Chemoattractant MCP-1 Research Articles

Cardioprotection by the adiponectin receptor agonist ALY688 in a preclinical mouse model of heart failure with reduced ejection fraction (HFrEF)

AimsAdiponectin has been shown to mediate cardioprotective effects and levels are typically reduced in patients with cardiometabolic disease. Hence, there has been intense interest in developing adiponectin-based therapeutics. The aim of this translational research study was to examine the functional significance of targeting adiponectin signaling with the adiponectin receptor agonist ALY688 in a mouse model of heart failure with reduced ejection fraction (HFrEF), and the mechanisms of cardiac remodeling leading to cardioprotection. Methods and resultsWild-type mice were subjected to transverse aortic constriction (TAC) to induce left ventricular pressure overload (PO), or sham surgery, with or without daily subcutaneous ALY688-SR administration. Temporal analysis of cardiac function was conducted via weekly echocardiography for 5 weeks and we observed that ALY688 attenuated the PO-induced dysfunction. ALY688 also reduced cardiac hypertrophic remodeling, assessed via LV mass, heart weight to body weight ratio, cardiomyocyte cross sectional area, ANP and BNP levels. ALY688 also attenuated PO-induced changes in myosin light and heavy chain expression. Collagen content and myofibroblast profile indicated that fibrosis was attenuated by ALY688 with TIMP1 and scleraxis/periostin identified as potential mechanistic contributors. ALY688 reduced PO-induced elevation in circulating cytokines including IL-5, IL-13 and IL-17, and the chemoattractants MCP-1, MIP-1β, MIP-1alpha and MIP-3α. Assessment of myocardial transcript levels indicated that ALY688 suppressed PO-induced elevations in IL-6, TLR-4 and IL-1β, collectively indicating anti-inflammatory effects. Targeted metabolomic profiling indicated that ALY688 increased fatty acid mobilization and oxidation, increased betaine and putrescine plus decreased sphingomyelin and lysophospholipids, a profile indicative of improved insulin sensitivity. ConclusionThese results indicate that the adiponectin mimetic peptide ALY688 reduced PO-induced fibrosis, hypertrophy, inflammation and metabolic dysfunction and represents a promising therapeutic approach for treating HFrEF in a clinical setting.

Restriction of interleukin-6 alters endothelial cell immunogenicity in an allogenic environment.

The microvascular endothelium of the renal transplant is the first site of graft interaction with the host immune system and is often injured in chronic Antibody Mediated Rejection (AMR). Microvascular inflammation is an independent determinant of AMR and heightens endothelial expression of HLA molecules thereby increasing the possibility of Donor Specific Antibody (DSA) binding. Endothelial cells produce IL-6 in the steady-state and this is increased by inflammation or by HLA-DR antibody binding in an allogeneic setting. Because IL-6 has been implicated in AMR, IL-6 blockade is currently under investigation as a therapeutic target. To further understand the role of IL-6 in endothelial cell immunogenicity, we have examined whether humanized antibody blockade of IL-6 altered endothelial cell interactions with allogeneic PBMC and after anti-HLA or DSA binding to endothelial cells in an in vitro human experimental model. Soluble factors, endothelial phenotype, Stat-3 activation, CD4+ -T differentiation, and C4d deposition were examined. Blockade of IL-6 reduced endothelial cell secretion of IL-6 and of the monocyte chemoattractant MCP-1. Pre-activation of endothelial cells by anti-HLA or DSA binding increased IL-6 secretion, that was further increased by concurrent binding of both antibodies and this was inhibited by IL-6 blockade. Activation of Stat-3 in CD4+ -T mediated by soluble factors produced in endothelial-PBMC interactions, and endothelial differentiation of CD4+ -T cell subsets (Th1, Th17, Treg), were impaired whereas activation of Complement by anti-HLA antibody binding remained unchanged by IL-6 blockade. Together, these data identify EC-mediated pro-inflammatory responses (T cell expansion, EC auto-activation, chemokine secretion) targeted by IL-6 blockade.

Differential involvement of LUBAC-mediated linear ubiquitination in intestinal epithelial cells and macrophages during intestinal inflammation.

Disruption of the intestinal epithelial barrier and dysregulation of macrophages are major factors contributing to the pathogenesis of inflammatory bowel diseases (IBDs). Activation of NF-κB and cell death are involved in maintaining intestinal homeostasis in a cell type-dependent manner. Although both are regulated by linear ubiquitin chain assembly complex (LUBAC)-mediated linear ubiquitination, the physiological relevance of linear ubiquitination to intestinal inflammation remains unexplored. Here, we used two experimental mouse models of IBD (intraperitoneal LPS and oral dextran sodium sulfate [DSS] administration) to examine the role of linear ubiquitination in intestinal epithelial cells (IECs) and macrophages during intestinal inflammation. We did this by deleting the linear ubiquitination activity of LUBAC specifically from IECs or macrophages. Upon LPS administration, loss of ligase activity in IECs induced mucosal inflammation and augmented IEC death. LPS-mediated death of LUBAC-defective IECs was triggered by TNF. IEC death was rescued by an anti-TNF antibody, and TNF (but not LPS) induced apoptosis of organoids derived from LUBAC-defective IECs. However, augmented TNF-mediated IEC death did not overtly affect the severity of colitis after DSS administration. By contrast, defective LUBAC ligase activity in macrophages ameliorated DSS-induced colitis by attenuating both infiltration of macrophages and expression of inflammatory cytokines. Decreased production of macrophage chemoattractant MCP-1/CCL2, as well as pro-inflammatory IL-6 and TNF, occurred through impaired activation of NF-κB and ERK via loss of ligase activity in macrophages. Taken together, these results indicate that both intraperitoneal LPS and oral DSS administrations are beneficial for evaluating epithelial integrity under inflammatory conditions, as well as macrophage functions in the event of an epithelial barrier breach. The data clarify the cell-specific roles of linear ubiquitination as a critical regulator of TNF-mediated epithelial integrity and macrophage pro-inflammatory responses during intestinal inflammation. © 2022 The Pathological Society of Great Britain and Ireland.

Deletion of macrophage Gpr101 disrupts their phenotype and function dysregulating host immune responses in sterile and infectious inflammation

We recently found that the G protein coupled receptor GPR101 mediates the phagocyte-directed pro-resolving activities of RvD5n-3 DPA (n-3 docosapentaenoic acid-derived Resolvin D5). Herein, we investigated the endogenous role of this pro-resolving receptor in modulating macrophage biology using a novel mouse line where the expression of Gpr101 was conditionally deleted in macrophages (MacGpr101KO). Peritoneal macrophages obtained from naïve MacGpr101KO mice displayed a marked shift in the expression of phenotypic and activation markers, including the Interleukin (IL)-10 and IL-23 receptors. Loss of Gpr101 on macrophages was also associated with a significant disruption in their cellular metabolism and a decreased ability to migrate towards the chemoattractant Mcp-1. The alterations in macrophage phenotype observed in Gpr101 deficient macrophages were maintained following inflammatory challenge. This was linked with an increased inflammatory response in the Gpr101 deficient animals and a reduced ability of phagocytes, including macrophages, to clear bacteria. Loss of Gpr101 on macrophages disrupted host pro-resolving responses to zymosan challenge with MacGpr101KO mice exhibiting significantly higher neutrophil numbers and a delay in the resolution interval when compared with control mice. These observations were linked with a marked dysregulation in peritoneal lipid mediator concentrations in Gpr101 deficient mice, with a downregulation of pro-resolving mediators including MaR2n-3 DPA, Resolvin (Rv) D3 and RvE3. Together these findings identify Gpr101 as a novel regulator of both macrophage phenotype and function, modulating key biological activities in both limiting the propagation of inflammation and expediting its resolution.

Restriction of interleukin-6 alters endothelial cell immunogenicity in an allogenic environment.

The microvascular endothelium of the renal transplant is the first site of graft interaction with the host immune system and is often injured in chronic Antibody Mediated Rejection (AMR). Microvascular inflammation is an independent determinant of AMR and heightens endothelial expression of human leukocyte antigen (HLA) molecules thereby increasing the possibility of Donor Specific Antibody (DSA) binding. Endothelial cells (ECs) produce IL-6 in the steady-state that is increased by inflammation or by HLA-DR antibody binding in an allogeneic setting. Because IL-6 has been implicated in AMR, IL-6 blockade is currently under investigation as a therapeutic target. To further understand the role of IL-6 in EC immunogenicity, we have examined whether humanized antibody blockade of IL-6 altered EC interactions with allogeneic PBMC and after anti-HLA or DSA binding to ECs in an in vitro human experimental model. Soluble factors, endothelial phenotype, Stat-3 activation, CD4+ -T differentiation and C4d deposition were examined. Blockade of IL-6 reduced EC secretion of IL-6 and of the monocyte chemoattractant MCP-1. Pre-activation of ECs by anti-HLA or DSA binding increased IL-6 secretion, that was further increased by concurrent binding of both antibodies and this was inhibited by IL-6 blockade. Activation of Stat-3 in CD4+ -T mediated by soluble factors produced in endothelial-PBMC interactions, and endothelial differentiation of CD4+ -T cell subsets (Th1, Treg), were impaired whereas activation of Complement by anti-HLA antibody binding remained unchanged by IL-6 blockade. Together, these data identify EC-mediated pro-inflammatory responses (T cell expansion, EC auto-activation, chemokine secretion) targeted by IL-6 blockade.

Gut dysbiosis is associated with acceleration of lupus nephritis

The gut microbiota (GM) exerts a strong influence over the host immune system and dysbiosis of this microbial community can affect the clinical phenotype in chronic inflammatory conditions. To explore the role of the GM in lupus nephritis, we colonized NZM2410 mice with Segmented Filamentous Bacteria (SFB). Gut colonization with SFB was associated with worsening glomerulonephritis, glomerular and tubular immune complex deposition and interstitial inflammation compared to NZM2410 mice free of SFB. With SFB colonization mice experienced an increase in small intestinal lamina propria Th17 cells and group 3 innate lymphoid cells (ILC3s). However, although serum IL-17A expression was elevated in these mice, Th17 cells and ILC3s were not detected in the inflammatory infiltrate in the kidney. In contrast, serum and kidney tissue expression of the macrophage chemoattractants MCP-1 and CXCL1 were significantly elevated in SFB colonized mice. Furthermore, kidney infiltrating F4/80+CD206+M2-like macrophages were significantly increased in these mice. Evidence of increased gut permeability or “leakiness” was also detected in SFB colonized mice. Finally, the intestinal microbiome of SFB colonized mice at 15 and 30 weeks of age exhibited dysbiosis when compared to uncolonized mice at the same time points. Both microbial relative abundance as well as biodiversity of colonized mice was found to be altered. Collectively, SFB gut colonization in the NZM2410 mouse exacerbates kidney disease, promotes kidney M2-like macrophage infiltration and overall intestinal microbiota dysbiosis.

Loss of Diacylglycerol Kinase α Enhances Macrophage Responsiveness.

The diacylglycerol kinases (DGKs) are a family of enzymes responsible for the conversion of diacylglycerol (DAG) to phosphatidic acid (PA). In addition to their primary function in lipid metabolism, DGKs have recently been identified as potential therapeutic targets in multiple cancers, including glioblastoma (GBM) and melanoma. Aside from its tumorigenic properties, DGKα is also a known promoter of T-cell anergy, supporting a role as a recently-recognized T cell checkpoint. In fact, the only significant phenotype previously observed in Dgka knockout (KO) mice is the enhancement of T-cell activity. Herein we reveal a novel, macrophage-specific, immune-regulatory function of DGKα. In bone marrow-derived macrophages (BMDMs) cultured from wild-type (WT) and KO mice, we observed increased responsiveness of KO macrophages to diverse stimuli that yield different phenotypes, including LPS, IL-4, and the chemoattractant MCP-1. Knockdown (KD) of Dgka in a murine macrophage cell line resulted in similar increased responsiveness. Demonstrating in vivo relevance, we observed significantly smaller wounds in Dgka-/- mice with full-thickness cutaneous burns, a complex wound healing process in which macrophages play a key role. The burned area also demonstrated increased numbers of macrophages. In a cortical stab wound model, Dgka-/- brains show increased Iba1+ cell numbers at the needle track versus that in WT brains. Taken together, these findings identify a novel immune-regulatory checkpoint function of DGKα in macrophages with potential implications for wound healing, cancer therapy, and other settings.

Abstract A095: Discovering the immune profiles of a novel antifolate receptor alpha IgE antibody associated with monocyte-mediated antitumor functions

Abstract Monoclonal antibodies are an established modality for the clinical management of many cancers. To date, all antibodies approved for cancer treatment are of the IgG class, and many are targeted towards non-solid tumors. We hypothesized that antibodies of the IgE class may be able to recruit and activate immune cells against tissue resident tumors based on very high affinity of IgE for cognate Fcε Receptors on potent effector cells such as masT-cells, monocytes and macrophages. A novel IgE-based antibody against folate receptor alpha (FRα) for ovarian carcinoma was developed and demonstrated to engender superior anti-tumor effector functions compared with those triggered by its IgG counterpart in different in vivo models of cancer. We have now translated this concept to a first-in-man clinical trial. We have previously reported that monocytes and macrophages are important IgE effector cells against tumors. However, the underlying mechanisms of how IgE exerts antitumor immunity by engaging these effector cells are insufficiently characterized. Here, we sought to investigate the immune profile signatures triggered by the engagement of tumor antigen-specific IgE with Fc-receptors on the surface of monocytes, a key immune effector cell recruited by IgE antibodies. In order to investigate the above aim, the following methods were utilized: Antibody dependenT-cell-mediated cytotoxicity (ADCC) and phagocytosis (ADCP) were measured by flow cytometry. Cell stimulation studies with cytokines or by cross-linking of IgE monoclonal antibodies on the surface of monocytes were conducted. Expression and secretion of immune mediators were assessed using quantitative-PCR and ELISA. Toxicity of cytokines towards immune and tumor cells were investigated by cell viability (MTS) assays. As a result, tumor antigen-specific IgE-mediated tumor cell cytotoxicity mediated by human monocytes correlated with increased levels of TNFα, MCP-1 and IL-10. Cross-linking of IgE, but not IgG, on the monocyte surface, triggered up-regulation of the proinflammatory mediator TNFα. TNFα stimulation of monocytes and of tumor cells triggered stimulation of MCP-1 by both cells. Neither cross-linking of IgE or IgG antibodies on tumor cells or monocytes, nor stimulation of these cells with TNFα or MCP-1, was sufficient to upregulate IL-10. However, IL-10 expression was induced by monocytic cells when stimulated by TNFα and MCP-1 in combination, and by stimulation with IL-10 in an autocrine manner. None of these stimulation conditions was sufficient to upregulate IL-10 in cancer cell lines of different origins. None of the three cytokines had direct cytotoxic effects towards immune or tumor cells. However, blockade of TNFα receptor on monocytes reduced ADCC and increased levels of the chemoattractant MCP-1 recruited monocytes in chemotaxis assays in vitro and macrophages in tumor lesions in a rat model of cancer treated with anti-tumor IgE. These findings suggest that IgE-dependent monocyte-mediated tumor cell cytotoxicity triggers TNFα, MCP-1 and IL-10, an axis normally associated with IgE-mediated anti-parasite immune functions. This cascade appears to be responsible for a prominent recruitment of monocytes and macrophages towards tumor cells but may not be solely responsible for IgE-mediated antitumor cytotoxic functions. Identifying the immune profiles involved in IgE-mediated monocyte antitumor mechanisms may help identify markers of effector cell activation and support the development of IgE class as a novel immuno-oncology strategy. Citation Format: Mano Nakamura, Heather J. Bax, James F. Spicer, Katie E. Lacy, Sophia Tsoka, Debra H. Josephs, Sophia Karagiannis. Discovering the immune profiles of a novel antifolate receptor alpha IgE antibody associated with monocyte-mediated antitumor functions [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A095.

Odnos inflamatornih endotelijalnih markera sa težinom astme kod dece

Objective. Monocyte chemoattractant protein-1 (MCP)-1 and soluble vascular cell adhesion molecule-1 (sVCAM-1) are key regulators of the inflammation sites infiltrated with monocytes. The aim of the study was to investigate the role of these molecules in children with asthma. Methods. The levels of MCP-1 and sVCAM-1 during asthma exacerbation on the background of therapy in children with various degrees of asthma severity as well as correlation relationships between MCP-1, sVCAM-1 and parameters of respiration function were determined. Results. The level of MCP-1 and sVCAM-1 in all groups of the examined patients at the period of exacerbation of the disease was significantly higher than in children of the control group. On the background of therapy the levels of MCP-1 and sVCAM-1were decreased in patients of all groups, regardless of asthma severity. Negative correlation relationships between MCP-1 and FEV1, PEF, VC, FVC, FEV1/FVC were revealed before and after the therapy. Conclusions. The increased levels of the abovementioned biomarkers were preserved on the seventh day of therapy. This testifies to the involvement of these chemokines into the formation and prolongation of the inflammatory process. The revealed negative correlation between MCP-1 and the main parameters of pulmonary function testified to participation of chemoattractant MCP-1 in chronic inflammation of the airways.

RA10.02: A NOVEL ROLE FOR THE COMPLEMENT CASCADE IN CHEMORADIATION THERAPY RESISTANT OESOPHAGEAL ADENOCARCINOMA

Abstract Background Resistance to chemoradiation therapy (CRT) in oesophageal adenocarcinoma (OAC) is an important clinical challenge. Elucidating underlying mechanisms of treatment resistance is crucial to improving survival rates. Methods OAC tumour biopsies were collected prior to neoadjuvant CRT. Tumour conditioned media was generated by culturing human explants for 24 h. Response to treatment was determined pathologically using the Mandard Grading System. C3, CFB and MCP-1 mRNA expression was assessed by qPCR. Secreted C3, C3a and MCP-1 were measured by ELISA. An isogenic model of radioresistant OAC was established by chronically irradiating OE33 cells with clinically-relevant doses of 2 Gy X-ray radiation. C5aR and C3aR expression was determined by flow cytometry. Results We demonstrate, for the first time, that the central factor of the complement cascade, complement component 3 (C3), is expressed in OAC tumours and is increased in pre-treatment OAC biopsies from patients (n = 13) who have a subsequent poor response to neoadjuvant CRT (P < 0.05). In addition, C3 is secreted from ex vivo human OAC tumour explants (n = 13) and correlates with levels of the monocyte chemoattractant MCP-1 (P < 0.008) and complement factor B (CFB) a component of the alternative pathway of complement activation (P < 0.0001). In vitro, radioresistant OE33 R cells had increased mRNA levels of C3 (P < 0.01), CFB (P < 0.05) and MCP-1 (P < 0.05) compared to radiosensitive OE33 P cells. OEE3 R cells expressed increased levels of secreted C3 and the anaphylatoxin C3a, compared to radiosensitive OE33P cells (both P < 0.001). Furthermore, we demonstrate for the first time that the anaphylatoxin receptor C5aR is expressed by OAC cells, suggesting that OAC cells can respond to complement anaphylatoxins. Conclusion This study highlights, for the first time, a novel role for the complement cascade in the resistance of OAC to CRT and highlights C3 as a novel predictive marker of response to CRT in OAC. Disclosure All authors have declared no conflicts of interest.

Adjuvant composition and delivery route shape immune response quality and protective efficacy of a recombinant vaccine for Entamoeba histolytica

Amebiasis caused by Entamoeba histolytica is the third leading cause of parasitic mortality globally, with some 100,000 deaths annually, primarily among young children. Protective immunity to amebiasis is associated with fecal IgA and IFN-γ in humans; however, no vaccine exists. We have previously identified recombinant LecA as a potential protective vaccine antigen. Here we describe the development of a stable, manufacturable PEGylated liposomal adjuvant formulation containing two synthetic Toll-like receptor (TLR) ligands: GLA (TLR4) and 3M-052 (TLR7/8). The liposomes stimulated production of monocyte/macrophage chemoattractants MCP-1 and Mip-1β, and Th1-associated cytokines IL-12p70 and IFN-γ from human whole blood dependent on TLR ligand composition and dose. The liposomes also demonstrated acceptable physicochemical compatibility with the recombinant LecA antigen. Whereas mice immunized with LecA and GLA-liposomes demonstrated enhanced antigen-specific fecal IgA titers, mice immunized with LecA and 3M-052-liposomes showed a stronger Th1 immune profile. Liposomes containing GLA and 3M-052 together elicited both LecA-specific fecal IgA and Th1 immune responses. Furthermore, the quality of the immune response could be modulated with modifications to the liposomal formulation based on PEG length. Compared to subcutaneous administration, the optimized liposome adjuvant composition with LecA antigen administered intranasally resulted in significantly enhanced fecal IgA, serum IgG2a, as well as systemic IFN-γ and IL-17A levels in mice. The optimized intranasal regimen provided greater than 80% protection from disease as measured by parasite antigen in the colon. This work demonstrates the physicochemical and immunological characterization of an optimized mucosal adjuvant system containing a combination of TLR ligands with complementary activities and illustrates the importance of adjuvant composition and route of delivery to enhance a multifaceted and protective immune response to amebiasis.

The Anti-Hiv Candidate Abx464 Dampens Intestinal Inflammation by Triggering Il-22 Production in Activated Macrophages

The progression of human immunodeficiency virus (HIV) is associated with mucosal damage in the gastrointestinal (GI) tract. This damage enables bacterial translocation from the gut and leads to subsequent inflammation. Dextran sulfate sodium (DSS-exposure) is an established animal model for experimental colitis that was recently shown to recapitulate the link between GI-tract damage and pathogenic features of SIV infection. The current study tested the protective properties of ABX464, a first-in-class anti-HIV drug candidate currently in phase II clinical trials. ABX464 treatment strongly attenuated DSS-induced colitis in mice and produced a long-term protection against prolonged DSS-exposure after drug cessation. Consistently, ABX464 reduced the colonic production of the inflammatory cytokines IL-6 and TNFα as well as that of the chemoattractant MCP-1. However, RNA profiling analysis revealed the capacity of ABX464 to induce the expression of IL-22, a cytokine involved in colitis tissue repair, both in DSS-treated mice and in LPS-stimulated bone marrow-derived macrophages. Importantly, anti-IL-22 antibodies significantly reduced the protective effect of ABX464 on colitis in DSS-treated mice. Because reduced IL-22 production in the gut mucosa is an established factor of HIV and DSS-induced immunopathogenesis, our data suggest that the anti-inflammatory properties of ABX464 warrant exploration in both HIV and inflammatory ulcerative colitis (UC) disease.

Abstract 212: Endothelium-Specific Deletion of Epsins Attenuates Atherosclerosis in ApoE-deficient Mouse Model Through Stabilization of IP3 Receptor 1 and Maintaining ER Homeostasis

Background: Atherosclerosis is caused in part by endothelial dysfunction due to ER stress. Understanding the mechanisms that cause ER stress and endothelial dysfunction are essential in establishing new therapies to treat atherosclerosis. Epsins are endocytic adaptor proteins that mediate the internalization of plasma membrane receptors. However, whether epsins affect atherosclerosis remains unknown. To this end, we assessed the hypothesis that endothelial epsins play a critical role in regulating atherosclerosis. Methods and Results: In an ApoE-deficient murine model, endothelial-specific double knockout mice (EC-iDKO/ApoE-/-) significantly attenuated atherosclerotic lesion and macrophage infiltration in the aortic root and arch compared to ApoE-/- mice fed Western diet for ten weeks. To elucidate underlying mechanisms, mass spectrometry analysis identified IP3R1 as one of major interactors of epsins upon exposure to oxLDL. We show that epsins bind IP3R1 and facilitate its degradation in the endothelium. Downregulation of IP3R1 by atherosclerosis promoting agents augmented ER stress in primary mouse aortic endothelial cells. In line with these findings from mouse studies, in human atherosclerotic lesion, IP3R1 is dramatically downregulated and decrease in IP3R1 expression is associated with increased ER stress. Conversely, loss of epsins stabilized IP3R1, attenuated ER stress, reduced expression of P-selectin, adhesion molecules, and chemoattractant MCP-1 in primary mouse aortic endothelial cells. Molecular mapping analysis revealed that epsin’s UIM domain is critical in facilitating the degradation of IP3R1. Delivery of a synthetic UIM peptide conjugated with atheroma-targeting Lyp-1 peptide significantly attenuated atherosclerosis in ApoE-/- animal models by stabilizing IP3R1 and maintaining ER homeostasis. Importantly, atherosclerotic lesion was restored in EC-iDKO/ApoE-/- mice by breeding them onto an endothelial-specific IP3R1 heterozygous background, suggesting that endothelial epsins promote atheroma progression in part by mediating IP3R1 degradation. Conclusions: Our data identified a novel mechanism of epsins in regulating atherosclerosis and provide a potential target to treat atherosclerosis.

Anti-Folate Receptor-α IgE but not IgG Recruits Macrophages to Attack Tumors via TNFα/MCP-1 Signaling.

IgE antibodies are key mediators of antiparasitic immune responses, but their potential for cancer treatment via antibody-dependent cell-mediated cytotoxicity (ADCC) has been little studied. Recently, tumor antigen-specific IgEs were reported to restrict cancer cell growth by engaging high-affinity Fc receptors on monocytes and macrophages; however, the underlying therapeutic mechanisms were undefined and in vivo proof of concept was limited. Here, an immunocompetent rat model was designed to recapitulate the human IgE-Fcε receptor system for cancer studies. We also generated rat IgE and IgG mAbs specific for the folate receptor (FRα), which is expressed widely on human ovarian tumors, along with a syngeneic rat tumor model expressing human FRα. Compared with IgG, anti-FRα IgE reduced lung metastases. This effect was associated with increased intratumoral infiltration by TNFα+ and CD80+ macrophages plus elevated TNFα and the macrophage chemoattractant MCP-1 in lung bronchoalveolar lavage fluid. Increased levels of TNFα and MCP-1 correlated with IgE-mediated tumor cytotoxicity by human monocytes and with longer patient survival in clinical specimens of ovarian cancer. Monocytes responded to IgE but not IgG exposure by upregulating TNFα, which in turn induced MCP-1 production by monocytes and tumor cells to promote a monocyte chemotactic response. Conversely, blocking TNFα receptor signaling abrogated induction of MCP-1, implicating it in the antitumor effects of IgE. Overall, these findings show how antitumor IgE reprograms monocytes and macrophages in the tumor microenvironment, encouraging the clinical use of IgE antibody technology to attack cancer beyond the present exclusive reliance on IgG. Cancer Res; 77(5); 1127-41. ©2017 AACR.

Inactivation of the miR-183/96/182 Cluster Decreases the Severity of Pseudomonas aeruginosa-Induced Keratitis.

PurposeThe microRNA-183/96/182 cluster (miR-183/96/182) plays important roles in sensory organs. Because the cornea is replete with sensory innervation, we hypothesized that miR-183/96/182 modulates the corneal response to bacterial infection through regulation of neuroimmune interactions.MethodsEight-week-old miR-183/96/182 knockout (ko) mice and their wild-type littermates (wt) were used. The central cornea of anesthetized mice was scarred and infected with Pseudomonas aeruginosa (PA), strain 19660. Corneal disease was graded at 1, 3, and 5 days postinfection (dpi). Corneal RNA was harvested for quantitative RT-PCR. Polymorphonuclear neutrophils (PMN) were enumerated by myeloperoxidase assays; the number of viable bacteria was determined by plate counts, and ELISA assays were performed to determine cytokine protein levels. A macrophage (Mϕ) cell line and elicited peritoneal PMN were used for in vitro functional assays.ResultsMicroRNA-183/96/182 is expressed in the cornea, and in Mϕ and PMN of both mice and humans. Inactivation of miR-183/96/182 resulted in decreased corneal nerve density compared with wt mice. Overexpression of miR-183/96/182 in Mϕ decreased, whereas knockdown or inactivation of miR-183/96/182 in Mϕ and PMN increased their capacity for phagocytosis and intracellular killing of PA. In PA-infected corneas, ko mice showed decreased proinflammatory neuropeptides such as substance P and chemoattractant molecules, MIP-2, MCP1, and ICAM1; decreased number of PMN at 1 and 5 dpi; increased viable bacterial load at 1 dpi, but decreased at 5 dpi; and markedly decreased corneal disease.ConclusionsMicroRNA-183/96/182 modulates the corneal response to bacterial infection through its regulation of corneal innervation and innate immunity.

Abstract 12274: Endothelium-specific Deletion of Epsins Attenuates Atherosclerosis Through Maintaining ER Homeostasis

Epsins are endocytotic proteins for the internalization of plasma membrane receptors. Our previous studies have suggested that epsins play an important role in tumorigenesis and lymphatic development by modulating VEGFR2, VEGFR3 or Wnt signaling. Whether epsins affect atherosclerosis remains unknown. In an ApoE-deficient murine model, endothelial-specific double knockout mice (EC-iDKO/ApoE-/-) significantly attenuated atherosclerotic lesion and macrophage infiltration in aortic root and aortic arch compared to ApoE-/- mice fed Western diet for ten weeks. Endothelium-dependent relaxation is significantly improved in epsin-deficient mice. Mechanistically, the expression of epsins is upregulated in atheroma and mouse endothelial cells in a pro-inflammatory condition, mirrored in human atherosclerotic specimen. Proteomics study using Mass spectrometry revealed that epsins bind IP3R1 and facilitate its degradation in endothelium under inflammatory condition. Downregulation of IP3R1 by athero-prone substances augmented ER stress and NFkB signaling, reversible by ER stress inhibitor 4-PBA. In line with the findings from mouse studies, IP3R1 is dramatically downregulated in human atherosclerotic endothelium. Loss of epsins stabilized IP3R1, and attenuated ER stress-dependent NFkB activation and inflammatory signaling as evidenced by the reduced expression of P-selectin, adhesion molecules, and chemoattractant MCP-1 in primary mouse aortic endothelial cells (MAEC), and reflected by in vitro rolling analysis between isolated MAEC and macrophage from mouse. Molecular modeling and mapping analysis revealed that epsin UIM is critical to facilitate the degradation of IP3R1. In conclusion, we have identified a novel molecular mechanism regarding epsins in the regulation of atherosclerosis, and our data suggest that epsins are a potential target to treat atherosclerosis.

Mas receptor deficiency exacerbates lipopolysaccharide-induced cerebral and systemic inflammation in mice

Beyond the classical actions of the renin-angiotensin system on the regulation of cardiovascular homeostasis, several studies have shown its involvement in acute and chronic inflammation. The G protein-coupled receptor Mas is a functional binding site for the angiotensin-(1–7); however, its role in the immune system has not been fully elucidated. In this study, we evaluated the effect of genetic deletion of Mas receptor in lipopolysaccharide (LPS)-induced systemic and cerebral inflammation in mice. Inflammatory response was triggered in Mas deficient (Mas−/−) and C57BL/6 wild-type (WT) mice (8–12 weeks-old) by intraperitoneal injection of LPS (5mg/kg). Mas−/− mice presented more intense hypothermia compared to WT mice 24h after LPS injection. Systemically, the bone marrow of Mas−/− mice contained a lower number of neutrophils and monocytes 3h and 24h after LPS injection, respectively. The plasma levels of inflammatory mediators KC, MCP-1 and IL-10 were higher in Mas−/− mice 24h after LPS injection in comparison to WT. In the brain, Mas−/− animals had a significant increase in the number of adherent leukocytes to the brain microvasculature compared to WT mice, as well as, increased number of monocytes and neutrophils recruited to the pia-mater. The elevated number of adherent leukocytes on brain microvasculature in Mas−/− mice was associated with increased expression of CD11b – the alpha-subunit of the Mac-1 integrin – in bone marrow neutrophils 3h after LPS injection, and with increased brain levels of chemoattractants KC, MIP-2 and MCP-1, 24h later. In conclusion, we demonstrated that Mas receptor deficiency results in exacerbated inflammation in LPS-challenged mice, which suggest a potential role for the Mas receptor as a regulator of systemic and brain inflammatory response induced by LPS.

Elevated sodium and dehydration stimulate inflammatory signaling in endothelial cells and promote atherosclerosis.

Cardiovascular diseases (CVDs) are a leading health problem worldwide. Epidemiologic studies link high salt intake and conditions predisposing to dehydration such as low water intake, diabetes and old age to increased risk of CVD. Previously, we demonstrated that elevation of extracellular sodium, which is a common consequence of these conditions, stimulates production by endothelial cells of clotting initiator, von Willebrand Factor, increases its level in blood and promotes thrombogenesis. In present study, by PCR array, using human umbilical vein endothelial cells (HUVECs), we analyzed the effect of high NaCl on 84 genes related to endothelial cell biology. The analysis showed that the affected genes regulate many aspects of endothelial cell biology including cell adhesion, proliferation, leukocyte and lymphocyte activation, coagulation, angiogenesis and inflammatory response. The genes whose expression increased the most were adhesion molecules VCAM1 and E-selectin and the chemoattractant MCP-1. These are key participants in the leukocyte adhesion and transmigration that play a major role in the inflammation and pathophysiology of CVD, including atherosclerosis. Indeed, high NaCl increased adhesion of mononuclear cells and their transmigration through HUVECs monolayers. In mice, mild water restriction that elevates serum sodium by 5 mmol/l, increased VCAM1, E-selectin and MCP-1 expression in mouse tissues, accelerated atherosclerotic plaque formation in aortic root and caused thickening or walls of coronary arteries. Multivariable linear regression analysis of clinical data from the Atherosclerosis Risk in Communities Study (n=12779) demonstrated that serum sodium is a significant predictor of 10 Years Risk of coronary heart disease. These findings indicate that elevation of extracellular sodium within the physiological range is accompanied by vascular changes that facilitate development of CVD. The findings bring attention to serum sodium as a risk factor for CVDs and give additional support to recommendations for dietary salt restriction and adequate water intake as preventives of CVD.

Abstract 1152: Gene profiling of multiple myeloma: MAPK pathway deregulation, which is regulated by PIM-1 and MOS, is associated with relapse within 6 months after autoSCT in MM patients

Abstract Substantial advances have been made in understanding the biology of multiple myeloma (MM) through the study of the bone marrow (BM) microenvironment. Dynamic interplay between bone marrow stromal cells (BMSCs) and MM cells regulated the relapsed MM after autologous bone marrow transplantation (ABMT). In this study, we performed gene expression profiling with microarray data to better dissect the molecular phenotypes and prognoses of relapsed multiple myeloma (MM). Using gene expression and clinical data, we applied gene expression signatures reflecting deregulation of oncogenic pathways to highlight molecular changes in bone marrow aspirate from 28 patients with relapsed MM. The patient subgroups were defined according to relapse-free interval, within 6 months versus more than 6 months. The microarray results revealed that dyregulation of MAPK pathway was associated with relapse within 6 months after ABMT. Among them, the expression of PIM-1 gene and MOS gene was higher in samples from patients with relapsed MM than MM cell lines (p=0.0037; p=0.0021). Also, IL-6, sIL-6R, and HGF expression in patients who relapsed within 6 months after ABMT was higher than the patients whose relapse-free interval were longer than 6 months. Treatment of shRNA PIM-1 gene and shRNA MOS gene in U266 and MOLP8 dramatically led to decreasing IL-6/sIL-6R mediated ERK phosphorylation and HGF-mediated MET phosphorylation. Similar results were noted for cluster genes for PIM-1/MOS. Especially, functional analysis of PIM-1 leads to inactivate the MAPK pathway through regulating p38 mediated signaling and Wnt/β-catenin pathway. Recent studies suggested that p38 activity in myeloma inhibits osteoblast differentiation and bone formation, but also enhances osteoclast maturation and bone resorption. p38 regulated the expression and secretion of the Wnt pathway antagonist DKK-1 and the monocyte chemoattractant MCP-1. Conclusionally, Our analysis suggested that MOS gene and PIM-1, which regulates MAPK pathway, is a noble prognostic marker for relapse of MM. The importance of the PIM-1-MOS-MAPK pathway as a prognostic marker in relapsed MM should be reassessed in the novel therapeutic agent era. Citation Format: Woo June Jung, Kwang-Sung Ahn, Chansu Lee, Youngil Koh, Hyun Jung Lee, Hyo Jung Kim, Hwi-Joong Yoon, Sung-Soo Yoon. Gene profiling of multiple myeloma: MAPK pathway deregulation, which is regulated by PIM-1 and MOS, is associated with relapse within 6 months after autoSCT in MM patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1152. doi:10.1158/1538-7445.AM2014-1152

A three-dimensional collagen construct to model lipopolysaccharide-induced activation of BV2 microglia.

BackgroundWe report a novel method of culturing microglia in three dimension (3D) using collagen as a substrate. By culturing microglia within a matrix, we aim to emulate the physical state of microglia embedded within parenchyma.MethodsBV2 microglia cell suspensions were prepared with type I collagen and cast into culture plates. To characterise the BV2 microglia cultured in 3D, the cultures were evaluated for their viability, cell morphology and response to lipopolysaccharide (LPS) activation. Conventional monolayer cultures (grown on uncoated and collagen-coated polystyrene) were set up concurrently for comparison.ResultsBV2 microglia in 3D collagen matrices were viable at 48 hrs of culture and exhibit a ramified morphology with multiplanar cytoplasmic projections. Following stimulation with 1 μg/ml LPS, microglia cultured in 3D collagen gels increase their expression of nitric oxide (NO) and CD40, indicating their capacity to become activated within the matrix. Up to 97.8% of BV2 microglia grown in 3D cultures gained CD40 positivity in response to LPS, compared to approximately 60% of cells grown in a monolayer (P < .05). BV2 microglia in 3D collagen gels also showed increased mRNA and protein expression of inflammatory cytokines IL-6, TNF-α and the chemoattractant MCP-1 following LPS stimulation.ConclusionsIn summary, BV2 microglia cultured in 3D collagen hydrogels exhibit multiplanar cytoplasmic projections and undergo a characteristic and robust activation response to LPS. This culture system is accessible to a wide range of analyses and provides a useful new in vitro tool for research into microglial activation.