Pro-inflammatory Niche Research Articles

P55 Additive regulatory effects of 1,25(OH)2D3 with anti-TNF and anti-IL-17A therapies in an ex vivo Th17-driven psoriatic arthritis synovitis model

Abstract Psoriatic arthritis (PsA) is an inflammatory disorder associated with psoriasis (PsO) and is part of the broader spectrum of psoriatic diseases. Persistent psoriatic synovitis causes joint pain, structural damage, and various systemic manifestations. Th17 cells, central to PsA pathogenesis, secrete pro-inflammatory cytokines (such as IL-17A, IL-22, IFN-γ and TNF-α) and interact with immune and stromal cells, notably synovial fibroblasts. When activated, synovial fibroblasts produce a broad range of pro-inflammatory cytokines (e.g. IL-6 and IL-8), matrix metalloproteinases (MMPs), and RANKL, facilitating immune response persistence, tissue destruction, and bone erosion. Understanding PsA pathogenesis has led to biological treatments targeting key pro-inflammatory cytokines, particularly TNFα and IL-17A. However, many patients experience suboptimal efficacy or treatment discontinuation. Additionally, vitamin D deficiency is prevalent among PsA patients, with low serum 25(OH)D levels (<20 ng/mL) linked to higher anti-TNF treatment discontinuation risk.1 We hypothesized that 1,25(OH)2D3 could provide additional value to cytokine-targeting therapies in PsA. CCR6+ CD25−/low memory Th17 cells were sorted from peripheral blood mononuclear cells of healthy donors and co-cultured with synovial fibroblasts derived from PsA patients for 3 days, with or without adalimumab (anti-TNFα antibody), secukinumab (anti-IL-17A antibody), or 1,25(OH)2D3. Immunoassays evaluated treatment efficacy. CCR6+ CD25−/low memory Th17 cells and PsA fibroblasts formed a pro-inflammatory feedback loop, significantly promoting pro-inflammatory niche development in the ex vivo synovitis model. Our results showed that blocking TNFα and/or IL-17A suppressed fibroblast activation, decreasing IL-6, IL-8, and MMPs. However, neither single nor dual inhibition of TNFα and IL-17A adequately regulated IFN-γ and IL-22 levels in the synovitis model. Individual 1,25(OH)2D3 treatment broadly suppressed pro-inflammatory cytokines and fibroblast activation, particularly IL-17A and IL-22, but not TNFα. When combined with biologics, 1,25(OH)2D3 demonstrated additive regulatory effects, comprehensively modulating major pro-inflammatory cytokines such as TNFα, IL-17A, IFN-γ and IL-22. Additional inhibition of fibroblast activation was observed for IL-6, IL-8 and MMPs. Moreover, 1,25(OH)2D3 boosted anti-inflammatory cytokine IL-10 production, providing a regulatory advantage in PsA treatment. To conclude, 1,25(OH)2D3 provided a comprehensive regulatory effect on Th17-induced synovitis, presenting a promising therapeutic strategy to improve PsA anti-cytokine therapy. Further research on supplementation, timing, dosage and outcome evaluation in both in vivo and clinical studies is needed.

ITGA5+ synovial fibroblasts orchestrate proinflammatory niche formation by remodelling the local immune microenvironment in rheumatoid arthritis

ObjectivesTo investigate the phenotypic heterogeneity of tissue-resident synovial fibroblasts and their role in inflammatory response in rheumatoid arthritis (RA).MethodsWe used single-cell and spatial transcriptomics to profile synovial cells and spatial...

CLUH functions as negative regulator of inflammation in human macrophages and determines ulcerative colitis pathogenesis.

Altered mitochondrial function without a well-defined cause has been documented in the patients with ulcerative colitis (UC). In our efforts to understand UC pathogenesis, we observed reduced expression of clustered mitochondrial homologue, CLUH, only in the active UC tissues compared to the unaffected areas from the same patient and healthy controls. Stimulation with bacterial toll like receptor (TLR) ligands similarly reduced CLUH expression in the human primary macrophages. Further, CLUH negatively regulated secretion of pro-inflammatory cytokines IL6, TNF-α and rendered a pro-inflammatory niche in the TLR stimulated macrophage. CLUH was further found to bind to mitochondrial fission protein DRP-1 and also regulated DRP-1 transcription in the human macrophages. In the TLR ligand stimulated macrophages, absence of CLUH led to enhanced DRP-1 availability for mitochondrial fission and smaller dysfunctional mitochondrial pool was observed. Mechanistically, this fissioned mitochondrial pool in turn enhanced mitochondrial ROS production, reduced mitophagy and lysosomal function in the CLUH knockout macrophages. Remarkably, our studies in the mice model of colitis with CLUH knockdown displayed exacerbated disease pathology. Taken together, this is the first report signifying the role of CLUH in UC pathogenesis, by means of regulating inflammation via maintaining mitochondrial-lysosomal functions in the human macrophages and intestinal mucosa.

Abstract PR02: RSK1 targeting impedes oncogenic driver and inflammatory cytokine signaling to attenuate myeloid neoplasms

Abstract Myeloproliferative neoplasms (MPNs), chronic myelomonocytic leukemia (CMML), and acute myeloid leukemia (AML) exist among a spectrum of myeloid malignancies, governed by critical genetic dependencies, and propagated by a ripe pro-inflammatory niche. We previously identified elevated RPS6KA1 (RSK1) expression in myeloid malignancies associated with poor overall survival. Here, we demonstrate that RSK1 plays a fundamental role in mediating inflammation and serves as a central signaling node driving leukemogenesis. We further evaluate a novel therapeutic regimen utilizing a first-in-class RSK1 inhibitor, PMD-026, currently evaluated in Phase 1/1b clinical trials for breast cancer, for use in myeloid malignancies. Plasma analysis of 144 MPN patients and RNA-seq of CD14+ monocytes from 54 MPN patients revealed enrichment of inflammatory cytokines and hyperactive NFκB signaling. RSK1 inhibition by shRNA or PMD-026 in THP-1 monocytic AML cells potently suppressed this NFκB node, decreased TNF and NFKB1 mRNA, and reduced luminescence in cells expressing a NFκB-luciferase reporter. Immunoblotting and cytokine mass cytometry revealed suppression of RelA/p65 phosphorylation with reduction of TNF, IL-6, IL-8, CCL3, and CCL4 in MPN and CMML CD14+ monocytes. We also observed that RSK1 inhibition attenuated NFκB activation by TNF, LPS, and Pam3CSK4, while also suppressing monocyte differentiation to M1 macrophages. In the MPL W515L myelofibrosis (MF) model, mice treated with PMD-026 had significant reduction in TNF, IL-6, IL-1b, CCL3, and CCL5 by multiplex cytokine profiling and diminished disease burden (reduction of leukocytosis, splenomegaly, and bone marrow fibrosis). Finally, amelioration of disease was observed across MF and sAML patient-derived xenograft (PDX) models with PMD-026 treatment. We extended our findings to FLT3-ITD de novo AML models, in which RSK1 inhibition led to lethality in FLT3-ITD cells and PMD-026 potently suppressed leukemic engraftment in a MV4-11 mouse xenograft. Both genetic and pharmacological inhibition of RSK1 reduced FLT3 phosphorylation and revealed a novel, bi-directional regulatory network. Moreover, we found that RSK1 perturbation also attenuated FLT3 expression and altered protein stability, which was not observed with FLT3 inhibitor quizartinib. Mechanistically, RSK1 modulated FLT3 dynamics through deubiquitinase USP1, which was downregulated upon RSK1 perturbation. USP1 perturbation led to cell death and phenocopied FLT3 suppression observed with RSK1 inhibition. We further identified elevated USP1 expression associated with worse survival outcomes across the TCGA LAML, BeatAML, and Leucegene cohorts, thus indicating the potential of USP1 as a novel biomarker. We uncover a role for targeting RSK1 as a two-pronged approach in reducing disease burden across myeloid malignancies via: 1) suppressing driver signaling, and 2) dampening the pro-inflammatory milieu. These findings highlight a critical dependency along the RSK1-USP1-FLT3 axis that is potentially exploitable with RSK1 inhibitors such as PMD-026. Citation Format: Tim Kong, Angelo Laranjeira, Stephen Oh. RSK1 targeting impedes oncogenic driver and inflammatory cytokine signaling to attenuate myeloid neoplasms [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr PR02.

Abstract 1254: Acetyl-coA distribution devote STAT3 related neuronal methylation phenotype and pro-inflammatory niche in gastric cancer

Abstract STAT3 activation is important in the development of gastric cancer (GCA), but its role in DNA methylation is not fully understood. STAT3-mediated DNA methylation is controlled by K685 acetylation, we therefore hypothesized that distribution of acetyl-CoA could affect the role of STAT3 in DNA methylation. Here, by methylation microarray from GCA patients and cells depleted with STAT3, we identified a STAT3-related CpG island methylator phenotype (CIMP), located at neuronal genes (neuronal CIMP). Further TCGA RNA-Seq identified neuronal CIMP+ve to be positively associated with the expression and higher cyto/mito ratio of acetyl-CoA synthetase (ACSS2). Intriguingly, patients with neuronal-CIMP +ve or higher cyto/mito ratio of ACSS were associated with a better survival, suggesting a different role of acetyl-STAT3. As STAT3 pathway plays a key role in chemokine response, we also analyze whether neuronal-CIMP contributes to anti-tumor immune response. Surprisingly, CIMP +ve patients correlated with pro-inflammatory niche, including activated CD4 + T cells and M1 macrophages. In conclusion, neuronal-CIMP may be a biomarker to predict patient’s immune responses in GCA. Citation Format: Jie-Ting Low, Yu-Ming Chuang, Yu-Ting Lee, Michael Wing-Yan Chan. Acetyl-coA distribution devote STAT3 related neuronal methylation phenotype and pro-inflammatory niche in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1254.

Helicase-like transcription factor-deletion from the tumor microenvironment in a cell line-derived xenograft model of colorectal cancer reprogrammed the human transcriptome-S-nitroso-proteome to promote inflammation and redirect metastasis.

Methylation of the HLTF gene in colorectal cancer (CRC) cells occurs more frequently in men than women. Progressive epigenetic silencing of HLTF in tumor cells is accompanied by negligible expression in the tumor microenvironment (TME). Cell line-derived xenografts (CDX) were established in control (Hltf+/+) and Hltf-deleted male Rag2-/-IL2rg-/- mice by direct orthotopic cell microinjection (OCMI) of HLTF+/+HCT116 Red-FLuc cells into the submucosa of the cecum. Combinatorial induction of IL6 and S100A8/A9 in the Hltf-deleted TME with ICAM-1 and IL8 in the primary tumor activated a positive feedback loop. The proinflammatory niche produced a major shift in CDX metastasis to peritoneal dissemination compared to controls. Inducible nitric oxide (iNOS) gene expression and transactivation of the iNOS-S100A8/A9 signaling complex in Hltf-deleted TME reprogrammed the human S-nitroso-proteome. POTEE, TRIM52 and UN45B were S-nitrosylated on the conserved I/L-X-C-X2-D/E motif indicative of iNOS-S100A8/A9-mediated S-nitrosylation. 2D-DIGE and protein identification by MALDI-TOF/TOF mass spectrometry authenticated S-nitrosylation of 53 individual cysteines in half-site motifs (I/L-X-C or C-X-X-D/E) in CDX tumors. POTEE in CDX tumors is both a general S-nitrosylation target and an iNOS-S100A8/A9 site-specific (Cys638) target in the Hltf-deleted TME. REL is an example of convergence of transcriptomic-S-nitroso-proteomic signaling. The gene is transcriptionally activated in CDX tumors with an Hltf-deleted TME, and REL-SNO (Cys143) was found in primary CDX tumors and all metastatic sites. Primary CDX tumors from Hltf-deleted TME shared 60% of their S-nitroso-proteome with all metastatic sites. Forty percent of SNO-proteins from primary CDX tumors were variably expressed at metastatic sites. Global S-nitrosylation of proteins in pathways related to cytoskeleton and motility was strongly implicated in the metastatic dissemination of CDX tumors. Hltf-deletion from the TME played a major role in the pathogenesis of inflammation and linked protein S-nitrosylation in primary CDX tumors with spatiotemporal continuity in metastatic progression when the tumor cells expressed HLTF.

Decoding Inflammatory Signals from the Extracellular Matrix for the Development of New Immunotherapies

An immune response to pathogen invasion is triggered upon detection of pathogenic molecules by innate immune sensors, whilst non-infectious threat is signalled by endogenous inflammatory stimuli. Although insights into pathogen pattern recognition have revealed a great deal about the molecular basis of host defence against infection, it is not well understood how endogenous molecules activate immunity. Previous work revealed how extracellular matrix molecules specifically induced during tissue damage and cellular stress, or associated with tumorigenesis, create a pro-inflammatory niche that enables resident stromal cells and infiltrating immune cells to survive and thrive, and within which matrix molecules directly activate site-specific inflammatory programmes. Our new data detail how matrix molecules are recognized as inflammatory triggers, the distinct immune signalling pathways they activate, and the importance of identifying pathologically relevant forms of matrix constituents to understanding their contribution to inflammation. Current work focuses on translating these discoveries into novel diagnostic tools, and developing new drugs that target the microenvironment to enable selective amelioration of aberrant ‘sterile’ inflammation in autoimmune and fibrotic diseases, and to re-educate immunity against tumors.

The Interplay between Slow-Cycling, Chemoresistant Cancer Cells and Fibroblasts Creates a Proinflammatory Niche for Tumor Progression.

Quiescent cancer cells are believed to cause cancer progression after chemotherapy through unknown mechanisms. We show here that human non-small cell lung cancer (NSCLC) cell line-derived, quiescent-like, slow-cycling cancer cells (SCC) and residual patient-derived xenograft (PDX) tumors after chemotherapy experience activating transcription factor 6 (ATF6)-mediated upregulation of various cytokines, which acts in a paracrine manner to recruit fibroblasts. Cancer-associated fibroblasts (CAF) underwent transcriptional upregulation of COX2 and type I collagen (Col-I), which subsequently triggered a slow-to-active cycling switch in SCC through prostaglandin E2 (PGE2)- and integrin/Src-mediated signaling pathways, leading to cancer progression. Both antagonism of ATF6 and cotargeting of Src/COX2 effectively suppressed cytokine production and slow-to-active cell cycling transition in SCC, withholding cancer progression. Expression of COX2 and Col-I and activation of Src were observed in patients with NSCLC who progressed while receiving chemotherapy. Public data analysis revealed significant association between COL1A1 and SRC expression and NSCLC relapse. Overall, these findings indicate that a proinflammatory niche created by the interplay between SCC and CAF triggers tumor progression. SIGNIFICANCE: Cotargeting COX2 and Src may be an effective strategy to prevent cancer progression after chemotherapy.

Substrate fiber alignment mediates tendon cell response to inflammatory signaling

Healthy tendon tissue features a highly aligned extracellular matrix that becomes disorganized with disease. Recent evidence suggests that inflammation coexists with early degenerative changes in tendon, and that crosstalk between immune-cells and tendon fibroblasts (TFs) can contribute to poor tissue healing. We hypothesized that a disorganized tissue architecture may predispose tendon cells to degenerative extracellular matrix remodeling pathways, particularly within a pro-inflammatory niche. This hypothesis was tested by analyzing human TFs cultured on electrospun polycaprolactone (PCL) mats with either highly aligned or randomly oriented fiber structures. We confirmed that fibroblast morphology, phenotype, and markers of matrix turnover could be significantly affected by matrix topography. More strikingly, the TF response to paracrine signals from polarized macrophages or by stimulation with pro-inflammatory cytokines featured significant downregulation of signaling related to extracellular synthesis, with significant concomitant upregulation of gene and protein expression of matrix degrading enzymes. Critically, this tendency towards degenerative re-regulation was exacerbated on randomly oriented PCL substrates. These novel findings indicate that highly aligned tendon cell scaffolds not only promote tendon matrix synthesis, but also play a previously unappreciated role in mitigating adverse resident fibroblast response within an inflammatory milieu. Statement of SignificanceUse of biomaterial scaffolds for tendon repair often results in tissue formation characteristic of scar tissue, rather than the highly aligned type-1 collagen matrix of healthy tendons. We hypothesized that non-optimal biomaterial surfaces may play a role in these outcomes, specifically randomly oriented biomaterial surfaces that unintentionally mimic structure of pathological tendon. We observed that disorganized scaffold surfaces do adversely affect early cell attachment and gene expression. We further identified that disorganized fiber surfaces can prime tendon cells toward pro-inflammatory signaling. These findings represent provocative evidence unstructured fiber surfaces may underlie inflammatory responses that drive aberrant collagen matrix turnover. This work could be highly relevant for the design of cell instructive biomaterial therapies that yield positive clinical outcomes.

Interleukin-36 receptor mediates the crosstalk between plasma cells and synovial fibroblasts.

The IL-1 family member IL-36α has proinflammatory and pathogenic properties in psoriasis. IL-36α binds to the IL-36 receptor leading to nuclear factor kappa B/mitogen activated protein kinase mediated cytokine release. The IL-36R antagonist prevents recruitment of IL-1 receptor accessory protein and therefore IL-36-dependent cell activation. In inflamed human tissue, we previously could show that resident Bcells and plasma cells (PC) express IL-36α. Further, fibroblast-like synoviocytes (FLS) produced proinflammatory cytokines upon IL-36α-stimulation. We hypothesize an IL-36-specific crosstalk between Bcells/PCs and FLS permitting a proinflammatory Bcell niche. Here, we firstly demonstrated that Bcell lines and Bcells from healthy donors express IL-36α and stimulation increased IL-36α in Bcells and primary plasmablasts/PCs. Moreover, FLS respond specifically to IL-36α by proliferation and production of matrix metalloproteinases via p38/HSP27 signaling. Importantly, IL-36R-deficiency abrogated IL-36α-induced production of inflammatory mediators in FLS and changed the intrinsic FLS-phenotype. Using an in vitro co-culture system, we could show that IL-36R-deficient FLS had a limited capacity to support PC survival compared to wild-type FLS. Hence, we demonstrated an IL-36R-dependent crosstalk between Bcells/PCs and FLS. Our data support the concept of initiation and maintenance of a proinflammatory niche by Bcells in the joints.

Intratumoral delivery of tumor antigen-loaded DC and tumor-primed CD4+ T cells combined with agonist α-GITR mAb promotes durable CD8+ T-cell-dependent antitumor immunity

ABSTRACT The progressive tumor microenvironment (TME) coordinately supports tumor cell expansion and metastasis, while it antagonizes the survival and (poly-)functionality of antitumor T effector cells. There remains a clear need to develop novel therapeutic strategies that can transform the TME into a pro-inflammatory niche that recruits and sustains protective immune cell populations. While intravenous treatment with tumor-primed CD4+ T cells combined with intraperitoneal delivery of agonist anti-glucocorticoid-induced TNF receptor (α-GITR) mAb results in objective antitumor responses in murine early stage disease models, this approach is ineffective against more advanced tumors. Further subcutaneous co-administration of a vaccine consisting of tumor antigen-loaded dendritic cells (DC) failed to improve the antitumor efficacy of this approach. Remarkably, these same three therapeutic agents elicited significant antitumor benefits when the antitumor CD4+ T cells and tumor antigen-loaded DC were co-injected directly into tumors along with intratumoral or intraperitoneal delivery of α-GITR mAb. This latter protocol induced the production of an array of antitumor cytokines and chemokines within the TME, supporting increased tumor-infiltration by antitumor CD8+ T cells capable of mediating tumor regression and extended overall survival.

Abstract 3668: Tumor-associated macrophages (TAMs) promote murine neuroblastoma tumor growth through upregulation of MYC and independent of IL6 expression

Abstract Background: The concept of tumor-promoting inflammation is a recognized enabling characteristic of cancers. Our group has demonstrated the significance of inflammation-related genes in poor outcome of children diagnosed with metastatic MYCN non-amplified neuroblastoma (NBL-NA). We investigated the effect of TAMs on neuroblastoma growth using a novel murine model. Methods: We have established a novel 100% penetrant NBL-NA murine model driven by SV40's Large T-antigen (NBL-Tag) and characterized expression of immune-related genes in tumors at various developmental stages. Single cell suspensions of murine tumors were analyzed using flow cytometry for determination of immune cells frequency and IL6 levels. NBL-Tag mice were crossed with IL6 knockout mice (NBL-Tag/IL6-/-) to investigate the role of IL6 in tumor pathogenesis. Results: NBL-Tag tumor growth coincided with IL6 levels becoming detectable and increasing in blood. Immune-related gene expression analysis of NBL-Tag adrenal glands in mice at pre-tumor development age (4, 8 and 12 week old) showed age-dependent increase in expression of IL6 and CCL2 (MCP1) compared to wild-type adrenal glands demonstrating early monocyte chemotaxis and establishment of a pro-inflammatory niche. FACS of tumors showed high expression of IL6, and infiltration by IL6-producing TAMs. In vitro co-culture of peritoneal macrophages from wild type mice increased proliferation of a NBL-Tag mice driven cell line (NBT2) by average of 50% over its basal rate, as measured by Brdu incorporation. This effect coincided with two-fold increase in IL6 protein level in the co-cultured media. However, the proliferative changes were only partially reversed by blocking IL6, suggesting other soluble factors also contribute to the observed effects. IL6 genetic ablation studies were conducted to generate NBL-Tag/IL6-/- mice, which convincingly demonstrated IL6 was not required for tumor growth and development. Macrophages from IL6-/- mice increased in vitro proliferation of tumor to similar levels as those from wild-type mice, while IHC staining of NBL-Tag/IL6-/- tumors revealed similar level of TAM infiltration and positivity for phospo-STAT3, indicating an IL6-independent pathway for STAT3 activation. Lastly, gene expression analyses revealed upregulation of MYC in tumor cells during co-culture and was confirmed by western analysis. Conclusions: This study demonstrates the recruitment and growth-promoting effects of TAMs in early pathogenesis of neuroblastoma in a novel MYCN non-amplified murine model. The macrophage and neuroblastoma cell cross-talk lead to tumor cell proliferation with phosphorylation of STAT3 and MYC upregulation in tumor cells. These effects were found to be independent of IL6 activity. Citation Format: Michael D. Hadjidaniel, Soheila Shirinbak, Sakunthala Muthugounder, Long Hung, Michael Sheard, Janahan Gnanachandran, Jin Kim, Richard Sposto, Hiroyuki Shimada, Shahab Asgharzadeh. Tumor-associated macrophages (TAMs) promote murine neuroblastoma tumor growth through upregulation of MYC and independent of IL6 expression. [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 3668. doi:10.1158/1538-7445.AM2014-3668

Congenitally acquired persistent lymphocytic choriomeningitis viral infection reduces neuronal progenitor pools in the adult hippocampus and subventricular zone.

Lymphocytic choriomeningitis virus (LCMV) can be transmitted through congenital infection, leading to persistent infection of numerous organ systems including the central nervous system (CNS). Adult mice persistently infected with LCMV (LCMV-cgPi mice) exhibit learning deficits, such as poor performance in spatial discrimination tests. Given that deficits in spatial learning have been linked to defects in adult neurogenesis, we investigated the impact of congenital LCMV infection on generation of neuroblasts from neural progenitor cells within neurogenic zones of adult mice. In LCMV-cgPi mice, QPCR and immunohistochemistry detected presence of LCMV glycoprotein-coding RNA and nucleoprotein in the hippocampal dentate gyrus and subventricular zone (SVZ), sites of neurogenesis that harbor populations of neuroblasts. Numbers of neuroblasts were reduced in LCMV-cgPi mice, as determined by IHC quantification, and analysis of BrdU incorporation by flow cytometry revealed lower numbers of BrdU-labeled neuroblasts. Additionally, TUNEL assays performed in situ showed increased numbers of apoptotic cells in the two neurogenic regions. Next, neurosphere cultures were infected in vitro with LCMV and differentiated to create a population of cells that consisted of both transit amplifying cells and neuroblasts. Immunocytochemical and TUNEL assays revealed increased numbers of TUNEL-positive cells that express nestin, suggesting that the drop in numbers of neuroblasts was due to a combination of impaired proliferation and apoptosis of progenitor cells. LCMV-cgPi mice exhibited transcriptional up-regulation several cytokines and chemokines, including gamma-interferon inducible chemokines CXCL9 and CXCL10. Chronic up-regulation of these chemokines can facilitate a pro-inflammatory niche that may contribute to defects in neurogenesis.

Major Vault Protein Regulates Class A Scavenger Receptor-mediated Tumor Necrosis Factor-α Synthesis and Apoptosis in Macrophages

Atherosclerosis is considered a disease of chronic inflammation largely initiated and perpetuated by macrophage-dependent synthesis and release of pro-inflammatory mediators. Class A scavenger receptor (SR-A) expressed on macrophages plays a key role in this process. However, how SR-A-mediated pro-inflammatory response is modulated in macrophages remains ill defined. Here through immunoprecipitation coupled with mass spectrometry, we reported major vault protein (MVP) as a novel binding partner for SR-A. The interaction between SR-A and MVP was confirmed by immunofluorescence staining and chemical cross-linking assay. Treatment of macrophages with fucoidan, a SR-A ligand, led to a marked increase in TNF-α production, which was attenuated by MVP depletion. Further analysis revealed that SR-A stimulated TNF-α synthesis in macrophages via the caveolin- instead of clathrin-mediated endocytic pathway linked to p38 and JNK, but not ERK, signaling pathways. Importantly, fucoidan invoked an enrichment of MVP in lipid raft, a caveolin-reliant membrane structure, and enhanced the interaction among SR-A, caveolin, and MVP. Finally, we demonstrated that MVP elimination ameliorated SR-A-mediated apoptosis in macrophages. As such, MVP may fine-tune SR-A activity in macrophages which contributes to the development of atherosclerosis.

English

Introduction The extracellular matrix is a complex, three-dimensional network of secreted molecules that provides structural support to tissues and environmental cues to the cells within. One particular subset of matrix molecules is specifically expressed upon tissue damage. These molecules contribute to effective tissue repair by orchestrating the behaviour of cells that mediate this process, and once the repair is complete, the expression of this matrix is down-regulated. Here, we focus on the recent data that highlights a direct role for injury-induced matrix molecules in driving inflammation upon tissue damage and propose that this matrix creates a specific microenvironment or ‘pro-inflammatory niche’ that is permissive for localized inflammation during repair. We also examine the evidence indicating that this niche exists, and persists, in the damaged joint of rheumatoid arthritis patients. Finally, we assess the data which demonstrate that these matrix molecules actively contribute to maintaining chronic inflammation during disease progression. Conclusion Together these findings imply that targeting the pro-inflammatory niche in the joint may provide a novel treatment strategy for rheumatoid arthritis. Introduction The extracellular matrix (ECM) is a complex organization of secreted molecules. The major constitutive components of the ECM include collagens, elastin, fibronectin, hyaluronic acid and proteoglycans. Together these molecules form a network that provides structural support for tissues and delivers environmental signals that regulate cell behaviour. However, there exists a unique subset of ECM molecules that are not constitutively expressed. Found at high levels during development, but absent from most healthy adult tissues, these proteins are specifically induced at sites of tissue injury. These molecules have been termed ‘matricellular proteins’ and include the CCN family (CCN1-6), galectins (Gal), fibulins, osteopontin (OPN), periostin, secreted protein acidic and rich in cysteine (SPARC), small leucine rich proteoglycans (SLPRs), tenascin-C (TNC) and thrombospondins-1 and -2 (TSP-1/2). Despite their structural diversity, these molecules possess a number of defining common features in addition to their distinct pattern of expression. They are key orchestrators of cell behaviour; this is mediated by their multimodular structure, which confers the ability to interact with a large number of diverse binding partners such as cell surface receptors, other ECM molecules, hormones, growth factors and cytokines. Moreover, they exert control over a wide range of cell functions in a highly contextand cell type-specific manner. Finally, although highly expressed during development, mice with targeted disruptions in these genes exhibit grossly normal phenotypes until subjected to tissue injury, whereupon they exhibit abnormal tissue repair. There is a large body of evidence supporting a major role for these proteins in driving the response to tissue injury1,2. The aim of this review is to discuss how the ECM drives persistent inflammation upon tissue damage to the joint in rheumatoid arthritis (RA). Discussion The authors have referenced some of their own studies in this review. These referenced studies have been conducted in accordance with the Declaration of Helsinki (1964), and the protocols of these studies have been approved by the relevant ethics committees related to the institution in which they were performed. All human subjects, in these referenced studies, gave informed consent to participate in these

Abstract 383: Interleukin-6 producing tumor associated macrophages (TAMs) increase MYC expression and promote tumor growth in a novel murine neuroblastoma model

Abstract The concept of tumor-promoting inflammation is a recognized enabling characteristic of cancers. Recent studies have demonstrated the prognostic significance of tumor-associated macrophages (TAM) in several cancers. Our group has also demonstrated the significance of TAMs and interleukin-6 receptor in predicting poor outcome in children diagnosed with metastatic MYCN non-amplified neuroblastoma (NBL-NA). Previous in vitro work has shown that co-culture of a human neuroblastoma MYCN non-amplified cell line with monocytes enhanced proliferation, an effect mediated in part by interleukin-6 (IL6). To address the mechanism by which inflammation contributes to tumorigenesis in neuroblastoma, we have established a novel, 100% penetrant, murine model of metastatic NBL-NA. The tumors of these transgenic mice (NBL-Tag) are detected by MRI only after 12 weeks of age, which coincide with IL6 levels becoming detectable and increasing in blood. DNA profiling of tumors reveal no amplification of the MYCN locus, while RNA profiling and FACS analyses of tumors show high expression of IL6, and infiltration by IL6-producing TAMs. Thus, a pro-inflammatory microenvironment mimicking that observed in human metastatic NBL-NA characterizes the growth of NBL-Tag murine tumors. Immune-related gene expression analysis of NBL-Tag adrenal glands in mice at pre-tumor development age (4, 8 and 12 weeks old) show age-dependent increase in expression of CCL2 (aka monocyte chemotactic protein 1) and fibronectin (FN1) compared to wildtype adrenal glands demonstrating early monocyte chemotaxis and establishment of a pro-inflammatory niche. In vitro co-culture of spleen or peritoneal macrophages from wildtype mice increases proliferation of a NBL-Tag cell line (NBT2) by 30 - 50% over its basal rate, as measured by Brdu incorporation. This effect coincides with two-fold increase in IL6 protein level in the co-cultured media. However, these proliferative changes do not require cell-cell contact and are only partially reversed by blocking IL6, suggesting other soluble factors also contribute to the observed effects. Co-cultured NBT2 cells also increase their expression of MYC by two-fold, providing insight into the tumor proliferative mechanism facilitated by TAMs. In summary, this study demonstrates the recruitment and growth-promoting effects of IL6 producing TAMs in early pathogenesis of neuroblastoma in a novel MYCN non-amplified murine model. The NBL-Tag mice provide a robust transgenic model to study neuroblastoma pathogenesis and provide a platform to assess therapies targeting tumor cells and tumor-associated inflammatory cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 383. doi:1538-7445.AM2012-383