A novel inherited CARD9 deficiency in an otherwise healthy woman with CNS candidiasis.

Patients with caspase-associated recruitment domain-9 (CARD9) deficiency are more likely to develop invasive fungal disease that affect CNS. However, the understanding of how Candida invades and persists in CNS is still limited. We here reported a 24-year-old woman who were previously immunocompetent and diagnosed with CNS candidiasis. A novel autosomal recessive homozygous CARD9 mutation (c.184 + 5G > T) from this patient was identified using whole genomic sequencing. Furthermore, we extensively characterized the impact of this CARD9 mutation on the host immune response in monocytes, neutrophils and CD4 + T cells, using single cell sequencing and in vitro experiments. Decreased pro-inflammatory cytokine productions of CD14 + monocyte, impaired Th17 cell differentiation, and defective neutrophil accumulation in CNS were found in this patient. In conclusion, this study proposed a novel mechanism of CNS candidiasis development. Patients with CNS candidiasis in absence of known immunodeficiencies should be analyzed for CARD9 gene mutation as the cause of invasive fungal infection predisposition.

Inherited CARD9 Deficiency Due to a Founder Effect in East Asia.

Autosomal recessive CARD9 deficiency can underly deep and superficial fungal diseases. We identified two Japanese patients, suffering from superficial and invasive Candida albicans diseases, carrying biallelic variants of CARD9. Both patients, in addition to another Japanese and two Korean patients who were previously reported, carried the c.820dup CARD9 variant, either in the homozygous (two patients) or heterozygous (three patients) state. The other CARD9 alleles were c.104G > A, c.1534C > T and c.1558del. The c.820dup CARD9 variant has thus been reported, in the homozygous or heterozygous state, in patients originating from China, Japan, or South Korea. The Japanese, Korean, and Chinese patients share a 10 Kb haplotype encompassing the c.820dup CARD9 variant. This variant thus originates from a common ancestor, estimated to have lived less than 4,000 years ago. While phaeohyphomycosis caused by Phialophora spp. was common in the Chinese patients, none of the five patients in our study displayed Phialophora spp.-induced disease. This difference between Chinese and our patients probably results from environmental factors. (161/250).

Listeria toxin promotes phosphorylation of the inflammasome adaptor ASC through Lyn and Syk to exacerbate pathogen expansion.

Inflammasome activation exacerbates infectious disease caused by pathogens such as Listeria monocytogenes, Staphylococcus aureus, and severe acute respiratory syndrome coronavirus 2. Although these pathogens activate host inflammasomes to regulate pathogen expansion, the mechanisms by which pathogen toxins contribute to inflammasome activation remain poorly understood. Here we show that activation of inflammasomes by Listeria infection is promoted by amino acid residue T223 of listeriolysin O (LLO) independently of its pore-forming activity. LLO T223 is critical for phosphorylation of the inflammasome adaptor ASC at amino acid residue Y144 through Lyn-Syk signaling, which is essential for ASC oligomerization. Notably, a Listeria mutant expressing LLO T223A is impaired in inducing ASC phosphorylation and inflammasome activation. Furthermore, the virulence of LLO T223A mutant is markedly attenuated in vivo due to impaired ability to activate the inflammasome. Our results reveal a function of a pathogen toxin that exacerbates infection by promoting phosphorylation of ASC.

Deficiency of CARMA3 attenuates the development of bleomycin induced pulmonary fibrosis.

BACKGROUND: Pulmonary fibrosis (PF) has attracted more and more attention due to its irreversibility and high mortality rate. Currently, there is no effective treatment option is available to reverse the disease. Caspase recruitment domain-containing membrane-associated guanylate kinase protein (CARMA3) has been recognized as a proinflammatory molecule involved in many lung diseases, such as Allergic airway inflammation and lung cancer. Bleomycin (Bleo), as an alkaline sugar peptide antibiotics, is often used as a first-line anti-tumor agent. Its toxic effect is to induce pulmonary fibrosis (PF) and its clinical symptoms, so it has been widely used in the construction of pulmonary fibrosis model. METHODS: Wild type mice (WT, n = 20) and CARMA3 knockout mice (CARMA3-KO, n = 20) were generated and injected with bleomycin or saline via trachea. The severity of fibrosis was evaluated by fibrosis markers and lung histological morphology. Furthermore, the amount of alveolar epithelial cells and inflammation in lung tissue were examined. Finally, epithelial-mesenchymal transition was further investigated. RESULTS: We found CARMA3 expression in the mice alveolar epithelial cells. And compared with WT mice, CARMA3-KO mice showed reduced deposition of collagen fibers, inflammation and destruction of alveolar epithelial cells in lung tissue. In addition, after bleomycin induction, the expressions of proinflammatory factors and collagen-related factors in CARMA3-KO mice were much lower than those in WT mice. The epithelial-mesenchymal transformation phenotype was also improved in CARMA3-KO mice compared to WT mice. CONCLUSION: Our Results shows that CARMA3 plays an important role in the pathogenesis of bleomycin-induced pulmonary fibrosis. CARMA3 could alleviate the fibrosis by improving inflammation, deposition of collagen and damage of alveolar epithelial cells, which revealed that CARMA3 may be a potential target for pulmonary fibrosis.

CARD9 Deficiency Increases Hippocampal Injury Following Acute Neurotropic Picornavirus Infection but Does Not Affect Pathogen Elimination.

Neurotropic viruses target the brain and contribute to neurologic diseases. Caspase recruitment domain containing family member 9 (CARD9) controls protective immunity in a variety of infectious disorders. To investigate the effect of CARD9 in neurotropic virus infection, CARD9-/- and corresponding C57BL/6 wild-type control mice were infected with Theiler's murine encephalomyelitis virus (TMEV). Brain tissue was analyzed by histology, immunohistochemistry and molecular analyses, and spleens by flow cytometry. To determine the impact of CARD9 deficiency on T cell responses in vitro, antigen presentation assays were utilized. Genetic ablation of CARD9 enhanced early pro-inflammatory cytokine responses and accelerated infiltration of T and B cells in the brain, together with a transient increase in TMEV-infected cells in the hippocampus. CARD9-/- mice showed an increased loss of neuronal nuclear protein+ mature neurons and doublecortin+ neuronal precursor cells and an increase in ß-amyloid precursor protein+ damaged axons in the hippocampus. No effect of CARD9 deficiency was found on the initiation of CD8+ T cell responses by flow cytometry and co-culture experiments using virus-exposed dendritic cells or microglia-enriched glial cell mixtures, respectively. The present study indicates that CARD9 is dispensable for the initiation of early antiviral responses and TMEV elimination but may contribute to the modulation of neuroinflammation, thereby reducing hippocampal injury following neurotropic virus infection.

In Vivo and In Vitro Impairments in T Helper Cell and Neutrophil Responses against Mucor irregularis in Card9 Knockout Mice.

Mucor irregularis is a frequently found fungus in Asia, especially China, and it causes primary cutaneous mucormycosis with a high rate of disfigurement. Caspase recruitment domain-containing protein 9 (Card9) is an essential adaptor molecule downstream of C-type lectin receptors. It mediates the activation of nuclear factor kappa B (NF-κB), regulates T helper 1 (Th1) and Th17 differentiation, and plays an important role in fungal immune surveillance. CARD9 deficiency correlates with the increased susceptibility to many fungal infections, including cutaneous mucormycosis caused by M. irregularis However, the underlying immunological mechanisms were not elucidated. Our study established a murine model of subcutaneous M. irregularis infection, and we isolated immune cells, including bone marrow-derived macrophages, bone marrow-derived dendritic cells, naive T cells, and neutrophils, from wild-type (WT) and Card9 knockout (Card9-/- ) mice to examine the antifungal effect of Card9 on M. irregularis in vivo and in vitroCard9-/- mice exhibited increased susceptibility to M. irregularis infection. Impaired local cytokine and chemokine production, NF-κB (p65) activation, and Th1/17 cell differentiation and partially impaired neutrophil-dependent antifungal immunity were observed in Card9-/- mice. This work enriches our knowledge of the relationship between CARD9 deficiency and mucormycosis.

Inherited CARD9 Deficiency in a Child with Invasive Disease Due to Exophiala dermatitidis and Two Older but Asymptomatic Siblings.

PURPOSE: Autosomal recessive CARD9 deficiency predisposes patients to invasive fungal disease. Candida and Trichophyton species are major causes of fungal disease in these patients. Other CARD9-deficient patients display invasive diseases caused by other fungi, such as Exophiala spp. The clinical penetrance of CARD9 deficiency regarding fungal disease is surprisingly not complete until adulthood, though the age remains unclear. Moreover, the immunological features of genetically confirmed yet asymptomatic individuals with CARD9 deficiency have not been reported. METHODS: Identification of CARD9 mutations by gene panel sequencing and characterization of the cellular phenotype by quantitative PCR, immunoblot, luciferase reporter, and cytometric bead array assays were performed. RESULTS: Gene panel sequencing identified compound heterozygous CARD9 variants, c.1118G>C (p.R373P) and c.586A>G (p.K196E), in a 4-year-old patient with multiple cerebral lesions and systemic lymphadenopathy due to Exophiala dermatitidis. The p.R373P is a known disease-causing variant, whereas the p.K196E is a private variant. Although the patient's siblings, a 10-year-old brother and an 8-year-old sister, were also compound heterozygous, they have been asymptomatic to date. Normal CARD9 mRNA and protein expression were found in the patient's CD14+ monocytes. However, these cells exhibited markedly impaired pro-inflammatory cytokine production in response to fungal stimulation. Monocytes from both asymptomatic siblings displayed the same cellular phenotype. CONCLUSIONS: CARD9 deficiency should be considered in previously healthy patients with invasive Exophiala dermatitidis disease. Asymptomatic relatives of all ages should be tested for CARD9 deficiency. Detecting cellular defects in asymptomatic individuals is useful for diagnosing CARD9 deficiency.

AIM2 in regulatory T cells restrains autoimmune diseases.

The inflammasome initiates innate defence and inflammatory responses by activating caspase-1 and pyroptotic cell death in myeloid cells1,2. It consists of an innate immune receptor/sensor, pro-caspase-1, and a common adaptor molecule, ASC. Consistent with their pro-inflammatory function, caspase-1, ASC and the inflammasome component NLRP3 exacerbate autoimmunity during experimental autoimmune encephalomyelitis by enhancing the secretion of IL-1ß and IL-18 in myeloid cells3-6. Here we show that the DNA-binding inflammasome receptor AIM27-10 has a T cell-intrinsic and inflammasome-independent role in the function of T regulatory (Treg) cells. AIM2 is highly expressed by both human and mouse Treg cells, is induced by TGFß, and its promoter is occupied by transcription factors that are associated with Treg cells such as RUNX1, ETS1, BCL11B and CREB. RNA sequencing, biochemical and metabolic analyses demonstrated that AIM2 attenuates AKT phosphorylation, mTOR and MYC signalling, and glycolysis, but promotes oxidative phosphorylation of lipids in Treg cells. Mechanistically, AIM2 interacts with the RACK1-PP2A phosphatase complex to restrain AKT phosphorylation. Lineage-tracing analysis demonstrates that AIM2 promotes the stability of Treg cells during inflammation. Although AIM2 is generally accepted as an inflammasome effector in myeloid cells, our results demonstrate a T cell-intrinsic role of AIM2 in restraining autoimmunity by reducing AKT-mTOR signalling and altering immune metabolism to enhance the stability of Treg cells.

Primary Cutaneous Aspergillosis in a Patient with CARD9 Deficiency and Aspergillus Susceptibility of Card9 Knockout Mice.

PURPOSE: We describe a case of primary cutaneous aspergillosis caused by Aspergillus fumigatus, and elucidate the underlying genetic and immunological mechanisms. MATERIALS AND METHODS: Routine clinical and laboratory investigations were performed. Whole-exome sequencing of the patient's DNA suggested the presence of a CARD9 mutation, which was confirmed by Sanger sequencing. Innate and adaptive immunological responses of patient-derived CARD9-deficient cells were evaluated with ELISA and flow cytometry. Cutaneous and pulmonary aspergillosis models were established in Card9 knockout (KO) mice, which were compared with wild-type and immunosuppressed mice, to explore the pathogenesis and Aspergillus susceptibility. RESULTS: A 45-year-old man presented with a 37-year history of skin lesions on his face. A diagnosis of primary cutaneous aspergillosis was made through histopathology, immunohistochemistry, and tissue culture. Sanger sequencing of CARD9 showed a homozygous frame-shift mutation (c.819_820insG, p.D274fsX60), which led to the lack of CARD9 expression. Peripheral blood mononuclear cells from the patient showed selective impairment of proinflammatory cytokines, and Th1-, Th17-, and Th22-associated responses upon fungus-specific stimulation. The cutaneous aspergillosis model established in Card9 KO mice presented with persistent infection, with fungal germs and short hyphae in tissue, consistent with the patient's lesions. Skin lesions in immunosuppressed mice were more severe, and led to death. Unlike our patient, Card9 KO mice were relatively susceptible to pulmonary aspergillosis, with reasons to be investigated. CONCLUSIONS: This is, to our knowledge, the first report that links cutaneous aspergillosis to CARD9 mutation. This work enriches both the phenotypic spectrum of CARD9 deficiencies and the genetic background of cutaneous aspergillosis.

CARD9 Deficiency in a Chinese Man with Cutaneous Mucormycosis, Recurrent Deep Dermatophytosis and a Review of the Literature.

Deficiency of caspase recruitment domain-containing protein 9 (CARD9) is an autosomal recessive primary immunodeficiency disorder, which typically predisposes immunocompetent individuals to single fungal infections and multiple fungal infections are very rare. We study an otherwise healthy 48-year-old man, who had been admitted to our hospital diagnosed with deep dermatophytosis caused by Trichophyton rubrum for three times at 29, 33 and 48 years old, respectively. At the age of 39 years, he suffered from cutaneous mucormycosis due to Mucor irregularis. Moreover, he had a long history of superficial fungal diseases and occasional oral candidiasis. Whole-exome sequencing revealed two compound heterozygous splicing variants in CARD9 gene, c. 184 + 5 G > T and c. 951G > A, confirmed by Sanger sequencing. Patients with recurrent fungal infections especially invasive fungal infections in the absence of known immunodeficiencies should be tested for CARD9 mutations.

ASC regulates platelet activation and contributes to thrombus formation independent of NLRP3 inflammasome.

BACKGROUND: Platelets are critical mediators of vascular homeostasis and thrombosis, and also contribute to the development of inflammation. NLRP3 inflammasome is a cytosolic multi-protein complex that consists of NLRP3, ASC and caspase-1, and regulates IL-1ß-mediated inflammation. METHOD AND RESULTS: Using two mouse models of thrombosis (i.e., occlusion of the middle cerebral artery and inferior vena cava), we found that thrombus formation was significantly enhanced in ASC-deficient (ASC-/-) mice, compared to that in wild-type (WT) and IL-1ß-/- mice. ASC deficiency had no effects on blood coagulation parameters (i.e., prothrombin time [PT] and activated partial thromboplastin time [APTT]). Platelets from WT mice express ASC, but neither NLRP3 nor caspase-1. ASC deficiency significantly enhanced the expression of P-selectin and GPIIb/IIIa in response to a GPVI agonist (collagen-related peptide [CRP]), but not to thrombin, in platelets. CRP induced ASC speck formation in WT platelets. ASC deficiency also enhanced cytosolic Ca2+ elevation and phosphorylation of ERK1/2 and Akt in platelets. CONCLUSION: Our results demonstrate that ASC negatively regulates GPVI signaling in platelets and enhances thrombus formation, independent of NLRP3 inflammasome and IL-1ß, and provide novel insights into the link between inflammation and thrombosis.

CARD9 mediates T cell inflammatory response in Coxsackievirus B3-induced acute myocarditis.

Cardiac inflammation in Coxsackievirus B3 (CVB3)-induced myocarditis is a consequence of viral-related cardiac injury and immune response. Caspase-associated recruitment domain 9 (CARD9) is a critical adaptor protein involved in transduction of signals from various innate pattern recognition receptors. In this study, the role of CARD9 in acute viral myocarditis was evaluated. CARD9-/- and C57BL/6 mice were infected with CVB3. On day 7 postinfection, myocardial tissue and blood samples were collected and examined. After CARD9 knockout, mRNA and protein levels of transforming growth factor-ß(TGF-ß), interleukin-17A(IL-17A), and CARD domain of B-cell CLL/lymphoma 10(BCL-10) in the myocardium were markedly lower in CARD9-/- mice than in C57BL/6 mice with CVB3-induced viral myocarditis. This trend was similar for the pathological scores for inflammation and serum levels of cytokines interleukin-6(IL-6), interleukin-10(IL-10), interferon -γ(IFN-γ), TGF-ß, and IL-17A. These results suggest that the CARD9-mediated secretion of pro-inflammatory cytokines plays an important role in the immune response to acute viral myocarditis.

Activation of ASC Inflammasome Driven by Toll-Like Receptor 4 Contributes to Host Immunity against Rickettsial Infection.

Rickettsiae are cytosolically replicating, obligately intracellular bacteria causing human infections worldwide with potentially fatal outcomes. We previously showed that Rickettsia australis activates ASC inflammasome in macrophages. In the present study, host susceptibility of ASC inflammasome-deficient mice to R. australis was significantly greater than that of C57BL/6 (B6) controls and was accompanied by increased rickettsial loads in various organs. Impaired host control of R. australis in vivo in ASC-/- mice was associated with dramatically reduced levels of interleukin 1ß (IL-1ß), IL-18, and gamma interferon (IFN-γ) in sera. The intracellular concentrations of R. australis in bone marrow-derived macrophages (BMMs) of TLR4-/- and ASC-/- mice were significantly greater than those in BMMs of B6 controls, highlighting the important role of inflammasome and these molecules in controlling rickettsiae in macrophages. Compared to B6 BMMs, TLR4-/- BMMs failed to secrete a significant level of IL-1ß and had reduced expression levels of pro-IL-1ß in response to infection with R. australis, suggesting that rickettsiae activate ASC inflammasome via a Toll-like receptor 4 (TLR4)-dependent mechanism. Further mechanistic studies suggest that the lipopolysaccharide (LPS) purified from R. australis together with ATP stimulation led to cleavage of pro-caspase-1 and pro-IL-1ß, resulting in TLR4-dependent secretion of IL-1ß. Taken together, these observations indicate that activation of ASC inflammasome, most likely driven by interaction of TLR4 with rickettsial LPS, contributes to host protective immunity against R. australis These findings provide key insights into defining the interactions of rickettsiae with the host innate immune system.

Deletion of haematopoietic Dectin-2 or CARD9 does not protect from atherosclerosis development under hyperglycaemic conditions.

BACKGROUND: C-type lectin receptors, including Dectin-2, are pattern recognition receptors on monocytes and macrophages that mainly recognize sugars and sugar-like structures present on fungi. Activation of C-type lectin receptors induces downstream CARD9 signalling, leading to the production of cytokines. We hypothesized that under hyperglycaemic conditions, as is the case in diabetes mellitus, glycosylated protein (sugar-like) structures activate C-type lectin receptors, leading to immune cell activation and increased atherosclerosis development. METHODS: Low-density lipoprotein receptor-deficient mice were lethally irradiated and transplanted with bone marrow from control wild-type, Dectin-2-/- or Card9-/- mice. After 6 weeks of recovery, mice received streptozotocin injections (50 mg/g BW; 5 days) to induce hyperglycaemia. After an additional 2 weeks, mice were fed a Western-type diet (0.1% cholesterol) for 10 weeks. RESULTS AND CONCLUSION: Deletion of haematopoietic Dectin-2 reduced the number of circulating Ly6Chi monocytes, increased pro-inflammatory cytokine production, but did not affect atherosclerosis development. Deletion of haematopoietic CARD9 tended to reduce macrophage and collagen content in atherosclerotic lesions, again without influencing the lesion size. Deletion of haematopoietic Dectin-2 did not influence atherosclerosis development under hyperglycaemic conditions, despite some minor effects on inflammation. Deletion of haematopoietic CARD9 induced minor alterations in plaque composition under hyperglycaemic conditions, without affecting lesion size.

CARD9 mediates dendritic cell-induced development of Lyn deficiency-associated autoimmune and inflammatory diseases.

CARD9 is an immune adaptor protein in myeloid cells that is involved in C-type lectin signaling and antifungal immunity. CARD9 is implicated in autoimmune and inflammatory-related diseases, such as rheumatoid arthritis, IgA nephropathy, ankylosing spondylitis, and inflammatory bowel disease (IBD). Given that Lyn-deficient (Lyn-/-) mice are susceptible to both autoimmunity and IBD, we investigated the immunological role of CARD9 in the development of these diseases using the Lyn-/- mouse model. We found that genetic deletion of CARD9 was sufficient to reduce the development of both spontaneous autoimmune disease as well as DSS- or IL-10 deficiency-associated colitis in Lyn-/- mice. Mechanistically, CARD9 was a vital component of the Lyn-mediated regulation of Toll-like receptor (TLR2 and TLR4) signaling in dendritic cells, but not in macrophages. In the absence of Lyn, signaling through a CD11b-Syk-PKCδ-CARD9 pathway was amplified, leading to increased TLR-induced production of inflammatory cytokines. Dendritic cell-specific deletion of CARD9 reversed the development of autoimmune and experimental colitis observed in dendritic cell-specific, Lyn-deficient mice. These findings suggest that targeting CARD9 may suppress the development of colitis and autoimmunity by reducing dendritic cell-driven inflammation.

Helicobacter pylori Exploits the NLRC4 Inflammasome to Dampen Host Defenses.

Helicobacter pylori colonizes the stomach of around 50% of humans. This chronic infection can lead to gastric pathologic conditions such as gastric ulcers and gastric adenocarcinomas. The strong inflammatory response elicited by H. pylori is characterized by the induction of the expression of several cytokines. Among those, IL-18 is found highly upregulated in infected individuals, and its expression correlates with the severity of gastric inflammation. IL-18 is produced as inactive proform and has to be cleaved by the multiprotein complex inflammasome to be active. In immune cells, the NLRC4 inflammasome, which is activated by flagellin or bacterial secretion systems, was shown to be dispensable for H. pylori-induced inflammasome activation. However, apart from immune cells, gastric epithelial cells can also produce IL-18. In this study, we analyzed the role of the NLRC4 inflammasome during H. pylori infection. Our results indicate that NLRC4 and a functional type IV secretion system are crucial for the production of IL-18 from human and murine gastric epithelial cells. In vivo, Nlrc4-/- mice failed to produce gastric IL-18 upon H. pylori infection. Compared with wild type mice, Nlrc4-/- mice controlled H. pylori better without showing strong inflammation. Moreover, H. pylori-induced IL-18 inhibits ß-defensin 1 expression in a NF-κB-dependent manner, resulting in higher bacterial colonization. At the same time, inflammasome activation enhances neutrophil infiltration, resulting in inflammation. Thus, NLRC4 inflammasome activation and subsequent IL-18 production favors bacterial persistence by inhibiting antimicrobial peptide production and, at the same time, contributes to gastric inflammation.

Crucial Role of NLRP3 Inflammasome in the Development of Peritoneal Dialysis-related Peritoneal Fibrosis.

Long-term peritoneal dialysis (PD) therapy leads to peritoneal inflammation and fibrosis. However, the mechanism underlying PD-related peritoneal inflammation and fibrosis remains unclear. NLRP3 inflammasome regulates the caspase-1-dependent release of interleukin-1ß and mediates inflammation in various diseases. Here, we investigated the role of NLRP3 inflammasome in a murine model of PD-related peritoneal fibrosis induced by methylglyoxal (MGO). Inflammasome-related proteins were upregulated in the peritoneum of MGO-treated mice. MGO induced parietal and visceral peritoneal fibrosis in wild-type mice, which was significantly reduced in mice deficient in NLRP3, ASC, and interleukin-1ß (IL-1ß). ASC deficiency reduced the expression of inflammatory cytokines and fibrotic factors, and the infiltration of macrophages. However, myeloid cell-specific ASC deficiency failed to inhibit MGO-induced peritoneal fibrosis. MGO caused hemorrhagic ascites, fibrin deposition, and plasminogen activator inhibitor-1 upregulation, but all of these manifestations were inhibited by ASC deficiency. Furthermore, in vitro experiments showed that MGO induced cell death via the generation of reactive oxygen species in vascular endothelial cells, which was inhibited by ASC deficiency. Our results showed that endothelial NLRP3 inflammasome contributes to PD-related peritoneal inflammation and fibrosis, and provide new insights into the mechanisms underlying the pathogenesis of this disorder.

CARD11 is dispensable for homeostatic responses and suppressive activity of peripherally induced FOXP3+ regulatory T cells.

FOXP3+ regulatory T (Treg) cells are essential for immunological tolerance and immune homeostasis. Despite a great deal of interest in modulating their number and function for the treatment of autoimmune disease or cancer, the precise mechanisms that control the homeostasis of Treg cells remain unclear. We report a new ENU-induced mutant mouse, lack of costimulation (loco), with atopic dermatitis and Treg cell deficiency typical of Card11 loss-of-function mutants. Three distinct single nucleotide variants were found in the Card11 introns 2, 10 and 20 that cause the loss of CARD11 expression in these mutant mice. These mutations caused the loss of thymic-derived, Neuropilin-1+ (NRP1+ ) Treg cells in neonatal and adult loco mice; however, residual peripherally induced NRP1- Treg cells remained. These peripherally generated Treg cells could be expanded in vivo by the administration of IL-2:anti-IL-2 complexes, indicating that this key homeostatic signaling axis remained intact in CARD11-deficient Treg cells. Furthermore, these expanded Treg cells could mediate near-normal suppression of activated, conventional CD4+ T cells, suggesting that CARD11 is dispensable for Treg cell function. In addition to shedding light on the requirements for CARD11 in Treg cell homeostasis and function, these data reveal novel noncoding Card11 loss-of-function mutations that impair the expression of this critical immune-regulatory protein.

Rare copy number variants in the genome of Chinese female children and adolescents with Turner syndrome.

Turner syndrome (TS) is a congenital disease caused by complete or partial loss of one X chromosome. Low bone mineral status is a major phenotypic characteristic of TS that can not be fully explained by X chromosome loss, suggesting other autosomal-linked mutations may also exist. Therefore, the present study aimed to detect potential genetic mutations in TS through examination of copy number variation (CNV). Seventeen patients with TS and 15 healthy volunteer girls were recruited. Array-based comparative genomic hybridization (a-CGH) was performed on whole blood genomic DNA (gDMA) from the 17 TS patients and 15 healthy volunteer girls to identify potential CNVs. The abnormal CNV of one identified gene (CARD11) was verified by quantitative PCR. All cases diagnosed had TS based on genotype examination and physical characteristics, including short stature and premature ovarian failure. Three rare CNVs, located individually at 7p22.3, 7p22.2, and Xp22.33, where six genes (TTYH3, AMZ1, GNA12, BC038729, CARD11, and SHOX (stature homeobox)) are located, were found in TS patients. Quantitative PCR confirmed the CNV of CARD11 in the genome of TS patients. Our results indicate that CARD11 gene is one of the mutated genes involved in TS disease. However, this CNV is rare and its contribution to TS phenotype requires further study.