Psoriasis is a chronic inflammatory skin disease with multiple genetic backgrounds, whose etiology and pathogenesis are still unclear. Complex T-cell immune imbalance has been demonstrated to play an important role in pathogenesis of psoriasis. This study reported that microRNA-126 (miR-126) expression was decreased in CD4⁺ T cells of both psoriasis patients and psoriasis-like mouse models and its expression was negatively correlated with the Psoriasis Area and Severity Index (PASI) score of psoriasis patients. Conditional Mir126 knockout in mouse CD4⁺ T cells can obviously aggravate the psoriasis-like dermatitis and promote T-helper (Th)1 and Th17 cells' infiltration in spleen of imiquimod (IMQ)-induced psoriasis-like mouse model. In addition, the mRNA expression of Il17a and Il17f were significantly increased in mouse naïve CD4⁺ T cells with Mir126 knockout after stimulating with CD3 and CD28. Compared with naïve CD4⁺ T cells, the expression of Mir126 was decreased in Th17 cells, and Mir126 knockout notably promoted the differentiation of naïve CD4⁺ T cells to Th17 cells as well as the mRNA expression of Il17a, Il17f, Rorc, and Il23R. Our results revealed that decreased miR-126 in psoriatic CD4⁺ T cells might accelerate the formation of skin lesions through promoting the differentiation of Th17 cells, thus suggesting a potential intervention target for psoriasis.

Keywords: CD4+ T cells; T-helper 17 cells; differentiation; microRNA-126; psoriasis.

Benzene is a common environmental carcinogen that induces leukemia. Studies suggest that metabolic disorder has a relationship with the toxicity of benzene. Pyruvate kinase M2 (PKM2) is a key rate-limiting enzyme in glycolysis. However, the upstream and downstream regulatory mechanisms of PKM2 in benzene-induced hematotoxicity and the therapeutic effects of targeting PKM2 in vivo are unclear. This study aims to provide insights into the new mechanism of benzene-induced hematotoxicity and reveal the therapeutic significance of targeting PKM2. Herein, we demonstrated that PKM2-dependent glycolysis contributes to benzene-induced hematotoxicity by regulating inflammation reaction. Mechanistically, acetylated proteomics revealed that 1,4-benzoquinone (1,4-BQ) induced acetylation of PKM2 at position K66, and this modification contributed to the increase of PKM2 expression and can be inhibited by inhibition of acetyltransferase GCN5. Meanwhile, the elevated PKM2 was shown to prompt the activation of nuclear phosphorylated Stat3 (p-Stat3) and IL17A. Clinically, pharmacological inhibition of PKM2 alleviated the blood toxicity induced by benzene, which was mainly characterized by an increase in routine blood parameters and improvement of hematopoietic imbalance. Besides, elevated PKM2 is a promising biomarker in people occupationally exposed to benzene. Overall, we identified PKM2/p-Stat3/IL-17A axis participates in the hematotoxicity of benzene, and targeting PKM2 has certain therapeutic implications in hematologic diseases.

Keywords: Acetylation; Benzene; Glycolysis; Hematotoxicity; Inflammation; PKM2.

The alveolar bone is a unique osseous tissue due to the presence of the teeth and the proximity of commensal oral microbes. Commensal microbe effects on alveolar bone homeostasis have been attributed to the oral microbiota, yet the impact of commensal gut microbes is unknown. Study purpose was to elucidate whether commensal gut microbes regulate osteoimmune mechanisms and skeletal homeostasis in alveolar bone. Male C57BL/6T germfree (GF) littermate mice were maintained as GF or monoassociated with segmented filamentous bacteria (SFB), a commensal gut bacterium. SFB has been shown to elicit broad immune response effects, including the induction of T_HIL17A immunity, which impacts the development and homeostasis of host tissues. SFB colonized the gut, but not oral cavity, and increased IL17A levels in the ileum and serum. SFB had catabolic effects on alveolar bone and non-oral skeletal sites, which was attributed to enhanced osteoclastogenesis. The alveolar bone marrow of SFB vs. GF mice had increased dendritic cells, activated helper T-cells, T_HHIL17A, or TNF to elucidate osteoblast-derived signaling factors contributing to the pro-osteoclastic phenotype in SFB mice. Treatment of RAW264.7 osteoclastic cells with supernatants from vehicle-stimulated SFB vs. GF osteoblasts recapitulated the osteoclast phenotype found in vivo. Supernatants from TNF-stimulated osteoblasts normalized RAW264.7 osteoclast endpoints across SFB and GF cultures, which was dependent on the induction of CXCL1 and CCL2. This report reveals that commensal gut microbes have the capacity to regulate osteoimmune processes in alveolar bone. Outcomes from this investigation challenge the current paradigm that alveolar bone health and homeostasis is strictly regulated by oral microbes.

Gaucher disease is a lysosomal storage disease, which happens due to mutations in GBA1/Gba1 that encodes the enzyme termed as lysosomal acid β-glucosidase. The major function of this enzyme is to catalyze glucosylceramide (GC) into glucose and ceramide. The deficiency of this enzyme and resultant abnormal accumulation of GC cause altered function of several of the innate and adaptive immune cells. For example, augmented infiltration of T cells contributes to the increased production of pro-inflammatory cytokines, (e.g., IFNγ, TNFα, IL6, IL12p40, IL12p70, IL23, and IL17A/F). This leads to tissue damage in a genetic mouse model (Gba1^9V/-) of Gaucher disease. The cellular mechanism(s) by which increased tissue infiltration of T cells occurs in this disease is not fully understood. Here, we delineate role of the CXCR3 receptor and its exogenous C-X-C motif chemokine ligand 9 (CXCL9) in induction of increased tissue recruitment of CD4⁺ T and CD8⁺ T cells in Gaucher disease. Intracellular FACS staining of macrophages (Mϕs) and dendritic cells (DCs) from Gba1^9V/- mice showed elevated production of CXCL9. Purified CD4⁺ T cells and the CD8⁺ T cells from Gba1^9V/- mice showed increased expression of CXCR3. Ex vivo and in vivo chemotaxis experiments showed CXCL9 involvement in the recruitment of Gba1^9V/- T cells. Furthermore, antibody blockade of the CXCL9 receptor (CXCR3) on T cells caused marked reduction in CXCL9- mediated chemotaxis of T cells in Gba1^9V/- mice. These data implicate abnormalities of the CXCL9-CXCR3 axis leading to enhanced tissue recruitment of T cells in Gaucher disease. Such results provide a rationale for blockade of the CXCL9/CXCR3 axis as potential new therapeutic targets for the treatment of inflammation in Gaucher disease.

Keywords: chemokine; chemokine receptor; inflammation; lysosomal storage disease.

Aims: Atherosclerosis is a chronic inflammatory disease of the vessel wall controlled by local and systemic immune responses. The role of interleukin-23 receptor (IL-23R), expressed in adaptive immune cells (mainly T helper 17 cells) and γδ T cells, in atherosclerosis is only incompletely understood. Here we investigated the vascular cell types expressing IL-23R and addressed the function of IL-23R and γδ T cells in atherosclerosis.

Method and results: IL-23R+ cells were frequently found in the aortic root in contrast to the aorta in low density lipoprotein receptor deficient IL-23R reporter mice (Ldlr-/-Il23rgfp/+), and mostly identified as γδ T cells that express IL-17 and GM-CSF. scRNA-seq confirmed γδ T cells as the main cell type expressing Il23r and Il17a in the aorta. Ldlr-/-Il23rgfp/gfp mice deficient in IL-23R showed a loss of IL-23R+ cells in the vasculature, and had reduced atherosclerotic lesion formation in the aortic root compared to Ldlr-/- controls after 6 weeks of high fat diet feeding. In contrast, Ldlr-/-Tcrδ-/- mice lacking all γδ T cells displayed unaltered early atherosclerotic lesion formation compared to Ldlr-/- mice. In both HFD-fed Ldlr-/-Il23rgfp/gfp and Ldlr-/-Tcrδ-/- mice a reduction in the plaque necrotic core area was noted as well as an expansion of splenic regulatory T cells. In vitro, exposure of bone marrow-derived macrophages to both IL-17A and GM-CSF induced cell necrosis, and necroptotic RIP3K and MLKL expression, as well as inflammatory mediators.

Conclusions: IL-23R+ γδ T cells are predominantly found in the aortic root rather than the aorta and promote early atherosclerotic lesion formation, plaque necrosis and inflammation at this site. Targeting IL-23R may thus be explored as a therapeutic approach to mitigate atherosclerotic lesion development.

Translational perspective: The mechanisms and cell types contributing to early inflammation and lesion formation are incompletely understood. Here we demonstrate that the aortic root harbors a population of IL23R-dependent γδ T cells that can release IL-17 and GM-CSF, and both cytokines together induce macrophage inflammation and necroptosis. IL-23R+ γδ T cells locally promote early lesion formation in the aortic root and contribute to the expansion of the necrotic core, a hallmark of vulnerable atherosclerotic lesions. Targeting IL-23R or IL-23 itself could thus be further explored as a therapeutic option in early atherosclerosis.

Detection of individual cytokines in routine biopsies from patients with inflammatory skin diseases has the potential to personalize diagnosis and treatment selection, but this approach has been limited by technical feasibility. We evaluate whether a chromogen-based RNA in situ hybridization approach can be used to detect druggable cytokines in psoriasis and atopic dermatitis. A series of psoriasis (n = 20) and atopic dermatitis (n = 26) biopsies were stained using RNA in situ hybridization for IL4, IL12B (IL-12/23 p40), IL13, IL17A, IL17F, IL22, IL23A (IL-23 p19), IL31, and TNF (TNF-α). NOS2 and IFNG, canonical psoriasis biomarkers, were also included. All 20 of the psoriasis cases were positive for IL17A, which tended to be the predominant cytokine, although some cases had relatively higher levels of IL12B, IL17F, or IL23A. The majority of cytokine expression in psoriasis was epidermal. A total of 22 of 26 atopic dermatitis cases were positive for IL13, also at varying levels; a subset of cases had significant IL4, IL22, or IL31 expression. Patterns were validated in independent bulk RNA-sequencing and single-cell RNA-sequencing datasets. Overall, RNA in situ hybridization for cytokines appears highly specific with virtually no background staining and may allow for individualized evaluation of treatment-relevant cytokine targets in biopsies from patients with inflammatory skin disorders.

Keywords: AD, atopic dermatitis; Cat, catalog; DC, dendritic cell; IHC, immunohistochemistry; ILC, innate lymphoid cell; KC, keratinocyte; LC, Langerhans cell; RISH, RNA in situ hybridization; RNA-seq, RNA sequencing; scRNA-seq, single-cell RNA sequencing.

Female NOD mice develop autoimmune diabetes spontaneously without extrinsic manipulation. Previously, we have shown that weekly administration of the prediabetic female NOD mice with the histone modifier Trichostatin A (TSA) prevented diabetes onset. Herein we show that T lymphocytes from diabetic mice transferred diabetes into immunodeficient NOD.scid recipients while those isolated from drug-treated mice displayed reduced disease-causing ability. Drug treatment also repressed T cell receptor-mediated IFN-γ transcription. Splenic CD4⁺ T-cells purified from prediabetic mice could be polarized into IFN-γ -producing Th1 and IL-17A-expressing Th17 subsets ex vivo. Adoptive transfer of these cells into immunocompromised NOD.scid mice caused diabetes comparably. Polarized Th1 cells were devoid of IL-17A-producing cells and did not transdifferentiate into Th17 cells in the spleen of immunodeficient recipients. However, polarized Th17 cell preparation had a few contaminant Th1 cells. Adoptive transfer of polarized Th17 cells into NOD.scid recipients led to IFN-γ transcription in recipient splenocytes. Notably, TSA treatment of prediabetic mice abolished the ability of CD4⁺ T-cells to differentiate into diabetogenic Th1 and Th17 cells ex vivo. This was accompanied by the absence of Ifng and Il17a transcription in the spleen of NOD.scid recipients receiving cells, respectively cultured under Th1 and Th17 polarizing conditions. Significantly, the histone modifier restored the ability of CD4⁺ but not CD8⁺ T-cells to undergo CD3-mediated apoptosis ex vivo in a caspase-dependent manner. These results indicate that the histone modifier bestowed protection against type 1 diabetes via negative regulation of signature lymphokines and restitution of self-tolerance in CD4⁺ T cells.

Keywords: Epigenetics; Self-tolerance; Th1 cells; Th17 cells; Trichostatin A; and Type 1 diabetes.

Background: Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS) mediated by autoimmunity. No objective clinical indicators are available for the diagnosis and prognosis of MS. Extracellular proteins are most glycosylated and likely to enter into the body fluid to serve as potential biomarkers. Our work will contribute to the in-depth study of the functions of extracellular proteins and the discovery of disease biomarkers.

Methods: MS expression profiling data of the human brain was downloaded from the Gene Expression Omnibus (GEO). Extracellular protein-differentially expressed genes (EP-DEGs) were screened by protein annotation databases. GO and KEGG were used to analyze the function and pathway of EP-DEGs. STRING, Cytoscape, MCODE and Cytohubba were used to construct a protein-protein interaction (PPI) network and screen key EP-DEGs. Key EP-DEGs levels were detected in the CSF of MS patients. ROC curve and survival analysis were used to evaluate the diagnostic and prognostic ability of key EP-DEGs.

Results: We screened 133 EP-DEGs from DEGs. EP-DEGs were enriched in the collagen-containing extracellular matrix, signaling receptor activator activity, immune-related pathways, and PI3K-Akt signaling pathway. The PPI network of EP-DEGs had 85 nodes and 185 edges. We identified 4 key extracellular proteins IL17A, IL2, CD44, IGF1, and 16 extracellular proteins that interacted with IL17A. We clinically verified that IL17A levels decreased, but Del-1 and resolvinD1 levels increased. The diagnostic accuracy of Del-1 (AUC: 0.947) was superior to that of IgG (AUC: 0.740) with a sensitivity of 82.4% and a specificity of 100%. High Del-1 levels were significantly associated with better relapse-free and progression-free survival.

Conclusion: IL17A, IL2, CD44, and IGF1 may be key extracellular proteins in the pathogenesis of MS. IL17A, Del-1, and resolvinD1 may co-regulate the development of MS and Del-1 is a potential biomarker of MS. We used bioinformatics methods to explore the biomarkers of MS and validated the results in clinical samples. The study provides a theoretical and experimental basis for revealing the pathogenesis of MS and improving the diagnosis and prognosis of MS.

Keywords: bioinformatics analysis; biomarkers; extracellular protein; protein-protein interactions; relapsing-remitting multiple sclerosis.

Cytokines are key modulators of immune response, and dysregulated production of proinflammatory and anti-inflammatory cytokines contributes to the pathogenesis of influenza A(H1N1)pdm09 virus infection. Cytokine production is impacted by single nucleotide polymorphisms (SNPs) in the genes coding for them. In the present study, SNPs in the IL6, TNFA, IFNG, IL17A, IL10, and TGFB were investigated for their association with disease severity and fatality in influenza A(H1N1)pdm09-affected patients with mild disease (n = 293) and severe disease (n = 86). Among those with severe disease, 41 patients had fatal outcomes. In a subset of the patients, levels of IL-2, IL-4, IL-6, IL-10, TNF, IFN-γ, and IL-17 were assayed in the plasma for their association with severe disease. The frequency of TNFA rs1800629 G/A allele was significantly higher in severe cases and survived severe cases group compared to that of those with mild infection (OR with 95% for mild vs. severe cases 2.95 (1.52-5.73); mild vs. survived severe cases 4.02 (1.84-8.82)). IL10 rs1800896-rs1800872 G-C haplotype was significantly lower (OR with 95% 0.34 (0.12-0.95)), while IL10 rs1800896-rs1800872 G-A haplotype was significantly higher (OR with 95% 12.11 (2.23-76.96)) in fatal cases group compared to that of the mild group. IL-6 and IL-10 levels were significantly higher in fatal cases compared to that of survived severe cases. IL-6 levels had greater discriminatory power than IL-10 to predict progression to fatal outcome in influenza A(H1N1)pdm09 virus-infected patients. To conclude, the present study reports the association of TNFA and IL10 SNPs with severe disease in Influenza A(H1N1)pdm09 virus-infected subjects. Furthermore, IL-6 levels can be a potential biomarker for predicting fatal outcomes in Influenza A(H1N1)pdm09 virus infected subjects.

Keywords: TNF-α; cytokines; gene polymorphisms; influenza A(H1N1)pdm09; interleukins.

Diacetyl (DA) is a highly reactive alpha diketone associated with flavoring-related lung disease. In rodents, acute DA vapor exposure can initiate an airway-centric, inflammatory response. However, this immune response has yet to be fully characterized in the context of flavoring-related lung disease progression. The following studies were designed to characterize the different T cell populations within the lung following repetitive DA vapor exposures. Sprague-Dawley rats were exposed to 200 parts-per-million DA vapor for 5 consecutive days × 6 h/day. Lung tissue and bronchoalveolar lavage fluid (BALF) were analyzed for changes in histology by H&E and Trichrome stain, T cell markers by flow cytometry, total BALF cell counts and differentials, BALF IL17a and total protein immediately, 1 and 2 weeks post-exposure. Lung histology and BALF cell composition demonstrated mixed, granulocytic lung inflammation with bronchial lymphoid aggregates at all time points in DA-exposed lungs compared to air controls. While no significant change was seen in percent lung CD3⁺, CD4⁺, or CD8⁺ T cells, a significant increase in lung CD4⁺CD25⁺ T cells developed at 1 week that persisted at 2 weeks post-exposure. Further characterization of this CD4⁺CD25⁺ T cell population identified Foxp3⁺ T cells at 1 week that failed to persist at 2 weeks. Conversely, BALF IL-17a increased significantly at 2 weeks in DA-exposed rats compared to air controls. Lung CD4⁺CD25⁺ T cells and BALF IL17a correlated directly with BALF total protein and inversely with rat oxygen saturations. Repetitive DA vapor exposure at occupationally relevant concentrations induced mixed, granulocytic lung inflammation with increased CD4⁺CD25⁺ T cells in the rat lung.

Keywords: 2,3-butanedione; CD4+CD25+ T cells; airways; bronchiolitis obliterans; diacetyl; flavorings-related lung disease; hazard potential; intoxication; lung; occupational hazard.

Background: Candida antigen injection is one of the most widely used intralesional immunotherapy in the treatment of warts. It acts through the induction of systemic immune response. The pattern of cytokines production may play an integral role in its mechanism of action.

Aim: To investigate the possible relation between serum levels of IL17 and MIF, and the clinical response to intralesional Candida antigen in multiple common warts.

Methods: A total of 90 patients with multiple common warts were divided into 2 groups. Sixty patients received intralesional Candida antigen injection into the largest wart, controlled against thirty patients who had intralesional saline, as placebo. The injection was done at a 2-week interval for 5 doses. Blood samples were obtained from both groups, and serum levels of IL17A and MIF were estimated at baseline and 2 weeks after the last session using ELISA kits.

Results: Complete clearance of warts was statistically higher in the Candida antigen group (40% of the patients) compared to the saline group (p < 0.05). The serum levels of IL17 had significantly declined from baseline, while the level of MIF had risen after intralesional Candida antigen injection, but not in the saline group. At a cutoff level of 316 pg/ml, IL17 had a sensitivity of 83.3% to predict therapeutic response.

Conclusion: IL17A and MIF may have possible roles in the mechanism of action of Candida antigen in the treatment of common warts. At a certain level, serum IL17A may be a potential predictor of response to treatment.

Keywords: Candida antigen; IL17; MIF; warts.

Genetic and immune factors play an important role in tuberculosis. Under different ethnicities and genetic backgrounds, different immune and inflammation-related gene polymorphisms may confer different susceptibility to tuberculosis. This study investigated the relationship between immune and inflammation-related gene polymorphism and susceptibility to tuberculosis in Xinjiang Uyghur population, China. In this case-control study, we enrolled 507 pulmonary tuberculosis patients and 454 healthy controls from Southern Xinjiang. single nucleotide polymorphism (SNP) genotyping was performed. The 12 SNPs of nine immune and inflammation-related genes (including TNF rs361525, IL6 rs2066992 and rs1524107, IL17A rs3748067, IL17F rs763780, VDR rs731236, rs2228570 and rs1544410, IFNGR1 rs1327474, P2RX7 rs3751143, CTAGE1 rs4331426 and Toll-like receptor 4 (TLR4) rs4986790) and their relationship with tuberculosis were evaluated. The T allele and TT genotype of IL-6 rs2066992 and rs1524107 increased the risk of active tuberculosis. The C allele of IFNGR1 rs1327474 was related to the reduced risk of tuberculosis in the Xinjiang Uyghur population. The G allele and AG/GG genotypes of TLR4 rs4986790 were associated with an increased risk of tuberculosis (p < .05). Furthermore, haplotype analysis found that the haplotype TT of interleukin (IL)-6 was a risk factor, whereas the CG type was a protective factor for active tuberculosis in the Xinjiang Uyghur population. There were three immune and inflammation-related genes (IL-6, IFNGR1 and TLR4) and a total of four SNPs (rs2066992, rs1524107, rs1327474 and rs4986790) related to the susceptibility of the Uyghur population to tuberculosis. Our findings may provide evidence for further understanding the mechanism of tuberculosis susceptibility in the Xinjiang Uyghur population.

Keywords: SNP; immune and inflammation-related gene; susceptibility; tuberculosis.

Antisynthetase syndrome (ASSD) is a rare multisystemic connective tissue disease affecting the skin, joints, muscles, and lungs, characterized by anti-aminoacyl transfer-RNA-synthetases (anti-tRNA) autoantibodies production, being anti-Jo1 the most frequent. We included one-hundred twenty-one ASSD patients and 340 healthy subjects (HS), and also, we divided the case group into anti-Jo1 and non-anti-Jo1. Two single nucleotide polymorphisms (SNPs) in the IL17A gene were evaluated. Anti-Jo1 was the most common anti-tRNA antibody in our cohort, and the most frequent tomographic pattern was non-specific interstitial pneumonia (NSIP). Anti-Jo1 ASSD patients had higher levels of creatine phosphokinase than the non-anti-Jo1 group. Significant differences in genotype frequencies with rs8193036/CC between anti-Jo1 vs. non-anti-Jo1 ASSD patients (p < 0.001), maintaining the association after Bonferroni correction (p = 0.002). Additionally, in the anti-Jo1 group vs. HS comparison, we found a statistically significant difference with the same SNP (p = 0.018, OR = 2.91, 95% CI = 1.15-7.35), maintaining the association after Bonferroni correction (p = 0.036). The rs8193036/CC genotype in IL17A is associated with ASSD patients with anti-Jo1. Also, anti-Jo1 and non-anti-Jo1 patients display differences in genotype frequencies.

Keywords: ASSD; IL17A; SNPs; anti-Jo1; anti-tRNA.

DNA methylation is an epigenetic mechanism controlling gene expression, and reduced methylation is associated with increased gene expression. We hypothesized that IL-17 cytokines are regulated by DNA methylation, are elevated in the circulation of preeclamptic women, and stimulate vascular neutrophil chemokine expression, which could account for vascular infiltration of neutrophils in preeclampsia. We found significantly reduced DNA methylation of IL17A, IL17E, and IL17F genes in omental arteries of preeclamptic women, significantly reduced methylation of IL2, which regulates IL-17-producing T-lymphocytes, and significantly reduced methylation of genes encoding neutrophil chemokines and TNFα receptors related to lymphocyte function. Maternal plasma levels of IL-17A were significantly elevated in the second trimester of preeclamptic pregnancy as compared to normal pregnancy. To test if methylation regulates IL-17 cytokines, a lymphocyte cell line (Jurkat) was cultured with a hypomethylating agent. Hypomethylation increased expression of IL17E (aka IL25), IL17F, and IL2. IL17A was not expressed by Jurkat cells. To test the potential role of IL-17 cytokines in vascular neutrophil infiltration associated with preeclampsia, human vascular smooth muscle cells were cultured with IL-17 cytokines. IL-17A, but not IL-17E or IL-17F, increased gene expression of neutrophil chemokines (IL-8, CXCL5, and CXCL6) that are increased in vascular smooth muscle of preeclamptic women. The monocyte chemokine, CCL-2, was not increased. TNFα also increased neutrophil chemokines. IL-17 cytokines are regulated by DNA methylation; IL-17A is elevated in preeclampsia and stimulates expression of neutrophil chemokines in vascular smooth muscle. IL-17A could be responsible for vascular infiltration of neutrophils in preeclampsia.

Keywords: Chemokines; DNA methylation; Interleukin-17; Neutrophils; Preeclampsia; TNFα.

Ulcerative colitis (UC) is a chronic idiopathic inflammatory disorder of colon. Costunolide, the main active constituent of Radix Aucklandiae, has been demonstrated to possess anti-inflammatory and immunomodulation activities. The aim of this study is to investigate the effect of costunolide on UC induced by dextran sulfate sodium (DSS). Results showed that oral administration of costunolide significantly improved the disease active index (DAI), rescued the reduction of colon length, downregulated myeloperoxidase (MPO) activity, alleviated the pathological changes, and decreased the levels of proinflammatory cytokines in colons of colitis mice. Costunolide also rebalanced Th17/Treg cells in colons, mesenteric lymph nodes and spleen, as indicated by decreased percentages of Th17 cells and reduced mRNA expressions of Rorc, Il17a. Interestingly, the in vitro experiment showed that no significant change in dendritic cell maturation, mRNA expressions of Ifng, Il6 and Treg cell differentiation, but a significant decreased Th17 cell differentiation was observed upon costunolide treatment. Deeper mechanistic studies showed that costunolide triggered the prolyl hydroxylase 2 (PHD2)-triggered proline hydroxylation-ubiquitination-proteasome degradation of HIF-1α, which in turn inactivated glycolytic process in Th17 rather than Treg cells. These findings clearly suggest that inhibition of HIF-1α-mediated glycolysis by costunolide is specifically responsible for Th17 cell differentiation and subsequent alleviation of UC and sets the stage for a new perspective on immune-metabolism therapy for colitis.

Keywords: Colitis; Costunolide; Glycolysis; HIF-1α; Th17 cell differentiation.

Background: Severe spermatogenic failure (SpF) represents the most extreme manifestation of male infertility, as it decreases drastically the semen quality leading to either severe oligospermia (SO, < 5 million spermatozoa/mL semen) or non-obstructive azoospermia (NOA, complete lack of sperm in the ejaculate without obstructive causes).

Objectives: The main objective of the present study is to analyze in the Iberian population the effect of 6 single-nucleotide polymorphisms (SNPs) previously associated with NOA in Han Chinese through genome-wide association studies (GWAS), and to establish their possible functional relevance in the development of specific SpF patterns.

Materials and methods: We genotyped 674 Iberian infertile men (including 480 NOA and 194 SO patients) and 1058 matched unaffected controls for the GWAS-associated variants PRMT6-rs12097821, PEX10-rs2477686, CDC42BPA-rs3000811, IL17A-rs13206743, ABLIM1-rs7099208, and SOX5-rs10842262. Their association with SpF, SO, NOA, and different NOA phenotypes was evaluated by logistic regression models, and their functional relevance was defined by comprehensive interrogation of public resources.

Results: ABLIM1-rs7099208 was associated with SpF under both the additive (OR=0.86, P=0.036) and dominant models (OR=0.78, P=0.026). The CDC42BPA-rs3000811 minor allele frequency was significantly increased in the subgroup of NOA patients showing maturation arrest (MA) of germ cells compared to the remaining NOA cases under the recessive model (OR=4.45, P=0.044). The PEX10-rs2477686 SNP was associated with a negative testicular sperm extraction (TESE) outcome under the additive model (OR=1.32, P=0.034). The analysis of functional annotations suggested that these variants affect the testis-specific expression of nearby genes and that lincRNA may play a role in SpF.

Conclusions: Our data support the association of 3 previously reported NOA risk variants in Asians (ABLIM1-rs7099208, CDC42BPA-rs3000811, and PEX10-rs2477686) with different manifestations of SpF in Iberians of European descent, likely by influencing gene expression and lincRNA deregulation.

Keywords: Genetic association study; azoospermia; impaired spermatogenesis; male infertility; non-obstructive; severe oligospermia.

Background: There is evidence to consider that the tumor microenvironment (TME) composition associates with antitumor immune response, and may predict the outcome of various non-Hodgkin lymphoma subtypes. However, in the case of mantle cell lymphoma (MCL), a rare and aggressive disease, there is lacking a detailed study of the TME components, as well as an integrative approach among them in patients' samples. Also, from the genetic point of view, it is known that single nucleotide variants (SNVs) in immune-response genes are among important regulators of immunity. At present, it is uncertain whether SNVs in candidate immune-response genes and the TME composition are able to alter the prognosis in MCL.

Methods: We assessed a detailed TME composition in 88 MCL biopsies using immunohistochemistry, which was automatically analyzed by pixel counting (Aperio system). We also genotyped SNVs located in candidate immune-response genes (IL12A, IL2, IL10, TGFB1, TGFBR1, TGFBR2, IL17A, IL17F) in 95 MCL patients. We tested whether the SNVs could modulate the respective protein expression and TME composition in the tumor compartment. Finally, we proposed survival models in rituximab-treated patients, considering immunohistochemical and SNV models.

Results: High FOXP3/CD3 ratios (p = 0.001), high IL17A levels (p = 0.003) and low IL2 levels (p = 0.03) were individual immunohistochemical predictors of poorer survival. A principal component, comprising high quantities of macrophages and high Ki-67 index, also worsened outcome (p = 0.02). In the SNV model, the CC haplotype of IL10 (p < 0.01), the GG genotype of IL2 rs2069762 (p = 0.02) and the AA+AG genotypes of TGFBR2 rs3087465 (p < 0.01) were independent predictors of outcome. Finally, the GG genotype of TGFB1 rs6957 associated with lower tumor TGFβ levels (p = 0.03) and less CD163+ macrophages (p = 0.01), but did not modulate patients' survival.

Conclusions: Our results indicate that the TME composition has relevant biological roles in MCL. In this setting, immunohistochemical detection of T-reg cells, IL17A and IL2, coupled with SNV genotyping in IL10, TGFBR2 and IL2, may represent novel prognostic factors in this disease, following future validations.

Keywords: Immunohistochemistry; Mantle cell lymphoma; Prognostic factors; SNVs; Tumor microenvironment.

Blinatumomab is a bispecific T-cell-engaging antibody approved for treatment of relapsed/refractory (r/r) ALL, with 40-50% complete response (CR)/CR with incomplete count recovery (CRi). Cytokine release syndrome (CRS) as a major adverse effect after blinatumomab therapy. Here, we evaluated the possible association between single nucleotide polymorphisms (SNPs) in cytokine genes, disease response, and CRS in r/r ALL patients who received blinatumomab between 2012 and 2017 at our center (n=66), using patients' archived DNA samples. With a median duration of 9.5 months (range:1-37), 37 patients (56.1%) achieved CR/CRi, 54 (81.8%) experienced CRS (G1: n=35, G2: n=14, G3: n=5), and 9 (13.6%) developed neurotoxicity. By multivariable analysis, after adjusting for high disease burden, one SNP on IL2 (rs2069762), Odds Ratio (OR)= 0.074 (95% CI: NE-0.43, p=0.01), and one SNP on IL17A (rs4711998), OR= 0.28 (95% CI: 0.078-0.92, p= 0.034) were independently associated with CR/CRi. None of the analyzed SNPs were associated with CRS. To our knowledge, this is the first study demonstrating a possible association between treatment response to blinatumomab and SNPs. Our hypothesis-generated data suggest a potential role for IL-17 and IL-2 in blinatumomab response and justify a larger confirmatory study, which may lead to personalized blinatumomab immunotherapy for B-ALL.

Keywords: acute lymphoblastic leukemia; blinatumomab; cytokine gene polymorphism.

Herpes zoster results from latent varicella zoster virus reactivation in the dorsal root ganglia, causing blistering rash along the dermatomal distribution and post-herpetic neuralgia. Increasing studies indicated that there may be a correlation between herpes zoster and COVID-19. Nevertheless, the detailed pathophysiological mechanism is still unclear. We used bioinformatic analyses to study the potential genetic crosstalk between herpes zoster and COVID-19. COVID-19 and herpes zoster were associated with a similar subset of genes involved in "cytokine-cytokine receptor interaction," "Jak-STAT signaling pathway," and "IL-17 signaling pathway," including TNF, IL10, ESR1, INFG, HLA-A, CRP, STAT3, IL6, IL7, and IL17A. Protein-protein interaction network assay showed that the combined gene set indicated a raised connectivity as compared to herpes zoster or COVID-19 alone, particularly the potentiated interactions with APOE, ARSA, CCR2, CCR5, CXCL13, EGFR, GAL, GP2, HLA-B, HLA-DRB1, IL5, TECTA, and THBS1, and these genes are related to "cytokine-cytokine receptor interaction". Augmented Th17 cell differentiation and the resulting enhanced IL-17 signaling were identified in both COVID-19 and herpes zoster. Our data suggested aberrant interleukin-17 signaling as one possible mechanism through which COVID-19 could raise the risk of herpes zoster.

Keywords: Cytokine; Jak-STAT; SARS-CoV-2; Varicella zoster virus.

Aims and methods: Accurate protein measurements using formalin-fixed biopsies are needed to improve disease characterisation. This feasibility study used targeted and global mass spectrometry (MS) to interrogate a spectrum of disease severities using 19 ulcerative colitis (UC) biopsies.

Results: Targeted assays for CD8, CD19, CD132 (interleukin-2 receptor subunit gamma/common cytokine receptor gamma chain), FOXP3 (forkhead box P3) and IL17RA (interleukin 17 receptor A) were successful; however, assays for IL17A (interleukin 17A), IL23 (p19) (interleukin 23, alpha subunit p19) and IL23R (interleukin 23 receptor) did not permit target detection. Global proteome analysis (4200 total proteins) was performed to identify pathways associated with UC progression. Positive correlation was observed between histological scores indicating active colitis and neutrophil-related measurements (R²=0.42-0.72); inverse relationships were detected with cell junction targets (R²=0.49-0.71) and β-catenin (R²=0.51-0.55) attributed to crypt disruption. An exploratory accuracy assessment with Geboes Score and Robarts Histopathology Index cut-offs produced sensitivities/specificities of 72.7%/75.0% and 100.0%/81.8%, respectively.

Conclusions: Pathologist-guided MS assessments provide a complementary approach to histological scoring systems. Additional studies are indicated to verify the utility of this novel approach.

Keywords: colitis; inflammatory bowel diseases; proteins.

Endothelial dysfunction (ED) is a key factor for the development of cardiovascular diseases. Due to its chronic, life-threatening nature, ED only can be studied experimentally in animal models. Therefore, this work was aimed to characterize a murine model of ED induced by a daily intraperitoneal administration of angiotensin II (AGII) for 10 weeks. Oxidative stress, inflammation, vascular remodeling, hypertension, and damage to various target organs were evaluated in treated animals. The results indicated that a chronic intraperitoneal administration of AGII increases the production of systemic soluble VCAM, ROS and ICAM-1 expression, and the production of TNFα, IL1β, IL17A, IL4, TGFβ, and IL10 in the kidney, as well as blood pressure levels; it also promotes vascular remodeling and induces non-alcoholic fatty liver disease, glomerulosclerosis, and proliferative retinopathy. Therefore, the model herein proposed can be a representative model for ED; additionally, it is easy to implement, safe, rapid, and inexpensive.

Kallistatin or kallikrein-binding protein (KBP) has been reported to regulate angiogenesis, inflammation and tumor progression. Autoimmune uveitis is a common, sight-threatening inflammatory intraocular disease. However, the roles of kallistatin in autoimmunity and autoreactive T cells are poorly investigated. Compared to non-uveitis controls, we found that plasma levels of kallistatin were significantly upregulated in patients with Vogt-Koyanagi-Harada (VKH) disease, one of the non-infectious uveitis. Using an experimental autoimmune uveitis (EAU) model induced by human interphotoreceptor retinoid-binding protein peptide 651-670 (hIRBP_651-670), we examined the effects of kallistatin on the pathogenesis of autoimmune diseases. Compared to wild type (WT) mice, kallistatin transgenic (KS) mice developed severe uveitis with dominant Th17 infiltrates in the eye. In addition, the proliferative antigen-specific T cells isolated from KS EAU mice produced increased levels of IL-17A, but not IFN-γ or IL-10 cytokines. Moreover, splenic CD4⁺ T cells from naïve KS mice expressed higher levels of Il17a mRNA compared to WT naïve mice. Under Th17 polarization conditions, KS mice exhibited enhanced differentiation of naïve CD4⁺ T cells into Th17 cells compared to WT controls. Together, our results indicate that kallistatin promotes Th17 differentiation and is a key regulator of aggravating autoinflammation in EAU. Targeting kallistatin might be a potential to treat autoimmune disease.

Keywords: Th17; autoimmune disease; experimental autoimmune uveitis; immunology; interphotoreceptor retinoid-binding protein; kallistatin; uveitis.

The human mesenchymal stem cell (hMSC) secretome has pleiotropic effects which underpin their therapeutic potential. hMSC serum-free conditioned media (SFCM) has been determined to contain a variety of cytokines with roles in regeneration and suppression of inflammation. Physiological oxygen (physoxia) has been demonstrated to impact upon a number of facets of hMSC biology and we hypothesized that the secretome would be similarly modified. We tested a range of oxygen conditions; 21% O₂ (air oxygen (AO)), 2% O₂ (intermittent hypoxia (IH)) and 2% O₂ Workstation (physoxia (P)) to evaluate their effect on hMSC secretome profiles. Total protein content of secretome was upregulated in IH and P (>3 fold vs AO) and IH (>1 fold vs P). Focused cytokine profiling indicated global upregulation in IH of all 31 biomolecules tested in comparison to AO and P with basic-nerve growth factor (bNGF) and granulocyte colony-stimulating factor (GCSF) (>3 fold vs AO) and bNGF and Rantes (>3 fold vs P) of note. Similarly, upregulation of interferon gamma-induced protein 10 (IP10) was noted in P (>3 fold vs AO). Interleukin-2 (IL2) and Rantes (in AO and P) and adiponectin, IL17a, and epidermal growth factor (EGF) (in AO only) were entirely absent or below detection limits. Quantitative analysis validated the pattern of IH-induced upregulation in vascular endothelial growth factor (VEGF), placental growth factor-1 (PIGF1), Tumor necrosis factor alpha (TNFa), IL2, IL4, and IL10 when compared to AO and P. In summary, modulation of environmental oxygen alters both secretome concentration and composition. This consideration will likely impact on delivering improved mechanistic understanding and potency effects of hMSC-based therapeutics.

Keywords: Secretome; mesenchymal stem cell; physoxia.

Klebsiella pneumoniae found in the normal flora of the human oral and intestinal tract mainly causes hospital-acquired infections but can also cause community-acquired infections. To date, most clinical trials of vaccines against K. pneumoniae have ended in failure. Furthermore, no single conserved protein has been identified as an antigen candidate to accelerate vaccine development. In this study, we identified five outer membrane proteins of K. pneumoniae, namely, Kpn_Omp001, Kpn_Omp002, Kpn_Omp003, Kpn_Omp004, and Kpn_Omp005, by using reliable second-generation proteomics and bioinformatics. Mice vaccinated with these five KOMPs elicited significantly higher antigen-specific IgG, IgG1, and IgG2a. However, only Kpn_Omp001, Kpn_Omp002, and Kpn_Omp005 were able to induce a protective immune response with two K. pneumoniae infection models. These protective effects were accompanied by the involvement of different immune responses induced by KOMPs, which included KOMPs-specific IFN-γ-, IL4-, and IL17A-mediated immune responses. These findings indicate that Kpn_Omp001, Kpn_Omp002, and Kpn_Omp005 are three potential Th1, Th2, and Th17 candidate antigens, which could be developed into multivalent and serotype-independent vaccines against K. pneumoniae infection.

Keywords: Klebsiella pneumoniae; outer membrane proteins; proteomics and bioinformatics; serotype-independent vaccines; vaccine.