Chemokine Cluster Research Articles

ScRNA-seq expression of IFI27 and APOC2 identifies four alveolar macrophage superclusters in healthy BALF.

Alveolar macrophages (AMs) reside on the luminal surface of the airways and alveoli, ensuring proper gas exchange by ingesting cellular debris and pathogens, and regulating inflammatory responses. Therefore, understanding the heterogeneity and diverse roles played by AMs, interstitial macrophages, and recruited monocytes is critical for treating airway diseases. We performed single-cell RNA sequencing on 113,213 bronchoalveolar lavage cells from four healthy and three uninflamed cystic fibrosis subjects and identified two MARCKS+LGMN+IMs, FOLR2+SELENOP+ and SPP1+PLA2G7+ IMs, monocyte subtypes, DC1, DC2, migDCs, plasmacytoid DCs, lymphocytes, epithelial cells, and four AM superclusters (families) based on the gene expression of IFI27 and APOC2 These four AM families have at least eight distinct functional members (subclusters) named after their differentially expressed gene(s): IGF1, CCL18, CXCL5, cholesterol, chemokine, metallothionein, interferon, and small-cluster AMs. Interestingly, the chemokine cluster further divides with each subcluster selectively expressing a unique combination of chemokines. One of the most striking observations, besides the heterogeneity, is the conservation of AM family members in relatively equal ratio across all AM superclusters and individuals. Transcriptional data and TotalSeq technology were used to investigate cell surface markers that distinguish resident AMs from recruited monocytes. Last, other AM datasets were projected onto our dataset. Similar AM superclusters and functional subclusters were observed, along with a significant increase in chemokine and IFN AM subclusters in individuals infected with COVID-19. Overall, functional specializations of the AM subclusters suggest that there are highly regulated AM niches with defined programming states, highlighting a clear division of labor.

Spatially Restricted T-cell Activation in Inflamed Tissues

Abstract Pathogen control requires T-cells to locate and make direct contact with antigen-presenting cells (APCs) for peripheral reactivation and delivery of effector molecules. The signals that orchestrate T-cell-APC interactions are poorly understood in inflamed peripheral tissues. Using intravital imaging (IVI), we have found that Th1 cell entry into the inflamed dermis is nucleated by perivascular CXCL10+ cellular clusters enriched for APCs. Th1 cells form more stable interactions with APCs within these clusters compared to outer regions. CXCL10+ clusters are amplified in a IFNγ-dependent manner, where CD4+ T-cell IFNγ release increases APC recruitment and CXCL10 expression. We hypothesize that these chemokine clusters serve as CXCL10 Peripheral Activation (PAC-10) niches, where Th1 cells achieve early peripheral activation away from the pathogen modulated milieu. We seek to determine the relationship between these spatially restricted PAC-10 niches, infection foci, and pathogen clearance. Using a combination of IVI, flow cytometry, and transcriptomics we characterized niche specific Th1 cells and niche cellular composition to determine if PAC-10 niches modulate Th1 cells for pathogen control. Initial studies reveal that niche specific Th1 cells have increased CD69 and IFNγ expression compared to non-niche counterparts. PAC-10 niches appear to have a spatially distinct innate cell population, likely with enhanced ability to support Th1 activation. Future studies will characterize the functional status of niche specific Th1 cells and evaluate the necessity of PAC-10 niches for pathogen control. These studies will inform therapeutic strategies to boost T-cell activation in infection or hinder activation in autoimmunity. Supported by NIH NIAID P01 AI02851 NIH NIAID R01 AI070826

Proteopathic tau primes and activates interleukin-1β via myeloid-cell-specific MyD88- and NLRP3-ASC-inflammasome pathway.

SUMMARYPathological hyperphosphorylation and aggregation of tau (pTau) and neuroinflammation, driven by interleukin-1β (IL-1β), are the major hallmarks of tauopathies. Here, we show that pTau primes and activates IL-1β. First, RNA-sequence analysis suggests paired-helical filaments (PHFs) from human tauopathy brain primes nuclear factor κB (NF-κB), chemokine, and IL-1β signaling clusters in human primary microglia. Treating microglia with pTau-containing neuronal media, exosomes, or PHFs causes IL-1β activation, which is NLRP3, ASC, and caspase-1 dependent. Suppression of pTau or ASC reduces tau pathology and inflammasome activation in rTg4510 and hTau mice, respectively. Although the deletion of MyD88 prevents both IL-1β expression and activation in the hTau mouse model of tauopathy, ASC deficiency in myeloid cells reduces pTau-induced IL-1β activation and improves cognitive function in hTau mice. Finally, pTau burden co-exists with elevated IL-1β and ASC in autopsy brains of human tauopathies. Together, our results suggest pTau activates IL-1β via MyD88- and NLRP3-ASC-dependent pathways in myeloid cells, including microglia.

CXCL10+ peripheral activation niches couple preferred sites of Th1 entry with optimal APC encounter.

SUMMARYCorrect positioning of T cells within infected tissues is critical for T cell activation and pathogen control. Upon tissue entry, effector T cells must efficiently locate antigen-presenting cells (APC) for peripheral activation. We reveal that tissue entry and initial peripheral activation of Th1 effector T cells are tightly linked to perivascular positioning of chemokine-expressing APCs. Dermal inflammation induces tissue-wide de novo generation of discrete perivascular CXCL10+ cell clusters, enriched for CD11c+MHC-II+ monocyte-derived dendritic cells. These chemokine clusters are “hotspots” for both Th1 extravasation and activation in the inflamed skin. CXCR3-dependent Th1 localization to the cluster micro-environment prolongs T-APC interactions and boosts function. Both the frequency and range of these clusters are enhanced via a T helper 1 (Th1)-intrinsic, interferon-gamma (IFNγ)-dependent positive-feedback loop. Thus, the perivascular CXCL10+ clusters act as initial peripheral activation niches, optimizing controlled activation broadly throughout the tissue by coupling Th1 tissue entry with enhanced opportunities for Th1-APC encounter.

CXCL10+ perivascular clusters nucleate Th1 cell tissue entry and activation in the inflamed skin

Abstract Efficient recruitment and correct positioning of T cells within infected peripheral tissues are crucial for pathogen clearance. Using a dual-chemokine reporter (Groom et Al. Immunity, 2012) mouse and intra-vital imaging, we identified a preferred tissue entry and accumulation site for effector Th1 cells, defined by clusters of CXCL9 and CXCL10 expressing hematopoietic cells, enriched with MHC-class-II+ cells, in restricted perivascular spaces within the inflamed dermis. Unbiased computational analysis of motility dynamics of antigen-specific Th1 cells within these clusters, revealed cell confinement characteristics. CXCR3 on Th1 cells and presence of cognate antigen were critical for persistence within clusters and T cell activation. Newly recruited Th1 cells strongly amplified CXCL9 and CXCL10 expression, mainly within skin monocyte-derived dendritic cells, in an antigen- and interferon-gamma-dependent manner, resulting in augmented chemokine clusters. Our data suggest a CXCR3-mediated mechanism of intrinsic amplification loop for optimal Th1 cell recruitment, positioning and activation in restricted antigen presentation sites within the inflamed skin.

Loss-of-Function Mutations in Dnmt3a and Tet2 Lead to Accelerated Atherosclerosis and Convergent Macrophage Phenotypes in Mice

Clonal hematopoiesis of indeterminate potential (CHIP) was recently identified as a major risk factor for development of both hematologic malignancies and atherosclerotic cardiovascular disease in humans. The most commonly mutated gene in CHIP, DNMT3A, is a de novo DNA methyltransferase. The second most commonly mutated gene is TET2, an enzyme which can lead to loss of DNA methylation, and thus is thought to have an opposing biochemical function to DNMT3A. Surprisingly, mutations in both genes lead to convergent phenotypes, such as clonal expansion of mutated stem cells, increased risk of malignant transformation, and increased risk of coronary heart disease. A molecular mechanism linking CHIP and cardiovascular disease has been explored only for loss of function mutations in the Tet2 gene (Jaiswal et al., NEJM 2017; Fuster et al., Science 2017). Here we tested the ability of null mutations in Dnmt3a to contribute to atherosclerosis in hypercholesteremic mice. We further explored the biological basis for this association through gene expression analyses and single-cell RNA sequencing.To model cardiovascular disease associated with DNMT3A-mutated CHIP, atherosclerosis-prone Ldlr-/- mice received bone marrow from Dnmt3a+/+ mice (WT), or from Dnmt3a-/- mice (KO) and WT mice in a 1:9 ratio to mimic a typical variant allele fraction observed in human CHIP. Mice then consumed a high-fat, high-cholesterol diet (HFD), and underwent assessment of atherosclerosis. At 9 weeks, mice that had received 10% Dnmt3a-/- bone marrow displayed an average lesion size that was 40% larger compared to mice receiving control marrow only (p=0.04). The increase in lesion size resembles that we previously observed in mice receiving Tet2-/- marrow (Jaiswal et al., NEJM 2017).De novo DNA methylation by Dnmt3a can alter gene expression. To elucidate how such changes may accelerate atherosclerosis, we first performed transcriptome analysis using bulk RNA sequencing of cholesterol-stimulated bone marrow derived macrophages (BMDM) from either WT or KO mice. BMDMs lacking Dnmt3a showed significantly augmented expression of genes belonging to the CXC chemokine cluster, Cxcl1, Cxcl2 and Cxcl3, as well as increases in mRNAs encoding canonical pro-inflammatory cytokines Il1b and Il6. These changes mirrored those we saw in macrophages lacking Tet2 (Jaiswal et al., NEJM 2017).We next asked how transcriptomic changes observed using the ex vivo BMDM system translated into the in vivo lesional environment. Single-cell RNA sequencing (10X Genomics) was performed on atherosclerotic aortae from mice that had been competitively transplanted with WT, Dnmt3a-/-, or Tet2-/- marrow at a 1:9 ratio. Clustering demonstrated broad changes in lesional immune cell composition in mice harboring CHIP. Lack of either Tet2 or Dnmt3a substantially expanded the myeloid compartment, containing cells that drive atherogenesis. A reciprocal reduction mainly affecting T lymphocyte populations accompanied this expansion. Within the myeloid cell compartment, Dnmt3a-/- or Tet2-/- donor cells, but not WT donor cells, gave rise to a distinct lesional macrophage population. These cells expressed markers associated with tissue-resident macrophages (Mrc1, Lyve1, F13a1), but also highly expressed several inflammatory mediators (Cxcl1, Pf4, Ccl2, Ccl7, Ccl8), and a characteristic set of transcription factors (Jun, Fos, Egr1).Overall, the present study reveals broad changes to the lesional cellular composition and transcriptome induced by the most common CHIP mutations, and provides novel insight into the mechanisms by which CHIP accelerates atherosclerosis. Despite exerting opposite catalytic functions, lack of Dnmt3a or of Tet2 function lead to a myriad of similar downstream transcriptomic and cellular changes. These results indicate that mutations in Dnmt3a and Tet2 accelerate atherosclerosis through convergent mechanisms. DisclosuresNo relevant conflicts of interest to declare.

Abstract A15: Immune-related changes in breast cancer tumor evolution

Abstract Immunotherapy is a highly promising therapeutic option in metastatic disease albeit only in a subset of patients possibly due to heterogeneity in the mechanisms by which tumors escape immune surveillance. Immune cells shape tumor evolution directly (e.g., anti-tumor immune response) and indirectly (e.g., changing the microenvironment) by selecting for cancer cells with certain properties. We hypothesized that the in situ (DCIS) to invasive ductal carcinoma (IDC) transition is a critical tumor progression step for immune escape in breast cancer that defines subsequent tumor evolution. In DCIS, cancer cells are physically separated from the stroma by the basement membrane and myoepithelial cell layer, and tumor-infiltrating leukocytes are rarely detected in direct contact with cancer cells. In contrast, in IDC, cancer cells and leukocytes are intermingled, thus, only cancer cells that can survive in this environment will play a role in disease progression. To dissect mechanisms of immune escape in breast cancer, we first analyzed the composition of leukocytes in normal breast tissues, DCIS, and IDC by polychromatic FACS. We found that DCIS and IDC contained significantly higher numbers of leukocytes, compared to normal breast, whereas in normal tissues more leukocytes were in the stromal than in the epithelial fraction. We also observed significant differences in the relative frequencies of several CD45+ cell types including increased neutrophils and decreased CD8+/CD4+ T cell ratios in tumors compared to normal stroma. Next, we analyzed the gene expression profiles of CD45+CD3+ T cells and found gene set enrichment of cytotoxic cells in DCIS including CD8+ T cells and NKT cells when compared to IDC. Conversely, we found enrichment for gene sets corresponding to regulatory T cells in IDC when compared to DCIS. Overall this suggested that DCIS had a more activated immune environment and IDC a more suppressed immune environment. We further explored this result by immunofluorescence (IF) and found fewer activated GZMB+CD8+ T cells in IDC than in DCIS, including a set of matched DCIS and locally recurrent IDC tissues. We also found that the TCR clonotype was more diverse in DCIS than in normal breast and IDCs. Interestingly we detected a few relatively frequent clones that were shared among different DCIS, one of which was previously shown to recognize a protein from the Epstein-Bar virus. To elucidate mechanisms of immune evasion in IDC, we performed IF analysis of immune checkpoint proteins PD-L1 and TIGIT and found significant differences between DCIS and IDC. TIGIT-expressing T cells were more slightly frequent in DCIS than in IDC. PD-L1 expression was higher in the epithelial cancer cells in triple negative IDC compared to DCIS, and amplification of CD274 (encoding PD-L1) was only detected in triple negative IDCs. Given the close proximity of ERBB2 (encoding HER2) to a cluster of genes encoding several chemokines, we analyzed the HER2+ samples from the TCGA. We found that co-amplification of 17q12 chemokine cluster (CC) with ERBB2 was enriched in HER+ER+ luminal-like tumors but not in the HER2+ER breast tumors. We also found higher expression of both T cell activation and inhibition-related genes in tumors that lack CC gain. Also by assessing tumor samples by multicolor FISH and IF, we determined that there is an inverse correlation between CC amplification and activation of CD8+ T cells. Overall our results show co-evolution of cancer cells and the immune microenvironment during tumor progression. Citation Format: Carlos R. Gil del Alcazar, SungJin Huh, Muhammad B. Ekram, Anne Trinh, Lin L. Liu, Francisco Beca, Zi Xiaoyuan, Misuk Kwak, Helga Bergholtz, Ying Su, Lina Ding, Lina Ding, Hege G. Russnes, Andrea L. Richardson, Kirsten Babski, Elizabeth Min Hui Kim, Charles H. McDonnell, III, Jon Wagner, Ron Rowberry, Gordon Freeman, Deborah Dillon, Therese Sorlie, Lisa M. Coussens, Judy E. Garber, Rong Fan, Kristie Bobolis, Joon Jeong, So Yeon Park, Franziska Michor, Kornelia Polyak. Immune-related changes in breast cancer tumor evolution [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A15.

Abstract A21: Characterization of the immune environment in the in situ to invasive breast carcinoma transition

Abstract Reactivation of immune responses against cancer cells—immunotherapy—is one of the few cancer therapies that can successfully eliminate even metastatic disease in a relatively nontoxic manner. However, its success has been limited to a subset of patients. For example, in breast cancer only ~20% of triple-negative breast cancer (TNBC) patients benefit from anti-PDL1 therapy. One reason for this limited success can be that different tumors evade the immune system via different mechanisms, which suggests that they may respond to different types of immunotherapies. Epithelial cancer cells in ductal carcinoma in situ (DCIS) are physically separated from the tumor-infiltrating leukocytes by the myoepithelial cell layer and the basement membrane, whereas in invasive ductal carcinoma (IDC), the epithelial cancer cells are intermingled with leukocytes. Therefore, we hypothesize that the DCIS to IDC transition is a key step in tumor progression as cancer cells are under different selection pressures, and only those that can evade the immune system can continue tumor progression, hence shaping subsequent tumor evolution. To dissect the role of leukocytes in the DCIS to IDC transition, we began by analyzing the composition and molecular profiles of leukocytes, with special emphasis on T cells, in normal breast tissues, DCIS, and IDC. We found that the relative frequency of leukocytes increases during tumor progression but the CD8/CD4 T cell ratio decreases. In addition, the gene expression profile of CD45+CD3+ T cells is different in DCIS compared to those isolated from normal breast tissue and IDCs. We found that gene set signatures corresponding to CD8+ T cells and NKT cells were enriched over regulatory T-cell signatures in DCIS compared to IDC. This result suggested that DCIS had a more activated immune environment compared to IDC. We further examined T-cell activation by immunofluorescence (IF) analysis and found a higher percentage of activated GZMB+CD8+ T cells in DCIS compared to IDC including a set of matched DCIS and locally recurrent IDC. We also found that the TCR clonotype was more diverse in DCIS than in IDCs. Interestingly, we detected a few relatively frequent clones that were shared among different DCIS patients, one of which was previously shown to recognize a protein from the Epstein-Bar virus. In order to dissect mechanisms of immune evasion in IDC, we analyzed immune checkpoint genes and proteins by FISH and IF. We found that TIGIT+ T cells were slightly more frequent in DCIS than in IDC. In triple-negative IDC, there was high expression of PD-L1 in epithelial cells and in 3/10 cases amplification of CD274 (encoding PD-L1), whereas DCIS had lower expression of PD-L1 and no amplification of CD274. To further elucidate mechanisms of immune evasion, we explored the significance of a cluster of genes encoding several chemokines that are located in close proximity of ERBB2 (encoding HER2). When analyzing the HER2+ samples from the TCGA, we found that coamplification of the 17q12 chemokine cluster (CC) with ERBB2 was enriched in HER2+ER+ luminal-like tumors, whereas there was either no gain or loss of the cluster in the HER2+ER breast tumors. Interestingly, we found higher expression of both T-cell activation and exhaustion-related genes in tumors that lack CC gain. Moreover, when assessing a cohort of HER2+ samples by multicolor FISH and IF, we found an inverse correlation between CC amplification and activation of CD8+ T cells. There was no correlation between CC amplification and recruitment of macrophages or myeloid-derived suppressor cells. Overall our results show coevolution of cancer cells and the immune microenvironment during tumor progression. Citation Format: Carlos R. Gil del Alcazar, SungJin Huh, Muhammad B. Ekram, Anne Trinh, Lin L. Liu, Francisco Beca, Zi Xiaoyuan, Misuk Kwak, Helga Bergholtz, Ying Su, Lina Ding, Hege G. Russnes, Andrea L. Richardson, Kirsten Babski, Elizabeth Min Hui Kim, Charles H. McDonnell, III, Jon Wagner, Ron Rowberry, Gordon J. Freeman, Deborah Dillon, Therese Sorlie, Lisa M. Coussens, Judy E. Garber, Rong Fan, Kristie Bobolis, D. Craig Allred, Joon Jeong, So Yeon Park, Franziska Michor, Kornelia Polyak. Characterization of the immune environment in the in situ to invasive breast carcinoma transition [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A21.

Identification of outcome-correlated serum cytokine and chemokine clusters in ovarian clear cell carcinoma.

5544Background: With the progress of immune-oncology, the immunological background of epithelial ovarian carcinoma (EOC) is gradually becoming understood. However, most of the studies have been foc...

Immune Escape in Breast Cancer During In Situ to Invasive Carcinoma Transition.

To investigate immune escape during breast tumor progression, we analyzed the composition of leukocytes in normal breast tissues, ductal carcinoma in situ (DCIS), and invasive ductal carcinomas (IDC). We found significant tissue and tumor subtype-specific differences in multiple cell types including T cells and neutrophils. Gene expression profiling of CD45+CD3+ T cells demonstrated a decrease in CD8+ signatures in IDCs. Immunofluorescence analysis showed fewer activated GZMB+CD8+ T cells in IDC than in DCIS, including in matched DCIS and recurrent IDC. T-cell receptor clonotype diversity was significantly higher in DCIS than in IDCs. Immune checkpoint protein TIGIT-expressing T cells were more frequent in DCIS, whereas high PD-L1 expression and amplification of CD274 (encoding PD-L1) was only detected in triple-negative IDCs. Coamplification of a 17q12 chemokine cluster with ERBB2 subdivided HER2+ breast tumors into immunologically and clinically distinct subtypes. Our results show coevolution of cancer cells and the immune microenvironment during tumor progression.Significance: The design of effective cancer immunotherapies requires the understanding of mechanisms underlying immune escape during tumor progression. Here we demonstrate a switch to a less active tumor immune environment during the in situ to invasive breast carcinoma transition, and identify immune regulators and genomic alterations that shape tumor evolution. Cancer Discov; 7(10); 1098-115. ©2017 AACR.See related commentary by Speiser and Verdeil, p. 1062This article is highlighted in the In This Issue feature, p. 1047.

Induction of the MCP chemokine cluster cascade in the periphery by cancer cell-derived Ccl3

The induction of localized pro-inflammatory niches in the periphery is instrumental in metastasis. In order to better understand how tumors engage distal sites and activate a pro-inflammatory response we utilized syngeneic breast cancers as a model and showed that soluble factors from the neoplastic epithelium activate the expression of the monocyte chemoattractive protein (MCP) chemokines of the mouse 11C cluster that include Ccl1, Ccl2, Ccl7, Ccl8, Ccl11 and Ccl12. Tissues such as the lungs and the brain, that are more prone to colonization by breast cancer cells, were more sensitive to MCP cluster chemokine induction than others such as the liver. Subsequent analyses involving chemokine arrays in breast cancer cells and media followed by functional validation assays in in vitro and in vivo identified the cytokine Ccl3 as the principle mediator of the communication between the neoplastic epithelium and the peripheral tissues in terms of MCP cluster chemokine induction. Our results show that MCP chemokines are activated in peripheral tissues of breast cancer-bearing mice, by a mechanism that involves breast cancer cell-derived Ccl3. Interference with the expression of cancer cell-derived Ccl3 may find application in the management of breast cancer metastases.

The Activation of IL-1-Induced Enhancers Depends on TAK1 Kinase Activity and NF-κB p65

The inflammatory gene response requires activation of the protein kinase TAK1, but it is currently unknown how TAK1-derived signals coordinate transcriptional programs in the genome. We determined the genome-wide binding of the TAK1-controlled NF-κB subunit p65 in relation to active enhancers and promoters of transcribed genes by chromatin immunoprecipitation sequencing (ChIP-seq) experiments. Out of 35,000 active enhancer regions, 410 H3K4me1-positive enhancers show interleukin 1 (IL-1)-induced H3K27ac and p65 binding. Inhibition of TAK1 or IKK2 or depletion of p65 blocked inducible enhancer activation and gene expression. As exemplified by the CXC chemokine cluster located on chromosome 4, the TAK1-p65 pathway also regulates the recruitment kinetics of the histone acetyltransferase CBP, of NF-κB p50, and of AP-1 transcription factors to both promoters and enhancers. This study provides a high-resolution view of epigenetic changes occurring during the IL-1 response and allows the genome-wide identification of a distinct class of inducible p65 NF-κB-dependent enhancers in epithelial cells.

The inflammatory cytokine TNFα cooperates with Ras in elevating metastasis and turns WT-Ras to a tumor-promoting entity in MCF-7 cells

BackgroundIn the present study we determined the relative contribution of two processes to breast cancer progression: (1) Intrinsic events, such as activation of the Ras pathway and down-regulation of p53; (2) The inflammatory cytokines TNFα and IL-1β, shown in our published studies to be highly expressed in tumors of >80% of breast cancer patients with recurrent disease.MethodsUsing MCF-7 human breast tumor cells originally expressing WT-Ras and WT-p53, we determined the impact of the above-mentioned elements and cooperativity between them on the expression of CXCL8 (ELISA, qRT-PCR), a member of a “cancer-related chemokine cluster” that we have previously identified. Then, we determined the mechanisms involved (Ras-binding-domain assays, Western blot, luciferase), and tested the impact of Ras + TNFα on angiogenicity (chorioallantoic membrane assays) and on tumor growth at the mammary fat pad of mice and on metastasis, in vivo.ResultsUsing RasG12V that recapitulates multiple stimulations induced by receptor tyrosine kinases, we found that RasG12V alone induced CXCL8 expression at the mRNA and protein levels, whereas down-regulation of p53 did not. TNFα and IL-1β potently induced CXCL8 expression and synergized with RasG12V, together leading to amplified CXCL8 expression. Testing the impact of WT-Ras, which is the common form in breast cancer patients, we found that WT-Ras was not active in promoting CXCL8; however, TNFα has induced the activation of WT-Ras: joining these two elements has led to cooperative induction of CXCL8 expression, via the activation of MEK, NF-κB and AP-1. Importantly, TNFα has led to increased expression of WT-Ras in an active GTP-bound form, with properties similar to those of RasG12V. Jointly, TNFα + Ras activities have given rise to increased angiogenesis and to elevated tumor cell dissemination to lymph nodes.ConclusionsTNFα cooperates with Ras in promoting the metastatic phenotype of MCF-7 breast tumor cells, and turns WT-Ras into a tumor-supporting entity. Thus, in breast cancer patients the cytokine may rescue the pro-cancerous potential of WT-Ras, and together these two elements may lead to a more aggressive disease. These findings have clinical relevance, suggesting that we need to consider new therapeutic regimens that inhibit Ras and TNFα, in breast cancer patients.

Expression of Ccl11 Associates with Immune Response Modulation and Protection against Neuroinflammation in Rats

Multiple sclerosis (MS) is a polygenic disease characterized by inflammation and demyelination in the central nervous system (CNS), which can be modeled in experimental autoimmune encephalomyelitis (EAE). The Eae18b locus on rat chromosome 10 has previously been linked to regulation of beta-chemokine expression and severity of EAE. Moreover, the homologous chemokine cluster in humans showed evidence of association with susceptibility to MS. We here established a congenic rat strain with Eae18b locus containing a chemokine cluster (Ccl2, Ccl7, Ccl11, Ccl12 and Ccl1) from the EAE- resistant PVG rat strain on the susceptible DA background and utilized myelin oligodendrocyte glycoprotein (MOG)-induced EAE to characterize the mechanisms underlying the genetic regulation. Congenic rats developed a milder disease compared to the susceptible DA strain, and this was reflected in decreased demyelination and in reduced recruitment of inflammatory cells to the brain. The congenic strain also showed significantly increased Ccl11 mRNA expression in draining lymph nodes and spinal cord after EAE induction. In the lymph nodes, macrophages were the main producers of CCL11, whereas macrophages and lymphocytes expressed the main CCL11 receptor, namely CCR3. Accordingly, the congenic strain also showed significantly increased Ccr3 mRNA expression in lymph nodes. In the CNS, the main producers of CCL11 were neurons, whereas CCR3 was detected on neurons and CSF producing ependymal cells. This corresponded to increased levels of CCL11 protein in the cerebrospinal fluid of the congenic rats. Increased intrathecal production of CCL11 in congenic rats was accompanied by a tighter blood brain barrier, reflected by more occludin+ blood vessels. In addition, the congenic strain showed a reduced antigen specific response and a predominant anti-inflammatory Th2 phenotype. These results indicate novel mechanisms in the genetic regulation of neuroinflammation.

Tumor-Promoting Circuits That Regulate a Cancer-Related Chemokine Cluster: Dominance of Inflammatory Mediators Over Oncogenic Alterations

Here, we investigated the relative contribution of genetic/signaling components versus microenvironmental factors to the malignancy phenotype. In this system, we took advantage of non-transformed fibroblasts that carried defined oncogenic modifications in Ras and/or p53. These cells were exposed to microenvironmental pressures, and the expression of a cancer-related chemokine cluster was used as readout for the malignancy potential (CCL2, CCL5, CXCL8, CXCL10). In cells kept in-culture, synergism between Ras hyper-activation and p53 dysfunction was required to up-regulate the expression of the chemokine cluster. The in vivo passage of RasHigh/p53Low-modified cells has led to tumor formation, accompanied by potentiation of chemokine release, implicating a powerful role for the tumor microenvironment in up-regulating the chemokine cluster. Indeed, we found that inflammatory mediators which are prevalent in tumor sites, such as TNFα and IL-1β, had a predominant impact on the release of the chemokines, which was substantially higher than that obtained by the oncogenic modifications alone, possibly acting through the transcription factors AP-1 and NF-κB. Together, our results propose that in the unbiased model system that we were using, inflammatory mediators of the tumor milieu have dominating roles over oncogenic modifications in dictating the expression of a pro-malignancy chemokine readout.

Widespread Hypomethylation Occurs Early and Synergizes with Gene Amplification during Esophageal Carcinogenesis

Although a combination of genomic and epigenetic alterations are implicated in the multistep transformation of normal squamous esophageal epithelium to Barrett esophagus, dysplasia, and adenocarcinoma, the combinatorial effect of these changes is unknown. By integrating genome-wide DNA methylation, copy number, and transcriptomic datasets obtained from endoscopic biopsies of neoplastic progression within the same individual, we are uniquely able to define the molecular events associated progression of Barrett esophagus. We find that the previously reported global hypomethylation phenomenon in cancer has its origins at the earliest stages of epithelial carcinogenesis. Promoter hypomethylation synergizes with gene amplification and leads to significant upregulation of a chr4q21 chemokine cluster and other transcripts during Barrett neoplasia. In contrast, gene-specific hypermethylation is observed at a restricted number of loci and, in combination with hemi-allelic deletions, leads to downregulatation of selected transcripts during multistep progression. We also observe that epigenetic regulation during epithelial carcinogenesis is not restricted to traditionally defined “CpG islands,” but may also occur through a mechanism of differential methylation outside of these regions. Finally, validation of novel upregulated targets (CXCL1 and 3, GATA6, and DMBT1) in a larger independent panel of samples confirms the utility of integrative analysis in cancer biomarker discovery.

Identification of Distinct Cytokine and Chemokine Clusters That Correlate with Outcome In B-Cell Chronic Lymphocytic Leukemia: Implications for Disease Pathogenesis

AbstractAbstract 1368The growth and accumulation of B-cell chronic lymphocytic leukemia (CLL) cells requires survival and migratory signals supplied by endogenously produced cytokines and chemokines. Multiplex cytokine and chemokine analyses revealed that CCL2, CCL3, CCL4, IL-1b, IL-2, IL-5, IL-6, IL-8, IL-10, IL-12, CXCL9, CXCL11, TNFa, IFNg, IFNa, and IL-17 levels are significantly elevated in the serum of CLL patients (84 total) as compared to healthy age-matched subjects (48 total; p<0.01 for all). Random Forest analysis was used to rank these serum factors with respect to their importance in discriminating CLL patients from normal controls. This approach revealed 8 cytokines and chemokines (CCL3, IL-10, IL-5, CCL4, CXCL11, IL-8, CCL2, and IL-17) that distinguish CLL and controls, with CCL3 being the discriminating factor with the highest weight. The Kaplan-Meier method and log-rank test were used to compare median survival in CLL patients with high (>median) versus low (<median) levels for each cytokine. This analysis revealed that high levels of CXCL10, CXCL11, and IL-1b independently correlated with poor survival (p<0.05). Unsupervised cluster analysis was performed to identify groups of cytokines and chemokines that might work in concert to modulate CLL cell biology. As expected, CLL patients and healthy subjects clustered together on different ends of the dendogram. Furthermore, CLL patients with short TFT clustered together, and expressed high levels of a discrete group of cytokines (CCL3, CCL4, CCL19, CXCL9, CXCL10, CXCL11, IFNg, IL-5, IL-10, and IL-12). We termed this group “Clus1”. Certain members of this group have previously been shown to relate to CLL pathogenesis. CCL3 and CCL4, inflammatory chemokines that regulate cell recruitment and activation, are secreted by CLL cells and trigger a cascade of events that supports the CLL survival. IL-10 and the CXCR3 ligands CXCL9, CXCL10, CXCL11, are elevated in CLL and may contribute anti-apoptotic and growth-promoting signals that promote CLL cell survival. But, our data show for the first time that this set of cytokines and chemokines may be functionally linked. Cluster analysis also revealed a group of CLL patients that had moderate levels of Clus1 in combination with a second discrete cluster of cytokines (“Clus2”; GM-CSF, IL-8, TNFa, and IL-6). Since this group of patients had a relatively long TFT, even in the presence of detectable levels of Clus1, we hypothesized that they might play a direct protective role in CLL or alternatively might serve to regulate the actions of Clus1 cytokines. Lastly, cluster analysis revealed a third group of patients that were characterized by elevated serum levels of another discrete set of cytokines and chemokines (“Clus3”; IL-1b, IL-2, IL-4, IL-15, IL-17, and IFNa). The Kaplan-Meier method and log-rank test were used to compare median survival in patients expressing higher levels of Clus1, Clus2, or Clus3 cytokines and combinations of these clusters. This analysis revealed that high serum levels of Clus1 cytokines independently correlated with shorter survival times (Clus1HI12.5 yr versus Clus1LOW >21.5 yr; p=0.01), while high serum levels of Clus3 cytokines independently correlated with longer survival times (Clus3HI 21.6 yr versus Clus3LOW 12.5 years; p=0.05). The levels of Clus2 did not correlate with CLL patient survival. Combination analyses revealed that patients with high levels of Clus1 in the absence of Clus2 had significantly shorter survival times (10 yr) relative to patients with high levels of Clus1 cytokines in the presence of Clus2 (10yr vs 16yr; p=0.0018). In contrast, there was no significant difference between survival in patients with high levels of Clus1 in either the presence or absence of Clus3HI. Taken together, this suggests a beneficial modulatory role of cluster 2 cytokines. CONCLUSIONS: Application of multiplex bead immunoassays has allowed us to identify a set of cytokines and chemokines that are elevated within the context of CLL, to rank these secreted factors with respect to their importance in discriminating CLL from controls, and to identify those factors whose expression correlates with CLL disease prognosis. Complementary bioinformatics analysis allowed identification of three distinct clusters of cytokines and chemokines whose integrated expression levels reflect different CLL patient outcomes. Disclosures:No relevant conflicts of interest to declare.

Innate immunity in ocular Chlamydia trachomatis infection: contribution of IL8 and CSF2 gene variants to risk of trachomatous scarring in Gambians

BackgroundTrachoma, a chronic keratoconjunctivitis caused by Chlamydia trachomatis, is the world's commonest infectious cause of blindness. Blindness is due to progressive scarring of the conjunctiva (trachomatous scarring) leading to in-turning of eyelashes (trichiasis) and corneal opacification. We evaluated the contribution of genetic variation across the chemokine and cytokine clusters in chromosomes 4q and 5q31 respectively to risk of scarring trachoma and trichiasis in a large case-control association study in a Gambian population.MethodsLinkage disequilibrium (LD) mapping was used to investigate risk effects across the 4q and 5q31 cytokine clusters in relation to the risk of scarring sequelae of ocular Ct infection. Disease association and epistatic effects were assessed in a population based study of 651 case-control pairs by conditional logistic regression (CLR) analyses.ResultsLD mapping suggested that genetic effects on risk within these regions mapped to the pro-inflammatory innate immune genes interleukin 8 (IL8) and granulocyte-macrophage colony stimulatory factor (CSF2) loci. The IL8-251 rare allele (IL8-251 TT) was associated with protection from scarring trachoma (OR = 0.29 p = 0.027). The intronic CSF2_27348 A allele in chromosome 5q31 was associated with dose dependent protection from trichiasis, with each copy of the allele reducing risk by 37% (p = 0.005). There was evidence of epistasis, with effects at IL8 and CSF2 loci interacting with those previously reported at the MMP9 locus, a gene acting downstream to IL8 and CSF2 in the inflammatory cascade.Conclusioninnate immune response SNP-haplotypes are linked to ocular Ct sequelae. This work illustrates the first example of epistatic effects of two genes on trachoma.

Inhibition of Lipopolysaccharide-Stimulated Chronic Obstructive Pulmonary Disease Macrophage Inflammatory Gene Expression by Dexamethasone and the p38 Mitogen-Activated Protein Kinase Inhibitor N-cyano-N′-(2-{[8-(2,6-difluorophenyl)-4-(4-fluoro-2-methylphenyl)-7-oxo-7,8-dihydropyrido[2,3-d] pyrimidin-2-yl]amino}ethyl)guanidine (SB706504)

p38 mitogen-activated protein kinase (MAPK) signaling is known to be increased in chronic obstructive pulmonary disease (COPD) macrophages. We have studied the effects of the p38 MAPK inhibitor N-cyano-N'-(2-{[8-(2,6-difluorophenyl)-4-(4-fluoro-2-methylphenyl)-7-oxo-7,8-dihydropyrido[2,3-d]-pyrimidin-2-yl]amino}ethyl)guanidine (SB706504) and dexamethasone on COPD macrophage inflammatory gene expression and protein secretion. We also studied the effects of combined SB706504 and dexamethasone treatment. Lipopolysaccharide (LPS)-stimulated monocyte derived macrophages (MDMs) and alveolar macrophages (AMs) were cultured with dexamethasone and/or SB706504. MDMs were used for gene array and protein studies, whereas tumor necrosis factor (TNF) alpha protein production was measured from AMs. SB706504 caused transcriptional inhibition of a range of cytokines and chemokines in COPD MDMs. The use of SB706504 combined with dexamethasone caused greater suppression of gene expression (-8.90) compared with SB706504 alone (-2.04) or dexamethasone (-3.39). Twenty-three genes were insensitive to the effects of both drugs, including interleukin (IL)-1beta, IL-18, and chemokine (CC motif) ligand (CCL) 5. In addition, the chromosome 4 chemokine cluster members, CXCL1, CXCL2, CXCL3, and CXCL8, were all glucocorticoid-resistant. SB706504 significantly inhibited LPS-stimulated TNFalpha production from COPD and smoker AMs, with near-maximal suppression caused by combination treatment with dexamethasone. We conclude that SB706504 targets a subset of inflammatory macrophage genes and when used with dexamethasone causes effective suppression of these genes. SB706504 and dexamethasone had no effect on the transcription of a subset of LPS-regulated genes, including IL-1beta, IL-18, and CCL5, which are all known to be involved in the pathogenesis of COPD.

Gender-specific influence of the chromosome 16 chemokine gene cluster on the susceptibility to Multiple Sclerosis

Macrophage-derived chemokine (MDC/CCL22) plays a role in Experimental Autoimmune Encephalomyelitis (EAE), the animal model of Multiple Sclerosis (MS). MDC/CCL22 gene is part of a chemokine cluster, which includes also thymus and Activation-Regulated Chemokine (TARC/CCL17). The frequency of the C/T and C/A Single Nucleotide Polymorphisms (SNPs) in the promoter and coding sequence of CCL22 as well as the C/T SNP in the promoter of CCL17 were determined in 370 patients with Multiple Sclerosis (MS) compared with 380 controls. A trend towards a decreased allelic frequency of the A allele of the CCL22 C/A SNP as well as of the T allele of the CCL17 C/T SNP was found in patients compared with controls. The frequency of the AT haplotype was significantly decreased in MS patients (P=0.017, OR: 0.49, CI: 0.28-0.87). Stratifying patients according to gender, the observed association was even more pronounced in male patients compared with male controls (P=0.004, OR=0.18, 95% CI: 0.06-0.50), whereas no significant differences were observed in females. Therefore, the presence of the AT haplotype in chromosome 16 chemokine cluster is likely to confer a decreased risk of developing MS, particularly in males.