Pharmacological Inhibition of CCR2 Signaling Exacerbates Exercise-Induced Inflammation Independently of Neutrophil Infiltration and Oxidative Stress

Although exercise-induced humoral factors known as exerkines benefit systemic health, the role of most exerkines has not been investigated. Monocyte chemoattractant protein-1 (MCP-1) is a representative chemokine whose circulating concentrations increase after exercise, and it is one of the exerkines. MCP-1 is a ligand for CC chemokine receptor 2 (CCR2), which is expressed on monocytes, macrophages, and muscle cells. However, there is no information on the role of CCR2 signaling in exercise. Therefore, to investigate the research question, we administrated CCR2 antagonist or PBS to mice to inhibit CCR2 signaling before and after exercise. Our results showed that CCR2 signaling inhibition promoted exercise-induced macrophage infiltration and inflammation 24 h after exercise in muscle. CCR2 signaling inhibition also exacerbated exercise-induced inflammation immediately after exercise in muscle. However, neutrophil infiltration and oxidative stress had no contribution to exercise-induced inflammation by CCR2 signaling inhibition. CCR2 signaling inhibition also exacerbated exercise-induced inflammation immediately after exercise in kidney, liver, and adipose tissues. To summarize, pharmacological inhibition of CCR2 signaling exacerbated exercise-induced inflammation independently of neutrophil infiltration and oxidative stress.

Immune Modulation as a Key Mechanism for the Protective Effects of Remote Ischemic Conditioning After Stroke

Remote ischemic conditioning (RIC), which involves a series of short cycles of ischemia in an organ remote to the brain (typically the limbs), has been shown to protect the ischemic penumbra after stroke and reduce ischemia/reperfusion (IR) injury. Although the exact mechanism by which this protective signal is transferred from the remote site to the brain remains unclear, preclinical studies suggest that the mechanisms of RIC involve a combination of circulating humoral factors and neuronal signals. An improved understanding of these mechanisms will facilitate translation to more effective treatment strategies in clinical settings. In this review, we will discuss potential protective mechanisms in the brain and cerebral vasculature associated with RIC. We will discuss a putative role of the immune system and circulating mediators of inflammation in these protective processes, including the expression of pro-and anti-inflammatory genes in peripheral immune cells that may influence the outcome. We will also review the potential role of extracellular vesicles (EVs), biological vectors capable of delivering cell-specific cargo such as proteins and miRNAs to cells, in modulating the protective effects of RIC in the brain and vasculature.

Regulation of Mating-Induced Increase in Female Germline Stem Cells in the Fruit Fly Drosophila melanogaster

In many insect species, mating stimuli can lead to changes in various behavioral and physiological responses, including feeding, mating refusal, egg-laying behavior, energy demand, and organ remodeling, which are collectively known as the post-mating response. Recently, an increase in germline stem cells (GSCs) has been identified as a new post-mating response in both males and females of the fruit fly, Drosophila melanogaster. We have extensively studied mating-induced increase in female GSCs of D. melanogaster at the molecular, cellular, and systemic levels. After mating, the male seminal fluid peptide [e.g. sex peptide (SP)] is transferred to the female uterus. This is followed by binding to the sex peptide receptor (SPR), which evokes post-mating responses, including increase in number of female GSCs. Downstream of SP-SPR signaling, the following three hormones and neurotransmitters have been found to act on female GSC niche cells to regulate mating-induced increase in female GSCs: (1) neuropeptide F, a peptide hormone produced in enteroendocrine cells; (2) octopamine, a monoaminergic neurotransmitter synthesized in ovary-projecting neurons; and (3) ecdysone, a steroid hormone produced in ovarian follicular cells. These humoral factors are secreted from each organ and are received by ovarian somatic cells and regulate the strength of niche signaling in female GSCs. This review provides an overview of the latest findings on the inter-organ relationship to regulate mating-induced female GSC increase in D. melanogaster as a model. We also discuss the remaining issues that should be addressed in the future.

Influence of the probiotic preparation Immunoflor on the physiological status of a young chicken of a productive herd of egg cross

The article presents the results of studies of the effectiveness of the use of the complex probiotic preparation Immunoflor in young hens of egg cross. In the course of the research work, it was found that the use of the probiotic preparation Immunoflor does not affect the clinical and physiological state of the body of young chickens, but at the same time reduces the incidence, mortality and increases the safety of the chickens by enriching and balancing the poultry diet, reducing the conversion feed, optimization of digestion, stimulation of the development of positive microflora in the gastrointestinal tract. The use of the complex probiotic preparation Immunoflor in the diet of young chickens at a dose of 15 g/t of water and 15 g/t of feed contributes to an increase in the number of erythrocytes, leukocytes and hemoglobin concentration, activation of cellular and humoral factors of nonspecific resistance of the body of chickens, providing a normal physiological state and homeostasis.

Plasma obtained following murine hindlimb ischemic conditioning protects against oxidative stress in zebrafish models through activation of nrf2a and downregulation of duox

Ischemia/reperfusion of organ systems in trauma patients with resuscitated hemorrhagic shock (HSR) contributes to tissue injury and organ dysfunction. Previous studies using a murine model of HSR showed that remote ischemic preconditioning (RIC) protected against organ injury and that the plasma was able to prevent neutrophil migration in a zebrafish tailfin-cut inflammation model. In this study, we hypothesized that RIC plasma inhibits neutrophil function through a decrease in reactive oxygen species (ROS) production via the upregulation of the transcription factor Nrf2 and downstream antioxidative genes. Plasma from mice subjected to RIC (4 cycles of 5-min hindlimb ischemia/reperfusion) was microinjected into zebrafish. The results show that RIC plasma caused a reduction of ROS generation in response to tail injury. In addition, RIC plasma protected the fish larvae in the survival studies when exposed to either H2O2 or LPS. Oxidative stress PCR Array showed that RIC plasma treatment led to upregulation of antioxidative related genes including hsp70, hmox1a, nqo1 as well as downregulation of duox, the producer of H2O2. To explore the role of nrf2 in RIC, RIC plasma from Nrf2 KO mice were injected to the zebrafish and showed no inhibitory effect on neutrophil migration. Moreover, knockdown of nrf2a attenuated the anti-inflammatory and protective effect of RIC plasma. The downregulation of duox and upregulation of hmox1a were confirmed to require the activation of nrf2a. Therefore, we show that the protective effect of RIC may be related to the elaboration of humoral factors which counter injury-induced ROS generation in a nrf2-dependent fashion.

Requirement of direct contact between chondrocytes and macrophages for the maturation of regenerative cartilage

Regenerative cartilage prepared from cultured chondrocytes is generally immature in vitro and matures after transplantation. Although many factors, including host cells and humoral factors, have been shown to affect cartilage maturation in vivo, the requirement of direct cell–cell contact between host and donor cells remains to be verified. In this study, we examined the host cells that promote cartilage maturation via cell–cell contact. Based on analysis of the transplanted chondrocytes, we examined the contribution of endothelial cells and macrophages. Using a semiclosed device that is permeable to tissue fluids while blocking host cells, we selectively transplanted chondrocytes and HUVECs or untreated/M1-polarized/M2-polarized RAW264.7 cells. As a result, untreated RAW264.7 cells induced cartilage regeneration. Furthermore, an in vitro coculture assay indicated communication between chondrocytes and RAW264.7 cells mediated by RNA, suggesting the involvement of extracellular vesicles in this process. These findings provide insights for establishing a method of in vitro cartilage regeneration.

CLL-Derived Exosomes Turn Endothelial Cells into IL-6 Producing Cells

CLL is characterized by gradual accumulation of mature appearing long-lived lymphocytes that travel in blood and reside in lymph nodes, spleen and bone marrow. In these sites, pro inflammatory humoral factors support the survival and proliferation of the neoplastic cells. Previous studies showed that levels of the proinflammatory cytokine IL-6 are at least 10 folds higher in patients with CLL compared with healthy individuals. Yet, which cells produce and secrete IL-6 and what triggers this cellular activity in CLL is unknown. Secreted by all types of cells, exosomes are nano-scaled particles that travel in blood and carry a cargo that at least partially reflects the molecular makeup of its cell of origin. Exosomes, including those originating from neoplastic cells, function as stable intercellular transport vehicles that deliver their cargo to cells that engulf them. For example, CLL-derived exosomes are taken up by mesenchymal stromal cells, transforming them to cancer associated fibroblasts. Given the appropriate stimulation, endothelial cells produce IL-6 which provides CLL cells with a survival advantage. Therefore, we hypothesized that CLL-exosomes turn endothelial cells into "IL-6-secreting cells". To test this hypothesis, we transfected vein-derived (HUVECs) and arterial-derived (HAOEC) endothelial cells with exosomes that we isolated from the peripheral blood of 45 treatment naïve patients. We found that endothelial cells take-up CLL-exosomes in a dose- and time- dependent manner. Since CLL cells are protected from apoptosis in IL-6 rich environment, we wondered whether CLL-exosomes turn endothelial cells into IL-6-producing cells. To test this, we exposed endothelial cells to CLL-exosomes and found 50% increase in IL-6 levels, suggesting that the endothelial-exposed cells produced and secreted IL-6. Subsequently, we filtered out this growth medium and added CLL cells to this IL-6 enriched medium. After 15 minutes, STAT3 became phosphorylated and there was 40% decrease in the rate of apoptosis, indicating that IL-6 activated STAT3-dependent anti-apoptotic pathway. Phosphor-proteomics analysis of endothelial cells that were loaded with CLL-exosomes revealed 23 phosphor-proteins that were upregulated. Network analysis unraveled the central role of phosphor-b-catenin. To test whether b-catenin induces IL-6 in these cells, we transfected HUVECs with a b-catenin containing plasmid. We found by ELISA 30% increase in the levels of IL-6 in the culture medium and by chromatin immunoprecipitation assay an increased binding of 3 transcription factors (NFkB, LEF/TCF, and CEBP) to the IL-6 promoter. Taken together, we found that CLL cells communicate with endothelial cells through exosomes that they release. Once these exosomes are taken up by endothelial, they turn them into IL-6 producing cells, which in turn contributes to CLL cells' survival. Disclosures No relevant conflicts of interest to declare.

693 Mucosal-associated invariant T (MAIT) cell regulation networks in anti-tumor immunity

BackgroundMAIT cells are MR1-restricted innate-like T cells that recognize non-peptide antigens including riboflavin derivates. They account for up to 10 % of circulating T cells, but they are further enriched at mucosal sites and the liver. On one hand, altered MAIT number and function have been reported in liver cancer with MAITs correlating with poor clinical outcome. On the other hand, we recently demonstrated that MAIT cells can potentially have anti-tumor activity suggesting them as a novel target for cancer immunotherapy. Yet, the cellular and humoral factors that determine MAIT cell fate in the context of malignancies remain largely unknown.MethodsHighly multiplexed immunofluorescence-based CODEX imaging and high-dimensional flow cytometry was used to analyze MAIT cell infiltration and phenotype in human HCC samples. We recently developed an experimental framework to manipulate MAIT cells in vivo using VitaminB2 synthesis pathway-derived antigen 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU) in combination with Toll-like receptor 9 agonist CpG. Next, we used murine models of orthotopic primary liver cancer and liver metastasis across two different mouse strains, to assess anti-tumor activity of MAIT cells. A series of pharmacological depletion experiments and genomic knockout mouse strains were used to identify additional effector immune cells and humoral factors mediating the anti-tumor effect.ResultsUsing flow cytometry and spatially resolved analysis of multiplexed CODEX microscopy images, we found impaired infiltration and altered phenotype of MAIT cells in human HCC tumors compared to unaffected liver tissue. Thus, we sought out to experimentally increase MAIT cell infiltration into liver cancers using murine models. Co-administration of 5-OP-RU + CpG induced a strong systemic in vivo expansion and activation of MAIT cells with Th1/NK-like polarization. We found MAIT cells to be potent orchestrators of anti-tumor function in vivo when activated by a combination of 5-OP-RU + CpG. MAIT-directed 5-OP-RU/CpG showed pronounced and consistent anti-tumor activity against different models of liver cancer and prolonged mouse survival. Importantly, such tumor inhibition was absent in MAIT-deficient MR1 k.o. mice but nor dependent on MR1 expression on tumor cells. Additional pharmacological depletion studies/genomic k.o. models helped to identify antigen presenting cells, downstream effector cells as well as co-stimulatory cytokines as critical components needed for MAIT-induced tumor suppression.ConclusionsMAIT cells are important players in cancer immunology and represent an attractive novel target for cancer immunotherapy. Fine-tuned, context-dependent mechanisms determine MAIT-cell fate in vivo as they undergo a phenotypic switch upon 5-OP-RU and CpG treatment enabling them to exert potent anti-tumor function.

INFLUENCE OF HOMEOSTATIC CYTOKINES – IL-7 AND IL-15 ON T-REGULATORY CELLS IN VITRO

Cytokines IL-7 and IL-15 are the most important humoral factors providing T-conventional cell pool reconstitution during homeostatic proliferation caused by lymphopenia. However, whether these cytokines can provide homeostatic maintenance and proliferation of T-regulatory (Treg) cells is largely unknown. Considering the association between homeostatic proliferation and the development of autoimmunity, we decided to investigate the ability of these factors to cause differentiation of Treg-cells into Th17-lymphocytes. Therefore, the purpose of this study was to investigate the influence of humoral factors of homeostatic proliferation (IL-7 and IL-15) on Treg-cells in vitro. The study used peripheral blood sampled from 22 healthy donors. PBMC fraction was isolated by Ficoll density gradient centrifugation. Proliferation was induced by IL-7, IL-15, and by a combination of IL-2 with anti-CD3-antibodies. The proliferation intensity of Tregs was evaluated by flow cytometry using CFSE in PBMC cultures by phenotype CD3+CD4+CD25+FoxP3+ and in the previously purified population of CD3+CD4+CD25+CD127lo-cells. In this case Treg-cells were obtained by immunomagnetic separation from PBMCs using a MACS Treg Isolation Kit. Also, the RORyt expression in CD3+CD4+CD25+FoxP3+-cells was evaluated during cultivation. Here, we have shown that IL-7 and IL-15 could support Treg-cells by number and phenotype. Also, we revealed that these factors provide FoxP3 expression in Treg-cells; meanwhile, stimulation with IL-2 + anti-CD3 can also cause induction of FoxP3 expression de novo in conventional CD4+ cells. Also, we have shown that IL-7 and IL-15 can cause lower-intensity proliferation of Treg-cells in comparison with IL-2 + anti-CD3. Herewith homeostatic cytokines didn’t have the ability to induce RORyt expression in both T-regulatory cells and CD4+ conventional T-lymphocytes. Thus, it has been shown that IL-7 and IL-15 can potentially participate in maintaining the total pool of Treg-cells during lymphopenia, when IL-2 deficiency occurs, without causing the induction of RORyt expression. However, how homeostatic cytokines affect the functional activity of Treg-cells remains unclear and requires further investigation.

Effects of remote ischemic preconditioning (RIPC) and chronic remote ischemic preconditioning (cRIPC) on levels of plasma cytokines, cell surface characteristics of monocytes and in-vitro angiogenesis: a pilot study

Remote ischemic preconditioning (RIPC) protects the heart against myocardial ischemia/reperfusion (I/R) injury and recent work also suggested chronic remote ischemic conditioning (cRIPC) for cardiovascular protection. Based on current knowledge that systemic immunomodulatory effects of RIPC and the anti-inflammatory capacity of monocytes might be involved in cardiovascular protection, the aim of our study was to evaluate whether RIPC/cRIPC blood plasma is able to induce in-vitro angiogenesis, identify responsible factors and evaluate the effects of RIPC/cRIPC on cell surface characteristics of circulating monocytes. Eleven healthy volunteers were subjected to RIPC/cRIPC using a blood pressure cuff inflated to > 200 mmHg for 3 × 5 min on the upper arm. Plasma and peripheral blood monocytes were isolated before RIPC (Control), after 1 × RIPC (RIPC) and at the end of 1 week of daily RIPC (cRIPC) treatment. Plasma concentrations of potentially pro-angiogenic humoral factors (CXCL5, Growth hormone, IGFBP3, IL-1α, IL-6, Angiopoietin 2, VEGF, PECAM-1, sTie-2, IL-8, MCSF) were measured using custom made multiplex ELISA systems. Tube formation assays for evaluation of in-vitro angiogenesis were performed with donor plasma, monocyte conditioned culture media as well as IL-1α, CXCL5 and Growth hormone. The presence of CD14, CD16, Tie-2 and CCR2 was analyzed on monocytes by flow cytometry. Employing in-vitro tube formation assays, several parameters of angiogenesis were significantly increased by cRIPC plasma (number of nodes, P < 0.05; number of master junctions, P < 0.05; number of segments, P < 0.05) but were not influenced by culture medium from RIPC/cRIPC treated monocytes. While RIPC/cRIPC treatment did not lead to significant changes of the median plasma concentrations of any of the selected potentially pro-angiogenic humoral factors, in-depth analysis of the individual subjects revealed differences in plasma levels of IL-1α, CXCL5 and Growth hormone after RIPC/cRIPC treatment in some of the volunteers. Nevertheless, the positive effects of RIPC/cRIPC plasma on in-vitro angiogenesis could not be mimicked by the addition of the respective humoral factors alone or in combination. While monocyte conditioned culture media did not affect in-vitro tube formation, flow cytometry analyses of circulating monocytes revealed a significant increase in the number of Tie-2 positive and a decrease of CCR2 positive monocytes after RIPC/cRIPC (Tie-2: cRIPC, P < 0.05; CCR2: RIPC P < 0.01). Cardiovascular protection may be mediated by RIPC and cRIPC via a regulation of plasma cytokines as well as changes in cell surface characteristics of monocytes (e.g. Tie-2). Our results suggest that a combination of humoral and cellular factors could be responsible for the RIPC/cRIPC mediated effects and that interindividual variations seem to play a considerable part in the RIPC/cRIPC associated mechanisms.