Regulatory Role of the RNA N6-Methyladenosine Modification in Immunoregulatory Cells and Immune-Related Bone Homeostasis Associated With Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a systemic autoimmune disease for which the etiology has not been fully elucidated. Previous studies have shown that the development of RA has genetic and epigenetic components. As one of the most highly abundant RNA modifications, the N6-methyladenosine (m6A) modification is necessary for the biogenesis and functioning of RNA, and modification aberrancies are associated with various diseases. However, the specific functions of m6A in the cellular processes of RA remain unclear. Recent studies have revealed the relationship between m6A modification and immune cells associated with RA. Therefore, in this review, we focused on discussing the functions of m6A modification in the regulation of immune cells and immune-related bone homeostasis associated with RA. In addition, to gain a better understanding of the progress in this field of study and provide the proper direction and suggestions for further study, clinical application studies of m6A modification were also summarized.

Current Preventions and Treatments of aGVHD: From Pharmacological Prophylaxis to Innovative Therapies

Graft versus host disease (GVHD) is one of the main causes of mortality and the reason for up to 50% of morbidity after hematopoietic stem cell transplantations (HSCT) which is the treatment of choice for many blood malignancies. Thanks to years of research and exploration, we have acquired a profound understanding of the pathophysiology and immunopathology of these disorders. This led to the proposition and development of many therapeutic approaches during the last decades, some of them with very promising results. In this review, we have focused on the recent GVHD treatments from classical chemical and pharmacological prophylaxis to more innovative treatments including gene therapy and cell therapy, most commonly based on the application of a variety of immunomodulatory cells. Furthermore, we have discussed the advantages and potentials of cell-free therapy as a newly emerging approach to treat GVHD. Among them, we have particularly focused on the implication of the TNFα-TNFR2 axis as a new immune checkpoint signaling pathway controlling different aspects of many immunoregulatory cells.

Immunoregulatory Cells in Myasthenia Gravis

Myasthenia gravis (MG) is a T cell-dependent, B-cell mediated autoimmune disease caused by antibodies against the nicotinic acetylcholine receptor or other components of the post-synaptic muscle endplate at the neuromuscular junction. These specific antibodies serve as excellent biomarkers for diagnosis, but do not adequately substitute for clinical evaluations to predict disease severity or treatment response. Several immunoregulatory cell populations are implicated in the pathogenesis of MG. The immunophenotype of these populations has been well-characterized in human peripheral blood. CD4+FoxP3+ regulatory T cells (Tregs) are functionally defective in MG, but there is a lack of consensus on whether they show numerical perturbations. Myeloid-derived suppressor cells (MDSCs) have also been explored in the context of MG. Adoptive transfer of CD4+FoxP3+ Tregs or MDSCs suppresses ongoing experimental autoimmune MG (EAMG), a rodent model of MG, suggesting a protective role of both populations in this disease. An imbalance between follicular Tregs and follicular T helper cells is found in untreated MG patients, correlating with disease manifestations. There is an inverse correlation between the frequency of circulating IL-10–producing B cells and clinical status in MG patients. Taken together, both functional and numerical defects in various populations of immunoregulatory cells in EAMG and human MG have been demonstrated, but how they relate to pathogenesis and whether these cells can serve as biomarkers of disease activity in humans deserve further exploration.

CXCL12 and IL7R as Novel Therapeutic Targets for Liver Hepatocellular Carcinoma Are Correlated With Somatic Mutations and the Tumor Immunological Microenvironment

The mechanism of liver hepatocellular carcinoma (LIHC) development in correlation with tumor microenvironments and somatic mutations is still being elucidated. This study aims to identify the potential molecular mechanisms and candidate biomarkers in response to tumor microenvironments and somatic mutations. Multiple bioinformatics analysis methods were applied to assess the tumor immunological microenvironment, differentially expressed genes, genetic function enrichment, immunocyte infiltration, regulatory network construction, and tumor mutational burden, and to identify DNA methylation sites. The immunological microenvironment features of ESTIMATE score (OS: p = 0.017, HR = 0.64; RFS: HR = 0.43, p < 0.001) have an important impact on the prognosis of LIHC patients. Cut-off by ESTIMATE score and prognostic information identified 666 DEGs (45 downregulated and 621 upregulated) that were linked with leukocyte migration and lymphocyte activation. In immunocyte infiltration analysis, NK cells (resting), M1 macrophages, CD8+ T cells, and regulatory T cells (Tregs), which are considered core immunoregulatory cells, exhibited significant differences between higher and lower ESTIMATE scores (overall survival and recurrence-free survival p-values < 0.01). Subsequently, further analysis of immunocyte-hub gene identification illustrated that the expression levels of CXCL12 and IL7R significantly correlated with core immunoregulatory cells and somatic mutations (CXCL12: p = 2.1E-06; IL7R: p = 0.001). This study provides new insight into our understanding of the mechanisms of immunocyte regulation and microenvironment involved in LIHC development as well as the effective biomarkers of CXCL12 and IL7R and core immunoregulatory cells, which may emerge as novel therapies for LIHC patients.

Gene expression of FOXP3, indoleamine 2,3-dioxygenase (IDO), IL10 and CSF1 in the uterus of cows that received an intrauterine infusion of maternal and paternal antigens

Sensitization with conceptus antigens has been shown to be useful for improving reproductive performance facilitating maternal acceptance of an allogeneic embryo through the induction of cytokines and immunoregulatory cells in the uterine microenvironment. As FOXP3, IDO, IL10 and CSF1 in the uterus are important on the recognition and development of embryos during early pregnancy, this study aimed to determine whether simultaneous or isolated administration of paternal (semen) and maternal (PBMCs) antigens in the uterus of cow, on the day of estrus, influence the gene expression of these cytokines. Forty crossbred cows were divided into four treatments: T0: Control; T1: Semen; T2: PBMCs (peripheral blood mononuclear cells) from another cow and T3: PBMCs+Semen. Antigens were administered into the uterine body on the estrus day (D0). Uterine biopsies designed for molecular analysis of gene expression were collected in vivo seven (D7) and fourteen (D14) days after immunostimulation. Transcripts from FOXP3, IDO, IL-10 and CSF-1 were detected in all RNA samples extracted from uterine biopsies. The semiquantitative analysis showed that none of the treatments caused significant increase in the expression of these genes. Furthermore, on D14 all treatments led to a decline in the number of CSF-1 transcripts; moreover, treatment with PBMCs+Semen also led to a drop in the abundance of IL-10 transcripts. Such results suggest that isolated or simultaneous administration of both antigens would not increase maternal tolerance to embryo alloantigens, nor would it create favorable conditions to its growth and pre-implantation development, at least regarding the effects mediated by these genes on D7 and D14 of the estrous cycle.

Immune Response in H. pylori-Associated Gastritis and Gastric Cancer

Helicobacter pylori (H. pylori) is the dominant member of the gastric microbiota and has infected more than half of the human population, of whom 5–15% develop gastric diseases ranging from gastritis and metaplasia to gastric cancer. These diseases always follow inflammation induced by cell surface and intracellular receptors and subsequent signaling, such as the NF-κB pathway and inflammasomes. Some types of immune cells are recruited to enforce an antibacterial response, which could be impeded by H. pylori virulence factors with or without a specific immune cell. Following decreased inflammation, neoplasm may appear with a little immune surveillance and may inhibit antitumor immunity. Therefore, the balance between H. pylori-associated inflammation and anti-inflammation is crucial for human health and remains to be determined. Here, we discuss multiple inflammation and immunoregulatory cells in gastritis and summarize the main immune evasion strategies employed by gastric cancer.

Integrated Immune Signature Analyses Identifies Evolution of Distinct Immunoregulatory Cell Populations Which Control Alloreactivity after Allogeneic HSCT

Background. Treatment failure after allogeneic haematopoietic stem-cell transplantation (AHST) using reduced-intensity conditioning (RIC) results from too much alloreactivity and harmful acute Graft-versus-Host Disease (aGVHD). Studies have identified many reconstituting immune cell subsets associated with development of clinical alloreactivity but the functionally dominant parameters at different time-points remain unknown. We therefore used mass cytometry (MS) to simultaneously assess multiple alloreactive and immunoregulatory cell populations to identify dominant immune reconstitution signatures associated with subsequent development of aGvHD after AHST. Methods. Phenotypic markers identifying more than 30 immune cell subsets known to influence alloreactivity were combined in a single MS panel. Peripheral blood from 58 patients with haematological cancers was analysed after T-replete HLA-matched RIC-AHST using uniform conditioning. Normalization of individual test samples spiked with CD45-barcoded healthy control cells was used to reduce batch effects. Complementary high-dimensional analytic tools were used to generate cellular profiles across the whole cohort and identify differences between patients grouped by subsequent development of aGVHD. Results. Unsupervised clustering analysis identified 40 phenotypically distinct T, B and NK cell clusters post-transplant. Significant batch effects were effectively reduced with a novel R-based algorithm normalising data to control cells. Cluster diversity analysis early post-transplant demonstrated lower cluster diversity in patients who subsequently developed aGvHD consistent with perturbation of phenotypic clusters in these patients. Two specific clusters were significantly different in abundance at D+30 in patients who went on to develop aGvHD and those who remained aGVHD-free. A cluster with a CD56brightCD16negCD27+/- regulatory NK cell (NKreg) phenotype was reduced in patients going on to develop aGvHD using both Phenograph and FlowSOM algorithms (p=0.001). These findings were validated by forward analysis using the CITRUS algorithm, revealing a similar differentiating cell population. CD56bright NKreg reconstitution was independent of CMV reactivation and did not impede reconstitution of WT1 and PR1 tumor-associated antigen-specific T cells. The reduction in NKreg in patients who subsequently developed aGvHD was accompanied by a significant increase in alloreactive CCR5+CD45RA-CCR7- CD4 effector memory T cells (Tem). We next used correlation analysis of cluster abundance across the whole cohort to identify all clusters contributing to the immune 'regulome' (those inversely correlated with alloreactive CD4 EM and/or CD8 EM T cell clusters). Notably, at D+30 the regulome consisted of 4 phenotypically distinct CD56bright NKreg clusters and a CD4+CD8+ double positive (DP) cluster, but not FOXP3+ CD4 regulatory T cells (Treg), Figure 1A Both the identity of differentiating clusters between patients subsequently developing aGVHD and those who remained aGvHD-free, and the dominant constituents of the regulome changed over time. By D+60 a CD56bright NKreg cluster (with a distinct phenotype to the differentiating cluster identified at D+30) and a DP T cell cluster were significantly reduced in patients subsequently developing aGvHD. The D+60 regulome consisted of multiple distinct CD56bright clusters, a DP T cell cluster and CD4 Treg, Figure 1B. Importantly by D+90 the immune regulome consisted of a reduced number of CD56bright NKreg clusters and increasing dominance of CD4Treg, Figure 1C. Conclusion. We show proof-of-concept that a novel acquisition and analysis pipeline can be applied to MS data to identify multiple immunoregulatory cells after AHST that contribute to the control of reconstituting alloreactive T cells. This approach identified a loss of NK cell-mediated control of alloreactive CD4 Tem cells as the dominant immune process preceding the development of aGvHD early post-transplant. Importantly, we show that specific immunoregulatory subsets are dominant at different time-points, with increasing influence of DP T cells and CD4 Treg at later time points. Our data provide mechanistic insight into the dynamic pattern of control of alloreactivity over time and show that strategies to expand or potentiate immunoregulatory cells to prevent aGvHD should be time-dependent. Disclosures Gribben: Acerta/Astra Zeneca: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding.

Macrophages in bacterial lung diseases: phenotype and functions (review)

This literature review is devoted to the analysis of the role of macrophages in the immunopathogenesis of infectious lung diseases of bacterial etiology. The article summarizes information about the origin of macrophages, their phenotypic and functional heterogeneity. The mechanisms of impaired protective function of innate immunity are associated with the polarization of the program of maturation and activation of macrophages in the direction to tolerogenic or immunoregulatory cells with phenotype of M2. Alveolar macrophages perform a variety of functions (from pro-inflammatory to regenerative) in the development of inflammation in the respiratory organs. Their inherent plasticity suggests that the same macrophages can change their phenotype and function depending on the microenvironment in the inflammatory focus at different stages of the disease. Understanding the mechanisms that regulate macrophage plasticity will be an important step towards realizing the potential of personalized immunomodulatory therapy.