Expression Of Inflammatory Response Genes Research Articles

Screening of Candidate Inflammatory Markers of Epithelial Cells in Hepatocellular Carcinoma Based on Integration Analysis of TCGA/ICGC Databases and Single-cell Sequencing.

Hepatocellular Carcinoma (HCC) is closely linked to inflammatory reactions, with chronic liver diseases acting as major risk factors. In the inflammatory microenvironment, repeated damage and repair of liver cells lead to genetic mutations, abnormal proliferation, and tumorigenesis. This study aimed to investigate the expression profile of specific cell clusters under inflammatory stimulation in HCC and identify potential therapeutic drugs. Comprehensive analysis of HCC transcriptome data and single-cell sequencing data from TCGA, ICGC, and GEO databases was conducted to explore the specific molecular mechanisms of epithelial cells. Virtual screening of natural compounds in the ZINC database and in vitro cell experiments were performed to identify drugs that regulate the expression of inflammatory factors in epithelial cells. Analysis of the single-cell dataset revealed cell clusters closely associated with HCC, notably Epithelial cells, Hepatocytes, MSC, and iPS cells, with Epithelial cells playing a pivotal role in HCC development. Further investigation of TCGA data unveiled 83 differentially expressed genes (DEGs) related to inflammatory responses in HCC. Intersection analysis of DEGs in epithelial cells and HCC DEGs identified 12 common DEGs, including ADRM1, ATP2B1, FZD5, GPC3, KIF1B, KLF6, LY6E, MET, NAMPT, SERPINE1, SPHK1, and SRI. Prognostic analysis revealed that CCL7, GPR132, ITGB8, PTAFR, SELL, and VIP were influential in the survival prognosis of HCC. A prognostic model based on the expression levels of these genes demonstrated an increased risk of HCC associated with higher differential expression of inflammatory response genes. Additionally, molecular dynamics simulations indicated that compounds NADH and Deferoxamine formed stable docking models with the inflammatory protein VIP, suggesting their potential as candidates for targeted therapy. Inflammatory factors CCL7, GPR132, ITGB8, PTAFR, SELL, and VIP influence the inflammatory cascade response in HCC epithelial cells, and their expression correlates with the survival prognosis of HCC patients. Interfering with VIP expression effectively suppresses proliferation, migration, and invasion of HCC cells, as well as inhibiting the occurrence of inflammatory cascade reactions, thus slowing down the progression of hepatocellular carcinoma. Furthermore, compounds NADH and Deferoxamine have the potential to target and bind to the inflammatory protein VIP, highlighting their relevance in potential HCC treatment.

Sevoflurane anesthesia reduces the expression of inflammatory response genes and β-site amyloid precursor protein-cleaving enzyme in hippocampi of diabetic mice.

Diabetes and inhaled anesthesia are associated with an increased likelihood of developing postoperative cognitive dysfunction in humans and animal models, but the mechanisms are unclear. This study aimed to investigate the effect and mechanism of sevoflurane anesthesia on cognitive function in diabetic (DM) mice. Spontaneously diabetic db/db and control db/m mice were subject to sevoflurane anesthesia or allowed to breathe air, respectively. The Morris water maze test as spatial learning and novel object recognition test as recognition memory were performed. The expression of inflammatory cytokines and neurotoxicity-related genes in the hippocampus of four groups was measured using real-time PCR. The expression level of neurotoxicity and neuroprotection-related proteins in DM mice hippocampus were estimated using Western blot assay. It is found that DM mice developed cognitive impairment; however, the cognitive impairment was not exacerbated in sevoflurane-exposed mice. Sevoflurane anesthesia led to a decrease in mRNA levels of inflammatory cytokines in DM mice hippocampi, including interleukin 17 (IL-17), C-C motif chemokine (CCL20), CCL7 as well as high mobility group box 1 and beta-site amyloid-β cleaving enzyme 1; and no effect was observed on the expression of neurotoxicity genes, including amyloid precursor protein, choline O-acetyltransferase, tumor necrosis factor, alpha-induced protein 1, B-cell lymphoma 2 and estrogen receptor 2. In addition, we observed elevated phosphorylation of cAMP response element-binding protein in DM mice exposed to sevoflurane anesthesia. In conclusion, sevoflurane did not exacerbate DM-associated cognitive impairment.

Veronica persica ameliorates acetaminophen-induced murine hepatotoxicity via attenuating oxidative stress and inflammation

Excess acetaminophen (APAP) commonly causes severe acute liver injury (ALI), characterized by oxidative stress, pro-inflammatory responses, and hepatocyte damage. Veronica persica (VP) is a traditional medicine with antioxidant and anti-inflammatory properties. There is a paucity of information on its medicinal value, especially its potential mechanisms for alleviating ALI. This study aimed to clarify the ameliorative effects and intracellular mechanisms of VP on APAP-induced ALI via attenuating oxidative stress and inflammation. Mice were given VP for 7 days before exposure to APAP (300 mg/kg). The HPLC and radical scavenging assay found that VP contains 12 phenolic acids and 6 flavonoids, as well as show robust antioxidant capacity. In the APAP-induced ALI model, pre-treatment with VP significantly reduces APAP-induced hepatotoxicity by observing improved hepatocyte pathological injury and further confirmed by serum biochemical indicator. Also, the reduction of TUNEL-positive regions and the regulation of Bcl-2-associated X protein indicated that VP attenuates hepatocytotoxicity. Moreover, VP pre-intervention inhibits the formation of liver pro-inflammatory cytokines, the expression of inflammatory response genes, and increases in myeloperoxidase (MPO) in APAP-exposed mice. The elevated reduced glutathione (GSH) levels and decreased oxidative stress markers indicate that VP reduces APAP-promoted oxidative stress. Further study revealed that VP inhibited the phosphorylation of NF-κB/STAT3 cascade, blocked ERK and JNK phosphorylation, and activated AMP-activated protein kinase (AMPK). To sum up, this study demonstrated that VP exists hepatoprotective abilities on APAP-induced ALI, primarily by suppressing the phosphorylation of NF-κB/STAT3 cascade and ERK-JNK and inducing AMPK activation to alleviate oxidative stress and inflammation.

Comparison of Transcriptomic Changes in Survivors of Exertional Heat Illness with Malignant Hyperthermia Susceptible Patients.

Exertional heat illness (EHI) is an occupational health hazard for athletes and military personnel-characterised by the inability to thermoregulate during exercise. The ability to thermoregulate can be studied using a standardised heat tolerance test (HTT) developed by The Institute of Naval Medicine. In this study, we investigated whole blood gene expression (at baseline, 2 h post-HTT and 24 h post-HTT) in male subjects with either a history of EHI or known susceptibility to malignant hyperthermia (MHS): a pharmacogenetic condition with similar clinical phenotype. Compared to healthy controls at baseline, 291 genes were differentially expressed in the EHI cohort, with functional enrichment in inflammatory response genes (up to a four-fold increase). In contrast, the MHS cohort featured 1019 differentially expressed genes with significant down-regulation of genes associated with oxidative phosphorylation (OXPHOS). A number of differentially expressed genes in the inflammation and OXPHOS pathways overlapped between the EHI and MHS subjects, indicating a common underlying pathophysiology. Transcriptome profiles between subjects who passed and failed the HTT (based on whether they achieved a plateau in core temperature or not, respectively) were not discernable at baseline, and HTT was shown to elevate inflammatory response gene expression across all clinical phenotypes.

RNA-seq analysis reveals prenatal alcohol exposure is associated with placental inflammatory cells and gene expression

BackgroundFetal alcohol spectrum disorders (FASD) are the most common preventable cause of birth defects and neurodevelopmental disorders worldwide. The placenta is the crucial interface between mother and fetus. Prenatal alcohol exposure (PAE) has been shown to alter placental structure and expression of genes in bulk placental tissue samples, but prior studies have not examined effects on placental cell-type composition or taken cell-type into consideration in transcriptome analyses. MethodsWe leveraged an existent placenta single-cell RNA-seq dataset to perform cell-type deconvolution of bulk placental RNA-seq data from 35 heavy drinking pregnant women and 33 controls in a prospective birth cohort in Cape Town, South Africa. We used bivariate analyses and multivariable adjusted linear regression models to assess the relation of PAE on inferred placental cell-type proportions. We also examined differential expression of inflammatory response genes and PAE, using multivariable adjusted linear models. ResultsDeconvolution analyses showed heterogeneous placenta cell-type composition in which stromal (27 %), endothelial (26 %) and cytotrophoblasts (18 %) were the predominant cell-types. PAE around conception was associated with a higher proportion of Hofbauer cells (B = 0.51, p = 0.035) in linear models adjusted for maternal age, infant sex, and gestational age. Among the 652 inflammatory genes examined, 35 were differential expressed in alcohol exposed placentas (FDR p < 0.05). ConclusionsOur findings suggest that heavy alcohol exposure during pregnancy can influence the proportion of fetal placental villi macrophages (Hofbauer cells) and increased expression of inflammatory genes. Future studies are needed to further characterize these effects and to assess the potential functional roles of placental inflammation in FASD.

The role of microglia in 67NR mammary tumor-induced suppression of brain responses to immune challenges in female mice.

It is poorly understood how solid peripheral tumors affect brain neuroimmune responses despite the various brain-mediated side effects and higher rates of infection reported in cancer patients. We hypothesized that chronic low-grade peripheral tumor-induced inflammation conditions microglia to drive suppression of neuroinflammatory responses to a subsequent peripheral immune challenge. Here, Balb/c murine mammary tumors attenuated the microglial inflammatory gene expression responses to lipopolysaccharide (LPS) and live Escherichia coli (E. coli) challenges and the fatigue response to an E. coli infection. In contrast, the inflammatory gene expression in response to LPS or a toll-like receptor 2 agonist of Percoll-enriched primary microglia cultures was comparable between tumor-bearing and -free mice, as were the neuroinflammatory and sickness behavioral responses to an intracerebroventricular interleukin (IL)-1β injection. These data led to the hypothesis that Balb/c mammary tumors blunt the neuroinflammatory responses to an immune challenge via a mechanism involving tumor suppression of the peripheral humoral response. Balb/c mammary tumors modestly attenuated select circulating cytokine responses to LPS and E. coli challenges. Further, a second mammary tumor/mouse strain model (E0771 tumors in C57Bl/6 mice) displayed mildly elevated inflammatory responses to an immune challenge. Taken together, these data indicate that tumor-induced suppression of neuroinflammation and sickness behaviors may be driven by a blunted microglial phenotype, partly because of an attenuated peripheral signal to the brain, which may contribute to infection responses and behavioral side effects reported in cancer patients. Finally, these neuroimmune effects likely vary based on tumor type and/or host immune phenotype.

Inflammatory response-based subtyping and potential therapeutic strategies for triple-negative breast cancer

Objective: Inflammatory response plays a crucial role in the development and treatment of cancer. However, the role of inflammatory response in triple-negative breast cancer (TNBC) remains unclear. Based on the heterogeneity of the inflammatory response, we classified TNBC, elucidated its subtype features, and revealed potential therapeutic strategies. Methods: We established inflammatory response subtyping based on the RNA sequencing data of TNBCs derived from a cohort at the Fudan University Shanghai Cancer Center (FUSCC). Next, we explored the features and potential therapeutic strategies for each subgroup by analyzing transcriptome data. Using a machine-learning method, we validated and generalized the TNBC inflammatory response subtypes in an external dataset. Results: A total of 360 TNBC samples and 88 normal tissues were collected from a cohort at FUSCC. Patients with TNBC were divided into four inflammatory response groups (IRGs) based on the expression of inflammatory response genes: high inflammatory response gene expression with pronounced pyroptosis phenotype and high immune cell infiltration (IRG 1), low inflammatory response gene expression and low immune cell infiltration (IRG 2), ITGB8 specific inflammatory response with a predominant proliferation phenotype (IRG 3), and low M1/M2 ratio with a marked angiogenesis phenotype (IRG 4). Relapse-free survival (RFS) was better in IRG 1 and 2 and worse in IRG 3 and 4. Owing to their poor prognosis, we mainly focused on IRG 3 and IRG 4 to investigate potential treatment strategies. ITGB8 was highly expressed in IRG 3; thus, targeting ITGB8 may be a potential therapeutic strategy for patients in IRG 3. IRG 4 had a lower M1/M2 ratio and a marked angiogenesis phenotype; therefore, therapeutic strategies, such as anti-angiogenesis or M2 to M1 repolarization of macrophages, could be recommended for these patients. Additionally, we validated and generalized the TNBC inflammatory response subtyping in an external dataset using a machine-learning method. Conclusion: TNBC patients with different inflammatory response subtypes have different characteristics and may need subtype-specific treatment strategies.

Regulation of inflammatory genes in decidual cells: Involvement of the bromodomain and extra-terminal family proteins.

The decidua undergoes proinflammatory activation in late pregnancy, promoting labor. Bromodomain and Extra-Terminal (BET) family proteins interact with acetylated histones and may control gene expression in inflammation. Here, we assessed whether BETs are involved in inflammatory gene regulation in human decidual cells. We have treated primary cultures of decidual stromal cells (DSCs) from term pregnancies with endotoxin (LPS) and measured the expression of a panel of pro-and anti-inflammatory genes. BET involvement was assessed using the selective BET inhibitors (+)-JQ1 and I-BET-762 or the negative control compound (-)-JQ1. Histone 3 and -4 acetylation and BETs binding at the target gene promoters were determined to assess whether these processes are involved in the actions of LPS, BETs, and BET inhibitors. LPS increased the expression of the proinflammatory (PTGS2, IL6, CXCL8/IL8, TNF) and the anti-inflammatory (IL10, IDO1) genes of the panel. The constitutively expressed inflammatory genes (PTGS1, PTGES) were unaffected. The BET inhibitors, but not the control compound, reduced the basal and LPS-induced expression of PTGS1, PTGS2, IL6, CXCL8/IL8, IL10, and IDO1. TNF expression was not changed by BET inhibition. The dominant BETs were Bromodomain-containing protein -2 (BRD2) and -4L (BRD4L) in DSCs. LPS increased histone 4 acetylation at the CXCL8/IL8 and TNF promoters and histone 3 and -4 acetylation at the IDO1 promoter, while (+)-JQ1 abrogated histone acetylation at several promoters. Overall, histone acetylation and promoter binding of BETs showed no consistent relationship with gene expression across the gene panel and the treatments. BET proteins, predominantly BRD2 and BRD4L, control critical pro- and anti-inflammatory genes in DSCs. TNF induction exemplifies a BET-independent pathway. Changing histone acetylation at the promoters is not a general obligatory requirement for inflammatory gene expression in response to LPS. BETs likely act at chromatin loci separate from the examined promoters. BET inhibitors may block decidual activation at labor.

LincRNA-Cox2 Regulates Smoke-induced Inflammation in Murine Macrophages.

Cigarette smoke (CS) exposure is a risk factor for many chronic diseases, including chronic obstructive pulmonary disease, but the mechanism by which smoke exposure can alter homeostasis and bring about chronic inflammation is poorly understood. Here, we showcase a novel role for smoke in regulating long noncoding RNAs, showing that it activates lincRNA-Cox2, which we previously characterized as functional in inflammatory regulation. Exposing lincRNA-Cox2 murine models to smoke invivo confirmed lincRNA-Cox2 as a regulator of inflammatory gene expression in response to smoke both systemically and within the lung. We also report that lincRNA-Cox2 negatively regulates genes in smoked bone marrow-derived macrophages exposed to LPS stimulation. In addition to the effects on long noncoding RNAs, we also report dysregulated transcription and splicing of inflammatory protein-coding genes in the bone marrow niche after CS exposure invivo. Collectively, this work provides insights into how innate immune signaling from gene expression to splicing is altered after invivo exposure to CS and highlights an important new role for lincRNA-Cox2 in regulating immune genes after smoke exposure.

Expression Profile of Inflammation Response Genes and Potential Regulatory Mechanisms in Dilated Cardiomyopathy.

Background The inflammatory response is important in dilated cardiomyopathy (DCM). However, the expression of inflammatory response genes (IRGs) and regulatory mechanisms in DCM has not been well characterized. Methods We analyzed 27,665 cells of single-cell RNA sequencing dataset of four DCM samples and two healthy controls (HC). IRGs among differentially expressed genes (DEGs) of active cell clusters were screened from the Molecular Signatures Database (MSigDB). The bulk sequencing dataset of 166 DCM patients and 166 HC was analyzed to explore the common IRGs. The biological functions of the IRGs were analyzed according to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. IRG-related transcription factors (TFs) were determined using the TRRUST database. The protein–protein interaction (PPI) network was constructed using the STRING database. Then, we established the noncoding RNA (ncRNA) regulatory network based on the StarBase database. Finally, the potential drugs that target IRGs were explored using the Drug Gene Interaction Database (DGIdb). Results The proportions of dendritic cells (DCs), B cells, NK cells, and T cells were increased in DCM patients, whereas monocytes were decreased. DCs expressed more IRGs in DCM. The GO and KEGG analyses indicated that the functional characteristics of active cells mainly focused on the immune response. Thirty-nine IRGs were commonly expressed among active cell cluster DEGs, bulk RNA DEGs, and inflammatory response-related genes. ETS1 plays an important role in regulation of IRG expression. The competing endogenous RNA regulatory network showed the relationship between ncRNA and IRGs. Sankey diagram showed that arachidonate 5-lipoxygenase (ALOX5) played a major role in regulation between TFs and potential drugs. Conclusion DCs infiltrate into the myocardium and contribute to the immune response in DCM. The transcription factor ETS1 plays an important role in regulation of IRGs. Moreover, ALOX5 may be a potential therapeutic target for DCM.

HMGA2 Maintains Hematopoietic Stem Cell Via Pleiotropic Regulation of the Transcription in Stress Conditions

High-mobility group AT-hook 2 (Hmga2), an epigenetic modifier, opens the chromatin and modulate the transcription. Hmga2 is highly expressed in fetal and adult hematopoietic stem cells (HSCs). Hmga2 over-expression has been shown to promote self-renewal of HSC, however the molecular mechanism of how Hmga2 enhanced the self-renewal of HSC is still unclear. In this study, we assessed the function of Hmga2 in HSCs in steady and stress conditions by utilizing new Hmga2 conditional knock-in (KI) mouse and Hmga2 conditional knock-out mouse, which were crossed with either Cre-ERT2 mouse or Vav1-iCre mouse. Hmga2 KI mice showed a mild elevation in platelet counts, but did not develop malignancies in one year observation period. We performed a competitive transplantation assay by using purified HSCs, and found that wild-type HSCs diminished the repopulating capacity at the tertiary transplantation, Hmga2 KI HSCs maintained higher chimerism in myeloid cells and platelets in the PB and HSCs in the BM. We found that Hmga2 KO cells reduced the repopulating capacity, compared to wild-type cells. Thus, the expression of Hmga2 is critical for the self-renewal of HSC upon the transplantation. By performing RNA-sequencing of HSCs in homeostatic condition, we found that Hmga2 KI HSCs showed positive enrichments in cell cycle and proliferative signature, but maintained a stem cell signature, compared to wild-type HSCs. Since Hmga2 has been shown to globally open the chromatin in neural stem cells, we performed ATAC-sequence analysis in HSCs. Notably, Hmga2 KI HSCs showed 539 opened and 387 closed chromatin in H3K27ac-marked active regulatory regions, compared to wild-type HSCs. Among these opened genes by Hmga2, we generated a virus vector for fifteen genes, which were highly expressed in Hmga2 KI HSCs, and found that ectopic expression of Igf2bp2, an RNA binding protein, increased self-renewal capacity of HSC, but did not induce the enhanced production of myeloid cells and platelets that were observed in Hmga2 KI cells, in in vitro and in vivo settings. Indeed, Hmga2-ChIP-sequencing revealed that Hmga2 was directly bound to a proximal region of the Igf2bp2 gene, and CRISPR/Cas9-mediated deletion of the Igf2bp2 gene canceled the enhanced self-renewal capacity of Hmga2 KI HSCs, indicating that the Hmga2-Igf2bp2 axis is critical for the self-renewal of HSC.We next assessed function of Hmga2 in stress hematopoiesis after in vivo treatment of 5-FU. Hmga2 KI mice showed faster recoveries of reduction of platelets in the PB and increased CD41+HSCs and megakaryocyte progenitors in the BM in twelve days, in which WT mice reduced numbers of those cells in this condition. RNA-sequencing revealed that Hmga2 KI HSCs maintained expression levels of genes in stem cell- and proliferation-signatures at 3 days and 6 days post 5-FU injection, compared to WT HSCs that reduced expression of stem cell genes but activated inflammatory response genes. In contrast, Hgma2 KO HSCs enhanced expression of inflammatory response genes post the 5-FU injection, indicating Hmga2 represses expression of inflammatory response genes in the stress condition. Indeed, Hmga2-ChIP-sequencing revealed that Hmga2 was bound to larger numbers of genes involved in inflammatory responses in Kit+ cells post the 5-FU treatment from those in the control cells. Given increased expression of and a post-translational modification of Hmga2 protein in Kit+ cells post the 5-FU treatment, the remodeling of Hmga2 binding regions was appeared to depend on the modification of Hmga2 at downstream of the stress signal. Thus, Hmga2 directly activates Igf2bp2 to enhance the self-renew of HSC, but also represses the inflammatory response, leading to the enhanced megakaryopoiesis in the stress condition. DisclosuresNo relevant conflicts of interest to declare.

Role of nuclear factor‐κB (NF‐κB) in the regulation of pro‐inflammatory IκB‐kinase‐ε (IKKε) gene expression in human pulmonary epithelial cells

Rationale Clinically, mild-to-moderate asthma is well-controlled by inhaled glucocorticoids. However, severe asthma is poorly controlled by this key pharmacological intervention. Understanding which pro-inflammatory genes and signalling pathways avoid repression by glucocorticoids represents an important step towards improving current pharmacological interventions. IκB-kinase-ε (IKKε) was shown to escape full repression by glucocorticoids in airway epithelial cells, the primary site of action for glucocorticoid therapies in asthma. IKKε is also highly homologous to the canonical IκB kinases known to activate a family of pro-inflammatory transcription factors: nuclear factor-κB (NF-κB). However, the role and regulation of IKKε is largely uncharacterized. This study aimed to elucidate the regulation of IKKε gene expression by NF-κB in airway epithelial cells. Methods Human pulmonary epithelial A549 or bronchial airway epithelial BEAS-2B cells were used to model gene expression in airway epithelial cells. Cells were treated with cytokines interleukin-1β (IL1B, 1 ng/ml), tumour necrosis factor-α (TNF, 10 ng/ml), and/or glucocorticoid (dexamethasone, 1µM). Western blotting, qPCR, and RNA-sequencing were performed to characterize protein and mRNA expression of IKKε. Cells harbouring an NF-κB driven luciferase reporter construct were used to assess overall NF-κB activity. Chromatin immunoprecipitation (ChIP) sequencing data from BEAS-2B cells was used to explore binding of NF-κB subunit RelA/p65 to DNA using the UCSC genome browser. Results In A549 cells, IKKε protein was upregulated by both and IL1B and TNF with highest expression observed at 24h post stimulus. This peak expression was significantly, albeit partially, repressed by the glucocorticoid, dexamethasone. IKKε mRNA expression peaked at 6h, with partial repression by dexamethasone observed at 24h. ChIP-seq data from BEAS-2B cells treated with TNF showed p65 binding to the promoter region of IKKε. This suggests regulation of IKKε expression by NF-κB. In A549 cells, silencing of p65 decreased the ability of IL1B to induce NF-κB reporter activity and also reduced IL1B-induced expression of IKKε. Likewise, adenoviral-mediated overexpression of IκBαΔN, a dominant inhibitor of NF-κB, markedly reduced IKKε mRNA expression that was induced by IL1B. Conclusions These data suggest that induction of IKKε expression by IL1B or TNF is dependent on NF-κB activity in pulmonary epithelial cells. This supports the conclusion that early onset NF-κB activity leads to the expression of inflammatory genes, such as IKKε, to activate later onset signal transduction cascades to prolong inflammatory gene expression in response to a given stimuli. As IKKε induction was only partially repressed by glucocorticoid, the induction of downstream effector processes may also be largely resistant to the repressive effects of glucocorticoid. These findings represent an important step in characterizing the role of IKKε in inflammatory signalling cascades and paves the way for interventions in severe asthma therapy that may be more able to repress the activity of this pro-inflammatory pathway.

Waterpipe smoke condensate influences epithelial to mesenchymal transition and interferes with the cytotoxic immune response in non-small cell lung cancer cell lines.

Waterpipe tobacco smoking (WPS) continues to spread globally and presents serious health hazards. The aim of the present study was to investigate the effects of treatment with WPS condensate (WPSC) on lung cell proliferation and plasticity as well as tumor cell recognition and killing by natural killer (NK) cells using cytotoxicity assays. The results indicated that exposure of normal and cancer lung cell lines to WPSC resulted in a decrease in their in vitro growth in a dose-dependent manner and it induced tumor senescence. In addition, WPSC selectively caused DNA damage as revealed by an increase in γH2AX and 53BP1 in tumor lung cells. To gain further insight into the molecular mechanisms altered by WPSC, we conducted a global comprehensive transcriptome analysis of WPSC-treated tumor cells. Data analysis identified an expression profile of genes that best distinguished treated and non-treated cells involving several pathways. Of these pathways, we focused on those involved in epithelial to mesenchymal transition (EMT) and stemness. Results showed that WPSC induced an increase in SNAI2 expression associated with EMT, ACTA2 and SERPINE2 were involved in invasion and CD44 was associated with stemness. Furthermore, WPSC exposure increased the expression of inflammatory response genes including CASP1, IL1B, IL6 and CCL2. While immune synapse formation between NK and WPSC-treated lung cancer target cells was not affected, the capacity of NK cells to kill these target cells was reduced. The data reported in the present study are, to the best of our knowledge, the first in vitro demonstration of WPSC effects on lung cellular parameters providing evidence of its potential involvement in tumor physiology and development.

Inflammatory B cells correlate with failure to checkpoint blockade in melanoma patients

ABSTRACT The understanding of the role of B cells in patients with solid tumors remains insufficient. We found that circulating B cells produced TNFα and/or IL-6, associated with unresponsiveness and poor overall survival of melanoma patients treated with anti-CTLA4 antibody. Transcriptome analysis of B cells from melanoma metastases showed enriched expression of inflammatory response genes. Publicly available single B cell data from the tumor microenvironment revealed a negative correlation between TNFα expression and response to immune checkpoint blockade. These findings suggest that B cells contribute to tumor growth via the production of inflammatory cytokines. Possibly, these B cells are different from tertiary lymphoid structure-associated B cells, which have been described to correlate with favorable clinical outcome of cancer patients. Further studies are required to identify and characterize B cell subsets and their functions promoting or counteracting tumor growth, with the aim to identify biomarkers and novel treatment targets.

Differential expression of inflammatory responsive genes between chronic periodontitis and periodontally affected bronchiectasis patients.

The study aimed to investigate differential expression of targeted inflammatory-immune responsive genes [LTA, LTB, TNFSF4, TNFSF11/RANKL, TNFSF13, TNFSF13B, TNFRSF11B/ Osteoprotegerin; OPG and GFPT1/GFA ] in gingival tissues of bronchiectasis patients having chronic periodontitis in North central Indian population. Gingival tissues were collected from 30 systemically healthy chronic periodontitis patients (CP), 30 bronchiectasis patients with chronic periodontitis (B+CP), 3 systemically healthy with healthy gingiva (healthy control; HC) and 3 bronchiectasis with healthy gingiva (bronchiectasis control; BC). Statistical analysis revealed 7 genes to be significantly upregulated on comparing CP with B+CP i.e LTA (P<0.0001) in B+CP while LTB (P<0.0001), TNFSF4 (P=0.0003), TNFSF11 (P<0.0001), TNFSF13 (P=0.0003), TNFSF13B (P<0.0001) and TNFRSF11B (P=0.0004) in CP group. LTA (Lymphotoxin A) gene could be a potential genetic marker in bronchiectasis patients with chronic periodontitis.

Autophagy Inhibits Grass Carp Reovirus (GCRV) Replication and Protects Ctenopharyngodon idella Kidney (CIK) Cells from Excessive Inflammatory Responses after GCRV Infection.

Autophagy is an essential and highly conserved process in mammals, which is critical to maintaining physiological homeostasis, including cell growth, development, repair, and survival. However, the understanding of autophagy in fish virus replication is limited. In this study, we found that grass carp reovirus (GCRV) infection stimulated autophagy in the spleen of grass carp (Ctenopharyngodon idella). Moreover, both Western blot (WB) analysis and fluorescent tracer tests showed that GCRV infection induced the enhancement of autophagy activation in Ctenopharyngodon idella kidney (CIK) cells. Autophagy inducer rapamycin and autophagy inhibitor 3-MA pretreatment can inhibit and promote the proliferation of GCRV, respectively. In addition, grass carp autophagy-related gene 5 (CiATG5)-induced autophagy, as well as rapamycin, showed effects on GCRV replication in CIK cells. Transcriptome analysis revealed that the total number of differentially expressed genes (DEGs) in CiATG5 overexpression groups was less than that of the control during GCRV infection. Enrichment analysis showed that CiATG5 overexpression induced the enhancement of autophagy, lysosome, phagosome, and apoptosis in the early stage of GCRV infection, which led to the clearance of viruses. In the late stage, steroid biosynthesis, DNA replication, terpenoid backbone biosynthesis, and carbon metabolism were upregulated, which contributed to cell survival. Moreover, signaling pathways involved in the immune response and cell death were downregulated in CiATG5 overexpression groups. Further study showed that CiATG5 repressed the expression of inflammatory response genes, including cytokines and type I interferons. Taken together, the results demonstrate that autophagy represses virus replication and attenuates acute inflammatory responses to protect cells.

BAF60a Deficiency in Vascular Smooth Muscle Cells Prevents Abdominal Aortic Aneurysm by Reducing Inflammation and Extracellular Matrix Degradation.

Currently, there are no approved drugs for abdominal aortic aneurysm (AAA) treatment, likely due to limited understanding of the primary molecular mechanisms underlying AAA development and progression. BAF60a-a unique subunit of the SWI/SNF (switch/sucrose nonfermentable) chromatin remodeling complex-is a novel regulator of metabolic homeostasis, yet little is known about its function in the vasculature and pathogenesis of AAA. In this study, we sought to investigate the role and underlying mechanisms of vascular smooth muscle cell (VSMC)-specific BAF60a in AAA formation. Approach and Results: BAF60a is upregulated in human and experimental murine AAA lesions. In vivo studies revealed that VSMC-specific knockout of BAF60a protected mice from both Ang II (angiotensin II)-induced and elastase-induced AAA formation with significant suppression of vascular inflammation, monocyte infiltration, and elastin fragmentation. Through RNA sequencing and pathway analysis, we found that the expression of inflammatory response genes in cultured human aortic smooth muscle cells was significantly downregulated by small interfering RNA-mediated BAF60a knockdown while upregulated upon adenovirus-mediated BAF60a overexpression. BAF60a regulates VSMC inflammation by recruiting BRG1 (Brahma-related gene-1)-a catalytic subunit of the SWI/SNF complex-to the promoter region of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) target genes. Furthermore, loss of BAF60a in VSMCs prevented the upregulation of the proteolytic enzyme cysteine protease CTSS (cathepsin S), thus ameliorating ECM (extracellular matrix) degradation within the vascular wall in AAA. Our study demonstrated that BAF60a is required to recruit the SWI/SNF complex to facilitate the epigenetic regulation of VSMC inflammation, which may serve as a potential therapeutic target in preventing and treating AAA.

Pathogenesis of Human Papillomaviruses Requires the ATR/p62 Autophagy-Related Pathway.

High-risk human papillomaviruses (HPVs) constitutively activate the ataxia telangiectasia and Rad3-related (ATR) DNA damage response pathway, and this is required for viral replication. In fibroblasts, activated ATR regulates transcription of inflammatory genes through its negative effects on the autophagosome cargo protein p62. In addition, suppression of p62 results in increased levels of the transcription factor GATA4, leading to cellular senescence. In contrast, in HPV-positive keratinocytes, we observed that activation of ATR resulted in increased levels of phosphorylated p62, which in turn lead to reduced levels of GATA4. Knockdown of ATR in HPV-positive cells resulted in decreased p62 phosphorylation and increased GATA4 levels. Transcriptome sequencing (RNA-seq) analysis of HPV-positive cells identified inflammatory genes and interferon factors as negative transcriptional targets of ATR. Furthermore, knockdown of p62 or overexpression of GATA4 in HPV-positive cells leads to inhibition of viral replication. These findings identify a novel role of the ATR/p62 signaling pathway in HPV-positive cells.IMPORTANCE High-risk human papillomaviruses (HPVs) infect epithelial cells and induce viral genome amplification upon differentiation. HPV proteins activate the ATR DNA damage repair pathway, and this is required for HPV genome amplification. In the present study, we show that HPV-induced ATR activation also leads to suppression of expression of inflammatory response genes. This suppression results from HPV-induced phosphorylation of the autophagosome cargo protein p62 which regulates the levels of the transcription factor GATA4. Activation of p62 in normal fibroblasts results in senescence, but this is not seen in HPV-positive keratinocytes. Importantly, knockdown of p62 or overexpression of GATA4 in HPV-positive cells abrogates viral replication. This study demonstrates that activation of ATR in HPV-positive cells triggers a p62-directed pathway inducing suppression of inflammatory gene expression independent of DNA repair and facilitating HPV replication.

CITED2 limits pathogenic inflammatory gene programs in myeloid cells.

Monocyte‐derived macrophages are the major innate immune cells that provide the first line of cellular defense against infections or injuries. These recruited macrophages at the site of inflammation are exposed to a broad range of cytokines that categorically incite a robust pro‐inflammatory response. However, macrophage pro‐inflammatory activation must be under exquisite control to avert unbridled inflammation. Thus, endogenous mechanisms must exist that rigorously preserve macrophage quiescence and yet, allow nimble pro‐inflammatory macrophage response with precise spatiotemporal control. Herein, we identify the CBP/p300‐interacting transactivator with glutamic acid/aspartic acid‐rich carboxyl‐terminal domain 2 (CITED2) as a critical intrinsic negative regulator of inflammation, which broadly attenuates pro‐inflammatory gene programs in macrophages. Our in vivo studies revealed that myeloid‐CITED2 deficiency significantly heightened macrophages and neutrophils recruitment to the site of inflammation. Our integrated transcriptomics and gene set enrichment analysis (GSEA) studies uncovered that CITED2 deficiency broadly enhances NFκB targets, IFNγ/IFNα responses, and inflammatory response gene expression in macrophages. Using complementary gain‐ and loss‐of‐function studies, we observed that CITED2 overexpression attenuate and CITED2 deficiency elevate LPS‐induced NFκB transcriptional activity and NFκB‐p65 recruitment to target gene promoter in macrophages. More importantly, blockade of NFκB signaling completely reversed elevated pro‐inflammatory gene expression in macrophages. Collectively, our findings show that CITED2 restrains NFκB activation and curtails broad pro‐inflammatory gene programs in myeloid cells.

Lysophosphatidylcholine acyltransferase 2 (LPCAT2) co-localises with TLR4 and regulates macrophage inflammatory gene expression in response to LPS

Despite extensive investigations, an effective treatment for sepsis remains elusive and a better understanding of the inflammatory response to infection is required to identify potential new targets for therapy. In this study we have used RNAi technology to show, for the first time, that the inducible lysophosphatidylcholine acyltransferase 2 (LPCAT2) plays a key role in macrophage inflammatory gene expression in response to stimulation with bacterial ligands. Using siRNA- or shRNA-mediated knockdown, we demonstrate that, in contrast to the constitutive LPCAT1, LPCAT2 is required for macrophage cytokine gene expression and release in response to TLR4 and TLR2 ligand stimulation but not for TLR-independent stimuli. In addition, cells transfected to overexpress LPCAT2 exhibited increased expression of inflammatory genes in response to LPS and other bacterial ligands. Furthermore, we have used immunoprecipitation and Western blotting to show that in response to LPS, LPCAT2, but not LPCAT1, rapidly associates with TLR4 and translocates to membrane lipid raft domains. Our data thus suggest a novel mechanism for the regulation of inflammatory gene expression in response to bacterial stimuli and highlight LPCAT2 as a potential therapeutic target for development of anti-inflammatory and anti-sepsis therapies.