN patients. Carriers with the T allele of rs10781533 and the C allele of rs3812552 also presented with severe segmental glomerulosclerosis lesions (p = 0.001 and 0.010, respectively). Additionally, rs10747047-C and rs10870077-C alleles were independently related to the poor prognosis of IgAN patients after adjustments for covariates (TT vs. CC hazard ratio [HR] = 0.138, 95% confidence interval [95% CI] = 0.022–0.871, p = 0.035; GG vs. CC HR = 0.321, 95% CI = 0.123, 0.836, p = 0.020, respectively). Conclusion: CARD9 variants are associated with disease severity and rapid disease progression for IgAN in a Chinese Han population.

A Critical Role for CARD9 in Intestinal Microbiota Modulation and Colorectal Malignancies

The adaptor protein Caspase Recruitment Domain Family Member 9 (CARD9) plays an indispensable role in innate immunity. Recent studies indicate that dysregulated CARD9 is a critical risk factor in the progression of colorectal cancer (CRC). This review provides novel insights into the functions of CARD9 in CRC, particularly in delineating its role in disrupting the host microbe balance, fueling gut microbiota metabolism and inducing systemic immunoglobulin G (IgG) antifungal antibodies. These pathways provide important information that can potentially be used for therapeutic innovation in developing potential vaccines for CRC.

CARD9 contributes to ovarian cancer cell proliferation, cycle arrest, and cisplatin sensitivity

BackgroundOvarian cancer recurrence and chemotherapy resistance are still urgent issues, and exploring the mechanisms of metastasis and chemotherapy resistance is beneficial to the development of therapeutic methods. Caspase recruitment domain family member 9 (CARD9) and homeobox B5 (HOXB5) are related and both are upregulated in ovarian cancer. This study aimed to define their functions in ovarian cancer cell proliferation, migration, and cisplatin sensitivity.ResultsThe levels of CARD9 were detected in acquired ovarian cancer tissues and cell lines. CARD9 was indeed abnormally upregulated in them. CARD9 knockdown significantly suppressed cell proliferation, colony formation, migration, cycle arrest, and cisplatin sensitivity. HOXB5 bound to the CARD9 promoter, and HOXB5 overexpression reversed the regulation by CARD9 knockdown in cells, as well as the activation of NF-κB signaling. This indicated that CARD9 was positively regulated by HOXB5 in ovarian cancer cells.ConclusionTogether, CARD9 is involved in ovarian cancer cell proliferation, migration, and cisplatin sensitivity via NF-κB signaling after transcriptional activation by HOXB5.

Identification of key genes for hypertrophic cardiomyopathy using integrated network analysis of differential lncRNA and gene expression.

ObjectiveHypertrophic cardiomyopathy (HCM) is a complex heterogeneous heart disease. Recent reports found that long non-coding RNAs (lncRNAs) play an important role in the progression of cardiovascular diseases. The present study aimed to identify the novel lncRNAs and messenger RNAs (mRNAs) and determine the key pathways involved in HCM.MethodsThe lncRNA and mRNA sequencing datasets of GSE68316 and GSE130036 were downloaded from the Gene Expression Omnibus (GEO) database. An integrated co-expression network analysis was conducted to identify differentially expressed lncRNAs and differentially expressed mRNAs in patients with HCM. Then, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were explored to identify the biological functions and signaling pathways of the co-expression network. Protein–protein interaction (PPI) and hub gene networks were constructed by using Cytoscape software. Plasma samples of patients with HCM and the GSE89714 dataset were used to validate the bioinformatics results.ResultsA total of 1,426 differentially expressed long non-coding RNAs (lncRNAs) and 1,715 differentially expressed mRNAs were obtained from GSE68316, of which 965 lncRNAs and 896 mRNAs were upregulated and 461 lncRNAs and 819 mRNAs were downregulated. A total of 469 differentially expressed lncRNAs and 2,407 differentially expressed mRNAs were screened from GSE130036, of which 183 lncRNAs and 1,283 mRNAs were upregulated and 286 lncRNAs and 1,124 mRNAs were downregulated. A co-expression network was constructed and contained 30 differentially expressed lncRNAs and 63 differentially expressed mRNAs, which were primarily involved in ‘G-protein beta/gamma-subunit complex binding,' ‘polyubiquitin modification-dependent protein binding,' ‘Apelin signaling pathway,' and ‘Wnt signaling pathway.' The 10 hub genes in the upregulated network [G Protein Subunit Alpha I2 (GNAI2), G Protein Subunit Alpha I1 (GNAI1), G Protein Subunit Alpha I3 (GNAI3), G Protein Subunit Gamma 2 (GNG2), G Protein Subunit Beta 1 (GNB1), G Protein Subunit Gamma 13 (GNG13), G Protein Subunit Gamma Transducin 1 (GNGT1), G Protein Subunit Gamma 12 (GNG12), AKT Serine/Threonine Kinase 1 (AKT1) and GNAS Complex Locus (GNAS)] and the 10 hub genes in the downregulated network [Nucleotide-Binding Oligomerization Domain Containing Protein 2 (NOD2), Receptor-Interacting Serine/Threonine Kinase 2 (RIPK2), Nucleotide-Binding Oligomerization Domain Containing Protein 1 (NOD1), Mitochondrial Antiviral Signaling Protein (MAVS), Autophagy Related 16-Like 1 (ATG16L1), Interferon Induced With Helicase C Domain 1 (IFIH1), Autophagy Related 5 (ATG5), TANK-Binding Kinase 1 (TBK1), Caspase Recruitment Domain Family Member 9 (CARD9), and von Willebrand factor (VWF)] were screened using cytoHubba. The expression of LA16c-312E8.2 and RP5-1160K1.3 in the plasma of patients with HCM was elevated, and the expression of the MIR22 host gene (MIR22HG) was decreased, which was consistent with our analysis, while the expression of LINC00324 and Small Nucleolar RNA Host Gene 12 (SNHG12) was not significantly different between the two groups. Verification analyses performed on GSE89714 showed the upregulated mRNAs of Chloride Voltage-Gated Channel 7 (CLCN7), N-Acetylglucosamine-1-Phosphate Transferase Subunit Gamma (GNPTG), Unk Like Zinc Finger (UNKL), Adenosine Monophosphate Deaminase 2 (AMPD2), GNAI3, WD Repeat Domain 81 (WDR81), and Serpin Family F Member 1 (SERPINF1) and downregulated mRNAs of TATA-Box Binding Protein Associated Factor 12 (TAF12) co-expressed with five crucial lncRNAs. Moreover, GNAI2, GNAI3, GNG12, and vWF were upregulated and GNAS was downregulated in the top 10 hub genes of upregulated and downregulated PPI networks.ConclusionThese findings from integrative biological analysis of lncRNA-mRNA co-expression networks explored the key genes and pathways and provide new insights into the understanding of the mechanism and discovering new therapeutic targets for HCM. Three differentially expressed pivotal lncRNAs (LA16c-312E8.2, RP5-1160K1.3, and MIR22HG) in the co-expression network may serve as biomarkers and intervention targets for the diagnosis and treatment of HCM.

Epidermal clearance of Candida albicans is mediated by IL-17 but independent of fungal innate immune receptors.

IL-17 plays important roles in host defense against Candida albicans at barrier surfaces and during invasive infection. However, the role of IL-17 in host defense after colonization of the epidermis, a main site of C. albicans infection, remains poorly understood. Using a murine model of epicutaneous candidiasis without skin abrasion, we found that skin inflammation triggered by epidermal C. albicans colonization was self-limiting with fungal clearance completed by day 7 after inoculation in wild-type mice or animals deficient in IL-17A or IL-17F. In contrast, marked neutrophilic inflammation in the epidermis and impaired fungal clearance were observed in mice lacking both IL-17A and IL-17F. Clearance of C. albicans was independent of Dectin-1, Dectin-2, CARD9 (caspase-recruitment domain family, member 9), TLR2 (Toll-like receptor 2) and MyD88 in the epidermal colonization model. We found that group 3 innate lymphoid cells (ILC3s) and γδT cells were the major IL-17 producers in the epicutaneous candidiasis model. Analyses of Rag2-/- mice and Rag2-/-Il2rg-/- mice revealed that production of IL-17A and IL-17F by ILC3s was sufficient for C. albicans clearance. Finally, we found that depletion of neutrophils impaired C. albicans clearance in the epidermal colonization model. Taken together, these findings indicate a critical and redundant function of IL-17A and IL-17F produced by ILC3s in host defense against C. albicans in the epidermis. The results also suggest that epidermal C. albicans clearance is independent of innate immune receptors or that these receptors act redundantly in fungal recognition and clearance.

TREM (Triggering Receptor Expressed on Myeloid Cells)-1 Inhibition Attenuates Neuroinflammation via PKC (Protein Kinase C) δ/CARD9 (Caspase Recruitment Domain Family Member 9) Signaling Pathway After Intracerebral Hemorrhage in Mice.

Intracerebral hemorrhage (ICH) is a devastating subtype of stroke with high mortality and disability. Inflammatory response promotes secondary brain injury after ICH. TREM (triggering receptor expressed on myeloid cells)-1 is a key regulator of inflammation. The aim of this study was to evaluate the role of TREM-1 in neuroinflammatory response after ICH in mice. CD1 mice (n=275) were used in this study. Mice were subjected to ICH by autologous blood injection. TREM-1 knockout CRISPR was administered intracerebroventricularly to evaluate the role of TREM-1 after ICH. A selective TREM-1 inhibitor, LP17, was administered intranasally 2 hours after ICH. To elucidate TREM-1 signaling pathway, CARD9 (caspase recruitment domain family member 9) activation CRISPR was administered with LP17 and TREM-1 activating anti-mouse TREM-1 monoclonal antibody (mAb) was administered with Rottlerin, a specific PKC (protein kinase C) δ inhibitor. Lastly, to evaluate the role of HMGB1 (high-mobility group box 1) in TREM-1 mediated microglia activation, glycyrrhizin, an inhibitor of HMBG1 was administered with TREM-1 activating mAb. Neurobehavioral test, brain water content, Western blot, immunofluorescence staining, and coimmunoprecipitation was performed. TREM-1 knockout reduced ICH-induced neurobehavioral deficits and neuroinflammatory response. The temporal expression of HMGB1, TREM-1, PKC δ, and CARD9 increased after ICH. TREM-1 was expressed on microglia. Intranasal administration of LP17 significantly decreased brain edema and improved neurobehavioral outcomes at 24 and 72 hours after ICH. LP17 promoted M2 microglia polarization and reduced proinflammatory cytokines after ICH, which was reversed with CARD9 activation CRISPR. TREM-1 mAb increased neurobehavior deficits, proinflammatory cytokines, and reduced M2 microglia after ICH, which was reversed with Rottlerin. HMBG1 interaction with TREM-1 increased after ICH, and glycyrrhizin reduced neuroinflammation and promoted M2 microglia which was reversed with TREM-1 mAb. This study demonstrated that TREM-1 enhanced neuroinflammation by modulating microglia polarization after ICH, and this regulation was partly mediated via PKC δ/CARD9 signaling pathway and increased HMGB1 activation of TREM-1.

Mechanistic understanding of the combined immunodeficiency in complete human CARD11 deficiency

Germline pathogenic variants impairing the caspase recruitment domain family member 11 (CARD11)-B cell chronic lymphocytic leukemia/lymphoma 10 (BCL10)-MALT1 paracaspase (MALT1) (CBM) complex are associated with diverse human diseases including combined immunodeficiency (CID), atopy, and lymphoproliferation. However, the impact of CARD11 deficiency on human B-cell development, signaling, and function is incompletely understood. This study sought to determine the cellular, immunological, and biochemical basis of disease for 2 unrelated patients who presented with profound CID associated with viral and fungal respiratory infections, interstitial lung disease, and severe colitis. Patients underwent next-generation sequencing, immunophenotyping by flow cytometry, signaling assays by immunoblot, and transcriptome profiling by RNA-sequencing. Both patients carried identical novel pathogenic biallelic loss-of-function variants in CARD11 (c.2509C>T; p.Arg837∗) leading to undetectable protein expression. This variant prevented CBM complex formation, severely impairing the activation of nuclear factor-κB, c-Jun N-terminal kinase, and MALT1 paracaspase activity in B and T cells. This functional defect resulted in a developmental block in B cells at the naive and type 1 transitional B-cell stage and impaired circulating T follicular helper cell (cTFH) development, which was associated with impaired antibody responses and absent germinal center structures on lymph node histology. Transcriptomics indicated that CARD11-dependent signaling is essential for immune signaling pathways involved in the development of these cells. Both patients underwent hematopoietic stem cell transplantations, which led to functional normalization. Complete human CARD11 deficiency causes profound CID by impairing naive/type 1 B-cell and cTFH cell development and abolishing activation of MALT1 paracaspase, NF-κB, and JNK activity. Hematopoietic stem cell transplantation functionally restores impaired signaling pathways.

TREM2 is a receptor for non-glycosylated mycolic acids of mycobacteria that limits anti-mycobacterial macrophage activation

Mycobacterial cell-wall glycolipids elicit an anti-mycobacterial immune response via FcRγ-associated C-type lectin receptors, including Mincle, and caspase-recruitment domain family member 9 (CARD9). Additionally, mycobacteria harbor immuno-evasive cell-wall lipids associated with virulence and latency; however, a mechanism of action is unclear. Here, we show that the DAP12-associated triggering receptor expressed on myeloid cells 2 (TREM2) recognizes mycobacterial cell-wall mycolic acid (MA)-containing lipids and suggest a mechanism by which mycobacteria control host immunity via TREM2. Macrophages respond to glycosylated MA-containing lipids in a Mincle/FcRγ/CARD9-dependent manner to produce inflammatory cytokines and recruit inducible nitric oxide synthase (iNOS)-positive mycobactericidal macrophages. Conversely, macrophages respond to non-glycosylated MAs in a TREM2/DAP12-dependent but CARD9-independent manner to recruit iNOS-negative mycobacterium-permissive macrophages. Furthermore, TREM2 deletion enhances Mincle-induced macrophage activation in vitro and inflammation in vivo and accelerates the elimination of mycobacterial infection, suggesting that TREM2-DAP12 signaling counteracts Mincle-FcRγ-CARD9-mediated anti-mycobacterial immunity. Mycobacteria, therefore, harness TREM2 for immune evasion.

A potential role of caspase recruitment domain family member 9 (Card9) in transverse aortic constriction-induced cardiac dysfunction, fibrosis, and hypertrophy.

Macrophage- and monocyte-derived cytokines are elevated in the myocardium of pressure-overloaded hearts, where they play critical roles in pathological remodeling. Caspase recruitment domain family member 9 (CARD9) regulates macrophage cytokine secretion, but its role in a transverse aortic constriction (TAC) model of pressure overload has not been evaluated. To investigate whether CARD9 may serve as a valuable therapeutic target, wild-type (WT) and CARD9-knockout mice were subjected to 3 months of TAC, and then cardiac function, hypertrophy, and fibrosis were analyzed. The expression of protein markers of myocardial autophagy and nuclear factor kappa B signaling was also investigated. At 1 month after TAC, cardiomyocyte contractile dynamics were measured in a separate cohort to further assess contractility and diastolic function. In WT but not CARD9-/- mice, TAC resulted in severe cardiomyocyte contractile dysfunction at 1 month and functional decrements in fractional shortening at 3 months in vivo. Furthermore, CARD9-/- mice did not develop cardiac fibrosis or hypertrophy. CARD9-/- mice also had decreased protein expression of inhibitor of κB kinase-α/β, decreased phosphorylation of p65, and increased expression of protein markers of autophagy. These findings suggest that CARD9 plays a role in pathological remodeling and cardiac dysfunction in mouse hearts subjected to TAC and should be investigated further.

Increase in Interleukin‐16, a Pro‐inflammatory Cytokine, and Other Distinct Markers of Lymphocytic Activation, in Kidney Tissue of Cats with Renal Dysfunction

Inflammation plays an important role in the initiation and subsequent progression of chronic kidney disease. An increase in inflammatory markers including cytokines as well as markers of oxidative stress is associated with reduced kidney function. Inflammatory markers and the underlying mechanisms that contribute to the pathophysiology of kidney disease are not clear and likely are multifactorial. Another feature associated with renal dysfunction and inflammatory response is the formation of calcium oxalate stones formed from the crystal deposits in the tubular epithelial cells. The aim of the present study was to investigate changes in gene expression in the renal cortex obtained from cats with kidney disease or calcium oxalate stone formers (CaOx) at necropsy in order to identify novel inflammatory biomarkers associated with renal dysfunction. At the time of death the circulating levels of creatinine as well as symmetric dimethyl arginine (SDMA), both markers of kidney decline in cats, were significantly higher in cats with renal disease (n=11) or stone‐forming cats (CaOx, n=12) when compared to controls (n=19). Using RNAseq in kidney tissue, we found a significant increase in the expression of IL‐16, a T‐cell chemoattractant, in both cats with kidney disease (6.72 fold) and stone formers (7.03 fold) compared to controls (both p<0.0001). There was also a significant increase in caspase recruitment domain family member 11 (CARD11), an activator of T and B‐lymphocytes and NFkB, in cats with kidney disease (9.2 fold) and stone formers (10 fold) compared to controls (p<0001). However, some differences between the kidney disease and CaOx groups were also observed. In cats with kidney disease there was an increased expression of LY9 (CD299), found in T and B‐cells and thymocytes, IL2Rg, a marker of lymphocyte activation, and SPP1 (Osteopontin), expressed in activated T and B‐cells (all >6‐fold) when compared to controls (p<0.0001) but such increased expression was not seen in CaOx cats. In summary, while IL‐16 was increased in both groups, our results indicate important molecular differences in the pathogenesis of kidney dysfunction in cats with kidney disease and stone formers, and that distinct lymphocyte markers may be involved in the persistent decline of kidney function. Our studies suggest that the optimal nutritional therapy to slow the progression of kidney dysfunction may be different for cats with kidney disease or stone formers.Support or Funding InformationThis study was funded by Hill’s Pet Nutrition, Inc

Caspase recruitment domain family member 9 expression is a promising biomarker in esophageal squamous cell carcinoma.

AimEsophageal squamous cell carcinoma (ESCC) is a refractory digestive organ cancer that requires better treatment strategies. We have recently reported that the antidiabetic drug metformin exerts antitumor effects on ESCC by inhibition of nuclear factor kappa B (NF‐κB) nuclear translocation. In the present study, we focused on caspase recruitment domain family member 9 (CARD9), an essential signal adapter in NF‐κB activation to examine whether it can be used as a prognostic factor in ESCC.MethodsWe investigated CARD9 expression immunohistochemically in clinical samples obtained from 93 patients with ESCC who underwent curative esophagectomy. CARD9 expression was analyzed for correlation with clinicopathological characteristics and ESCC prognosis. The molecular effects were investigated by knocking down ESCC cells. Comprehensive RNA expression changes in these ESCC cells were detected by next‐generation sequencing (NGS).ResultsHigh CARD9 expression is significantly correlated with advanced tumor depth (P < .001), positive lymph node metastasis (P = .005) and advanced stage (P = .001). Kaplan‐Meier method and the log‐rank test showed that overall survival (OS) and disease‐free survival (DFS) were significantly poor in the high CARD9 expression group (OS: P = .027, DFS: P = .005). Univariate and multivariate analysis showed that high CARD9 expression is a significant poor prognostic factor for DFS. Cell proliferation and migration were suppressed by CARD9 knockdown. NGS detected altered the expression of some RNAs including maternally expressed 3 (MEG3).ConclusionHigh CARD9 expression is significantly associated with poor prognosis. Therefore, CARD9 expression may be a prospective prognostic biomarker in ESCC.

Genome-Wide Association Study of H/L Traits in Chicken.

Simple SummaryWith the continuous increase of intensive agriculture, the poultry industry has developed rapidly. Concurrently, diseases such as avian influenza, salmonella, and Newcastle disease have brought huge losses to the poultry industry. The traditional method of disease prevention and treatment includes vaccinations, but these have been linked to concerns associated with expense and meat safety. To solve these problems, genetic breeding methods can be used. In this paper, a genome-wide association analysis was linked to heterophil/lymphocyte ratio disease-resistance traits as a means through which disease damage can be mitigated.Presently, the heterophil-to-lymphocyte (H/L) ratio is being studied extensively as a disease resistance trait. Through intricate mechanisms to identify and destroy pathogenic microorganisms, heterophils play a pivotal role in the immune defense systems of avian species. To reveal the genetic basis and molecular mechanisms affecting the H/L ratio, phenotypic and H/L data from 1650 white feather chicken broilers were used in performing a genome-wide association study. A self-developed, chicken-specific 55K chip was used for heterophils, lymphocytes, and H/L classification, according to individual genomic DNA profiles. We identified five significant single nucleotide polymorphisms (SNPs) when the genome-wide significance threshold was set to 5% (p < 2.42 × 10−6). A total of 15 SNPs obtained seemingly significant levels (p < 4.84 × 10−5). Gene annotation indicated that CARD11 (Caspase recruitment domain family member 11), BRIX1 (Biogenesis of ribosomes BRX1), and BANP (BTG3 associated nuclear protein) play a role in H/L-associated cell regulation and potentially constitute candidate gene regions for cellular functions dependent on H/L ratios. These results lay the foundation for revealing the genetic basis of disease resistance and future marker-assisted selection for disease resistance.

Deletion of hematopoietic Dectin-2 or CARD9 does not protect against atherosclerotic plaque formation in hyperlipidemic mice

Inflammatory reactions activated by pattern recognition receptors (PRRs) on the membrane of innate immune cells play an important role in atherosclerosis. Whether the PRRs of the C-type lectin receptor (CLR) family including Dectin-2 may be involved in the pathogenesis of atherosclerosis remains largely unknown. Recently, the CLR-adaptor molecule caspase recruitment domain family member 9 (CARD9) has been suggested to play a role in cardiovascular pathologies as it provides the link between CLR activation and transcription of inflammatory cytokines as well as immune cell recruitment. We therefore evaluated whether hematopoietic deletion of Dectin-2 or CARD9 reduces inflammation and atherosclerosis development. Low-density lipoprotein receptor (Ldlr)-knockout mice were transplanted with bone marrow from wild-type, Dectin-2- or Card9-knockout mice and fed a Western-type diet containing 0.1% (w/w) cholesterol. After 10 weeks, lipid and inflammatory parameters were measured and atherosclerosis development was determined. Deletion of hematopoietic Dectin-2 or CARD9 did not influence plasma triglyceride and cholesterol levels. Deletion of hematopoietic Dectin-2 did not affect atherosclerotic lesion area, immune cell composition, ex vivo cytokine secretion by peritoneal cells or bone marrow derived macrophages. Unexpectedly, deletion of hematopoietic CARD9 increased atherosclerotic lesion formation and lesion severity. Deletion of hematopoietic CARD9 did also not influence circulating immune cell composition and peripheral cytokine secretion. Besides a tendency to a reduced macrophage content within these lesions, plasma MCP-1 levels decreased upon WTD feeding. Deletion of hematopoietic Dectin-2 did not influence atherosclerosis development in hyperlipidemic mice. The absence of CARD9 unexpectedly increased atherosclerotic lesion size and severity, suggesting that the presence of CARD9 may protect against initiation of atherosclerosis development.

Immune Evasion of Mycobacteria Via TREM2&amp;nbsp;Through Induction of Permissive Macrophages And&amp;nbsp;Suppression Of&amp;nbsp;Mincle/CARD9-Induced Anti-Mycobacterial Immunity

Mycobacterial cell-wall glycolipids elicit an anti-mycobacterial immune response via FcRγ-associated C-type lectin receptors, including Mincle, and caspase-recruitment domain family member 9 (CARD9). Additionally, mycobacteria harbor immuno-evasive cell-wall lipids associated with virulence and latency; however, their mechanism of action remains unclear. Here, we show that the DAP12-associated triggering receptor expressed on myeloid cells 2 (TREM2) recognizes mycobacterial cell-wall mycolic acid (MA)-containing lipids and suggest a mechanism by which mycobacteria control host immunity via TREM2. Macrophages responded to glycosylated MA-containing lipids in a Mincle/FcRγ/CARD9-dependent manner to produce inflammatory cytokines and recruit inducible nitric oxide synthase (iNOS)-positive mycobactericidal macrophages. Conversely, macrophages responded to non-glycosylated MAs in a TREM2/DAP12-dependent but CARD9-independent manner to recruit iNOS-negative mycobacterium-permissive macrophages. Furthermore, TREM2 deletion enhanced Mincle-induced macrophage activation in vitro and inflammation in vivo and accelerated the elimination of mycobacterial infection, suggesting that TREM2/DAP12 signaling counteracts Mincle/FcRg/CARD9-mediated anti-mycobacterial immunity. Mycobacteria, therefore, harness TREM2 to evade the host immunity.