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.

Carma3 Protects from Liver Injury by Preserving Mitochondrial Integrity in Liver Sinusoidal Endothelial Cells.

Carma3 is an intracellular scaffolding protein that can form complex with Bcl10 and Malt1 to mediate G protein-coupled receptor- or growth factor receptor-induced NF-κB activation. However, the in vivo function of Carma3 has remained elusive. Here, by establishing a Con A-induced autoimmune hepatitis model, we show that liver injury is exacerbated in Carma3 -/- mice. Surprisingly, we find that the Carma3 expression level is higher in liver sinusoidal endothelial cells (LSECs) than in hepatocytes in the liver. In Carma3 -/- mice, Con A treatment induces more LSEC damage, accompanied by severer coagulation. In vitro we find that Carma3 localizes at mitochondria and Con A treatment can trigger more mitochondrial damage and cell death in Carma3-deficient LSECs. Taken together, our data uncover an unrecognized role of Carma3 in maintaining LSEC integrity, and these results may extend novel strategies to prevent liver injury from toxic insults.

Intestinal CD8+ tissue-resident memory T cells: From generation to function.

Tissue-resident memory T cells (Trm), and particularly the CD8+ subset, have been shown to play a pivotal role in protection against infections and tumors. Studies in animal models and human tissues have highlighted that, while a core functional program is shared by Trm at all anatomical sites, distinct tissues imprint unique features through specific molecular cues. The intestinal tissue is often the target of pathogens for local proliferation and penetration into the host systemic circulation, as well as a prominent site of tumorigenesis. Therefore, promoting the formation of Trm at this location is an appealing therapeutic option. The various segments composing the gastrointestinal tract present distinctive histological and functional characteristics, which may reflect on the imprinting of unique functional features in the respective Trm populations. What these features are, and whether they can effectively be harnessed to promote local and systemic immunity, is still under investigation. Here, we review how Trm are generated and maintained in distinct intestinal niches, analyzing the required molecular signals and the models utilized to uncover them. We also discuss evidence for a protective role of Trm against infectious agents and tumors. Finally, we integrate the knowledge obtained from animal models with that gathered from human studies.

Linear Ubiquitination of RIPK1 on Lys612 Regulates Systemic Inflammation via Preventing Cell Death.

Receptor-interacting protein kinase-1 (RIPK1) is a master regulator of the TNF-α-induced cell death program. The function of RIPK1 is tightly controlled by posttranslational modifications, including linear ubiquitin chain assembly complex-mediated linear ubiquitination. However, the physiological function and molecular mechanism by which linear ubiquitination of RIPK1 regulates TNF-α-induced intracellular signaling remain unclear. In this article, we identified Lys627 residue as a major linear ubiquitination site in human RIPK1 (or Lys612 in murine RIPK1) and generated Ripk1K612R/K612R mice, which spontaneously develop systemic inflammation triggered by sustained emergency hematopoiesis. Mechanistically, without affecting NF-κB activation, Ripk1K612R/K612R mutation enhances apoptosis and necroptosis activation and promotes TNF-α-induced cell death. The systemic inflammation and hematopoietic disorders in Ripk1K612R/K612R mice are completely abolished by deleting TNF receptor 1 or both RIPK3 and Caspase-8. These data suggest the critical role of TNF-α-induced cell death in the resulting phenotype in Ripk1K612R/K612R mice. Together, our results demonstrate that linear ubiquitination of RIPK1 on K612 is essential for limiting TNF-α-induced cell death to further prevent systemic inflammation.

Intrinsic Abnormalities of Keratinocytes Initiate Skin Inflammation through the IL-23/T17 Axis in a MALT1-Dependent Manner.

Increasing evidence has supported the crucial role of CARD14 in the pathogenesis of psoriasis, whereas the precise cellular signaling involved in skin physiopathology remains poorly understood. In this article, we show that neither genetic ablation of Il17a nor elimination of T cells was sufficient to restrain the skin inflammation in a CARD14-E138A-mutation-induced psoriasis-like mouse model, whereas depletion of Il23, which extremely blocked the IL-23/T17 axis, was more effective. Targeting CBM complex by conditional deletion of MALT1 or BCL10 in keratinocytes abrogated both the cutaneous and systemic inflammation of heterozygous Card14 E138A/+ mice. Selective inactivation of keratinocyte-specific MALT1 proteolytic activity strongly ameliorated the Card14 E138A/+- and Card14 ΔQ136/+-induced skin disease, which was reproduced by using the imiquimod-induced mouse model. Together, our results suggest a sequence of events under CARD14-mutation-induced psoriasis condition that keratinocyte-intrinsic activation of CBM complex initiates the skin inflammation depending on the IL-23/T17 axis. Targeting keratinocytes by inactivation of MALT1 paracaspase activity might be a promising therapeutic target for early psoriasis treatment.

Malt1 Protease Is Critical in Maintaining Function of Regulatory T Cells and May Be a Therapeutic Target for Antitumor Immunity.

The paracaspase Malt1 is a key molecule in mediating Ag receptor-induced NF-κB activation in lymphocytes, but the role of Malt1 in the function of regulatory T (Treg) cells is still unclear. In this article, we reported that specific deletion of Malt1 in Treg cells would lead to Scurfy-like lethal autoimmune disease, which was caused by Treg cell dysfunction but not number loss. Interestingly, Foxp3CreMalt1fl/C472A mice, in which Malt1 protease was specifically inactivated in Treg cells, also displayed spontaneous inflammatory disorders, with severe hair loss and skin hyperplasia. Consistently, Foxp3CreMalt1fl/C472A mice showed enhanced antitumor response because of their decreased function and infiltration of Treg cells, as well as reduced CD8+ T cell exhaustion. Gene expression profiling analysis revealed dysregulated expression pattern of Treg effector genes upon Malt1 deletion or its protease inactivation. Together, our data unraveled a critical role of Malt1, especially its protease activity, in maintaining homeostasis and function of Treg cells.

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 ß-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.

Prokaryotic Expression of Bioactive Zinc Transporter 8 Antigens and Detection of Diabetes Specific Autoantibodies in a Single Dot Immunogold Filtration Assay.

BACKGROUND: Type 1 diabetes mellitus is an autoimmune disease, and islet autoantibodies secreted by auto-reactive plasma cells are diagnostic indicators of the immune processes. Autoantibodies to zinc transporter 8 (ZnT8) have been identified as a novel reliable biomarker for the prediction, diagnosis, monitoring, and prognosis of autoimmune diabetes, complementing the panel of existing diagnostic autoantibodies. Although the enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay are the most frequently used testing methods, they do not allow simultaneous detection of multiple autoantibodies. Another obstacle is the cost of ZnT8 production for antibody assays. This study aimed to develop a cost-effective expression system for the production of two ZnT8 C-terminal fragments containing main ZnT8 antigen epitopes and establish an improved reliable and rapid assay for the detection of anti-ZnT8 antibodies with a potential to simultaneously measure multiple autoantibodies. METHODS: The coding codons of the human ZnT8 were optimized for prokaryotic expression and the mutation was achieved using site-directed mutagenesis. A total of 42 newly diagnosed type 1 diabetes patients (16 males and 26 females) and 100 healthy controls (57 males and 43 females) were enrolled for sera. The dot immunogold filtration assay (DIGFA) was evaluated by comparing with ELISA as the "gold standard". RESULTS: Two ZnT8 antigens (arginine and tryptophan ZnT8 at position 325) were successfully produced. We established a rapid DIGFA method for the simultaneous detection of anti-ZnT8 antibodies, with the sensitivity, specificity, accuracy, Youden index, and positive and negative likelihood ratio being 64.3%, 96.4%, 85.7%, 0.607, 18.0, and 0.370, respectively, and the results did not significantly differ from those for ELISA (p = 0.22). CONCLUSIONS: These results demonstrate that the pColdII expression system is suitable for the production of bioactive ZnT8 antigens and that DIGFA can be a rapid, reliable, and highly specific method for the detection of ZnT8 antibodies, which can be potentially applied to identify a panel of diabetes-specific autoantibodies simultaneously.

Association between SLC30A8 rs13266634 Polymorphism and Type 2 Diabetes Risk: A Meta-Analysis.

BACKGROUND: Accumulating but inconsistent data about the role of rs13266634 variant of SLC30A8 in type 2 diabetes have been reported, partly due to small sample sizes and non-identical ethnicity. MATERIAL AND METHODS: We searched PubMed and Cochrane Library to identify eligible studies and extract data of baseline characteristics, genotype count, odds ratio (OR), and 95% confidence interval (CI). Both adjusted OR with 95% CI and genotype counts were employed to assess the association. Genotype data were further pooled to provide estimates under different genetic models and the most appropriate model was determined. Sensitivity and cumulative analysis were conducted to assure the strength of results. RESULTS: Fifty-five datasets of 39 studies (including 38 of 24 with genotype count) were included. Significant associations were found in allelic contrasts using adjusted ORs and raw genotype count, respectively, overall in Asian and European populations (overall: OR=1.147/1.157, 95% CI 1.114-1.181/1.135-1.180; Asian: OR=1.186/1.165, 95% CI 1.150-1.222/1.132-1.198; European: OR=1.100/1.151, 95% CI 1.049-1.153/1.120-1.183; All p=0.00), but not in African populations (African: OR=1.255/1.111, 95% CI 0.964-1.634/0.908-1.360, p=0.091/0.305). Further analysis with genotype count under different genetic models all showed that individuals with CC genotype had 33.0% and 16.5% higher risk of type 2 diabetes than those carrying TT and CT genotypes, respectively, under the most likely codominant model. Cumulative analysis indicated gradually improved precision of estimation after studies accumulated. CONCLUSIONS: Our results suggest that rs13266634 may be an important genetic factor of type 2 diabetes risk among Asian and European but not African populations.

Declined plasma sfrp5 concentration in patients with type 2 diabetes and latent autoimmune diabetes in adults.

OBJECTIVE: Secreted frizzled-related protein 5 (sfrp5), like adiponectin, has been identified as a novel insulin-sensitising and anti-inflammatory adipokine. Our objective was to determine whether differences of circulating plasma sfrp5 concentration exist among type 2 diabetes (T2D), latent autoimmune diabetes in adults (LADA) and healthy population. METHODS: Enzyme-linked immuno sorbent assay was employed to detect the circulating sfrp5 level in plasma, and other lab tests such as fasting glucose and creatinine were also examined. Correlation analysis between sfrp5 and characteristics of subjects was conducted IBM SPSS Statistics and GraphPad Prism. RESULTS: Circulating sfrp5 level was significantly decreased in T2D and LADA patients plasma compared with that in healthy control (14.14±11.91ng/mL, 14.82±11.27ng/mL, 22.98±12.36ng/mL, respectively), although no differences was observed between LADA and T2D groups. Furthermore, we found sfrp5 was correlated with homeostasis model assessment of insulin resistance (HOMA-IR), diabetes duration and BMI. Finally we found sfrp5 was still negatively correlated with HOMA-IR after being adjusted for disease duration and BMI(r= -0.315, P< 0.05). CONCLUSIONS: Our results support a role for SFRP5 as a protective factor in the pathogenesis of autoimmune diabetes and facilitate a novel aspect for diabetes research.

Local antigen encounter promotes generation of tissue-resident memory T cells in the large intestine.

Upon infection, CD8+ T cells that have been primed in the draining lymph nodes migrate to the invaded tissue, where they receive cues prompting their differentiation into tissue-resident memory cells (Trm), which display niche-specific transcriptional features. Despite the importance of these cells, our understanding of their molecular landscape and the signals that dictate their development remains limited, particularly in specific anatomical niches such as the large intestine (LI). Here, we report that LI Trm-generated following oral infection exhibits a distinct transcriptional profile compared to Trm in other tissues. Notably, we observe that local cues play a crucial role in the preferential establishment of LI Trm, favoring precursors that migrate to the tissue early during infection. Our investigations identify cognate antigen recognition as a major driver of Trm differentiation at this anatomical site. Local antigen presentation not only promotes the proliferation of effector cells and memory precursors but also facilitates the acquisition of transcriptional features characteristic of gut Trm. Thus, antigen recognition in the LI favors the establishment of Trm by impacting T cell expansion and gene expression.

Enhancing diabetic wound healing with a pH/glucose dual-responsive hydrogel for ROS clearance and antibacterial activity.

Currently, the treatment of diabetic wounds in clinical practice is still unsatisfactory due to the risks of oxidative damage and bacterial infection during the healing process. An optimal wound dressing should exhibit robust capabilities in scavenging reactive oxygen species (ROS) and combatting bacterial growth. In this study, we utilized borax as a crosslinker and prepared a pH/glucose dual-responsive composite hydrogel based on poly(vinyl alcohol) (PVA), sodium alginate (SA), and tannic acid (TA). This hydrogel, loaded with cerium dioxide, serves as an effective ROS scavenger, promoting wound closure by reducing the level of ROS in the wound area. Additionally, the hydrogel can release the antibacterial drug ofloxacin in response to the low pH and high glucose microenvironment in infected wounds. Results from skin defect model in diabetic mice demonstrated this ROS-scavenging and antibacterial hydrogel can suppress inflammation and accelerate wound healing. In summary, our work provides a new perspective on a local and stimulus-responsive drug delivery strategy for treating diabetic wounds.

Intestinal carbapenem-resistant Klebsiella pneumoniae undergoes complex transcriptional reprogramming following immune activation.

Carbapenem-resistant Klebsiella pneumoniae (CR-Kp) is a significant threat to public health worldwide. The primary reservoir for CR-Kp is the intestinal tract. There, the bacterium is usually present at low density but can bloom following antibiotic treatment, mostly in hospital settings. The impact of disturbances in the intestinal environment on the fitness, survival, expansion, and drug susceptibility of this pathogen is not well-understood, yet it may be relevant to devise strategies to tackle CR-Kp colonization and infection. Here, we adopted an in vivo model to examine the transcriptional adaptation of a CR-Kp clinical isolate to immune activation in the intestine. We report that as early as 6 hours following host treatment with anti-CD3 antibody, CR-Kp underwent rapid transcriptional changes including downregulation of genes involved in sugar utilization and amino acid biosynthesis and upregulation of genes involved in amino acid uptake and catabolism, antibiotic resistance, and stress response. In agreement with these findings, treatment increased the concentration of oxidative species and amino acids in the mouse intestine. Genes encoding for proteins containing the domain of unknown function (DUF) 1471 were strongly upregulated, however their deletion did not impair CR-Kp fitness in vivo upon immune activation. Transcription factor enrichment analysis identified the global regulator cAMP-Receptor Protein, CRP, as a potential orchestrator of the observed transcriptional signature. In keeping with the recognized role of CRP in regulating utilization of alternative carbon sources, crp deletion in CR-Kp resulted in strongly impaired gut colonization, although this effect was not amplified by immune activation. Thus, following intestinal colonization, which occurs in a CRP-dependent manner, CR-Kp can rapidly respond to immune cues by implementing a well-defined and complex transcriptional program whose direct relevance toward bacterial fitness warrants further investigation. Additional analyses utilizing this model may identify key factors to tackle CR-Kp colonization of the intestine.

Study of tractor side tilt operation on intervertebral disc injury between L4 and L5 in drivers.

The tilting of the cab seat when the tractor is in deep ploughing operation changes the sitting position of the driver, which may accelerate lumbar spine injury. This paper adopts the musculoskeletal model and the finite element model of the lumbar L4-L5 segment to predict the maximum Von-Mises stress and maximum strain of the driver's lumbar L4-L5 segment intervertebral disc. In this study, we used 3D motion capture to obtain the driver's spine position spatial data when the tractor tilted at different angles. A tractor-driver musculoskeletal model and a finite element model of the lumbar spine L4-L5 segments were created in AnyBody™ and Abaqus, respectively. The tractor-driver musculoskeletal model was used to calculate the load of the driver's lumbar spine L4-L5 segment at different angles of tractor tilt, which was used as the load condition of the finite element model of the lumbar spine L4-L5 segment, and then the influence of tractor tilt angle and vibration on the driver's lumbar spine L4-L5 disc was studied. The results show that the maximum Von-Mises stress and maximum strain of the driver's lumbar L4-L5 intervertebral disc will increase due to the tilt. The maximum Von-Mises stress occurs in the annulus II, and the maximum strain occurs in the upper end plate of the intervertebral disc. With the occurrence of tilt, the position of the maximum Von-Mises stress changes, which can lead to disc injury to the driver, and vibration may exacerbate this injury.

Effect of seat tilting on biomechanics of L4 in tractor drivers.

The tilting of the tractors' seat during deep tillage operations affects the drivers' sitting position, which can lead to lumbar spine injuries. To investigate the effect of seat tilting on the driver's L4 biomechanics, we built a tractor-driver musculoskeletal model in AnyBody™. The maximum activity of the driver's erector spinae at different tilted angles were measured by sEMG and compared with the simulation results to validate the model. The spatial position of the driver's spine at different tilted angles were obtained by 3 D motion capture. The model simulated the driver's spine posture during the actual tilt and investigated the effects of different tilted angles and vibration on the biomechanics of the driver's L4 . The results showed that as the tilt angle of the tractor increased, the load on the driver's L4 also increased, especially the shear force increased at a faster rate than the axial and normal forces, with the shear force on the driver's L4 increasing from 0 N to 138.7 N when the tractor was tilted from 0° to 15°. When vibration was applied to the musculoskeletal model, the maximum value of the shear force on the driver became progressively greater as the angle of tilt of the tractor increased. Overall, tilting the tractor can have a large impact on the biomechanics of the driver's L4, and tilting the tractor may be an important cause of lumbar spine injuries for tractor drivers.

Improving insulin self-injection accuracy in patients with diabetes mellitus through a nursing project.

BACKGROUND: Non-standardized insulin injection has an impact on the efficacy of glucose control. OBJECTIVES: The aim of the study was to explore the effectiveness of a nursing project in improving the insulin self-injection accuracy of diabetes mellitus patients. MATERIAL AND METHODS: A total of 200 type 2 diabetes patients who received insulin therapy with an insulin pen were recruited at the First Affiliated Hospital of Army Medical University (Chongqing, China). Patients were randomly assigned to a control (n = 100) or intervention (n = 100) group. Conventional health education was conducted in the control group, while a nursing project and conventional health education were undertaken in the intervention group. The following parameters were analyzed between the 2 groups: standardized insulin pen use at admission and discharge, glycosylated hemoglobin (HbA1c), time in range (TIR), and adipose hyperplasia incidence rate 6 months after discharge. RESULTS: Concerning standardized insulin self-injection, the intervention group was superior to the control group, and the difference between the 2 groups was statistically significant (p < 0.05). The HbA1c levels (p = 0.000), TIR (p = 0.005) and adipose hyperplasia incidence rate 6 months after discharge (p = 0.000) all improved in the intervention group compared to the control group. CONCLUSIONS: The application of the nursing project effectively improved the efficacy of glucose control in diabetes mellitus patients.

Recombinant VirB5 protein as a potential serological marker for the diagnosis of bovine brucellosis.

The molecular tag vaccine against Brucella abortus and serological testing are the main methods of prevention of brucellosis used currently. They can discriminate vaccinated animals and humans from those naturally infected. In this study, we constructed a gene deletion mutant strain, B. abortus S19 virB5 with a molecular tag. Recombinant VirB5 was expressed and purified for evaluation as a diagnostic reagent for bovine brucellosis. In total, 400 sera samples were tested using a VirB5 antigen-based enzyme-linked immunosorbent assay (ELISA) and the results were compared with those of the standard tube agglutination test (SAT). This showed that the sensitivity was 88.2%, specificity was 97.8% and accuracy was 94.8%. Recombinant VirB5 could also be used to discriminate B. abortus-infected mice from mice infected with the B. abortus S19 virB5 mutant strain. It was concluded that recombinant VirB5 could be used as a potential antigen and serological marker for the diagnosis of bovine brucellosis.

Knockout of immunotherapy prognostic marker genes eliminates the effect of the anti-PD-1 treatment.

The efficacy of immunotherapy is largely patient-specific due to heterogeneity in tumors. Combining statistic power from a variety of immunotherapies across cancer types, we found four biological pathways significantly correlated with patient survival following immunotherapy. The expression of immunotherapy prognostic marker genes (IPMGs) in these pathways can predict the patient survival with high accuracy not only in the TCGA cohort (89.36%) but also in two other independent cohorts (80.91%), highlighting that the activity of the IPMGs can reflect the sensitivity of the tumor immune microenvironment (TIME) to immunotherapies. Using mouse models, we show that knockout of one of the IPMGs, MALT1, which is critical for the T-cell receptor signaling, can eliminate the antitumor effect of anti-PD-1 treatment completely by impairing the activation of CD8+ T cells. Notably, knockout of another IPMG, CLEC4D, a C-type lectin receptor that expressed on myeloid cells, also reduced the effect of anti-PD-1 treatment potentially through maintaining the immunosuppressive effects of myeloid cells. Our results suggest that priming TIME via activating the IPMGs may increase the response rate and the effect of immune checkpoint blockers.

Molecular characterization, structural analysis and determination of host range of a novel bacteriophage LSB-1.

BACKGROUND: Bacteriophages (phages) are widespread in the environment and play a crucial role in the evolution of their bacterial hosts and the emergence of new pathogens. RESULTS: LSB-1, a reference coliphage strain, was classified as a member of the Podoviridae family with a cystic form (50 ± 5 nm diameter) and short tail (60 ± 5 nm long). The double stranded DNA was about 30 kilobase pairs in length. We identified its host range and determined the gp17 sequences and protein structure using shotgun analysis and bioinformatics technology. CONCLUSIONS: Coliphage LSB-1 possesses a tailspike protein with endosialidase activity which is probably responsible for its specific enteroinvasive E.coli host range within the laboratory.