Extracellular CIRP Induces an Inflammatory Phenotype in Pulmonary Fibroblasts via TLR4
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ABSTRACT:
PROVIDER: PRJNA738147 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR14820200_1.fastq.gz | Fastqsanger.gz | |||
| SRR14820200_2.fastq.gz | Fastqsanger.gz | |||
| SRR14820201_1.fastq.gz | Fastqsanger.gz | |||
| SRR14820201_2.fastq.gz | Fastqsanger.gz | |||
| SRR14820202_1.fastq.gz | Fastqsanger.gz |
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Project description:The wild type (WT) and TLR4 -/- pulmonary fibroblasts were treated with phosphate-buffered saline (PBS), 1 µg/mL recombinant mouse (rm) CIRP, 2 ng/mL rmTGF-ß1 (R&D Systems), or rmCIRP plus rmTGF-ß1. The cell lysates collected at 24 hours were used for high throughput mRNA sequencing.
2021-07-15 | GSE178255 | GEO
Project description:Extracellular cold-inducible RNA-binding protein (eCIRP), a new damage-associated molecular pattern (DAMP), has been recently shown to play a critical role in promoting the development of bleomycin-induced pulmonary fibrosis. Although fibroblast activation is a critical component of the fibrotic process, the direct effects of eCIRP on fibroblasts have never been examined. We studied eCIRP's role in the induction of inflammatory phenotype in pulmonary fibroblasts and its connection to bleomycin-induced pulmonary fibrosis in mice. We found that eCIRP causes the induction of proinflammatory cytokines and differentially expression-related pathways in a TLR4-dependent manner in pulmonary fibroblasts. Our analysis further showed that the accessory pathways MD2 and Myd88 are involved in the induction of inflammatory phenotype. In order to study the connection of the enrichment of these pathways in priming the microenvironment for pulmonary fibrosis, we investigated the gene expression profile of lung tissues from mice subjected to bleomycin-induced pulmonary fibrosis collected at various time points. We found that at day 14, which corresponds to the inflammatory-to-fibrotic transition phase after bleomycin injection, TLR4, MD2, and Myd88 were induced, and the transcriptome was differentially enriched for genes in those pathways. Furthermore, we also found that inflammatory cytokines gene expressions were induced, and the cellular responses to these inflammatory cytokines were differentially enriched on day 14. Overall, our results show that eCIRP induces inflammatory phenotype in pulmonary fibroblasts in a TLR4 dependent manner. This study sheds light on the mechanism by which eCIRP induced inflammatory fibroblasts, contributing to pulmonary fibrosis.
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Project description:The successful repair of alveolar epithelial injury is required to restore the integrity of gas exchanging regions of the lung and preserve organ function. Severe pulmonary fibrosis is the result of repeated episodes of epithelial injury, activation of fibroblasts, and matrix accumulation. Thus, impaired alveolar epithelial progenitor cell renewal could contribute to the progression of fibrosis. We provide evidence that expression of TLR4 and hyaluronan (HA) on Type 2 alveolar epithelial cells (AEC2s) is necessary for self-renewal. Either deletion of TLR4 or HA synthase 2 leads to impaired regeneration of AEC2s, severe fibrosis and mortality, in part due to blunted production of IL-6. AEC2s from patients with pulmonary fibrosis have reduced cell surface HA, and impaired renewal capacity, suggesting that interactions between HA and TLR4 are key regulators of lung stem cell renewal, repair of lung injury and that severe pulmonary fibrosis is the result of epithelial stem cell failure. We used microarrays to detail the gene expression of AEC2 cells from WT and TLR4-/- mice.
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Project description:Toll like receptor 4 (TLR4), an innate immunity gene, is involved in responses to several pulmonary agonists including endotoxin (LPS; Poltorak et al.,1998), ozone (O3 ,Kleeberger et. al., 2001), Pseudomonas aeruginosa (Faure et al, 2004), and hyperoxia (Zhang et al, 2005). TLR4 appears to partially mediate the response to LPS- and O3-induced lung injury, however, TLR4 is protective for prevention of injury in Pseudomonas aeruginosa infection and against acute lung injury (hyperoxia). The mechanism behind this protection is unclear. We previously demonstrated that TLR4 was also protective against BHT-induced chronic inflammation and tumor promotion (Bauer et al, 2005). C.C3H-Tlr4Lps-d (BALBLps-d) mice, congenic for a 10 cM region of C3H/HeJ chromosome 4 that contains Tlr4 (Vogel et al, 1994), have a missence mutation that renders TLR4 dysfunctional. The Tlr4 mutation likely abrogates signaling by disrupting a direct point of contact with other signaling molecules (Akira S, Takeda K. Toll-like receptor signalling. Nat Rev Immunol 2004;4(7):499-511.). Bronchoalveolar lavage fluid (BALF) alveolar macrophages, lymphocytes, and total protein content were significantly elevated in BALBLps-d mice compared to BALB/c (BALB; Tlr4 sufficient) mice following chronic BHT (Bauer et al., 2005). BALBLps-d mice also had a significant increase in tumor multiplicity (60%) over that of BALB mice in response to an MCA/BHT tumor promotion protocol. While this was the first model to demonstrate a protective role for TLR4 in chronic lung inflammation and tumorigenesis, the downstream mechanism regulating this protective response remains unknown. Using Affymetrix microarray analysis followed by GeneSpring and Ingenuity pathway analyses, we herein identified known and novel downstream pathways and their interactions that may be involved in the protective mechanism elicited by TLR4. We therefore hypothesize that these pathways and interactions amongst the genes identified during the tumor promotion/chronic inflammation stage are in part influencing the differential strain response observed during tumorigenesis. Keywords: time course, tumor study Protocol 1 - 3 biological replicates after chronic dosing in each mouse strain Protocol 2 - multiple replicates after MCA/BHT tumor progression model used
2009-12-12 | E-GEOD-8504 | biostudies-arrayexpress
Project description:Toll like receptor 4 (TLR4), an innate immunity gene, is involved in responses to several pulmonary agonists including endotoxin (LPS; Poltorak et al.,1998), ozone (O3 ,Kleeberger et. al., 2001), Pseudomonas aeruginosa (Faure et al, 2004), and hyperoxia (Zhang et al, 2005). TLR4 appears to partially mediate the response to LPS- and O3-induced lung injury, however, TLR4 is protective for prevention of injury in Pseudomonas aeruginosa infection and against acute lung injury (hyperoxia). The mechanism behind this protection is unclear. We previously demonstrated that TLR4 was also protective against BHT-induced chronic inflammation and tumor promotion (Bauer et al, 2005). C.C3H-Tlr4Lps-d (BALBLps-d) mice, congenic for a 10 cM region of C3H/HeJ chromosome 4 that contains Tlr4 (Vogel et al, 1994), have a missence mutation that renders TLR4 dysfunctional. The Tlr4 mutation likely abrogates signaling by disrupting a direct point of contact with other signaling molecules (Akira S, Takeda K. Toll-like receptor signalling. Nat Rev Immunol 2004;4(7):499-511.). Bronchoalveolar lavage fluid (BALF) alveolar macrophages, lymphocytes, and total protein content were significantly elevated in BALBLps-d mice compared to BALB/c (BALB; Tlr4 sufficient) mice following chronic BHT (Bauer et al., 2005). BALBLps-d mice also had a significant increase in tumor multiplicity (60%) over that of BALB mice in response to an MCA/BHT tumor promotion protocol. While this was the first model to demonstrate a protective role for TLR4 in chronic lung inflammation and tumorigenesis, the downstream mechanism regulating this protective response remains unknown. Using Affymetrix microarray analysis followed by GeneSpring and Ingenuity pathway analyses, we herein identified known and novel downstream pathways and their interactions that may be involved in the protective mechanism elicited by TLR4. We therefore hypothesize that these pathways and interactions amongst the genes identified during the tumor promotion/chronic inflammation stage are in part influencing the differential strain response observed during tumorigenesis. Keywords: time course, tumor study
2009-12-12 | GSE8504 | GEO
Project description:Background: Human lung adenocarcinoma (ADC), the predominant subtype of lung cancer, are at a disadvantage as the disease lacks available biomarkers and is not usually diagnosed until its later stages. We previously demonstrated that Tlr4-mutantmice were more susceptible to BHT-induced pulmonary inflammation (bronchoalveolar lavage macrophages and lymphocytes) and tumorigenesis in comparison to Tlr4-sufficient control mice. Differential transcriptional profiles were also noted in the whole lung tissue of the mice. The study objective was to elucidate the mechanisms underlying the protective effect of Tlr4 including differences in specific innate immune cell populations, their functional responses, and the influence of these cellular differences on the growth of ACD progenitor cell types. Methods: Following butylated hydroxytoluene (BHT) treatment (tumor promoter;4 weekly ip.injections), BALB (Tlr4-sufficient) and C.C3-Tlr4Lps-d/J (BALBLpsd; Tlr4 mutant) lungs were lavaged, processed for flow cytometry, and frozen for molecular analysis (ELISAs for IGF1, KC, Raybiotech Mouse Cytokine Array). BALF-derived macrophages were collected for phagocytosis and efferocytosis assays. BALF macrophage RNA was also isolated and utilized for Affymetrix gene arrays and real-time quantitative PCR. Enrichment of bronchiolar Clara cells, an ADC progenitor cell, was performed and cells enumerated. Femur bone marrow cells were differentiated into macrophages, and co-cultured with C10 cells, a type II cell line, for a wound healing assay. Results: Tlr4-deficiency resulted in significantly increased numbers of innate immune cells in whole lung tissue in response to BHT, including macrophages, PMNs, myeloid-derived suppressor cells (MDSC)s and dendritic cells (DCs), in comparison to Tlr4-sufficient mice (p<05). Macrophage functionality was also affected. BHT treated Tlr4-mutant mice demonstrated enhanced phagocytic and efferocytic abilities and wound healing in comparison to macrophages from Tlr4-sufficient and control treated mice (P<0.05). Additionally, Clara cell proliferation was significantly increased in Tlr4-mutant compared to Tlr4-sufficient mice in response to BHT. Pulmonary cytokine, chemokine and growth factor protein expression also significantly differed among the strains and within macrophages, gene expression and cell surface markers combined demonstrated a more plastic macrophage phenotype in the Tlr4-mutant mice. The transcriptome study identified immune pathways important in inflammation, such as phagosome. Conclusion: There are distinct pulmonary innate immune cell populations, with particular emphasis on macrophages, that are likely influencing the enhanced tumor promotion observed in Tlr4-mutant mice. Though not as striking, the pulmonary lymphoid populations may also be affecting the promotion environment, if only via their influence upon the innate immune cells. Future studies will investigate the origins of the macrophages and continued delineation of the lymphoid cell populations. Two strains of mice were used for these studies. The C.C3-Tlr4Lps-d/J (BALBLpsd) with a dominant negative Tlr4 on a BALB/c background and the BALB/cJ mice with a sufficient Tlr4. Total RNA was extracted from bronchoalveolar lavage fluid macrophages 3 days following a chronic BHT protocol. (oil controls vs BHT treated for both strains. BALB oil and BHT treatment n=3 mice per treatment group; BALBLpsd oil controls n=2 per treatment group; BALBLpsd BHT treated n = 3 per treatment group). Therefore a total of 13 arrays with one BHT exposure times and oil (vehicle) controls.
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Project description:Background: Human lung adenocarcinoma (ADC), the predominant subtype of lung cancer, are at a disadvantage as the disease lacks available biomarkers and is not usually diagnosed until its later stages. We previously demonstrated that Tlr4-mutantmice were more susceptible to BHT-induced pulmonary inflammation (bronchoalveolar lavage macrophages and lymphocytes) and tumorigenesis in comparison to Tlr4-sufficient control mice. Differential transcriptional profiles were also noted in the whole lung tissue of the mice. The study objective was to elucidate the mechanisms underlying the protective effect of Tlr4 including differences in specific innate immune cell populations, their functional responses, and the influence of these cellular differences on the growth of ACD progenitor cell types. Methods: Following butylated hydroxytoluene (BHT) treatment (tumor promoter;4 weekly ip.injections), BALB (Tlr4-sufficient) and C.C3-Tlr4Lps-d/J (BALBLpsd; Tlr4 mutant) lungs were lavaged, processed for flow cytometry, and frozen for molecular analysis (ELISAs for IGF1, KC, Raybiotech Mouse Cytokine Array). BALF-derived macrophages were collected for phagocytosis and efferocytosis assays. BALF macrophage RNA was also isolated and utilized for Affymetrix gene arrays and real-time quantitative PCR. Enrichment of bronchiolar Clara cells, an ADC progenitor cell, was performed and cells enumerated. Femur bone marrow cells were differentiated into macrophages, and co-cultured with C10 cells, a type II cell line, for a wound healing assay. Results: Tlr4-deficiency resulted in significantly increased numbers of innate immune cells in whole lung tissue in response to BHT, including macrophages, PMNs, myeloid-derived suppressor cells (MDSC)s and dendritic cells (DCs), in comparison to Tlr4-sufficient mice (p<05). Macrophage functionality was also affected. BHT treated Tlr4-mutant mice demonstrated enhanced phagocytic and efferocytic abilities and wound healing in comparison to macrophages from Tlr4-sufficient and control treated mice (P<0.05). Additionally, Clara cell proliferation was significantly increased in Tlr4-mutant compared to Tlr4-sufficient mice in response to BHT. Pulmonary cytokine, chemokine and growth factor protein expression also significantly differed among the strains and within macrophages, gene expression and cell surface markers combined demonstrated a more plastic macrophage phenotype in the Tlr4-mutant mice. The transcriptome study identified immune pathways important in inflammation, such as phagosome. Conclusion: There are distinct pulmonary innate immune cell populations, with particular emphasis on macrophages, that are likely influencing the enhanced tumor promotion observed in Tlr4-mutant mice. Though not as striking, the pulmonary lymphoid populations may also be affecting the promotion environment, if only via their influence upon the innate immune cells. Future studies will investigate the origins of the macrophages and continued delineation of the lymphoid cell populations.
2016-07-14 | GSE68067 | GEO
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Project description:Human macrophages secrete extracellular vesicles loaded with numerous immunoregulatory proteins. Here we employed high throughput quantitative proteomics to characterize the modulation of vesicle-mediated protein secretion during non-canonical caspase-4/5-dependent inflammasome activation. We show that human macrophages activate robust caspase-4- dependent extracellular vesicle secretion upon transfection of LPS, and this process is also partially dependent on NLRP-3 and caspase-5. Similar effect occurs with delivery of the LPS with E. coli-derived outer membrane vesicles. Moreover, sensitization of the macrophages through TLR4 prior to LPS transfection dramatically augments the EV-mediated protein secretion. Our data demonstrate that this process differs significantly from ATP-induced vesiculation, and is dependent on autocrine interferon signal associated with TLR4 activation. TLR4 activation preceding the non-canonical inflammasome activation significantly enhances vesicle-mediated secretion of inflammasome components caspase-1, ASC and lytic cell death effectors GSDMD, MLKL and NINJ1, suggesting that inflammatory EV transfer may exert paracrine effects in recipient cells. Moreover, using bioinformatic methods, we identify 15-deoxy-delta-12,14-prostaglandin J2 and parthenolide as inhibitors of caspase-4-mediated inflammation and vesicle secretion, indicating potential new therapeutic potential of these anti-inflammatory drugs.
2022-12-01 | PXD033002 | Pride
Project description:We previously identified toll-like receptor 4 (Tlr4) as a candidate gene responsible for ozone (O3)-induced pulmonary hyperpermeability and inflammation. The objective of this study was to determine the mechanism through which TLR4 modulates O3-induced pulmonary responses and to utilize transcriptomics to determine TLR4 effector molecules. C3H/HeJ (HeJ; Tlr4 mutant) and C3H/HeOuJ (OuJ; Tlr4 normal), mice were exposed continuously to 0.3 ppm O3 or filtered air for 6, 24, 48 or 72 hr. Affymetrix Mouse430A_MOE gene arrays were used to analyze lung homogenates from HeJ and OuJ mice followed using a bioinformatic analysis. Inflammation was assessed by bronchoalveolar lavage and molecular analysis by ELISA, immunoblotting, and transcription factor activity. TLR4 signals through both the MYD88-dependent and independent pathways in OuJ mice, which involves MAP kinase activation, NF-kappaB, AP-1, and KC. Microarray analyses identifiedTLR4 responsive genes for strain and time in OuJ versus HeJ mice (p<0.05). One significantly upregulated cluster of genes in OuJ were the heat shock proteins (Hspa1b; Hsp70), Hsp90ab1). Furthermore, O3-induced expression of HSP70 protein was increased in OuJ compared to HeJ mice following 24-48 h O3. Moreover, BAL polymorphonuclear leukocytes (PMN) and total protein were significantly reduced in response to O3 in Hspa1a/Hspa1btm1Dix (Hsp70-/-) compared to Hsp70+/+ mice (p<0.05). TLR4 signaling (MYD88-dependent), ERK1/2, AP-1 activity, and KC protein content were also significantly reduced after O3 exposure in Hsp70-/- compared to Hsp70+/+ mice (p<0.05). These studies suggest that HSP70 is involved in the regulation of O3-induced lung inflammation through the TLR4 pathway and provide evidence that HSP70 is an endogenous in vivo TLR4 ligand.
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