Secreted immune metabolites that mediate immune cell communication and function.

Metabolites are emerging as essential factors for the immune system that are involved in both metabolic circuits and signaling cascades. Accumulated evidence suggests that altered metabolic programs initiated by the activation and maturation of immune cell types are accompanied by the delivery of various metabolites into the local environment. We propose that, in addition to protein/peptide ligands, secreted immune metabolites (SIMets) are essential components of immune communication networks that fine-tune immune responses under homeostatic and pathological conditions. We summarize recent advances in our understanding of SIMets and discuss the potential mechanisms by which some metabolites engage in immunological responses through receptor-, transporter-, and post-translational-mediated regulation. These insights may contribute to understanding physiology and developing effective therapeutics for inflammatory and immune-mediated diseases.

OMIP-101: 27-color flow cytometry panel for immunophenotyping of major leukocyte populations in fixed whole blood.

This 27-color flow cytometry antibody panel allows broad immune-profiling of major leukocyte subsets in human whole blood (WB). It includes lineage markers to identify myeloid and lymphoid cell populations including granulocytes, monocytes, myeloid dendritic cells (mDCs), natural killer (NK) cells, NKT-like cells, B cells, conventional CD4 and CD8 T cells, γδ T cells, mucosa-associated invariant T (MAIT) cells and innate lymphoid cells (ILC). To further characterize each of these populations, markers defining stages of cell differentiation (CCR7, CD27, CD45RA, CD127, CD57), cytotoxic potential (perforin, granzyme B) and cell activation/proliferation (HLA-DR, CD38, Ki-67) were included. This panel was developed for quantifying absolute counts and phenotyping major leukocyte populations in cryopreserved, fixed WB collected from participants enrolled in large multi-site tuberculosis (TB) vaccine clinical trials. This antibody panel can be applied to profile major leukocyte subsets in other sample types such as fresh WB or peripheral blood mononuclear cells (PBMCs) with only minor additional optimization.

Blood-based molecular and cellular biomarkers of early response to neoadjuvant PD-1 blockade in patients with non-small cell lung cancer.

BACKGROUND: Despite the improved survival observed in PD-1/PD-L1 blockade therapy, a substantial proportion of cancer patients, including those with non-small cell lung cancer (NSCLC), still lack a response. METHODS: Transcriptomic profiling was conducted on a discovery cohort comprising 100 whole blood samples, as collected multiple times from 48 healthy controls (including 43 published data) and 31 NSCLC patients that under treatment with a combination of anti-PD-1 Tislelizumab and chemotherapy. Differentially expressed genes (DEGs), simulated immune cell subsets, and germline DNA mutational markers were identified from patients achieved a pathological complete response during the early treatment cycles. The predictive values of mutational markers were further validated in an independent immunotherapy cohort of 1661 subjects, and then confirmed in genetically matched lung cancer cell lines by a co-culturing model. RESULTS: The gene expression of hundreds of DEGs (FDR p < 0.05, fold change < -2 or > 2) distinguished responders from healthy controls, indicating the potential to stratify patients utilizing early on-treatment features from blood. PD-1-mediated cell abundance changes in memory CD4 + and regulatory T cell subset were more significant or exclusively observed in responders. A panel of top-ranked genetic alterations showed significant associations with improved survival (p < 0.05) and heightened responsiveness to anti-PD-1 treatment in patient cohort and co-cultured cell lines. CONCLUSION: This study discovered and validated peripheral blood-based biomarkers with evident predictive efficacy for early therapy response and patient stratification before treatment for neoadjuvant PD-1 blockade in NSCLC patients.

Temperature impacts the bovine ex vivo immune response towards Mycoplasmopsis bovis.

Although cattle are the mammalian species with most global biomass associated with a huge impact on our planet, their immune system remains poorly understood. Notably, the bovine immune system has peculiarities such as an overrepresentation of γδ T cells that requires particular attention, specifically in an infectious context. In line of 3R principles, we developed an ex vivo platform to dissect host-pathogen interactions. The experimental design was based on two independent complementary readouts: firstly, a novel 12-14 color multiparameter flow cytometry assay measuring maturation (modulation of cell surface marker expression) and activation (intracellular cytokine detection) of monocytes, conventional and plasmacytoid dendritic cells, natural killer cells, γδ T cells, B and T cells; secondly, a multiplex immunoassay monitoring bovine chemokine and cytokine secretion levels. The experiments were conducted on fresh primary bovine blood cells exposed to Mycoplasmopsis bovis (M. bovis), a major bovine respiratory pathogen. Besides reaffirming the tight cooperation of the different primary blood cells, we also identified novel key players such as strong IFN-γ secreting NK cells, whose role was so far largely overlooked. Additionally, we compared the host-pathogen interactions at different temperatures, including commonly used 37 °C, ruminant body temperature (38-38.5 °C) and fever (≥ 39.5 °C). Strikingly, working under ruminant physiological temperature influenced the capacity of most immune cell subsets to respond to M. bovis compared to 37 °C. Under fever-like temperature conditions the immune response was impaired compared to physiological temperature. Our experimental approach, phenotypically delineating the bovine immune system provided a thorough vision of the immune response towards M. bovis and the influence of temperature towards that immune response.

Mesenchymal stem cell-regulated miRNA-mRNA landscape in acute-on-chronic liver failure.

BACKGROUND: Acute-on-chronic liver failure (ACLF) is a major challenge in the field of hepatology. While mesenchymal stem cell (MSC) therapy can improve the prognosis of patients with ACLF, the molecular mechanisms through which MSCs attenuate ACLF remain poorly understood. We performed global miRNA and mRNA expression profiling via next-generation sequencing of liver tissues from MSC-treated ACLF mice to identify important signaling pathways and major factors implicated in ACLF alleviation by MSCs. METHODS: Carbon tetrachloride-induced ACLF mice were treated with saline or mouse bone marrow-derived MSCs. Mouse livers were subjected to miRNA and mRNA sequencing. Related signal transduction pathways were obtained through Gene Set Enrichment Analysis. Functional enrichment, protein-protein interaction, and immune infiltration analyses were performed for the differentially expressed miRNA target genes (DETs). Hub miRNA and mRNA associated with liver injury were analyzed using LASSO regression. The expression levels of hub genes were subjected to Pearson's correlation analysis and verified using RT-qPCR. The biological functions of hub genes were verified in vitro. RESULTS: The tricarboxylic acid cycle and peroxisome proliferator-activated receptor pathways were activated in the MSC-treated groups. The proportions of liver-infiltrating NK resting cells, M2 macrophages, follicular helper T cells, and other immune cells were altered after MSC treatment. The expression levels of six miRNAs and 10 transcripts correlated with the degree of liver injury. miR-27a-5p was downregulated in the mouse liver after MSC treatment, while its target gene E2f2 was upregulated. miR-27a-5p inhibited E2F2 expression, suppressed G1/S phase transition and proliferation of hepatocytes, in addition to promoting their apoptosis. CONCLUSIONS: This is the first comprehensive analysis of miRNA and mRNA expression in the liver tissue of ACLF mice after MSC treatment. The results revealed global changes in hepatic pathways and immune subpopulations. The miR-27a-5p/E2F2 axis emerged as a central regulator of the MSC-induced attenuation of ACLF. The current findings improve our understanding of the molecular mechanisms through which MSCs alleviate ACLF.

Analysis of Immune Cell Subsets in Peripheral Blood from Patients with Engineered Stone Silica-Induced Lung Inflammation.

Silicosis caused by engineered stone (ES-silicosis) is an emerging worldwide issue characterized by inflammation and fibrosis in the lungs. To our knowledge, only a few reports have investigated leukocyte/lymphocyte subsets in ES-silicosis patients. The present study was designed to explore the proportions of the main lymphocyte subsets in ES-silicosis patients stratified into two groups, one with simple silicosis (SS) and the other with a more advanced state of the disease, defined as progressive massive fibrosis (PMF). The proportions of B (memory and plasmablasts) cells, T (helper, cytotoxic, regulatory) cells, and natural killer (NK) (regulatory and cytotoxic) cells were investigated by multiparameter flow cytometry in 91 ES-silicosis patients (53 SS patients and 38 PMF patients) and 22 healthy controls (HC). Although the total number of leukocytes did not differ between the groups studied, lymphopenia was observed in patients compared to healthy controls. Compared with those in healthy controls, the proportions of memory B cells, naïve helper T cells, and the CD4+/CD8+ T cells' ratio in the peripheral blood of patients with silicosis were significantly decreased, while the percentages of plasma cells, memory helper T cells, and regulatory T cells were significantly increased. For the NK cell subsets, no significant differences were found between the groups studied. These results revealed altered cellular immune processes in the peripheral blood of patients with ES-silicosis and provided further insight into silicosis pathogenesis.

Neo-peripheral adaptive immune score predicts neoadjuvant chemotherapy for locally advanced breast cancer.

PURPOSE: Whether peripheral immune cell subsets can predict pathological complete response (pCR) in breast cancer patients remains to be elucidated. We aimed to dissect the relationship between peripheral immune cell subsets and pCR. METHODS: Two hundred and twenty-six eligible patients from two prospective clinical trials (SHPD001 and SHPD002) in China were randomly divided into a training cohort and a validation cohort. The breast cancer subtypes in this study included hormone receptor (HR)-positive/human epidermal growth factor receptor 2 (HER2)-negative (n = 95), HER2-positive (n = 100), and triple negative (n = 31) breast cancer. We defined the "Neo-Peripheral Adaptive Immune Score" for neoadjuvant chemotherapy (neoPAI Score) based on the percentages of CD4 + T cells, CD8 + T cells, B cells, and the CD4 + /CD8 + ratio in peripheral blood. We also evaluated the ability of the neoPAI Score derived from tumor-infiltrating immune cells (TIICs) to predict survival by employing The Cancer Genome Atlas-Breast Cancer (TCGA-BRCA) database. RESULTS: In the training cohort, multivariate analysis showed that HR status [odds ratio (OR) 0.325; 95% confidence interval (CI) 0.135-0.761; P = 0.010], HER2 status (OR 2.657; 95% CI 1.266-5.730; P = 0.011), Ki67 index (OR 3.191; 95% CI 1.509-6.956; P = 0.003), histological grade (OR 2.297; 95% CI 1.031-5.290; P = 0.045) and neoPAI Score (OR 4.451; 95% CI 1.608-13.068; P = 0.005) were independent predictors of pCR. In the validation cohort, histological grade (OR 3.779; 95% CI 3.793-1.136 × 103; P = 0.008) and neoPAI Score (OR 90.828; 95% CI 3.827-9.843 × 103; P = 0.019) were independent predictors of pCR. The Immune Model that integrated the neoPAI Score was more accurate in predicting pCR than the Clinical Model that exclusively contained clinicopathological parameters in both cohorts. In TCGA-BRCA database, the neoPAI Score constructed from TIICs can predict the progression-free interval (P = 0.048) of breast cancer. CONCLUSION: The neoPAI Score defined by the percentages of peripheral immune cell subsets could be used as a potential biomarker for neoadjuvant chemotherapy efficacy.

OMIP-94: Twenty-four-color (thirty-marker) panel for deep immunophenotyping of immune cells in human peripheral blood.

This newly established 24-color (30-marker) panel focuses on the characterization of the main human immune cell subtypes and was optimized for the analysis of human whole blood using a full spectrum flow cytometer. The panel covers all main leukocyte populations: neutrophils, eosinophils and basophils, monocytes (with additional subsets), dendritic cells, innate lymphoid cells and lymphocytes. As for lymphocytes, this panel includes CD4+ T helper, Treg cells, and CD8+ cytotoxic T cells. Further T cells subsets are included with special focus on invariant T cells: γδ T cells (including δ2TCR variant), invariant NKT cells and MAIT (mucosal-associated invariant T cells) cells. Additionally, total B cells (including Bregs and plasmocytes), NK cells, and NKT cells are included. For the overall check of activation status of the analyzed immune cells we used HLA-DR, CD38, CD57, CD69, PD-1, and CD94. In addition, we used CD62L, CD45RA, CD27, and CD39 to describe the differentiation status of these cells. The panel was designed to maximize the information that can be obtained from surface markers in order to avoid the need for fixation and permeabilization steps. The presented multimarker panel offers the possibility to discover new immune cell subtypes which in patients and in cohort studies may lead to the identification of altered immune phenotypes and might give a link to immune system based or to certain other diseases. This panel was developed for a full spectrum flow cytometer equipped with a minimum of three lasers. We developed this panel using healthy human fresh blood, however it was also successfully used for staining of isolated human peripheral blood mononuclear cells (PBMC).

Platelet is an unfavorable prognostic biomarker and associated with leukemia stem cells and immunomodulatory factors in acute myeloid leukemia.

Many clinical features, besides cytogenetic and molecular abnormalities, can affect the prognosis of the patients with acute myeloid leukemia (AML). Within this context it remains unclear if and how platelet counts affect the outcome of AML patients. In the present study, we examined the platelet counts at diagnosis in 633 newly diagnosed adult patients with AML from January 2010 to April 2021, and divided the cases into the group with low level of platelet counts (≤30×109/L, n=316) and high level of platelet counts (>30×109/L, n=317) according to the median platelet counts. We then validated the prognostic significance and potential mechanism of platelet counts on the relevance of spectral features for diagnostic risk stratification, initial induction therapy response, treatment effect maintenance, long-term survival, leukemia stem cells (LSCs) proportion, immunomodulatory cytokines level and immune cell subsets proportion. The results suggested that AML patients with a high level of platelet counts at diagnosis were associated with a high-risk molecular cytogenetic stratification, low complete remission (CR) rate, poor leukemia free survival (LFS), high proportion of LSCs, high level of transforming growth factor-ß (TGF-ß) and interleukin-1ß (IL-1ß), high proportion of regulatory T cells (Tregs) and monocytic myeloid-derived suppressor cells (M-MDSCs). It was demonstrated that platelet might be an unfavorable prognostic biomarker and was associated with LSCs and immunomodulatory cytokines as well as immune cell subsets in AML.

Main lymphocyte subpopulations in cerebrospinal fluid and peripheral blood in HIV-1 subtypes C and B.

HIV-1 subtype C (HIV-1C) shows reduced Tat protein chemoattractant activity compared with HIV-1B. The impact of HIV-1C Tat on the chemotaxis of the main lymphocyte subpopulations in the cerebrospinal fluid (CSF) and the peripheral blood (PB) is unclear. We hypothesized that there would be a lower frequency of specific lymphocyte subpopulations CD3+ or CD19+ in CSF in HIV-1C than in HIV-1B. The objectives were to detect the differences in the proportions of main lymphocyte subpopulations in CSF and PB, between people with HIV (PWH) and HIV-1-uninfected volunteers (PWoH) and in HIV-1B and HIV-1C. Lymphocyte immunophenotyping was studied in CSF and paired PB samples of PWH (n = 22) and PWoH (n = 14). Lymphocytes were analyzed within the CD45+ gated region. The proportions of CSF CD3+CD4+, CD3+CD8+, and CD3-CD19+ lymphocytes in CSF were comparable in HIV-1B and C. There was an increase in the proportion of CD3+CD8+ cells and a decrease in CD3+CD4+ T cells (ps = 0.016) in the CSF samples of the PWH compared with the PWoH group. In the PWH group, both CD3+CD4+ and CD3+CD8+ lymphocytes were significantly higher in the CSF than in the PB (p = 0.047 and 0.005). The proportion of CD3+CD4+ was lower and that of CD3+CD8+ was higher in the CSF samples of the aviremic group than that of HIV-negative control (p = 0.0008 and < 0.0001, respectively). HIV-1C Tat substitution (C30S) did not interfere with the CNS migration of the main lymphocyte subpopulations. This is the first study to evaluate these lymphocytes in CSF and PB of HIV-1C compared with HIV-1B.

Immunological characteristics and effect of cyclosporin in patients with immune thrombocytopenia.

OBJECTIVE: Immune thrombocytopenia (ITP) is well-known as an antibody-mediated autoimmune disease, and it is easy to get response but often turns to relapse or refractory. Cyclosporin is a traditional immunosuppressant and had a good effect on ITP patients. In this paper, we retrospectively analyze the immunological characteristics and therapeutic effect of cyclosporin in 220 patients with ITP. METHODS: All newly diagnosed ITP patients in the Department of Hematology, Tianjin Medical University General Hospital from June 2018 to December 2020 were enrolled and divided into four groups according to the expression of autoantibodies and the occurrence of prodromal infection. The basic data and immune indexes of ITP patients in each group were collected. The clinical immunological characteristics of patients in each group and the therapeutic effect of cyclosporin in each group were analyzed. RESULTS: Multi-autoantibody ITP patients were more likely to have low serum albumin and high gamma globulin, and the ratio of albumin to globulin decreased. In addition, the level of IgA and IgG increased and the level of complement C3 and C4 decreased more frequently than those in other groups. The number of CD3+T lymphocytes, especially CD3+CD4+T lymphocytes, decreased in ANA+ITP patients. The number of CD16+CD56+NK cells, pDC/DC ratio, and pDC/mDC ratio were higher than those in other groups. The expression of IL-6 and the proportion of CD19+B lymphocytes increased in two groups of ITP patients with abnormal autoantibodies. The patients of pro-infected group were more likely to suffer from coagulation disorder. After treatment with cyclosporin, the response rate increased and the 3-month relapse rate decreased in all ITP patients, and the therapeutic effect of patients with high megakaryocyte number was significantly higher than that of patients with low megakaryocyte number. The impact factors that influence the effect of glucocorticoid and(or) IVIG were the number of CD3+CD8+T lymphocytes, CD4/CD8 cell ratio, and the number of CD19+B lymphocytes. The independent impact factor of cyclosporin therapeutic response rate was the number of CD3+T lymphocytes. CONCLUSIONS: ITP is a heterogeneous disease, recurrence may occur during or rapidly after treatment. Cyclosporine included treatment can improve the effective rate of ITP and reduce the relapse rate within 3 months. The number of CD3+T lymphocytes was the only impact factor that influence the therapeutic effect of cyclosporin in ITP patients.

Modulation of Immune Cell Subsets by Hepatitis C Virus and Antiviral Therapy in Early Virological Response HCV Genotype 4-Infected Patients with Compensated Liver Disease.

BACKGROUND: Resolution of chronic hepatitis C virus (HCV) infection requires a complicated interaction between immune cell subsets. The effect of antiviral therapy on immune cell subsets remains to be defined. This study aimed to investigate the absolute count of certain immune cell subsets during therapy with pegylated interferon-α and ribavirin (PegIFN/RBV). MATERIALS AND METHODS: Sixty HCV genotype 4-infected patients with compensated liver disease were treated with PegIFN/RBV therapy for 52 weeks. Efficacy was measured by studying the early virological response (EVR) at post-therapy week 12. Absolute counts of mature T cells, T helper cells, T cytotoxic cells, activated T cells, natural killer cells, natural killer/T (NKT) cells, B cells, and T regulatory cells (Treg), and the ratio of T helper to T cytotoxic cells were longitudinally analyzed by flow cytometry throughout the treatment and follow-up course. RESULTS: Of the 60 genotype 4-infected subjects, 39 (65%) had EVR and 21 (35%) were non-EVR patients. In the first part of this study, there were significantly lower mean absolute count values of mature T, T cytotoxic, B, and NKT cells. Also, we detected statistically significantly lower mean values for the percentages of T cytotoxic, NKT, Treg, and activated T cells of HCV-infected patients at baseline values when compared with healthy subjects. After the initiation of PegIFN/RBV therapy, frequencies of T helper cells, activated T cells, Treg cells, B cells, and T helper:T cytotoxic ratio were found to be significantly lower in EVR patients than in non-EVR patients (p < 0.05). In contrast, frequencies of T cytotoxic and NKT cells were significantly increased in EVR patients when compared to non-EVR patients (p < 0.05). CONCLUSION: These results suggest a pattern of higher levels of T cytotoxic and NKT cells, and lower levels of T helper, activated T, Treg, and B cell populations in patients who respond favorably to PegIFN/RBV therapy.

Cytokine and immune cell profiling in the cerebrospinal fluid of patients with neuro-inflammatory diseases.

BACKGROUND: Cytokines play multiple roles during neuro-inflammatory processes and several cytokines have been studied in the context of specific diseases. This study provides a comprehensive picture of cerebrospinal fluid (CSF) changes during neuro-inflammation by analyzing multiple cytokines in combination with immune cell subsets and standard CSF parameters. METHODS: Using multiplex assays, we simultaneously measured 36 cytokines (CCL1-3, CCL7, CCL8, CCL11, CCL13, CCL19, CCL20, CCL22-27, CXCL1, CXCL2, CXCL5, CXCL6, CXCL8, CXCL9, CXCL11-13, CXCL16, CX3CL1, IL2, IL4, IL6, IL10, IL16, GM-CSF, IFNγ, MIF, TNFα, and MIB1ß) in the CSF and serum of 75 subjects. Diagnoses included clinically isolated syndrome and relapsing-remitting multiple sclerosis (MS, n = 18), secondary progressive MS (n = 8), neuro-syphilis (n = 6), Lyme neuro-borreliosis (n = 13), bacterial and viral meningitis (n = 20), and patients with non-inflammatory neurological diseases (NIND, n = 10). Cytokine concentrations were correlated with CSF standard parameters and CSF immune cell subsets (CD4 and CD8 T cells, B cells, plasmablasts, monocytes, and NK cells) quantified by flow cytometry. RESULTS: We observed increased levels of multiple cytokines (26/36) in patients with neuro-inflammatory diseases when compared to NIND that consistently correlated with CSF cell count and QAlbumin. Most CSF cytokine concentrations correlated with each other, but correlations between CSF and serum values were scarce (3/36). Within the CSF compartment, CXCL13 showed a strong association with B cells when analyzing all patients, as well as patients with an intact blood-brain barrier (BBB). NK cells positively correlated with CSF concentrations of multiple cytokines (22/36) when analyzing all patients. These correlations were maintained when looking at patients with a disrupted BBB but not detectable in patients with an intact BBB. CONCLUSIONS: Under conditions of neuro-inflammation, multiple CSF cytokines are regulated in parallel and most likely produced locally. A combined increase of CSF CXCL13 levels and B cells occurs under conditions of an intact BBB. Under conditions of a disrupted BBB, CSF NK cells show significantly increased values and seem to have a major contribution to overall inflammatory processes, reflected by a strong correlation with multiple cytokines. Future studies are necessary to address the exact kinetics of these cytokines during neuro-inflammation and their relation to specific diseases phenotypes.

Multiparameter Flow Cytometry Assay for Quantification of Immune Cell Subsets, PD-1 Expression Levels and PD-1 Receptor Occupancy by Nivolumab and Pembrolizumab.

We report the development and validation of a 12 parameter immunofluorescence flow cytometry method for the sensitive determination of cell concentrations, their expression of PD-1, and PD-1 receptor occupancy. Cell subsets include CD4+ and CD8+ -T-cells, B-cells, natural killer cells, classical-, intermediate- and non-classical monocytes, and myeloid- and plasmacytoid dendritic cells. Cells were isolated from peripheral blood by density gradient centrifugation. The validation parameters included specificity, linearity, limit of quantification, precision, biological within- and between subject variations. The lower limit of quantification was 5.0% of PD-1+ cells. Samples were stable for at least 153 days of storage at -80°C. The clinical applicability of the method was demonstrated in 11 advanced cancer patients by the successful determination of immune cell concentrations, relative number of PD-1+ immune cells, and number of PD-1 molecules per immune cell. Shortly after infusion of nivolumab, receptor occupancy on CD8+ -T-cells was 98%. Similar values were found predose cycle 2, suggesting receptor occupancy remained high throughout the entire cycle. © 2019 International Society for Advancement of Cytometry.

Glycolithocholic acid increases the frequency of circulating Tregs through constitutive androstane receptor to alleviate postmenopausal osteoporosis.

BACKGROUND AND PURPOSE: Gut microbiota and their metabolic activity are important regulators of host immunity. However, the role of gut microbiota and their metabolic activity-mediated osteoimmunity in postmenopausal osteoporosis (PMO) remains unknown. This study aimed to explore the role of gut microbiota and their metabolic activity in PMO. EXPERIMENTAL APPROACH: 16S rDNA sequencing was used for analyzing the gut microbiota diversity of patients with PMO and rat models, and a targeted metabolism study was performed for analyzing metabolite levels. Flow cytometry was used for analyzing the frequency of immune cells. Micro-CT was used for analyzing bone damage in rat models. Fecal microbiota transplantation was performed for exploring the therapeutic effect of the gut microbiota on PMO. CD4+ T cells were co-cultured with bone marrow mesenchymal stem cells for evaluating their molecular mechanisms. KEY RESULTS: Patients with PMO exhibited reduced gut microbiota diversity, and fecal glycolithocholic acid (GLCA) levels correlated with the degree of osteoporosis. GLCA levels in the gut were positively correlated with the frequency of circulating Tregs in ovariectomized rats. Restoration of the gut microbiota alleviated osteoporosis in ovariectomized rats. Circulating GLCA augmented CD4+ T cell differentiation into Tregs via constitutive androstane receptors. The increased frequency of Tregs further promoted the osteogenic differentiation of bone marrow mesenchymal stem cells to alleviate osteoporosis. CONCLUSION AND IMPLICATIONS: GLCA alleviated PMO by increasing the frequency of circulating Tregs, acting via the constitutive androstane receptor. This study reveals a new strategy for the treatment of PMO, with GLCA as a potential drug candidate.

Clinical and immunological characteristics and prognosis of patients with autoantibody negative dermatomyositis: a case control study.

OBJECTIVES: Myositis-specific antibodies (MSAs) and myositis-associated antibodies (MAAs) are associated with distinctive dermatomyositis (DM) clinical phenotypes. The aim of this study is to explicate the clinical and immunological features of MSAs-negative DM patients. METHODS: A total of 515 individuals diagnosed with DM was screened from 2013 to 2022 and 220 DM patients were enrolled in this retrospective cohort. Clinical and laboratory data of these patients were analyzed. RESULTS: MSAs-negative DM patients were categorized into two groups: MAAs-negative (MSAs (-)/MAAs (-)) group and MAAs-positive (MSAs (-)/MAAs (+)) group. The percentage of Raynaud's phenomenon (P=0.026) was higher in the MSAs (-)/MAAs (+) DM patients than the MSAs-positive DM patients and MSAs (-)/MAAs (-) DM patients. The proportion of rapidly progressive interstitial lung disease (RP-ILD) in the MSAs-negative DM patients was lower than that in the MSAs-positive group. The MSAs (-)/MAAs (+) group had a higher proportion of organizing pneumonia and usual interstitial pneumonia (P=0.011), and elevated eosinophils in their bronchoalveolar lavage fluid (P=0.008). Counts of lymphocytes (P=0.001) and CD16+CD56+ natural killer (NK) cells (P=0.012) were higher in the MSAs-negative group. Additionally, the percentage of CD4+TNFα+ (P=0.040), CD4+IFNγ+ (P=0.037), and CD4+IL-2+ (P=0.018) cells among total CD4+ T cells were higher in the MSA-negative DM patients compared with the MSAs-positive DM patients. Besides, MSAs-negative patients demonstrated a more favorable prognosis than MSAs-positive patients. Multivariable regression analysis identified advanced onset age, higher level of carcinoembryonic antigen (CEA), and RP-ILD as risk factors for mortality in DM patients. CONCLUSIONS: Compared with MSAs-positive group, MSAs-negative DM patients suffered less from organ involvement compared with MSAs-positive group and tend to have better prognosis. Key Points MSAs-negative DM patients exhibited distinct characteristics in comparison with MSAs-positive DM patients:   • The MSAs (-)/MAAs (+) DM patients demonstrated a higher prevalence of organizing pneumonia (OP) and usual interstitial pneumonia (UIP), and elevated eosinophil counts in bronchoalveolar lavage fluid.   • CEA levels were lower in MSAs-negative patients compared with MSAs-positive patients.   • Elevated counts of lymphocytes and CD16+CD56+ NK cells were identified in the MSAs-negative patients. Additionally, proportions of CD4+TNFα+, CD4+IFNγ+, and CD4+IL-2+ cells among total CD4+ T cells were higher in the MSAs-negative DM patients compared with DM MSAs-positive DM patients.   • MSAs-negative DM patients had a more favorable prognosis than MSAs-positive DM patients. A multivariable regression analysis revealed the advanced onset age, high CEA levels, and RP-ILD were risk factors for mortality in DM patients.

A Pilot Study on the Effects of Exercise Training on Cardiorespiratory Performance, Quality of Life, and Immunologic Variables in Long COVID.

Objectives: Fatigue is a prominent feature of long COVID (LC) and may be related to several pathophysiologic mechanisms, including immune hyperstimulation. Aerobic endurance exercise training may be a useful therapy, with appropriate attention to preventing post-exertional malaise. Methods: Fourteen participants completed a pilot study of aerobic exercise training (twenty 1.5 h sessions of over 10 weeks). Cardiorespiratory fitness, 6 min walk distance, quality of life, symptoms, 7-day physical activity, immunophenotype, and inflammatory biomarkers were measured before and after exercise training. Results: The participant characteristics at baseline were as follows: 53.5 ± 11.6 yrs, 53% f, BMI 32.5 ± 8.4, 42% ex-smokers, 15.1 ± 8.8 months since initial COVID-19 infection, low normal pulmonary function testing, V.O2peak 19.3 ± 5.1 mL/kg/min, 87 ± 17% predicted. After exercise training, participants significantly increased their peak work rate (+16 ± 20 W, p = 0.010) and V.O2peak (+1.55 ± 2.4 mL/kg/min, p = 0.030). Patients reported improvements in fatigue severity (-11%), depression (-42%), anxiety (-29%), and dyspnea level (-46%). There were no changes in 6MW distance or physical activity. The circulating number of CD3+, CD4+, CD19+, CD14++CD16, and CD16++CD14+ monocytes and CD56+ cells (assessed with flow cytometry) increased with acute exercise (rest to peak) and was not diminished or augmented by exercise training. Plasma concentrations of TNF-α, IL-6, IL-8, IL-10, INF-γ, and INF-λ were normal at study entry and not affected by training. Conclusions: Aerobic endurance exercise training in individuals with LC delivered beneficial effects on cardiorespiratory fitness, quality of life, anxiety, depression, and fatigue without detrimental effects on immunologic function.

Gut microbiota-derived metabolites associate with circulating immune cell subsets in unexplained recurrent spontaneous abortion.

Currently, the precise causes of over 40 % of recurrent spontaneous abortion (RSA) cases cannot be identified, leading to the term "unexplained RSA" (URSA). Through an exploration of the gut microbiota, metabolites, and immune cell subsets in URSA, this study establishes a link between gut microbiota-derived metabolites and immune cells. The results indicate reduced diversity in the gut microbiota of URSA. Targeted metabolomic analyses reveal decreased levels of gut microbiota-derived deoxycholic acid (DCA), glycolithocholic acid (GLCA), acetate, propionate, and butyrate in URSA. Furthermore, elevated frequencies of Th1, Th17, and plasma B cells, along with decreased frequencies of Tregs and Bregs, are observed in the peripheral blood of URSA. The results demonstrate correlations between the levels of gut microbiota-derived bile acids and short-chain fatty acids and the frequencies of various immune cell subsets in circulation. Collectively, this study uncovers an association between gut microbiota-derived metabolites and circulating immune cell subsets in URSA.

Investigating the complex interplay between fibroblast activation protein α-positive cancer associated fibroblasts and the tumor microenvironment in the context of cancer immunotherapy.

Introduction: This study investigates the role of Fibroblast Activation Protein (FAP)-positive cancer-associated fibroblasts (FAP+CAF) in shaping the tumor immune microenvironment, focusing on its association with immune cell functionality and cytokine expression patterns. Methods: Utilizing immunohistochemistry, we observed elevated FAP+CAF density in metastatic versus primary renal cell carcinoma (RCC) tumors, with higher FAP+CAF correlating with increased T cell infiltration in RCC, a unique phenomenon illustrating the complex interplay between tumor progression, FAP+CAF density, and immune response. Results: Analysis of immune cell subsets in FAP+CAF-rich stromal areas further revealed significant correlations between FAP+ stroma and various T cell types, particularly in RCC and non-small cell lung cancer (NSCLC). This was complemented by transcriptomic analyses, expanding the range of stromal and immune cell subsets interrogated, as well as to additional tumor types. This enabled evaluating the association of these subsets with tumor infiltration, tumor vascularization and other components of the tumor microenvironment. Our comprehensive study also encompassed cytokine, angiogenesis, and inflammation gene signatures across different cancer types, revealing heterogeneous cellular composition, cytokine expressions and angiogenic profiles. Through cytokine pathway profiling, we explored the relationship between FAP+CAF density and immune cell states, uncovering potential immunosuppressive circuits that limit anti-tumor activity in tumor-resident immune cells. Conclusions: These findings underscore the complexity of tumor biology and the necessity for personalized therapeutic and patient enrichment approaches. The insights gathered from FAP+CAF prevalence, immune infiltration, and gene signatures provide valuable perspectives on tumor microenvironments, aiding in future research and clinical strategy development.

Differences in leucocytes and inflammation-based indices among critically ill patients owing to SARS-CoV-2 variants during several successive waves of COVID-19 pandemic.

BACKGROUND/AIM: Inflammatory indices are useful informative markers in assessing the severity of the COVID-19 disease course; however, their involvements during series waves of SARS-CoV-2 virus outbreaks in critical patients with COVID-19 remain unclear. Hence, we aimed to ascertain the changing dynamics of the combined inflammatory indices (NLR, dNLR, CLR, LMR, PLR, SII, and SIRI) and their associations with clinical outcomes in severe COVID-19 patients during serial waves of SARS-CoV-2. PATIENTS AND METHODS: We retrospectively enrolled 163 severe COVID-19 patients admitted to the ICU during six SARS-CoV-2 waves. RESULTS: We found that most of patients admitted to the ICU were from the fourth wave. Patients in the fourth wave were considerably younger and had the highest percentage of ARDS than other waves. The highest CRP was found in the first wave, while the lowest in patients admitted in the sixth wave. Although most of the COVID-19 waves were marked with leukocytosis, neutrophilia, and lymphocytopenia, the lowest of both NLR and dNLR were found in the fourth wave "Delta wave" and the lowest of both CLR and SII were observed in "Omicron wave". Interestingly, during most of the COVID-19 waves, the derived combined inflammatory ratio NLR, dNLR, CLR, SII and SIRI were sustained at high levels in fatal cases at the last day of hospitalization, while these indices declined in the alive group at the end of ICU hospitalization. No major difference was identified in lymphocyte count between admission and the last day of hospitalization in both deceased and recovered COVID-19 patients during Delta and Omicron waves. Moreover, patients admitted in the Omicron wave had less severe disease compared to those admitted in the Delta wave. The Kaplan-Meier analysis revealed no significant difference in survival rates or the probability of respiratory failure between six successive COVID-19 waves. CONCLUSION: Taken together, our results showed marked differences in the alteration of nonspecific inflammation and damage in the adaptive immune response during the six serial SARS-CoV-2 waves. Considering the inflammatory response of infectious diseases, embedding inflammatory indices informative markers into routine clinical testing offers the potential to mitigate the impact of future pandemics of COVID-19 and other infectious diseases.