Bone Marrow-derived Macrophages In Response Research Articles

Pro-Inflammatory Implications of 2-Hydroxypropyl-β-cyclodextrin Treatment.

Lifestyle- and genetically induced disorders related to disturbances in cholesterol metabolism have shown the detrimental impact of excessive cholesterol levels on a plethora of pathological processes such as inflammation. In this context, two-hydroxypropyl-β-cyclodextrin (CD) is increasingly considered as a novel pharmacological compound to decrease cellular cholesterol levels due to its ability to increase cholesterol solubility. However, recent findings have reported contra-indicating events after the use of CD questioning the clinical applicability of this compound. Given its potential as a therapeutic compound in metabolic inflammatory diseases, in this study, we evaluated the inflammatory effects of CD administration in the context of cholesterol-induced metabolic inflammation in vivo and in vitro. The inflammatory and cholesterol-depleting effects of CD were first investigated in low-density lipoprotein receptor knockout (Ldlr-/) mice that were transplanted with Npc1nih or Npc1wt bone marrow and were fed either regular chow or a high-fat, high-cholesterol (HFC) diet for 12 weeks, thereby creating an extreme model of lysosomal cholesterol-induced metabolic inflammation. In the final three weeks, these mice received daily injections of either control (saline) or CD subcutaneously. Subsequently, the inflammatory properties of CD were investigated in vitro in two macrophage cell lines and in murine bone marrow-derived macrophages (BMDMs). While CD administration improved cholesterol mobilization outside lysosomes in BMDMs, an overall pro-inflammatory profile was observed after CD treatment, evidenced by increased hepatic inflammation in vivo and a strong increase in cytokine release and inflammatory gene expression in vitro in murine BMDMs and macrophages cell lines. Nevertheless, this CD-induced pro-inflammatory profile was time-dependent, as short term exposure to CD did not result in a pro-inflammatory response in BMDM. While CD exerts desired cholesterol-depleting effects, its inflammatory effect is dependent on the exposure time. As such, using CD in the clinic, especially in a metabolic inflammatory context, should be closely monitored as it may lead to undesired, pro-inflammatory side effects.

Macrophage LC3‐associated phagocytosis is an immune defense against Streptococcus pneumoniae that diminishes with host aging

Streptococcus pneumoniae is a leading cause of pneumonia and invasive disease, particularly, in the elderly. S. pneumoniae lung infection of aged mice is associated with high bacterial burdens and detrimental inflammatory responses. Macrophages can clear microorganisms and modulate inflammation through two distinct lysosomal trafficking pathways that involve 1A/1B-light chain 3 (LC3)-marked organelles, canonical autophagy, and LC3-associated phagocytosis (LAP). The S. pneumoniae pore-forming toxin pneumolysin (PLY) triggers an autophagic response in nonphagocytic cells, but the role of LAP in macrophage defense against S. pneumoniae or in age-related susceptibility to infection is unexplored. We found that infection of murine bone-marrow-derived macrophages (BMDMs) by PLY-producing S. pneumoniae triggered Atg5- and Atg7-dependent recruitment of LC3 to S. pneumoniae-containing vesicles. The association of LC3 with S. pneumoniae-containing phagosomes required components specific for LAP, such as Rubicon and the NADPH oxidase, but not factors, such as Ulk1, FIP200, or Atg14, required specifically for canonical autophagy. In addition, S. pneumoniae was sequestered within single-membrane compartments indicative of LAP. Importantly, compared to BMDMs from young (2-mo-old) mice, BMDMs from aged (20- to 22-mo-old) mice infected with S. pneumoniae were not only deficient in LAP and bacterial killing, but also produced higher levels of proinflammatory cytokines. Inhibition of LAP enhanced S. pneumoniae survival and cytokine responses in BMDMs from young but not aged mice. Thus, LAP is an important innate immune defense employed by BMDMs to control S. pneumoniae infection and concomitant inflammation, one that diminishes with age and may contribute to age-related susceptibility to this important pathogen.

Toll‐like receptor 4 antagonist FP7 alleviates lipopolysaccharide‐induced septic shock via NF‐kB signaling pathway

Septic shock is the most severe complication of sepsis occurs when body has an overwhelming response to infection, making it the most prevalent cause of deaths in surgical intensive-care units. Therefore, it is urgent to understand its pathogenesis and develop new therapeutic candidate drugs for septic shock. Here, we explored the effect of FP7, an antagonist of Toll-like receptor 4 (TLR4), in the septic shock. First, we injected mice with FP7 and found that FP7 had no effect on immune cells. Then, bone marrow-derived macrophages (BMDMs) isolated from mice were pretreated with FP7 followed by lipopolysaccharide (LPS) stimulation, and FP7 specifically suppressed LPS-induced inflammatory responses in BMDMs via Nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) signaling pathway, with no effect on other TLRs-mediated inflammations. Finally, we injected mice with LPS and D-galactosamine to induce septic shock, followed by the treatment of FP7, and FP7 significantly increased survival rate, improved lung necrosis, and inhibited the secretions of proinflammatory cytokines in the mice with septic shock. Therefore, our study suggested that FP7 had a protective role in septic shock and it might serve as a promising therapeutic candidate drug to treat septic shock.

The lung microenvironment shapes a dysfunctional response of alveolar macrophages in aging.

Alveolar macrophages orchestrate the response to viral infections. Age-related changes in these cells may underlie the differential severity of pneumonia in older patients. We performed an integrated analysis of single-cell RNA-Seq data that revealed homogenous age-related changes in the alveolar macrophage transcriptome in humans and mice. Using genetic lineage tracing with sequential injury, heterochronic adoptive transfer, and parabiosis, we found that the lung microenvironment drove an age-related resistance of alveolar macrophages to proliferation that persisted during influenza A viral infection. Ligand-receptor pair analysis localized these changes to the extracellular matrix, where hyaluronan was increased in aged animals and altered the proliferative response of bone marrow-derived macrophages to granulocyte macrophage colony-stimulating factor (GM-CSF). Our findings suggest that strategies targeting the aging lung microenvironment will be necessary to restore alveolar macrophage function in aging.

Macrophage LC3-associated phagocytosis is an immune defense against Streptococcus pneumoniae that diminishes with host aging

Streptococcus pneumoniae is a leading cause of pneumonia and invasive disease, particularly, in the elderly. S. pneumoniae lung infection of aged mice is associated with high bacterial burdens and detrimental inflammatory responses. Macrophages can clear microorganisms and modulate inflammation through two distinct lysosomal trafficking pathways that involve 1A/1B-light chain 3 (LC3)-marked organelles, canonical autophagy, and LC3-associated phagocytosis (LAP). The S. pneumoniae pore-forming toxin pneumolysin (PLY) triggers an autophagic response in nonphagocytic cells, but the role of LAP in macrophage defense against S. pneumoniae or in age-related susceptibility to infection is unexplored. We found that infection of murine bone-marrow-derived macrophages (BMDMs) by PLY-producing S. pneumoniae triggered Atg5- and Atg7-dependent recruitment of LC3 to S. pneumoniae-containing vesicles. The association of LC3 with S. pneumoniae-containing phagosomes required components specific for LAP, such as Rubicon and the NADPH oxidase, but not factors, such as Ulk1, FIP200, or Atg14, required specifically for canonical autophagy. In addition, S. pneumoniae was sequestered within single-membrane compartments indicative of LAP. Importantly, compared to BMDMs from young (2-mo-old) mice, BMDMs from aged (20- to 22-mo-old) mice infected with S. pneumoniae were not only deficient in LAP and bacterial killing, but also produced higher levels of proinflammatory cytokines. Inhibition of LAP enhanced S. pneumoniae survival and cytokine responses in BMDMs from young but not aged mice. Thus, LAP is an important innate immune defense employed by BMDMs to control S. pneumoniae infection and concomitant inflammation, one that diminishes with age and may contribute to age-related susceptibility to this important pathogen.

Identification of cGAS as an innate immune sensor of extracellular bacterium Pseudomonas aeruginosa.

SummaryCyclic GMP-AMP synthase (cGAS) is reported essential for detecting intracellular bacteria. However, it remains to be determined whether and how cGAS is involved in extracellular bacterial infection. Here, we report that cGAS is essential for mediating type I interferon (IFN) production in infection by multiple extracellular pathogens, including Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus aureus. In addition, the canonical cGAS-stimulator of interferon gene (STING)-IFN axis is required for protecting mice from P. aeruginosa-induced mouse acute pulmonary infection, confirmed in cGAS pathway-specific gene deficiency mouse models. cGAS−/− and STING−/− mice exhibited reduced type I IFNs production, excessive inflammatory response accompanied with decreased resistance to P. aeruginosa challenge. Unfolded protein response was also modulated by cGAS through IRF3 and type I IFNs under P. aeruginosa infection. Collectively, these findings uncover the importance of cGAS in initiating immune responses against extracellular bacterial infection.

HtrA2 is required for inflammatory responses in BMDMs via controlling TRAF2 stability in collagen-induced arthritis

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease characterized by the destruction of cartilage and bone. The present study aims to investigate the role of HtrA serine peptidase 2 (HtrA2) in the collagen-induced arthritis. The expressions of HtrA2 were determined in the database BioGPS and bone marrow-derived macrophages (BMDMs). The populations of myeloid and lymphoid cells were determined in wild type and HtrA2 knockout (HtrA2MKO) mice using flow cytometry. In addition, the expressions of pro-inflammatory cytokines (Il6, Tnf, and Il1β) were determined in the activated BMDMs from wild type (WT) and HtrA2MKO mice. STRING database was used to predict the interactive proteins of HtrA2 and Co-Immunoprecipitation was used to confirm these interactions. A collagen-induced arthritis model was established to investigate the effects of HtrA2 on the arthritis symptoms. It was found that HtrA2 reduction was associated with the activation of myeloid cells. Interestingly, HtrA2 deficiency did not affect the development of myeloid and lymphoid cells. Further studies demonstrated that HtrA2 deficiency suppressed the production of pro-inflammatory cytokines in BMDMs induced by lipopolysaccharide or CpG. Co-Immunoprecipitation results demonstrated that HtrA2 enhanced the stability of TNF receptor-associated factor 2 (TRAF2). HtrA2 participated in the activation of the inflammatory response in a collagen-induced arthritis model. In summary, HtrA2 modulates inflammatory responses in BMDMs by controlling TRAF2 stability in a collagen-induced arthritis mouse model.

HUWE1 mediates inflammasome activation and promotes host defense against bacterial infection.

The mechanism by which inflammasome activation is modulated remains unclear. In this study, we identified an AIM2-interacting protein, the E3 ubiquitin ligase HUWE1, which was also found to interact with NLRP3 and NLRC4 through the HIN domain of AIM2 and the NACHT domains of NLRP3 and NLRC4. The BH3 domain of HUWE1 was important for its interaction with NLRP3, AIM2, and NLRC4. Caspase-1 maturation, IL-1β release, and pyroptosis were reduced in Huwe1-deficient bone marrow-derived macrophages (BMDMs) compared with WT BMDMs in response to stimuli to induce NLRP3, NLRC4, and AIM2 inflammasome activation. Furthermore, the activation of NLRP3, NLRC4, and AIM2 inflammasomes in both mouse and human cells was remarkably reduced by treatment with the HUWE1 inhibitor BI8622. HUWE1 mediated the K27-linked polyubiquitination of AIM2, NLRP3, and NLRC4, which led to inflammasome assembly, ASC speck formation, and sustained caspase-1 activation. Huwe1-deficient mice had an increased bacterial burden and decreased caspase-1 activation and IL-1β production upon Salmonella, Francisella, or Acinetobacter baumannii infection. Our study provides insights into the mechanisms of inflammasome activation as well as a potential therapeutic target against bacterial infection.

Necroptosis Mediates Cigarette Smoke-Induced Inflammatory Responses in Macrophages.

IntroductionCigarette smoke (CS)-induced inflammation in macrophages is involved in the pathological process of chronic obstructive pulmonary disease (COPD). Necroptosis, which is a form of programmed necrosis, has a close relationship with robust inflammation, while its roles in COPD are unclear.Materials and MethodsNecroptosis markers were measured in mouse alveolar macrophages and cultured bone marrow-derived macrophages (BMDMs). Necroptosis inhibitors were used to block necroptosis in BMDMs, and inflammatory cytokines were detected. We further explored the related signaling pathways.ResultsIn this study, we demonstrated the way in which necroptosis, in addition to its upstream and downstream signals, regulates CS-induced inflammatory responses in macrophages. We observed that CS exposure caused a significant increase in the levels of necroptosis markers (receptor interacting kinases [RIPK] 1 and 3) in mouse alveolar macrophages and BMDMs. Pharmacological inhibition of RIPK1 or 3 caused a significant suppression in CS extract (CSE)-induced inflammatory cytokines, chemokine ligands (CXCL) 1 and 2, and interleukin (IL)-6 in BMDMs. CSE-induced necroptosis was regulated by mitochondrial reactive oxygen species (mitoROS), which also promoted inflammation in BMDMs. Furthermore, necroptosis regulated CSE-induced inflammatory responses in BMDMs, most likely through activation of the nuclear factor-κB pathway.ConclusionTaken together, our results demonstrate that mitoROS-dependent necroptosis is essential for CS-induced inflammation in BMDMs and suggest that inhibition of necroptosis in macrophages may represent effective therapeutic approaches for COPD patients.

Microtubule-Mediated NLRP3 Inflammasome Activation Is Independent of Microtubule-Associated Innate Immune Factor GEF-H1 in Murine Macrophages

Inflammasomes are intracellular multiple protein complexes that mount innate immune responses to tissue damage and invading pathogens. Their excessive activation is crucial in the development and pathogenesis of inflammatory disorders. Microtubules have been reported to provide the platform for mediating the assembly and activation of NLRP3 inflammasome. Recently, we have identified the microtubule-associated immune molecule guanine nucleotide exchange factor-H1 (GEF-H1) that is crucial in coupling microtubule dynamics to the initiation of microtubule-mediated immune responses. However, whether GEF-H1 also controls the activation of other immune receptors that require microtubules is still undefined. Here we employed GEF-H1-deficient mouse bone marrow-derived macrophages (BMDMs) to interrogate the impact of GEF-H1 on the activation of NLRP3 inflammasome. NLRP3 but not NLRC4 or AIM2 inflammasome-mediated IL-1β production was dependent on dynamic microtubule network in wild-type (WT) BMDMs. However, GEF-H1 deficiency did not affect NLRP3-driven IL-1β maturation and secretion in macrophages. Moreover, α-tubulin acetylation and mitochondria aggregations were comparable between WT and GEF-H1-deficient BMDMs in response to NLRP3 inducers. Further, GEF-H1 was not required for NLRP3-mediated immune defense against Salmonella typhimurium infection. Collectively, these findings suggest that the microtubule-associated immune modulator GEF-H1 is dispensable for microtubule-mediated NLRP3 activation and host defense in mouse macrophages.

Effect of Porphyromonas gingivalis lipopolysaccharide on innate immune responses in bone-marrow derived macrophages and osteoclasts

Objective To analyze the immune responses of bone-marrow derived macrophages and osteoclasts to lipopolysaccharide from Porphyromonas gingivalis (Pg-LPS) so as to provide reference for host immunomodulatory therapy of periodontitis. Methods Bone marrow mononuclear cells from C57BL/6 mice were obtained and induced into bone marrow macrophages (BMM) and osteoclasts (OC) by conditioned medium. The BMMs and OCs were divided into blank control group and Pg-LPS treated experimental group. Pg-LPS (10 μg/ml) was used to stimulate BMM and OC. The mRNA and protein expression of TLR-2 and TLR-4 and secretion of inflammatory cytokines were detected, respectively. Furthermore, the effect of Pg-LPS on the differentiation of BMM into OC was evaluated. Results Mouse bone marrow mononuclear cells were successfully induced into BMM and mature OC. In the presence of Pg-LPS, the gene level of TLR-2 in BMM was significantly up-regulated by (41.41±13.07) folds (P 0.05). Moreover, the productions of tumor necrosis factor-α and interleukin-6 in response to LPS in BMM and OC remarkably increased (P<0.01). Lower cytokine expression was observed in OC than that in BMM (P<0.01). In addition, Pg-LPS obviously inhibited the number and size of OC formation, with a reduction in area percentage of approximately 47%. Conclusions Pg-LPS generated a robust immune inflammatory response to BMM, while OC had a relatively weaker immune response to the Pg-LPS. Pg-LPS might inhibite the differentiation of BMM into OC. Key words: Macrophages; Osteoclasts; Immune response

Circadian pharmacological effects of berberine on chronic colitis in mice: Role of the clock component Rev-erbα

Berberine, initially isolated from Rhizoma Coptidis (Huanglian in Chinese), is a drug used to treat gastrointestinal disorders such as colitis. Here we uncovered a time-varying berberine effect on chronic colitis in mice, and investigated a potential role of the clock protein Rev-erbα in this timing effect. Berberine activity toward Rev-erbα was determined by luciferase reporter, Gal4-cotransfection assay and target gene expression analyses. Chronic colitis was induced by feeding mice with dextran sulfate sodium in drinking water. Colitis severity and pharmacological effects of berberine were assessed by measuring myeloperoxidase and malondialdehyde activities as well as the levels of inflammatory factors (IL-1β, IL-6, IL-18 and Ccl2). Berberine significantly inhibited Bmal1 (−2000/+100 bp)- and Nlrp3 (−1310/+100 bp)-Luc reporter activities, and dose-dependently decreased cellular expressions of both Bmal1 and Nlrp3. Also, it enhanced the transcriptional repressor activity of Rev-erbα in the Gal4 chimeric assay. These data indicated berberine as a Rev-erbα agonist. As expected, berberine attenuated inflammatory responses in BMDMs (bone marrow-derived macrophages) and in colitis mice. However, the anti-inflammatory effects of berberine were lost in BMDMs derived from Rev-erbα-deficient mice. Furthermore, chronic colitis displayed a diurnal rhythmicity in disease severity and its diurnal pattern was in an opposite phase to that of Rev-erbα expression, supporting a direct control of colitis by Rev-erbα. Moreover, berberine effects on chronic colitis were dosing time-dependent. ZT10 dosing generated a better treatment outcome compared to ZT2. This was because colitis was less severe and Rev-erbα expression was much higher at ZT10 than at ZT2. In conclusion, circadian pharmacological effects of berberine on chronic colitis were mainly contributed by diurnal rhythms of both disease severity and Rev-erbα (as a drug target). The findings may have implications for chronotherapeutic practice on colitis or related diseases.

Tissue-type plasminogen activator selectively inhibits multiple toll-like receptors in CSF-1-differentiated macrophages.

Tissue-type plasminogen activator (tPA) is a major activator of fibrinolysis, which also attenuates the pro-inflammatory activity of lipopolysaccharide (LPS) in bone marrow-derived macrophages (BMDMs) and in vivo in mice. The activity of tPA as an LPS response modifier is independent of its proteinase activity and instead, dependent on the N-methyl-D-aspartate Receptor (NMDA-R), which is expressed by BMDMs. The major Toll-like receptor (TLR) for LPS is TLR4. Herein, we show that enzymatically-inactive (EI) tPA blocks the response of mouse BMDMs to selective TLR2 and TLR9 agonists, rapidly reversing IκBα phosphorylation and inhibiting expression of TNFα, CCL2, interleukin-1β, and interleukin-6. The activity of EI-tPA was replicated by activated α2-macroglobulin, which like EI-tPA, signals through an NMDA-R-dependent pathway. EI-tPA failed to inhibit cytokine expression by BMDMs in response to agonists that target the Pattern Recognition Receptors (PRRs), NOD1 and NOD2, providing evidence for specificity in the function of EI-tPA. Macrophages isolated from the peritoneal space (PMs), without adding eliciting agents, expressed decreased levels of cell-surface NMDA-R compared with BMDMs. These cells were unresponsive to EI-tPA in the presence of LPS. However, when PMs were treated with CSF-1, the abundance of cell-surface NMDA-R increased and the ability of EI-tPA to neutralize the response to LPS was established. We conclude that the anti-inflammatory activity of EI-tPA is selective for TLRs but not all PRRs. The ability of macrophages to respond to EI-tPA depends on the availability of cell surface NMDA-R, which may be macrophage differentiation-state dependent.

Gem-Difluorobisarylic derivatives: design, synthesis and anti-inflammatory effect

IntroductionNew fluorinated diaryl ethers and bisarylic ketones were designed and evaluated for their anti-inflammatory effects in primary macrophages.MethodsThe synthesis of the designed molecules started from easily accessible and versatile gem-difluoro propargylic derivatives. The desired aromatic systems were obtained using Diels–Alder/aromatization sequences and this was followed by Pd-catalyzed coupling reactions and, when required, final functionalization steps. Both direct inhibitory effects on cyclooxygenase-1 or -2 activities, protein expression of cyclooxygenase-2 and nitric oxide synthase-II and the production of prostaglandin E2, the pro-inflammatory nitric oxide and interleukin-6 were evaluated in primary murine bone marrow-derived macrophages in response to lipopolysaccharide. Docking of the designed molecules in cyclooxygenase-1 or -2 was performed.ResultsOnly fluorinated compounds exerted anti-inflammatory activities by lowering the secretion of interleukin-6, nitric oxide, and prostaglandin E2, and decreasing the protein expression of inducible nitric oxide synthase and cyclooxygenase-2 in mouse primary macrophages exposed to lipopolysaccharide, as well as cyclooxygenase activity for some inhibitors with different efficiencies depending on the R-groups. Docking observation suggested an inhibitory role of cyclooxygenase-1 or -2 for compounds A3, A4 and A5 in addition to their capacity to inhibit nitrite, interleukin-6, and nitric oxide synthase-II and cyclooxygenase-2 expression.ConclusionThe new fluorinated diaryl ethers and bisarylic ketones have anti-inflammatory effects in macrophages. These fluorinated compounds have improved potential anti-inflammatory properties due to the fluorine residues in the bioactive molecules.

Curcumin-activated autophagy plays a negative role in its anti-osteoclastogenic effect

Background/purposeIt remains unclear what role curcumin plays in the autophagy of osteoclast precursors (OCPs) during osteoclastogenesis, since some researchers found that curcumin has the ability to inhibit osteoclastogenesis. While others have considered it as an autophagy activator. This study aimed to determine the effect of curcumin-regulated autophagy on osteoclastogenesis. ResultsThe results revealed that direct administration of curcumin enhanced the OCP autophagy response in bone marrow-derived macrophages (BMMs). Curcumin could also abate RANKL's stimulatory effect on OCP autophagy and osteoclastogenesis. Autophagic suppression related to pharmacological inhibitors or gene silencing could further enhance the inhibitory effect of curcumin on osteoclastogenesis. As expected, curcumin ameliorated ovariectomy (OVX)-induced bone loss and its effect could be promoted by an autophagy inhibitor (chloroquine). ConclusionsIn conclusion, curcumin can directly enhance the autophagic activity of OCPs, which inhibits its anti-osteoclastogeneic effects. Autophagy inhibition-based drugs are expected to enhance curcumin's efficacy in treating osteoporosis.

Promotion of macrophage activation by Tie2 in the context of the inflamed synovia of rheumatoid arthritis and psoriatic arthritis patients.

ObjectiveTo examine the role of Tie2 signalling in macrophage activation within the context of the inflammatory synovial microenvironment present in patients with RA and PsA.MethodsClinical responses and macrophage function were examined in wild-type and Tie2-overexpressing (Tie2-TG) mice in the K/BxN serum transfer model of arthritis. Macrophages derived from peripheral blood monocytes from healthy donors, RA and PsA patients, and RA and PsA synovial tissue explants were stimulated with TNF (10 ng/ml), angiopoietin (Ang)-1 or Ang-2 (200 ng/ml), or incubated with an anti-Ang2 neutralizing antibody. mRNA and protein expression of inflammatory mediators was analysed by quantitative PCR, ELISA and Luminex.ResultsTie2-TG mice displayed more clinically severe arthritis than wild-type mice, accompanied by enhanced joint expression of IL6, IL12B, NOS2, CCL2 and CXCL10, and activation of bone marrow-derived macrophages in response to Ang-2 stimulation. Ang-1 and Ang-2 significantly enhanced TNF-induced expression of pro-inflammatory cytokines and chemokines in macrophages from healthy donors differentiated with RA and PsA SF and peripheral blood-derived macrophages from RA and PsA patients. Both Ang-1 and Ang-2 induced the production of IL-6, IL-12p40, IL-8 and CCL-3 in synovial tissue explants of RA and PsA patients, and Ang-2 neutralization suppressed the production of IL-6 and IL-8 in the synovial tissue of RA patients.ConclusionTie2 signalling enhances TNF-dependent activation of macrophages within the context of ongoing synovial inflammation in RA and PsA, and neutralization of Tie2 ligands might be a promising therapeutic target in the treatment of these diseases.

A loss-of-function single nucleotide polymorphism in cytochrome b5 reductase 3 amplifies the response of bone marrow-derived macrophages to high salt

Abstract Introduction High dietary salt intake is a risk factor for hypertension and autoimmune diseases. Recent studies show high salt intake can result in high-concentration interstitial salt accumulation. In macrophages, similar concentrations induce pro-inflammatory gene expression and amplify activation by LPS. African Americans, a notably salt-sensitive population, have an exclusively high frequency (~23%) for a missense single nucleotide polymorphism (SNP) in cytochrome b5 reductase 3 (CYB5R3), an enzyme important for oxidative stress resistance. We have shown this SNP, T117S, decreases enzymatic activity. Given macrophages’ sizable productive capacity for reactive oxygen species (ROS), we hypothesize T117S expression amplifies salt-mediated pro-inflammatory gene expression and ROS production. Results Culture of bone marrow-derived macrophages (BMDMs) from WT or T117S-expressing mice in high salt medium (+51mM NaCl) increased expression of pro-inflammatory genes iNOS, IL-6, and IL-1β. However, the increase was significantly greater in T117S BMDMs. High salt also increased hydrogen peroxide (H2O2) production in both groups, but more so in T117S BMDMs. Of note, pretreatment with inhibitors targeting p38, a MAP kinase involved in the inflammatory response, or NOX2, the major macrophage superoxide source, ablated salt-mediated H2O2 production in both groups. Conclusions We show presence of loss-of-function variant T117S CYB5R3 in macrophages enhances salt-mediated pro-inflammatory gene expression and ROS production in a p38 and NOX2-dependent manner. This suggests CYB5R3 may be involved in the response to high salt and raises the possibility that T117S carriers may have an exacerbated response to high salt intake.

Hyperglycemia-Triggered ATF6-CHOP Pathway Modulates Inflammatory Responses by β-Catenin Signaling in Liver Ischemia/Reperfusion Injury

Background: Hyperglycemia is an adverse risk factor for hepatic ischemia reperfusion injury (IRI); however, the underlying mechanism remains unclear. Herein, we addressed the question of whether and how hyperglycemia triggered endoplasmic reticulum (ER) stress and affected liver IRI. Methods: Diabetic patients and streptozotocin (STZ)-induced diabetic mice were involved in vivo. Bone marrow-derived macrophages (BMDMs) were used in vitro. Findings: The ER stress-ATF6-CHOP pathway was specifically activated in liver tissues and Kuppfer cells (KCs) from diabetic patients and STZ-induced diabetic mice. The ER stress inhibitor, 4-phenylbutyrate (PBA), attenuated hyperglycemia-related liver IRI, and decreased TLR4-related pro-inflammatory responses. Furthermore, CHOP gene knockout (CHOP-/-) had markedly reduced hyperglycemia-related liver IRI and inflammatory responses. Importantly, expression of β-catenin, was effectively inhibited, accompanied by activation of ATF6-CHOP signaling in diabetic mice, and was almost restored in liver tissues from PBA-treated or CHOP-/- diabetic mice. Furthermore, β-catenin small interfering RNA (siRNA) targeting KCs abrogated the CHOP-/--related protective effects against hyperglycemia-related liver IRI. High glucose (HG) specifically activated ATF6-CHOP signaling, inhibited β-catenin expression, and enhanced TLR4-related inflammatory responses in BMDMs in vitro; these effects were partly reversed in PBA- or CHOP siRNA-treated/CHOP-/- BMDMs. However, the anti-inflammatory functions of CHOP-/- were almost abolished by β-catenin siRNA in BMDMs under HG conditions. The above results were furthermore confirmed using a chimeric mice carrying WT or CHOP-/- BMDMs by BMDMs transplantation. Interpretation: This study revealed hyperglycemia specifically triggers ER stress-ATF6-CHOP signaling, inhibits β-catenin activity, promotes inflammation, and exacerbates liver IRI, which might provide the rationale for novel therapeutic strategies aimed at managing diabetic-related liver surgery. Funding: This study was supported by the Foundation of Jiangsu Collaborative Innovation Center of Biomedical Functional Materials, the Priority Academic Program Development of Jiangsu Higher Education Institutions, the National Natural Science Foundation of China (81400650, 814700901, 81273261, and 81270583), and Basic research program-Youth Fund Project of Jiangsu Province (BK20140092). Declaration of Interest: All authors declare no conflict of interests. Ethical Approval: Informed consent was obtained from all participants, and the study was approved by the local ethics committee of Nanjing Medical University. The animal protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing Medical University (Protocol Number IACUC-1702001).

Inhibition of the hepatic Nlrp3 protects dopaminergic neurons via attenuating systemic inflammation in a MPTP/p mouse model of Parkinson\u2019s disease

BackgroundParkinson’s disease (PD) is a neurodegenerative disorder with progressive loss of dopaminergic (DA) neurons. Systemic inflammation is shown to initiate and exacerbate DA neuronal degeneration in the substantia nigra. The infiltration and transformation of immune cells from the peripheral tissues are detected in and around the affected brain regions of PD patients. Our previous studies demonstrated the crucial role that microglial Nod-like receptor protein (NLRP) 3 inflammasome plays in the pathogenesis of PD. Nevertheless, the direct linkage between peripheral inflammation and DA neuron death remains obscure.MethodsIn the present study, we detected the NLRP3 expressions in the midbrain, liver, and bone marrow-derived macrophages in response to 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) acute and chronic challenge. We then used a tail vein injection of Nlrp3-siRNA wrapped with lentivirus to explore the potential influence of hepatic NLRP3 inflammasome-mediated inflammation on neuronal injury in a mouse model of PD via immunohistochemistry, ELISA, and Western blotting analysis.ResultsWe showed that siNlrp3 downregulated the NLRP3 protein expression and inhibited the activation of NLRP3 inflammasomes in mice livers. The tail vein injection of LV3-siNlrp3 reduced the liver pro-inflammatory cytokine production, which subsequently alleviated MPTP-triggered microglial activation and DA neuron loss in the midbrain. These findings indicated that inhibition of hepatic NLRP3 inflammasome weakens inflammatory cytokines spreading into the brain and delays the progress of neuroinflammation and DA neuronal degeneration.ConclusionThis study gives us an insight into the direct linkage between liver inflammation and DA neuron damage in the pathogenesis of PD and provides the potential target of NLRP3 for developing novel drugs for PD therapy.

K63-Linked Polyubiquitination on TRAF6 Regulates LPS-Mediated MAPK Activation, Cytokine Production, and Bacterial Clearance in Toll-Like Receptor 7/8 Primed Murine Macrophages.

Post viral infection bacterial pneumonia is a major cause of morbidity and mortality associated with both seasonal and pandemic influenza virus illness. Despite much efforts put into the discovery of mechanisms of post viral–bacterial infections and their complications in recent years, the molecular mechanisms underlying the increased susceptibility to bacterial infection remain poorly understood. In this study, we focused on the pathways regulating immune responses in murine macrophages and modeled post viral–bacterial infections through pretreatment of bone marrow-derived macrophages (BMDMs) with a toll-like receptor (TLR) 7/8 ligand (R848) and subsequent challenge with TLR2/4 agonists to mimic bacterial infection. We found R848-primed BMDMs upon subsequent exposure to TLR2/4 ligands respond with enhanced inflammatory cytokine production, especially IL-6 and TNF-α. The enhanced cytokine production in R848-primed BMDMs in response to TLR2/4 was due to increased TGF-β-activated kinase (TAK) 1 phosphorylation with subsequent activation of ERK and p38 MAPKs. Furthermore, we identified that R848 priming leads to increased K63-linked polyubiquitination on TRAF6. K63-linked polyubiquitination on TRAF6 is a signal leading to enhanced activation of downstream pathways including TAK1. Importantly, R848-primed BMDMs infected with live bacteria exhibited decreased bacterial clearance. Small-molecule enhancer of rapamycin 3, an ubiquitin ligase inhibitor reversed the K63-linked polyubiquitination on TRAF6 in R848-primed BMDMs and subsequently decreased TAK1 and MAPK phosphorylation, and cytokine production as well as reversed the decreased bacterial clearance capacity of BMDMs. Our study may provide a novel molecular target to alleviate post viral–bacterial infections.