Local Assessment of the Immunohistochemical Expression of Foxp3 + Regulatory T Lymphocytes in the Different Pathological Forms Associated with Bovine Paratuberculosis

Background Mycobacterium avium subsp. paratuberculosis infected animals show a variety of granulomatous lesions: from focal forms, restricted to the gut-associated lymphoid tissue (GALT), related to latency and resistance, to diffuse lesions, with abundant (multibacillary) or scant (paucibacillary) bacteria, seen in clinical stages. Factors that determine the response to the infection and responsible for the occurrence of the different types of lesion are still not fully determined. It has been seen that regulatory T cells (Treg) play an important role in the progress of various diseases where they act on the limitation of the immunopathology associated with the immune response. In the case of paratuberculosis (PTB) the role of Treg lymphocytes in the immunity against Map is far away to be completely understood; therefore, several studies addressing this subject have appeared recently. The aim of this work was to assess, by immunohistochemical methods, the presence of Foxp3+ T lymphocytes in intestinal samples with different types of lesions seen in cows with PTB. Methods Intestinal samples of twenty cows showing the different pathological forms of PTB were evaluated: uninfected controls (n = 5), focal lesions (n = 5), diffuse paucibacillary (n = 5) and diffuse multibacillary (n = 5) forms. Foxp3+ lymphocyte distribution was assessed by differential cell count in intestinal lamina propria (LP), gut-associated lymphoid tissue (GALT) and mesenteric lymph node (MLN). Results A significant increase in the number of Foxp3+ T cells was observed in all infected animals, but this increase was only significant in cows with focal lesions and, to a lesser extent, in animals with diffuse paucibacillary forms. The former showed the highest numbers, significantly different from those found in cows with diffuse lesions, where no differences were noted between the two forms. No specific distribution pattern was observed within the granulomatous lesions in any of the groups. Conclusions The increase of Foxp3+ T cells in focal forms, that have been associated with latency or resistance to infection, suggest an anti-inflammatory action of these cells at these stages, helping to prevent exacerbation of the inflammatory response, as occurs in diffuse forms, responsible for the appearance of clinical signs.

Local cellular immune response in chronic periodontitis

Microflora of the oral cavity forms a biofilm that induces response of immune system at the mucous membranes. Transition to periodontal lesion is provided by certain classes of resident mucosal immune cells and inflammatory/immune cells migrating to the periodont. In periodontal diseases, Th1, Th2, Th17, Treg are detected. T regulatory cells (Tregs) are proven to comprise the main anti-inflammatory cell population. Th17 cells and Treg cells play an important role in osteoclast differentiation. IL-17 secreted by Th17 cells affects osteoclastogenesis and may induce macrophages to enhance the local inflammatory response. In this regard, the aim of our work was to identify the local immune cells in oral cavity which are associated with severity of chronic generalized periodontitis. The oral cavity cells from 58 persons aged 38-65 years of both sexes in their mature age with a diagnosis of «chronic periodontitis» were examined by means of flow cytofluorometry. When determining levels of CD64+CD16+CD14- neutrophils in the patients with periodontitis of different severity, a statistically significant increase of this cell population was revealed upon development of this disease. In mild cases of periodontitis, a significant increase of relative CD64+CD16+CD14- neutrophil contents was revealed (Me = 36.16%, p < 0.05) compared to the control group (Me = 7.7%, Q0.25 = 2.4%, Q0.75 = 12%). When assessing relative numbers of CD14+ monocytes in periodontitis of various severity, we revealed a significant increase in the number of these cells in severe cases. When studying levels of regulatory T lymphocytes (CD4+CD25+CD127low) in periodontitis of different severity, we revealed significantly decreased amounts of this cell population during development of the disease. In mild cases of periodontitis, a decreased level of CD4+CD25+CD127low cells (p < 0.05, Me = 1356 cells/ml) was revealed, as compared with control group (Me = 10666 cells/ml). Although the concentration of CD4+CD25+CD127low (Me = 4709 cells/ml) in the patients with moderate periodontitis was higher than the values in milder cases, the range of the main values was comparable and lower, than in control group. In severe periodontitis, a significantly decreased concentration of regulatory T lymphocytes was revealed (Me = 2637 cells/ml). These results confirm the anti-inflammatory regulatory function of Tregs. Understanding the osteo-immune mechanisms of bone remodeling control will help to understand the pathophysiology of accelerated bone loss observed in severe chronic periodontitis.

Dynamics of the cytokine profile in achieving the therapeutic effect of allergen-specific immunotherapy in children with allergic rhinitis and bronchial asthma

Allergen-specific immunotherapy is a pathogenetic method for the treatment of IgE-mediated allergic diseases.Objective. To evaluate effectiveness of allergen-specific immunotherapy in 471 children with allergic rhinitis and bronchial asthma, as well to analyze the dynamics of the cytokine profile at baseline and 1 year after the start of treatment in 86 children. The research was supported by a grant from the International Scientific Council for Young Scientists of Kazan State Medical University.Results. Analysis of the data obtained demonstrated that allergen-specific immunotherapy is a highly effective method of treating children with allergic rhinitis and atopic bronchial asthma. The dynamics of the level of the studied cytokines during treatment indirectly indicates an increase in the functional activity of adaptive subpopulations of regulatory T-lymphocytes (T-reg) and B-lymphocytes (B-reg) with a decrease in the activity of Th2 lymphocytes, which may indicate the reconstruction of the immune response and possible disease-modifying effect.

CD73-Mediated Immunosuppression Is Linked to a Specific Fibroblast Population That Paves the Way for New Therapy in Breast Cancer

Background: Cancer-associated fibroblasts (CAF) are heterogeneous with multiple functions in breast cancer. Recently, we identified a specific CAF subpopulation (referred to as CAF-S1), which promotes immunosuppression and immunotherapy resistance. Methods and Results: Here, by studying a large collection of human samples, we highlight the key function of CD73/NT5E in CAF-S1-mediated immunosuppression in breast cancer. We first reveal that CD73 protein level specifically accumulates in CAF-S1 in breast cancer patients. Interestingly, infiltration of regulatory T lymphocytes (Tregs) is significantly correlated with CD73 expression in stroma but not in epithelium, indicating that CD73 contributes to immunosuppression when expressed in CAF-S1 and not in tumor cells. By performing functional assays based on relevant systems using primary CAF-S1 isolated from patients, we demonstrate that CAF-S1 increase the content in both PD-1+ and CTLA-4+ Tregs. Importantly, the use of a blocking anti-CD73 antibody on CAF-S1 reduces CAF-S1-mediated immunosuppression by preventing expression of these immune checkpoints on Tregs. Conclusions: Our data support the potential clinical benefit of using both anti-CD73 and immune-checkpoint inhibitors in breast cancer patients for inhibiting CAF-S1-mediated immunosuppression and enhancing anti-tumor immune response.

Regulatory T lymphocytes/Th17 lymphocytes imbalance in autism spectrum disorders: evidence from a meta-analysis

Background Immune system dysfunction has been proposed to play a critical role in the pathophysiology of autism spectrum disorders (ASD). Conflicting reports of lymphocyte subpopulation abnormalities have been described in numerous studies of patients with ASD. To better define lymphocytes abnormalities in ASD, we performed a meta-analysis of the lymphocyte profiles from subjects with ASD. Methods We used the PRISMA recommendations to query PubMed, Embase, PsychoINFO, BIOSIS, Science Direct, Cochrane CENTRAL, and Clinicaltrials.gov for terms related to clinical diagnosis of ASD and to lymphocytes’ populations. We selected studies exploring lymphocyte subpopulations in children with ASD. The search protocol has been registered in the international Prospective Register of Systematic Reviews (CRD42019121473). Results We selected 13 studies gathering 388 ASD patients and 326 healthy controls. A significant decrease in the CD4+ lymphocyte was found in ASD patients compared to controls [− 1.51 (95% CI − 2.99; − 0.04) p = 0.04] (I2 = 96% [95% CI 94.6, 97.7], p < 0.01). No significant difference was found for the CD8+ T, B and natural killer lymphocytes. Considering the CD4+ subpopulation, there was a significant decrease in regulatory T lymphocytes (Tregs) in ASD patients (n = 114) compared to controls (n = 107) [− 3.09 (95% CI − 4.41; − 1.76) p = 0.0001]; (I2 = 90.9%, [95% CI 76.2, 96.5], p < 0.0001) associated with an increase oin the Th17 lymphocytes (ASD; n = 147 controls; n = 128) [2.23 (95% CI 0.79; 3.66) p = 0,002] (I2 = 95.1% [95% CI 90.4, 97.5], p < 0.0001). Limitations Several factors inducing heterogeneity should be considered. First, differences in the staining method may be responsible for a part in the heterogeneity of results. Second, ASD population is also by itself heterogeneous, underlying the need of studying sub-groups that are more homogeneous. Conclusion Our meta-analysis indicates defects in CD4+ lymphocytes, specifically decrease oin Tregs and increase in Th17 in ASD patients and supports the development of targeted immunotherapies in the field of ASD.

Vitamin D and the Immune System

In the past few decades, various novel actions of vitamin D have been discovered. The mechanism of action of calcitriol or vitamin D is mediated by the Vitamin D receptor (VDR), a subfamily of nuclear receptors, which acts as a transcription factor in the target cells after formation of a heterodimer with the retinoid X receptor (RXR). As the VDR has been found in virtually all cell types, vitamin D exerts multiple actions on different tissues. Vitamin D has important immunomodulatory actions, which includes enhancement of the innate immune system and inhibition of the adaptative immune responses. These actions are associated with an increase in production of interleukin (IL)-4 by T helper (Th)-2 lymphocytes and the up-regulation of regulatory T lymphocytes. Vitamin D can regulate the immune responses in secondary lymphoid organs as well as in target organs through a number of mechanisms. Vitamin D inhibits the expression of APC cytokines, such as interleukin-1 (IL-1), IL-6, IL-12, and tissue necrosis factor- α (TNF-α) and decreases the expression of a set of major histocompatibility complex (MCH) class II cell surface proteins in macrophages. Vitamin D also inhibits B cell differentiation and antibody production. These actions reflect an important role of Vitamin D in balancing the immune system.

Abstract P393: Fabrication Of Immunomodulatory Hydrogels For Cardiac Repair After Acute Myocardial Infaction

Introduction: The balance of pro- and anti-inflammatory processes is tightly linked to left ventricular remodeling after myocardial infarction. Immune activation also plays a key role in rejection of transplanted allogeneic stem cells. In this study, we present the design, fabrication and characterization of immunomodulatory chitosan-based hydrogels for cardiac repair after myocardial infarction. Methods: Chitosan hydrogels conjugated with small immunomodulatory molecules were synthesized through a thermogelation process. Resultant hydrogels were characterized using scanning electron microscopy and Fourier-transformed infrared spectroscopy. Human mesenchymal stem cells were encapsulated into the hydrogels and biocompatibility was assessed after one week using fluorescence microscopy and a colorimetric assay. Immunomodulatory activity was assessed after co-culture with human T-lymphocytes using flow cytometry for CD4+IFN-γ+ pro-inflammatory and CD4+CD25+FoxP3+ regulatory T-lymphocytes. Results: Small immunomodulatory molecules were successfully integrated into chitosan hydrogels. Physico-chemical characterization revealed no significant changes to the 3D structure and porosity of hydrogels. The addition of 10μM atorvastatin or 10μM rosuvastatin did not result in significant cytotoxicity to encapsulated mesenchymal stem cells at 3 or 7 days. Addition of statins resulted in marked suppression of CD4+ T-lymphocyte proliferation (Control 25.1 Fold, Atorvastatin 1.0 Fold, Rosuvastatin 2.3 Fold, p<0.001) and activation (CD4+IFN-γ+ Population: Control 87.1%, Rosuvastatin 23.7%, p<0.001) after stimulation. No differences were seen in percentages of CD4+CD25+FoxP3+ regulatory T-lymphocytes (Control 5.5%, Rosuvastatin 5.7%, ns). Conclusion: A biocompatible immunomodulatory hydrogel was created through integration of atorvastatin and rosuvastatin into a chitosan hydrogel. Experiments are currently underway in vivo to examine its usefulness for stem cell delivery and reducing adverse left ventricular remodeling after myocardial infarction.

Annexin A1 as a Regulator of Immune Response in Cancer

Annexin A1 is a 37 kDa phospholipid-binding protein that is expressed in many tissues and cell types, including leukocytes, lymphocytes and epithelial cells. Although Annexin A1 has been extensively studied for its anti-inflammatory activity, it has been shown that, in the cancer context, its activity switches from anti-inflammatory to pro-inflammatory. Remarkably, Annexin A1 shows pro-invasive and pro-tumoral properties in several cancers either by eliciting autocrine signaling in cancer cells or by inducing a favorable tumor microenvironment. Indeed, the signaling of the N-terminal peptide of AnxA1 has been described to promote the switching of macrophages to the pro-tumoral M2 phenotype. Moreover, AnxA1 has been described to prevent the induction of antigen-specific cytotoxic T cell response and to play an essential role in the induction of regulatory T lymphocytes. In this way, Annexin A1 inhibits the anti-tumor immunity and supports the formation of an immunosuppressed tumor microenvironment that promotes tumor growth and metastasis. For these reasons, in this review we aim to describe the role of Annexin A1 in the establishment of the tumor microenvironment, focusing on the immunosuppressive and immunomodulatory activities of Annexin A1 and on its interaction with the epidermal growth factor receptor.

Features of the immunoreactivity T and B lymphocytes subpopulations and cytokine imbalance in patients with hepatosplenomegaly of different etiology

The aim was to study the mechanisms of immunological dysregulation of cytokine and immunoglobulin production, changes in the CD expression of T and B lymphocyte subpopulations in patients with hepatosplenomegaly of different etiology. Materials and methods. We examined 73 patients with liver cirrhosis complicated by portal hypertension, hepatosplenomegaly, and bleeding from phlebectasia. We identified three groups of patients depending on the triggering factors of cirrhosis: the first (I) group – HBV/HCV; the second (II) group – CMV/VEB; the third (III) group – hereditary enzymopathies. The study material was lymphocytes and blood serum. We used the methods of ELISA, immunofluorescence and flow cytometry. Results. An increase in the concentration of IgA and IgM was revealed against the background of normal number of CD22+ B lymphocytes with HBV/HCV (I group), high level of IgM and their producers, B lymphocytes, with CMV/VEB (II group), in group III with hereditary enzymopathies, the concentration of all immunoglobulins was normal with an increased content of B lymphocytes. Multidirectional changes in the content of cytokines were revealed: in group I the synthesis of anti-inflammatory cytokines IL-4, IL-10 and in group II – pro-inflammatory IL-1β, INF-γ, TNF-α dominated; in group III the concentration of IL-6 and vascular growth factor (VEGF) was maximally increased. The number of activated CD3+CD4+CD25+ T cells was reduced in groups I and II – by 2.3 and 2.0 times respectively, in group III – increased by 1.2 times. The number of regulatory T lymphocytes CD3+CD4+CD25+CD127neg was reduced by half in I and II groups. Expression of co-stimulatory molecules CD3+CD4+CD28+ was low in all groups and the maximum decrease was in group III. In patients with HCV/HBV, an increase in the expression of the late activation marker of lymphocytes CD3+HLA-DR+ by an average of 63 % was noted. Conclusions. The revealed immune disorders in hepatosplenomegaly of different etiology are characterized by multidirectional changes. Approaches to the treatment of these patients should be complex, taking into account the trigger factors that cause dysregulation of immune responses, which leads to further destruction, and focuses at remodeling target organs.