An NGS-based assay for accurate detection and quantification of immune gene expression in mouse tumor models.

Tumor microenvironment (TME) is a complex dynamic system with many tumor-interacting components including tumor-infiltrating leukocytes (TILs), cancer associated fibroblasts, blood vessels, and other stromal constituents. It intrinsically affects tumor development and pharmacology of oncology therapeutics, particularly immune-oncology (IO) treatments. Accurate measurement of TME is therefore of great importance for understanding the tumor immunity, identifying IO treatment mechanisms, developing predictive biomarkers, and ultimately, improving the treatment of cancer. Here, we introduce a mouse-IO NGS-based (NGSmIO) assay for accurately detecting and quantifying the mRNA expression of 1080 TME related genes in mouse tumor models. The NGSmIO panel was shown to be superior to the commonly used microarray approach by hosting 300 more relevant genes to better characterize various lineage of immune cells, exhibits improved mRNA and protein expression correlation to flow cytometry, shows stronger correlation with mRNA expression than RNAseq with 10x higher sequencing depth, and demonstrates higher sensitivity in measuring low-expressed genes. We describe two studies; firstly, detecting the pharmacodynamic change of interferon-γ expression levels upon anti-PD-1: anti-CD4 combination treatment in MC38 and Hepa 1-6 tumors; and secondly, benchmarking baseline TILs in 14 syngeneic tumors using transcript level expression of lineage specific genes, which demonstrate effective and robust applications of the NGSmIO panel.

The Role of Interferon-γ in Autoimmune Polyendocrine Syndrome Type 1.

BACKGROUND: Autoimmune polyendocrine syndrome type 1 (APS-1) is a life-threatening, autosomal recessive syndrome caused by autoimmune regulator (AIRE) deficiency. In APS-1, self-reactive T cells escape thymic negative selection, infiltrate organs, and drive autoimmune injury. The effector mechanisms governing T-cell-mediated damage in APS-1 remain poorly understood. METHODS: We examined whether APS-1 could be classified as a disease mediated by interferon-γ. We first assessed patients with APS-1 who were participating in a prospective natural history study and evaluated mRNA and protein expression in blood and tissues. We then examined the pathogenic role of interferon-γ using Aire-/-Ifng-/- mice and Aire-/- mice treated with the Janus kinase (JAK) inhibitor ruxolitinib. On the basis of our findings, we used ruxolitinib to treat five patients with APS-1 and assessed clinical, immunologic, histologic, transcriptional, and autoantibody responses. RESULTS: Patients with APS-1 had enhanced interferon-γ responses in blood and in all examined autoimmunity-affected tissues. Aire-/- mice had selectively increased interferon-γ production by T cells and enhanced interferon-γ, phosphorylated signal transducer and activator of transcription 1 (pSTAT1), and CXCL9 signals in multiple organs. Ifng ablation or ruxolitinib-induced JAK-STAT blockade in Aire-/- mice normalized interferon-γ responses and averted T-cell infiltration and damage in organs. Ruxolitinib treatment of five patients with APS-1 led to decreased levels of T-cell-derived interferon-γ, normalized interferon-γ and CXCL9 levels, and remission of alopecia, oral candidiasis, nail dystrophy, gastritis, enteritis, arthritis, Sjögren's-like syndrome, urticaria, and thyroiditis. No serious adverse effects from ruxolitinib were identified in these patients. CONCLUSIONS: Our findings indicate that APS-1, which is caused by AIRE deficiency, is characterized by excessive, multiorgan interferon-γ-mediated responses. JAK inhibition with ruxolitinib in five patients showed promising results. (Funded by the National Institute of Allergy and Infectious Diseases and others.).

Single-Cell Transcriptomic Analysis of Epstein-Barr Virus-Associated Hemophagocytic Lymphohistiocytosis.

Epstein-Barr virus (EBV) infection can lead to infectious mononucleosis (EBV-IM) and, more rarely, EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH), which is characterized by a life-threatening hyperinflammatory cytokine storm with immune dysregulation. Interferon-gamma (IFNγ) has been identified as a critical mediator for primary HLH; however, the detailed role of IFNγ and other cytokines in EBV-HLH is not fully understood. In this study, we used single-cell RNA sequencing to characterize the immune landscape of EBV-HLH and compared it with EBV-IM. Three pediatric patients with EBV-HLH with different backgrounds, one with X-linked lymphoproliferative syndrome type 1 (XLP1), two with chronic active EBV disease (CAEBV), and two patients with EBV-IM were enrolled. The TUBA1B + STMN1 + CD8 + T cell cluster, a responsive proliferating cluster with rich mRNA detection, was explicitly observed in EBV-IM, and the upregulation of SH2D1A-the gene responsible for XLP1-was localized in this cluster. This proliferative cluster was scarcely observed in EBV-HLH cases. In EBV-HLH cases with CAEBV, upregulation of LAG3 was observed in EBV-infected cells, which may be associated with an impaired response by CD8 + T cells. Additionally, genes involved in type I interferon (IFN) signaling were commonly upregulated in each cell fraction of EBV-HLH, and activation of type II IFN signaling was observed in CD4 + T cells, natural killer cells, and monocytes but not in CD8 + T cells in EBV-HLH. In conclusion, impaired responsive proliferation of CD8 + T cells and upregulation of type I IFN signaling were commonly observed in EBV-HLH cases, regardless of the patients' background, indicating the key features of EBV-HLH.

The Myc-associated zinc finger protein epigenetically controls expression of interferon-γ-stimulated genes by recruiting STAT1 to chromatin.

The MYC-Associated Zinc Finger Protein (MAZ) plays important roles in chromatin organization and gene transcription regulation. Dysregulated expression of MAZ causes diseases, such as glioblastoma, breast cancer, prostate cancer, and liposarcoma. Previously, it has been reported that MAZ controls the proinflammatory response in colitis and colon cancer via STAT3 signaling, suggesting that MAZ is involved in regulating immunity-related pathways. However, the molecular mechanism underlying this regulation remains elusive. Here, we investigate the regulatory effect of MAZ on interferon-gamma (IFN-γ)-stimulated genes via STAT1, a protein that plays an essential role in immune responses to viral, fungal, and mycobacterial pathogens. We demonstrate that about 80% of occupied STAT1-binding sites colocalize with occupied MAZ-binding sites in HAP1/K562 cells after IFN-γ stimulation. MAZ depletion significantly reduces STAT1 binding in the genome. By analyzing genome-wide gene expression profiles in the RNA-Seq data, we show that MAZ depletion significantly suppresses a subset of the immune response genes, which include the IFN-stimulated genes IRF8 and Absent in Melanoma 2. Furthermore, we find that MAZ controls expression of the immunity-related genes by changing the epigenetic landscape in chromatin. Our study reveals an important role for MAZ in regulating immune-related gene expression.

Transcriptional Changes in Radiation-Induced Lung Injury: A Comparative Analysis of Two Radiation Doses for Preclinical Research.

In a recent stereotactic body radiation therapy animal model, radiation pneumonitis and radiation pulmonary fibrosis were observed at around 2 and 6 weeks, respectively. However, the molecular signature of this model remains unclear. This study aimed to examine the molecular characteristics at these two stages using RNA-seq analysis. Transcriptomic profiling revealed distinct transcriptional patterns for each stage. Inflammatory response and immune cell activation were involved in both stages. Cell cycle processes and response to type II interferons were observed during the inflammation stage. Extracellular matrix organization and immunoglobulin production were noted during the fibrosis stage. To investigate the impact of a 10 Gy difference on fibrosis progression, doses of 45, 55, and 65 Gy were tested. A dose of 65 Gy was selected and compared with 75 Gy. The 65 Gy dose induced inflammation and fibrosis as well as the 75 Gy dose, but with reduced lung damage, fewer inflammatory cells, and decreased collagen deposition, particularly during the inflammation stage. Transcriptomic analysis revealed significant overlap, but differences were observed and clarified in Gene Ontology and KEGG pathway analysis, potentially influenced by changes in interferon-gamma-mediated lipid metabolism. This suggests the suitability of 65 Gy for future preclinical basic and pharmaceutical research connected with radiation-induced lung injury.

Interferon-gamma signaling promotes cartilage regeneration after injury.

Osteoarthritis is a common chronic disease and major cause of disability and chronic pain in ageing populations. In this pathology, the entire joint is involved, and the regeneration of articular cartilage still remains one of the main challenges. Here, we investigated the molecular mechanisms underlying cartilage regeneration in young mice using a full-thickness cartilage injury (FTCI) model. FTCI-induced cartilage defects were created in the femoral trochlea of young and adult C57BL/6 mice. To identify key molecules and pathways involved in the early response to cartilage injury, we performed RNA sequencing (RNA-seq) analysis of cartilage RNA at 3 days after injury. Young mice showed superior cartilage regeneration compared to adult mice after cartilage injury. RNA-seq analysis revealed significant upregulation of genes associated with the immune response, particularly in the IFN-γ signaling pathway and qRT-PCR analysis showed macrophage polarization in the early phase of cartilage regeneration (3 days) in young mice after injury, which might promote the removal of damaged or necrotic cells and initiate cartilage regeneration in response to injury. IFN-γR1- and IFN-γ-deficient mice exhibited impaired cartilage regeneration following cartilage injury. DMM-induced and spontaneous OA phenotypes were exacerbated in IFN-γR1-/- mice than in wild-type mice. Our data support the hypothesis that IFN-γ signaling is necessary for cartilage regeneration, as well as for the amelioration of post-traumatic and age-induced OA.

[Effect of LAG3 deficiency on natural killer cell function and hepatic fibrosis in mice infected with Echinococcus multilocularis].

OBJECTIVE: To investigate the effect of LAG-3 deficiency (LAG3-/-) on natural killer (NK) cell function and hepatic fibrosis in mice infected with Echinococcus multilocularis. METHODS: C57BL/6 mice, each weighing (20 ± 2) g, were divided into the LAG3-/- and wild type (WT) groups, and each mouse in both groups was inoculated with 3 000 E. multilocularis protoscoleces via the hepatic portal vein. Mouse liver and spleen specimens were collected 12 weeks post-infection, sectioned and stained with sirius red, and the hepatic lesions and fibrosis were observed. Mouse hepatic and splenic lymphocytes were isolated, and flow cytometry was performed to detect the proportions of hepatic and splenic NK cells, the expression of CD44, CD25 and CD69 molecules on NK cell surface, and the secretion of interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), interleukin (IL)-4, IL-10 and IL-17A. RESULTS: Sirius red staining showed widening of inflammatory cell bands and hyperplasia of fibrotic connective tissues around mouse hepatic lesions, as well as increased deposition of collagen fibers in the LAG3-/-group relative to the WT group. Flow cytometry revealed lower proportions of mouse hepatic (6.29% ± 1.06% vs. 11.91% ± 1.85%, P < 0.000 1) and splenic NK cells (4.44% ± 1.22% vs. 5.85% ± 1.10%, P > 0.05) in the LAG3-/- group than in the WT group, and the mean fluorescence intensity of CD44 was higher on the surface of mouse hepatic NK cells in the LAG3-/- group than in the WT group (t = -3.234, P < 0.01), while no significant differences were found in the mean fluorescence intensity of CD25 or CD69 on the surface of mouse hepaticNK cells between the LAG3-/- and WT groups (both P values > 0.05). There were significant differences between the LAG3-/- and WT groups in terms of the percentages of IFN-γ (t = -0.723, P > 0.05), TNF-α (t = -0.659, P > 0.05), IL-4 (t = -0.263, P > 0.05), IL-10 (t = -0.455, P > 0.05) or IL-17A secreted by mouse hepatic NK cells (t = 0.091, P > 0.05), and the percentage of IFN-γ secreted by mouse splenic NK cells was higher in the LAG3-/- group than in the WT group (58.40% ± 1.64% vs. 50.40% ± 4.13%; t = -4.042, P < 0.01); however, there were no significant differences between the two groups in terms of the proportions of TNF-α (t = -1.902, P > 0.05), IL-4 (t = -1.333, P > 0.05), IL-10 (t = -1.356, P > 0.05) or IL-17A secreted by mouse splenic NK cells (t = 0.529, P > 0.05). CONCLUSIONS: During the course of E. multilocularis infections, LAG3-/- promotes high-level secretion of IFN-γ by splenic NK cells, which may participate in the reversal the immune function of NK cells, resulting in aggravation of hepatic fibrosis.

Interferon-γ and infectious diseases: Lessons and prospects.

Infectious diseases continue to claim many lives. Prevention of morbidity and mortality from these diseases would benefit not just from new medicines and vaccines but also from a better understanding of what constitutes protective immunity. Among the major immune signals that mobilize host defense against infection is interferon-γ (IFN-γ), a protein secreted by lymphocytes. Forty years ago, IFN-γ was identified as a macrophage-activating factor, and, in recent years, there has been a resurgent interest in IFN-γ biology and its role in human defense. Here we assess the current understanding of IFN-γ, revisit its designation as an "interferon," and weigh its prospects as a therapeutic against globally pervasive microbial pathogens.

Association of the IFNG +874T/A Polymorphism with Symptomatic COVID-19 Susceptibility.

Tumor necrosis factor (TNF) and interferon-gamma (IFNγ) are important inflammatory mediators in the development of cytokine storm syndrome (CSS). Single nucleotide polymorphisms (SNPs) regulate the expression of these cytokines, making host genetics a key factor in the prognosis of COVID-19. In this study, we investigated the associations of the TNF -308G/A and IFNG +874T/A polymorphisms with COVID-19. We analyzed the frequencies of the two polymorphisms in the control groups (CG: TNF -308G/A, n = 497; IFNG +874T/A, n = 397), a group of patients with COVID-19 (CoV, n = 222) and among the subgroups of patients with nonsevere (n = 150) and severe (n = 72) COVID-19. We found no significant difference between the genotypic and allelic frequencies of TNF -308G/A in the groups analyzed; however, both the frequencies of the high expression genotype (TT) (CoV: 13.51% vs. CG: 6.30%; p = 0.003) and the *T allele (CoV: 33.56% vs. CG: 24. 81%; p = 0.001) of the IFNG +874T/A polymorphism were higher in the COVID-19 group than in the control group, with no differences between the subgroups of patients with nonsevere and severe COVID-19. The *T allele of IFNG +874T/A (rs2430561) is associated with susceptibility to symptomatic COVID-19. These SNPs provided valuables clues about the potential mechanism involved in the susceptibility to developing symptomatic COVID-19.

Genetic liability to elevated circulating IP-10, IFNγ and SCGFß levels in relation to thoracic aortic aneurysm: A mendelian randomization study.

Inflammation is associated with thoracic aortic aneurysm (TAA) but the effects of each circulating inflammatory factor on TAA remain unclear. In this study, we explored the relationship between circulating inflammatory factors and TAA risk using Mendelian randomization (MR) approach based on summary statistics from the latest genome-wide association study (GWAS) of 41 circulating inflammatory factors in 8293 Finns and a GWAS involving 1351 TAA cases and 18,295 controls of European ancestry. In univariable MR, higher interferon gamma-induced protein 10 (IP-10) levels, higher interferon gamma (IFNγ) levels and higher stem cell growth factor beta (SCGFß) levels were associated with an increased risk of TAA (OR = 1.37, 95 % CI = 1.17-1.59, p = 7.42 × 10-5; OR = 1.43, 95 % CI = 1.19-1.74, p = 2.04 × 10-4; OR = 1.27, 95 % CI = 1.09-1.48, p = 2.40 × 10-3, respectively). In multivariable MR, the patterns of associations for the three cytokines remained adjusting for each other or smoking, but were attenuated differently with adjustment for other cardiovascular risk factors, especially for lipids and body mass index. Bidirectional MR approach did not identify any significant associations between cytokines and risk factors. Our results indicated that circulating cytokines may play mediation roles in the pathogenesis of TAA. Further studies are needed to determine whether these biomarkers can be used to prevent and treat TAA.

Implications of IFNγ SNP rs2069705 in primary Sjögren's syndrome: transcriptional activation and B cell infiltration.

Primary Sjögren's syndrome (pSS) is characterized by its autoimmune nature. This study investigates the role of the IFNγ SNP rs2069705 in modulating the susceptibility to pSS. Differential expression of IFNγ and BAFF was analyzed using the GEO database's mRNA microarray GSE84844. Genotyping of the IFNγ SNP rs2069705 was conducted via the dbSNP website. The JASPAR tool was used for predicting transcription factor bindings. Techniques such as dual-luciferase reporter assays, Chromatin immunoprecipitation, and analysis of a pSS mouse model were applied to study gene and protein interactions. A notable increase in the mutation frequency of IFNγ SNP rs2069705 was observed in MNCs from the exocrine glands of pSS mouse models. Bioinformatics analysis revealed elevated levels of IFNγ and BAFF in pSS samples. The model exhibited an increase in both CD20+ B cells and cells expressing IFNγ and BAFF. Knocking down IFNγ resulted in lowered BAFF expression and less lymphocyte infiltration, with BAFF overexpression reversing this suppression. Activation of the Janus kinase (JAK)/STAT1 pathway was found to enhance transcription in the BAFF promoter region, highlighting IFNγ's involvement in pSS. In addition, rs2069705 was shown to boost IFNγ transcription by promoting interaction between its promoter and STAT4. SNP rs2069705 in the IFNγ gene emerges as a pivotal element in pSS susceptibility, primarily by augmenting IFNγ transcription, activating the JAK/STAT1 pathway, and leading to B-lymphocyte infiltration in the exocrine glands.NEW & NOTEWORTHY The research employed a combination of bioinformatics analysis, genotyping, and experimental models, providing a multifaceted approach to understanding the complex interactions in pSS. We have uncovered that the rs2069705 SNP significantly affects the transcription of IFNγ, leading to altered immune responses and B-lymphocyte activity in pSS.

TNF and IFNγ-induced cell death requires IRF1 and ELAVL1 to promote CASP8 expression.

TNFα and IFNγ (TNF/IFNγ) synergistically induce caspase-8 activation and cancer cell death. However, the mechanism of IFNγ in promoting TNF-initiated caspase-8 activation in cancer cells is poorly understood. Here, we found that in addition to CASP8, CYLD is transcriptionally upregulated by IFNγ-induced transcription factor IRF1. IRF1-mediated CASP8 and CYLD upregulation additively mediates TNF/IFNγ-induced cancer cell death. Clinically, the expression levels of TNF, IFNγ, CYLD, and CASP8 in melanoma tumors are increased in patients responsive to immune checkpoint blockade (ICB) therapy after anti-PD-1 treatment. Accordingly, our genetic screen revealed that ELAVL1 (HuR) is required for TNF/IFNγ-induced caspase-8 activation. Mechanistically, ELAVL1 binds CASP8 mRNA and extends its stability to sustain caspase-8 expression both in IFNγ-stimulated and in basal conditions. Consequently, ELAVL1 determines death receptors-initiated caspase-8-dependent cell death triggered from stimuli including TNF and TRAIL by regulating basal/stimulated caspase-8 levels. As caspase-8 is a master regulator in cell death and inflammation, these results provide valuable clues for tumor immunotherapy and inflammatory diseases.

[Effect of signal peptide optimized by site-directed mutagenesis on the function of chimeric antigen receptor T cells].

Signal peptides (SP) are involved in regulating the secretion level and transmembrane translocation of chimeric antigen receptors (CAR), which is crucial for CAR-T cells. This study aimed to optimize the SP sequence by site-directed mutagenesis and investigate its impact on the killing function of CD19-CAR-T. Firstly, CAR vectors targeting CD19 containing wild-type SP (SP-wtY) or two mutant SP (SP-muK or SP-muR) were constructed using gene synthesis and molecular cloning techniques. The successfully constructed vector was packaged with lentivirus, and T cells were infected. The transfection efficiency of T cells was detected by flow cytometry, while the killing effect on target cells was assessed using the calcein release method. The secretion levels of cytokines interferon-γ (IFN-γ) and interferon-α (TNF-α) were measured using enzyme linked immunosorbent assay (ELISA). The results showed that successful construction of recombinant lentivirus plasmids with wild type and signal peptide mutation. After the transferring the lentivirus into T cells, the transfection efficiency of CD19-CAR carrying three signal peptides (SP-wtY, SP-muK, or SP-muR) were 33.9%, 35.5%, and 36.8%, respectively. Further killing assay showed that the tumor-killing effect of SP-muR cells was significantly higher than that of SP-muK and SP-wtY cells. When the ratio of effector to target was 10:1, the secretion levels of cytokines IFN-γ and TNF-α of CAR-T cells of the SP-muR group were significantly higher than those in SP-muK and SP-wtY groups. In summary, this study revealed that increasing the N-terminal positive charge of the signal peptide can improve the expression efficiency of CAR and promote the killing of CD19+ target cells. These findings provide a scientific basis the optimization and clinical application of CAR structure.

IL-32 production from lung adenocarcinoma cells is potentially involved in immunosuppressive microenvironment.

Interleukin 32 (IL-32) is a proinflammatory cytokine secreted from several kinds of cancer cells. In the present study, we investigated the significance of IL-32 in lung adenocarcinoma by immunohistochemistry and bioinformatics analysis. IL-32 was positive in cancer cells of 21 cases (9.2%) of total 228 cases. Increased IL-32 gene expression was linked to worse clinical course in TCGA analysis, however, IL-32 expression in immunohistochemistry was not associated to clinical course in our cohort. It was also found that high IL-32 expression was seen in cases with increased lymphocyte infiltration. In vitro studies indicated that IFN-γ induced gene expression of IL-32 and PD1-ligands in lung adenocarcinoma cell lines. IL-32, especially IL-32ß, also induced overexpression of PD1-ligands in human monocyte-derived macrophages. Additionally, Cancer-cell-derived IL-32 was elevated by stimulation with anticancer agents. In conclusion, IL-32 potentially induced by inflammatory conditions and anticancer therapy and contribute to immune escape of cancer cells via development the immunosuppressive microenvironment. IL-32 might be a target molecule for anti-cancer therapy.

Characterization of type II IFNs and their receptors in a cyprinid fish, the blunt snout bream Megalobrama amblycephala.

Type II interferons (IFNs) are a key class of molecules regulating innate and adaptive immunity in vertebrates. In the present study, two members of the type II IFNs, IFN-γ and IFNγ-rel, were identified in the blunt snout bream (Megalobrama amblycephala). The open reading frame (ORF) of IFN-γ and IFNγ-rel was found to have 564 bp and 492 bp, encoding 187 and 163 amino acids, with the first 26 and 24 amino acids being the signal peptide, respectively. IFN-γ and IFNγ-rel genes showed a high degree of similarity to their zebrafish homologues, being 76.9 % and 58.9 %, respectively. In the phylogenetic tree, IFN-γ and IFNγ-rel were clustered with homologous genes in cyprinids. In blunt snout bream, IFN-γ and IFNγ-rel were constitutively expressed in trunk kidney, head kidney, spleen, liver, heart, muscle, gill, intestine and brain and were significantly up-regulated by poly (I:C) induction in head kidney, spleen, liver, gill and intestine. Using recombinant proteins of IFN-γ and IFNγ-rel, the surface plasmon resonance (SPR) results showed that IFN-γ was bound to CRFB6, CRFB13 and CRFB17, but mainly to CRFB6 and CRFB13, whereas IFN-γrel bound mainly to CRFB17 and had no affinity with CRFB6. These results contribute to a better understanding on type II IFNs and their receptor usage in teleost fish.

Inhalable extracellular vesicle delivery of IL-12 mRNA to treat lung cancer and promote systemic immunity.

Lung carcinoma is one of the most common cancers and has one of the lowest survival rates in the world. Cytokines such as interleukin-12 (IL-12) have demonstrated considerable potential as robust tumour suppressors. However, their applications are limited due to off-target toxicity. Here we report on a strategy involving the inhalation of IL-12 messenger RNA, encapsulated within extracellular vesicles. Inhalation and preferential uptake by cancer cells results in targeted delivery and fewer systemic side effects. The IL-12 messenger RNA generates interferon-γ production in both innate and adaptive immune-cell populations. This activation consequently incites an intense activation state in the tumour microenvironment and augments its immunogenicity. The increased immune response results in the expansion of tumour cytotoxic immune effector cells, the formation of immune memory, improved antigen presentation and tumour-specific T cell priming. The strategy is demonstrated against primary neoplastic lesions and provides profound protection against subsequent tumour rechallenge. This shows the potential for locally delivered cytokine-based immunotherapies to address orthotopic and metastatic lung tumours.

Hyperglycemia modulates M1/M2 macrophage polarization in chronic diabetic patients with pulmonary tuberculosis infection.

Increased susceptibility to bacterial infections like tuberculosis (TB) is one of the complications of type 2 diabetes, however the underlying mechanisms remains poorly characterized. To explore how chronic hyperglycemia in diabetes affects progression of active TB, we examined mRNA expression of M1 (proinflammatory) and M2 (anti-inflammatory) cytokines/markers, in monocyte-derived macrophages obtained from patients with PTB + DM (pulmonary TB + diabetes mellitus type 2), patients with DM alone, patients with PTB alone, and healthy individuals (controls). Our findings indicate a dysregulated cytokine response in patients with both PTB and DM, characterized by decreased expression levels of interferon-gamma (IFN-γ) and inducible nitric oxide synthase (iNOS), along with increased expression levels of interleukin-1 beta (IL-1ß) and CD206. Furthermore, we observed a positive correlation of IL-1ß and CD206 expression with levels of glycosylated hemoglobin (HbA1c) in both PTB + DM and DM groups, while IFN-γ showed a positive correlation with HbA1c levels, specifically in the PTB + DM group. Additionally, M1 cytokines/markers, IL-1ß and iNOS were found to be significantly associated with the extent of sputum positivity in both PTB and PTB + DM groups, suggesting it to be a function of increased bacterial load and hence severity of infection. Our data demonstrates that tuberculosis in individuals with PTB + DM is characterized by altered M1/M2 cytokine responses, indicating that chronic inflammation associated with type 2 diabetes may contribute to increased immune pathology and inadequate control of tuberculosis infection.

Cytokines signatures and susceptibility to cutaneous leishmaniasis in patients from Sistan and Baluchestan province of Iran.

BACKGROUND: Cutaneous leishmaniasis (CL) is a complex, multifactorial disease that results from environmental factors such as parasite polymorphism, phlebotomine vectors, and host genetic factors. Some studies have identified specific genetic factors that may be associated with cutaneous leishmaniasis. The objective of this research was to resolve the association of 8 cytokine polymorphisms, including TNF-α -308 A/G (rs 1800629), TNF-α -238 A/G (rs 361525), TGF-ß1 -509 T/C (rs 1800469), TGF-ß1+ 915 G/C (rs 1800471), IFN-γ -874 T/A (rs 2430561), IFN-γ -179 G/A (rs 2069709), IL-10 -819 C/T (rs 1800871), and IL-10 -592 A/C (rs 1800872) with susceptibility to CL. METHODS: A total of 152 patients with designated CL and 100 healthy controls were selected from those referred to Sistan and Baluchestan hospitals. CL was diagnosed by microscopic examination of Giemsa-stained samples and culture. Leishmania species were identified using ITS2 gene PCR amplification with universal primers. Genetic polymorphism was determined by the ARMS PCR method on extracted genomic DNA of individuals. Eight SNPs cytokines were genotyped. RESULTS: Most of the Genotypic and allelic frequency comparisons between patients with CL and healthy subjects showed no difference, except 3. Individual SNP analysis showed highest association of TGF-ß1 -509 (rs1800469) -CC genotype (P = 0.03, OR = 7.05, 95 % CI = 3.3-15) with 5.7-fold increase, IFN-γ -874 (rs 2430561) -AA genotype (P = 0.04, OR = 4.72, 95 % CI = 1.6-14) with 4.2-fold increase, and IL10 -819 (rs1800871) -CC genotype (P = 0.05, OR = 3.63, 95 % CI = 2.5-5.3) with 1.9-fold increase, with CL. Odds ratios (ORs) and 95 % confidence intervals (CIs) were evaluated to assess the association power. CONCLUSION: Our results conclude that rs1800469 (TGF-ß1), rs2430561 (INF-γ), and rs1800872 (IL10) polymorphisms are associated with CL in southeastern Iranian people.

Interventing mitochondrial PD-L1 suppressed IFN-γ-induced cancer stemness in hepatocellular carcinoma by sensitizing sorafenib-induced ferroptosis.

Evidence recently showed that pleiotropic cytokine interferon-gamma (IFN-γ) in the tumor microenvironment (TME) plays a positive role in hepatocellular carcinoma (HCC) progression through the regulation of liver cancer stem cells (LCSCs) in HCC. The present study explored the role and potential mechanism of mitochondrial programmed cell death-ligand 1 (PD-L1) and its regulation of ferroptosis in modulating the cancer stemness of LCSCs. It was shown that mimicking TME IFN-γ exposure increased the LCSCs ratio and cancer stemness phenotypes in HCC cells. IFN-γ exposure inhibited sorafenib (Sora)-induced ferroptosis by enhancing glutathione peroxidase 4 (GPX4) expression as well reactive oxygen species (ROS) and lipid peroxidation (LPO) generation in LCSCs. Furthermore, IFN-γ exposure upregulated PD-L1 expression and its mitochondrial translocation, inducing dynamin-related protein 1 (Drp1)-dependent mitochondrial fission and correlating with glycolytic metabolism reprogramming in LCSCs. The genetic intervention of PD-L1 promoted ferroptosis-dependent anti-tumor effects of Sora, reduced glycolytic metabolism reprogramming, and inhibited cancer stemness of HCC in vitro and in vivo. Our results revealed a novel mechanism that IFN-γ exposure-induced mitochondrial translocation of PD-L1 enhanced glycolytic reprogramming to mediate the GPX4-dependent ferroptosis resistance and cancer stemness in LCSCs. This study provided new insights into the role of mitochondrial PD-L1-Drp1-GPX4 signal axis in regulating IFN-γ exposure-associated cancer stemness in LCSCs and verified that PD-L1-targeted intervention in combination with Sora might achieve promising synergistic anti-HCC effects.

Dictamnine Ameliorates DNFB-Induced Atopic Dermatitis Like Skin Lesions in Mice by Inhibiting M1 Macrophage Polarization and Promoting Autophagy.

Autophagy and M1 macrophage polarization play important roles in the regulation of inflammation in atopic dermatitis (AD). Dictamnine is one of the main ingredients in Cortex Dictamni, a widely used traditional Chinese medicine for the treatment of dermatitis. In the present study, we investigated the anti-inflammatory effects of dictamnine on AD like skin lesions and M1 macrophage polarization. A 2,4-dinitrofluorobenzene (DNFB) triggered AD like skin lesions models in mice was established to identify the ameliorative effects of dictamnine on AD in vivo. In addition, an M1 macrophage polarization model was co-stimulated by lipopolysaccharide (LPS) and interferon-γ (IFN-γ) using phorbol myristate acetate (PMA) differentiated THP-1 cells, to investigate the effect of dictamnine on promoting autophagy and inhibiting inflammatory factor release. Dictamnine suppressed DNFB-induced skin inflammation by inhibiting M1 macrophage polarization, up-regulating the expression of microtubule-associated protein 1A/1B-light chain 3 (LC3) expression, and promoting macrophage autophagy at inflammatory sites. Dictamnine also could reduce the release of interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1), and interleukin-8 (IL-8), and down-regulate the mRNA expression of these genes in LPS-IFN-γ triggered M1 polarized macrophages. Dictamnine ameliorates AD like skin lesions by inhibiting M1 macrophage polarization and promoting autophagy. Hence, dictamnine is expected to be a potential therapeutic candidate for AD.