Gastric cancer-derived mesenchymal stem cells regulate the M2 polarization of macrophages within gastric cancer microenvironment via JAK2/STAT3 signaling pathway / 中华肿瘤杂志

Objective: To investigate the role and mechanism of tumor-derived mesenchymal stem cells in regulating the M2 polarization of macrophages within gastric cancer microenvironment. Methods: Gastric cancer tissues and the adjacent non-cancerous tissues were collected from patients underwent gastric cancer resection in the First People's Hospital of Lianyungang during 2018. In our study, THP-1-differentiated macrophages were co-cultured with gastric cancer-derived mesenchymal stem cells (GC-MSCs). Then, the M2 subtype-related gene, the markers expressed on cell surface and the cytokine profile were analyzed by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), flow cytometry and Luminex liquid chip, respectively. The key cytokines mediating the inducing effect of GC-MSCs on macrophage polarization into the M2 subtype were detected and screened by Luminex liquid chip, which were further confirmed by the neutralizing antibody test. The expressions of macrophage proteins involved in M2 polarization-related signaling pathways under the different co-culture conditions of GC-MSCs were detected by western blot. Results: In Mac+ GC-MSC-culture medium (CM) group, the expression levels of Ym-1 and Fizz-1 (1.53±0.32 and 13.22±1.05, respectively), which are markers for M2 subtype, were both significantly higher than those of Mac group (1.00±0.05 and 1.21±0.38, respectively, P<0.05). The level of iNOS in Mac+ GC-MSC-CM group (0.60±0.41) was significantly lower than that of Mac group (1.06±0.38, P=0.023). In Mac+ GC-MSC-Transwell (TW) group, the expression levels of Ym-1 and Fizz-1 (1.47±0.09 and 13.16±2.77, respectively) were both significantly higher than those of Mac group (1.00±0.05 and 1.21±0.38, respectively, P<0.05). The level of iNOS in Mac+ GC-MSC-CM group (0.56±0.03) was significantly lower than that of Mac group (1.06±0.38, P=0.026). The ratios of CD163(+) /CD204(+) cells in Mac+ GC-MSC-CM and Mac+ GC-MSC-TW groups (3.80% and 4.40%, respectively) were both remarkably higher than that of Mac group (0.60%, P<0.05). The expression levels of IL-10, IL-6, MCP-1 and VEGF in Mac+ GC-MSC-CM group were (592.60±87.52), (1 346.80±64.70), (11 256.00±29.03) and (1 463.90±66.67) pg/ml, respectively, which were significantly higher than those of Mac group [(41.03±2.59), (17.35±1.79), (5 213.30±523.71) and (267.12±12.06) pg/ml, respectively, P<0.05]. The levels of TNF-α, IP-10, RANTES and MIP-1α were (95.57±9.34), (410.48±40.68), (6 967.30±1.29) and (1 538.70±283.04) pg/ml, which were significantly lower than those of Mac group [(138.01±24.31, (1 298.60±310.50), (14 631.00±4.21) and (6 633.20±1.47) pg/ml, respectively, P<0.05]. The levels of IL-6 and IL-8 in GC-MSCs [(11 185.02±2.82) and (12 718.03±370.17) pg/ml, respectively] were both strikingly higher than those of MSCs from adjacent non-cancerous gastric cancer tissues [(270.71±59.38) and (106.04±32.84) pg/ml, repectively, P<0.05]. The ratios of CD86(+) cells in Mac+ IL-6-blocked-GC-MSC-CM and Mac+ IL-8-blocked-GC-MSC-CM groups (28.80% and 31.40%, respectively) were both higher than that of Mac+ GC-MSC-CM group (24.70%). Compared to Mac+ GC-MSC-CM group (13.70%), the ratios of CD204(+) cells in Mac+ IL-6-blocked-GC-MSC-CM and Mac+ IL-8-blocked-GC-MSC-CM groups (9.90% and 8.70%, separately) were reduced. The expression levels of p-JAK2 and p-STAT3, which are proteins of macrophage M2 polarization-related signaling pathway, in Mac+ GC-MSC-CM group (0.86±0.01 and 1.08±0.01, respectively) were significantly higher than those of Mac group (0.50±0.01 and 0.82±0.01, respectively, P<0.05). The expression levels of p-JAK2 in Mac+ IL-6-blocked-GC-MSC-CM group (0.47±0.02) were significantly lower those that of Mac+ GC-MSC-CM group (0.86±0.01, P<0.05). The expression levels of p-JAK2 and p-STAT3 in Mac+ IL-8-blocked-GC-MSC-CM group (0.50±0.01 and 0.85±0.01, respectively) were both significantly lower than those of Mac+ GC-MSC-CM group (0.86±0.01 and 1.08±0.01, P<0.05). The expression levels of p-JAK2 and p-STAT3 in Mac+ IL-6/IL-8-blocked-GC-MSC-CM group (0.37±0.01 and 0.65±0.01, respectively) were both significantly lower than those of Mac+ GC-MSC-CM group (0.86±0.01 and 1.08±0.01, P<0.05). Conclusion: GC-MSCs promote the activation of JAK2/STAT3 signaling pathway in macrophages via high secretions of IL-6 and IL-8, which subsequently induce the macrophage polarization into a pro-tumor M2 subtype within gastric cancer microenvironment.

Integrated metabolism and epigenetic modifications in the macrophages of mice in responses to cold stress / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

The negative effects of low temperature can readily induce a variety of diseases. We sought to understand the reasons why cold stress induces disease by studying the mechanisms of fine-tuning in macrophages following cold exposure. We found that cold stress triggers increased macrophage activation accompanied by metabolic reprogramming of aerobic glycolysis. The discovery, by genome-wide RNA sequencing, of defective mitochondria in mice macrophages following cold exposure indicated that mitochondrial defects may contribute to this process. In addition, changes in metabolism drive the differentiation of macrophages by affecting histone modifications. Finally, we showed that histone acetylation and lactylation are modulators of macrophage differentiation following cold exposure. Collectively, metabolism-related epigenetic modifications are essential for the differentiation of macrophages in cold-stressed mice, and the regulation of metabolism may be crucial for alleviating the harm induced by cold stress.

Proteomic characterization of four subtypes of M2 macrophages derived from human THP-1 cells / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Macrophages are widely distributed immune cells that contribute to tissue homeostasis. Human THP-1 cells have been widely used in various macrophage-associated studies, especially those involving pro-inflammatory M1 and anti-inflammatory M2 phenotypes. However, the molecular characterization of four M2 subtypes (M2a, M2b, M2c, and M2d) derived from THP-1 has not been fully investigated. In this study, we systematically analyzed the protein expression profiles of human THP-1-derived macrophages (M0, M1, M2a, M2b, M2c, and M2d) using quantitative proteomics approaches. The commonly and specially regulated proteins of the four M2 subtypes and their potential biological functions were further investigated. The results showed that M2a and M2b, and M2c and M2d have very similar protein expression profiles. These data could serve as an important resource for studies of macrophages using THP-1 cells, and provide a reference to distinguish different M2 subtypes in macrophage-associated diseases for subsequent clinical research.

Effect of IL-33 on pyroptosis of macrophages in mice with sepsis via NF-κB/p38 MAPK signaling pathway

ABSTRACT Purpose To demonstrate the effect of IL-33 on the macrophage pyroptosis in mice with sepsis through the NF-kB/p38 MAPK signal pathway. Methods In total, 24 C57BL/6 mice were divided into the sham operation group (sham) and the cecal ligation and puncture group (CLP). After CLP, 24 IL-33-/- mice were divided into the IL-33-/- group and the IL-33-/- intervention group. The latter group was intraperitoneally injected with IL-33. Mouse mortality was observed after CLP. Macrophage apoptosis in peritoneal lavage fluid was detected by flow cytometry. Serum inflammatory factor level was detected by ELISA. Apoptotic protein expression and NF-κB/p38 MAKP signaling pathway protein expression were detected by qRT-PCR and Western blot. Results Knocking out IL-33 significantly reduced the mortality of CLP mice, as well as the mRNA expression of IL-33 and the levels of serum inflammatory factors, including IL-33, IL-1β, and IL-18. It also reduced the rate of macrophage apoptosis and the expression of the apoptotic protein caspase-1 p10; increased the expression of IκBα; and reduced the protein expression of NF-κB and p38 MAPK. These effects were reversed after exogenous injection of IL-33. Conclusions IL-33 can increase the level of macrophage pyroptosis in mice with sepsis (by activating the NF-kB/p38MAPK signal pathway) and the mortality of these mice.

Polarized activation affects iron metabolism in macrophages / 生理学报

The aim of this study was to investigate the effects of polarization program on the ability of macrophages to regulate iron metabolism. M1 and M2 macrophages were propagated in vitro from porcine alveolar macrophages 3D4/2 and polarized by cytokines. The 3D4/2 macrophages were treated with 20 ng/mL interferon gamma (IFN-γ) and 10 ng/mL interleukin-4 (IL-4) combined with 10 ng/mL macrophage colony-stimulating factor (M-CSF) to induce polarization to M1 and M2, respectively. After incubation for 24 h, the expression levels of inflammatory factors and iron-metabolism genes were determined using real-time qPCR, Western bot and immunofluorescence. The M1/M2 macrophages culture media supernatant was collected and used to treat porcine intestinal epithelial cells IPEC-J2. The proliferation ability of IPEC-J2 was detected using CCK-8 assay kit. Following exogenous addition of ammonium ferric citrate (FAC) to M1/M2 macrophages, the phagocytic function of macrophages was detected using fluorescein isothiocyanate-dextran (FITC-dextran) and flow cytometry. The results showed that, compared with control, M1 macrophages had higher mRNA levels of iron storage proteins (ferritin heavy and light polypeptide, i.e. FtH and FtL), hepcidin and lipocalin-2, as well as iron content. Moreover, iron enhanced the ability of M1 macrophages to phagocytize FITC-dextran. There was no significant change in these mRNA expression levels in M2 macrophages, but the mRNA expression levels of ferroportin and transferrin receptor were up-regulated. In addition, the conditioned media supernatant from M2 macrophages promoted cell proliferation of IPEC-J2. These findings indicate that M1 macrophages tend to lock iron in the cell and reduce extracellular iron content, thereby inhibiting the proliferation of extracellular bacteria. While M2 macrophages tend to excrete iron, which contributes to the proliferation of surrounding cells and thus promotes tissue repair.

Bone marrow-derived mesenchymal stem cells modulate autophagy in RAW264.7 macrophages via the phosphoinositide 3-kinase/protein kinase B/heme oxygenase-1 signaling pathway under oxygen-glucose deprivation/restoration conditions / 中华医学杂志(英文版)

BACKGROUND@#Autophagy of alveolar macrophages is a crucial process in ischemia/reperfusion injury-induced acute lung injury (ALI). Bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent cells with the potential for repairing injured sites and regulating autophagy. This study was to investigate the influence of BM-MSCs on autophagy of macrophages in the oxygen-glucose deprivation/restoration (OGD/R) microenvironment and to explore the potential mechanism.@*METHODS@#We established a co-culture system of macrophages (RAW264.7) with BM-MSCs under OGD/R conditions in vitro. RAW264.7 cells were transfected with recombinant adenovirus (Ad-mCherry-GFP-LC3B) and autophagic status of RAW264.7 cells was observed under a fluorescence microscope. Autophagy-related proteins light chain 3 (LC3)-I, LC3-II, and p62 in RAW264.7 cells were detected by Western blotting. We used microarray expression analysis to identify the differently expressed genes between OGD/R treated macrophages and macrophages co-culture with BM-MSCs. We investigated the gene heme oxygenase-1 (HO-1), which is downstream of the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling pathway.@*RESULTS@#The ratio of LC3-II/LC3-I of OGD/R treated RAW264.7 cells was increased (1.27 ± 0.20 vs. 0.44 ± 0.08, t = 6.67, P  < 0.05), while the expression of p62 was decreased (0.77 ± 0.04 vs. 0.95 ± 0.10, t = 2.90, P  < 0.05), and PI3K (0.40 ± 0.06 vs. 0.63 ± 0.10, t = 3.42, P  < 0.05) and p-Akt/Akt ratio was also decreased (0.39 ± 0.02 vs. 0.58 ± 0.03, t = 9.13, P  < 0.05). BM-MSCs reduced the LC3-II/LC3-I ratio of OGD/R treated RAW264.7 cells (0.68 ± 0.14 vs. 1.27 ± 0.20, t = 4.12, P  < 0.05), up-regulated p62 expression (1.10 ± 0.20 vs. 0.77 ± 0.04, t = 2.80, P  < 0.05), and up-regulated PI3K (0.54 ± 0.05 vs. 0.40 ± 0.06, t = 3.11, P  < 0.05) and p-Akt/Akt ratios (0.52 ± 0.05 vs. 0.39 ± 0.02, t = 9.13, P  < 0.05). A whole-genome microarray assay screened the differentially expressed gene HO-1, which is downstream of the PI3K/Akt signaling pathway, and the alteration of HO-1 mRNA and protein expression was consistent with the data on PI3K/Akt pathway.@*CONCLUSIONS@#Our results suggest the existence of the PI3K/Akt/HO-1 signaling pathway in RAW264.7 cells under OGD/R circumstances in vitro, revealing the mechanism underlying BM-MSC-mediated regulation of autophagy and enriching the understanding of potential therapeutic targets for the treatment of ALI.

Inhibition of nitric oxide production of activated mice peritoneal macrophages is independent of the Toxoplasma gondii strain

BACKGROUND Toxoplasma gondii causes toxoplasmosis and is controlled by activated macrophages. However, infection of macrophages by tachyzoites induces TGF-β signaling (TGF-s) inhibiting nitric oxide (NO) production. NO inhibition may be a general escape mechanism of distinct T. gondii strains. OBJECTIVES To evaluate in activated macrophages the capacity of T. gondii strains of different virulence and genetics (RH, type I; ME-49, type II; VEG, type III; P-Br, recombinant) to evade the NO microbicidal defense system and determine LC3 loading to the parasitophorous vacuole. METHODS Activated peritoneal macrophages were infected with the different T. gondii strains, NO-production was evaluated by the Griess reagent, and inducible nitric oxide synthase expression, TGF-s, and LC3 localisation assayed by immunofluorescence. FINDINGS Only RH persisted in macrophages, while VEG was more resistant than P-Br and ME-49. All strains induced TGF-s, degradation of inducible nitric oxide synthase, and NO-production inhibition from 2 to 24 h of infection, but only RH sustained these alterations for 48 h. By 24 h of infection, TGF-s lowered in macrophages infected by ME-49, and P-Br, and NO-production recovered, while VEG sustained TGF-s and NO-production inhibition longer. LC3 loading to parasitophorous vacuole was strain-dependent: higher for ME-49, P-Br and VEG, lower for RH. All strains inhibited NO-production, but only RH sustained this effect probably because it persisted in macrophages due to additional evasive mechanisms as lower LC3 loading to parasitophorous vacuole. MAIN CONCLUSIONS These results support that T. gondii can escape the NO microbicidal defense system at the initial phase of the infection, but only the virulent strain sustain this evasion mechanism.

Apamin-induced alterations in J774 1. 6 macrophage metabolism / [Alterações induzidas por apamina no metabolismo de macrófagos J774 1. 6]

Among the immune system cells, macrophages have an important role. Apamin, a bee venom constituent, is important in the defense of these insects. Thus, we aimed to evaluate the metabolism of J774 1.6 macrophage cell line when exposed to isolated and purified apamin, using cytotoxicity tests by MTT reduction and analysis by flow cytometry (apoptosis / necrosis, production of reactive oxygen species (ROS), membranous lipoperoxidation (LPO), electrical potential of the mitochondrial membrane (mMP) and DNA fragmentation). None of the tested concentrations (10 to 100µg/mL) were cytotoxic according to MTT reductions. Apoptosis rates decreased at concentrations of 2.5, 5.0, and 10.0µg/mL (P<0.05), while necrosis rates increased (P<0.05). However, rates of healthy cells at the highest tested concentration (10µg/mL) did not differ from control (P>0.05). Apamin did not alter ROS, LPO, or DNA fragmentation. Therefore, all analyzed concentrations (1.25 to 10µg/mL) decreased mMP. Such decrease in apoptosis might be due to a suppression of mitochondrial pro-apoptotic messengers, as this peptide causes no oxidative stress, lipid peroxidation, and DNA damage. Highly sensitive techniques are majorly important for proper interpretation of cellular toxicity mechanisms, combined with routine laboratory methods.(AU)

Das células do sistema imunológico, macrófagos desempenham um papel fundamental. Apamina, constituinte do veneno de abelhas, é importante na defesa destas. Objetivou-se avaliar o metabolismo da linhagem de macrófagos J774 1.6 expostos à apamina isolada e purificada, avaliando-se citotoxicidade por redução de MTT e análise por citometria de fluxo (apoptose / necrose, produção de espécies reativas de oxigênio (EROs), lipoperoxidação membranosa (LPO), potencial elétrico da membrana mitocondrial (MMP) e fragmentação do DNA). Nenhuma concentração testada (10 a 100µg / mL) foi citotóxica. As taxas de apoptose diminuíram nas concentrações 2,5, 5,0 e 10,0µg / mL (P<0,05), enquanto as de necrose aumentaram (P<0,05). Entretanto, as taxas de células saudáveis na maior concentração testada (10µg / mL) não diferiram do controle (P>0,05). A apamina não alterou as ERO, a LPO nem a fragmentação do DNA. Portanto, todas as concentrações analisadas (1,25 a 10µg / mL) diminuíram a mMP. Tal diminuição na apoptose pode ser por uma supressão de mensageiros pró-apoptóticos mitocondriais, já que este peptídeo não causa estresse oxidativo, peroxidação lipídica nem dano ao DNA. Técnicas altamente sensíveis são importantes para adequada interpretação dos mecanismos de citotoxicidade.(AU)

Research progress on macrophage in radiation induced lung injury / 浙江大学学报·医学版

Radiation-induced lung injury (RILI), including acute radiation pneumonitis and chronic radiation-induced pulmonary fibrosis (RIPF), is a side effect of radiotherapy for lung cancer and esophageal cancer. Pulmonary macrophages, as a kind of natural immune cells maintaining lung homeostasis, play a key role in the whole pathological process of RILI. In the early stage of RILI, classically activated M1 macrophages secrete proinflammatory cytokines to induce inflammation and produce massive reactive oxygen species (ROS) through ROS-induced cascade to further impair lung tissue. In the later stage of RILI, alternatively activated M2 macrophages secrete profibrotic cytokines to promote the development of RIPF. The roles of macrophage in the pathogenesis of RILI and the related potential clinical applications are summarized in this review.

Relationship between PI3K/mTOR/RhoA pathway-regulated cytoskeletal rearrangements and phagocytic capacity of macrophages

The objective of this study was to investigate the relationship between PI3K/mTOR/RhoA signaling regulated cytoskeletal rearrangements and phagocytic capacity of macrophages. RAW264.7 macrophages were divided into four groups; blank control, negative control, PI3K-RNAi, and mTOR-RNAi. The cytoskeletal changes in the macrophages were observed. Furthermore, the phagocytic capacity of macrophages against Escherichia coli is reported as mean fluorescence intensity (MFI) and percent phagocytosis. Transfection yielded 82.1 and 81.5% gene-silencing efficiencies against PI3K and mTOR, respectively. The PI3K-RNAi group had lower mRNA and protein expression levels of PI3K, mTOR, and RhoA than the blank and negative control groups (Р<0.01). The mTOR-RNAi group had lower mRNA and protein levels of mTOR and RhoA than the blank and the negative control groups (Р<0.01). Macrophages in the PI3K-RNAi group exhibited stiff and inflexible morphology with short, disorganized filopodia and reduced number of stress fibers. Macrophages in the mTOR-RNAi group displayed pronounced cellular deformations with long, dense filopodia and an increased number of stress fibers. The PI3K-RNAi group exhibited lower MFI and percent phagocytosis than blank and negative control groups, whereas the mTOR-RNAi group displayed higher MFI and percent phagocytosis than the blank and negative controls (Р<0.01). Before and after transfection, the mRNA and protein levels of PI3K were both positively correlated with mTOR and RhoA (Р<0.05), but the mRNA and protein levels of mTOR were negatively correlated with those of RhoA (Р<0.05). Changes in the phagocytic capacity of macrophages were associated with cytoskeletal rearrangements and were regulated by the PI3K/mTOR/RhoA signaling pathway.

Perillyl alcohol has antibacterial effects and reduces ROS production in macrophages

Abstract Natural products have emerged as a rich source of bioactive compounds for adjunctive treatments of many infectious and inflammatory conditions, including periodontitis. Among the monoterpenes with significant biological properties, there is the perillyl alcohol (POH), which can be found in several essential oils and has shown immunomodulatory properties in recent studies, which may be interesting in the treatment of non-neoplastic inflammatory disorders. Objective To determine the antibacterial and immune modulatory activities of the POH. Methodology The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the POH for two significant Gram-negative periodontal pathogens were determined by macrodilution and subculture, respectively. Cell proliferation and cytotoxicity in RAW 264.7 macrophages were determined by Trypan Blue and mitochondrial enzymatic activity assay. The modulation of reactive oxygen species (ROS) was analyzed by flow cytometry and expression of TNF and arginase-1 by real-time PCR. Results The POH was effective against P. gingivalis (ATCC 33277) and F. nucleatum (ATCC 25586) with MIC= MBC=1600 μM. No cytotoxicity up to 100 µM was observed on macrophages. The cell proliferation was inhibited from 48 hours at 100 μM (p<0.05) and 250 μM (p<0.01). The POH increased ROS production at both 10 μM and 100 μM (p<0.05) in unstimulated cells. The PMA-induced ROS production was not affected by POH, whereas 100 μM significantly reduced lipopolysaccharide-induced (LPS-induced) ROS. The expression of TNF was not affected by POH in unstimulated cells or in cells polarized to M1 phenotype, whereas both concentrations of POH reduced (p<0.05) the expression of arginase-1 in M2-polarized macrophages. Conclusion The POH has antibacterial activity against periodontal pathogens and reduced proliferation of murine macrophages without significant cytotoxicity at concentrations up to 100 μM. In addition, the POH reduced the LPS-induced ROS and the expression of arginase-1 in M2-polarized macrophages.

Analysis of macrophage subtypes and annexin A1 expression in lesions of patients with cutaneous leishmaniasis

Abstract INTRODUCTION: Cutaneous leishmaniasis is caused by protozoa of the genus Leishmania and transmission occurs through the bite of sandflies. It is an infectious disease, which affects skin and mucosa. The aim was to quantify the macrophages M1 and M2 and the annexin A1 expression in the skin lesions of patients with cutaneous leishmaniasis. METHODS: Skin biopsies from patients (n = 50) were analyzed and classified according to the lesion type as: exudative cellular reaction, exudative granulomatous reaction, exudative necrotic reaction, exudative necrotic-granulomatous reaction. Using the immunofluorescence technique, macrophages were identified by CD163 marker, differentiated by anti-MHCII and anti-CD206 antibodies, and annexin A1 expression was determined by arbitrary unit (a.u.) densitometry. RESULTS: In M1 macrophages, a greater expression of this protein was observed in the exudative cellular reaction type lesions (136.3 ± 2.6 a.u., assuming mean and standard derivation) when compared to the expression in the lesions of exudative granulomatous reaction, exudative necrotic reaction and exudative necrotic-granulomatous reaction patients (108.0 ± 2.3, 121.6 ± 3.2 and 124.7 ± 2.4 a.u., respectively). Regarding M2 macrophages, it was observed that patients with exudative cellular reaction lesion also had a higher expression of this protein (128.8 ± 2.6 a.u.), when compared to the expression in the lesions of exudative granulomatous reaction, exudative necrotic reaction and exudative necrotic-granulomatous reaction patients (105.6 ± 2, 113.9 ± 2.8, 114.3 ± 2.1 a.u., respectively). CONCLUSIONS: These data suggest that annexin A1 is assisting macrophages in the phagocytosis process of patients with exudative cellular reaction lesion type.

A hiperglicemia altera o perfil inflamatório e imunometabólico de macrófagos derivados de medula óssea de camundongos / Hyperglycemia alters the inflammatory and immunometabolic profile of mouse bone marrow derived macrophages

O diabetes mellitus é um grupo heterogêneo de distúrbios metabólicos caracterizado pela hiperglicemia. Indivíduos diabéticos possuem maior susceptibilidade a infecções comparado a indivíduos sadios e a hiperglicemia é um dos principais fatores que contribuem para isso, em parte, por alterar a resposta imune. Sendo assim, os macrófagos, como células essenciais para a resposta inflamatória, podem apresentar importante papel na resposta imune alterada de indivíduos diabéticos. Neste estudo, investigamos como a hiperglicemia modula os macrófagos derivados da medula óssea (BMDMs) sob um estímulo inflamatório. Para realizar este estudo, os BMDMs de camundongos C57BL/6 machos não diabéticos e diabéticos (60 mg/kg de aloxana, iv) (CEUA / FCF / USP-488) foram cultivados sob condições normais de glicose (5,5 mM) e alta concentração de glicose (25 mM ou 40 mM) e estimuladas ou não com lipopolissacarídeo (LPS, 100 ng/mL). Em comparação com os BMDMs dos camundongos não diabéticos, os BMDMs dos camundongos diabéticos estimulados com LPS apresentaram menor expressão de CD38 no tempo basal e após 24 horas, além de menor expressão de receptor do tipo Toll (TLR)-4 na superfície celular, menor capacidade fagocítica e redução na secreção de óxido nítrico, lactato, fator de necrose tumoral- e interleucina (IL)-10, porém apresentaram maior expressão de CD80, CD86 e MHC-II, maior consumo de oxigênio e maior fosforilação em quinase ativada por estresse/quinase Jun-amino-terminal (SAPK/JNK) subunidade p46 e em quinase regulada por sinal extracelular (ERK) subunidade p42, proteína quinase B (AKT) e proteína quinase C (PKC)-δ assim como maior secreção de IL-6. Quando os BMDMs dos camundongos não diabéticos foram cultivados sob condições de alta concentração de glicose in vitro e estimulados com LPS, a expressão de TLR4 e os níveis de óxido nítrico e peróxido de hidrogênio foram reduzidos. Por outro lado, os BMDMs diabéticos que também foram cultivados em alta concentração de glicose in vitro apresentaram níveis aumentados de lactato e fosforilação reduzida em AKT e PKC-δ, porém apresentaram fosforilação aumentada em p46 SAPK/JNK. A alta concentração de glicose parece modificar o comportamento dos macrófagos, afetando diferentes aspectos dos BMDMs diabéticos e não diabéticos sob estímulo de LPS, assim a hiperglicemia deixa um legado de glicose, induzindo uma memória glicêmica, alterando o estado basal dos macrófagos, modificando a via de sinalização do TLR4 contribuindo para a susceptibilidade de indivíduos diabéticos a infecções

Diabetes mellitus is a heterogeneous group of metabolic disorders characterized by hyperglycemia. Diabetic individuals are more susceptible to infections compared to healthy subjects, and hyperglycemia is one of the major contributing factors, partly because they alter the immune response. Thus, macrophages, as essential cells for the inflammatory response, may play an important role in the altered immune response of diabetic individuals. In this study, we investigated how hyperglycemia modulates bone marrow derived macrophages (BMDMs) under an inflammatory stimulus. To perform this study, BMDMs from non-diabetic male and diabetic C57BL/6 mice (60 mg / kg aloxane, iv) (CEUA / FCF / USP-488) were cultured under normal glucose conditions (5.5 mM) and high glucose concentration (25 mM or 40 mM) and stimulated or not with lipopolysaccharide (LPS, 100 ng / ml). Compared to non-diabetic mice BMDMs, the BMDMs of LPS-stimulated diabetic mice showed lower expression of CD38 at baseline and after 24 hours, as well as lower Toll-like receptor (TLR)-4 on the cell surface, lower secretion of lactate, tumor necrosis factor-, and interleukin (IL)-10, but showed higher expression of CD80, CD86 and MHC-II, higher oxygen consumption and greater phosphorylation in stress-activated kinase/Jun-amino-terminal kinase (SAPK / JNK) p46 subunit and in extracellular signal regulated kinase (ERK) p42 subunit, protein kinase B (AKT) and protein kinase C (PKC)-δ as well as higher secretion of IL-6. When the BMDMs of nondiabetic mice were cultured under conditions of in vitro high glucose concentration and stimulated with LPS, the levels of TLR4 expression, nitric oxide and hydrogen peroxide were reduced. On the other hand, diabetic BMDMs that were also cultured in high glucose concentration of glucose in vitro showed increased levels of lactate and reduced phosphorylation in AKT and PKC-δ, but showed increased phosphorylation in p46 SAPK/JNK. A high glucose concentration seems to modify the behavior of macrophages, affecting different aspects of diabetic and non-diabetic BMDMs under the same LPS stimulus. Hyperglycemia leaves a glucose legacy, inducing a glycemic memory, altering the basal state of macrophages, modifying the TLR4 signaling pathway, and may play a key role in the high susceptibility of diabetic individuals to infections

Immunostimulatory activity in mammalian cells of 3, 5-dihydroxy-7-methoxyflavanone ((-)-alpinone) isolated from Heliotropium huascoense resinous exudate / Actividad inmunoestimulante en células de mamífero de 3, 5-dihidroxi-7-metoxiflavanona ((-)-alpinona) aislada del exudado resinoso de Heliotropium huascoense

The flavonoid 3,5-dihydroxy-7-methoxyflavanone ((-)-alpinone) isolated from sticky resinous exudate of Heliotropium huascoense was evaluated as immunostimulatory in mammalian cells . Preliminary observations had showed that (-)-alpinone had increased the expression levels of pro-inflammatory cytokine transcripts in salmonid. Due to high morbidity and mortality that infectious diseases cause in humans, we evaluate the effect of (-)-alpinone as an immunostimulant in mammalian cells. Reactive oxygen species (ROS) are produced by macrophages activators for the destruction of pathogens; we evaluated (-)-alpinone effect in ROS generation and the proliferation of macrophages. The results showed that proliferation in Raw 264.7 cells treated with 10 and 25 µg/mL of (-)-alpinone had a significant increase in macrophage proliferation. In relation to ROS formation, cells treated with 1 and 5 µg/mL of (-)-alpinone, induce ROS formation in macrophages.

El flavonoide 3,5-dihidroxi-7-metoxiflavanona ((-)-alpinona) aislado del exudado resinoso de Heliotropium huascoense se evaluó como inmunoestimulador en células de mamíferos. Resultados preliminares habían demostrado que (-)-alpinona aumentaba los niveles de expresión de transcritos de citoquinas proinflamatorias en salmónidos. Debido a la alta morbilidad y mortalidad que causan las enfermedades infecciosas en los humanos, evaluamos el efecto de (-)-alpinona como inmunoestimulante en células de mamíferos. Dado que las especies de oxígeno reactivo (ROS) son producidas por macrófagos activados para la destrucción de patógenos, se evaluó el efecto de (- )-alpinona en la generación de ROS y la proliferación de macrófagos. Los resultados mostraron que la proliferación en células Raw264.7 tratadas con 10 y 25 µg / mL del flavonoíde tuvo un aumento significativo en la proliferación de macrófagos. En relación con la formación de ROS, las células tratadas con 1 y 5 µg/mL de (-)-alpinona, inducen la formación de ROS en los macrófagos.

Pro-inflammatory Analysis of Macrophages in Contact with Titanium Particles and Porphyromonas gingivalis

Abstract During insertion of titanium dental implants, particles may shear from the implant to the periimplant region causing osteolysis, and their association with bacteria can exacerbate the inflammatory reaction. However, the association of a high invasive bacterium from the oral cavity, Porphyromonas gingivalis (Pg), and titanium particles remains unknown. This study evaluated pro-inflammatory reaction of human macrophages in contact with micro and nanoparticles of titanium associated with Porphyromonas gingivalis lipopolysaccharide (PgLPS). THP-1 cell were used and treated for 12, 24 and 48 h following 6 groups: Control(C), PgLPS (L); Microparticles (M); Nanoparticles (N); PgLPS and microparticles (LM); PgLPS and nanoparticles (LN). The following assays were carried out: i) cell viability using MTS, ii) cell morphology by SEM and iii) expression of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β) and interleukin-6 (IL-6) by qRT-PCR and ELISA. For statistics two-way ANOVA followed by Tukey's test was used (p<0.05). After treatment, cells presented similar viability and morphology demonstrating that the treatments were not able to induce cell death. Gene expression was significantly higher for TNF-α and IL1-β after 12 h, and for IL-6 after 24 h in the N and LN groups. Cytokine production over time was an ascending curve for TNF-α with the peak at 48 h and IL1-β and IL-6 had a straight line among the time points, although cells from N group presented a significant production of IL-6 at 48 h. In conclusion, these results suggest that titanium nanoparticles stimulate stronger pro-inflammatory response in macrophages, independent of their association with LPS from P.gingivalis.

Resumo Durante a inserção de implantes dentários partículas de titânio podem ser liberadas na região peri-implantar levando ao processo de osteólise e a associação com a bactéria pode exacerbar ainda mais a reação inflamatória. Entretanto, a associação de uma bactéria altamente invasiva da cavidade oral, Porphyromonas gingivalis (Pg) e partículas de titânio ainda não foi investigada. Este estudo avaliou a reação pró-inflamatória de macrófagos humanos em contato com micro e nanopartículas de titânio associada a lipopolissacarídeo P. gingivalis (PgLPS). As células THP-1 foram utilizadas e tratadas durante 12, 24 e 48 h nos 6 seguintes grupos: Controle (C), PgLPS (L); micropartículas (M); nanopartículas (N); PgLPS e micropartículas (LM); PgLPS e nanopartículas (LN). Em seguida foram realizados os seguintes ensaios: i) a viabilidade celular utilizando MTS, ii) a morfologia celular por MEV e iii) expressão do fator de necrose tumoral alfa (TNF-α), interleucina-1 beta (IL-1β) e interleucina 6 (IL-6) por qRT-PCR e ELISA. Como estatística foi realizado o teste ANOVA two-way seguido pelo teste de Tukey (p<0,05). Após o tratamento, as células apresentaram viabilidade e morfologia semelhantes, demonstrando que os tratamentos não foram capazes de induzir a morte celular. A expressão de genes foi significativamente mais elevada para o TNF-α e IL1-β após 12h, e para a IL-6 após 24 horas em N e grupos de LN. A produção de citocinas em relação ao tempo representou uma curva ascendente para o TNF-α com o pico em 48 h, enquanto que para IL1-β e IL-6 se apresentou como uma linha reta com relação ao tempo, exceto pelo grupo N que foi significativo para IL-6 em 48 h . Conclui-se, a partir destes resultados, que as nanopartículas de titânio produziram o maior estímulo na resposta pró-inflamatória nos macrófagos, independente da sua associação com LPS de P. gingivalis.

Inhibitory effects on the production of inflammatory mediators and reactive oxygen species by Mori folium in lipopolysaccharide-stimulated macrophages and zebrafish

ABSTRACT Mori folium, the leaf of Morus alba L. (Moraceae), has been traditionally used for various medicinal purposes from ancient times to the present. In this study, we examined the effects of water extract of Mori folium (WEMF) on the production of inflammatory mediators, such as nitric oxide (NO) and prostaglandin E2 (PGE2), and reactive oxygen species (ROS) in lipopolysaccharide (LPS)-stimulated murine RAW 264.7 macrophages. Our data indicated that WEMF significantly suppressed the secretion of NO and PGE2 in RAW 264.7 macrophages without any significant cytotoxicity. The protective effects were accompanied by a marked reduction in their regulatory gene expression at the transcription level. WEMF attenuated LPS-induced intracellular ROS production in RAW 264.7 macrophages. It inhibited the nuclear translocation of the nuclear factor-kappa B p65 subunit and the activation of mitogen-activated protein kinases in LPS-treated RAW 264.7 macrophages. Furthermore, WEMF reduced LPS-induced NO production and ROS accumulation in zebrafish. Although more efforts are needed to fully understand the critical role of WEMF in the inhibition of inflammation, the findings of the present study may provide insights into the approaches for Mori folium as a potential therapeutic agent for inflammatory and antioxidant disorders.

Oxidative stress in sepsis. Possible production of free radicals through an erythrocyte-mediated positive feedback mechanism

Abstract Background: Sepsis is an illness with a high morbidity for which no effective treatment exists. Its treatment has a high cost because it usually requires an intensive care unit and expensive antibiotics. The present study focus in the production of reactive oxygen species in the early stages of sepsis. This study aimed at investigating the production of reactive oxygen specie during the inflammatory response in patients with sepsis. Methods: Reactive oxygen specie production and insoluble myeloperoxidase obtained from fresh whole blood were measured by photon counting chemiluminescence in the blood of 18 septic patients and 12 healthy individuals. Modified red blood cells were evaluated by staining of blood smears. The production of reactive oxygen species by macrophages and polymorphonuclear leukocytes put into contact with modified red blood cells were also assessed by photon counting chemiluminescence. Results: The appearance of oxidatively modified erythrocytes, which is an evidence of oxidative stress, was supported by the detection of reactive oxygen species and insoluble myeloperoxidase in the whole blood of all septic patients. Peroxynitrite was the main reactive oxygen species found in the whole blood. Oxidatively modified erythrocytes activated phagocytic cells in vitro, leading to the considerable production of free radicals. Conclusion: It was found that sepsis led to a high oxidative stress and to extensive modification of erythrocytes. It is proposed that a positive feedback mechanism, involving the activation of circulating leukocytes by these modified erythrocytes would maintain the pro-oxidative state even after the disappearance of bacteria.

Effects of chitin and sepia ink hybrid sponge on the healing of burning wound rats and its impact on macrophages in vitro

PURPOSE: To detect whether chitin and sepia ink sponge (CS) can promote wound healing and elevate impact of CS on phagocytosis ability of macrophages. METHODS: Forty-eight rats were assigned to four groups: Normal group (Normal), negative control group (Con), chitin and sepia ink sponge group (CS) and positive control Surgicel Gauze(r) group (SG). Deep second-degree burn model was created in rats. Wound area was recorded by digital imaging and determined using Image J software. Samples were collected and kept at -80oC on 3d, 7d, 14d and 21d for cytokines detecting. Transforming growth factor (TGF)-β1, interleukin (IL)-6, matrix metalloproteinase (MMP)-1, hydroxyproline (Hyp) and macrophage activity reflected by tumor necrosis factor (TNF)-α were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Comparing to Con and SG, scabs in CS group fell off and basically healed on 21 day. TGF-β1, IL-6, MMP-1 and Hyp were significantly increased by CS and SG comparing to Con (p < 0.05), CS had more apparently adjustment on TGF-β1 and MMP-1 compared to SG; results in vitro indicated CS significantly promoted phagocytosis ability of macrophages reflected in TNF-α (p < 0.05). CONCLUSION: CS improved wound healing through exerting significant influences on secretion of kinds of cytokines and activating macrophages.

Glucagon-like peptide 1 improves insulin resistance in vitro through anti-inflammation of macrophages

Glucagon-like peptide 1 (GLP-1), a kind of gut hormone, is used in the treatment of type 2 diabetes (T2D). Emerging evidence indicates that GLP-1 has anti-inflammatory activity. Chronic inflammation in the adipose tissue of obese individuals is a cause of insulin resistance and T2D. We hypothesized that GLP-1 analogue therapy in patients with T2D could suppress the inflammatory response of macrophages, and therefore inhibit insulin resistance. Our results showed that GLP-1 agonist (exendin-4) not only attenuated macrophage infiltration, but also inhibited the macrophage secretion of inflammatory cytokines including TNF-β, IL-6, and IL-1β. Furthermore, we observed that lipopolysaccharide (LPS)-induced macrophage conditioned media could impair insulin-stimulated glucose uptake. This effect was compensated by treatment with the conditioned media from macrophages treated with the combination of LPS and exendin-4. It was also observed that exendin-4 directly inhibited the activation of NF-κB in macrophages. In conclusion, our results indicated that GLP-1 improved inflammatory macrophage-derived insulin resistance by inhibiting NF-κB pathway and secretion of inflammatory cytokines in macrophages. Furthermore, our observations suggested that the anti-inflammatory effect of GLP-1 on macrophages can contribute to GLP-1 analogue therapy of T2D.

Expression of annexin A1 in Leishmania-infected skin and its correlation with histopathological features

ABSTRACTINTRODUCTION:The aim of this study was quantify annexin A1 expression in macrophages and cluster of differentiation 4 (CD4) + and cluster of differentiation 8 (CD8)+ T cells from the skin of patients with cutaneous leishmaniasis (n=55) and correlate with histopathological aspects.METHODS:Infecting species were identified by polymerase chain reaction-restriction fragment length polymorphism, and expression of annexin A1 was analyzed by immunofluorescence.RESULTS:All patients (n = 55) were infected with Leishmania braziliensis . Annexin A1 was expressed more abundantly in CD163 + macrophages in infected skin (p < 0.0001) than in uninfected skin. In addition, macrophages in necrotic exudative reaction lesions expressed annexin A1 at higher levels than those observed in granulomatous (p < 0.01) and cellular lesions p < 0.05). This difference might be due to the need to clear both parasites and necrotic tissue from necrotic lesions. CD4 + cells in cellular lesions expressed annexin A1 more abundantly than did those in necrotic (p < 0.05) and granulomatous lesions (p < 0.01). Expression in CD8 + T cells followed the same trend. These differences might be due to the pervasiveness of lymphohistiocytic and plasmacytic infiltrate in cellular lesions.CONCLUSIONS:Annexin A1 is differentially expressed in CD163 + macrophages and T cells depending on the histopathological features of Leishmania -infected skin, which might affect cell activation.