Gasdermin D promotes development of intestinal tumors through regulating IL-1ß release and gut microbiota composition.

The interplay between gut microbiota and host is crucial for maintaining host health. When this balance is broken, various diseases can arise, including colorectal cancer (CRC). However, the mechanism by which gut microbiota and host interactions mediate CRC development remains unclear. Here, we found that Gasdermin D (GSDMD), an inflammasome effector responsible for forming membrane pores to mediate cell pyroptosis, was upregulated in both human and mouse intestinal tumor samples. GSDMD deficiency significantly suppressed intestinal tumor development in Apcmin/+ mice, a spontaneous CRC mouse model. Apcmin/+Gsdmd-/- mice exhibited reduced IL-1ß release in the intestine, and the administration of recombinant mouse IL-1ß partially restored intestinal tumor development in Apcmin/+Gsdmd-/- mice. Moreover, 16s rRNA sequencing showed a substantial increase in Lactobacillus abundance in the feces of Apcmin/+Gsdmd-/- mice compared to Apcmin/+ mice. Concurrently, Kynurenine (Kyn), a metabolite derived from host tryptophan (Trp) metabolism, was significantly decreased in the feces of Apcmin/+Gsdmd-/- mice, as shown by metabolite analysis. Additionally, Kyn levels were inversely correlated with Lactobacillus abundance. Furthermore, the administration of exogenous Kyn also promoted intestinal tumor development in Apcmin/+Gsdmd-/- mice. Thus, GSDMD promotes spontaneous CRC development through increasing IL-1ß release and Kyn production. Our data suggest an association between GSDMD, gut microbiota, the host Trp/Kyn pathway, and CRC development.

A Reference Profile of N-Glycosylation for Human Kidney and the Identification of Cell-Cell Interactions between Parietal Epithelial Cells and Capillary Endothelial Cells by Single-Cell Glycosylation-Sequencing.

BACKGROUND: N-glycosylation is one of the most common posttranslational modifications in humans, and these alterations are associated with kidney diseases. METHODS: A novel technological approach, single-cell N-acetyllactosamine sequencing (scLacNAc-seq), was applied to simultaneously detect N-glycosylation expression and the transcriptome at single-cell resolution in three human kidney tissues from zero-time biopsy. Cell clusters, glycation abundance in each cell cluster, functional enrichment analysis, cell-cell crosstalk, and pseudotime analysis were applied. RESULTS: Using scLacNAc-seq, 24,247 cells and 22 cell clusters were identified, and N-glycan abundance in each cell was obtained. Transcriptome analysis revealed a close connection between capillary endothelial cells (CapECs) and parietal epithelial cells (PECs). PECs and CapECs communicate with each other through several pairs of ligand receptors (e.g., TGFB1-EGFR, GRN-EGFR, TIMP1-FGFR2, VEGFB-FLT1, ANGPT2-TEK, and GRN-TNFRSF1A). Finally, a regulatory network of cell-cell crosstalk between PECs and CapECs was constructed, which is involved in cell development. CONCLUSIONS: We here, for the first time, constructed the glycosylation profile of 22 cell clusters in the human kidney from zero-time biopsy. Moreover, cell-cell communication between PECs and CapECs through the ligand-receptor system may play a crucial regulatory role in cell proliferation.

Serum metabolomics analysis reveals metabolite profile and key biomarkers of idiopathic membranous nephropathy.

Background: Idiopathic membranous nephropathy (IMN) is an organ-specific autoimmune disease with multiple and complex pathogenic mechanisms. Currently, renal biopsy is considered the gold standard for diagnosing membranous nephropathy. However, there were limitations to the renal puncture biopsy, such as the relatively high cost, longer time consuming, and the risk of invasive procedures. We investigated the profile of serum metabolites in IMN patients based on the UHPLC-QE-MS metabolomics technique for exploring the potential disease biomarkers and clinical implementation. Methods: In our research, we collected serum samples from healthy control (n = 15) and IMN patients (n = 25) to perform metabolomics analysis based on the UHPLC-QE-MS technique. Result: We identified 215 differentially expressed metabolites (DEMs) between the IMN and healthy control (HC) groups. Furthermore, these DEMs were significantly identified in histidine metabolism, arginine and proline metabolism, pyrimidine metabolism, purine metabolism, and steroid hormone biosynthesis. Several key DEMs were significantly correlated with the level of clinical parameters, such as serum albumin, IgG, UTP, and cholesterol. Among them, dehydroepiandrosterone sulfate (DHEAS) was considered the reliable diagnostic biomarker in the IMN group. There was an increased abundance of actinobacteria, phylum proteobacteria, and class gammaproteobacterial in IMN patients for host-microbiome origin analysis. Conclusion: Our study revealed the profiles of DEMs from the IMN and HC groups. The result demonstrated that there were disorders of amino acids, nucleotides, and steroids hormones metabolism in IMN patients. The down-regulation of DHEAS may be associated with the imbalance of the immune environment in IMN patients. In host-microbiome origin analysis, the gut microbiota and metabolite disturbances were present in IMN patients.

NFE2L3 as a Potential Functional Gene Regulating Immune Microenvironment in Human Kidney Cancer.

With the increasing incidence and mortality of renal cancer, it is pressing to find new biomarkers and drug targets for diagnosis and treatment. However, as one negative upstream regulator of p53, the prognostic and immunological role of NFE2L3 in renal cancer is still barely known. We investigated the expression, prognostic value, and relevant pathways of NFE2L3 using the datasets from public databases, including The Cancer Genome Atlas Program (TCGA), Genotype-Tissue Expression (GTEx), Cancer Cell Line Encyclopedia (CCLE), and UALCAN. Furthermore, we analyzed the relationship between NFE2L3 expression and the immune microenvironment using distinct methods. We found that NFE2L3 was higher expressed in kidney renal clear cell carcinoma (KIRC) and kidney renal papillary cell carcinoma (KIRP) tissues than adjacent normal tissues. Additionally, we identified NFE2L3 as one survival-related factor for KIRC and KIRP. The enrichment analyses revealed that NFE2L3 was associated with a variety of immune-relevant pathways in KIRC and related to the infiltration ratios of 17 types of immune cells in KIRC patients. Ultimately, we demonstrated nine significantly enriched mutations, such as TP53 and MET, in NFE2L3-expression-changing groups. The elevated expression of NFE2L3 in renal cancerous tissues versus normal tissues is associated with poor outcomes in patients. Besides, NFE2L3 has a role in the regulation of the immune microenvironment in renal cancer patients. The findings of our study provide a potential prognostic biomarker and a new drug target for renal cancer.

Human IL-17 and TNF-α Additively or Synergistically Regulate the Expression of Proinflammatory Genes, Coagulation-Related Genes, and Tight Junction Genes in Porcine Aortic Endothelial Cells.

Immune rejection is the major limitation for porcine xenograft survival in primate recipients. Proinflammatory cytokines play important roles in immune rejection and have been found to mediate the pathological effects in various clinical and experimental transplantation trials. IL-17 and TNF-α play critical pathological roles in immune disorders, such as psoriasis and rheumatoid arthritis. However, the pathological roles of human IL-17 (hIL-17) and human TNF-α (hTNF-α) in xenotransplantation remain unclear. Here we found that hIL-17 and hTNF-α additively or synergistically regulate the expression of 697 genes in porcine aortic endothelial cells (PAECs). Overall, 415 genes were found to be synergistically regulated, while 282 genes were found to be additively regulated. Among these, 315 genes were upregulated and 382 genes were downregulated in PAECs. Furthermore, we found that hIL-17 and hTNF-α additively or synergistically induced the expression of various proinflammatory cytokines and chemokines (e.g., IL1α, IL6, and CXCL8) and decreased the expression of certain anti-inflammatory genes (e.g., IL10). Moreover, hIL-17 plus hTNF-α increased the expression of IL1R1 and IL6ST, receptors for IL1 and IL6, respectively, and decreased anti-inflammatory gene receptor expression (IL10R). hIL-17 and hTNF-α synergistically or additively induced CXCL8 and CCL2 expression and consequently promoted primary human neutrophil and human leukemia monocytic cell migration, respectively. In addition, hIL-17 and hTNF-α induced pro-coagulation gene (SERPINB2 and F3) expression and decreased anti-coagulation gene (TFPI, THBS1, and THBD) expression. Additionally, hIL-17 and hTNF-α synergistically decreased occludin expression and consequently promoted human antibody-mediated complement-dependent cytotoxicity. Interestingly, hTNF-α increased swine leukocyte antigen (SLA) class I expression; however, hIL-17 decreased TNF-α-mediated SLA-I upregulation. We concluded that hIL-17 and hTNF-α likely promote the inflammatory response, coagulation cascade, and xenoantibody-mediated cell injury. Thus, blockade of hIL-17 and hTNF-α together might be beneficial for xenograft survival in recipients.

Dysregulated Microbiota-Driven Gasdermin D Activation Promotes Colitis Development by Mediating IL-18 Release.

The balance between gut microbiota and host is critical for maintaining host health. Although dysregulation of the gut microbiota triggers the development of various inflammatory diseases, including colitis, the molecular mechanism of microbiota-driven colitis development is largely unknown. Here, we found that gasdermin D (GSDMD) was activated during acute colitis. In the dextran sulfate sodium (DSS)-induced colitis model, compared to wild-type mice, Gsdmd-deficient mice had less colitis severity. Mechanistically, GSDMD expression in intestinal epithelial cells (IECs), but not infiltrating immune cells, was critical for GSDMD-mediated colitis progression. Moreover, commensal Escherichia coli (E. coli) largely overgrew during colitis, and then the dysregulated commensal E. coli mediated GSDMD activation. Furthermore, the activated GSDMD promoted the release of interleukin-18 (IL-18), but not the transcript or maturation level of IL-18, which in turn mediated goblet cell loss to induce colitis development. Thus, GSDMD promotes colitis development by mediating IL-18 release, and the microbiota can mediate colitis pathogenesis through regulation of GSDMD activation. Our results provide a potential molecular mechanism by which the microbiota-driven GSDMD activation contributes to colitis pathogenesis.

Proliferative glomerulonephritis with monoclonal immunoglobulin G deposits in a young woman: A case report.

BACKGROUND: Proliferative glomerulonephritis with monoclonal immunoglobulin G (IgG) deposits (PGNMID) is a newly recognized rare disease. The renal pathology is characterized by prominent manifestations of membranous hyperplasia, which are easy to misdiagnose. The clinical symptoms are severe. Massive proteinuria and hypoproteinemia are conspicuous, and most patients are accompanied by renal insufficiency and microscopic hematuria. CASE SUMMARY: A 27-year-old woman was admitted to a hospital for macroscopic hematuria and proteinuria 4 years prior, and renal biopsy in the hospital suggested moderate-to-severe mesangial proliferating glomerulonephritis (MsPGN). She had taken a glucocorticoid, cyclophosphamide, mycophenolate mofetil, and other treatments and achieved brief partial remission. Recently, the patient visited our hospital due to massive proteinuria. Repeated renal biopsy and re-evaluation of the first biopsy obtained 4 years previously revealed monoclonal immunoglobulin deposition in the glomeruli. A bone marrow examination was performed to exclude hematologic malignancy, and a diagnosis of PGNMID was established. The patient showed remission after four cycles of a bortezomib + cyclophosphamide + dexamethasone scheme. CONCLUSION: PGNMID is usually misdiagnosed as MsPGN or membranoproliferative glomerulonephritis. Although it often occurs in middle-aged and elderly individuals, it cannot be readily excluded in young people, even when serum immunofixation electrophoresis is negative. IgG subtype and light chain staining are necessary when this disease is highly suspected. An accurate diagnosis at the earliest stage may avoid the overuse of glucocorticoids and immunosuppressants.

Systemic lupus erythematosus and antineutrophil cytoplasmic antibody-associated vasculitis overlap syndrome in a 77-year-old man: A case report.

BACKGROUND: Systemic lupus erythematosus (SLE) and antineutrophil cytoplasmic antibody-associated vasculitis (AAV) are classically thought to cause renal impairment and small vessel vasculitis with different pathophysiologies. Their overlap constitutes a rare rheumatologic disease. To date, only dozens of such cases with biopsy-proven glomerulonephritis have been reported worldwide typically in women of childbearing age. Here, we present a unique clinical case due to its rarity and individualized treatment of a Chinese man in his eighth decade of life. CASE SUMMARY: A 77-year-old man was admitted to several hospitals for shortness of breath and received nonspecific treatments over the past 3 years. As his symptoms were not completely relieved, he visited our hospital for further treatment. Laboratory examinations revealed kidney dysfunction, severe anaemia, hypocom-plementemia, glomerular proteinuria, and microscopic haematuria. Antinuclear antibodies, as well as anti-dsDNA antibodies, were positive. Computed tomography of the chest showed right pleural effusion. Renal biopsy was performed, and histology suggested crescentic glomerulonephritis, pauci-immune type. After treatment with plasmapheresis, glucocorticoid, and cyclo-phosphamide, the disease was in remission, and the patient remained in a stable condition for over 3 years post-hospital discharge. CONCLUSION: Due to its complexity and rarity, SLE and AAV overlap syndrome is easily misdiagnosed. An accurate diagnosis and treatment at the earliest stage may significantly improve the condition and reduce irreversible organ injury.

Generation of Systemic Lupus Erythematosus Patient-Derived Induced Pluripotent Stem Cells from Blood.

Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease characterized by the production of multiple autoimmune antibodies and potentially involves any organ or tissue with a broad range of clinical manifestations. Conventional therapy still utilizes glucocorticoids and immunosuppressants. However, some patients show inadequate responses to glucocorticoids and immunosuppression, which may induce secondary immune dysfunction and severe infection as well as lead to an increased tumor risk. The lack of in vitro models has hampered progress in understanding and treating SLE. Patient-derived induced pluripotent stem cells (iPSCs) may provide a unique opportunity for modeling in vitro diseases as well as a platform for drug screening in individual patients. We isolated peripheral blood mononuclear cells from blood to explore the establishment of an in vitro model platform for SLE and directly purified CD34+ cells and seeded them for expansion. CD34+ cells were forced to express seven pluripotency factors, OCT4, SOX2, NANOG, LIN28, c-MYC, KLF4, and SV40LT, through transduction in lentiviral vectors. The morphological characteristics of induced pluripotent stem-like cells, such as prominent nucleoli and a high nucleus-to-cytoplasm ratio, were observed. The pluripotency of established SLE patient-derived iPSCs was confirmed by the expression of embryonic stem cell (ESC) markers and the ability of cells to differentiate into multiple cell lines. SLE patient-derived iPSCs exhibited human ESC properties, including morphology; growth characteristics; expression of pluripotency, genes, and surface markers; and teratoma formation. In conclusion, we generated SLE patient-derived iPSCs and validated their pluripotency. This study is a first but critical step that can provide a model platform for research aimed at understanding the SLE mechanism, which may lead to the discovery of new targets or compounds for the treatment of this disease.

Goodpasture syndrome and hemorrhage after renal biopsy: A case report.

BACKGROUND: Goodpasture syndrome (GS) is a rare disease, the morbidity of which is estimated to be 0.5-0.8 per million per year. Hemorrhage is the most serious complication in renal biopsy. Despite the fact that both GS and hemorrhage after renal biopsy are rare, it has not been reported that they are likely to occur in the same patient. CASE SUMMARY: A 30-year-old man with diffuse pulmonary hemorrhage and rapid progressive renal function caused by anti-glomerular basement membrane disease presented atypical symptoms without hemoptysis, accompanied by life-threatening hypoxemia. Plasmapheresis was performed, and glucocorticoids and cyclophosphamide were administered. The patient started to show signs of improvement. Percutaneous renal biopsy is an appropriate diagnostic measure that is commonly safe, but this patient experienced hemorrhage after operation, thus necessitating embolization of the renal artery to stop the bleeding. The patient's condition was improved, and the serum anti-glomerular basement membrane antibody level was 106 AU/mL (normal range: < 24 AU/mL) and slowly decreased. His discharge medications were oral daily prednisone (30 mg) and continued maintenance hemodialysis. CONCLUSION: GS is a rare organ-specific autoimmune disease that is invariably ubiquitous in the lung and kidney areas. Renal biopsy is the appropriate procedure for the treatment of GS disease, although it is an invasive measure.

TNF-α promotes human antibody-mediated complement-dependent cytotoxicity of porcine endothelial cells through downregulating P38-mediated Occludin expression.

BACKGROUND: The major limitation of organ transplantation is the shortage of available organs. Xenotransplantation is considered to be an effective way to resolve the problem. Immune rejection is a major hurdle for the successful survival of pig xenografts in primate recipients. Cytokines play important roles in inflammation and many diseases including allotransplantation, however, their roles in xenotransplantation have been less well investigated. METHODS: We assessed the role of several cytokines in xenotransplantation using an in vitro model of human antibody-mediated complement-dependent cytotoxicity (CDC). Porcine aortic endothelial cells (PAECs) and porcine iliac endothelial cells (PIECs) were selected as target cells. The complement regulators (CD46, CD55 and CD59) and junction protein genes were assessed by real-time PCR, flow cytometry, or western-blotting assay. Flow cytometry assay was also used to evaluate C3 and C5b-9 deposition, as well as the extent of human IgM and IgG binding to PIECs. Gene silencing was used to reduce genes expression in PIECs. Gene overexpression was mediated by adenovirus or retrovirus. RESULTS: Recombinant human TNF-α increased the cytotoxicity of PAECs and PIECs in a human antibody-mediated CDC model. Unexpectedly, we found that the expression of complement regulators (CD46, CD55 and CD59) increased in PIECs exposed to human TNF-α. Human TNF-α did not modify C3 or C5b-9 deposition on PIECs. The extent of human IgM and IgG binding to PIECs was not affected by human TNF-α. Human TNF-α decreased the expression of Occludin in PIECs. Gene silencing and overexpression assay suggested that Occludin was required for human TNF-α-mediated cytotoxicity of PIECs in this model. P38 gene silencing or inhibition of P38 signaling pathway with a specific inhibitor, SB203580, inhibited the reduction of Occludin expression induced by TNF-α, and suppressed TNF-α-augmented cytotoxicity of PIECs. CONCLUSION: Our data suggest that human TNF-α increases the cytotoxicity of porcine endothelial cells in a human antibody-mediated CDC model by downregulating P38-dependent Occludin expression. Pharmacologic blockade of TNF-α is likely to increase xenograft survival in pig-to-primate organ xenotransplantation.

c-Jun N-terminal kinases differentially regulate TNF- and TLRs-mediated necroptosis through their kinase-dependent and -independent activities.

Tumor necrosis factor (TNF) and Toll-like receptor (TLR)3/TLR4 activation trigger necroptotic cell death through downstream signaling complex containing receptor-interacting protein kinase 1 (RIPK1), RIPK3, and pseudokinase mixed lineage kinase-domain-like (MLKL). However, the regulation of necroptotic signaling pathway is far less investigated. Here we showed that c-Jun N-terminal kinases (JNK1 and JNK2) displayed kinase-dependent and -independent functions in regulating TNF- and TLRs-mediated necroptosis. We found that RIPK1 and RIPK3 promoted cell-death-independent JNK activation in macrophages, which contributed to pro-inflammatory cytokines production. Meanwhile, blocking the kinase activity of JNK dramatically reduced TNF and TLRs-induced necroptotic cell death. Consistently, inhibition of JNK activity protected mice from TNF-induced death and Staphylococcus aureus-mediated lung damage. However, depletion of JNK protein using siRNA sensitized macrophages to necroptosis that was triggered by LPS or poly I:C but still inhibited TNF-induced necroptosis. Mechanistic studies revealed that RIPK1 recruited JNK to the necrosome complex and their kinase activity was required for necrosome formation and the phosphorylation of MLKL in TNF- and TLRs-induced necroptosis. Loss of JNK protein consistently suppressed the phosphorylation of MLKL and necrosome formation in TNF-triggered necroptosis, but differentially promoted the phosphorylation of MLKL and necrosome formation in poly I:C-triggered necroptosis by promoting the oligomeration of TRIF. In conclusion, our findings define a differential role for JNK in regulating TNF- and TLRs-mediated necroptosis by their kinase or scaffolding activities.

Porcine IL-6, IL-1ß, and TNF-α regulate the expression of pro-inflammatory-related genes and tissue factor in human umbilical vein endothelial cells.

Whether porcine cytokines are induced after pig-to-primate xenotransplantation and activate human cells remains unknown. First, we investigated the regulation of porcine IL-6, IFN-γ, IL-1ß, and TNF-α in xenotransplantation using an in vitro model in which porcine aortic endothelial cells (PAECs) and porcine peripheral blood mononuclear cells (PBMCs) were stimulated with human serum. Downstream cytokines/chemokines were monitored. Pro-inflammatory cytokines (IL-6, IFN-γ, and IL-1ß) and chemokines (IL-8, MCP-1, and CXCL2) were upregulated in the both cell types. TNF-α was induced 10-fold in PAECs, but not in PBMCs. Then, we assessed the role of porcine IL-6, IFN-γ, IL-1ß, and TNF-α in xenotransplantation using western blotting and real-time PCR. Human umbilical vein endothelial cells (HUVECs) were selected as the target cells. Signaling pathways and downstream genes, such as those related to adhesion, inflammation, and coagulation, and chemokines were investigated. Porcine IL-1ß and TNF-α significantly activated NF-κB and P38, and STAT3 was activated by porcine IL-6 in HUVECs. The adhesion genes (E-selectin, VCAM-1, and ICAM-1), inflammatory cytokines (IL-6, IL-1ß, and TNF-α), chemokines (MCP-1 and IL-8), and the pro-coagulation gene (tissue factor) were upregulated by porcine IL-1ß and TNF-α. Porcine IL-6 increased the expression of ICAM-1, IL-6, MCP-1, and tissue factor, but decreased IL-8 expression slightly. Surprisingly, porcine IFN-γ could not activate STAT1 or regulate the expression of any of the above genes in HUVECs. In conclusion, these findings suggest that porcine IL-6, IL-1ß, and TNF-α activate HUVECs and regulate downstream genes expression, which may promote inflammation and coagulation response after xenotransplantation.