Lipopolysaccharide Stimulation Research Articles

Investigating the role of GPNMB in modulating LPS mediated inflammation in progranulin‐deficient macrophages

AbstractBackgroundProgranulin (PGRN) is a glycosylated holoprotein that is critical to endo‐lysosomal health and regulating inflammatory responses over the lifespan. Insufficient PGRN due to nonsense mutations in the GRN gene has been linked to increased risk of developing Frontotemporal Dementia (FTD) and Neuronal Ceroid Lipofuscinosis (NCL‐11). The precise mechanism(s) behind the onset and progression of disease is unclear, and no disease‐modifying treatment exists. Previous work has identified increased glycoprotein non‐metastatic B (GPNMB) in both the cortex and plastma of Grn−/− mice relative to Grn+/+ mice, and our lab has recently identified different GPNMB expression in PGRN‐deficient immune cell populations, but it remains unclear how this increase in GPNMB modifies the inflammatory response(s) with PGRN deficits. This work seeks to investigate GPNMB expression over the course of the immune response in PGRN‐deficient cells, and the extent to which GPNMB modulates inflammation.MethodTo investigate the role(s) of GPNMB in PGRN‐deficient myeloid cells, we isolated peritoneal macrophages (pMacs) from Grn+/+,and Grn−/− mice and quantified GPNMB protein before and after lipopolysaccharide (LPS) stimulation with western blot. We also determined the extent to which exposure to the GPNMB extracellular domain (ECD) modifies LPS‐elicited markers of inflammation in both Grn+/+and Grn−/‐ pMacs via qPCR. Finally, we determined how markers of inflammation were altered in pMacs from Grn−/−Gpmnb−/‐ relative to Grn+/+and Grn−/‐ with and without LPS stimulation.ResultWe determined that the additional loss of GPNMB in Grn−/−Gpmnb−/− pMacs altered the expression of inflammatory markers after LPS challenge relative to both Grn−/−and Grn+/+ pMacs. We also discovered that GPNMB is dynamically regulated during and after LPS challenge in both Grn+/+ and Grn−/− pMacs. Finally, we determined that the addition of GPNMB ECD before and during LPS treatment ameliorated the altered expression of inflammatory markers during LPS challenge.ConclusionThe increase of GPNMB expression in Grn−/‐ myeloid cells is a significant modulator of inflammatory responses and may be a potential target in the development of therapeutics.

Lipopolysaccharide (LPS) stimulation of Pancreatic Ductal Adenocarcinoma (PDAC) and macrophages activates the NLRP3 inflammasome that influences the levels of pro-inflammatory cytokines in a co-culture model

ABSTRACT Modified macrophages, tumor-associated macrophages (TAMs), are key contributors to the survival, growth, and metastatic behavior of pancreatic ductal adenocarcinoma (PDAC) cells. Central to the role of inflammation and TAMs lies the NLRP3 inflammasome. This study investigated the effects of LPS-stimulated inflammation on cell proliferation, levels of pro-inflammatory cytokines, and the NLRP3 inflammasome pathway in a co-culture model using PDAC cells and macrophages in the presence or absence of MCC950, a NLRP3-specific inhibitor. The effects of LPS-stimulated inflammation were tested on two PDAC cell lines (Panc 10.05 and SW 1990) co-cultured with RAW 264.7 macrophages. Cell proliferation was determined using the MTT assay. Levels of pro-inflammatory cytokines, IL-1β, and TNF-α were determined by ELISA. Western blot analyses were used to examine the expression of NLRP3 in both PDAC cells and macrophages. The co-culture and interaction between PDAC cell lines and macrophages led to pro-inflammatory microenvironment under LPS stimulation as evidenced by high levels of secreted IL-1β and TNF-α. Inhibition of the NLRP3 inflammasome by MCC950 counteracted the effects of LPS stimulation on the regulation of the NLRP3 inflammasome and pro-inflammatory cytokines in PDAC and macrophages. However, MCC950 differentially modified the viability of the metastatic vs primary PDAC cell lines. LPS stimulation increased PDAC cell viability by regulating the NLRP3 inflammasome and pro-inflammatory cytokines in the tumor microenvironment of PDAC cells/macrophages co-cultures. The specific inhibition of the NLRP inflammasome by MCC950 effectively counteracted the LPS-stimulated inflammation.

Expression of proBDNF/p75NTR in peripheral blood lymphocytes of patients with sepsis and its impact on lymphocyte differentiation.

Sepsis is a life-threatening organ dysfunction caused by the host's imbalanced response to infection. Due to lack of effective treatments, it has always been the difficulty and focus of clinical treatment of sepsis. Studies have shown that pro-brain-derived neurotrophic factor (proBDNF) binds to the high-affinity total neurotrophic factor p75 neurotrophin receptor (p75NTR), which activates downstream signaling cascades and disrupts immunological inflammation and plays an important role in the progression of sepsis. This study aims to explore the expression changes of lymphocyte-derived proBDNF/p75NTR in patients with sepsis and its effect on lymphocyte differentiation. From the healthy donors (control group, n=40) and sepsis patients (sepsis group, n=40) admitted to the hospital for the first time, peripheral blood samples and blood routine clinical detection indicators were obtained. By using flow cytometry, the proportion of lymphocyte subsets and their expression of proBDNF/p75NTR were examined. The peripheral blood lymphocytes were isolated from the control group and incubated with lipopolysaccharide (LPS). Flow cytometry analysis technology was used to detect the expression of proBDNF/p75NTR on LPS-treated lymphocyte subsets. On this basis, we investigated the effects on lymphocyte differentiation by inhibiting p75NTR. White blood cell count, neutrophil count, and neutrophil percentage of the patients in the sepsis group at admission were significantly higher than those in the control group; on the contrary, lymphocyte count and lymphocyte percentage in the sepsis group were lower than those in the control group (all P<0.001). The patients in the sepsis group had considerably greater neutrophil/lymphocyte and monocyte/lymphocyte ratios than those in the control group (both P<0.05). In the peripheral blood of sepsis patients, proBDNF expression was upregulated on CD19+ B cells, whereas p75NTR expression was elevated on B cells, CD4+ T cells, and CD8+ T cells (all P<0.05). ProBDNF/p75NTR expression was upregulated by LPS stimulation in vitro in peripheral blood cells of the control group (P<0.05), and this tendency was similar to the expression alterations in peripheral lymphocytes of the sepsis group. Inhibition of p75NTR increased CD4+ T cell and CD19+ B cell percentages, cytokine expression of IL-4 and IL-10, and reduced IL-1β and IL-6 production (all P<0.05). The immunosuppressive state of sepsis patients is indicated by a reduction in lymphocyte count and an increase in the proportion of inactive neutrophils. ProBDNF/p75NTR expression is upregulated in the peripheral blood lymphocytes of sepsis patients, and p75NTR inhibition may control lymphocyte differentiation involved in sepsis progression.

Distinct Cellular, Transcriptional, and Mechanistic Properties Determine HSC Fate during Emergency Granulopoiesis

Emergency granulopoiesis (EG) is the process responsible for the rapid and enhanced production of granulocytes during acute infections. Traditionally, EG was understood as a cellular mechanism mostly initiated and supported by myeloid progenitors, however, recent data suggest that hematopoietic stem cells (HSCs) might also participate in the process. To investigate whether and how HSCs contribute to the initial steps of EG, we performed scRNA-seq and ATAC-seq analysis of sorted murine HSCs 4 hours after in vivo lipopolysaccharide (LPS) administration, mimicking an acute bacterial infection. Strikingly, we observed radical transcriptional changes between HSCs isolated from PBS control and LPS treated mice, mostly marked by alterations in HSC lineage bias. We identified a steady state lymphoid-biased subpopulation of HSCs, marked by expression of Procr (CD201). Following LPS stimulation, the CD201 expression was lost and this population was transcriptionally rewired to a myeloid-biased HSC population. Accordingly, ATACseq data corroborated opening of myeloid-bias loci upon LPS administration rather than steady-state lymphoid-biased loci. Further, we confirmed the loss of CD201 expression in HSCs by flow cytometry in mice after LPS and G-CSF treatment, as well as Candida albicans infection, suggesting that the loss of CD201 expression is a general event during EG induced by different stimuli. Interestingly, the CD201 downregulation in HSCs was independent of the master regulator of EG C/EBPβ, while we observed the contribution of the TLR4-MyD88 signaling axis. Next, we functionally validated our scRNA-seq results in vitro and in vivo. When cultivated under myeloid differentiation conditions, the CD201- HSCs gave rise to mature granulocytes, whereas CD201+ HSCs remained rather immature. In transplantation settings, CD201+ HSCs showed increased engraftment and preferential bias to produce lymphoid cells, while CD201- HSC showed decreased engraftment ability and a bias towards myeloid production. Surprisingly, LPS challenge of BM chimeras transplanted with lymphoid-biased CD201+ HSCs led to EG response marked by rapid loss of lymphoid-biased multipotent progenitors (MPPs) and the expansion of myeloid-biased MPPs, thus confirming the lympho-myeloid switch observed in the scRNA-seq data. Interestingly, the treatment of WT mice with CD201 blocking antibody was able to partially impair the lympho-myeloid switch, suggesting that CD201 plays an active role in HSCs during EG. Mechanistically, we observed that both CD201+ and CD201- HSCs rely on different signaling pathways under EG. While CD201+ HSCs express higher levels of TLR4 and have higher activation of NF-κB signaling upon in vitro LPS stimulation, the CD201- HSCs express higher levels of G-CSF-R and have higher activation of pSTAT3 signaling upon G-CSF-stimulation. Moreover, while the LIP isoform of C/EBPβ, important for cell proliferation, is present in both CD201+ and CD201- HSCs, the LAP/LAP* isoform, important for myeloid differentiation, is present only in the CD201- HSCs fraction. Altogether, our data suggest that EG is supported by a population of HSCs which upon pathogen sensing undergo a radical transcriptional rewiring that promotes their myeloid output. Initially, the pathogen is directly sensed by TLR4 on the surface of a steady state lymphoid-biased CD201+ HSCs, causing a rapid activation of the downstream NF-κB signaling pathway. Subsequently, the lymphoid-myeloid transcriptional switch, marked by the loss of CD201 expression, occurs and EG is then supported by myeloid-biased CD201- HSCs. CD201- HSCs respond to the infection in an indirect manner through G-CSF-R on their surface and exhibit enhanced pSTAT3 activation and elevated LAP/LAP* C/EBPβ isoform, cellular mechanisms known to promote myeloid differentiation and granulocytic production. In conclusion, the switch from CD201+ to CD201- HSCs facilitates both fast and sustained EG leading to the supply of new granulocytes to fight the infection. This detailed understanding of the distinct cellular, transcriptional, and mechanistic properties that determine HSC fate during emergency granulopoiesis opens new venues to potentially modulate granulocytic production in certain clinical conditions such as sepsis. This work was partially supported by a GACR 22-18300S, GAUK 327722, and IMG institutional funding RVO 68378050.

HT-6184 Inhibits the Formation of the NLRP3 Inflammasome in Human Monocytic THP-1 Cells

Background The NLRP3 inflammasome is a multiprotein complex composed of several proteins, including NLRP3, ASC, Nek-7, and pro-caspase-1. Many immune cell populations express the NLRP3 inflammasome. In the presence of various exogenous stimuli such as lipopolysaccharide (LPS) or extracellular ATP, the NLRP3 inflammasome components assemble and become activated, releasing pro-inflammatory cytokines IL-1β and IL-18. One of the critical steps in inflammasome assembly is the recruitment of ASC and the formation of ASC protein aggregates called specks. These specks are large enough to be measured through microscopy, and the ASC speck count indicates NLRP3 activation. The release of IL-1β and IL-18 and the upregulation of NLRP3 inflammasome component genes have been observed in many inflammatory and hematological disorders, suggesting the NLRP3 pathway is involved in the disease pathogenesis of many diseases. Purpose HT-6184 is a small molecule that blocks the assembly of the NLRP3 inflammasome through allosteric binding to the protein Nek7. Here we demonstrate the ability of HT-6184 to modulate NLRP3 inflammasome formation by measuring ASC specks using a human monocytic THP-1 cell line engineered to express a GFP-labeled ASC protein. Methods THP1-ASC-GFP cells were cultured in RPMI 1640 with 10% heat-inactivated FBS, 100 μg/mL normocin, and 100 μg/mL Pen-Strep. Cells were seeded at 400,000 cells per well in a 96-well plate and left overnight. Cells were then treated with vehicle or LPS for 3 hours. Following LPS stimulation, cells were treated with vehicle or HT-6184 for 2 hours. After incubation, cells were treated with 5 μM Nigericin and imaged in 3-minute intervals for 1 hour. The images were analyzed for specks. Results By treating THP1-ASC-GFP with LPS and nigericin, we induced the formation of ASC specks and visualized them using fluorescent microscopy. Cells treated with 200 nM HT-6184 had significantly fewer ASC specks than the LPS and nigericin-treated control. ASC speck inhibition by HT-6184 was dose-dependent, as inhibition of speck formation decreased with lower doses of HT-6184. Conclusions HT-6184 effectively prevents the formation of ASC specks in THP-1-ASC-GFP cells. Since THP-1 cells are a monocytic cell line, this data suggests our ability to inhibit the NLRP3 pathway in hematological disorders effectively. By preventing inflammasome formation and activation, we expect to be able to reduce disease burden in hematological diseases.

Multi-Omic Profiling of Macrophages Lacking Tet2 or Dnmt3a Reveals Mechanisms of Hyper-Inflammation in Clonal Hematopoiesis

Introduction Clonal Hematopoiesis of Indeterminate Potential (CHIP) is a source of age-associated inflammation, caused by somatic mutations in bone marrow stem cells that promote clonal expansion. The functional impact of CHIP mutations on macrophages derived from these stem cells is linked to increased cardiovascular disease risk among other diseases. Interestingly, CHIP-associated mutations frequently occur in genes with opposing functions: DNMT3A and TET2, enzymes responsible for DNA methylation and demethylation, respectively. While it is known that DNMT3A KO and TET2 KO in macrophages lead to hyperinflammatory responses, the underlying mechanism remains poorly understood how opposing mutations lead to a similar phenotype. Methods To evaluate how DNMT3A and TET2 contribute to inflammatory responses in the context of CHIP, we exposed bone marrow derived macrophages from Dnmt3a KO, Tet2 KO, and C57BL/6 Vav1-Cre wild type (WT) control mice to 0.5 ng/mL of the inflammatory stimuli lipopolysaccharide (LPS). We then measured gene responses with RNA-seq replicates across 12 time points from 0 to 24 hours, paired with single cell RNA-seq (scRNA-seq), ATAC-seq, and 50x-coverage whole genome enzymatic methyl-seq (EM-seq) for multiple time points. Results Although the composition and kinetics of the LPS-induced expression program were similar in all genotypes, Dnmt3a KO and Tet2 KO had significantly greater changes in gene expression and chromatin accessibility by 2 hours compared to WT, particularly for a large subset of inflammatory response genes, like Il1b, Il6, Cxcl1, Tnfsf9, and Il23a. The promoter regions of the 278 genes significantly overexpressed (adjusted p &amp;lt; 0.05) for both KOs at 2 hours compared to WT were enriched with binding sites of the inflammatory pioneer factor AP-1 (p = 0.001). Inflammatory signals were observed even before stimulation, suggesting altered DNA methylation in each KO predisposed cells to hyperinflammatory states. Bulk RNA-seq found the gene set ‘IFNA response’ from MSigDB Hallmarks collection to be most enriched at baseline in both KOs compared to WT (FDR &amp;lt; 0.0007). At baseline Dnmt3a KO and Tet2 KO had 1,566 and 441 over-expressed genes, respectively (adjusted p &amp;lt; 0.05). scRNA-seq similarly showed a baseline inflammatory population enriched with type 1 IFN response gene expression that contained more cells with measurable baseline expression of inflammatory genes for both KOs. ATAC-seq identified highly significant enrichment of AP-1 binding sites at baseline and at 2 hours in the 2,270 regions more accessible in Dnmt3a KO compared to WT (p = 1x10 -40), as well as the 2,148 regions less accessible in Tet2 KO compared to WT (p = 1x10 -41). Although LPS stimulation did not lead to sites with altered CpG methylation using a genome-wide significance threshold, methylation globally differed between genotypes at baseline especially for promoter and enhancer regions, and this differential methylation directly explained some of the hyper-responsive chromatin accessibility and gene expression. For example, the promoter region of Cxcl1 for both KOs was significantly hypomethylated and more accessible at baseline, with these chromatin accessibility and associated expression differences becoming even larger at 2 hours of LPS stimulation. In general, DNA methylation and chromatin accessibility were rarely concordantly altered in both KOs at regions near genes with differential expression. For adjusted p-value &amp;lt; 0.05, Dnmt3a KO had 236,062 differentially hypomethylated CpG segments and 1,023 hypermethylated, while Tet2 KO had 7,488 hypomethylated and 4,482 hypermethylated segments. Conclusions Our integrative multi-omic analysis demonstrates that Dnmt3a and Tet2 modulate DNA methylation to limit inflammatory gene expression in healthy macrophages. We find loss of Dnmt3a or Tet2 activity in macrophages alters baseline DNA methylation at different regions, separately influencing the transcription factor binding landscape of AP-1, and inducing similar baseline over-expression of type 1 interferon response genes. Upon stimulation, these differences lead to higher inflammatory gene expression, possibly through more cooperation with transcription factors NF-κB and IRFs. Targeting AP-1 and type 1 interferon may be a therapeutic avenue for blocking the pathogenic role of CHIP for many inflammatory diseases of aging.

Elucidating the Role of Tolerogenic Dendritic Cells in Immune Tolerance and Disease Progression in Multiple Myeloma

Introduction: Previous research has demonstrated that programmed death-ligand 1 (PD-L1) in multiple myeloma (MM) cells not only suppresses anti-tumor immune responses but also enhances malignant potential through the PD-L1-PD-1 interaction [Ishibashi M, et al. Cancer Immunol Res. 2016]. However, it has not yet been shown that monotherapy and combination therapies utilizing PD-1/PD-L1 antibodies are sufficiently effective for MM. One reason for this is the absence of myeloma-specific cytotoxic T lymphocytes induction in the MM tumor microenvironment. In certain cancers, tumor-infiltrating dendritic cells (DCs) undergo conversion into tolerogenic DCs, which are crucial for inducing and maintaining T-cell tolerance, although this remains unclear in MM. Thus, this study aimed to investigate the function and induction mechanisms of tolerogenic DCs in the MM microenvironment. Materials and Methods: (1) Flow cytometry (FCM) was employed to assess the two conventional DC (cDC; lineage negative, HLA-DR +CD11c high) subsets, cDC1 (CD141 + cDC) and cDC2 (CD1c + cDC), in bone marrow (BM) samples obtained from MM patients (n=17) and healthy controls (n=13). (2) CD14 + monocytes were cultured with IL-4 and GM-CSF to generate monocyte-derived DCs (moDCs). Simultaneously, moDCs were co-cultured with MM cell lines using a trans-well system. The characteristics and functions of moDCs were analyzed using real-time PCR, RNA-sequencing (RNA-seq), western blotting, ELISA, and FCM after lipopolysaccharide stimulation. Results: The frequency of cDC1 subsets was significantly lower in MM patients compared to controls, while the major cDC2 subset did not differ. Similarly, when moDCs were co-cultured with MM cells, moDC numbers were markedly reduced. These co-cultured moDCs exhibited downregulated expression of activation markers CD80, CD86, and CD83, along with decreased phosphorylation of ERK and S6K. Conversely, these moDCs upregulated the expression of immunosuppressive factors IL-10, ARG1, and NOS2 through increased transcription factor C/EBPβ. Moreover, these moDCs inhibited T-cell proliferation, activation, and interferon-γ production in comparison to control moDCs, indicating the presence of immature and tolerogenic phenotypes. However, these phenotypes were not observed in moDCs co-cultured with soluble factors secreted from MM and stromal cells. Treatment with AZD3965 improved the phenotype of moDCs co-cultured with MM cells, reversing their suppressive effect on T-cell proliferation. AZD3965 targets monocarboxylate transporter 1 (MCT1) in MM cells, thereby suppressing extracellular lactate accumulation and acidic pH in the MM microenvironment. Furthermore, RNA-seq analysis comparing moDCs co-cultured with and without MM cells revealed downregulation of gene sets associated with activated DCs in moDCs co-cultured with MM. Notably, moDCs co-cultured with MM showed high enrichment of gene sets related to lysosome biogenesis and NOD-like receptor signaling. This suggests that inflammasome activation in these moDCs leads to caspase-1 activation, resulting in increased levels of mature IL-1β and IL-18 in their cell culture supernatants. Additionally, co-culturing CD38-positive MM cells with CD203a/CD73-positive HS-5 stromal cells and NAD + resulted in the generation of adenosine in the cell culture supernatant. Adenosine promotes the immaturation of mature DCs; however, inflammasome activation does not occur in these DCs. Conclusions: Patients with MM exhibit a reduced frequency of cDC1 subsets, which are crucial for cross-presentation to CD8 + T cells. moDCs in acid pH conditions derived from MM cells display an immature phenotype and impaired function due to decreased DC activation signals. Furthermore, inflammasome activation promotes the secretion of IL-1β and IL-18, contributing to pro-tumorigenic inflammation in the MM microenvironment. Moreover, adenosine generated via the CD38/CD203a/CD73 adenosinergic pathway promotes the immaturation and dysfunction of moDCs. Thus, the characteristics of tolerogenic DCs may vary depending on the MM microenvironment. These findings highlight the induction of tolerogenic DCs by the metabolic microenvironment in MM, leading to immune tolerance and disease progression.

Effects of dietary N-carbamylglutamate supplementation on the modulation of microbiota and Th17/Treg balance-related immune signaling after lipopolysaccharide challenge.

This study aimed to evaluate the effects of N-carbamylglutamate (NCG) on piglets' growth performance and immune response, and to unravel the mechanisms of such effects. In a 2 × 2 factorial design including diet (with or without NCG) and immunological challenge (saline or lipopolysaccharide (LPS)), 24 piglets were randomly distributed into four groups. After being fed a basic diet or a NCG-supplemented diet for 21 days, piglets were administered LPS or saline intraperitoneally. The results showed that NCG increased the average daily gain and average daily feed intake, and the feed conversion ratio of piglets, and alleviated the adverse effects of LPS stimulation on intestinal morphology. At the phylum level, NCG reversed the increase in the abundance of Firmicutes and the reduction in that of Actinomycete caused by LPS stimulation. At the genus level, NCG increased the abundance of Lactobacillus, Blautia, norank_Butyricicoccaceae, Subdoligranulum, and Ruminococcus_gauvreauii_group, and LPS decreased the abundance of Escherichia-Shigella and Ruminococcus_gauvreauii_group. The short-chain fatty acid content was increased by NCG, but LPS reduced its content. N-Carbamylglutamate also inhibited significantly the LPS-induced increase in the relative expression of signal transducer and activator of transcription (STAT) 3, related orphan receptor (RAR) c, and pro-inflammatory cytokines, and the decrease in the relative expression of STAT5, forkhead box P3, IL-10 and transforming growth factor beta 1 mRNA. A significant correlation was found between intestinal microbiota and inflammatory cytokines and short-chain fatty acids. N-Carbamylglutamate can improve piglets' growth performance. It can also attenuate LPS-induced intestinal inflammation by modulating microbiota and Th17/Treg balance-related immune signaling pathways. © 2023 Society of Chemical Industry.

Accelerated Oral Healing by Angelica gigas Nakai from Hot Melt Extrusion Technology: An In Vitro Study.

Background and Objectives: In spite of the oral environment being healing-prone, its dynamic changes may affect wound healing. The purpose of this study was to assess the oral wound healing effect of Angelica gigas Nakai (AG) prepared by hot-melt extrusion. Materials and Methods: Human gingival fibroblast (HGF) cells were treated with AG or AG via hot-melt extrusion (AGH) for 24 h to determine the optimal concentration. For evaluating the anti-inflammatory effect of AG and AGH, a nitric oxide assay was performed under lipopolysaccharide (LPS) stimulation. The wound-healing effects of AG and AGH were evaluated using cell proliferation/migration assays and wound-healing marker expression through qRT-PCR. Results: Both AG and AGH showed no cytotoxicity on HGH cells. Regarding nitric oxide production, AGH significantly decreased LPS-induced nitric oxide production (p < 0.05). AGH showed a significantly positive result in the cell proliferation/cell migration assay compared with that in AG and the control. Regarding wound healing marker expression, AGH showed significantly greater VEGF and COL1α1 expression levels than those in the others (p < 0.05), whereas α-SMA expression was significantly different among the groups. Conclusions: Within the limits of this study, AGH accelerated oral wound healing in vitro.

Molecular characterization, expression analysis and subcellular location of the members of STAT family from spotted seabass (Lateolabrax maculatus)

The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway is a pervasive intracellular signal transduction pathway, involving in biological processes such as cell proliferation, differentiation, apoptosis and immune regulation. In this study, seven STAT genes, STAT1, STAT1-like, STAT2, STAT3, STAT4, STAT5a and STAT5b, were identified and characterized in spotted seabass (Lateolabrax maculatus). Analyses of multiple sequence alignment, genomic organization, phylogeny and conserved synteny were conducted to infer the evolutionary conservation of these genes in the STAT family. The results of the bioinformatics analysis assumed that STAT1 and STAT1-like might be homologous to STAT1a and STAT1b, respectively. Furthermore, the expression of the seven genes were detected in eight tissues of healthy spotted seabass, which revealed that they were expressed in a variety of tissues, mainly in gill, spleen and muscle, and extremely under-expression in liver. The expression of the seven genes in gill, head-kidney, spleen and intestine were significantly induced by lipopolysaccharide (LPS) or Edwardsiella tarda challenge. The expression of most of the LmSTATs were up-regulated, and the highest expression levels at 12 h after LPS stimulation, however, the LmSTATs were down-regulated by E. tarda infection. The results of subcellular localization show that the native LmSTAT1, LmSTAT1-like, LmSTAT2, LmSTAT3 and LmSTAT5a were localized in the cytoplasm, but they were translocated into the nucleus after LPS stimulation. Whereas, LmSTAT4 and LmSTAT5b were translocation into the nucleus whether with LPS stimulation or not. Overall, this is the first study to systematically revealed the localization of STAT members in fish, and indicated that LmSTATs participate in the process of protecting the host from pathogens invasion in the form of entry into nucleus.

Bisdemethoxycurcumin alleviates LPS-induced acute lung injury via activating AMPKα pathway

ObjectiveInflammation and oxidative stress contribute to the pathogenesis of acute lung injury (ALI), and subsequently result in rapid deterioration in health. Considering the indispensable role of bisdemethoxycurcumin (BDMC) in inflammation and oxidative stress, the present study aims to examine the effect of BDMC on sepsis-related ALI.MethodsC57BL/6 mice were administered with BDMC (100 mg/kg) or an equal volume of vehicle, and then injected with lipopolysaccharides (LPS) to induce ALI. We assessed the parameters of lung injury, inflammatory response and oxidative stress in lung tissues. Consistently, the macrophages with or without BDMC treatment were exposed to LPS to verify the effect of BDMC in vitro.ResultsBDMC suppressed LPS-induced lung injury, inflammation and oxidative stress in vivo and in vitro. Mechanistically, BDMC increased the phosphorylation of AMPKα in response to LPS stimulation, and AMPK inhibition with Compound C almost completely blunted the protective effect of BDMC in LPS-treated mice and macrophages. Moreover, we demonstrated that BDMC activated AMPKα via the cAMP/Epac pathway.ConclusionOur study identifies the protective effect of BDMC against LPS-induced ALI, and the underlying mechanism may be related to the activation of cAMP/Epac/AMPKα signaling pathway.

Multi-omics analysis reveals the molecular regulatory network underlying the prevention of Lactiplantibacillus plantarum against LPS-induced salpingitis in laying hens

BackgroundSalpingitis is one of the common diseases in laying hen production, which greatly decreases the economic outcome of laying hen farming. Lactiplantibacillus plantarum was effective in preventing local or systemic inflammation, however rare studies were reported on its prevention against salpingitis. This study aimed to investigate the preventive molecular regulatory network of microencapsulated Lactiplantibacillus plantarum (MLP) against salpingitis through multi-omics analysis, including microbiome, transcriptome and metabolome analyses.ResultsThe results revealed that supplementation of MLP in diet significantly alleviated the inflammation and atrophy of uterus caused by lipopolysaccharide (LPS) in hens (P < 0.05). The concentrations of plasma IL-2 and IL-10 in hens of MLP-LPS group were higher than those in hens of LPS-stimulation group (CN-LPS group) (P < 0.05). The expression levels of TLR2, MYD88, NF-κB, COX2, and TNF-α were significantly decreased in the hens fed diet supplemented with MLP and suffered with LPS stimulation (MLP-LPS group) compared with those in the hens of CN-LPS group (P < 0.05). Differentially expressed genes (DEGs) induced by MLP were involved in inflammation, reproduction, and calcium ion transport. At the genus level, the MLP supplementation significantly increased the abundance of Phascolarctobacterium, whereas decreased the abundance of Candidatus_Saccharimonas in LPS challenged hens (P < 0.05). The metabolites altered by dietary supplementation with MLP were mainly involved in galactose, uronic acid, histidine, pyruvate and primary bile acid metabolism. Dietary supplementation with MLP inversely regulates LPS-induced differential metabolites such as LysoPA (24:0/0:0) (P < 0.05).ConclusionsIn summary, dietary supplementation with microencapsulated Lactiplantibacillus plantarum prevented salpingitis by modulating the abundances of Candidatus_Saccharimonas, Phascolarctobacterium, Ruminococcus_torques_group and Eubacterium_hallii_group while downregulating the levels of plasma metabolites, p-tolyl sulfate, o-cresol and N-acetylhistamine and upregulating S-lactoylglutathione, simultaneously increasing the expressions of CPNE4, CNTN3 and ACAN genes in the uterus, and ultimately inhibiting oviducal inflammation.

Aitongping patch could alleviate cancer pain via suppressing microglia activation and modulating the miR-150-5p/CXCL12 signaling.

We aimed to investigate the pharmacological effects and mechanisms of the Aitongping formula for treating cancer pain. We enrolled 60 cancer patients with Numeric Rating Scale above 4 and grouped them randomly as a Control group (N= 30) and a Patch group (N= 30). We also established bone cancer mice models via tumor implantation. And the animal groups were established as a Sham group, a tumor cell implantation (TCI) group, a TCI + Patch group, and a Patch group. After the validation of successful tumor implantation, we identified candidate miRNAs and genes that were dysregulated in TCI mice and compared their expressions between different mice groups. We also observed the effect of Aitongping patch in vitro in mice primary microglia. The time to disease progression and cancer stability were prolonged by Aitongping patch in cancer patients. And the daily morphine dose was lower, and patients' quality of life was improved in the Patch group. Moreover, Aitongping patch alleviated cancer pain and inhibited microglia activation after the successful implantation of bone tumor in TCI mice. We also observed the dysregulation of miR-150-5p and chemokine CXC motif ligand 12 (CXCL12) mRNA in TCI mice. And CXCL12 was found to be targeted by miR-150-5p. Aitongping patch was found to upregulate miR-150-5p and downregulate CXCL12 in vivo and in vitro. Aitongping patch could alleviate cancer pain via suppressing microglia activation, and the downregulation of miR-150-5p, as well as the upregulation of CXCL12 mRNA and protein, induced by tumor implantation or lipopolysaccharide stimulation, was restored by Aitongping treatment.

Proteomic Profiling of Early Secreted Proteins in Response to Lipopolysaccharide-Induced Vascular Endothelial Cell EA.hy926 Injury.

Sepsis is a crucial public health problem with a high mortality rate caused by a dysregulated host immune response to infection. Vascular endothelial cell injury is an important hallmark of sepsis, which leads to multiple organ failure and death. Early biomarkers to diagnose sepsis may provide early intervention and reduce risk of death. Damage-associated molecular patterns (DAMPs) are host nuclear or cytoplasmic molecules released from cells following tissue damage. We postulated that DAMPs could potentially be a novel sepsis biomarker. We used an in vitro model to determine suitable protein-DAMPs biomarkers for early sepsis diagnosis. Low and high lipopolysaccharide (LPS) doses were used to stimulate the human umbilical vein endothelial cell line EA.hy926 for 24, 48, and 72 h. Results showed that cell viability was reduced in both dose-dependent and time-dependent manners. Cell injury was corroborated by a significant increase in lactate dehydrogenase (LDH) activity within 24 h in cell-conditioned medium. Secreted protein-DAMPs in the supernatant, collected at different time points within 24 h, were characterized using shotgun proteomics LC-MS/MS analysis. Results showed that there were 2233 proteins. Among these, 181 proteins from the LPS-stimulated EA.hy926 at 1, 12, and 24 h were significantly different from those of the control. Twelve proteins were up-regulated at all three time points. Furthermore, a potential interaction analysis of predominant DAMPs-related proteins using STITCH 5.0 revealed the following associations with pathways: response to stress; bacterium; and LPS (GO:0080134; 0009617; 0032496). Markedly, alpha-2-HS-glycoprotein (AHSG or fetuin-A) and lactotransferrin (LTF) potentially presented since the first hour of LPS stimulation, and were highly up-regulated at 24 h. Taken together, we reported proteomic profiling of vascular endothelial cell-specific DAMPs in response to early an in vitro LPS stimulation, suggesting that these early damage-response protein candidates could be novel early biomarkers associated with sepsis.

TMEM16A deficiency in alveolar type 2 epithelial cells protected against endoplasmic reticulum stress-induced ferroptosis during acute lung injury

Transmembrane protein 16A (TMEM16A) is one of the members of the ten-member family of “transmembrane protein 16”, playing critical roles in infection and solid organ injury. Acute lung injury (ALI) is a devastating disease which could be triggered by sepsis, trauma, and ischemia reperfusion. However, molecular mechanisms contributing to ALI are poorly understood at presently. In this study, we investigated the role of TMEM16A in sepsis-induced ALI using TMEM16A-deficient mice. Sepsis-induced ALI model was established by intratracheal injection of lipopolysaccharide (LPS). Our results showed that LPS stimulation significantly upregulated the expression levels of TMEM16A in lung tissues and in alveolar epithelial type II (AT2) cells. Knockout of TMEM16A in AT2 cells significantly improved pulmonary function and alleviated lung pathological injury in LPS-treated mice. Meanwhile, TMEM16A deficiency also inhibited endoplasmic reticulum (ER) stress and ferroptosis in AT2 cells from LPS-treated mice. In vitro experiments further demonstrated that ER stress and ferroptosis were inhibited after TMEM16A was knocked out. Furthermore, we used ER stress inducer thapsigargin to induce ER stress in TMEM16A-null AT2 cells and found that the induction of ER stress abolished the inhibition of ferroptosis by TMEM16A deficiency in LPS-treated AT2 cells. Finally, we disclosed that pharmacological inhibition of TMEM16A by shikonin also showed similar therapeutic effect on LPS-induced ALI in vivo. In conclusion, TMEM16A deficiency in AT2 cells could alleviate sepsis-induced ALI by decreasing ER stress-induced ferroptosis during ALI.

Myristoylated, alanine-rich C-kinase substrate (MARCKS) regulates toll-like receptor 4 signaling in macrophages

MARCKS (myristoylated alanine-rich C-kinase substrate) is a membrane-associated protein expressed in many cell types, including macrophages. MARCKS is functionally implicated in cell adhesion, phagocytosis, and inflammation. LPS (lipopolysaccharide) triggers inflammation via TLR4 (toll-like receptor 4).The presence of MARCKS and the formation of phospho-MARCKS in various cell types have been described, but the role(s) of MARCKS in regulating macrophage functions remain unclear. We investigated the role of MARCKS in inflammation. Confocal microscopy revealed that MARCKS and phospho-MARCKS increased localization to endosomes and the Golgi apparatus upon LPS stimulation.CRISPR-CAS9 mediated knockout of MARCKS in macrophages downregulated the production of TNF and IL6, suggesting a role for MARCKS in inflammatory responses. Our comprehensive proteomics analysis together with real-time metabolic assays comparing LPS-stimulation of WT and MARCKS knock-out macrophages provided insights into the involvement of MARCKS in specific biological processes including innate immune response, inflammatory response, cytokine production, and molecular functions such as extracellularly ATP-gated cation channel activity, electron transfer activity and oxidoreductase activity, uncovering specific proteins involved in regulating MARCKS activity upon LPS stimulation. MARCKS appears to be a key regulator of inflammation whose inhibition might be beneficial for therapeutic intervention in inflammatory diseases.

Transcriptomic analysis reveals that RasGEF1b deletion alters basal and LPS-induced expression of genes involved in chemotaxis and cytokine responses in macrophages

Ras guanine nucleotide exchange factor member 1b (RasGEF1b) of the RasGEF/CDC25 domain-containing family is preferentially expressed by macrophages. However, information is lacking about its role in macrophage function. In this study, we generated mice with ubiquitous deletion of Rasgef1b and used RNA-seq-based transcriptomics to compare the global gene expression in wild-type and knock-out primary bone-marrow-derived macrophages under basal conditions and after lipopolysaccharide (LPS) treatment. Transcriptional filtering identified several genes with significantly different transcript levels between wild-type and knock-out macrophages. In total, 49 and 37 differentially expressed genes were identified at baseline and in LPS-activated macrophages, respectively. Distinct biological processes were significantly linked to down-regulated genes at the basal condition only, and largely included chemotaxis, response to cytokines, and positive regulation of GTPase activity. Importantly, validation by RT-qPCR revealed that the expression of genes identified as down-regulated after LPS stimulation was also decreased in the knock-out cells under basal conditions. We used a luciferase-based reporter assay to showcase the capability of RasGEF1b in activating the Serpinb2 promoter. Notably, knockdown of RasGEF1b in RAW264.7 macrophages resulted in impaired transcriptional activation of the Serpinb2 promoter, both in constitutive and LPS-stimulated conditions. This study provides a small collection of genes that shows relative expression changes effected by the absence of RasGEF1b in macrophages. Thus, we present the first evidence that RasGEF1b mediates the regulation of both steady-state and signal-dependent expression of genes and propose that this GEF plays a role in the maintenance of the basal transcriptional level in macrophages.

The expression, function, and network regulation of circDNAJB6 in chicken macrophages under lipopolysaccharide (LPS) stimulation

Circular RNAs (circRNA) originate from back-splicing events that link a downstream 5'splice site to an upstream 3′ splice site. Circular RNA has been shown to be involved in gene expression, interacting with microRNA and RNA binding proteins to affect transcription, splicing, translation, and other processes. However, little is known about the potential function of chicken circRNAs that trigger the pathogenesis. In a previous study, a circular RNA DNAJB6 (circDNAJB6) was identified as a typical covalently closed circular RNA that is abundant in chicken macrophages upon bacterial infection. It was identified that circDNAJB6 was formed by reverse splicing of exons 2 to 5 of the DNAJB6 gene by PCR amplification, Sanger sequencing, and RNase R exonuclease treatment. Moreover, circDNAJB6 had ability to exacerbate the lipopolysaccharides (LPS) induced cellular injury via reducing cell viability, increasing NO product and pro-inflammatory cytokines. In addition, bioinformatic analysis showed that five miRNAs were identified to interact with circDNAJB6, potentially targeting 75 genes, which were significantly enriched in the pathways of autophagy-animal and MAPK signaling. This study has provided and broadened a better understanding the function of circDNAJB6, which may exert potential biomarkers and act as potential targets for the treatment of bacterial infection.

Chronic Allergen Exposure Contributes to Steroid Resistance via Increased Phosphorylation of Glucocorticoid Receptors S226 and p38 MAPK in a Mouse Model of Asthma

Chronic allergen exposure can significantly induce p38 mitogen-activated protein kinase (MAPK) activation in asthma. p38 MAPK is involved in steroid resistance through phosphorylation of glucocorticoid receptors (GR) at S226. This study aims to investigate whether chronic allergen exposure can induce steroid resistance and whether it is associated with p38 MAPK activation in asthma.&#x0D; A mouse model of asthma was prepared by sensitizing and challenging mice with chronic ovalbumin (OVA) exposure. Key features of allergic asthma, encompassing bronchial hyperresponsiveness, pathology of lung tissues, cytokine profiles of inflammation in bronchoalveolar lavage fluid (BALF), and serum immunoglobulin (Ig)E concentration were evaluated. Furthermore, suppressive effects of corticosteroid on the splenocytes under stimulation of lipopolysaccharides, glucocorticoid receptor (GR) DNA binding ability of splenocytes, expression of GRα and phosphorylation of GR s226 in splenocytes, and p38 MAPK phosphorylation in splenocytes and lung tissues were determined.&#x0D; Chronic OVA exposure substantially induced airway hypersensitivity, leading to increased inflammatory infiltration in lung tissues. Additionally, it resulted in elevated levels of interleukin (IL)-4, IL-5, and IL-6 in BALF, as well as heightened levels of IgE in serum. Furthermore, OVA exposure substantially enhanced p38 MAPK phosphorylation in lung tissues. It also weakened the suppressive impacts of corticosteroids on splenocytes, impaired the GR DNA binding ability, and led to an enhanced phosphorylated state of GR S226 and p38 MAPK in splenocytes.&#x0D; Taken together, chronic allergen exposure contributes to steroid resistance in asthma, which is linked to an increased phosphorylated state of GR S226 and p38 MAPK.&#x0D;

High-Content Image-Based Screening and Deep Learning for the Detection of Anti-Inflammatory Drug Leads.

We developed a high-content image-based screen that utilizes the pro-inflammatory stimulus lipopolysaccharide (LPS) and murine macrophages (RAW264.7) with the goal of enabling the identification of novel anti-inflammatory lead compounds. We screened 2,259 bioactive compounds with annotated mechanisms of action (MOA) to identify compounds that block the LPS-induced phenotype in macrophages. We utilized a set of seven fluorescence microscopy probes to generate images that were used to train and optimize a deep neural network classifier to distinguish between unstimulated and LPS-stimulated macrophages. The top hits from the deep learning classifier were validated using a linear classifier trained on individual cells and subsequently investigated in a multiplexed cytokine secretion assay. All 12 hits significantly modulated the expression of at least one cytokine upon LPS stimulation. Seven of these were allosteric inhibitors of the mitogen-activated protein kinase kinase (MEK1/2) and showed similar effects on cytokine expression. This deep learning morphological assay identified compounds that modulate the innate immune response to LPS and may aid in identifying new anti-inflammatory drug leads.