Bone Marrow-derived Macrophages In Response Research Articles

TAK1-binding proteins (TAB)2 and TAB3 are redundantly required for TLR-induced cytokine production in macrophages.

Transforming growth factor-β-activated kinase 1 (TAK1) plays a pivotal role in innate and adaptive immunity. TAK1 is essential for the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB pathways downstream of diverse immune receptors, including Toll-like receptors (TLRs). Upon stimulation with TLR ligands, TAK1 is activated via recruitment to lysine 63-linked polyubiquitin chain through TAK1-binding proteins (TAB) 2 and TAB3. However, the physiological importance of TAB2 and TAB3 in macrophages is still controversial. A previous study has shown that mouse bone marrow-derived macrophages (BMDMs) isolated from mice double deficient for TAB2 and TAB3 produced tumor necrosis factor (TNF)-α and interleukin (IL)-6 to the similar levels as control wild-type BMDMs in response to TLR ligands such as lipopolysaccharide (LPS) or Pam3CSK4, indicating that TAB2 and TAB3 are dispensable for TLR signaling. In this study, we revisited the role of TAB2 and TAB3 using an improved mouse model. We observed a significant impairment in the production of pro-inflammatory cytokines and chemokine in LPS- or Pam3CSK4-treated BMDMs deficient for both TAB2 and TAB3. Double deficiency of TAB2 and TAB3 resulted in the decreased activation of NF-κB and MAPK pathways as well as the slight decrease in TAK1 activation in response to LPS or Pam3CSK4. Notably, the TLR-mediated expression of inhibitor of NF-κB (IκB)ζ was severely compromised at the protein and mRNA levels in the TAB2/TAB3 double-deficient BMDMs, thereby impeding IL-6 production. Our results suggest that TAB2 and TAB3 play a redundant and indispensable role in TLR signaling pathway.

Preliminary study on the effect of Echinococcus multilocaris on phenotypic transformations of glucose metabolism and polarization types in macrophages

To investigate the effects of Echinococcus multilocularis on the phenotypic transformations of glucose metabolism, polarization types and inflammatory responses in macrophages, so as to provide insights into elucidation of echinococcosis pathogenesis. Bone marrow cells were isolated from C57BL/6J mice at ages of 6 to 8 weeks, and induced into bone marrow-derived macrophages (BMDMs) with mouse macrophage colony-stimulating factor (M-CSF), which served as controls (BMDMs-M0). BMDMs-M0 induced M2 macrophages by interleukin-4 for 24 hours served as the IL-4 induction group, and BMDMs-M0 co-cultured with 2.4 ng/mL E. multilocularis cystic fluid (CF) served as the BMDM-CF co-culture group, while BMDMs-M0 co-cultured with E. multilocularis protoscolex (PSC) at a ratio of 500:1 served as the BMDM-PSC co-culture group. The types of polarization of BMDMs co-cultured with E. multilocularis CF and PSC were analyzed using flow cytometry, and the expression of macrophage markers, inflammatory factors, and glucose metabolism-related enzymes was quantified using fluorescent quantitative real-time PCR (qPCR) and Western blotting assays. There were significant differences among the four groups in terms of Arginase-1 (Arg1) (F = 1 457.00, P < 0.000 1), macrophages-derived C-C motif chemokine 22 (Ccl22) (F = 22 203.00, P < 0.000 1), resistin-like α (Retnla) (F = 151.90, P < 0.000 1), inducible nitric oxide synthase (iNOS) (F = 107.80, P < 0.001), hexokinase (HK) (F = 9 389.00, P < 0.000 1), pyruvate kinase (PK) (F = 641.40, P < 0.001), phosphofructokinase 1 (PFK1) (F = 43.97, P < 0.01), glucokinase (GK) (F = 432.50, P < 0.000 1), pyruvate dehydrogenase kinases1 (PDK1) (F = 737.30, P < 0.000 1), lactic dehydrogenase (LDH) (F = 3 632.00, P < 0.000 1), glucose transporter 1 (GLUT1) (F = 532.40, P < 0.000 1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (F = 460.00, P < 0.000 1), citrate synthase (CS) (F = 5 642.00, P < 0.01), glycogen synthase1 (GYS1) (F = 273.30, P < 0.000 1), IL-6 (F = 1 823.00, P < 0.000 1), IL-10 (F = 291.70, P < 0.000 1), IL-1β (F = 986.60, P < 0.000 1), and tumor necrosis factor (TNF)-α (F = 334.80, P < 0.000 1) and transforming growth factor (TGF)-β mRNA expression (F = 163.30, P < 0.001). The proportion of M2 macrophages was significantly higher than that of M1 macrophages in the BMDM-PSC co-culture group [(22.87% ±1.48%) vs. (1.70% ±0.17%); t = 24.61, P < 0.001], and the proportion of M2 macrophages was significantly higher than that of M1 macrophages in the BMDM-CF co-culture group [(20.07% ±0.64%) vs. (1.93% ±0.25%); t = 45.73, P < 0.001]. The mRNA expression of M2 macrophages markers Arg1, Ccl22 and Retnla was significantly higher in the BMDM-CF and BMDM-PSC co-culture groups than in the control group (all P values < 0.01), and no significant difference was seen in the mRNA expression of the M1 macrophage marker iNOS among the three groups (P > 0.05), while qPCR assay quantified higher mRNA expression of key glycolytic enzymes HK, PK and PFK, as well as inflammatory factors IL-10, IL-1β, TNF-α and TGF-β in the BMDM-CF and BMDM-PSC co-culture groups than in the control group (all P values < 0.01). Western blotting assay determined higher HK, PK and PFK protein expression in the BMDM-PSC co-culture group than in the control group (all P values < 0.05), and qPCR quantified higher GLUT1, GAPDH and IL-6 mRNA expression in the BMDM-CF co-culture group than in the control group (all P values < 0.05), while higher HK, PK and PFK protein and mRNA expression (all P values < 0.01), as well as lower IL-6 and TNF-α and higher TGF-β mRNA expression (both P values < 0.05) was detected in the IL-4 induction group than in the control group. Glycolytic stress test showed no significant difference in the extracellular acidification rate (ECAR) of mouse BMDM among the control group, IL-4 induction group and BMDM-PSC co-culture group (F = 124.4, P < 0.05), and a higher ECAR was seen in the BMDM-PSC co-culture group and a lower ECAR was found in the IL-4 induction group than in the control group (both P values < 0.05). Treatment of E. multilocularis CF or PSC mainly causes polarization of BMDM into M2 macrophages, and phenotypic transformation of glucose metabolism into high-energy and high-glycolytic metabolism, and affects inflammatory responses in BMDM.

Activation of Mouse Bone Marrow-Derived Macrophages in Response to Antibodies

Activation of Mouse Bone Marrow-Derived Macrophages in Response to Antibodies

Activation of Mouse Bone Marrow-Derived Macrophages in Response to Antibodies

Activation of Mouse Bone Marrow-Derived Macrophages in Response to Antibodies

Actinidia eriantha polysaccharide exerts adjuvant activity by targeting linc-AAM

Actinidia eriantha polysaccharide (AEPS) is a potent adjuvant with dual Th1 and Th2 potentiating activity. linc-AAM has been previously proved to facilitate the expression of immune response genes (IRGs) in AEPS-activated RAW264.7 macrophages. However, its role in mediating adjuvant activity of AEPS remains to be elucidated. In this study, bone marrow-derived macrophages (BMDMs) from wide-type (WT) and linc-AAM knockout C57BL/6J mice treated with AEPS were subjected to transcriptome sequencing and bioinformatic analysis. linc-AAM deficiency inhibited M1 and M2 immune responses in BMDMs induced by AEPS. In mechanisms, AEPS facilitated the expression of IRGs and activated BMDMs through NF-κB−linc-AAM−JAK/STAT axis. Furthermore, linc-AAM knockout inhibited cytokine and chemokine production, immune cell recruitment as well as immune cell migration to draining lymph nodes at peritoneal cavity in mice induced by AEPS. More importantly, linc-AAM deletion reduced the adjuvant activity of APES on antigen-specific cellular and humoral immune responses to ovalbumin in mice. This study has for the first time demonstrated the role of lncRNAs in regulating the adjuvant activity of polysaccharides and its mechanisms. These findings expanded current knowledge on the mechanism of action of adjuvant and provide a new target for the design and development of vaccine adjuvants.

Dietary fat differentially modulates the response of bone marrow-derived macrophages to TLR4 and NOD2 agonists.

Consumption of diets high in fat has been linked to the development of obesity and related metabolic complications. Such associations originate from the enhanced, chronic, low-grade inflammation mediated by macrophages in response to translocated bacteria, bacterial products, or dietary constituents such as fatty acids (FAs). Nucleotide-binding Oligomerization Domain 2 (NOD2) senses muramyl dipeptide (MDP), a component of bacterial peptidoglycan. The inability to sense peptidoglycan through NOD2 has been demonstrated to lead to dysbiosis, increased bacterial translocation, inflammation and metabolic dysfunction. Currently, it is unknown how consumption of HFDs with different FA compositions might influence NOD2-dependent responses. In this study, we subjected WT mice to a control diet or to HFDs comprised of various ratios of unsaturated to saturated fats and determined the macrophage response to TLR4 and NOD2 agonists. A HFD with equal ratios of saturated and unsaturated fats enhanced subsequent responsiveness of macrophages to LPS but not to MDP. However, a high-unsaturated fat diet (HUFD) or a high-saturated fat diet (HSFD) both decreased the responsiveness to NOD2 agonists compared to that observed in control diet (CD) fed mice. These data suggest that dietary fatty acid composition can influence the subsequent macrophage responsiveness to bacterial products.

Mkp-1 deficiency strengthens immune defense against fungal infection

Abstract Fungal infections are a serious threat to people with a compromised immune system. The recognition of fungal pathogens relies on pathways mediated by TLRs and C-type lectin receptors. Activation of these TLR and C-type lectin receptor pathways converge on both NF-κB and MAPKs to initiate cytokine and chemokine production, leading to phagocyte recruitment and pathogen elimination. Mkp-1 is a negative feedback regulator of the p38 and JNK MAPKs. To understand the role of Mkp-1 in immune defense against fungal pathogens, we assessed the effects of Mkp-1 deficiency on the immune response to C. albicans, the most common fungal pathogen. Contrary to our previous observation with E. coli or bacterial products such as LPS, Mkp1−/− mice were protected from a lethal dose of C. albicans. In response to C. albicans challenge, Mkp1−/− mice produced greater amounts of TNF-α, had lower kidney fungal burdens and smaller fungal lesions. In wildtype mice large number of neutrophils were found in the kidneys, forming clusters surrounding fungal lesions. In contrast, far less neutrophils were found in the kidneys of Mkp-1−/− mice, although these neutrophils appeared to express greater levels of neutrophil elastase and form tighter clusters surrounding the fungi. Upon stimulation with heat-killed C. albicans yeasts and hyphae Mkp-1−/− bone marrow-derived macrophages (BMDM) also produced greater amounts of TNF-α and had higher p38 and JNK activities than did WT BMDM. TNF-α production in both wildtype and Mkp-1−/− BMDM in response to heat-killed C. albicans was attenuated by the pharmacological inhibitors of MEK1, JNK, p38, and Syk. Taken together, our results suggest that during fungal infection Mkp-1 acts to hamper the immune response against fungal pathogens. *The studies were supported by NIH grants (AI113930 and AI124029 to Y.L., and AI121196 and AI123253 to J.Z.).

Nlrp12 deficiency alters gut microbiota and ameliorates Faslpr -mediated systemic autoimmunity in male mice.

NLRP12 has dual roles in shaping inflammation. We hypothesized that NLRP12 would modulate myeloid cells and T cell function to control systemic autoimmunity. Contrary to our hypothesis, the deficiency of Nlrp12 in autoimmune-prone B6.Faslpr/lpr mice ameliorated autoimmunity in males but not females. Nlrp12 deficiency dampened B cell terminal differentiation, germinal center reaction, and survival of autoreactive B cells leading to decreased production of autoantibodies and reduced renal deposition of IgG and complement C3. In parallel, Nlrp12 deficiency reduced the expansion of potentially pathogenic T cells, including double-negative T cells and T follicular helper cells. Furthermore, reduced pro-inflammatory innate immunity was observed, where the gene deletion decreased in-vivo expansion of splenic macrophages and mitigated ex-vivo responses of bone marrow-derived macrophages and dendritic cells to LPS stimulation. Interestingly, Nlrp12 deficiency altered the diversity and composition of fecal microbiota in both male and female B6/lpr mice. Notably, however, Nlrp12 deficiency significantly modulated small intestinal microbiota only in male mice, suggesting that the sex differences in disease phenotype might be gut microbiota-dependent. Together, these results suggest a potential pathogenic role of NLRP12 in promoting systemic autoimmunity in males. Future studies will investigate sex-based mechanisms through which NLRP12 differentially modulates autoimmune outcomes.

Myeloid miR-155 plays a limited role in antibacterial defense during Klebsiella-derived pneumosepsis and is dispensable for lipopolysaccharide- or Klebsiella-induced inflammation in mice.

MicroRNA-155 (miR-155) plays a crucial role in regulating host inflammatory responses during bacterial infection. Previous studies have shown that constitutive miR-155 deficiency alleviates inflammation while having varying effects in different bacterial infection models. However, whether miR-155 in myeloid cells is involved in the regulation of inflammatory and antibacterial responses is largely elusive. Mice with myeloid cell specific miR-155 deficiency were generated to study the in vitro response of bone marrow-derived macrophages (BMDMs), alveolar macrophages (AMs) and peritoneal macrophages (PMs) to lipopolysaccharide (LPS), and the in vivo response after intranasal or intraperitoneal challenge with LPS or infection with Klebsiella (K.) pneumoniae via the airways. MiR-155 deficient macrophages released less inflammatory cytokines than control macrophages upon stimulation with LPS in vitro. However, the in vivo inflammatory cytokine response to LPS or K. pneumoniae was not affected by myeloid miR-155 deficiency. Moreover, bacterial outgrowth in the lungs was not altered in myeloid miR-155 deficient mice, but Klebsiella loads in the liver of these mice were significantly higher than in control mice. These data argue against a major role for myeloid miR-155 in host inflammatory responses during LPS-induced inflammation and K. pneumoniae-induced pneumosepsis but suggest that myeloid miR-155 contributes to host defense against Klebsiella infection in the liver.

Cell volume restriction by mercury chloride reduces M1-like inflammatory response of bone marrow-derived macrophages.

Dysregulation of macrophages in the pro-inflammatory (M1) and anti-inflammatory (M2) sub-phenotypes is a crucial element in several inflammation-related diseases and injuries. We investigated the role of aquaporin (AQP) in macrophage polarization using AQP pan-inhibitor mercury chloride (HgCl2). Lipopolysaccharides (LPSs) induced the expression of AQP-1 and AQP-9 which increased the cell size of bone marrow-derived macrophages. The inhibition of AQPs by HgCl2 abolished cell size changes and significantly suppressed M1 polarization. HgCl2 significantly reduced the activation of the nuclear factor kappa B (NF-κB) and p38 mitogen-activated protein kinase (MAPK) pathways and inhibited the production of IL-1β. HgCl2 attenuated LPS-induced activation of mitochondria and reactive oxygen species production and autophagy was promoted by HgCl2. The increase in the light chain three II/light chain three I ratio and the reduction in PTEN-induced kinase one expression suggests the recycling of damaged mitochondria and the restoration of mitochondrial activity by HgCl2. In summary, the present study demonstrates a possible mechanism of the AQP inhibitor HgCl2 in macrophage M1 polarization through the restriction of cell volume change, suppression of the p38 MAPK/NFκB pathway, and promotion of autophagy.

Protein phosphatase 2A regulates xanthine oxidase-derived ROS production in macrophages and influx of inflammatory monocytes in a murine gout model.

Background: Gout is a common arthritis, due to deposition of monosodium urate (MSU) crystals which results in IL-1β secretion by tissue-resident macrophages. Xanthine oxidase (XO) catalyzes uric acid (UA) production and in the process, reactive oxygen species (ROS) are generated which contributes to NLRP3 inflammasome activation. Protein phosphatase 2A (PP2A) may be involved in regulating inflammatory pathways in macrophages. The objective of this study was to investigate whether PP2A regulates gout inflammation, mediated by XO activity modulation. We studied UA and ROS generations in MSU stimulated murine bone marrow derived macrophages (BMDMs) in response to fingolimod phosphate, a PP2A activator, and compared its anti-inflammatory efficacy to that of an XO inhibitor, febuxostat. Methods: BMDMs were stimulated with MSU, GM-CSF/IL-1β or nigericin ± fingolimod (2.5μM) or febuxostat (200μM) and UA levels, ROS, XO, and PP2A activities, Xdh (XO) expression and secreted IL-1β levels were determined. PP2A activity and IL-1β in MSU stimulated BMDMs ± N-acetylcysteine (NAC) (10μM) ± okadaic acid (a PP2A inhibitor) were also determined. M1 polarization of BMDMs in response to MSU ± fingolimod treatment was assessed by a combination of iNOS expression and multiplex cytokine assay. The in vivo efficacy of fingolimod was assessed in a murine peritoneal model of acute gout where peritoneal lavages were studied for pro-inflammatory classical monocytes (CMs), anti-inflammatory nonclassical monocytes (NCMs) and neutrophils by flow cytometry and IL-1β by ELISA. Results: Fingolimod reduced intracellular and secreted UA levels (p < 0.05), Xdh expression (p < 0.001), XO activity (p < 0.001), ROS generation (p < 0.0001) and IL-1β secretion (p < 0.0001), whereas febuxostat enhanced PP2A activity (p < 0.05). NAC treatment enhanced PP2A activity and reduced XO activity and PP2A restoration mediated NAC's efficacy as co-treatment with okadaic acid increased IL-1β secretion (p < 0.05). Nigericin activated caspase-1 and reduced PP2A activity (p < 0.001) and fingolimod reduced caspase-1 activity in BMDMs (p < 0.001). Fingolimod reduced iNOS expression (p < 0.0001) and secretion of IL-6 and TNF-α (p < 0.05). Fingolimod reduced CMs (p < 0.0001), neutrophil (p < 0.001) and IL-1β (p < 0.05) lavage levels while increasing NCMs (p < 0.001). Conclusion: Macrophage PP2A is inactivated in acute gout by ROS and a PP2A activator exhibited a broad anti-inflammatory effect in acute gout in vitro and in vivo.

Substitution of the SERCA2 Cys674 reactive thiol accelerates atherosclerosis by inducing endoplasmic reticulum stress and inflammation.

The cysteine674 (C674) thiol of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 is easily and irreversibly oxidized under atherosclerotic conditions. However, the contribution of the C674 thiol redox status in the development of atherosclerosis remains unclear. Our goal was to elucidate the possible mechanism involved. Heterozygous SERCA2 C674S knock-in mice in which half of the C674 was substituted by serine (S674) were used to mimic the removal of the reactive C674 thiol, which occurs under pathological conditions. Bone marrow-derived macrophages (BMDMs) and cardiac endothelial cells (ECs) were used for intracellular Ca2+ , macrophage adhesion, and protein expression analysis. The whole aorta and aortic root were isolated for histological analysis. Cell culture studies suggest the partial substitution of SERCA2 C674 increased intracellular Ca2+ levels and induced ER stress in both BMDMs and ECs. The release of proinflammatory factors and macrophage adhesion increased in SKI BMDMs. In ECs, overexpression of S674 induced endothelial inflammation and promoted macrophage recruitment. SKI mice developed more severe atherosclerotic plaque and macrophage accumulation. Additionally, 4-phenyl butyric acid, an ER stress inhibitor, suppressed ER stress and inflammatory responses in BMDMs and ECs, and alleviated atherosclerosis in SKI mice. The substitution of SERCA2 C674 thiol accelerates the development of atherosclerosis by inducing ER stress and inflammation. Our findings highlight the importance of SERCA2 C674 redox state in the context of atherosclerosis and open up a novel therapeutic strategy to combat atherosclerosis.

Indole supplementation ameliorates MCD-induced NASH in mice

Indole is a microbiota metabolite that functions to protect against obesity-associated non-alcoholic fatty liver disease. The present study examined the extent to which indole supplementation alleviates the severity of non-alcoholic steatohepatitis (NASH), which is the advanced form of non-alcoholic fatty liver disease. In C57BL/6J mice, feeding a methionine- and choline-deficient diet (MCD) resulted in significant weight loss, overt hepatic steatosis, and massive aggregations of macrophages in the liver compared with control diet-fed mice. Upon indole supplementation, the severity of MCD-induced hepatic steatosis and inflammation, as well as liver fibrosis, was significantly decreased compared with that of MCD-fed and control-treated mice. In vitro, indole treatment caused significant decreases in lipopolysaccharide-induced proinflammatory responses in hepatocytes incubated with either basal or MCD-mimicking media. However, indole treatment only significantly decreased lipopolysaccharide-induced proinflammatory responses in bone marrow-derived macrophages incubated with basal, but not MCD-mimicking media. These differential effects suggest that, relative to the responses of macrophages to indole, the responses of hepatocytes to indole appeared to make a greater contribution to indole alleviation of NASH, in particular liver inflammation. While indole supplementation decreased liver expression of desmin in MCD-fed mice, treatment of LX2 cells (a line of hepatic stellate cells) with indole also decreased the expression of various markers of hepatic stellate cell fibrogenic activation. Lastly, indole supplementation decreased intestinal inflammation in MCD-fed mice, suggesting that decreased intestinal inflammation also was involved in indole alleviation of NASH. Collectively, these results demonstrate that indole supplementation alleviates MCD-induced NASH, which is attributable to, in large part, indole suppression of hepatocyte proinflammatory responses and hepatic stellate cell fibrogenic activation, as well as intestinal proinflammatory responses.

Fibrinogen-like protein 2 deficiency inhibits virus-induced fulminant hepatitis through abrogating inflammatory macrophage activation.

BACKGROUNDHeterogeneous macrophages play an important role in multiple liver diseases, including viral fulminant hepatitis (VFH). Fibrinogen-like protein 2 (FGL2) is expressed on macrophages and regulates VFH pathogenesis; however, the underlying mechanism remains unclear. AIMTo explore how FGL2 regulates macrophage function and subsequent liver injury during VFH. METHODSMurine hepatitis virus strain 3 (MHV-3) was used to induce VFH in FGL2-deficient (Fgl2-/-) and wild-type (WT) mice. The dynamic constitution of hepatic macrophages was examined. Adoptive transfer of Fgl2-/- or WT bone marrow-derived macrophages (BMDMs) into WT recipients with macrophages depleted prior to infection was carried out and the consequent degree of liver damage was compared. The signaling cascades that may be regulated by FGL2 were detected in macrophages.RESULTSFollowing MHV-3 infection, hepatic macrophages were largely replenished by proinflammatory monocyte-derived macrophages (MoMFs), which expressed high levels of FGL2. In Fgl2-/- mice, the number of infiltrating inflammatory MoMFs was reduced compared with that in WT mice after viral infection. Macrophage depletion ameliorated liver damage in WT mice and further alleviated liver damage in Fgl2-/- mice. Adoptive transfer of Fgl2-/- BMDMs into macrophage-removed recipients significantly reduced the degree of liver damage. Inhibition of monocyte infiltration also significantly ameliorated liver damage. Functionally, Fgl2 deletion impaired macrophage phagocytosis and the antigen presentation potential and attenuated the proinflammatory phenotype. At the molecular level, FGL2 deficiency impaired IRF3, IRF7, and p38 phosphorylation, along with NF-κB activation in BMDMs in response to viral infection. CONCLUSIONInfiltrated MoMFs represent a major source of hepatic inflammation during VFH progression, and FGL2 expression on MoMFs maintains the proinflammatory phenotype via p38-dependent positive feedback, contributing to VFH pathogenesis.

NOX1 mediates metabolic heart disease in mice and is upregulated in monocytes of humans with diastolic dysfunction.

Microvascular inflammation plays an important role in the pathogenesis of diastolic dysfunction (DD) and metabolic heart disease. NOX1 is expressed in vascular and immune cells and has been implicated in the vascular pathology of metabolic disease. However, its contribution to metabolic heart disease is less understood. NOX1-deficient mice (KO) and male wild-type (WT) littermates were fed a high-fat high-sucrose diet (HFHS) and injected streptozotocin (75 mg/kg i.p.) or control diet (CTD) and sodium citrate. Despite similar weight gain and increase in fasting blood glucose and insulin, only WT-HFHS but not KO-HFHS mice developed concentric cardiac hypertrophy and elevated left ventricular filling pressure. This was associated with increased endothelial adhesion molecule expression, accumulation of Mac-2-, IL-1β-, and NLRP3-positive cells and nitrosative stress in WT-HFHS but not KO-HFHS hearts. Nox1 mRNA was solidly expressed in CD45+ immune cells isolated from healthy mouse hearts but was negligible in cardiac CD31+ endothelial cells. However, in vitro, Nox1 expression increased in response to lipopolysaccharide (LPS) in endothelial cells and contributed to LPS-induced upregulation of Icam-1. Nox1 was also upregulated in mouse bone marrow-derived macrophages in response to LPS. In peripheral monocytes from age- and sex-matched symptomatic patients with and without DD, NOX1 was significantly higher in patients with DD compared to those without DD. NOX1 mediates endothelial activation and contributes to myocardial inflammation and remodelling in metabolic disease in mice. Given its high expression in monocytes of humans with DD, NOX1 may represent a potential target to mitigate heart disease associated with DD.

Francisella tularensis Outer Membrane Vesicles Participate in the Early Phase of Interaction With Macrophages.

Francisella tularensis is known to release unusually shaped tubular outer membrane vesicles (OMV) containing a number of previously identified virulence factors and immunomodulatory proteins. In this study, we present that OMV isolated from the F. tularensis subsp. holarctica strain FSC200 enter readily into primary bone marrow-derived macrophages (BMDM) and seem to reside in structures resembling late endosomes in the later intervals. The isolated OMV enter BMDM generally via macropinocytosis and clathrin-dependent endocytosis, with a minor role played by lipid raft-dependent endocytosis. OMVs proved to be non-toxic and had no negative impact on the viability of BMDM. Unlike the parent bacterium itself, isolated OMV induced massive and dose-dependent proinflammatory responses in BMDM. Using transmission electron microscopy, we also evaluated OMV release from the bacterial surface during several stages of the interaction of Francisella with BMDM. During adherence and the early phase of the uptake of bacteria, we observed numerous tubular OMV-like protrusions bulging from the bacteria in close proximity to the macrophage plasma membrane. This suggests a possible role of OMV in the entry of bacteria into host cells. On the contrary, the OMV release from the bacterial surface during its cytosolic phase was negligible. We propose that OMV play some role in the extracellular phase of the interaction of Francisella with the host and that they are involved in the entry mechanism of the bacteria into macrophages.

Gut microbiota involved in leptospiral infections.

Leptospirosis is a re-emerging zoonotic disease worldwide. Intestinal bleeding is a common but neglected symptom in severe leptospirosis. The regulatory mechanism of the gut microbiota on leptospirosis is still unclear. In this study, we found that Leptospira interrogans infection changed the composition of the gut microbiota in mice. Weight loss and an increased leptospiral load in organs were observed in the gut microbiota-depleted mice compared with those in the control mice. Moreover, fecal microbiota transplantation (FMT) to the microbiota-depleted mice reversed these effects. The phagocytosis response and inflammatory response in bone marrow-derived macrophages and thioglycolate-induced peritoneal macrophages were diminished in the microbiota-depleted mice after infection. However, the phagocytosis response and inflammatory response in resident peritoneal macrophage were not affected in the microbiota-depleted mice after infection. The diminished macrophage disappearance reaction (bacterial entry into the peritoneum acutely induced macrophage adherence to form local clots and out of the fluid phase) led to an increased leptospiral load in the peritoneal cavity in the microbiota-depleted mice. In addition, the impaired capacity of macrophages to clear leptospires increased leptospiral dissemination in Leptospira-infected microbiota-depleted mice. Our study identified the microbiota as an endogenous defense against L. interrogans infection. Modulating the structure and function of the gut microbiota may provide new individualized preventative strategies for the control of leptospirosis and related spirochetal infections.

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.