Chronic Phases Of Inflammation Research Articles

Impact of Interleukin-6 Activation and Arthritis on Epidermal Growth Factor Receptor (EGFR) Activation in Sensory Neurons and the Spinal Cord.

In tumor cells, interleukin-6 (IL-6) signaling can lead to activation of the epidermal growth factor receptor (EGFR), which prolongs Stat3 activation. In the present experiments, we tested the hypothesis that IL-6 signaling activates EGFR signaling in peripheral and spinal nociception and examined whether EGFR localization and activation coincide with pain-related behaviors in arthritis. In vivo in anesthetized rats, spinal application of the EGFR receptor blocker gefitinib reduced the responses of spinal cord neurons to noxious joint stimulation, but only after spinal pretreatment with IL-6 and soluble IL-6 receptor. Using Western blots, we found that IL-6-induced Stat3 activation was reduced by gefitinib in microglial cells of the BV2 cell line, but not in cultured DRG neurons. Immunohistochemistry showed EGFR localization in most DRG neurons from normal rats, but significant downregulation in the acute and most painful arthritis phase. In the spinal cord of mice, EGFR was highly activated mainly in the chronic phase of inflammation, with localization in neurons. These data suggest that spinal IL-6 signaling may activate spinal EGFR signaling. Downregulation of EGFR in DRG neurons in acute arthritis may limit nociception, but pronounced delayed activation of EGFR in the spinal cord may be involved in chronic inflammatory pain.

ScRNA-seq and flow cytometry based analysis reveals that chronic heart failure associates with enhanced activation and clonal expansion of T cells with predictive autoreactive capacity

Abstract Background Chronic heart failure (HF) is marked by adverse remodelling and chronic inflammation causing impaired function of the heart and poor prognosis. While much is known about the immune response immediately after MI, little is known of the role of the immune system in chronic HF. Our aim is to elucidate the response of T cells to HF. Methods and Results In a pilot study assessing healthy controls (HC) and HF patients (n=16), scRNA-seq showed an enhanced T cell activation profile in HF patients combined with an increased capacity for adhesion and antigen-presentation on monocytes. A confirmation cohort of n=180 HC and HF patients using flow cytometry of peripheral blood confirmed an increase of T cell memory/effector cells (HF=65%, HC=53% activated T cells), representing activated and stimulated T cells, and a reduction in the non-active naïve T cell compartment in HF patients. Correspondingly, APCs showed a higher immune stimulatory profile (e.g. HLA-DR, ICAM-1, TREM-1). Immune secretome analysis further supports the chronic inflammatory phase by significantly increased serum levels of sICAM-1, IL6 and TNFRI. Given that T cells showed strong activation profiles in HF patients, we investigated whether T cells are preferentially clonally expanded in the same patients. Bulk TCR sequencing revealed that TCR diversity was significantly reduced (p=0.03) along with clonal expansion of T cells in HF patients. To assess what these clonally expanding T cells might be targeting, we utilized epitope prediction tools to identify 22 unique human-derived epitopes found only in HF patients. ScRNA-seq of CD45+ sorted cardiac-derived and circulating cells of the same HF patient (N=1) versus healthy controls (n=4) showed clonal expansion of TCRs in the cardiac tissue and blood in HF and epitope prediction confirmed epitopes found in bulk TCR sequencing. We then assessed the T cell response in a time course post-myocardial infarction in mice with scRNA-seq. We found an accumulation of specifically pro-inflammatory (Th17 T cells in the heart at days 14, 28 and 48 post-MI. Dimensional reduction analysis of T cells from all time points showed that T cells appearing at days 28 and 48 post-MI were more pro-inflammatory than at the earlier time points. To assess the impact of immune dysregulation in HF on cardiac cells, we cultured the secretome of HF or HC immune cells with endothelial cells. We found that HF secretomes decreased viability of endothelial cells and increased monocyte adhesion to stimulated endothelial cells (p=0.005). Additionally, the immune regulatory molecule PDL-1 was downregulated in HF secretome treated endothelial cells (p=0.014). Conclusion This study suggests that chronic HF may drive persistent T cell activation and clonal expansion, with potential cardiac-relevant autoimmune implications causing or promoting adverse remodelling. These data also provide additional insights for therapeutic cardio-immunological interventions.

Transgenic disruption of endogenous glucocorticoid signaling in osteoblasts does not alter long-term K/BxN serum transfer-induced arthritis

BackgroundDisruption of glucocorticoid (GC) signaling in osteoblasts results in a marked attenuation of acute antibody-induced arthritis. The role of endogenous GCs in chronic inflammatory arthritis is however not fully understood. Here, we investigated the impact of endogenous GC signaling in osteoblasts on inflammation and bone integrity under chronic inflammatory arthritis by inactivating osteoblastic GC signaling in a long-term K/BxN serum transfer-induced induced arthritis (STIA) model.MethodsIntracellular GC signaling in osteoblasts was disrupted by transgenic (tg) overexpression of 11beta-hydroxysteroid dehydrogenase type 2 (11ß-HSD2). Inflammatory arthritis was induced in 5-week-old male tg mice and their wild type (WT) littermates by intraperitoneal (i.p.) injection of K/BxN serum while controls (CTRLs) received phosphate-buffered saline (PBS). In a first cohort, K/BxN STIA was allowed to abate until the endpoint of 42 days (STIA). To mimic rheumatic flares, a second cohort was additionally injected on days 14 and 28 with K/BxN serum (STIA boost). Arthritis severity was assessed daily by clinical scoring and ankle size measurements. Ankle joints were assessed histopathologically. Systemic effects of inflammation on long bone metabolism were analyzed in proximal tibiae by micro-computed tomography (μCT) and histomorphometry.ResultsAcute arthritis developed in both tg and WT mice (STIA and STIA boost) and peaked around day 8. While WT STIA and tg STIA mice showed a steady decline of inflammation until day 42, WT STIA boost and tg STIA boost mice exhibited an arthritic phenotype over a period of 42 days. Clinical arthritis severity did not differ significantly between WT and tg mice, neither in the STIA nor in the STIA boost cohorts. Correspondingly, histological indices of inflammation, cartilage damage, and bone erosion showed no significant difference between WT and tg mice on day 42. Histomorphometry revealed an increased bone turnover in tg CTRL and tg STIA boost compared to WT CTRL and WT STIA boost animals, respectively.ConclusionsIn contrast to the previously reported modulating effects of endogenous GC signaling in osteoblasts during acute K/BxN STIA, this effect seems to perish during the chronic inflammatory and resolution phase. These findings indicate that endogenous GC signaling in osteoblasts may mainly be relevant during acute and subacute inflammatory processes.

Highly specific neutrophil-mediated delivery of albumin nanoparticles to ectopic lesion for endometriosis therapy

Endometriosis is an estrogen-dependent chronic inflammatory disease. Hormonal and surgical treatments are the most commonly used clinical therapies, but they have many sides effects or are traumatic to the body. Therefore, specific drugs for endometriosis treatment are urgently needed to develop. In this study, we identified two features of endometriosis, namely the continuous recruitment of neutrophils into the ectopic lesions and the higher uptake of glucose by ectopic cells. For the above features, we designed a glucose oxidase-loaded bovine serum albumin nanoparticle (BSA-GOx-NPs) that is inexpensive and facilitates large-scale production. After injection, BSA-GOx-NPs were high specifically delivered to ectopic lesions in a neutrophil-dependent manner. Furthermore, BSA-GOx-NPs deplete glucose and induce apoptosis in the ectopic lesions. Whereupon BSA-GOx-NPs produced excellent anti-endometriosis effects when administrated in both acute and chronic inflammatory phases. These results reveal for the first time that the neutrophil hitchhiking strategy is effective in chronic inflammatory disease and provide a non-hormonal and easy-to-achieve approach for endometriosis treatment.

Sex differences in the therapeutic effect of unaltered versus NFκB sensing IL-4 over-expressing mesenchymal stromal cells in a murine model of chronic inflammatory bone loss

Wear particles from joint arthroplasties induce chronic inflammation associated with prolonged upregulation of nuclear factor kappa-B (NF-κB) signaling in macrophages and osteoclasts, which leads to osteolysis and implant loosening. Mesenchymal stromal cell (MSC)-based therapy showed great potential for immunomodulation and mitigation of osteolysis in vivo, especially in the chronic phase of inflammation. We previously generated genetically modified MSCs that secrete the anti-inflammatory cytokine interleukin 4 (IL-4) in response to NF-κB activation (NFκB-IL-4 MSCs). However, whether the impact of sexual difference in the internal environment can alter the therapeutic effects of IL-4 over-secreting MSCs that simultaneously mitigate prolonged inflammation and enhance bone formation remains unknown. This study investigated the therapeutic effects of unaltered MSCs versus NFκB-IL-4 MSCs in mitigating chronic inflammation and enhancing bone formation in male and female mice. The murine model was established by continuous infusion of polyethylene particles contaminated with lipopolysaccharide (cPE) into the medullary cavity of the distal femur for 6 weeks to induce chronic inflammation. Unaltered MSCs or NFκB-IL-4 MSCs were infused into the femoral intramedullary cavity in sex-matched groups beginning 3 weeks after primary surgery. Femurs were harvested at 6 weeks, and bone marrow density was measured with micro-computational tomography. Numbers of osteoclast-like cells, osteoblasts, and macrophages were evaluated with histochemical and immunofluorescence staining. cPE infusion resulted in severe bone loss at the surgery site, increased tartrate-resistant acid phosphatase positive osteoclasts and M1 pro-inflammatory macrophages, and decreased alkaline phosphatase expression. MSC-based therapy effectively decreased local bone loss and polarized M1 macrophages into an M2 anti-inflammatory phenotype. In females, unaltered MSCs demonstrated a larger impact in enhancing the osteogenesis, but they demonstrated similar anti-inflammatory effects compared to NFκB-IL-4 MSCs. These results demonstrated that local inflammatory bone loss can be effectively modulated via MSC-based treatments in a sexually dimorphic manner, which could be an efficacious therapeutic strategy for treatment of periprosthetic osteolysis in both genders.

Experimental Arthritis Inhibits Adult Hippocampal Neurogenesis in Mice.

Background: Adult-born neurons of the hippocampal dentate gyrus play a role in specific forms of learning, and disturbed neurogenesis seems to contribute to the development of neuropsychiatric disorders, such as major depression. Neuroinflammation inhibits adult neurogenesis, but the effect of peripheral inflammation on this form of neuroplasticity is ambiguous. Objective: Our aim was to investigate the influence of acute and chronic experimental arthritis on adult hippocampal neurogenesis and to elucidate putative regulatory mechanisms. Methods: Arthritis was triggered by subcutaneous injection of complete Freund’s adjuvant (CFA) into the hind paws of adult male mice. The animals were killed either seven days (acute inflammation) or 21 days (chronic inflammation) after the CFA injection. Behavioral tests were used to demonstrate arthritis-related hypersensitivity to painful stimuli. We used in vivo bioluminescence imaging to verify local inflammation. The systemic inflammatory response was assessed by complete blood cell counts and by measurement of the cytokine/chemokine concentrations of TNF-α, IL-1α, IL-4, IL-6, IL-10, KC and MIP-2 in the inflamed hind limbs, peripheral blood and hippocampus to characterize the inflammatory responses in the periphery and in the brain. In the hippocampal dentate gyrus, the total number of newborn neurons was determined with quantitative immunohistochemistry visualizing BrdU- and doublecortin-positive cells. Microglial activation in the dentate gyrus was determined by quantifying the density of Iba1- and CD68-positive cells. Results: Both acute and chronic arthritis resulted in paw edema, mechanical and thermal hyperalgesia. We found phagocytic infiltration and increased levels of TNF-α, IL-4, IL-6, KC and MIP-2 in the inflamed hind paws. Circulating neutrophil granulocytes and IL-6 levels increased in the blood solely during the acute phase. In the dentate gyrus, chronic arthritis reduced the number of doublecortin-positive cells, and we found increased density of CD68-positive macrophages/microglia in both the acute and chronic phases. Cytokine levels, however, were not altered in the hippocampus. Conclusions: Our data suggest that acute peripheral inflammation initiates a cascade of molecular and cellular changes that eventually leads to reduced adult hippocampal neurogenesis, which was detectable only in the chronic inflammatory phase.

Intestinal inflammation-associated hypersensitivity is attenuated in a DSS model of colitis in Sigma-1 knockout C57BL/6 mice

Sigma-1 receptors (σ1R) have been implicated in several pain pathways. We assessed the implication of σ1Rs in the development of intestinal inflammation and inflammation-associated referred hypersensitivity in a model of colitis in σ1R knockout (KO) mice. Colitis was induced with dextran sulfate sodium (DSS) in wild type (WT) and σ1R KO mice. The development of referred mechanical hypersensitivity (von Frey test) was assessed. Colonic and spinal changes in expression of immune- and sensory-related markers were also investigated (RT-qPCR/Western blot). Absence of σ1Rs had little impact in colitis generation and progression, although during the chronic phase a reduction in edema and a down-regulation of iNOS gene expression was observed. In σ1R KO mice, inflammation-associated hypersensitivity was significantly attenuated (paw) or completely prevented (abdomen). During colitis, in WT mice, changes in the colonic expression of nociceptive markers were observed during the acute and chronic phases of inflammation. Although σ1R KO mice showed similar regulation in the acute phase, an attenuated response was observed during the chronic phase of colitis. These differences were especially relevant for CB2 and TRPV1 receptors, which could play an important role in σ1-mediated regulation of sensitivity. No changes were detected on ERK phosphorylation at the level of the lumbosacral spinal cord. In summary, intestinal inflammation-associated referred hyperalgesia was reduced (paw) or absent (abdomen) in σ1R KO mice, thus confirming an important role for σ1R in the development of colitis-associated hypersensitivity. These results identify σ1Rs as a possible therapeutic target for the treatment of hypersensitivity associated to intestinal inflammation.

The CysLT2R receptor mediates leukotriene C4-driven acute and chronic itch

Acute and chronic itch are burdensome manifestations of skin pathologies including allergic skin diseases and atopic dermatitis, but the underlying molecular mechanisms are not well understood. Cysteinyl leukotrienes (CysLTs), comprising LTC4, LTD4, and LTE4, are produced by immune cells during type 2 inflammation. Here, we uncover a role for LTC4 and its signaling through the CysLT receptor 2 (CysLT2R) in itch. Cysltr2 transcript is highly expressed in dorsal root ganglia (DRG) neurons linked to itch in mice. We also detected CYSLTR2 in a broad population of human DRG neurons. Injection of leukotriene C4 (LTC4) or its nonhydrolyzable form NMLTC4, but neither LTD4 nor LTE4, induced dose-dependent itch but not pain behaviors in mice. LTC4-mediated itch differed in bout duration and kinetics from pruritogens histamine, compound 48/80, and chloroquine. NMLTC4-induced itch was abrogated in mice deficient for Cysltr2 or when deficiency was restricted to radioresistant cells. Itch was unaffected in mice deficient for Cysltr1, Trpv1, or mast cells (WSh mice). CysLT2R played a role in itch in the MC903 mouse model of chronic itch and dermatitis, but not in models of dry skin or compound 48/80- or Alternaria-induced itch. In MC903-treated mice, CysLT levels increased in skin over time, and Cysltr2-/- mice showed decreased itch in the chronic phase of inflammation. Collectively, our study reveals that LTC4 acts through CysLT2R as its physiological receptor to induce itch, and CysLT2R contributes to itch in a model of dermatitis. Therefore, targeting CysLT signaling may be a promising approach to treat inflammatory itch.

Different Effects of Intramedullary Injection of Mesenchymal Stem Cells During the Acute vs. Chronic Inflammatory Phase on Bone Healing in the Murine Continuous Polyethylene Particle Infusion Model.

Chronic inflammation is a common feature in many diseases of different organ systems, including bone. However, there are few interventions to mitigate chronic inflammation and preserve host tissue. Previous in vitro studies demonstrated that preconditioning of mesenchymal stem cells (pMSCs) using lipopolysaccharide and tumor necrosis factor-α polarized macrophages from a pro-inflammatory to an anti-inflammatory phenotype and increased osteogenesis compared to unaltered MSCs. In the current study, we investigated the local injection of MSCs or pMSCs during the acute versus chronic inflammatory phase in a murine model of inflammation of bone: the continuous femoral intramedullary polyethylene particle infusion model. Chronic inflammation due to contaminated polyethylene particles decreased bone mineral density and increased osteoclast-like cells positively stained with leukocyte tartrate resistant acid phosphatase (TRAP) staining, and resulted in a sustained M1 pro-inflammatory macrophage phenotype and a decreased M2 anti-inflammatory phenotype. Local injection of MSCs or pMSCs during the chronic inflammatory phase reversed these findings. Conversely, immediate local injection of pMSCs during the acute inflammatory phase impaired bone healing, probably by mitigating the mandatory acute inflammatory reaction. These results suggest that the timing of interventions to facilitate bone healing by modulating inflammation is critical to the outcome. Interventions to facilitate bone healing by modulating acute inflammation should be prudently applied, as this phase of bone healing is temporally sensitive. Alternatively, local injection of MSCs or pMSCs during the chronic inflammatory phase may be a potential intervention to mitigate the adverse effects of contaminated particles on bone.

Characteristics of inflammation process in monocrotaline-induced pulmonary arterial hypertension in rats

ObjectiveA growing evidence demonstrates that inflammation is a major contributor to the pathogenesis of pulmonary arterial hypertension (PAH). However, blocking inflammation has only been shown to be of minor clinical benefit due to a lack of understanding of the precise inflammation present in PAH. Thus, the present study aimed to investigate characteristics of inflammatory process in PAH induced by monocrotaline (MCT) in rats. MethodsAdult male Sprague-Dawley rats received a single dose of MCT (50 mg/kg, ip), and the occurrence of PAH and inflammation biomarkers were measured at 3, 6, 9, 12, 15, 18, 21, 24, 27 and 30 days after MCT injection. ResultsFrom the 6th day after the injection of MCT, the mean pulmonary artery pressure gradually increased and doubled on the 30th day, accompanied by right ventricular hypertrophy and pulmonary arterial remodeling in a time-dependent manner. In the first 6 days after MCT treatment, only pro-inflammatory cytokines TNF-α, IL-1β increased, which was defined as acute inflammatory phase, after that, both pro-inflammatory factors TNF-α, IL-1β, IL-6, IL-12 and anti-inflammatory factors Arg1, IL-10, TGF-β increased, which was defined as chronic inflammatory phase. The M1/M2 macrophage ratios in lung and alveolar lavage fluid were elevated on the 6th and 30th day, moreover, which were higher on the 6th than 30th day, and the PI3K/Akt signaling pathway increased along with the progression of PAH and correlated with pro-inflammatory proteins, which revealed also to some extent the characteristics of inflammation of PAH induced by MCT. ConclusionThe course of PAH induced by MCT injection is progressive with persistent inflammation, which is defined as acute inflammatory phase within 6 days after MCT treatment, after that, is defined as chronic inflammatory phase.

Understanding the Potential Role and Delivery Approaches of Nitric Oxide in Chronic Wound Healing Management.

Nitric oxide (NO) is a promising pharmaceutical component that has vasodilator, anti-bacterial, and wound healing activities. Chronic ulcers are non-healing disorders that are generally associated with distortion of lower limbs. Among the severe consequence derivatives of these diseases are the problems of chronic wound progression. NO, which is categorized as the smallest gaseous neurotransmitter, has beneficial effects in different phases of chronic inflammation. The defensive mechanism of NO is found useful in several severe conditions, such as gestational healing, gastrointestinal healing, and diabetic healing. The current review presents an updated collection of literature about the role of NO in chronic ulcers due to the prevalence of diabetes, DPN, and diabetic foot ulcers, and because of the lack of available effective treatments to directly address the pathology contributing to these conditions, novel treatments are being sought. This review also collects information about deficiency of NO synthase in diabetic patients, leading to a lack of vascularization of the peripheral nerves, which causes diabetic neuropathy, and this could be treated with vasodilators such as nitric oxide. Apart from the pharmacological mechanism of NO, the article also reviewed and analyzed to elucidate the potential of transdermal delivery of NO for the treatment of chronic ulcers.

Exploring antiulcer and anti-inflammatory activities of methanolic leaves extract of an Indian mistletoe Helicantes elasticus (Desv.) Danser

The potential of methanolic leaves extract of H. elasticus in the mitigation of ulcer and inflammation was evaluated in this work. Ethanol and pylorus ligation induced rats were used for the screening of ulcer, whereas the anti-inflammatory activity against acute and chronic phases of inflammation was screened using carrageenan-induced paw edema and cotton pellet induced granuloma in rats. The H. elasticus (200 and 400 mg/kg p.o.) was administered to the ethanol and pylorus ligation induced rats. Gastric volume, pH, free acidity and total acidity were estimated in pylorus ligation induced ulcers of rats. Treatment with H. elasticus showed a reduction in ulcer index against ethanol and pylorus ligation induced ulcerations. The antiulcer activity may be due to the antisecretory and cytoprotective effects of the extract. Moreover, it also showed a highly significant (P ≤ 0.001) reduction in carrageenan and cotton pellet induced inflammations. This investigation suggests that H. elasticus leaves possess anti-inflammatory activity and promotes ulcer protection.

536-P: Protein Tyrosine Phosphatase 1B Inhibitor MSI-1436 Promotes M2 Macrophage Polarization and Decreases Oxidative Stress in Human Monocytes under High-Glucose Conditions

Peripheral neuropathy plays a role in chronic diabetic foot ulcers (DFUs), also leading to an imbalance in inflammation, as pro-inflammatory macrophages (M1) have difficulty switching to an anti-inflammatory phenotype (M2) contributing to a chronic inflammatory phase and healing impairment. Protein tyrosine phosphatase 1B (PTP1B), in the skin, is over expressed in diabetes and DFUs. We aimed to evaluate the role of PTP1B inhibition in macrophage under high glucose conditions. THP-1 monocytic cells were differentiated into macrophages, treated with the PTP1B inhibitor MSI-1436 (1 µM) and stimulated with LPS for 24h, under high glucose (25 mM) or normoglycemic conditions (5.5 mM). Cell viability was measured by MTT assay. The numbers of M1 and M2 macrophages were evaluated by immunocytochemistry by double staining with CD68 and TNF-α for M1 macrophages, and CD68 and CD163 for M2 macrophages. In addition, pro-inflammatory cytokine expression was analyzed by qRT-PCR. Oxidative stress was assessed by the DCFH-DA assay, heme oxygenase-1 (HO-1) enzyme levels were also measured by immunocytochemistry and Western blot. The treatment with MSI-1436 did not alter THP-1 cell viability. In inflammatory conditions (LPS), THP-1 cells grown under HG and treated with MSI-1436 showed an increase in M2 macrophages and a reduction in M1 macrophages (246 ± 26% and 102 ± 9%, p<0.05, of LPS treated cells, respectively). In addition, PTP1B inhibition induced a decrease in pro-inflammatory cytokine expression, IL-6, IL-1β and MCP-1, a decrease in ROS levels (78 ± 10% of LPS treated cells) and an increase in HO-1 expression (290 ± 23%, p<0.05, of LPS treated cells). Inhibition of PTP1B in macrophages, under high glucose conditions, promotes a significant reduction in the inflammatory environment and oxidative stress via increase of HO-1 expression, suggesting that PTP1B is a potential therapeutic target for the treatment of chronic DFUs. Disclosure A. Figueiredo: None. E.C. Leal: None. D. Santos: None. G. Gasiunaite: None. M. Delibegovic: None. E. Carvalho: None. Funding Diabetes UK; Portuguese Foundation for Science and Technology; European Foundation for the Study of Diabetes; Portuguese Society of Diabetology

Herpesvirus Entry Mediator Binding Partners Mediate Immunopathogenesis of Ocular Herpes Simplex Virus 1 Infection.

Ocular herpes simplex virus 1 (HSV-1) infection leads to an immunopathogenic disease called herpes stromal keratitis (HSK), in which CD4+ T cell-driven inflammation contributes to irreversible damage to the cornea. Herpesvirus entry mediator (HVEM) is an immune modulator that activates stimulatory and inhibitory cosignals by interacting with its binding partners, LIGHT (TNFSF14), BTLA (B and T lymphocyte attenuator), and CD160. We have previously shown that HVEM exacerbates HSK pathogenesis, but the involvement of its binding partners and its connection to the pathogenic T cell response have not been elucidated. In this study, we investigated the role of HVEM and its binding partners in mediating the T cell response using a murine model of ocular HSV-1 infection. By infecting mice lacking the binding partners, we demonstrated that multiple HVEM binding partners were required for HSK pathogenesis. Surprisingly, while LIGHT-/-, BTLA-/-, and CD160-/- mice did not show differences in disease compared to wild-type mice, BTLA-/- LIGHT-/- and CD160-/- LIGHT-/- double knockout mice showed attenuated disease characterized by decreased clinical symptoms, increased retention of corneal sensitivity, and decreased infiltrating leukocytes in the cornea. We determined that the attenuation of disease in HVEM-/-, BTLA-/- LIGHT-/-, and CD160-/- LIGHT-/- mice correlated with a decrease in gamma interferon (IFN-γ)-producing CD4+ T cells. Together, these results suggest that HVEM cosignaling through multiple binding partners induces a pathogenic Th1 response to promote HSK. This report provides new insight into the mechanism of HVEM in HSK pathogenesis and highlights the complexity of HVEM signaling in modulating the immune response following ocular HSV-1 infection.IMPORTANCE Herpes simplex virus 1 (HSV-1), a ubiquitous human pathogen, is capable of causing a progressive inflammatory ocular disease called herpes stromal keratitis (HSK). HSV-1 ocular infection leads to persistent inflammation in the cornea resulting in outcomes ranging from significant visual impairment to complete blindness. Our previous work showed that herpesvirus entry mediator (HVEM) promotes the symptoms of HSK independently of viral entry and that HVEM expression on CD45+ cells correlates with increased infiltration of leukocytes into the cornea during the chronic inflammatory phase of the disease. Here, we elucidated the role of HVEM in the pathogenic Th1 response following ocular HSV-1 infection and the contribution of HVEM binding partners in HSK pathogenesis. Investigating the molecular mechanisms of HVEM in promoting corneal inflammation following HSV-1 infection improves our understanding of potential therapeutic targets for HSK.

Folate receptor-targeted positron emission tomography of experimental autoimmune encephalomyelitis in rats

BackgroundFolate receptor-β (FR-β) is a cell surface receptor that is significantly upregulated on activated macrophages during inflammation and provides a potential target for folate-based therapeutic and diagnostic agents. FR-β expression in central nervous system inflammation remains relatively unexplored. Therefore, we used focally induced acute and chronic phases of experimental autoimmune encephalomyelitis (EAE) to study patterns of FR-β expression and evaluated its potential as an in vivo imaging target.MethodsFocal EAE was induced in rats using heat-killed Bacillus Calmette-Guérin followed by activation with complete Freund’s adjuvant supplemented with Mycobacterium tuberculosis. The rats were assessed with magnetic resonance imaging and positron emission tomography/computed tomography (PET/CT) at acute (14 days) and chronic (90 days) phases of inflammation. The animals were finally sacrificed for ex vivo autoradiography of their brains. PET studies were performed using FR-β-targeting aluminum [18F]fluoride-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid conjugated folate ([18F]AlF-NOTA-folate, 18F-FOL) and 18 kDa translocator protein (TSPO)-targeting N-acetyl-N-(2-[11C]methoxybenzyl)-2-phenoxy-5-pyridinamine (11C-PBR28). Post-mortem immunohistochemistry was performed using anti-FR-β, anti-cluster of differentiation 68 (anti-CD68), anti-inducible nitric oxide synthase (anti-iNOS), and anti-mannose receptor C-type 1 (anti-MRC-1) antibodies. The specificity of 18F-FOL binding was verified using in vitro brain sections with folate glucosamine used as a blocking agent.ResultsImmunohistochemical evaluation of focal EAE lesions demonstrated anti-FR-β positive cells at the lesion border in both acute and chronic phases of inflammation. We found that anti-FR-β correlated with anti-CD68 and anti-MRC-1 immunohistochemistry; for MRC-1, the correlation was most prominent in the chronic phase of inflammation. Both 18F-FOL and 11C-PBR28 radiotracers bound to the EAE lesions. Autoradiography studies verified that this binding took place in areas of anti-FR-β positivity. A blocking assay using folate glucosamine further verified the tracer’s specificity. In the chronic phase of EAE, the lesion-to-background ratio of 18F-FOL was significantly higher than that of 11C-PBR28 (P = 0.016).ConclusionOur EAE results imply that FR-β may be a useful target for in vivo imaging of multiple sclerosis-related immunopathology. FR-β-targeted PET imaging with 18F-FOL may facilitate the monitoring of lesion development and complement the information obtained from TSPO imaging by bringing more specificity to the PET imaging armamentarium for neuroinflammation.

Study of mast cells in appendicitis: possible significance

Background: To study the mean mast cell count per square mm field in different layers of appendix and to correlate the relationship of these with progression of disease and extent of fibrosis. Also to study the possible significance of mast cell counts with different histological changes and relation with clinical presentation. Material and Methods: A total of 120 cases of appendix comprised of 20 cases of normal appendix which served as the control and 100 cases of appendicitis in different phase of evolution of the disease were studied for mast cell counts and its significance was assessed in correlation with the histopathological diagnosis as per the evolution of the disease. Results: Mast cell counts in submucosa were always higher then in mucosa or in muscular propria both in normal and in pathological appendicitis. The increased mast cell count in the mucosal layer was statistically significant in organizing phase than in chronic appendicitis. Submucosal mast cell count did not show any statistically significant difference between different stages of appendicitis and normal appendicitis. The mast cell count in muscularis propria showed statistically significant increase in chronic appendicitis as well as in early acute phase. In appendicitis with grade 2,3 and 4 histology, the total mean mast cell count/sq mm was much higher than in range in grade 1 and grade 5. Conclusion: In acute phase of inflammation there is decrease in the mast cell. In chronic phase of inflammation there is increase in the mast cell counts. A significant increase of mast cells in muscularis propria during early acute phase of appendicitis which was observed in present study has not been described in literature. This finding needs further evaluation to explain the role of mast cells in the development of early acute inflammatory response.

HMGB1 Causes Anemia of Inflammation By Modulating Erythropoietin Signal Transduction

The mechanisms underlying the development of erythropoietin (EPO)-refractory anemia in the setting of chronic inflammatory states are largely unknown. Elevated levels of the classical inflammatory mediators decrease red cell output. However, pathologic concentrations of many of these molecules do not persist beyond the acute phase, indicating that specific mediators are likely to play a role in the anemia associated with chronic inflammation. High mobility group box protein 1 (HMGB1) is a potent alarmin able to induce tissue injury during the acute and chronic phases of inflammation, and recently, shown to contribute to anemia in a murine model of sepsis. Here, we show that HMGB1 directly inhibits erythropoiesis by modulating EPO signal transduction in human erythroid cells through a newly identified HMGB1 receptor, which is surprisingly the erythropoietin receptor (EPOR). Surface plasmon resonance (SPR) reveals that HMGB1 binds the extracellular domain of EPOR (Kd = 130nM) with an affinity comparable to that of EPO. Cysteine residues contained within the A- and B-box domains of HMGB1 that have previously been shown to mediate HMGB1-receptor interactions are also responsible for the EPOR-HMGB1 interaction since a mutant form of HMGB1 lacking these cysteine residues (i.e. 3S HMGB1) fails to bind the EPOR. Cell-based assays suggest that the direct binding of HMGB1 to the EPOR and the subsequent degradation of EPOR accounts for altered EPO signaling by HMGB1. Biologically, HMGB1 reduces the phosphorylation of intracellular EPO effectors including JAK2 (2-fold reduction), STAT5 (4-fold), and ERK1/2 (4-fold). Decreased effector phosphorylation is not due to the increased activity of SHP1/2 phosphatases further implicating inhibition at the receptor level. Loss of EPO signaling due to HMGB1 binding results in decreased erythroid proliferation of differentiated CD34+ cells at the EPO-dependent stages of erythropoiesis: Day 14: 1.03x108 ± 4.67x107 cells/mL vs 1.87x106 ± 9.70x105 cells/mL, vehicle vs HMGB1, respectively. In addition, HMGB1 decreases the numbers of colony forming unit-erythroid (CFU-E) progenitors by 60%, and these progenitors fail to undergo terminal erythroid differentiation with a block at the basophilic erythroblast stage and apoptosis of late-stage erythroblasts as determined by flow cytometric analysis of annexin V staining. To understand the consequences of HMGB1-EPOR interactions on the EPO-induced transcriptome, RNA-sequencing was performed on purified human CFU-E dosed with HMGB1 and EPO. HMGB1 reduces the expression of known EPO target genes (ERFE, CISH, EGR1), and concomitantly, upregulates a number of unique transcripts (ETS2, VMP1, NFKBIZ) suggesting that HMGB1-EPOR interactions may alter receptor conformation in manner that differentially activates the EPOR and consequently, gene expression. Finally, in a mouse model of sepsis survival, bone marrow-derived erythroid precursor cells contain diminished phosphorylated STAT5 levels at a time when elevated HMGB1 plasma concentrations are observed, thereby demonstrating that the loss of EPO signal transduction also occurs in vivo. Taken together, our work identifies HMGB1 as a novel inhibitor of EPO signaling through its interaction with the EPOR, and strongly implicates HMGB1 as a previously undiscovered effector of EPO-refractory anemia associated with chronic inflammation. DisclosuresNo relevant conflicts of interest to declare.

Regenerative Medicine Therapies for Targeting Neuroinflammation After Stroke.

Inflammation is a major pathological event following ischemic stroke that contributes to secondary brain tissue damage leading to poor functional recovery. Following the initial ischemic insult, post-stroke inflammatory damage is driven by initiation of a central and peripheral innate immune response and disruption of the blood-brain barrier (BBB), both of which are triggered by the release of pro-inflammatory cytokines and infiltration of circulating immune cells. Stroke therapies are limited to early cerebral blood flow reperfusion, and whilst current strategies aim at targeting neurodegeneration and/or neuroinflammation, innovative research in the field of regenerative medicine aims at developing effective treatments that target both the acute and chronic phase of inflammation. Anti-inflammatory regenerative strategies include the use of nanoparticles and hydrogels, proposed as therapeutic agents and as a delivery vehicle for encapsulated therapeutic biological factors, anti-inflammatory drugs, stem cells, and gene therapies. Biomaterial strategies—through nanoparticles and hydrogels—enable the administration of treatments that can more effectively cross the BBB when injected systemically, can be injected directly into the brain, and can be 3D-bioprinted to create bespoke implants within the site of ischemic injury. In this review, these emerging regenerative and anti-inflammatory approaches will be discussed in relation to ischemic stroke, with a perspective on the future of stroke therapies.

P041 GLIAL CONNEXIN-43 REGULATES NEURO-IMMUNE INTERACTIONS IN THE MOUSE COLON

The enteric nervous system regulates gut reflexes. Our prior data show that enteric glia regulate the activity and survival of enteric neurons in health and disease through mechanisms that involve the release of mediators through connexin-43 (Cx43) hemichannels. Given that immune cells express receptors for glial mediators released via Cx43, we hypothesized that enteroglial signaling involving Cx43 contributes to immune cell recruitment. Cx43 was ablated in glial cells in hGFAP::CreERT2+/–/Cx43f/f mice (TG) and we assessed the effect of acute and chronic inflammation driven by dextran sodium sulfate (DSS). Acute colitis (aDSS) was induced by 2% DSS in drinking water for 1 week while chronic colitis (cDSS) was driven by intermittent exposure to DSS (1 week on/ 1 off) for 3 weeks. Body weight and macroscopic damage were used to assess the inflammation, and immunohistochemistry was used to determine numbers of neurons, glia and immune cells. Cytokine profiles were assayed by plate array. Our results show that DSS caused weight loss and macroscopic tissue damage, but not significant neurodegeneration in the myenteric plexus of either TG mice or littermate controls (wt). cDSS drove a significant increase in CD45+ and CD68+ immune cells in wt mice (P=0.013 and 0.013, 2-way ANOVA, water-DSS comparison) that was not observed in TG animals (P=0.127 and 0.982, 2-way ANOVA, water-DSS comparison). In addition, loss of enteroglial Cx43 affected the colonic production of cytokines: about 2-fold reduction of IL-2, IL-7, IL-9, and MIP-1a (P<0.05, 2-way ANOVA, genotype comparison), and protected against the aDSS-induced increase of M-CSF in the transgenic animals [P=0.030 (wt) vs 0.904 (TG), 2-way ANOVA, water-DSS comparison]. Together, our findings show that enteric glia contribute to immune cell recruitment during the chronic phase of inflammation through mechanisms that involve Cx43. These novel findings could be used to design novel therapeutics for inflammatory bowel disease.

Abstract 492: Chronic inflammation contributes to neoplastic progression in prostate tissue: a needle biopsy study

Abstract The relationship between inflammation and prostate cancer has not been established, although chronic inflammation has frequently been identified in prostate biopsies, radical prostatectomy and benign prostatic hyperplasia specimens. In the peripheral zone of the prostate, sometimes adjacent to foci of high-grade PIN and cancer, certain morphologic changes are often identified, which may represent active and terminal phases of chronic inflammation. These changes are designated as proliferative inflammatory atrophy (PIA) and post atrophic hyperplasia (PAH), and their morphology is well documented. In our previous studies, we have identified chronic inflammation as a putative contributor to neoplastic progression in prostate epithelial cells, and hypothesized that its adverse effects were related to an increase in Bcl2, a survival protein involved in cell survival and carcinogenesis. We hypothesize that changes in the stromal microenvironment, characterized by infiltration of immune cells, with generation of reactive oxygen species, can induce oxidative stress in the surrounding proliferating epithelium and cause permanent genomic alterations. In the present study, we focused on several key proteins involved in the inflammatory process, COX2 and iNOS; cell survival, Bcl2 and GSTPπ; and evaluated immunohistochemical expression of alpha-methylacyl coenzyme A racemase (AMACR) and basal cell-specific markers 34βE12 and/or p63 to evaluate possible neoplastic alterations in epithelial cells in an inflammatory environment. We evaluated 16 prostate core needle biopsy specimens that exhibited the presence of chronic inflammation as well as PIA and PAH lesions along with immunohistochemical staining for various markers was performed in each set of biopsies. The integrity of the basal layer was maintained in the area of chronic inflammation with high expression of p63 in 72% of these cells. Approximately 68% of luminal cells expressed high to moderate levels of iNOS and COX-2, whereas 55% of these cells express modest levels of GSTP1 and Bcl2. Interestingly, prostatic glands near the areas of chronic inflammation in the PIA lesions exhibit high AMACR expression in luminal cells and weak to patchy p63 expression in basal cells, which was associated with increased expression of the inflammatory markers COX2 and iNOS, as well as loss in pro-survival signal GSTP1 and Bcl2 in the adjacent luminal cells. These neoplastic alterations were observed in 6/16 (38%) of the needle biopsy specimens. Our findings suggest that basal cells undergo alterations in a setting of chronic inflammation. This is important because basal cells are considered to be progenitor cells capable of differentiating into secretory luminal cells, but under the influence of chronic inflammation, they may instead transform into the neoplastic cells that characterize high grade prostatic intraepithelial neoplasia and prostatic adenocarcinoma. Citation Format: Shardul C. Soni, Michael Glover, Qinghu Ren, Gregory T. MacLennan, Pingfu Fu, Sanjay Gupta. Chronic inflammation contributes to neoplastic progression in prostate tissue: a needle biopsy study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 492. doi:10.1158/1538-7445.AM2017-492