Immune Polarization Research Articles

232 - SOCS1 S-Nitrosation in the nucleus is a switch of macrophage polarization and function

Macrophages play a critical role in inflammation initiation, progression and resolution due to their capacity to assume both pro- and anti-inflammatory phenotypes. The molecular mechanisms that regulate these transitions are of great clinical interest but remain poorly understood. We discovered that suppressor of cytokine signaling-1, an enzyme that targets and degrades RelA, is susceptible to S-nitrosation on cisteines 147 and 179 specifically in the nucleus. Nitrosation in the nucleus is effected by nitric oxide synthase-1 (NOS1) in macrophages. Nitrosation of SOCS1 prevents its interaction with RelA, and the formation of the pro-inflammatory p65-p50 NFkB heterodimer. In NOS1 knockout macrophages, SOCS1 is not nitrosated upon activation of the inflammatory response leading to rapid degradation of RelA/p65 with the accumulation of p50. Accumulation of p50 promotes homodimerization (p50-p50) leading to anti-inflammatory gene transcription. Interestingly, transplantation of NOS1 knockout bone marrow-derived macrophages (BMDM) into the inflammation prone lungs of SOCS1fl/fl.LysMCre mice is protective against LPS-induced tissue injury. In addition, levels of pro-inflammatory cytokine production in these lungs are markedly reduced after LPS challenge suggesting NOS1KO BMDM have the capacity to suppress pro-inflammatory immune polarization and associated tissue injury in a model of chronic inflammatory lung disease. Overall our results indicate that SOCS1 acts as a switch between pro- and anti-inflammatory macrophage polarization regulated by NOS1-mediated nitrosation.

Absence of Regulatory T Cells Causes Phenotypic and Functional Switch in Murine Peritoneal Macrophages.

Tissue macrophages are important components of tissue homeostasis and inflammatory pathologies. In the peritoneal cavity, resident macrophages interact with a variety of immune cells and can exhibit broad range of phenotypes and functions. Forkhead-box-P3 (FOXP3)+ regulatory T cells (Tregs) play an indispensable role in maintaining immunological tolerance, yet whether, and how the pathological condition that results from the lack of functional Tregs affects peritoneal macrophages (PM) is largely unknown. We used FOXP3-deficient scurfy (Sf) mice to investigate PM behavior in terms of the missing crosstalk with Tregs. Here, we report that Treg deficiency induced a marked increase in PM numbers, which was reversed after adoptive transfer of CD4+ T cells or neutralization of macrophage colony-stimulating factor. Ex vivo assays demonstrated a pro-inflammatory state of PM from Sf mice and signs of excessive activation and exhaustion. In-depth immunophenotyping of Sf PM using single-cell chipcytometry and transcriptome analysis revealed upregulation of molecules involved in the initiation of innate and adaptive immune responses. Moreover, upon transfer to non-inflammatory environment or after injection of CD4+ T cells, PM from Sf mice reprogramed their functional phenotype, indicating remarkable plasticity. Interestingly, frequencies, and immune polarization of large and small PM subsets were dramatically changed in the FOXP3-deficient mice, suggesting distinct origin and specialized function of these subsets in inflammatory conditions. Our findings demonstrate the significant impact of Tregs in shaping PM identity and dynamics. A better understanding of PM function in the Sf mouse model may have clinical implication for the treatment of immunodysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, and other forms of immune-mediated enteropathies.

Porcine Reproductive and Respiratory Syndrome Virus Type 1.3 Lena Triggers Conventional Dendritic Cells 1 Activation and T Helper 1 Immune Response Without Infecting Dendritic Cells.

Porcine Reproductive and Respiratory Syndrome virus (PRRSV) is an arterivirus responsible for highly contagious infection and huge economic losses in pig industry. Two species, PRRSV-1 and PRRSV-2 are distinguished, PRRSV-1 being more prevalent in Europe. PRRSV-1 can further be divided in subtypes. PRRSV-1.3 such as Lena are more pathogenic than PRRSV-1.1 such as Lelystad or Flanders13. PRRSV-1.3 viruses trigger a higher Th1 response than PRRSV-1.1, although the role of the cellular immune response in PRRSV clearance remains ill defined. The pathogenicity as well as the T cell response inductions may be differentially impacted according to the capacity of the virus strain to infect and/or activate DCs. However, the interactions of PRRSV with in vivo-differentiated-DC subtypes such as conventional DC1 (cDC1), cDC2, and monocyte-derived DCs (moDC) have not been thoroughly investigated. Here, DC subpopulations from Lena in vivo infected pigs were analyzed for viral genome detection. This experiment demonstrates that cDC1, cDC2, and moDC are not infected in vivo by Lena. Analysis of DC cytokines production revealed that cDC1 are clearly activated in vivo by Lena. In vitro comparison of 3 Europeans strains revealed no infection of the cDC1 and cDC2 and no or little infection of moDC with Lena, whereas the two PRRSV-1.1 strains infect none of the 3 DC subtypes. In vitro investigation of T helper polarization and cytokines production demonstrate that Lena induces a higher Th1 polarization and IFNγ secretion than FL13 and LV. Altogether, this work suggests an activation of cDC1 by Lena associated with a Th1 immune response polarization.

Commitment to Aerobic Glycolysis Sustains Immunosuppression of Human Mesenchymal Stem Cells.

Human mesenchymal stem cells (hMSCs) promote endogenous tissue repair in part by coordinating multiple components of the host immune system in response to environmental stimuli. Recent studies have shown that hMSCs are metabolically heterogeneous and actively reconfigure metabolism to support the biochemical demands of tissue repair. However, how hMSCs regulate their energy metabolism to support their immunomodulatory properties is largely unknown. This study investigates hMSC metabolic reconfiguration during immune activation and provides evidence that the hMSC metabolic state significantly influences their immunomodulatory properties. Specifically, hMSC immune polarization by interferon‐gamma (IFN‐γ) treatment leads to remodeling of hMSC metabolic pathways toward glycolysis, which is required to sustain the secretion of immunosuppressive factors. IFN‐γ exposure also inhibited mitochondrial electron transport activity, and the accumulation of mitochondrial reactive oxygen species plays an important signaling role in this metabolic reconfiguration. The results also show that activation of the Akt/mTOR signaling pathway is required for metabolic reconfiguration during immune polarization and that interruption of these metabolic changes alters the immune response in IFN‐γ licensed hMSCs. The results demonstrate the potential of altering hMSC metabolism to enhance their immunomodulatory properties and therapeutic efficacy in various diseases. Stem Cells Translational Medicine 2019;8:93–106

The frequency of specific contact allergies is reduced in patients with psoriasis.

Earlier studies suggested an inverse association between contact allergy and psoriasis, although the results of clinical studies have been inconsistent. Gene expression studies in human contact allergy focusing on immune responses revealed nickel being an inducer of T helper (Th)1/Th17 and some Th22 immune polarization, whereas fragrances were found to promote a Th2-dominated immune activation. To investigate the epidemiological association between contact allergy and psoriasis in a large multicentre cohort and to analyse the sensitization profile to specific allergens in these patients. Retrospective analysis of patch-tested patients from 56 departments of dermatology (1996-2015), including 2387 patients with psoriasis and 161989 control patients. All patients with atopic dermatitis were excluded from both groups. The odds ratio (OR) for contact allergy was calculated to be 0·55 in patients with psoriasis (95% confidence interval 0·50-0·59). Logistic regression analyses with several independent variables indicated a 'protective effect' of having psoriasis for most allergens, independently of age, sex, affected body site and patch test indication. Fragrance mix II (OR 0·36) and lanolin alcohols (OR 0·38) were found to be among the least common allergens in patients with psoriasis. In contrast, the frequency of contact dermatitis to nickel was only marginally affected in patients with psoriasis compared with controls (OR 0·75). The inverse association between psoriasis and allergic contact sensitization is likely to be not exclusively mediated by psoriasis itself. The polarization of the activated immune response by specific allergens may influence the occurrence and significance of contact allergies in underlying immune-mediated diseases, eventually even beyond the skin.

MicroRNAs: Mediators and Therapeutic Targets to Airway Hyper Reactivity After Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) is the most important pathogen correlated to the first-time infant wheezing and later recurrence after its primary infection. RSV infection promotes the bronchial smooth muscle sensitivity to leukotrienes (LTs) in acute stage, causes the extensive inflammatory reaction and the aggregation of Th2-like cells during respiratory tract obstruction. Infants and young children infected with RSV exhibit an increased susceptibility to the exposure of exogenous allergens, easy to suffer from the recurrent wheezing, which prompts that the body is still in a state of inflammation or immunological bias. However, the pathological mechanism is unclear. The recent researches demonstrate that abnormal expression of non-coding microRNAs (miRNAs) can be detected from the peripheral blood and airway tract epithelial of RSV infected infants, which participate the regulation of immune cells polarization and LTs synthesis. Improving the immune tolerance can significantly relieve the airway inflammation and broncho-spasm caused by RSV. In this review, we discuss recent advances in understanding the mechanism of RSV-induced inflammatory reaction and immune dysfunction leading to airway hyper-reactivity. Further, we summarize the potential molecular basis that, in this process, miRNAs, which are produced by airway epithelial cells or peripheral blood mononuclear cells, directly or in the form of exosome to regulate the inflammation programs as well as the function, differentiation and proliferation of immune cells. miRNAs may become a potential bio-marker of detecting severe RSV infection and a novel target of early intervention and therapeutic strategy in recurrent wheezing or asthma related to RSV infection.

Ca2+-Activated K+ Channel KCa3.1 as a Therapeutic Target for Immune Disorders.

In lymphoid and myeloid cells, membrane hyperpolarization by the opening of K+ channels increases the activity of Ca2+ release-activated Ca2+ (CRAC) channels and transient receptor potential (TRP) Ca2+ channels. The intermediate-conductance Ca2+-activated K+ channel KCa3.1 plays an important role in cell proliferation, differentiation, migration, and cytokine production in innate and adaptive immune systems. KCa3.1 is therefore an attractive therapeutic target for allergic, inflammatory, and autoimmune disorders. In the past several years, studies have provided new insights into 1) KCa3.1 pharmacology and its auxiliary regulators; 2) post-transcriptional and proteasomal regulation of KCa3.1; 3) KCa3.1 as a regulator of immune cell migration, cytokine production, and phenotypic polarization; 4) the role of KCa3.1 in the phosphorylation and nuclear translocation of Smad2/3; and 5) KCa3.1 as a therapeutic target for cancer immunotherapy. In this review, we have assembled a comprehensive overview of current research on the physiological and pathophysiological significance of KCa3.1 in the immune system.

Mitochondrial fidelity and metabolic agility control immune cell fate and function.

Remodeling of mitochondrial metabolism plays an important role in regulating immune cell fate, proliferation, and activity. Furthermore, given their bacterial ancestry, disruption in mitochondrial fidelity leading to extravasation of their content initiates and amplifies innate immune surveillance with a myriad of physiologic and pathologic consequences. Investigations into the role of mitochondria in the immune system have come to the fore, and appreciation of mitochondrial function and quality control in immune regulation has enhanced our understanding of disease pathogenesis and identified new targets for immune modulation. This mitochondria-centered Review focuses on the role of mitochondrial metabolism and fidelity, as well as the role of the mitochondria as a structural platform, for the control of immune cell polarity, activation, and signaling. Mitochondria-linked disease and mitochondrially targeted therapeutic strategies to manage these conditions are also discussed.

Chemokine CCL20 plasmid improves protective efficacy of the Montanide ISA™ 206 adjuvanted foot-and-mouth disease vaccine in mice model

This study aimed to investigate the chemokine CCL20, a macrophage inflammatory protein-3 alpha, for adjuvant potential in inactivated foot-and-mouth disease (FMD) vaccine. Groups of mice were injected intramuscularly with either murine CCL20 DNA or CCL20 protein two days ahead of the immunization with Montanide ISA206 adjuvanted inactivated FMD vaccine and humoral and cellular immune responses were measured in post-vaccinal sera. We demonstrated that the mice immunized with CCL20 plasmid plus FMD vaccine showed earlier and significantly (p < 0.05) higher neutralizing antibody responses compared to the mice vaccinated with CCL20 protein plus FMD vaccine. In fact, CCL20 as a protein did not show any adjuvant effect and the immune responses induced in this group were comparable to that of the mice vaccinated with FMD vaccine alone. All the vaccination groups showed serum IgG1 and IgG2 antibody responses; however, the mice vaccinated with CCL20 plasmid plus FMD vaccine showed significantly (p < 0.05) higher IgG1 and IgG2 responses and the responses remained high at all-time points post vaccination, although not always statistically significant. Upon restimulation of the vaccinated splenocytes with the inactivated FMD viral antigen, significantly (p < 0.05) higher IFN-γ and IL-2 levels in culture supernatants were found in animals vaccinated with the CCL20 plasmid plus FMD vaccine, which is indicative of the TH1 type of cellular immunity. On challenge with the homologous FMD virus on 28th day post immunization, CCL20 plasmid plus FMD vaccine showed complete protection (100%) while animals immunized with CCL20 protein plus FMD vaccine or FMD vaccine alone showed 66% protection. In summary, we show that prior injection of CCL20 plasmid improved protective efficacy of the inactivated FMD vaccine and thus offers a valuable strategy to modulate the efficacy and polarization of specific immunity against inactivated vaccines.

Visceral leishmaniasis: A novel nuclear envelope protein ‘nucleoporins-93 (NUP-93)’ from Leishmania donovani prompts macrophage signaling for T-cell activation towards host protective immune response

The shift of macrophage and T-cell repertoires towards proinflammatory cytokine signalling ensures the generation of host-protective machinery that is otherwise compromised in cases of the intracellular Leishmania parasite. Different groups have attempted to restore host protective immunity. These vaccine candidates showed good responses and protective effects in murine models, but they generally failed during human trials. In the present study, we evaluated the effect of 97 kDa recombinant nucleoporin-93 of Leishmania donovani (rLd-NUP93) on mononuclear cells in healthy and treated visceral leishmaniasis (VL) patients and on THP-1 cell lines. rLd-NUP93 stimulation increased the expression of the early lymphocyte activation marker CD69 on CD4+ and CD8+ T cells. The expression of the host protective pro-inflammatory cytokines IFN-γ, IL-12 and TNF-α was increased, with a corresponding down-regulation of IL-10 and TGF-β upon rLd-NUP93 stimulation. This immune polarization resulted in the up-regulation of NF-κB p50 with scant expression of SMAD-4. Augmenting lymphocyte proliferation upon priming with rLd-NUP93 ensured its potential for activation and generation of strong T-cell mediated immune responses. This stimulation extended the leishmanicidal activity of macrophages by releasing high amounts of reactive oxygen species (ROS). Further, the leishmanicidal activity of macrophages was intensified by the elevated production of nitric oxide (NO). The fact that this antigen was earlier reported in circulating immune complexes of VL patients highlights its antigenic importance. In addition, in silico analysis suggested the presence of MHC class I and II-restricted epitopes that proficiently trigger CD8+ and CD4+ T-cells, respectively. This study reported that rLd-NUP93 was an effective immunoprophylactic agent that can be explored in future vaccine design.

The metabolic axis of macrophage and immune cell polarization.

ABSTRACTThe extracellular space of solid tumors ranges from being well-nurtured to being completely ischemic and can serve as a source of intratumoral heterogeneity, determining the behavior and molecular profiles of malignant and stromal cells. Here, we discuss how the metabolic tumor microenvironment modulates the phenotypes of the immune cells that infiltrate tumors, with an emphasis on tumor-associated macrophages. These cells constitute a diverse population that has pro-tumoral and anti-inflammatory properties, and are likened to anti-inflammatory ‘M2’ macrophages. Recent findings show how different metabolic microenvironments specify an array of phenotypic changes in macrophages. In tumors, extracellular metabolite levels vary predictably according to proximity to the vasculature, and phenotypic changes in tumor-associated macrophages and in other immune cells are also predictable. We speculate that this ‘metabolic axis’ of macrophage polarization modulates – and is modulated by – the response to inflammatory cues, creating a wide variety of possible phenotypic states. Understanding how extracellular metabolites influence cell phenotypes allows us to predict how tumor-associated macrophages and other tumor cells might change, with the aim of harnessing this predictability for therapy. Overall, we describe an emerging picture in which chemokines, growth factors and the metabolic tumor microenvironment act together to determine the phenotypes of tumor-infiltrating immune cells.

Abstract 1687: Checkpoint inhibitor signatures across endometrial cancer histologies

Abstract Background: Therapies targeting immune checkpoints have revolutionized cancer treatment. However, biomarkers that effectively stratify patients to these therapies are required. Endometrial carcinomas consist of several histologies and a subset harbor hypermutation/microsatellite instability (MSI) that has been associated with response to PD-1 inhibition. The purpose of this study was to investigate the immune checkpoint landscape across endometrial cancer subtypes and ascertain whether more complex signatures indicate directions for combination immune therapies in efforts to improve patient response. Methods: With IRB approval, 60 diagnostic endometrial tumor samples were obtained from 2013-2017 consisting of MSI low grade endometrioid (n=11), microsatellite stable (MSS) low grade endometrioid (n=11), high grade endometrioid (n=16), carcinosarcoma (n=11), and uterine serous carcinoma (n=11) histologies. Immune RNA expression signatures were evaluated using a Luminex platform that evaluated 142 gene targets, including inflammation mediators, immune checkpoints, immune cell type and immune cell polarity markers. Expression of RNA for genes encoding CTLA-4, PD-1, PD-L1, LAG-3, and TIM-3 were quantified and correlated with clinical outcomes utilizing parametric and non-parametric testing. These signatures will be further investigated with multispectral imaging, including proximity analysis. Results: Cohort characteristics included 32 patients with stage 1A, 9 patients with stage 1B, 6 with stage II, 8 with stage III and 5 with stage IV disease. The median age at diagnosis was 66 years. RNA expression for CTLA-4, PD-1, PD-L1, LAG-3, and TIM-3 were compared across the various endometrial carcinoma histologies. While there were no significant differences noted between carcinosarcoma and serous carcinoma, levels of TIM-3 and CTLA-4 expression were significantly elevated in endometrioid carcinoma (P&amp;lt;0.01, P&amp;lt;0.001 respectively). When evaluating expression within endometrioid subtypes, high grade tumors were independently associated with elevated checkpoint expression (LAG-3 (P=0.04), PD-L1 (P=0.04), and CTLA-4 (P=0.04)), as was the presence of MSI (TIM-3 (P=0.02), PD-L1 (P=0.01), and CTLA-4 (P=0.014). When both grade and MSI were factored together, PD-1 was significantly elevated in MSI high grade versus MSI low grade cancers (P=0.04). RNA expression levels for PD-L1 and PD-L2 were also significantly elevated in endometrioid cancers. Preliminary analysis suggests that recurrent tumors demonstrate altered levels of immune checkpoint signatures compared to those that do not. Conclusion: A complex landscape of regulatory immune receptors was found across endometrial cancer histologies, with particular relevance in endometrioid carcinomas that was grade and MSI-dependent. These data suggest directions for combined immune blockade therapy in this cancer type. Citation Format: Amanda Ramos, Sarah Fortin, Victoria Melchert, David Jenkins, Whitfield Growdon, Darrell Borger. Checkpoint inhibitor signatures across endometrial cancer histologies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1687.

Metabolic Stress in the Immune Function of T Cells, Macrophages and Dendritic Cells.

Innate and adaptive immune cells from myeloid and lymphoid lineages resolve host infection or cell stress by mounting an appropriate and durable immune response. Upon sensing of cellular insults, immune cells become activated and undergo rapid and efficient functional changes to unleash biosynthesis of macromolecules, proliferation, survival, and trafficking; unprecedented events among other mammalian cells within the host. These changes must become operational within restricted timing to rapidly control the insult and to avoid tissue damage and pathogen spread. Such changes occur at high energy cost. Recent advances have established that plasticity of immune functions occurs in distinct metabolic stress features. Evidence has accumulated to indicate that specific metabolic signatures dictate appropriate immune functions in both innate and adaptive immunity. Importantly, recent studies have shed light on whether successfully manipulating particular metabolic targets is sufficient to modulate immune function and polarization, thereby offering strong therapeutic potential for various common immune-mediated diseases, including inflammation and autoimmune-associated diseases and cancer. In this review, we detail how cellular metabolism controls immune function and phenotype within T cells and macrophages particularly, and the distinct molecular metabolic programming and targets instrumental to engage this regulation.

Differential expression and evolution of three tandem, interleukin-1 receptor-like 1 genes in rainbow trout (Oncorhynchus mykiss)

Interleukin-1 receptor-like 1 (Il1rl1 or ST2), a member of the interleukin-1 receptor family, has pleiotropic roles including tissue homeostasis, inflammation, immune polarization, and disease resistance in mammals. A single orthologue was previously described in salmonid fish; however, a recently improved genome assembly of rainbow trout (Oncorhynchus mykiss) revealed three adjacent, tandem il1rl1 orthologues on chromosome Omy 03. Here, we report the genomic organization and evolution of the three il1rl1 genes (il1rl1α, il1rl1β, il1rl1γ), and use both RNA-seq and gene-specific qPCR methods to quantify expression patterns. Nucleotide sequence homology between the three genes is >95% and each predicted protein contains three IG/IG-like domains, a transmembrane region and a TIR domain. The amino acid sequence homology of the rainbow trout il1rl1 genes are highly related to two functional copies in Atlantic and Coho salmon (∼94%) but relatively low (22–26%) with avian and mammalian species. Transcript abundance measured by RNA-seq in 15 tissues of healthy adult rainbow trout indicate constitutive expression of each gene. In whole body lysates, il1rl1α was shown to have >20 fold mRNA expression compared to il1rl1β and il1rl1γ as measured by qPCR assays specific to il1rl1α or il1rl1γ, as well as a multi-gene qPCR assay (il1rl1α,β,γ). Unrooted phylogenetic trees grouped the rainbow trout il1rl1 genes apart from other interleukin-1 receptor family genes and genomic comparisons identify preserved synteny between mammals, birds and salmonids albeit a pseudogene is present in both Atlantic salmon and Coho salmon. Phylogenetic analyses suggest that the three genes arose by tandem duplication but are inconclusive whether these events occurred prior-to or after salmonid speciation. These findings further the understanding of interleukin receptor family evolution and their contribution to teleost immune function.

DNA immunization site determines the level of gene expression and the magnitude, but not the type of the induced immune response.

Optimization of DNA vaccine delivery improves the potency of the immune response and is crucial to clinical success. Here, we inquired how such optimization impacts the magnitude of the response, its specificity and type. BALB/c mice were DNA-immunized with two model immunogens, HIV-1 protease and reverse transcriptase by intramuscular or intradermal injections with electroporation. DNA immunogens were co-delivered with DNA encoding luciferase. Delivery and expression were monitored by in vivo bioluminescence imaging (BLI). The endpoint immune responses were assessed by IFN-γ/IL-2 FluoroSpot, multiparametric flow cytometry and antibody ELISA. Expression and immunogenicity were compared in relation to the delivery route. Regardless of the route, protease generated mainly IFN-γ, and reverse transcriptase, IL-2 and antibody response. BLI of mice immunized with protease- or reverse transcriptase/reporter plasmid mixtures, demonstrated significant loss of luminescence over time. The rate of decline of luminescence strongly correlated with the magnitude of immunogen-specific response, and depended on the immunogenicity profile and the immunization route. In vitro and in vivo BLI-based assays demonstrated that intradermal delivery strongly improved the immunogenicity of protease, and to a lesser extent, of reverse transcriptase. Immune response polarization and epitope hierarchy were not affected. Thus, by changing delivery/immunogen expression sites, it is possible to modulate the magnitude, but not the type or fine specificity of the induced immune response.

Treatment with IL-19 improves locomotor functional recovery after contusion trauma to the spinal cord.

IL-19 skews the immune response towards a Th2 type and appears to stimulate angiogenesis. In the current study, we tested if IL-19 treatment could reduce secondary injury and improve functional recovery after contusion spinal cord injury (SCI). Firstly, mice were given a moderate-severe thoracic SCI at the T9-10 level and expression of IL-19 and its receptor was measured in the injured spinal cord. Then SCI mice were treated with mouse recombinant IL-19 and its blocking antibody to investigate the therapeutic effect of IL-19. Protein expression of IL-19 and its receptor IL-20R1 and IL-20R2 was up-regulated in the injured spinal cord of mice. IL-19 treatment promoted the recovery of locomotor function dose-dependently and reduced loss of motor neurons and microglial and glial activation following SCI. Treatment of SCI mice with IL-19 attenuated macrophage accumulation, reduced protein levels of TNF-α and CCL2 and promoted Th2 response and M2 macrophage activation in the injured region. Treatment of SCI mice with IL-19 promoted angiogenesis through up-regulating VEGF in the injured region. Treatment of SCI mice with IL-19 up-regulated HO-1 expression and decreased oxidative stress in the injured region. The beneficial effect of IL-19 was abolished by coadministration of the blocking antibody. Additionally, IL-19 deficiency in mice delayed the recovery of locomotor function following SCI. IL-19 treatment reduced secondary injuries and improved locomotor functional recovery after contusion SCI, through diverse mechanisms including immune cell polarization, angiogenesis and anti-oxidative responses.

Hyaluronan: A Master Switch Between Vascular Homeostasis and Inflammation.

Hyaluronan (HA) matrix responds quickly to changing conditions during atherogenesis and atheroprogression. Our evolving understanding of HA’s specific functions at the interface of inflammatory responses and phenotypic control of mesenchymal cells in the vessel wall points to its importance in inflammation and atherosclerosis. Additionally, the enzymes that mediate HA biosynthesis and degradation are potential target molecules for novel therapies, as are signal-transducing HA receptors: deciphering the HA matrix, thus, has significant translational possibilities. HA is glycosaminoglycan polymer without covalent binding to a core protein. A unique feature of HA is its de novo synthesis at the plasma membrane by HA synthases (HAS1–3) from UDP (uridine diphosphate)-sugar precursors. The chemical structure of HA is simple and comprises repeating disaccharides of β(1,4)- N -acetyl-d-glucosamine and β(1,3)-d-glucuronic acid that form an unbranched and unmodified polymer. This structure is conserved across all mammalian species. The biological functions of HA are complex and highly context-specific because as soon as HA is extruded to the extracellular space, it is subject to complex interactions with HA-binding proteins (hyaladherins). The function of the pericellular HA matrix depends on the particular hyaladherins present and their engagement with HA receptors, such as CD44, RHAMM (receptor of HA-mediated motility), and Lyve-1. The resulting interactions modulate the adhesion, migration, and proliferation of smooth muscle cells (SMC), fibroblasts, and other cell types present in vascular tissues. In parallel, HA complexes also influence important aspects of immune cell polarization, differentiation, and function. Beyond the immediate pericellular microenvironment, HA is also integrated into structures such as basement membranes, is present in circulation, and can function as a signaling molecule to activate HA receptors. The molecular weight of HA has critical functional importance in vivo. In the steady state, longer, high-molecular-weight HA polymers support homeostasis and immune surveillance. On injury, however, HA …

Abstract 232: Development and Validation of a Mathematical Model Representing Complex Cellular Interactions Within the Artery Wall: in Silico Approach to Test Targeted Interventions

Atherosclerotic cardiovascular diseases remain the number one cause of morbidity and mortality despite significant advances in lipid management. Complex cellular interactions occur within the artery wall requiring timely infiltration/egress of immune cells and lipoproteins within a changing inflammatory milieu that determine the progression of an atherosclerotic plaque. Lack of detailed understanding of the multiple processes involved and their potential interactions has hindered development of targeted therapies. The objective of this study was to develop a computational model of the sequential influx of immune cells in response to a trigger to permit a system-level analyses of the processes involved using ordinary differential equations (ODEs). Thioglycollate induced peritonitis was used as a model to examine the infiltration of immune cells and phenotypic polarization of macrophages in response to a stimulus. Peritoneal exudates obtained at 10 different time-points over 7 days were analyzed by FACS to determine the distribution of neutrophils, macrophages and Ly6C Hi (M1) or Ly6C Lo (M2) polarization. Weighted least squares of the different parameters were used to calibrate the model developed in our laboratory (Panel A) to simulate inflammation/repair during sepsis using the Levenberg-Marquardt algorithm. Panels B-D show the “match” of the experimental data to the calibrated model demonstrating the validity of this model. Since the ODEs are derived from a combination of known and hypothesized kinetics of the biological system, model the changes in physiological variables over time and are based on biological interactions, our calibrated mathematical model will permit the evaluation of changes in one or more targeted parameters in silico on immune cell distribution/phenotype. Efficacy of various macrophage-specific interventions (e.g., reduction in cholesterol content or inflammation) can be predicted prior to preclinical experimentation.

Hemolysis and immune regulation.

Hemolytic anemias caused by premature destruction of red blood cells occur in many disorders including hemoglobinopathies, autoimmune conditions, during infection or following reaction to drugs or transfusions. Recent studies which will be reviewed here have uncovered several novel mechanisms by which hemolysis can alter immunological functions and increase the risk of severe complications in hemolytic disorders. Plasma-free heme can induce the formation of neutrophil extracellular traps (NETs) through reactive oxygen species signaling. Although NETs protect the host against infections, in patients with sickle disease, they are associated with vaso-occlusive crises. Heme may increase host susceptibility to infections by inducing heme oxygenase 1 (HO-1) in immature neutrophils, thereby inhibiting oxidative burst required for clearance of engulfed bacteria. In addition, heme impairs macrophage phagocytosis and microbial clearance through inhibition of cytoskeletal remodeling. Hemolysis can also favor anti-inflammatory immune cell polarization by inhibiting dendritic cell maturation necessary for effector T-cell responses, inducing differentiation of monocytes into red pulp macrophages, important for iron recycling from senescent erythrocytes, and driving regulatory T-cell expansion through modulation of HO-1 expression in nonclassical monocytes. Hemolysis breakdown products show remarkable effects on the regulation of immune cell differentiation and function.

Emerging Concepts of Adaptive Immunity in Leprosy.

Leprosy is a chronic intracellular infection caused by the acid-fast bacillus, Mycobacterium leprae. The disease chiefly affects the skin, peripheral nerves, mucosa of the upper respiratory tract, and the eyes. The damage to peripheral nerves results in sensory and motor impairment with characteristic deformities and disability. Presently, the disease remains concentrated in resource-poor countries in tropical and warm temperate regions with the largest number of cases reported from India. Even though innate immunity influences the clinical manifestation of the disease, it is the components of adaptive immune system which seem to tightly correlate with the characteristic spectrum of leprosy. M. leprae-specific T cell anergy with bacillary dissemination is the defining feature of lepromatous leprosy (LL) patients in contrast to tuberculoid leprosy (TT) patients, which is characterized by strong Th1-type cell response with localized lesions. Generation of Th1/Th2-like effector cells, however, cannot wholly explain the polarized state of immunity in leprosy. A comprehensive understanding of the role of various regulatory T cells, such as Treg and natural killer T cells, in deciding the polarized state of T cell immunity is crucial. Interaction of these T cell subsets with effector T cells like Th1 (IFN-γ dominant), Th2 (interluekin-4 dominant), and Th17 (IL-17+) cells through various regulatory cytokines and molecules (programmed death-1/programmed death ligand-1) may constitute key events in dictating the state of immune polarization, thus controlling the clinical manifestation. Studying these important components of the adaptive immune system in leprosy patients is essential for better understanding of immune function, correlate(s) the immunity and mechanism(s) of its containment.