Regulation Of Chemokines Research Articles

Cyclosporine a directly affects human and mouse b cell migration in vitro by disrupting a hIF-1 \u03b1dependent, o2 sensing, molecular switch

BackgroundHypoxia is a potent molecular signal for cellular metabolism, mitochondrial function, and migration. Conditions of low oxygen tension trigger regulatory cascades mediated via the highly conserved HIF-1 α post-translational modification system. In the adaptive immune response, B cells (Bc) are activated and differentiate under hypoxic conditions within lymph node germinal centers, and subsequently migrate to other compartments. During migration, they traverse through changing oxygen levels, ranging from 1-5% in the lymph node to 5-13% in the peripheral blood. Interestingly, the calcineurin inhibitor cyclosporine A is known to stimulate prolyl hydroxylase activity, resulting in HIF-1 α destabilization and may alter Bc responses directly. Over 60% of patients taking calcineurin immunosuppressant medications have hypo-gammaglobulinemia and poor vaccine responses, putting them at high risk of infection with significantly increased morbidity and mortality.ResultsWe demonstrate that O 2 tension is a previously unrecognized Bc regulatory switch, altering CXCR4 and CXCR5 chemokine receptor signaling in activated Bc through HIF-1 α expression, and controlling critical aspects of Bc migration. Our data demonstrate that calcineurin inhibition hinders this O 2 regulatory switch in primary human Bc.ConclusionThis previously unrecognized effect of calcineurin inhibition directly on human Bc has significant and direct clinical implications.

Circadian Regulator CLOCK Recruits Immune-Suppressive Microglia into the GBM Tumor Microenvironment.

Glioblastoma (GBM) is a lethal brain tumor containing a subpopulation of glioma stem cells (GSC). Pan-cancer analyses have revealed that stemness of cancer cells correlates positively with immunosuppressive pathways in many solid tumors, including GBM, prompting us to conduct a gain-of-function screen of epigenetic regulators that may influence GSC self-renewal and tumor immunity. The circadian regulator CLOCK emerged as a top hit in enhancing stem-cell self-renewal, which was amplified in about 5% of human GBM cases. CLOCK and its heterodimeric partner BMAL1 enhanced GSC self-renewal and triggered protumor immunity via transcriptional upregulation of OLFML3, a novel chemokine recruiting immune-suppressive microglia into the tumor microenvironment. In GBM models, CLOCK or OLFML3 depletion reduced intratumoral microglia density and extended overall survival. We conclude that the CLOCK-BMAL1 complex contributes to key GBM hallmarks of GSC maintenance and immunosuppression and, together with its downstream target OLFML3, represents new therapeutic targets for this disease. SIGNIFICANCE: Circadian regulator CLOCK drives GSC self-renewal and metabolism and promotes microglia infiltration through direct regulation of a novel microglia-attracting chemokine, OLFML3. CLOCK and/or OLFML3 may represent novel therapeutic targets for GBM.This article is highlighted in the In This Issue feature, p. 327.

Sickle Cell Anemia Patients Display an Intricate Cellular and Serum Biomarker Network Highlighted by TCD4+CD69+ Lymphocytes, IL-17/MIP-1β, IL-12/VEGF, and IL-10/IP-10 Axis.

Background Sickle cell anemia (SCA) is associated with a chronic proinflammatory state characterized by elevated leukocyte count, mortality from severe recurrent infections, and subsequent vasoocclusive complications with leukocyte adhesion to the endothelium and increased plasma levels of inflammatory cytokines. The immune system has a close connection with morbidity in SCA, but further studies are needed to uncover the involvement of innate and adaptive immunities in modulating the SCA physiopathology. We performed measurements of the frequency of innate and adaptive immunity cells, cytokines, chemokines, and growth factors and immunophenotyping of Toll-like receptor and adhesion molecule expression in the blood of SCA patients and healthy donors to evaluate the different profiles of these biomarkers, the relationship among them, and their correlation to laboratory records and death risk. Material and Methods. Immunophenotyping of cells, Toll-like receptors, and adhesion molecules were performed from peripheral blood samples of SCA patients and healthy donors by flow cytometry and cytokine/chemokine/growth factor measurement by the Luminex technique performed from the serum of the same subjects. Results Cells of adaptive immunity such as IL-12, IL-17, and IL-10 cytokines; IL-8, IP-10, MIP-1α, MIP-1β, and RANTES chemokines; and VEGF, FGF-basic, and GM-CSF growth factors were higher in SCA patients than healthy donors regardless of any laboratorial and clinical condition. However, high death risk appears to have relevant biomarkers. Conclusion In the SCA pathophysiology at steady state, there is a broad immunological biomarker crosstalk highlighted by TCD4+CD69+ lymphocytes, IL-12 and IL-17 inflammatory and IL-10 regulatory cytokines, MIP-1α, MIP-1β, and IP-10 chemokines, and VEGF growth factor. High expression of TLR2 in monocytes and VLA-4 in TCD8+ lymphocytes and high levels of MIP-1β and RANTES appear to be relevant in high death risk conditions. The high reticulocytosis and high death risk conditions present common correlations, and there seems to be a balance by the Th2 profile.

Interplaying of regulatory T-cells and related chemokines in immune thrombocytopenic purpura patients

BACKGROUND: Chronic immune thrombocytopenic purpura (ITP) is an immune-mediated bleeding disorder, in which platelets are opsonized by autoantibodies directed against platelet surface membrane glycoproteins, and prematurely cleared and destructed by Fc-receptors on the surface of macrophages in the reticuloendothelial system.OBJECTIVES: This work is designed to show the contribution of lymphocyte subsets and platelet destruction in adult chronic ITP and role of infection.MATERIALS AND METHODS: Transforming growth factor-β1 (TGF-β1) was measured using ELISA, and the frequency of regulatory T-cell (Treg) profile (CD4+CD25+CD127−) was investigated by FCM in blood samples of 50 Iraqi ITP patients (35 on-treatment ITP patients and 15 newly diagnosed) along with 20 age-matched healthy people that act as controls, as well as all patients were breath tested for detecting Helicobacter pylori using urea breath test. The study was carried out in the National Center of Hematology, Mustansiriyah University.RESULTS: The results showed that although there was a significant reduction in Treg number in ITP patients compared with the control individuals (P < 0.001), the effect of treatment has shown a restored count of Tregs in comparison to the newly diagnosed ones (P = 0.002), while the assessment of cytokine serum level revealed that TGF-β1 was significantly increased (P = 0.001) in the on-treatment group of patients (TGF-β1 = 3.24 ± 0.3 ng/μl) in comparison with the nontreated group of patients (TGF-β1 = 1.75 ± 0.2 ng/μl). However, it was still significantly (P < 0.001) less than their values in the apparently healthy individuals (TGF-β1 = 9.0 ± 0.2 ng/μ). Moreover, 25 out of 50 (50%) showed positive results for the presence of H. pylori.CONCLUSION: The present study revealed that Treg and its cytokines may play a fundamental role in the pathophysiology of adult chronic ITP since they contribute to the maintenance of peripheral immune tolerance. However, a causal link between H. pylori infection and ITP diseases is considerable.

Effects of Electroacupuncture on the Regulation of Chemokine Induced Spinal Activation of Microglia in the Rat Model of Neuropathic Pain

Effects of Electroacupuncture on the Regulation of Chemokine Induced Spinal Activation of Microglia in the Rat Model of Neuropathic Pain

Lipopolysaccharide-dependent transcriptional regulation of PU.1 in microglial cells

PU.1 is a pioneer transcription factor and a master regulator of the myeloid lineage. However, the role of PU.1 in lipopolysaccharide-dependent microglial activation has yet to be investigated. So, this study was conducted to determine the effects of PU.1 in LPS-induced activation of microglial cells. We knocked out PU.1 in murine BV-2 cells using the CRISPR-Cas9 system to investigate the role of PU.1 in the expression of immune-related genes. We performed RNA sequencing (RNA-seq) of PU.1 KO and BV-2 cells to analyze the gene expression patterns in PU.1 KO cells and compare them to those in wild-type BV-2 cells. The validation of differential expressions was achieved by qRT-PCR. To explore this regulatory role of PU.1, ChIP sequencing for PU.1 and H3K27Ac was performed. The sequencing result was further confirmed by ChIP-qPCR. RNA sequencing and subsequent bioinformatic analysis revealed that the expression of most of the immune-related genes was suppressed in the absence of PU.1. Proinflammatory chemokine genes were differentially expressed in LPS-treated PU.1 KO cells. The ChIP sequencing result followed by ChIP-qPCR revealed a LPS-mediated increase in the enrichment of PU.1 binding in pro-inflammatory chemokine gene promoters and enhancer regions in wild-type BV-2 cells. There was no enrichment of PU.1 in PU.1 KO cells. The above-mentioned results suggest that PU.1 is directly involved in regulating the immune response and that this regulation of inflammatory chemokines is LPS-dependent. We hope that PU.1 would be an option for limiting neurodegeneration in a diverse range of neurological disorders.

Bleomycin hydrolase regulates the release of chemokines important for inflammation and wound healing by keratinocytes

Bleomycin hydrolase (BLMH) is a well-conserved cysteine protease widely expressed in several mammalian tissues. In skin, which contains high levels of BLMH, this protease is involved in the degradation of citrullinated filaggrin monomers into free amino acids important for skin hydration. Interestingly, the expression and activity of BLMH is reduced in patients with atopic dermatitis (AD) and psoriasis, and BLMH knockout mice acquire tail dermatitis. Apart from its already known function, we have discovered a novel role of BLMH in the regulation of inflammatory chemokines and wound healing. We show that lowered BLMH levels in keratinocytes result in increased release of the pro-inflammatory chemokines CXCL8 and GROα, which are upregulated in skin from AD patients compared to healthy individuals. Conditioned media from keratinocytes expressing low levels of BLMH increased chemotaxis by neutrophils and caused a delayed wound healing in the presence of low-level TNFα. This defective wound healing was improved by blocking the shared receptor of CXCL8 and GROα, namely CXCR2, using a specific receptor antagonist. Collectively, our results present a novel function of BLMH in regulating the secretion of chemokines involved in inflammation and wound healing in human keratinocytes.

Chemokine regulation of inflammation during respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) can cause severe lower respiratory tract infections especially in infants, immunocompromised individuals and the elderly and is the most common cause of infant hospitalisation in the developed world. The immune responses against RSV are crucial for viral control and clearance but, if dysregulated, can also result in immunopathology and impaired gas exchange. Lung immunity to RSV and other respiratory viruses begins with the recruitment of immune cells from the bloodstream into the lungs. This inflammatory process is controlled largely by chemokines, which are small proteins that are produced in response to innate immune detection of the virus or the infection process. These chemokines serve as chemoattractants for granulocytes, monocytes, lymphocytes and other leukocytes. In this review, we highlight recent advances in the field of RSV infection and disease, focusing on how chemokines regulate virus-induced inflammation.

Dual Programmed Death Receptor-1 and Vascular Endothelial Growth Factor Receptor-2 Blockade Promotes Vascular Normalization and Enhances Antitumor Immune Responses in Hepatocellular Carcinoma.

Activation of the antitumor immune response using programmed death receptor-1 (PD-1) blockade showed benefit only in a fraction of patients with hepatocellular carcinoma (HCC). Combining PD-1 blockade with antiangiogenesis has shown promise in substantially increasing the fraction of patients with HCC who respond to treatment, but the mechanism of this interaction is unknown. We recapitulated these clinical outcomes using orthotopic-grafted or induced-murine models of HCC. Specific blockade of vascular endothelial receptor 2 (VEGFR-2) using a murine antibody significantly delayed primary tumor growth but failed to prolong survival, while anti-PD-1 antibody treatment alone conferred a minor survival advantage in one model. However, dual anti-PD-1/VEGFR-2 therapy significantly inhibited primary tumor growth and doubled survival in both models. Combination therapy reprogrammed the immune microenvironment by increasing cluster of differentiation 8-positive (CD8+ ) cytotoxic T cell infiltration and activation, shifting the M1/M2 ratio of tumor-associated macrophages and reducing T regulatory cell (Treg) and chemokine (C-C motif) receptor 2-positive monocyte infiltration in HCC tissue. In these models, VEGFR-2 was selectively expressed in tumor endothelial cells. Using spheroid cultures of HCC tissue, we found that PD-ligand 1 expression in HCC cells was induced in a paracrine manner upon anti-VEGFR-2 blockade in endothelial cells in part through interferon-gamma expression. Moreover, we found that VEGFR-2 blockade increased PD-1 expression in tumor-infiltrating CD4+ cells. We also found that under anti-PD-1 therapy, CD4+ cells promote normalized vessel formation in the face of antiangiogenic therapy with anti-VEGFR-2 antibody. We show that dual anti-PD-1/VEGFR-2 therapy has a durable vessel fortification effect in HCC and can overcome treatment resistance to either treatment alone and increase overall survival in both anti-PD-1 therapy-resistant and anti-PD-1 therapy-responsive HCC models.

P5393Deficiency of the long noncoding RNA NEAT1 disturbs T cell and monocyte-macrophage lineage differentiation and functions and results in systemic inflammation with high circulating interferon levels

Abstract Background Inflammation is a key driver of atherosclerosis and myocardial infarction (MI), and beyond proteins and microRNAs, long noncoding RNAs (lncRNAs) are implicated in inflammation control. To obtain further information on the role of lncRNAs in the context of atherosclerosis, we analyzed transcriptome maps of circulating immune cells (PBMCs) of post-MI patients in whom the lncRNA NEAT1 was suppressed. Here, we report immune disturbances in murine NEAT1 knockout models with wildtype or ApoE−/− genetic background. Methods and results RNA-sequencing (RNA-seq) of PBMCs from post-MI patients revealed profound transcriptome disturbances compared to healthy controls. Among these, NEAT1 suppression was notable since it affected the most highly expressed lncRNA as part of a molecular circuit also encompassing chemokines and interleukins. We used NEAT1−/− mice to evaluate whether NEAT1 depletion per se may cause immune dysfunction. NEAT1−/− splenocytes displayed enhanced baseline ROS production, and RNA-seq identified anomalous expression and regulation of chemokines/ receptors, innate immunity genes, TNF and caspases. FACS revealed displayed anomalous Treg and TH cell differentiation in NEAT1−/− spleens vs. wildtype (WT). Beyond grossly altered transcriptome, NEAT1−/− bone marrow derived macrophages (BMDMs) responded to LPS with increased (p&lt;0.001) ROS production, enhanced baseline phagocytic activity (p&lt;0.001), and attenuated proliferation (p=0.001). FACS revealed deregulated monocyte-macrophage differentiation in NEAT1−/− bone marrow and blood. Further, NEAT1−/− mice displayed aortic wall CD68+ cell infiltration and there was evidence of myocardial inflammation which could lead to severe and potentially life-threatening structural damage in some of these animals. This observation suggests that even stochastic activation of the highly unstable NEAT1−/− immune system may trigger uncontrolled pathogenic cascades, explaining the survival disadvantage of NEAT1−/− mice. In addition to these studies on homozygous NEAT1−/− deficiency in WT background, we obtained data on mice with partial i.e. heterozygous NEAT1−/+ deficiency on ApoE−/− background. Analysis of this new NEAT1−/+ ApoE−/− strain indicates that even partial NEAT1 deficiency leads to systemic inflammation with high IFN-gamma levels, when the animals are exposed to immune stress e.g. high LDL cholesterol. Conclusions Regarding the monocyte-enriched NEAT1 suppressed in post-MI PBMCs, the data from NEAT1−/− and NEAT1−/+ ApoE−/− mice document NEAT1 as a key immune system coordinator whose deficiency affects monocyte-macrophage and T cell differentiation and functions and renders the immune system unstable and highly vulnerable to immune stress. Since in patients NEAT1 is part of a molecular circuit persistently deregulated post-MI, too, it appears reasonable to further search for new therapeutic targets within this circuit, taking advantage of the described genetic animal models.

Axl is a key regulator of intestinal γδ T-cell homeostasis.

Gut-homing γδ T cells are induced by chemokines and cell adhesion molecules and play a critical role in homeostasis and mucosal immunity; however, little is known regarding their upstream regulators. We investigated the role of Axl as a specific regulator of chemokines and cell adhesion molecule in the distribution of intestinal γδ T cells. The population of γδ T-cell receptor-positive cells including Vγ1 and Vγ7 subsets was remarkably increased in the intraepithelial lymphocytes of Axl-/- mice compared with those of wild-type (WT) mice. An increased number of migrated γδ T cells were observed in the coculture with intraepithelial cells from Axl-/- mice. The mRNA expression level of chemokine (C-C motif) ligand (CCL) 25 was specifically higher in the small intestine of Axl-/- mice than in WT mice. In adoptive transfer, the migration of both thymic and extrathymic γδ T cells was increased in Axl-/- mice. The activation of Axl signaling down-regulated CCL25 expression via ERK signaling pathway and reduced the population of γδ T cells. Systemic dissemination was suppressed in Axl-/- mice infected with Salmonella typhimurium. Thus, our findings suggest that Axl plays a critical role in regulating the migration of γδ T cells for the maintenance of homeostasis and bacterial resistance.-Kim, S.-M., Park, M., Yee, S.-M., Ji, K.-Y., Lee, E.-H., Nguyen, T.-V., Nguyen, T. H.-L., Jang, J., Kim, E.-M., Choi, H.-R., Yun, C.-H., Kang, H.-S. Axl is a key regulator of intestinal γδ T-cell homeostasis.

Deletion of the RNA regulator HuR in tumor-associated microglia and macrophages stimulates anti-tumor immunity and attenuates glioma growth.

Glioblastoma is a malignant brain tumor that portends a poor prognosis. Its resilience, in part, is related to a remarkable capacity for manipulating the microenvironment to promote its growth and survival. Microglia/macrophages are prime targets, being drawn into the tumor and stimulated to produce factors that support tumor growth and evasion from the immune system. Here we show that the RNA regulator, HuR, plays a key role in the tumor-promoting response of microglia/macrophages. Knockout (KO) of HuR led to reduced tumor growth and proliferation associated with prolonged survival in a murine model of glioblastoma. Analysis of tumor composition by flow cytometry showed that tumor-associated macrophages (TAMs) were decreased, more polarized toward an M1-like phenotype, and had reduced PD-L1 expression. There was an overall increase in infiltrating CD4+ cells, including Th1 and cytotoxic effector cells, and a concomitant reduction in tumor-associated polymorphonuclear myeloid-derived suppressor cells. Molecular and cellular analyses of HuR KO TAMs and cultured microglia showed changes in migration, chemoattraction, and chemokine/cytokine profiles that provide potential mechanisms for the altered tumor microenvironment and reduced tumor growth in HuR KO mice. In summary, HuR is a key modulator of pro-glioma responses by microglia/macrophages through the molecular regulation of chemokines, cytokines, and other factors. Our findings underscore the relevance of HuR as a therapeutic target in glioblastoma.

Adenovirus‑mediated overexpression of bone morphogenetic protein‑9 promotes methionine choline deficiency‑induced non‑alcoholic steatohepatitis in non‑obese mice

Liver inflammation and macrophage infiltration are critical steps in the progression of non-alcoholic fatty liver to the development of non-alcoholic steatohepatitis. Bone morphogenetic protein-9 is a cytokine involved in the regulation of chemokines and lipogenesis. However, the function of bone morphogenetic protein-9 in non-alcoholic steatohepatitis is still unknown. The present study hypothesized that bone morphogenetic protein-9 may contribute to steatohepatitis in mice fed a methionine choline deficiency diet (MCD). C57BL/6 mice overexpressing bone morphogenetic protein-9 and control mice were fed the MCD diet for 4 weeks. Liver tissue and serum samples were obtained for subsequent measurements. Bone morphogenetic protein-9 overexpression exacerbated steatohepatitis in mice on the MCD diet, as indicated by liver histopathology, increased serum alanine aminotransferase activity, aspartate transaminase activity, hepatic inflammatory gene expression and M1 macrophage recruitment. Although bone morphogenetic protein-9 overexpression did not affect the expression of pro-fibrogenic genes, including Collagen I (α)1 or matrix metalloproteinase (MMP) 9, it did upregulate the expression of transforming growth factor-β and plasminogen activator inhibitor 1, and downregulated the expression of MMP2. The above results indicate that bone morphogenetic protein-9 exerts a pro-inflammatory role in MCD diet-induced non-alcoholic steatohepatitis.

Abstract 3765: Somatic JAK-STAT mutations in subtypes of aggressive B-cell lymphomas with DLBCL morphology

Abstract Aberrant activation of the JAK-STAT pathway is a hallmark of a variety of lymphomas and can alter the lymphoma cells secretome and the composition of the tumor microenvironment (TME). The up-regulation of the immune regulatory chemokine CCL17 has been shown to be mediated by STATs-dependent mechanism in primary mediastinal B cell lymphoma (PMBCL). Here, we assembled a cohort of 340 R-CHOP-treated aggressive lymphomas with diffuse large B cell lymphoma (DLBCL) morphology to investigate JAK-STAT mutations (targeted gene sequencing), copy number (CN) alterations (SNP arrays), gene expression (RNAseq) and TME composition (IHC). The cohort was evaluated using FISH for BCL2, BCL6 and MYC rearrangements and the molecular classification assay, Lymph2Cx, to distinguish ABC vs GBC vs double-hit lymphomas with DLBCL morphology (DH-DLBCL). Among the 317 evaluable cases, 26 were DH-DLBCLs, 101 ABC, 155 GCB and 35 unclassified. In addition, we used the recently published Lymph3Cx assay (Mottok A. et al., Blood 2018) to identify cases with a molecular PMBCL (mPMBCL) expression signature within the GCB-DLBCL group (6.5%, 10/155 cases). Twenty-five of 155 GCB cases (16.1%) were classified as ‘uncertain PMBCL/DLBCL’. Mutational analysis of all cases (n=340) revealed the presence of JAK-STAT pathway mutations at the following frequencies: SOCS1 21.6%, STAT6 4.8%, IL4R 5.3% and 9p24 amplification 11.3%. These mutations were significantly enriched in the mPMBCL (n=10) group compared to the bona fide GCB (n=118) and intermediate phenotype group (uncertain PMBCL/DLBCL) (n=25) (p&amp;lt;0.05). The relative frequency of any JAK-STAT mutations was 100% (10/10) in mPMBCL, 29.2% (35/118) in bona fide GCB and 56% (14/25) in uncertain PMBCL/DLBCL. Among the GCB samples with follow-up data (n=107, incl. bona fide GCB and mPMBCL), cases carrying mutations in IL4R (n=11, 10.3%) had an inferior disease-specific survival (p=0.011) and time to progression (p=0.0048) after R-CHOP therapy. Interestingly, IL4R mutations were also detected in the DH-DLBCL group with an incidence of 11.5% (3/26 cases). Mutations in the extracellular and transmembrane domains of IL4R resulted in gain-of-function mutations leading to constitutive activation of the JAK-STAT pathway in vitro. IL4R gain-of-function increased CCL17 expression through a STAT6-dependent mechanism in vitro. Similarly, gene expression analysis of primary patient samples carrying activating IL4R mutations displayed increased CCL17expression (p&amp;lt;0.05), which positively correlated with the level of the T-regulatory marker FOXP3 (p&amp;lt;0.05) by IHC. In summary, aberrant JAK-STAT activation driven by alterations, such as activating IL4R mutations, plays a significant role in altering chemokine expression profiles and TME changes. Our data strongly suggest the biological and clinical relevance of Lymph3Cx to define a subgroup of aggressive lymphoma harboring a molecular PMBCL signature. Citation Format: Elena Vigano, Gerben Duns, Daisuke Ennishi, Clementine Sarkozy, Bruce Woolcock, Faith Cheung, Elizabeth Chavez, Stacy S. Hung, Katsuyoshi Takata, Anja Mottok, Randy Gascoyne, Kerry J. Savage, Ryan Morin, David W. Scott, Christian Steidl. Somatic JAK-STAT mutations in subtypes of aggressive B-cell lymphomas with DLBCL morphology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3765.

Systematic Identification and Analysis of Expression Profiles of mRNAs and Incrnas in Macrophage Inflammatory Response.

Long noncoding RNAs (lncRNAs), once thought to be transcriptional noise, have been recently shown to regulate a variety of biological processes. However, their roles in the inflammatory response are largely unexplored. In this study, we performed high-throughput sequencing to identify the profiles of mRNA and lncRNA transcriptomes in response to lipopolysaccharide (LPS) stimulation, followed by a comprehensive bioinformatics analysis. We found a total of 325 lncRNAs and 1,187 mRNAs to be significantly dysregulated in RAW264.7 cells stimulated with LPS (fold change >4.0 or <0.25, false discovery rate <0.01). Further validation with qRT-PCR demonstrated that Cd40 and Traf1 mRNAs were significantly upregulated, whereas Slc43a2 and Ccnd1 were downregulated in RAW264.7 cells treated with LPS. Gene ontology (GO) analysis indicated that the altered mRNAs and lncRNAs were mainly involved in the immune response, inflammation response, chemokine receptor binding, protein binding, and regulation of cytokine production. KEGG pathway analysis showed that altered lncRNAs and mRNAs were significantly enriched in immune- and inflammation-related signaling pathways, such as Herpes simplex infection, cytokine-cytokine receptor interaction, and TNF, PI3K-Akt, MAPK, NF-κB, and JAK-STAT signaling pathways. lncRNA-mRNA network analysis showed that the coexpression network profile for mRNAs and lncRNAs from the immune category consisted of 93 network nodes and 145 connections among 70 differentially expressed mRNAs and 23 dysregulated lncRNAs, suggesting that lncRNAs play an important role in the regulation of functional mRNA expression in LPS-induced inflammation.

A potential role of TLR2 in xenograft rejection of porcine iliac endothelial cells: An in vitro study.

Porcine vascular endothelial cells are a major participant in xenograft rejection. The Toll-like receptor 2 (TLR2) pathway plays an important role in both innate and adaptive immunity. The specific role of TLR2 in the response to a xenograft has not been reported. Whether the TLR2 pathway in pig vascular endothelial cells is involved in acute rejection needs to be investigated, and the mechanism is explored. We used a modified antibody-dependent complement-mediated cytotoxicity (ADCC) assay to conduct in vitro experiments. In porcine iliac artery endothelial cells (PIECs), siRNA was used to knock down the expression of TLR2, CXCL8, and CCL2. The effect of human serum or inactivated human serum on the expression of TLR2 was analyzed by real-time PCR and Western blotting, and transwell assays were used to assess the chemotactic efficiency of PIECs on human monocyte-macrophages (THP-1 cells) and human neutrophils. The downstream signaling pathways activated by human serum were detected by Western blotting, and the regulation of proinflammatory chemokines and cytokines by TLR2 signaling was assessed by real-time PCR and ELISA. TLR2 was significantly upregulated in PIECs after exposure to human serum, and porcine proinflammatory chemokines, CXCL8 and CCL2, were induced, at least partially, in a TLR2-dependent pattern; the upregulated chemokines participated in the chemotaxis of human neutrophils and THP-1 cells across the species barrier. (i) TLR2 is significantly upregulated in PIECs by human serum, (ii) the elevated TLR2 participates in the chemotaxis of inflammatory cells through the secretion of chemokine CCL2 and CXCL8, and (iii) blockade of TLR2 would be beneficial for xenograft survival.

Atypical Chemokine Receptor 1 (DARC/ACKR1) in Breast Tumors Is Associated with Survival, Circulating Chemokines, Tumor-Infiltrating Immune Cells, and African Ancestry.

Tumor-specific immune response is an important aspect of disease prognosis and ultimately impacts treatment decisions for innovative immunotherapies. The atypical chemokine receptor 1 (ACKR1 or DARC) gene plays a pivotal role in immune regulation and harbors several single-nucleotide variants (SNV) that are specific to sub-Saharan African ancestry. Using computational The Cancer Genome Atlas (TCGA) analysis, case-control clinical cohort Luminex assays, and CIBERSORT deconvolution, we identified distinct immune cell profile-associated DARC/ACKR1 tumor expression and race with increased macrophage subtypes and regulatory T cells in DARC/ACKR1-high tumors. In this study, we report the clinical relevance of DARC/ACKR1 tumor expression in breast cancer, in the context of a tumor immune response that may be associated with sub-Saharan African ancestry. Briefly, we found that for infiltrating carcinomas, African Americans have a higher proportion of DARC/ACKR1-negative tumors compared with white Americans, and DARC/ACKR1 tumor expression is correlated with proinflammatory chemokines, CCL2/MCP-1 (P <0.0001) and anticorrelated with CXCL8/IL8 (P <0.0001). Sub-Saharan African-specific DARC/ACKR1 alleles likely drive these correlations. Relapse-free survival (RFS) and overall survival (OS) were significantly longer in individuals with DARC/ACKR1-high tumors (P <1.0 × 10-16 and P <2.2 × 10-6, respectively) across all molecular tumor subtypes. DARC/AKCR1 regulates immune responses in tumors, and its expression is associated with sub-Saharan African-specific alleles. DARC/ACKR1-positive tumors will have a distinct immune response compared with DARC/AKCR1-negative tumors. This study has high relevance in cancer management, as we introduce a functional regulator of inflammatory chemokines that can determine an infiltrating tumor immune cell landscape that is distinct among patients of African ancestry.

Regulation of Chemokines and Cytokines by Histone Deacetylases and an Update on Histone Decetylase Inhibitors in Human Diseases.

Histone acetyltransferases (HATs) and histone deacetylases (HDACs) counteract with each other to regulate gene expression by altering chromatin structure. Aberrant HDAC activity was reported in many human diseases including wide range of cancers, viral infections, cardiovascular complications, auto-immune diseases and kidney diseases. HDAC inhibitors are small molecules designed to block the malignant activity of HDACs. Chemokines and cytokines control inflammation, immunological and other key biological processes and are shown to be involved in various malignancies. Various HDACs and HDAC inhibitors were reported to regulate chemokines and cytokines. Even though HDAC inhibitors have remarkable anti-tumor activity in hematological cancers, they are not effective in treating many diseases and many patients relapse after treatment. However, the role of HDACs and cytokines in regulating these diseases still remain unclear. Therefore, understanding exact mechanisms and effector functions of HDACs are urgently needed to selectively inhibit them and to establish better a platform to combat various malignancies. In this review, we address regulation of chemokines and cytokines by HDACs and HDAC inhibitors and update on HDAC inhibitors in human diseases.

Heme oxygenase-1 induced by desoxo-narchinol-A attenuated the severity of acute pancreatitis via blockade of neutrophil infiltration

Heme oxygenase-1 (HO-1) has an anti-inflammatory action in acute pancreatitis (AP). However, its mechanism of action and natural compounds/drugs to induce HO-1 in pancreas are not well understood. In this study, we investigated the regulatory mechanisms of HO-1 during AP using desoxo-narchinol-A (DN), the natural compound inducing HO-1 in the pancreas. Female C57/BL6 Mice were intraperitoneally injected with supramaximal concentrations of cerulein (50 μg/kg) hourly for 6 h to induce AP. DMSO or DN was administered intraperitoneally, then mice were sacrificed 6 h after the final cerulein injection. Administration of DN increased pancreatic HO-1 expression through activation of activating protein-1, mediated by mitogen-activated protein kinases. Furthermore, DN treatment reduced the pancreatic weight-to-body weight ratio as well as production of digestive enzymes and pro-inflammatory cytokines. Inhibition of HO-1 by tin protoporphyrin IX abolished the protective effects of DN on pancreatic damage. Additionally, DN treatment inhibited neutrophil infiltration into the pancreas via regulation of chemokine (C-X-C motif) ligand 2 (CXCL2) by HO-1. Our results suggest that DN is an effective inducer of HO-1 in the pancreas, and that HO-1 regulates neutrophil infiltration in AP via CXCL2 inhibition.

Inflammatory and regulatory CCL and CXCL chemokine and cytokine cellular responses in patients with patent Mansonella perstans filariasis.

Mansonella perstans (Mp) filariasis is present in large populations in sub-Saharan Africa, and to what extent patent Mp infection modulates the expression of immunity in patients, notably their cellular cytokine and chemokine response profile, remains not well known. We studied the spontaneous and inducible cellular production of chemokines (C-X-C motif) ligand 9 (CXCL9) [monokine induced by interferon (IFN)-γ (MIG)], CXCL-10 [inducible protein (IP)-10], chemokine (C-C motif) ligand 24 (CCL24) (eotaxin-2), CCL22 [macrophage-derived chemokine (MDC)], CCL13 [monocyte chemotactic protein-4 (MCP-4)], CCL18 [pulmonary and activation-regulated chemokine (PARC)], CCL17 [thymus- and activation-regulated chemokine (TARC)] and interleukin (IL)-27 in mansonelliasis patients (Mp-PAT) and mansonelliasis-free controls (CTRL). Freshly isolated peripheral mononuclear blood cells (PBMC) were stimulated with helminth, protozoan and bacterial antigens and mitogen [phytohaemagglutinin (PHA)]. PBMC from Mp-PAT produced spontaneously (without antigen stimulation) significantly higher levels of eotaxin-2, IL-27, IL-8, MCP-4 and MDC than cells from CTRL, while IFN-γ-IP-10 was lower in Mp-PAT. Helminth antigens activated IL-27 and MCP-4 only in CTRL, while Ascaris antigen, Onchocerca antigen, Schistosoma antigen, Entamoeba antigen, Streptococcus antigen, Mycobacteria antigen and PHA stimulated MIG release in CTRL and Mp-PAT. Notably, Entamoeba antigen and PHA strongly depressed (P<0·0001) eotaxin-2 (CCL24) production in both study groups. Multiple regression analyses disclosed in Mp-PAT and CTRL dissimilar cellular chemokine and cytokine production levels being higher in Mp-PAT for CCL24, IL-27, IL-8, MCP-4, MDC and PARC (for all P<0·0001), at baseline (P<0·0001), in response to Entamoeba histolytica strain HM1 antigen (EhAg) (P<0·0001), Onchocerca volvulus adult worm-derived antigen (OvAg) (P=0·005), PHA (P<0·0001) and purified protein derivative (PPD) (P<0·0001) stimulation. In Mp-PAT with hookworm co-infection, the cellular chemokine production of CXCL10 (IP-10) was diminished. In summary, the chemokine and cytokine responses in Mp-PAT were in general not depressed, PBMC from Mp-PAT produced spontaneously and selectively inducible inflammatory and regulatory chemokines and cytokines at higher levels than CTRL and such diverse and distinctive reactivity supports that patent M. perstans infection will not polarize innate and adaptive cellular immune responsiveness in patients.