Induction Of CCL2 Research Articles

Commensal gut bacterium critically regulates alveolar bone homeostasis

AbstractThe alveolar bone is a unique osseous tissue due to the presence of the teeth and the proximity of commensal oral microbes. Commensal microbe effects on alveolar bone homeostasis have been attributed to the oral microbiota, yet the impact of commensal gut microbes is unknown. Study purpose was to elucidate whether commensal gut microbes regulate osteoimmune mechanisms and skeletal homeostasis in alveolar bone. Male C57BL/6T germfree (GF) littermate mice were maintained as GF or monoassociated with segmented filamentous bacteria (SFB), a commensal gut bacterium. SFB has been shown to elicit broad immune response effects, including the induction of TH17/IL17A immunity, which impacts the development and homeostasis of host tissues. SFB colonized the gut, but not oral cavity, and increased IL17A levels in the ileum and serum. SFB had catabolic effects on alveolar bone and non-oral skeletal sites, which was attributed to enhanced osteoclastogenesis. The alveolar bone marrow of SFB vs. GF mice had increased dendritic cells, activated helper T-cells, TH1 cells, TH17 cells, and upregulated Tnf. Primary osteoblast cultures from SFB and GF mice were stimulated with vehicle-control, IL17A, or TNF to elucidate osteoblast-derived signaling factors contributing to the pro-osteoclastic phenotype in SFB mice. Treatment of RAW264.7 osteoclastic cells with supernatants from vehicle-stimulated SFB vs. GF osteoblasts recapitulated the osteoclast phenotype found in vivo. Supernatants from TNF-stimulated osteoblasts normalized RAW264.7 osteoclast endpoints across SFB and GF cultures, which was dependent on the induction of CXCL1 and CCL2. This report reveals that commensal gut microbes have the capacity to regulate osteoimmune processes in alveolar bone. Outcomes from this investigation challenge the current paradigm that alveolar bone health and homeostasis is strictly regulated by oral microbes.Commensal microbe effects on alveolar bone homeostasis have been attributed to the oral microbiota, yet the impact of commensal gut microbes is unknown. Studies performed with segmented filamentous bacteria (SFB) monoassociated mice revealed commensal gut microbes modulate osteoimmune responses and skeletal homeostasis in alveolar bone. This work challenges the current paradigm that alveolar bone health is strictly regulated by oral microbes.

The renal inflammatory network of nephronophthisis.

Renal ciliopathies are the leading cause of inherited kidney failure. In autosomal dominant polycystic kidney disease (ADPKD), mutations in the ciliary gene PKD1 lead to the induction of CCL2, which promotes macrophage infiltration in the kidney. Whether or not mutations in genes involved in other renal ciliopathies also lead to immune cells recruitment is controversial. Through the parallel analysis of patients' derived material and murine models, we investigated the inflammatory components of nephronophthisis (NPH), a rare renal ciliopathy affecting children and adults. Our results show that NPH mutations lead to kidney infiltration by neutrophils, macrophages and T cells. Contrary to ADPKD, this immune cell recruitment does not rely on the induction of CCL2 in mutated cells, which is dispensable for disease progression. Through an unbiased approach, we identified a set of inflammatory cytokines that are upregulated precociously and independently of CCL2 in murine models of NPH. The majority of these transcripts is also upregulated in NPH patient renal cells at a level exceeding those found in common non-immune chronic kidney diseases. This study reveals that inflammation is a central aspect in NPH and delineates a specific set of inflammatory mediators that likely regulates immune cell recruitment in response to NPH genes mutations.

Angiotensin-(1-7)/MasR axis promotes migration of monocytes/macrophages with a regulatory phenotype to perform phagocytosis and efferocytosis.

Nonphlogistic migration of macrophages contributes to the clearance of pathogens and apoptotic cells, a critical step for the resolution of inflammation and return to homeostasis. Angiotensin-(1-7) [Ang-(1-7)] is a heptapeptide of the renin-angiotensin system that acts through Mas receptor (MasR). Ang-(1-7) has recently emerged as a novel proresolving mediator, yet Ang-(1-7) resolution mechanisms are not fully determined. Herein, Ang-(1-7) stimulated migration of human and murine monocytes/macrophages in a MasR-, CCR2-, and MEK/ERK1/2–dependent manner. Pleural injection of Ang-(1-7) promoted nonphlogistic mononuclear cell influx alongside increased levels of CCL2, IL-10, and macrophage polarization toward a regulatory phenotype. Ang-(1-7) induction of CCL2 and mononuclear cell migration was also dependent on MasR and MEK/ERK. Of note, MasR was upregulated during the resolution phase of inflammation, and its pharmacological inhibition or genetic deficiency impaired mononuclear cell recruitment during self-resolving models of LPS pleurisy and E. coli peritonitis. Inhibition/absence of MasR was associated with reduced CCL2 levels, impaired phagocytosis of bacteria, efferocytosis, and delayed resolution of inflammation. In summary, we have uncovered a potentially novel proresolving feature of Ang-(1-7), namely the recruitment of mononuclear cells favoring efferocytosis, phagocytosis, and resolution of inflammation. Mechanistically, cell migration was dependent on MasR, CCR2, and the MEK/ERK pathway.

Intrinsic Antiviral Activity of Optineurin Prevents Hyperproliferation of a Primary Herpes Simplex Virus Type 2 Infection.

Very little knowledge exists on virus-specific host cell intrinsic mechanisms that prevent hyperproliferation of primary HSV type 2 (HSV-2) genital infections. In this study, we provide evidence that the Nemo-related protein, optineurin (OPTN), plays a key role in restricting HSV-2 infection both in vitro and in vivo. Contrary to previous reports regarding the proviral role of OPTN during Sendai virus infection, we demonstrate that lack of OPTN in cells causes enhanced virus production. OPTN deficiency negatively affects the host autophagy response and results in a marked reduction of CCL5 induction. OPTN knockout (OPTN-/-) mice display exacerbated genital disease and dysregulated T cell frequencies in infected tissues and lymph nodes. A human transcriptomic profile dataset provides further credence that a strong positive correlation exists between CCL5 upregulation and OPTN expression during HSV-2 genital infection. Our findings underscore a previously unknown OPTN/CCL5 nexus that restricts hyperproliferative spread of primary HSV-2 infection, which may constitute an intrinsic host defense mechanism against herpesviruses in general.

Disruption of monocyte-macrophage differentiation and trafficking by a heme analog during active inflammation.

Heme metabolism is a key regulator of inflammatory responses. Cobalt protoporphyrin IX (CoPP) is a heme analog and mimic that potently activates the NRF2/heme oxygenase-1 (HO-1) pathway, especially in monocytes and macrophages. We investigated the influence of CoPP on inflammatory responses using a murine model of colitis. Surprisingly, conditional deletion of myeloid HO-1 did not impact the colonic inflammatory response or the protective influence of CoPP in the setting of dextran sodium sulphate-induced colitis. Rather, we reveal that CoPP elicits a contradictory shift in blood myeloid populations relative to the colon during active intestinal inflammation. Major population changes include markedly diminished trafficking of CCR2+Ly6Chi monocytes to the inflamed colon, despite significant mobilization of this population into circulation. This resulted in significantly diminished colonic expansion of monocyte-derived macrophages and inflammatory cytokine expression. These findings were linked with significant induction of systemic CCL2 leading to a disrupted CCL2 chemoattractant gradient towards the colon and concentration-dependent suppression of circulating monocyte CCR2 expression. Administration of CoPP also induced macrophage differentiation toward a MarcohiHmox1hi anti-inflammatory erythrophagocytic phenotype, contributing to an overall decreased inflammatory profile. Such findings redefine protective influences of heme metabolism during inflammation, and highlight previously unreported immunosuppressive mechanisms of endogenous CCL2 induction.

IM-4 Impact of oHSV activated NOTCH signaling in tumor microenvironment and its impact on anti-tumor immunity

Oncolytic herpes simplex virus-1 (oHSV) is novel FDA-approved immunotherapy for advanced melanoma patients in US. Also, oHSV is recently approved for the treatment of recurrent GBM in Japan. We have shown that oHSV treatment of GBM cells induces NICD cleavage and NOTCH activation in adjacent uninfected glioma cells via HSV-1 microRNA-H16 (Otani Y and Yoo JY, Clin Cancer Res, 2020), however, the consequences of NOTCH on immunotherapy in GBM is unknow. Here we have investigated the impact of oHSV-induced NOTCH signaling on the tumor microenvironment (TME). Analysis of TCGA GBM data and experimental murine models revealed NOTCH induced immunosuppressive myeloid cell recruitment and limited anti-tumor immunity. In oHSV treated tissue, viral infection educated tumor associated macrophages to secrete CCL2 which recruited monocytic myeloid derived suppressor cell (MDSC) that attenuated anti-tumor immunity. Consistent with this, CCL2 induction was also observed in serum of recurrent GBM patients treated with oHSV (NCT03152318). Importantly, blockade of NOTCH signaling reduced the oHSV induced immunosuppressive environment and activated a CD8 dependent anti-tumor memory response. These findings present the opportunities for combination therapies that can help improve therapeutic benefit and anti-tumor immunity in GBM.

MicroRNA-mediated calcineurin signaling activation induces CCL2, CCL3, CCL5, IL8, and chemotactic activities in 4,4′-methylene diphenyl diisocyanate exposed macrophages

Occupational exposure to 4,4′-methylene diphenyl diisocyanate (MDI), the most widely used monomeric diisocyanate, is one of the leading causes of occupational asthma (OA). Previously, we identified microRNA (miR)-206-3p/miR-381-3p-mediated PPP3CA/calcineurin signalling regulated iNOS transcription in macrophages and bronchoalveolar lavage cells (BALCs) after acute MDI exposure; however, whether PPP3CA/calcineurin signalling participates in regulation of other asthma-associated mediators secreted by macrophages/BALCs after MDI exposure is unknown. Several asthma-associated, macrophage-secreted mediator mRNAs from MDI exposed murine BALCs and MDI-glutathione (GSH) conjugate treated differentiated THP-1 macrophages were analysed using RT-qPCR. Endogenous IL1B, TNF, CCL2, CCL3, CCL5, and TGFB1 were upregulated in MDI or MDI-GSH conjugate exposed BALCs and macrophages, respectively. Calcineurin inhibitor tacrolimus (FK506) attenuated the MDI-GSH conjugate-mediated induction of CCL2, CCL3, CCL5, and CXCL8/IL8 but not others. Transfection of either miR-inhibitor-206-3p or miR-inhibitor-381-3p in macrophages induced chemokine CCL2, CCL3, CCL5, and CXCL8 transcription, whereas FK506 attenuated the miR-inhibitor-206-3p or miR-inhibitor-381-3p-mediated effects. Finally, MDI-GSH conjugate treated macrophages showed increased chemotactic ability to various immune cells, which may be attenuated by FK506. In conclusion, these results indicate that MDI exposure to macrophages/BALCs may recruit immune cells into the airway via induction of chemokines by miR-206-3p and miR-381-3p-mediated calcineurin signalling activation.

Hepatoprotective activity and free radical scavenging against induction of CCl4 in an experimental model using dendronaphthya crude extract loaded chitosan nanocarrier

The encapsulation of soft coral Dendronephthya gigantea (SCD) in biopolymers could be attributed to the nature of this extract. In the present work, chitosan nanoparticles (NCS) as natural biopolymer was prepared using sodium tripolyphosphate (TPP) via ionic gelation method and then encapsulated with SCD. Firstly, GC-MS was used to determine the components of the SCD. More than Forty-components of SCD, constituting 89.94%, were identified. Secondly, to enhance the entrapment efficiency and encapsulating degree, polyvinyl alcohol (PVA) was used to cap the whole product of NSC@SCD. The entrapment efficiency of PVA-capped NSC@SCD is higher than that of NSC@SCD and reached about 96%. Physical advanced tools such as TEM, DLS, and SEM were utilized to confirm the particle shape for the formed nanocomposite, affirming that the particles originated in a spherical shape with no noticeable agglomeration. Besides, PVA was found to protect the produced nanocomposite from agglomeration and exhibit the latter homogenous structure. After that, rats with approximately the same weight were divided into seven groups and treated with different formulas to evaluate hepatoprotective activity against induction of CCl4 of the prepared nanocomposites loaded with SCD. Form the obtained resultants, it can be observed that the nanocomposite has a positive effect on the treated rats via improving in the values of catalase activity (CA), reduced glutathione (GSH), and total antioxidant capacity (TAC) with a significant decrease in malondialdehyde (MDA) values. It was also observed that the level of liver enzymes closely resembles the untreated rat group. The obtained data also proved that nanocomposite based on NSC@SCD exhibited high albumin and total protein level. Thus, It can be concluded that the extract (SCD) loaded chitosan nanoparticles (NSC) is considered a promising formula to be used in medicinal domains.

Twist1 in podocytes ameliorates podocyte injury and proteinuria by limiting CCL2-dependent macrophage infiltration.

The transcription factor Twist1 regulates several processes that could impact kidney disease progression, including epithelial cell differentiation and inflammatory cytokine induction. Podocytes are specialized epithelia that exhibit features of immune cells and could therefore mediate unique effects of Twist1 on glomerular disease. To study Twist1 functions in podocytes during proteinuric kidney disease, we employed a conditional mutant mouse in which Twist1 was selectively ablated in podocytes (Twist1-PKO). Deletion of Twist1 in podocytes augmented proteinuria, podocyte injury, and foot process effacement in glomerular injury models. Twist1 in podocytes constrained renal accumulation of monocytes/macrophages and glomerular expression of CCL2 and the macrophage cytokine TNF-α after injury. Deletion of TNF-α selectively from podocytes had no impact on the progression of proteinuric nephropathy. By contrast, the inhibition of CCL2 abrogated the exaggeration in proteinuria and podocyte injury accruing from podocyte Twist1 deletion. Collectively, Twist1 in podocytes mitigated urine albumin excretion and podocyte injury in proteinuric kidney diseases by limiting CCL2 induction that drove monocyte/macrophage infiltration into injured glomeruli. Myeloid cells, rather than podocytes, further promoted podocyte injury and glomerular disease by secreting TNF-α. These data highlight the capacity of Twist1 in the podocyte to mitigate glomerular injury by curtailing the local myeloid immune response.

Anti-tumor immunity in mismatch repair-deficient colorectal cancers requires type I IFN-driven CCL5 and CXCL10.

Colorectal cancers (CRCs) deficient in DNA mismatch repair (dMMR) contain abundant CD8+ tumor-infiltrating lymphocytes (TILs) responding to the abundant neoantigens from their unstable genomes. Priming of such tumor-targeted TILs first requires recruitment of CD8+ T cells into the tumors, implying that this is an essential prerequisite of successful dMMR anti-tumor immunity. We have discovered that selective recruitment and activation of systemic CD8+ T cells into dMMR CRCs strictly depend on overexpression of CCL5 and CXCL10 due to endogenous activation of cGAS/STING and type I IFN signaling by damaged DNA. TIL infiltration into orthotopic dMMR CRCs is neoantigen-independent and followed by induction of a resident memory-like phenotype key to the anti-tumor response. CCL5 and CXCL10 could be up-regulated by common chemotherapies in all CRCs, indicating that facilitating CD8+ T cell recruitment underlies their efficacy. Induction of CCL5 and CXCL10 thus represents a tractable therapeutic strategy to induce TIL recruitment into CRCs, where local priming can be maximized even in neoantigen-poor CRCs.

Screening of secondary metabolites and immunostimulation potential of poly herbal formoulation (Asparagus racemosus, Mucuna pruriens, Anacyclus pyrethrum and Tribulus terristeris) in Wistar Albino rats

Immune system is a defensive mechanism system which is responsible to defend against several foreign invading agents that harm individual health. It generates several types of cells which have ability to recognize, eliminate and kill all the undesirable agents. The immune system works by inhibition, multiplication, amplification, expression and induction response at different phases of immune system. Hence immunology is the most advance and recent biomedical research system which covers the prevention and treatment of wide range of medical disorders. In this study, Immunomodulatory activity of Polyherbal formulation was determined. First of all, plants methanolic extracts of Asparagus racemosus, Mucuna pruriens, Anacyclus pyrethrum, Tribulus terrestris were prepared. The methanolic extracts of each plant were taken separately in equal amount and mixed to prepare the Polyherbal formulation (PHF). Screening test was applied on PHF for quantitative analysis. Hemolytic activity, thrombolytic activity and antioxidant activity were determined on experimental animals. To demonstrate the effect of standard Immunol drug, CCl4 and polyherbal formulation extract on hematological parameters. The methanolic extract of PHF was dissolved in distilled water and the dose was selected at 100, 200 and 300 mg/kg body weight. Blood sample collected in EDTA as anticoagulant were processed to determine the effect of PHP on selected hematological parameters including hemoglobin (Hb), red blood cell counts (RBCs), white blood cells (WBCs) count and Platelets count. Liver enzymes like transaminases (ALT and AST), uric acid, urea and creatinine were determined by automated photometric method to evaluate the drug safety. Serum samples were using for the estimation of Immunoglobulin M and Immunoglobulin G using enzyme linked immunosorbent assay (ELISA). Further, the C-reactive protein (CRP) was also estimated using standard protocol. Phytochemical screening of methanolic extract of polyherbal formulation showed the presence of alkaloids, tannins, flavonoids, phenols and cardiac glycosides. Bioactive constituent’s alkaloids, flavonoids and phenols were present in higher concentration. The maximum hemolysis was seen at a dose of 800μg/ml (40 ± 0.38). The methanolic extracts of PHF were investigated for its thrombolytic activity. Addition of 800μl of streptokinase as positive control (30,000 IU/ ml) to clots and successive incubation for 90 minutes at 37 oC showed 74.53±1.11% lysis of clot. Distilled water was considered as negative control which exhibit insignificant percentage of lysis of clot (3.74±0.51). In the study, the methanolic extract of PHF showed highest thrombolytic activity at 800μg/ml concentrations (41±0.38) when compared with streptokinase (positive control). When all the groups were compared the serum level ALT, AST, TAS, TOS and OSI 50 index, a statistically significant difference was observed between the groups. A variety of antioxidant function approaches have been suggested. During the study, it was found that induction of CCl4 increases the number of altered macrophages. However, the administration of Polyherbal extract in CCl4 intoxicated animals, the number of altered macrophages came to level. Administration of polyherbal formulation extract in control mice also showed significant reduction of changed macrophages. Immunomodulatory drugs are substances that can alter the immune function of an individual, are called immunostimulants if their immune responses are enhanced, or are called immunosuppressant’s if they decrease. These medications are most widely used in infections, allergies and cancer infections. Modern medical treatment is also far-reaching because of the economic limitations in developed countries including Pakistan. For immunomodular operations, just a few plants have been tested. The analysis indicates that many medicinal plants and marine products have immunomodulatory function, however there is little proof that their use in clinical practice is not allowed. In future work in herbal medicine, therefore, immunomodulatory agents should become more relevant.

Keratinocyte autophagy enables the activation of keratinocytes and fibroblastsand facilitates wound healing

ABSTRACT Macroautophagy/autophagy is a cellular catabolic process that is implicated in several physiological and pathological processes. However, the role of epidermal autophagy in wound healing remains unknown. Here, using mice with genetic ablation of the essential Atg5 (autophagy related 5) or Atg7 (autophagy related 7) in their epidermis to inhibit autophagy, we show that keratinocyte autophagy regulates wound healing in mice. Wounding induces the expression of autophagy genes in mouse skin. Epidermis-specific autophagy deficiency inhibits wound closure, re-epithelialization, keratinocyte proliferation and differentiation, dermal granulation tissue formation, and infiltration of immune cells including macrophages, neutrophils, and mast cells, while it does not affect angiogenesis. Using cytokine array screening, we found that autophagy deficiency inhibits the transcription and production of the cytokine CCL2/MCP-1 by TNF. At the molecular level, TNF induces autophagic flux and the expression of autophagy genes through NFKB in epidermal keratinocytes. TNF promotes CCL2 transcription through the autophagy-AMPK-BRAF-MAPK1/3/ERK-activator protein 1 (AP1) pathway. Indeed, treating mice with recombinant CCL2 can reverse the effect of autophagy deficiency in keratinocytes. At the cellular level, we found that CCL2 induction via autophagy in keratinocytes is required not only for keratinocyte migration and proliferation but also for dermal fibroblast activation. Our findings demonstrate a critical role of epidermal autophagy in wound healing in vivo and elucidate a critical molecular machinery coordinating keratinocyte-fibroblast interaction in skin repair. Abbreviations: ACTA2/α-SMA: actin alpha 2, smooth muscle; ACTB: β-actin; ADGRE1: adhesion G protein-coupled receptor E1; AMPK: AMP-activated protein kinase; AP1: activator protein 1; AP1-RE: AP1 response element; ATG: autophagy-related; ATG16L1: autophagy related 16 like 1; BECN1: beclin 1; BRAF: B-Raf proto-oncogene, serine/threonine kinase; C5: complement C5; CCL2/MCP-1: C-C motif chemokine ligand 2; CCL3: C-C motif chemokine ligand 3; CK: cytokeratin; cKO: conditional knockout; CRTC1: CREB-regulated transcription coactivator 1; CXCL1: C-X-C motif chemokine ligand 1; CXCL2: C-X-C motif chemokine ligand 2; ECM: extracellular matrix; EGF: epidermal growth factor; FGF7: fibroblast growth factor 7; GABARAPL2: GABA type A receptor associated protein like 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HBEGF: heparin binding EGF like growth factor; HPRT1: hypoxanthine phosphoribosyltransferase 1; IHC: immunohistochemical; IL1B: interleukin 1 beta; KRT10: keratin 10; KRT14: keratin 14; MAP1LC3B/LC3B-I/II: microtubule-associated protein 1 light chain 3 beta; MAPK1/3/ERK: mitogen-activated protein kinase 1/3; MKI67/Ki-67: marker of proliferation; MPO: myeloperoxidase; NFKB: NF-kappa B, nuclear factor kappa-light-chain-enhancer of activated B cells; NFKB-RE: NFKB response element; PDGF: platelet-derived growth factor; PECAM1: platelet and endothelial cell adhesion molecule 1; PRKAA1: protein kinase AMP-activated catalytic subunit alpha 1; RELA/p65: RELA proto-oncogene, NFKB subunit; shCON: small hairpin negative control; siNC: negative control; siRNA: small interfering RNA; SP1: sp1 transcription factor; SQSTM1/p62: sequestosome 1; TGFA: transforming growth factor alpha; TGFB1: transforming growth factor beta 1; TIMP1: TIMP metallopeptidase inhibitor 1; TNF/TNF-alpha: tumor necrosis factor; TREM1: triggering receptor expressed on myeloid cells 1; WT: wild-type

Abstract 6679: Immunophenotypic and transcriptome analyses of CT26 and 4T1 murine tumor models following anti-CTLA-4 treatment

Abstract Model selection for evaluation of immuno-oncology agents is often aided by both response to therapy and changes in immune cell subsets following treatment. To identify underlying factors that influence differences in therapeutic response, CT26 colorectal and 4T1-Luc (4T1) breast tumor models, which are immunologically warm and cold, respectively, were treated with anti-cytotoxic T-lymphocyte associated protein 4 (CTLA-4). Flow cytometry and transcriptome analysis were used to describe treatment-related changes in immune cell infiltrate, TIL activation status, cytokine/granzyme B production, and overall changes in gene expression. Response to anti-CTLA-4 therapy was distinct between these models. Tumor growth inhibition was observed in CT26 (74%), but not 4T1. In CT26, CD8+ and NK T cell infiltration increased, while M2 macrophages decreased. In 4T1 tumors, only increased NKT cell infiltration was noted after treatment, and no demonstrable changes were observed in any other cell type or activation marker. Several pharmacodynamic changes in immune cell activation were observed in CT26. Notably, CT26-derived CD4+, CD8+, and NK T cell subsets had increased IFNγ production (70%, 25%, and 17%, respectively, versus control). An increase in polyfunctional T cells producing both IFNγ and TNFα was also noted for CT26 and was correlated with slower tumor growth. Granzyme B was reduced and CD107a was increased in CT26-derived NK T cells, suggesting functionally active T cells. This was also supported by upregulation of genes in the cytotoxicity, antigen presentation, and NFkappaB pathways. Following CT26 treatment with anti-CTLA-4, transcriptome analysis with the nCounter® Mouse PanCancer IO 360 Panel (NanoString) showed an 8-10 fold induction of several T cell-related genes as well as an 8-14 fold induction of several chemokines and their receptors. These data complement the flow cytometry data and support stimulation of T cell activation and recruitment to CT26 tumors following anti-CTLA-4 treatment. In contrast, analysis of 4T1 tumors indicated only a 2 to 3-fold induction of CCL4, CCL11, and Granzyme A after treatment. Interestingly, genes in the matrix remodeling pathway were upregulated in 4T1 tumors upon anti-CTLA-4 treatment. In summary, CT26 tumors were responsive to anti-CTLA-4 treatment, showing increased polyfunctional T cell recruitment, NK T cell activity, and reduced M2 macrophage infiltration, as well as marked upregulation of several genes involved in T cell recruitment and activation in tumor. In contrast, 4T1 tumors did not display any remarkable response to therapy, changes to TIL infiltrates, and only limited changes to gene expression with treatment. Collectively, these results suggest tumor profiles following treatment can provide insight into potential targets of interest and are important for preclinical model selection. Citation Format: Sheri R. Barnes, David Draper, Sarah E. Church, Maryland Rosenfeld Franklin. Immunophenotypic and transcriptome analyses of CT26 and 4T1 murine tumor models following anti-CTLA-4 treatment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6679.

The endocannabinoid anandamide has an anti-inflammatory effect on CCL2 expression in vascular smooth muscle cells.

Endocannabinoids are important lipid-signaling mediators. Both protective and deleterious effects of endocannabinoids in the cardiovascular system have been reported but the mechanistic basis for these contradicting observations is unclear. We set out to identify anti-inflammatory mechanisms of endocannabinoids in the murine aorta and in human vascular smooth muscle cells (hVSMC). In response to combined stimulation with cytokines, IL-1β and TNFα, the murine aorta released several endocannabinoids, with anandamide (AEA) levels being the most significantly increased. AEA pretreatment had profound effects on cytokine-induced gene expression in hVSMC and murine aorta. As revealed by RNA-Seq analysis, the induction of a subset of 21 inflammatory target genes, including the important cytokine CCL2 was blocked by AEA. This effect was not mediated through AEA-dependent interference of the AP-1 or NF-κB pathways but rather through an epigenetic mechanism. In the presence of AEA, ATAC-Seq analysis and chromatin-immunoprecipitations revealed that CCL2 induction was blocked due to increased levels of H3K27me3 and a decrease of H3K27ac leading to compacted chromatin structure in the CCL2 promoter. These effects were mediated by recruitment of HDAC4 and the nuclear corepressor NCoR1 to the CCL2 promoter. This study therefore establishes a novel anti-inflammatory mechanism for the endogenous endocannabinoid AEA in vascular smooth muscle cells. Furthermore, this work provides a link between endogenous endocannabinoid signaling and epigenetic regulation.

Exposure to 4,4′‐Methylene Diphenyl Diisocyanate Upregulates Chemokines CCL2, CCL3, CCL5, and IL‐8 Transcription Through Activation of Calcineurin/NFAT Signaling in Macrophages

Exposure to the most widely used monomericdiisocyanate, 4,4′‐methylene diphenyl diisocyanate (MDI), in the occupational setting may lead to the development of occupational asthma (OA). Currently, the underlying molecular mechanism(s) by which MDI induces OA remain an active area of research. Airway macrophage dysfunctions such as decreased phagocytosis and efferocytosis as well as increased production of reactive oxygen species (ROS), nitric oxide(NO) and immune mediators, etc., play important roles in asthma pathogenesis. Previously, we demonstrated that the downregulation of microRNA (miR)‐206‐3p and miR‐381‐3p mediated activation of the Calcineurin/nuclear factors of activated T‐cells (NFATs) signaling pathway which induced endogenous inducible nitric oxide synthase (iNOS) transcription in macrophages and bronchoalveolarlavage cells (BALCs) after acute MDI exposure. However, whether MDI may affect expression of other asthma‐associated immune mediators in macrophages/BALCs and the underlying molecular mechanisms driving these effects are currently unknown. We hypothesize that MDI exposure induces several asthma‐associated mediators through Calcineurin/NFAT signaling activation in macrophages. We determined the expression of macrophage‐secreted, asthma‐associated mediators including cytokines (IL1‐β, TNF‐α, and IL‐6); chemokines (CCL2, CCL3, CCL5, CCL11, and IL‐8), and growth factors (GM‐CSF and TGF‐β) from both MDI‐exposed murine BALCs and MDI‐glutathione(GSH) conjugate‐treated differentiated THP‐1 macrophages using RT‐qPCR. Both in vivo murine MDI aerosol and in vitro MDI‐GSH exposures result in the upregulation of endogenous IL1‐β, TNF‐α, IL‐6, CCL2, CCL3, CCL5, and TGF‐β. Treatment of THP‐1 macrophages with 1 μM Tacrolimus (FK506), a specific Calcineurin/NFAT signaling inhibitor, blocked the induction of CCL2, CCL3, CCL5, and IL‐8 expression by MDI‐GSH conjugates but not IL1‐β, TNF‐α, IL‐6, and TGF‐β. These results indicate that Calcineurin/NFAT signaling activation plays an important role for CCL2, CCL3, CCL5, and IL‐8 transcriptional upregulation following MDI exposure in macrophage. These results suggest MDI exposure may result in recrutement of many different cell types such as eosinophils, T‐cells, monocytes, macrophages, etc., into the airway by induction of chemokines transcription via Calcineurin/NFAT signaling activation in the macrophages/BALCs.Support or Funding InformationThis work was supported by intramural funds from the National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention.

Synergistic induction of CCL5, CXCL9 and CXCL10 by IFN-γ and NLRs ligands on human fibroblast-like synoviocytes—A potential immunopathological mechanism for joint inflammation in rheumatoid arthritis

Interferon-γ (IFN-γ) is traditionally regarded as a proinflammatory cytokine by virtue of its strong macrophage activating potential and its association with Th1 driven immune responses. NOD1 and NOD2 are cytoplasmic receptors that can initiate the initial immune response by sensing bacterial components or danger signals. In this study, we investigated the immunopathological roles of IFN-γ and NOD1, 2 ligands iE-DAP/MDP on the activation of fibroblast-like synoviocytes (FLS) in RA. FLS constitutively express functional NOD1 and NOD2, and the gene and protein expression of NOD1 and NOD2 could be enhanced by the treatment with IFN-γ. The synergistic effect was observed in the combined treatment of IFN-γ and NOD1 ligand iE-DAP or NOD2 ligand MDP on the release of CCL5, CXCL9 and CXCL10 from FLS, and its effect was in a dose-dependent manner. The co-stimulation which IFN-γ combined with iE-DAP/MDP could abolish the inhibition of CXCL8 level by IFN-γ alone. Further investigations showed that synergistic effects on the production of CCL5, CXCL9 and CXCL10 in FLS stimulated by IFN-γ and iE-DAP/MDP were differentially regulated by intracellular activation of NF-κB, p38MAPK and ERK pathways. In conclusion, our data confirmed the inflammatory effect of IFN-γ and iE-DAP/MDP on human FLS for the first time and therefore provided a new insight into the IFN-γ combined with NOD1 or NOD2 activated immunopathological mechanisms mediated by distinct intracellular signal transduction in joint inflammation of RA.

Elevated circulating CCL2 in prostate cancer patients.

201 Background: Monocyte chemoattractant protein 1 (CCL2 or MCP-1), a chemokine secreted by monocytic cells, is critical in recruiting Treg and MDSCs into the tumor microenvironment and in regulating prostate cancer (PCa) migration and proliferation. In this study, we examined circulating CCL2 levels in healthy vs PCa patients and used an in vitro coculture model to identify the source of the elevated CCL2. Methods: Serum CCL2 concentrations were evaluated via ELISA in 59 patients (19 health controls, 20 “treatment naïve” PCa, 20 mCRPC). Monocytic leukemia cells (U937) were either directly cocultured with PC3 PCa cell line or cultured in the PC3 conditioned medium (CM). The induction of CCL2 mRNA in the cultures was examined by qPCR. The secretion of CCL2 into cell culture supernatants was evaluated via human cytokine array. Neutralizing antibodies to several overexpressed inflammatory cytokines in PC3 cells were added into the PC3 CM to evaluate the contribution of these inflammatory cytokines to CCL2 induction. Results: Circulating CCL2 concentrations were significantly higher in prostate cancer patient serum compared to control patient serum (p=4.4e-6) (Table). To understand the potential source of elevated CCL2, we grew U937 and PC3 in coculture and evaluated with qPCR, revealing that while CCL2 was not expressed in PC3 cells, it was expressed at very low levels in U937 cells. Interestingly, coculture of PC3 with U937 increased CCL2 mRNA expression by over 10-fold, and the result was confirmed at protein levels by human cytokine array. Our results also indicated that IL-6 and GM-SCF were the two major cytokines released by PCa cells to induce CCL2 mRNA in U937 cells and MEK and JAK-STAT signaling were crucial for CCL2 induction. Conclusions: Prostate cancer cells induce CCL2 secretion from monocytes in an IL-6 and GM-CSF dependent manner. Given the critical role of CCL2 in mediating immunosuppressive tumor microenvironments, our study highlighted the CCL2 Concentrations in PCa vs Healthy Serum.[Table: see text]

Glycated albumin stimulates expression of inflammatory cytokines in muscle cells

The effects of glycated albumin on the expression of inflammatory cytokines in differentiated myotubes were investigated. Glycated albumin stimulates the expression of TNF α, IL-1β, IL-6 and CCL-2 both at the mRNA and protein levels via the receptor of AGEs. Various cytokines demonstrated different kinetics of stimulation by glycated albumin. At a high glucose concentration, the stimulation effect was more pronounced than at a low one. At physiological concentrations of albumin and fructosamine, the stimulation effect of glycated albumin on inflammatory cytokine expression in myotubes was also observed. The induction of expression of all studied cytokines was sensitive to the inhibitors of JNK, p38 MAPK, MEK1/2, Src family protein kinases and NF-κB. At the same time, the induction of TNFα and IL-1β was diminished by the Ca2+/calmodulin-dependent protein kinase inhibitor, whereas the induction of IL-6 and CCL-2 was reduced by the inhibitor of phosphoinositide 3-kinase. Possible implications of observed stimulation of cytokine expression by glycated albumin in the development of diabetes mellitus symptoms are discussed.

Reduction of CD11b+ myeloid suppressive cells augments anti-neuroblastoma immune response induced by the anti-GD2 antibody ch14.18/CHO

ABSTRACT Neuroblastoma (NB) still remains a major challenge in pediatric oncology. We recently showed CD11b+-dependent upregulation of the PD-1/PD-L1 checkpoint on NB cells treated with the chimeric anti-GD2 antibody (Ab) ch14.18/CHO. Here, we report effects of reduction of CD11b+ myeloid suppressive cells on ch14.18/CHO immunotherapy against NB. Flow cytometry, immunohistochemistry and RT-PCR were used to assess tumor infiltrating leukocytes and expression of myeloid suppressive cell-associated genes. XTT assay was used to show impact of 5-FU on tumor and effector cells. Antitumor effects of the combined treatment with ch14.18/CHO and reduction of myeloid suppressive cells were evaluated in a syngeneic NB mouse model. Tumor tissue of untreated mice showed a strong infiltration by CD11b+ cells (53% of all tumor infiltrating leukocytes). RT-PCR analysis of tumors revealed strong expression of the myeloid suppressive cell-associated genes analyzed with the strongest induction of M-CSFr, CCL2, IL-1β, IL-4, IL-6 r, IL-8, Arg1, and NOS2. Compared to controls, application of anti-CD11b Ab resulted in reduction of both CD11b+ cells in tumors and expression of myeloid suppressive cell-associated genes as well as delayed tumor growth and prolonged survival. These effects could be further improved by 5-FU. Importantly, the combinatorial immunotherapy with ch14.18/CHO and 5-FU showed the strongest antitumor effects and superior survival rates. In conclusion, reduction of immune suppressive myeloid cells augments anti-NB efficacy of a ch14.18/CHO-based immunotherapy representing a new effective treatment strategy against GD2-positive cancers.

Benzalkonium chloride-induced direct and indirect toxicity on corneal epithelial and trigeminal neuronal cells: proinflammatory and apoptotic responses in vitro

Benzalkonium chloride (BAK), a quaternary ammonium compound widely used as disinfecting agent as well as preservative in eye drops is known to induce toxic effects on the ocular surface with inflammation and corneal nerve damage leading to dry eye disease (DED) in the medium-to-long term. The aim of this study was to evaluate in vitro the toxicity of a conditioned medium produced by corneal epithelial cells previously exposed to BAK (BAK-CM) on trigeminal neuronal cells. A human corneal epithelial (HCE) cell line was exposed to 5.10−3% BAK (i.e. 0.005% BAK) for 15 min and let recover for 5 h to prepare a BAK-CM. This BAK concentration is the lowest one found in eye drops. After this recovery period, BAK effect on HCE cells displayed cytotoxicity, morphological alteration, apoptosis, oxidative stress, ATP release, CCL2 and IL6 gene induction, as well as an increase in CCL2, IL-6 and MIF release. Next, a mouse trigeminal ganglion primary culture was exposed to the BAK-CM for 2 h, 4 h or 24 h. Whereas BAK-CM did not alter neuronal cell morphology, or induced neuronal cytotoxicity or oxidative stress, BAK-CM induced gene expression of Fos (neuronal activation marker), Atf3 (neuronal injury marker), Ccl2 and Il6 (inflammatory markers). Two and 4 h BAK-CM exposure promoted a neuronal damage (ATF-3, phospho-p38 increases; phospho-Stat3 decreases) while 24 h-BAK-CM exposure initiated a prosurvival pathway activation (phospho-p44/42, phospho-Akt increases; ATF-3, GADD153, active Caspase-3 decreases). In conclusion, this in vitro model, simulating paracrine mechanisms, represents an interesting tool to highlight the indirect toxic effects of BAK or any other xenobiotic on corneal trigeminal neurons and may help to better understand the cellular mechanisms that occur during DED pathophysiology.