LPS-treated Mouse Macrophages Research Articles

Transcriptome analysis reveals modulation of differentially expressed genes in LPS-treated mouse macrophages (RAW264.7cells) by grouper (Epinephelus coioides) Epinecidin-1.

The marine antimicrobial peptide Epinecidin (Epi)-1 has been shown to exert direct antimicrobial and immunomodulatory actions in teleost, mammalian and avian organisms. For instance, Epi-1 can suppress bacterial endotoxin lipolysachcharide (LPS)-induced proinflammatory cytokines in RAW264.7 murine macrophages. However, it remains unknown how Epi-1 might broadly affect non-activated and LPS-activated macrophages. To address this question, we performed a comparative transcriptomic analysis of non-treated and LPS-treated RAW264.7cells in the presence and absence of Epi-1. Gene enrichment analysis was conducted on filtered reads, followed by GO and KEGG analyses. The results showed that Epi-1 treatment modulated pathways and genes associated with nucleoside binding, intramolecular oxidoreductase activity, GTPase activity, peptide antigen binding, GTP binding, ribonucleoside/nucleotide binding, phosphatidylinositol binding and phosphatidylinositol-4-phosphate binding. Based on the GO analysis results, we performed real-time PCR at different treatment times to compare expression levels of selected proinflammatory cytokines, anti-inflammatory cytokines, MHC, proliferation and differentiation genes. Epi-1 decreased expression of the proinflammatory cytokines, TNF-α, IL-6 and IL-1β, and it increased the anti-inflammatory cytokine TGFβ and Sytx1. MHC-associated genes, GM7030, Arfip1, Gpb11 and Gem, were induced by Epi-1, which is expected to enhance the immune response against LPS. Immunoglobulin-associated Nuggc was also upregulated by Epi-1. Finally, we found that Epi-1 downregulated the expression of host defense peptides CRAMP, Leap2 and BD3. Taken together, these findings suggest that Epi-1 treatment induces orchestrated changes in the transcriptome of LPS-stimulated RAW264.7cells.

Streptococcus thermophilus Attenuates Inflammation in Septic Mice Mediated by Gut Microbiota.

Sepsis is a life-threatening organ dysfunction condition caused by a dysregulated host response to infection and lack of effective treatment method. Supplementation of probiotics has emerged as a potential biotherapy for inflammatory diseases in recent years, but its role in protecting viscera against the damage caused by sepsis and the underlying mechanism is poorly understood. Streptococcus thermophilus 19 is one of the most well-studied probiotics, which is selected in this study among seven strains isolated from homemade yogurt due to its optimal ability of suppressing the inflammation response in vitro. It showed significant decrease in the expression of TNF-α, IL-1β, and IL-6 in the co-culture of S. thermophilus 19 and LPS-treated mouse macrophage. The effect of S. thermophilus 19 in mice and the response of mice gut microbiota were subsequently investigated. In LPS-induced septic mouse model, S. thermophilus 19 was highly resistant to LPS and exhibited significantly decreased expressions of inflammatory factors compared to LPS-treated mice. A MiSeq-based 16S rDNA sequence analysis revealed that the decrease of gut microbial diversity in mice intraperitoneally injected with 1 mg/ml LPS were mitigated by the administration of S. thermophilus 19. Fusobacterium significantly decreased during the development of sepsis and rose again after supplement strain 19, while Flavonifractor showed the opposite trend, which demonstrated these two genera were the key bacteria that may function in the mice gut microbiota for alleviation of LPS-induced inflammation reaction. To conclude, S. thermophilus 19 may be a potential candidate for novel biotherapeutic interventions against inflammation caused by sepsis.

Miniaturized Paper-Supported 3D Cell-Based Electrochemical Sensor for Bacterial Lipopolysaccharide Detection.

Sensitive detection of lipopolysaccharides (LPSs), which are present on the outer wall of Gram-negative bacteria, is important to reflect the degree of bacterial contamination in food. For indirect assessment of the LPS content, a miniaturized electrochemical cell sensor consisting of a screen-printed paper electrode, a three-dimensional cells-in-gels-in-paper culture system, and a conductive jacket device was developed for in situ detection of nitric oxide released from LPS-treated mouse macrophage cells (Raw264.7). Nafion/polypyrrole/graphene oxide with excellent selectivity, high conductivity, and good biocompatibility functionalized on the working electrode via electrochemical polymerization could enhance sensing. Raw264.7 cells encapsulated in the alginate hydrogel were immobilized on a Nafion/polypyrrole/graphene oxide/screen-printed carbon electrode in paper fibers as a biorecognition element. Differential impulse voltammetry was employed to record the current signal as-influenced by LPS. Results indicated that LPS from Salmonella enterica serotype Enteritidis caused a significant increase in peak current, varying from 1 × 10-2 to 1 × 104 ng/mL, dose-dependently. This assay had a detection limit of 3.5 × 10-3 ng/mL with a linear detection range of 1 × 10-2 to 3 ng/mL. These results were confirmed by analysis of nitric oxide released from Raw264.7 via the Griess method. The miniaturized sensor was ultimately applied to detect LPSs in fruit juice samples. The results indicated that the method exhibited high recovery and relative standard deviation lower than 2.65% and LPSs in samples contaminated with 102-105 CFU/mL bacteria could be detected, which proved the practical value of the sensor. Thus, a novel, low-cost, and highly sensitive approach for LPS detection was developed, providing a method to assess Gram-negative bacteria contamination in food.

<i>Streptococcus thermophilus</i> Attenuates Inflammation in Septic Mice Mediated by Gut Microbiota

Sepsis is a life-threatening organ dysfunction symptom caused by a dysregulated host response to infection and lack of effective treatment method. Supplementation of probiotic is emerged as a potential biotherapy for inflammatory diseases recent years, but its role in protecting viscera against the damage caused by sepsis and the underlying mechanism is poorly understood. Streptococcus thermophilus 19 is one of the well-studied probiotics, which is selected in this study among seven strains isolated from homemade yogurt since its optimal ability of suppressing the inflammation response in vitro. It showed significant decrease the expression of TNF-α, IL-1β and IL-6 in the co-culture of S. thermophilus 19 and LPS-treated mouse macrophage. The effect of S. thermophilus 19 in mice and the response of mice gut microbiota were investigated subsequently. In LPS-induced septic mouse model, S. thermophilus 19 was highly resistant to LPS and exhibited significantly decreased expressions of inflammatory factors compared to LPS-treated mice. A MiSeq-based 16S rDNA sequence analysis revealed that the decrease of gut microbial diversity in mice intraperitoneally injected with 1 mg/ml LPS were mitigated by the administration of S. thermophilus 19. Fusobacterium significantly decreased during the development of sepsis and raised again after supplement strain 19 and while Flavonifractor showed opposite trend, which demonstrated these two genera were the key bacteria may function in the mice gut microbiota for alleviation of LPS-induced inflammation reaction. To conclude, S. thermophilus 19 may be a potential candidate for novel biotherapeutic interventions product against inflammation caused by sepsis. Funding Statement: This work was supported by National Natural Science Foundation of China (81530064, 81801913, 81601680 and 81701902). Declaration of Interests: The authors declare that they have no conflict of interests Ethics Approval Statement: All procedures and protocols used in this study conform to the institutional guidelines and were approved by the Ethics Committee of the Fourth Military Medical University.

Effects of shokyo (Zingiberis Rhizoma) and kankyo (Zingiberis Processum Rhizoma) on prostaglandin E2 production in lipopolysaccharide-treated mouse macrophage RAW264.7 cells.

We previously reported that shokyo and kankyo, which are water-extracted fractions of ginger, reduced LPS-induced PGE2 production in human gingival fibroblasts. In this study, we examined the effects of these herbs on LPS-treated mouse macrophage RAW264.7 cells. Both shokyo and kankyo reduced LPS-induced PGE2 production in a concentration-dependent manner. Shokyo and kankyo did not inhibit cyclooxygenase (COX) activity, nor did they alter the expression of molecules in the arachidonic acid cascade. In addition, these herbs did not alter NF-κB p65 translocation into nucleus, or phosphorylation of p65 or ERK. These results suggest that shokyo and kankyo inhibit cPLA2 activity. Although 6-shogaol produced similar results to those of shokyo and kankyo, the concentration of 6-shogaol required for the reduction of PGE2 production were higher than those of 6-shogaol in shokyo and kankyo. Therefore, several gingerols and shogaols other than 6-shogaol may play a role in the reduction of LPS-induced PGE2 production. Thus, 6-shogaol, and other gingerols and shogaols inhibit cPLA2 activity and reduce LPS-induced PGE2 production via a different mechanism from traditional anti-inflammatory drugs. Moreover, kampo medicines that contain shokyo or kankyo are considered to be effective for inflammatory diseases.

Induction of inducible nitric oxide synthase (iNOS) in Porphyromonas gingivalis LPS-treated mouse macrophage cell line (RAW264.7) requires Toll-like receptor 9.

The aim of this study is to investigate the involvement of TLR9 in the regulation of iNOS expression and nitric oxide (NO) production in Porphyromonas gingivalis LPS-treated mouse macrophages. Mouse macrophage cell line (RAW264.7) was transfected with siRNAs against TLR9 and then stimulated with P. gingivalis LPS. At indicated time points, the activated cells were lysed. Gene and protein expression of iNOS were determined by RT-PCR and immunoblotting, respectively. The level of nitric oxide (NO) production in the supernatant of the activated cells was determined by Griess reaction assay. Depletion of TLR9 in mouse macrophages demonstrated the markedly decreased iNOS gene and protein expression by P. gingivalis LPS compared to those of the wild-type or control siRNA transfected cells. In consistent with these results, the level of NO secretion was also significantly diminished in TLR9-depleted cells after challenged with P. gingivalis LPS. These results indicate that TLR9 involves in the regulation of the iNOS expression and the NO secretion in P. gingivalis LPS-treated macrophages.

IFN Regulatory Factor 2 Inhibits Expression of Glycolytic Genes and Lipopolysaccharide-Induced Proinflammatory Responses in Macrophages.

Rapid initiation and timely resolution of inflammatory response in macrophages are synergistic events that are known to be equally critical to optimal host defense against pathogen infections. However, the regulation of these processes, in particular by a specific cellular metabolic program, has not been well understood. In this study, we found that IFN regulatory factor 2 (IRF2) underwent an early degradation in a proteasome-mediated pathway in LPS-treated mouse macrophages, followed by a later recovery of the expression via transactivation. We showed that IRF2 was anti-inflammatory in that knockdown of this protein promoted the production of LPS-induced proinflammatory mediators. Mechanistically, although IRF2 apparently did not target the proximal cytoplasmic signaling events upon LPS engagements, it inhibited HIF-1α-dependent expression of glycolytic genes and thereby cellular glycolysis, sequential events necessary for the IRF2 anti-inflammatory activity. We found that macrophages in endotoxin tolerant state demonstrated deficiency in LPS-augmented glycolysis, which was likely caused by failed downregulation of IRF2 and the ensuing upregulation of the glycolytic genes in these cells. In contrast to observations with LPS, knockdown of IRF2 decreased IL-4-induced macrophage alternative activation. The pro-IL-4 activity of IRF2 was dependent on KLF4, a key mediator of the alternative activation, which was transcriptionally induced by IRF2. In conclusion, our data suggest that IRF2 is an important regulator of the proinflammatory response in macrophages by controlling HIF-1α-dependent glycolytic gene expression and glycolysis. This study also indicates IRF2 as a novel therapeutic target to treat inflammatory disorders associated with dysregulations of macrophage activations.

Resveratrol ‐ induced Browning of White Adipose Tissue: Macrophages in the Loop

Brown adipose tissue has been a recent target for obesity prevention and therapy. Identification of UCP1‐positive 'beige' adipocytes in the white adipose tissue (WAT) upon exposure to cold led multiple studies to investigate the mechanisms involved in the 'browning' of WAT. Adipose tissue microenvironment is comprised of large number of macrophages in obese than in lean individuals. However, the relative contribution of macrophages to the pathogenesis of obesity is not clearly understood. Recently, the thermogenic beige fat circuitry was identified to contain alternatively activated macrophages (M2‐Macrophages). Furthermore, catecholamine production by M2‐macrophages has been implicated in adaptive thermogenesis. In the current study, we investigated the novel mechanism through which a well‐studied anti‐obesity phytochemical resveratrol (RSV) induces browning of WAT. We observed that RSV significantly increased IL10 secretion; a signature cytokine of M2‐macrophages, at both transcriptional and translational level in LPS‐treated mouse macrophage cell line (RAW264.7). Further, RSV rescued RAW264.7 cells from LPS‐induced cell death. Additionally, RSV induced catecholamine secretion by RAW264.7 cells through upregulation of tyrosine hydroxylase in a time and dose dependent manner. Taken together, these studies suggest that RSV‐induced change in the polarity of macrophages leading to elevated levels of catecholamines will induce browning of WAT and contributes to the anti‐obesity effects of RSV. This work was supported by the CSO Intramural Funds from the Philadelphia College of Osteopathic Medicine ‐ School of Pharmacy.

Lipopolysaccharide Induces Anandamide Synthesis in Macrophages via CD14/MAPK/Phosphoinositide 3-Kinase/NF-κB Independently of Platelet-activating Factor

Macrophage-derived endocannabinoids have been implicated in endotoxin (lipopolysaccharide (LPS))-induced hypotension, but the endocannabinoid involved and the mechanism of its regulation by LPS are unknown. In RAW264.7 mouse macrophages, LPS (10 ng/ml) increases anandamide (AEA) levels >10-fold via CD14-, NF-kappaB-, and p44/42-dependent, platelet-activating factor-independent activation of the AEA biosynthetic enzymes, N-acyltransferase and phospholipase D. LPS also induces the AEA-degrading enzyme fatty acid amidohydrolase (FAAH), and inhibition of FAAH activity potentiates, whereas actinomycin D or cycloheximide blocks the LPS-induced increase in AEA levels and N-acyltransferase and phospholipase D activities. In contrast, cellular levels of the endocannabinoid 2-arachidonoylglycerol (2-AG) are unaffected by LPS but increased by platelet-activating factor. LPS similarly induces AEA, but not 2-AG, in mouse peritoneal macrophages where basal AEA levels are higher, and the LPS-stimulated increase in AEA is potentiated in cells from FAAH-/- as compared with FAAH+/+ mice. Intravenous administration of 107 LPS-treated mouse macrophages to anesthetized rats elicits hypotension, which is much greater in response to FAAH-/- than FAAH+/+ cells and is susceptible to inhibition by SR141716, a cannabinoid CB1 receptor antagonist. We conclude that AEA and 2-AG synthesis are differentially regulated in macrophages, and AEA rather than 2-AG is a major contributor to LPS-induced hypotension.

Triptolide, a novel diterpenoid triepoxide from Tripterygium wilfordii Hook. f., suppresses the production and gene expression of pro-matrix metalloproteinases 1 and 3 and augments those of tissue inhibitors of metalloproteinases 1 and 2 in human synovial fibroblasts.

Various extracts of the Chinese herbal remedy Tripterygium wilfordii Hook. f. (TWHF) have been reported to be therapeutically efficacious in rheumatoid arthritis (RA) in China, but their mechanism of action remains unclear. We investigated the effect of triptolide, a diterpenoid triepoxide from TWHF, on the production of pro-matrix metalloproteinase 1 (proMMP-1; or procollagenase 1 or pro-interstitial collagenase 1), proMMP-3 (or prostromelysin 1), tissue inhibitors of metalloproteinases (TIMPs), and proinflammatory cytokines in human synovial fibroblasts and J774A.1 mouse macrophages. Human synovial fibroblasts and mouse macrophages were cultured with interleukin-1alpha (IL-1alpha) or lipopolysaccharide (LPS) in the presence or absence of triptolide. The production of proMMPs 1 and 3, TIMPs 1 and 2, cyclooxygenase 1 (COX-1) and COX-2, prostaglandin E2 (PGE2), IL-1beta, and IL-6 was assayed by Western blot analysis and enzyme-linked immunosorbent assay. Gene expression of proMMPs 1 and 3, TIMPs 1 and 2, COX-1 and COX-2, IL-1alpha, IL-1beta, tumor necrosis factor alpha (TNFalpha), and IL-6 was also monitored by Northern blot analysis and reverse transcriptase-polymerase chain reaction. Triptolide suppressed the IL-1alpha-induced production of proMMPs 1 and 3 and decreased their messenger RNA levels in human synovial fibroblasts. In contrast, the IL-1alpha-induced gene expression and production of TIMPs 1 and 2 were further augmented by triptolide in the synovial cells. Triptolide also inhibited the IL-1alpha-induced production of PGE2 by selectively suppressing the gene expression and production of COX-2, but not those of COX-1. In addition, triptolide suppressed the LPS-induced production of PGE2 in mouse macrophages. Furthermore, the gene expression of IL-1alpha, IL-1beta, TNFalpha, and IL-6, as well as the production of IL-1beta and IL-6, were inhibited by triptolide in the LPS-treated mouse macrophages. We have demonstrated for the first time that the therapeutic effects of TWHF in RA are due in part to the novel chondroprotective effect of triptolide via the direct suppression of the production of proMMPs 1 and 3 and the simultaneous up-regulation of TIMPs in IL-1-treated synovial fibroblasts. Triptolide's interference with gene expression of proinflammatory cytokines and its known inhibitory effects on PGE2 production are also probably very effective.

Endotoxin-induced upregulation of type I interleukin-1 receptor mRNA expression in hepatocytes of mice: role of cytokines.

Interleukin-1 (IL-1) signal is transduced through the type I IL-1 receptor (IL-1RI). Although regulation of IL-1R expression has been extensively studied in vitro, little is known about it in vivo. By using RT-PCR analysis, we investigated the regulation of the IL-1RI mRNA expression level in various organs of mice at 2, 6, and 24 h following lipopolysaccharide (LPS) administration. IL-1RI mRNA expression in response to LPS appeared to be different in various organs. As a marked and sustained increase of IL-1RI mRNA expression in the liver was observed, we investigated the mechanism of the upregulation. IL-1, IL-6, and tumor necrosis factor (TNF) all increased the mRNA expression in the liver when administrated in vivo. In situ hybridization revealed that upregulation of IL-1R mRNA was observed in parenchymal liver cells (hepatocytes) in response to LPS administration. When primary cultured hepatocytes were treated in vitro, IL-1, IL-6, conditioned medium from LPS-treated mouse macrophages, and serum from LPS-treated mouse upregulated IL-1RI mRNA expression, but LPS, TNF, and prostaglandin E2 failed to do so. Therefore, these results suggest that the upregulation of IL-1RI mRNA in the hepatocytes by LPS administration is mediated by cytokines, especially by IL-1 and IL-6. The results also indicate that the regulation is different in different organs, and microenvironmental factors may be important.

Role of transcription factor NF-kappa B/Rel in induction of nitric oxide synthase.

The promoter of the murine gene encoding inducible nitric oxide synthase (iNOS) contains an NF-kappa B site beginning 55 base pairs upstream of the TATA box, designated NF-kappa Bd. Reporter constructs containing truncated promoter regions, when transfected into macrophages, revealed that NF-kappa Bd is necessary to confer inducibility by bacterial lipopolysaccharide (LPS). Oligonucleotide probes containing NF-kappa Bd plus the downstream 9 or 47 base pairs bound proteins that rapidly appeared in the nuclei of LPS-treated macrophages. The nuclear proteins bound to both probes in an NF-kappa Bd-dependent manner, but binding was resistant to cycloheximide only for the shorter probe. The proteins binding both probes reacted with antibodies against p50 and c-rel but not RelB; those binding the shorter probe also reacted with anti-RelA (p65). Pyrrolidine dithiocarbamate, which acts as a specific inhibitor of NF-kappa B, blocked both the activation of the NF-kappa Bd-binding proteins and the production of NO in LPS-treated macrophages. Thus, activation of NF-kappa B/Rel is critical in the induction of iNOS by LPS. However, additional, newly synthesized proteins contribute to the NF-kappa Bd-dependent transcription factor complex on the iNOS promoter in LPS-treated mouse macrophages.