Murine Peritoneal Macrophages Research Articles

Critical role of TLR2 in triggering protective immunity with cyclophilin entrapped in oligomannose-coated liposomes against Neospora caninum infection in mice

Neospora caninum is an intracellular protozoan parasite closely related to Toxoplasma gondii. N. caninum is thought to be a major cause of abortion in cattle worldwide. Given the current situation of drastic economic losses and a lack of efficient control strategies against such parasites, the challenge to develop potent vaccine candidates and technologies remains. We investigated the immune stimulating activity of N. caninum cyclophilin (NcCyp) with and without a formulation with oligomannose-coated-liposomes (OML) as the potential adjuvant. NcCyp-OML activated NF-κB in RAW 264.7 cells and triggered interleukin (IL)-12p40 production from murine peritoneal macrophages. In BALB/c mice, immunization with NcCyp-OML was associated with the production of specific antibodies (IgG1 and IgG2a). The specific antibody (IgG1) against naked NcCyp was also observed after the challenge infection, but it was significantly lower than those of NcCyp-OML. Moreover, significant cellular immune responses were induced, including spleen cell proliferation and interferon-gamma production. The immunization of mice with NcCyp-OML, and to lesser extent with naked NcCyp, induced significant protection against challenge with a lethal dose of N. caninum compared with the PBS control group. This protection was associated with a higher survival rate, slight changes in body weight, and lower clinical score of mice. In addition, the significant protective efficacy of NcCyp-OML was confirmed in another mouse strain, male C57BL/6 mice. The current study revealed the marked contribution of Toll-like receptor 2 (TLR2) to the protective immunity triggered by NcCyp-OML because higher numbers of TLR2−/− mice succumbed to a lethal dose of N. caninum compared with C57BL/6 mice. Moreover, prominent spleen cell proliferation and IFN-γ production was induced in NcCyp-OML-immunized mice by a TLR2-dependent mechanism.

Anti-inflammatory potential of pyocyanin in LPS-stimulated murine macrophages

Context: Pyocyanin is a typical Pseudomonas aeruginosa virulence factor, a common Gram-negative rod responsible for a wide range of severe nosocomial infections. There is evidence indicating that pyocyanin has multiple biological activities, but little is known about anti-inflammatory properties.Objective: This study investigated pyocyanin effect on nitric oxide and cytokine production in lipopolysaccharide (LPS)-activated murine peritoneal macrophages.Materials and methods: Macrophages were incubated in the presence and absence of pyocyanin (1, 5, 10, 50, and 100 µM) with and without LPS (1 µg/mL). Nitric oxide production was determined by Griess reagent and tumor necrosis factor (TNF)-α and interleukin (IL)-1β production was assessed by enzyme-linked immunosorbent assay. In addition, pyocyanin effects on zymosan A-induced peritonitis in mice were evaluated.Results: Pyocyanin (5 and 10 µM) decreased nitric oxide, TNF-α, and IL-1β production independent of macrophage death. On the other hand, in vivo, pyocyanin (5 mg/kg) was not able to affect leukocyte migration into the site of inflammation.Discussion and conclusion: Thus, our findings suggest that pyocyanin exerts anti-inflammatory effects on murine peritoneal macrophages, downregulating nitric oxide, TNF-α, and IL-1β levels, which seems to be independent of cell migration. These effects may represent a mechanism of immune evasion; nevertheless more detailed studies should be performed to confirm this hypothesis.

Assessment of Survival and Replication of Brucella spp. in Murine Peritoneal Macrophages.

Brucella spp. are bacteria that naturally infect a variety of domesticated and wild animals leading to abortions and infertility, and these bacteria are also capable of causing debilitating human infections, which often result from human exposure to infected animals and animal products. The brucellae are intracellular pathogens that reside in host cells, including macrophages and dendritic cells, and it is paramount for the pathogenesis of Brucella that the bacteria are able to survive and replicate in these host cells. The methods outlined in this chapter can be employed to study the interactions between Brucella strains and primary murine peritoneal macrophages.

Isolation and Stimulation of Peritoneal Macrophages withApoptotic Jurkat Cells to Produce IL-10.

Clearance of apoptotic cells by macrophages is critical to ensuring cellular homeostasis and suppression of autoimmunity. Macrophage recognition of apoptotic cells triggers an anti-inflammatory response, which is mediated by the release of IL-10, TGF-β etc. with concurrent inhibition of pro-inflammatory cytokines (such as TNFα, IL-12, IL-1β). To characterize cytokine profile produced by macrophages during phagocytosis of apoptotic cells, we developed an effective, more physiologic system using isolated murine peritoneal macrophages and T-lymphocyte cell line Jurkat as a source of apoptotic cells. Apoptosis of Jurkat cells is induced with staurosporine, a protein kinase C (PKC) inhibitor and detected by Annexin V/propidium iodide staining. This in vitro assay demonstrates that murine peritoneal macrophages produce large amounts of IL-10 following exposure to apoptotic Jurkat cells.

Antileishmanial in vitro activity of essential oil from Myrciaria plinioides, a native species from Southern Brazil

In South American folk medicine members of the genus Myrciaria are used for the treatment of malaria, diarrhoea, asthma, inflammation and post-partum uterine cleansing. The aim of this work was to evaluate its antileishmanial properties (in vitro) of essential oil derived from leaves of Myrciaria plinioides D. Legrand, a plant species that is native in South of Brazil. The essential oil was obtained by hydro-distillation using fresh leaves of M. plinioides. The chemical composition of this essential oil (MPEO, M. plinioides essential oil) was determined by gas chromatography coupled to mass spectrometry (GC‑MS). MPEO was assayed in vitro for antileishmanial properties against promastigotes of Leishmania amazonensis and Leishmania infantum, and for cytotoxicity against murine peritoneal macrophages. The MPEO comprised 66 components and was rich in oxygenated sesquiterpenes (82.66%) containing spathulenol (21.12%) as its major constituent. The MPEO was effective against L. amazonensis with IC50 value of 14.16 ± 7.40 µg/mL, while against L. infantum the IC50 value was higher with 101.50 ± 5.78 µg/mL. The MPEO showed significant activity against L. amazonensis, and presented a selectivity index (SI) of 6.60. The results suggest that the essential oil from leaves of M. plinioides is a promising source for new antileishmanial agents against L. amazonensis.

Immunostimulating and Antimetastatic Effects of Polysaccharides Purified from Ginseng Berry.

Ginseng root has been used in traditional oriental medicine for the enhancement of immune system function. The immunostimulatory effects of ginseng berry polysaccharides, however, remain unclear. Effects of polysaccharides from ginseng berry on the activation of natural killer (NK) cells and inhibition of tumors are reported. A crude polysaccharide was isolated from ginseng berry as a ginseng berry polysaccharide portion (GBPP) and was further fractionated using gel filtration chromatography to obtain the three polysaccharide fractions GBPP-I, -II and -III. GBPP-I consisted of mainly galactose (46.9%) and arabinose (27.5%). GBPP-I showed a high dose-dependent anticomplementary activity. Stimulation of murine peritoneal macrophages by GBPP-I showed the greatest enhancement of interleukin (IL)-6 and IL-12 and tumor necrosis factor (TNF)- production. In addition, an ex vivo assay of natural killer (NK) cell activity showed that oral ( administration of GBPP-I significantly increased NK cell cytotoxicity in YAC-1 tumor cells and production of granzyme B. Prophylactic intravenous ( and administration of GBPP-I significantly and dose-dependently inhibited lung metastatic activity in B16BL6 melanoma cells. Depletion of NK cells after injection of rabbit anti-asialo GM1 partially abolished the inhibitory effect of GBPP-I on lung metastasis, indicating that NK cells play an important role in anticancer effects. GBPP-I exerts a strong immune-enhancing activity and can prevent cancer metastasis through activation of NK cells and other immune-related cells.

Anti-inflammatory effects of the protein hydrolysate and peptide fractions isolated from Salvia hispanica L. seeds

ABSTRACT The aim of this work was to evaluate the potential anti-inflammatory effect of protein hydrolysate and peptide fractions from Salvia hispanica L. seeds. Protein isolate was obtained using defatted flour of mucilage-free seeds. Hydrolysis process was conducted by enzymatic digestion. The hydrolysate was fractionated using ultrafiltration membranes to obtain the peptide fractions (<1, 1-3, 3-5, 5-10, and >10 kDa). Protein derivatives were evaluated by in vitro activation of murine peritoneal macrophages. The anti-inflammatory activity was determined as NO production, H2O2 release and pro- and anti-inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-10) production. All the peptides exerted an anti-inflammatory activity, but peptide fraction between 1 - 3 kDa showed the highest anti-inflammatory effect. This fraction was evaluated on in vivo murine models of TPA-induced ear edema and DNFB-induced delayed-type hypersensitivity, exhibiting inhibitory effects. Hence, the results demonstrated that protein derivatives from S. hispanica L. seeds have in vitro and in vivo anti-inflammatory effects.

Characterization of M1 and M2 polarization phenotypes in peritoneal macrophages after treatment with graphene oxide nanosheets.

Macrophages play a key role in nanoparticle removal and are primarily responsible for their uptake and trafficking in vivo. Due to their functional plasticity, macrophages display a spectrum of phenotypes between two extremes indentified as pro-inflammatory M1 and reparative M2 macrophages, characterized by the expression of specific cell surface markers and the secretion of different cytokines. The influence of graphene oxide (GO) nanosheets functionalized with poly(ethylene glycol-amine) and labelled with fluorescein isothiocyanate (FITC-PEG-GO) on polarization of murine peritoneal macrophages towards M1 and M2 phenotypes was evaluated in basal and stimulated conditions by flow cytometry and confocal microscopy through the expression of different cell markers: CD80 and iNOS as M1 markers, and CD206 and CD163 as M2 markers. Although FITC-PEG-GO did not induce M1 or M2 macrophage polarization after 24 and 48 h in basal conditions, this nanomaterial decreased the percentage of M2 reparative macrophages. We have also compared control macrophages with macrophages that have or have not taken up FITC-PEG-GO after treatment with these nanosheets (GO+ and GO- cells, respectively). The CD80 expression diminished in GO+ macrophages after 48 h of GO treatment but the CD206 expression in GO+ population showed higher values than in both GO- population and control macrophages. In the presence of pro-inflammatory stimuli (LPS and IFN-γ), a significant decrease of CD80+ cells was observed after treatment with GO. This nanomaterial also induced significant decreases of CD206+ and CD163+ cells in the presence of reparative stimulus (IL-4). The CD80, iNOS and CD206 expression was lower in both GO- and GO+ cells than in control macrophages. However, higher CD163 expression was obtained in both GO- and GO+ cells in comparison with control macrophages. All these facts suggest that FITC-PEG-GO uptake did not induce the macrophage polarization towards the M1 pro-inflammatory phenotype, promoting the control of the M1/M2 balance with a slight shift towards M2 reparative phenotype involved in tissue repair, ensuring an appropriate immune response to these nanosheets.

Bavachin attenuates LPS-induced inflammatory response and inhibits the activation of NLRP3 inflammasome in macrophages

BackgroundBavachin is a natural product isolated from Psoralea corylifolia L. that has been applied as a traditional medicine in Asian countries. However, the anti-inflammatory effects of bavachin on LPS-induced inflammation and NLRP3 inflammasome activation by macrophages remain unclear. PurposeWe investigated the anti-inflammatory effects of bavachin on LPS-activated murine macrophage cell line J774A.1 cells and murine peritoneal macrophages. MethodsJ774A.1 cells and murine peritoneal macrophages were pre-treated with bavachin following LPS treatment. The concentrations of NO, PGE2, IL-6 and IL-12p40 in cell culture supernatant were analyzed. The expressions of iNOS, COX-2, mPGES-1 and MAPKs were analyzed using Western blotting, while NF-κB activity was detected using promoter reporter assay. To examine the activation of NLRP3 inflammasome, J774A.1 cells were incubated with LPS, and then treated with bavachin following treatment with ATP. The concentration of IL-1β in the cell culture supernatant was measured. The expressions of NLRP3, ASC, caspase-1 and IL-1β were analyzed using Western blotting. The formation of inflammasome complex was observed by immunofluorescence microscopy. ResultsBavachin suppressed LPS-induced NO and PGE2 production, and decreased iNOS and mPGES-1 expression. Bavachin also reduced LPS-induced IL-6 and IL-12p40 production and decreased the activation of MAPKs and NF-κB. Additionally, bavachin suppressed NLRP3 inflammasome-derived IL-1β secretion, decreased caspase-1 activation, repressed mature IL-1β expression, and inhibited inflammasome complex formation. Furthermore, bavachin also suppressed the production of NO, IL-6 and IL-12p40 by LPS-stimulated murine peritoneal macrophages. ConclusionOur experimental results indicated anti-inflammatory effects of bavachin exhibit attenuation of LPS-induced inflammation and inhibit activation of NLRP3 inflammasome in macrophages. These results suggest that bavachin might have potential in treating inflammatory and autoimmune diseases.

Berberis lycium Royle fruit extract mitigates oxi-inflammatory stress by suppressing NF-κB/MAPK signalling cascade in activated macrophages and Treg proliferation in splenic lymphocytes.

Although Berberis plant species have been advocated as immune modulators, information regarding their mechanism(s) of action is limited. Therefore, in the present study we assessed the efficacy of Berberis lycium Royle fruit extract (BLFE) in the attenuation of lipopolysaccharide (LPS)-induced oxi-inflammatory aggravation and concanavalin A (Con-A)-induced proliferation in murine peritoneal macrophages and lymphocytes, respectively. BLFE strongly suppressed production of the oxidative and inflammatory effector molecules nitric oxide (NO), reactive oxygen species (ROS), inducible nitric oxide synthase (iNOS), inflammatory cytokines (TNF-α/IL-6/IL-1β/IFN-γ) as well as chemokines (MCP-1 and RANTES), with a concomitant enhancement in heme oxygenase-1 (HO-1) and IL-10 levels. Subsequent mechanistic analysis revealed that BLFE strongly inhibited the phosphorylation of IκBα as well as MAPKs such as extracellular signal-regulated kinase (ERK), p38 MAPK, and c-Jun NH2-terminal kinase (JNK), thereby directly resulting in the suppression of nuclear factor-κB (NF-ĸB) and c-Jun activation, ultimately culminating in the observed attenuation of inflammatory molecules. Additionally, BLFE appeared to mitigate Con-A-induced proliferation of Tregs (CD3+ CD4+ CD25+) thereby suggesting its modulatory effects on adaptive immune cells. UPLC-DAD-ESI-QTOF-MS/MS of BLFE revealed the presence of major bioactive phenolics and alkaloids including chlorogenic acid, rutin, catechin and quercetin 3-D-galactoside, berberine and magnoflorine, which could have synergistically contributed to the above findings. Overall, this work provides evidence that BLFE may be effective in the mitigation of inflammatory disorders, especially those associated with NF-κB/MAPK activation.

8-Hydroxy-2-(1H-1,2,3-triazol-1-yl)-1,4-naphtoquinone derivatives inhibited P2X7 Receptor-Induced dye uptake into murine Macrophages.

Extracellular adenosine 5'-triphosphate (ATP) triggers the P2X7 receptor (P2X7R) ionic channel to stimulate the release of the interleukin-IL-1β cytokine into macrophages. The current study explored the reaction of six structurally diverse triazole derivatives on P2X7-mediated dye uptake into murine peritoneal macrophages. P2X7R activity determined by ATP-evoked fluorescent dye uptake. Triazole derivatives toxicity measured using dextran rhodamine exclusion based colorimetric assay. A740004 and BBG, both P2X7R antagonist, inhibited ATP-induced dye uptake. In contrast, the derivatives 5a, 5b, 5e, and 5f did not diminish P2X7R activity in concentrations until 100 µM. 5c and 5d analogs caused a potent inhibitory activity on P2X7-induced dye uptake. Dextran Rhodamine exclusion measurements after 24 h of continuous treatment with triazole derivatives indicated a moderated toxicity for all molecules. In conclusion, this study showed that a series of new hybrid 1,2,3-triazolic naphthoquinones reduces P2X7R-induced dye uptake into murine macrophages. In silico analysis indicates a good pharmacokinetic profile and molecular docking results of these analogs indicate the potential to bind into an allosteric site located into the P2X7R pore and juxtaposed with the ATP binding pocket. In this manner, the compounds 5c and 5d may be used as a scaffold for new P2X7R inhibitors with reduced toxicity, and good anti-inflammatory activity.

Absence of Regulatory T Cells Causes Phenotypic and Functional Switch in Murine Peritoneal Macrophages.

Tissue macrophages are important components of tissue homeostasis and inflammatory pathologies. In the peritoneal cavity, resident macrophages interact with a variety of immune cells and can exhibit broad range of phenotypes and functions. Forkhead-box-P3 (FOXP3)+ regulatory T cells (Tregs) play an indispensable role in maintaining immunological tolerance, yet whether, and how the pathological condition that results from the lack of functional Tregs affects peritoneal macrophages (PM) is largely unknown. We used FOXP3-deficient scurfy (Sf) mice to investigate PM behavior in terms of the missing crosstalk with Tregs. Here, we report that Treg deficiency induced a marked increase in PM numbers, which was reversed after adoptive transfer of CD4+ T cells or neutralization of macrophage colony-stimulating factor. Ex vivo assays demonstrated a pro-inflammatory state of PM from Sf mice and signs of excessive activation and exhaustion. In-depth immunophenotyping of Sf PM using single-cell chipcytometry and transcriptome analysis revealed upregulation of molecules involved in the initiation of innate and adaptive immune responses. Moreover, upon transfer to non-inflammatory environment or after injection of CD4+ T cells, PM from Sf mice reprogramed their functional phenotype, indicating remarkable plasticity. Interestingly, frequencies, and immune polarization of large and small PM subsets were dramatically changed in the FOXP3-deficient mice, suggesting distinct origin and specialized function of these subsets in inflammatory conditions. Our findings demonstrate the significant impact of Tregs in shaping PM identity and dynamics. A better understanding of PM function in the Sf mouse model may have clinical implication for the treatment of immunodysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, and other forms of immune-mediated enteropathies.

2-Aryl-quinazolin-4(3H)-ones as an inhibitor of leishmania folate pathway: In vitro biological evaluation, mechanism studies and molecular docking

To identify new agents for the American Cutaneous Leishmaniasis treatment, a series of 2-aryl-quinazolin-4(3H)-ones were tested against L. mexicana, L. braziliensis and L. amazonensis parasites as potential inhibitor of folic metabolism pathway. In general, the L. braziliensis and L. mexicana promastigote parasites were more sensitive to the action of the quinazolinones than L. amazonensis. The most active derivatives showed low-micromolar EC50 ranging from 4 to 10 μM, being 1.3 to 4 fold more potent than glucantime reference drug. A complete in vitro evaluation on intracellular amastigote, axenic amastigote and murine peritoneal macrophage were performed for the most active derivatives. The compounds 2j, 2h, 2t and 2u displayed acceptable responses against intracellular amastigote compared to reference drug, excellent antileishmanial activities against axenic amastigote (LD50 ranging from 1 to 4 μM) and relative low toxicities on peritoneal macrophages. To validate the efficacy of these four derivatives, an in vitro evaluation was performed against an antimony-resistant amastigote strain; identifying to 2h and 2u as promising antileishmanial leads for further pharmacokinetics and in vivo studies. Experimental mechanism assays putted in evidences that the most active compounds act as folate inhibitor. A tentative molecular docking on pteridine reductase 1 (PTR1) enzyme showed that the most active quinazolinones 2j and 2t are located in almost identical place compared with methotrexate reference into active site.

Role of Toll-like receptor 4/oxidant-coupled activation in regulating the biosynthesis of omega-3 polyunsaturated fatty acid derivative resolvin D1 in primary murine peritoneal macrophage

We have previously shown that reactive oxygen species (ROS) as prooxidants can activate Toll-like receptor 4 (TLR4) with the potential to initiate, propagate and maintain “sterile” inflammation of innate immunity, which plays a mediatory role in a host of human disease states. We now present new evidence that ROS can also activate TLR4 to counter the inflammatory phenotype by increasing the production of resolvin D1 (RvD1), which is a specialized anti-inflammatory and pro-resolving lipid mediator. We used primary murine peritoneal macrophages (pM) derived from both TLR4-WT and TLR4-KO mice as a cellular model. We used potassium peroxychromate (PPC) as a direct in vitro source of exogenous ROS. PPC treatment increased intracellular ROS levels, which decreased intracellular total antioxidant capacity, thus suggesting an enhanced cellular oxidative stress. PPC and LPS-EK (a TLR4-specific agonist) increased pro-inflammatory TNFα production with noeffect on IL-10, an anti-inflammatory cytokine. Treatment with the prooxidant increased the expression of 12 lipoxygenase (12-LOX) and 5-lipoxygenase (5-LOX) only in pM derived from TLR4 WT but not in pM from TLR4-KO mice. 5-LOX and 12-LOX are the key enzymes in the RvD1 biosynthetic pathway. In addition, PPC increased the expression of RvD1 receptor, a member of G-protein-coupled receptor only in pM from TLR4-WT mice. Our data support the involvement of TLR4-mediated oxidant-induced pro-inflammatory phenotypes that are in opposition to the production of anti-inflammatory/pro-resolution phenotypes in macrophages. Now, we show that through TLR4 activation, exogenous oxidants can play a role both in producing proinflammatory phenotypes at the same time that it enhances resolution of inflammation to maintain a state of cellular homeostasis and prevent tissue damage/disease.

Transfer of miRNA in Macrophage-Derived Exosomes Induces Drug Resistance in Pancreatic Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is known for its resistance to gemcitabine, which acts to inhibit cell growth by termination of DNA replication. Tumor-associated macrophages (TAM) were recently shown to contribute to gemcitabine resistance; however, the exact mechanism of this process is still unclear. Using a genetic mouse model of PDAC and electron microscopy analysis, we show that TAM communicate with the tumor microenvironment via secretion of approximately 90 nm vesicles, which are selectively internalized by cancer cells. Transfection of artificial dsDNA (barcode fragment) to murine peritoneal macrophages and injection to mice bearing PDAC tumors revealed a 4-log higher concentration of the barcode fragment in primary tumors and in liver metastasis than in normal tissue. These macrophage-derived exosomes (MDE) significantly decreased the sensitivity of PDAC cells to gemcitabine, in vitro and in vivo This effect was mediated by the transfer of miR-365 in MDE. miR-365 impaired activation of gemcitabine by upregulation of the triphospho-nucleotide pool in cancer cells and the induction of the enzyme cytidine deaminase; the latter inactivates gemcitabine. Adoptive transfer of miR-365 in TAM induced gemcitabine resistance in PDAC-bearing mice, whereas immune transfer of the miR-365 antagonist recovered the sensitivity to gemcitabine. Mice deficient of Rab27 a/b genes, which lack exosomal secretion, responded significantly better to gemcitabine than did wildtype. These results identify MDE as key regulators of gemcitabine resistance in PDAC and demonstrate that blocking miR-365 can potentiate gemcitabine response.Significance: Harnessing macrophage-derived exosomes as conveyers of antagomiRs augments the effect of chemotherapy against cancer, opening new therapeutic options against malignancies where resistance to nucleotide analogs remains an obstacle to overcome. Cancer Res; 78(18); 5287-99. ©2018 AACR.

Effect of Galectin-9/Tim-3 pathway on the polarization of M1/M2 subtype in murine macrophages induced by lipopolysaccharide

To investigate the meaning and molecular mechanisms of Galectin-9/T-cell immunoglobulin mucin-3 (Tim-3) pathway on lipopolysaccharide (LPS) induced murine macrophage M1/M2 subtype polarization. The murine peritoneal macrophages RAW264.7 were cultured in vitro until the cells had matured with 80%-90% fusion rate. (1) The cells were cultured in serum-free medium and treated with 0 (blank control), 0.01, 0.1, 1, 10 and 100 mg/L LPS for 24 hours. Real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR) or Western Blot was used to determine the expressions of M1 macrophage markers such as interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS) and M2 macrophage markers such as arginase-1 (Arg-1), leukocyte differentiation antigen 206 (CD206), as well as Tim-3 and Galectin-9 in the cells. (2) The other mice peritoneal macrophages were divided into blank control group (cultured in serum-free DMEM medium for 24 hours), LPS treatment group (cultured in serum-free DMEM medium containing 0.1 mg/L LPS for 24 hours) and α-lactose pretreatment group (pretreated with serum-free DMEM containing 40 μmol/L Galectin-9 signal antagonist 1 hour before LPS stimulation). Over closed Galectin-9 signal was used to verify the role of Galectin-9 in macrophage M1/M2 subtype polarization. (1) After stimulation with low concentrations of LPS (0.01 mg/L, 0.1 mg/L) for 24 hours, the expression of M1 markers was only slightly increased such as iNOS mRNA or not significantly changed such as IL-6 mRNA in macrophages, while the expressions of M2 markers such as Arg-1 mRNA and CD206 mRNA were significantly increased and peaked at LPS concentrations of 0.1 mg/L and 0.01 mg/L [compared with blank control group: Arg-1 mRNA (2-ΔΔCt) was 1.85±0.07 vs. 1.00±0.02, CD206 mRNA (2-ΔΔCt) was 2.03±0.11 vs.1.00±0.05, both P < 0.01]. With the increase of LPS concentration, the expressions of IL-6 mRNA and iNOS mRNA continued to increase, while the expressions of Arg-1 mRNA and CD206 mRNA were gradually decreased, and the macrophage M1/M2 subtype polarization status changed. At the same time, the level of Tim-3 protein in macrophages was significantly up-regulated after stimulation with 0.01 mg/L LPS as compared with that of blank control group (Tim-3/GAPDH: 0.84±0.04 vs. 0.69±0.02, P < 0.01), peaked at LPS concentrations of 0.1 mg/L, and then decreased with increasing LPS concentration. The intracellular Galectin-9 and supernatant secreted Galectin-9 (s-Galectin-9) protein levels showed no significant change after stimulation with low concentrations of LPS (0.01 mg/L, 0.1 mg/L), while then gradually decreased with the increase of LPS concentration. (2) Compared with blank control group, the mRNA expressions of M1 marker iNOS and M2 markers Arg-1 and CD206 were significantly increased in LPS treatment group, but IL-6 mRNA level was not changed significantly. The mRNA levels of IL-6 and iNOS were further elevated after pretreatment with α-lactose as compared with that of the LPS treatment group [IL-6 mRNA (2-ΔΔCt): 1.44±0.02 vs. 1.14±0.11, iNOS mRNA (2-ΔΔCt): 2.45±0.04 vs. 2.01±0.08, both P < 0.01], while the mRNA levels of Arg-1 and CD206 were significantly decreased [Arg-1 mRNA (2-ΔΔCt): 0.75±0.01 vs. 1.85±0.02, CD206 mRNA (2-ΔΔCt): 0.58±0.02 vs. 2.03±0.14, both P < 0.01]. Meanwhile, the blocking of Galectin-9 signaling could also reduce the extracellular s-Galectin-9 (compared with LPS treatment group: s-Galectin-9/GAPDH was 0.10±0.01 vs. 0.23±0.02, P < 0.01), down-regulated the expressions of Tim-3 and Galectin-9 (Tim-3/GAPDH: 0.28±0.01 vs. 0.43±0.01, Galectin-9/GAPDH: 0.21±0.01 vs. 0.43±0.01, both P < 0.01). LPS regulates macrophage M1/M2 subtype polarization via Galectin-9/Tim-3 signaling pathway. Low-doses of LPS can limit the development of inflammation by accommodating the expression and secretion of Galectin-9 to polarize macrophages to M2. High-doses of LPS promotes the development of inflammation by down-regulating the expression and secretion of Galectin-9 to polarize macrophages to M1.

Characterization of human peritoneal monocyte/macrophage subsets in homeostasis: Phenotype, GATA6, phagocytic/oxidative activities and cytokines expression

Peritoneal macrophages play a critical role in the control of infectious and inflammatory diseases. Although recent progress on murine peritoneal macrophages has revealed multiple aspects on their origin and mechanisms involved in their maintenance in this compartment, little is known on the characteristics of human peritoneal macrophages in homeostasis. Here, we have studied by flow cytometry several features of human peritoneal macrophages obtained from the peritoneal cavity of healthy women. Three peritoneal monocyte/macrophage subsets were established on the basis of CD14/CD16 expression (CD14++CD16−, CD14++CD16+ and CD14highCD16high), and analysis of CD11b, CD11c, CD40, CD62L, CD64, CD80, CD86, CD116, CD119, CD206, HLA-DR and Slan was carried out in each subpopulation. Intracellular expression of GATA6 and cytokines (pro-inflammatory IL-6 and TNF-α, anti-inflammatory IL-10) as well as their phagocytic/oxidative activities were also analyzed, in an attempt to identify genuine resident peritoneal macrophages. Results showed that human peritoneal macrophages are heterogeneous regarding their phenotype, cell complexity and functional abilities. A direct relationship of CD14/CD16 expression, intracellular content of GATA6, and activation/maturation markers like CD206 and HLA-DR, support that the CD14highCD16high subset represents the mature phenotype of steady-state human resident peritoneal macrophages. Furthermore, increased expression of CD14/CD16 is also related to the phagocytic activity.

Luteolin Regulates Macrophage Polarization via the PI3K/Akt Pathway to Inhibit the Apoptosis Stimulated by Angiotensin II.

The aim of this study was to investigate anti-apoptotic effects of luteolin on angiotensin II-stimulated murine peritoneal macrophages and to explore its mechanisms. The viability and cytotoxicity of murine peritoneal macrophages were assessed using the Cell Counting Kit-8 assay and measuring lactate dehydrogenase levels, respectively. Apoptotic rates were determined using Annexin V/propidium iodide staining. Protein expression was examined by western blotting, and markers of macrophage phenotypes were analyzed by flow cytometry and ELISA. Luteolin decreased the apoptotic rate of angiotensin II-stimulated macrophages. This effect was associated with increased Bcl-2 and caspase-3 levels as well as decreased Bax and cleaved caspase-3 levels. Additionally, luteolin reduced the expression of M1 macrophage phenotype markers (IL-6, TNF-α, iNOS, CD16/32) and increased the expression of M2 macrophage phenotype markers (Dectin-1, IL-10, Arg-1, CD206). Moreover, luteolin blocked Akt phosphorylation on residues 308 and 473, which were up-regulated in presence of angiotensin II. The effects of luteolin were similar to those of LY294002, a specific PI3K/Akt pathway inhibitor. These results indicated that luteolin has anti-apoptotic effects on angiotensin II-stimulated macrophages via macrophage polarization, which might be associated with PI3K/Akt signaling.

CXCL13 produced by macrophages due to Fli1 deficiency may contribute to the development of tissue fibrosis, vasculopathy and immune activation in systemic sclerosis.

CXCL13, a chemokine for B cells, follicular T cells, T helper 17 cells, and regulatory T cells, is reported to contribute to the development of systemic sclerosis (SSc), reflecting aberrant activation of immune system. To better understand the role of CXCL13 in SSc, we investigated the influence of Fli1 deficiency, a potential predisposing factor of this disease, on CXCL13 expression and assessed the clinical correlation of serum CXCL13 levels by multivariate regression analysis. Haploinsufficient loss of Fli1 remarkably induced CXCL13 expression in murine peritoneal macrophages, while gene silencing of FLI1 did not affect the expression of CXCL13 in human dermal fibroblasts and human dermal microvascular endothelial cells. Serum CXCL13 levels were elevated in SSc patients compared with healthy controls and correlated positively with skin score and negatively with pulmonary function test results. SSc patients with elevated serum CXCL13 levels had longer disease duration, diffuse cutaneous involvement, interstitial lung disease (ILD), heart involvement, pulmonary arterial hypertension, Raynaud's phenomenon, pitting scars, digital ulcers, telangiectasia, and high serum IgG levels more frequently than the other patients. In particular, serum CXCL13 levels were associated with ILD and digital ulcers by multivariate regression analysis. Taken together, these results indicate that CXCL13 expression is upregulated by Fli1 deficiency in macrophages, potentially contributing to the development of tissue fibrosis, vasculopathy and immune activation in SSc, especially ILD and digital ulcers.

Differential effect of Androctonus australis hector venom components on macrophage KV channels: electrophysiological characterization.

Neurotoxins of scorpion venoms modulate ion channels. Voltage-gated potassium (KV) channels regulate the membrane potential and are involved in the activation and proliferation of immune cells. Macrophages are key components of the inflammatory response induced by scorpion venom. The present study was undertaken to investigate the effect of Androctonus australis hector (Aah) venom on KV channels in murine resident peritoneal macrophages. The cytotoxicity of the venom was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) -based assay and electrophysiological recordings were performed using the whole-cell patch clamp technique. High doses of Aah venom (50, 125, 250 and 500µg/ml) significantly decreased cell viability, while concentrations of 0.1-25µg/ml were not cytotoxic towards peritoneal macrophages. Electrophysiological data revealed a differential block of KV current between resting and LPS-activated macrophages. Aah venom significantly reduced KV current amplitude by 62.5 ± 4.78% (n = 8, p < 0.05), reduced the use-dependent decay of the current, decreased the degree of inactivation and decelerated the inactivation process of KV current in LPS-activated macrophages. Unlike cloned KV1.5channels, Aah venom exerted a similar blocking effect on KV1.3 compared to KV current in LPS-activated macrophages, along with a hyperpolarizing shift in the voltage dependence of KV1.3 inactivation, indicating a direct mechanism of current inhibition by targeting KV1.3 subunits. The obtained results, demonstrating that Aah venom differentially targets KV channels in macrophages, suggest differentialoutcomesfor their inhibitions, and that further investigations of scorpion venom immunomodulatory potential are required.