STAT3 In Macrophages Research Articles

MiR-144/451 attenuates lipopolysaccharide-induced lung inflammation by downregulating Rac1 and STAT-3 in macrophages.

MicroRNAs have been shown to play a critical role in lung inflammatory diseases. Here, we report that knocking out miR-144/451 in mice exacerbates lipopolysaccharide (LPS)-induced lung inflammation. The lung inflammation in mice was induced by intratracheal instillation of LPS. Loss-of-function experiments demonstrated that miR-144/451 gene knockout (KO) increased LPS-induced lung inflammation and oxidant stress compared with wild-type (WT) mice, as manifested by increased total bronchoalveolar lavage fluid cells and neutrophil counts, elevated TNF-α and IL-6 levels in bronchoalveolar lavage fluid, enhanced myeloperoxidase activity, and reduced catalase and glutathione peroxidase activity in lung tissues. We also found that LPS significantly decreased miR-451 expression in lung tissues and macrophages; while miR-451 overexpression in LPS-induced RAW264.7 cells remarkably reduced TNF-α and IL-6 levels as well as reactive oxygen species (ROS) production, suggesting a feedback loop might exist in inflammatory cells. Rac1 mRNA and protein levels were downregulated in miR-451-overexpressed RAW264.7 cells. Ex vivo stimulation experiments, performed using alveolar macrophages isolated from miR-144/451 KO mice, confirmed that Rac1 inhibitor alleviated levels of TNF-α and ROS in response to LPS stimulation compared with WT controls. Luciferase reporter assay demonstrated that STAT-3 is a direct target of miR-451. STAT-3 protein levels were elevated in miR-144/451 KO macrophages. LPS treatment also resulted in higher phosphorylation levels of STAT-3 in macrophages from KO mice than in WT cells. Our study identified miR-144/451 as an anti-inflammatory factor in LPS-induced lung inflammation that acts by downregulating Rac1 and STAT-3.

Negative Regulation of RIG-I by Tim-3 Promotes H1N1 Infection

ABSTRACT The mechanisms by which retinoic acid-inducible gene I (RIG-I), a critical RNA virus sensor, is regulated in many biological and pathological processes remain to be determined. Here, we demonstrate that T cell immunoglobulin and mucin protein-3 (Tim-3), an immune checkpoint inhibitor, mediates infection tolerance by suppressing RIG-I-type I interferon pathway. Overexpression or blockade of Tim-3 affects type I interferon expression, virus replication, and tissue damage in mice following H1N1 infection. Tim-3 signaling decreases RIG-I transcription via STAT1 in macrophages and promotes the proteasomal dependent degradation of RIG-I by enhancing K-48-linked ubiquitination via the E3 ligase RNF-122. Silencing RIG-I reversed Tim-3 blockage-mediated upregulation of type I interferon in macrophages. We thus identified a new mechanism through which Tim-3 mediates the immune evasion of H1N1, which may have clinical implications for the treatment of viral diseases.

Glucosamine regulates macrophage function in heart failure.

Glucosamine regulates macrophage function in heart failure.

MiR-29b regulates Th2 cell differentiation in asthma by targeting inducible B7-H3 and STAT3.

BackgroundMicroRNAs play an important role in T cell responses. However, how microRNAs regulate Th cells in asthma remains poorly defined.ObjectiveIn this study, we investigated the mechanism and pathways of miR‐29b regulating Th cells in asthma, in order to find new targets for asthma.MethodsWe detected miR‐29b, B7‐H3 and STAT3 in the peripheral blood of children with asthma, explored the relationship between these molecules and their effects on T cells through in vitro cell culture, and verified it by animal model.ResultsMiR‐29b levels were decreased in the peripheral blood mononuclear cells from children with asthma. Vitro studies found that the expression of miR‐29b in macrophages was decreased and the expression of B7‐H3 and STAT3 was increased after house dust mite (HDM) stimulation. After down‐regulation of miR‐29b in macrophages, the expressions of B7‐H3 and STAT3 in macrophages were increased and T cells differentiate into Th2 cells. After the addition of B7‐H3 or STAT3 antibodies, the differentiation of naive T cells into Th2 cells was reduced. In OVA induced mice asthmatic model, after the up‐regulation of miR‐29b in lung, the expression of B7‐H3 and STAT3 decreased in the lung tissues of mice, and the expression of Th2 cells and type II cytokine decreased simultaneously. The pathological changes of lung tissues were also alleviated.ConclusionThe expression of miR‐29b is decreased in asthmatic children. MiR‐29b can inhibit Th2 cell differentiation by inhibiting B7‐H3 and STAT3 pathways at the same time, and reduce asthmatic immune inflammation.

Cardiotrophin Like Cytokine Factor 1 (CLCF1) alleviates bone loss in osteoporosis mouse models by suppressing osteoclast differentiation through activating interferon signaling and repressing the nuclear factor-κB signaling pathway.

A growing body of evidence suggests that immune factors that regulate osteoclast differentiation and bone resorption might be promising therapeutic agents for the treatment of osteoporosis. The expression of CLCF1, an immune cell-derived molecule, has been reported to be reduced in patients with postmenopausal osteoporosis. This suggests that it may be involved in bone remodeling. Thus, we explored the functional role of CLCF1 in osteoclastogenesis and bone loss associated with osteoporosis. Surprisingly, the administration of recombinant CLCF1 repressed excessive bone loss in ovariectomized mice and prevented RANKL-induced bone loss in calvarial mouse model. Likewise, the addition of recombinant CLCF1 to RANKL-stimulated monocytes resulted in a significant suppression in the number of differentiated osteoclasts with small resorption areas being observed on dentine slices in vitro. At the same dosage, CLCF1 did not exhibit any detectable negative effects on the differentiation of osteoblasts. Mechanistically, the inhibition of osteoclast differentiation by the CLCF1 treatment appears to be related to the activation of interferon signaling (IFN) and the suppression of the NF-κB signaling pathway. Interestingly, the expression of the main components of IFN-signaling namely, STAT1 and IRF1, was detected in macrophages as early as 1h after stimulation with CLCF1. Consistent with these results, the blockade of STAT1 in macrophages abolished the inhibitory effect of CLCF1 on osteoclast differentiation in vitro. These collective findings point to a novel immunoregulatory function of CLCF1 in bone remodeling and highlight it as a potentially useful therapeutic agent for the treatment of osteoporosis.

Anti-inflammatory effects of miR-150 are associated with the downregulation of STAT1 in macrophages following lipopolysaccharide treatment.

Sepsis is a condition that is associated with high rates of mortality. It is characterized by serious systemic inflammatory responses induced by pathogenic invasion. Although microRNA-150 (miR-150) has been previously reported to be involved in the modulation of sepsis, the underlying molecular mechanism in sepsis remains poorly understood. In the present study, the human monocytic cell line THP-1 was treated with LPS to mimic sepsis in vitro, following which miR-150 and STAT1 expression were measured using reverse transcription-quantitative PCR or western blotting. Secretion of inflammatory cytokines interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α) into the medium were measured by ELISA. The potential relationship between STAT1 and miR-150 was determined using dual-luciferase reporter and RNA immunoprecipitation assays. miR-150 expression was found to be was downregulated by LPS treatment in THP-1 cells in both dose- and time-dependent manners. LPS treatment also induced IL-1β, IL-6 and TNF-α secretion in a manner that could be inhibited by miR-150 overexpression and enhanced by transfection with the miR-150 inhibitor. miR-150 was revealed to directly target STAT1 by negatively regulating its expression. In addition, STAT1 expression was demonstrated to be upregulated by LPS treatment. STAT1 overexpression reversed the inhibitory effects of miR-150 overexpression on IL-1β, IL-6 and TNF-α secretion whilst STAT1 knockdown attenuated IL-1β, IL-6 and TNF-α secretion induced by miR-150 inhibitor transfection. In conclusion, the present study suggested that miR-150 regulates the inflammatory response in macrophages following LPS challenge by regulating the expression of STAT1.

TRIM59 suppresses NO production by promoting the binding of PIAS1 and STAT1 in macrophages

Macrophages, which can secret various inflammation mediators, have an essential role in tumor growth and metastasis. However, the mechanism(s) to regulate the production of inflammation mediator is not completely clear. Here we found that TRIM 59 could inhibit the production of NO and the expression of inducible nitric oxide synthase (iNOS), cytochrome c oxidase subunit2 (COX2) and TNFα. TRIM59 mediated suppression on nitric oxide (NO) production is through inhibiting the activation of JAK2-STAT1 signal pathway. In response to LPS, TRIM59 in macrophages was translocated from cytoplasm to nucleus and directly bound with STAT1. During this process, TRIM59 could recruit much more PIAS1 to bind with STAT1 to suppress the activation of STAT1. Finally, TRIM59 modified macrophages could promote tumor growth. Thus, TRIM59 mediated suppression on NO production by promoting the binding of PIAS1 and STAT1 in macrophages may regulate tumor growth.

Upregulation of miR-196b-5p attenuates BCG uptake via targeting SOCS3 and activating STAT3 in macrophages from patients with long-term cigarette smoking-related active pulmonary tuberculosis

BackgroundCigarette smoking (CS) triggers an intense and harmful inflammatory response in lungs mediated by alveolar and blood macrophages, monocytes, and neutrophils and is closely associated with prevalence of tuberculosis (TB). The risk of death in patients with long-term cigarette smoking-related pulmonary tuberculosis (LCS-PTB) is approximately 4.5 times higher than those with nonsmoking pulmonary tuberculosis (N-PTB). However, the mechanisms underlying the harmful inflammatory responses in the setting of LCS-PTB have not been well documented.Methods28 cases LCS-PTB patients, 22 cases N-PTB patients and 20 cases healthy volunteers were enrolled in this study. Monocytes were isolated from peripheral blood mononuclear cells. Differentiated human MDM and U937 cell were prepared with M-CSF and PMA stimulation, respectively. The miR-196b-5p, STAT1, STAT3, STAT4, STAT5A, STAT5B, STAT6, SOCS1 and SOCS3 mRNA expression were detected by qRT-PCR. Western blot was performed according to SOCS1, SOCS3, and pSTAT3 expression. The mycobacterial uptake by MDMs from different groups of patients after Bacillus Calmette–Guérin (BCG) infection and agomir-196b-5p or antagomir-196b-5p transfection were used by flow cytometry analysis. Human IL-6, IL-10 and TNF-α levels on the plasma and cell culture supernatant samples were measured using ELISA. For dual-luciferase reporter assay, the SOCS3 3′-UTR segments, containing the binding elements of miR-196b-5p or its mutant versions were synthesized as sense and antisense linkers.ResultsIn this study, we found that IL-6, TNF-α production, SOCS3 mRNA expression were downregulated, while miR-196b-5p and STAT3 mRNA expression were upregulated in monocytes from LCS-PTB patients as compared to N-PTB patients. Meanwhile, we demonstrated that miR-196b-5p could target SOCS3 and activate STAT3 signaling pathway, which may possibly contribute to attenuation of BCG uptake and decrease in IL-6 and TNF-α production in macrophages.ConclusionsOur findings revealed that CS exposure regulates inflammatory responses in monocyte/macrophages from LCS-PTB patients via upregulating miR-196b-5p, and further understanding of the specific role of miR-196b-5p in inflammatory responses mightfacilitate elucidating the pathogenesis of LCS-PTB, thus leading to the development of new therapeutic strategies for PTB patients with long-term cigarette smoking.

Ox-LDL increases microRNA-29a transcription through upregulating YY1 and STAT1 in macrophages.

Macrophages and oxidized low-density lipoprotein (ox-LDL) have been verified playing vital roles in the pathogenesis of atherosclerosis (AS). Previous studies demonstrated that microRNA-29a (miR-29a) was upregulated in many atherogenic process and cells, thus acting as an important participant in AS. But the detailed regulation mechanism of miR-29a in AS has not been fully understood. In our study, we demonstrated a positive feedback loop of ox-LDL-SRA-miR-29a. Furthermore, we found that YY1 and STAT1 were upregulated in ox-LDL-stimulating macrophages followed by translocation in the nucleus and binding to the transcriptional promoter region of miR-29a, thus leading to the increase of miR-29a expression. In addition, we demonstrated that JAK1/2 signaling was involved in miR-29a upregulation. Finally, we found that miR-29a played important roles in the secretion of proinflammation factors and lipid uptake in macrophages. We uncovered the molecular mechanism and provide novel insights into the function and regulatory network of miR-29a expression regulated by ox-LDL in macrophages.

Early Secreted Antigenic Target of 6-kDa of Mycobacterium tuberculosis Stimulates IL-6 Production by Macrophages through Activation of STAT3

As early secreted antigenic target of 6 kDa (ESAT-6) of Mycobacterium tuberculosis (Mtb) is an essential virulence factor and macrophages are critical for tuberculosis infection and immunity, we studied ESAT-6 stimulated IL-6 production by macrophages. ESAT-6 stimulated significantly higher IL-6 secretion by murine bone marrow derived macrophages (BMDM) compared to culture filtrate protein 10 kDa (CFP10) and antigen 85A. Polymyxin B, an LPS blocker, did not affect ESAT-6 stimulated macrophage IL-6 production. ESAT-6 but not Pam3CSK4 induced IL-6 by TLR2 knockout BMDM. ESAT-6 induced phosphorylation and DNA binding of STAT3 and this was blocked by STAT3 inhibitors but not by rapamycin. STAT3 inhibitors suppressed ESAT-6-induced IL-6 transcription and secretion without affecting cell viability. This was confirmed by silencing STAT3 in macrophages. Blocking neither IL-6Rα/IL-6 nor IL-10 affected ESAT-6-induced STAT3 activation and IL-6 production. Infection of BMDM and human macrophages with Mtb with esat-6 deletion induced diminished STAT3 activation and reduced IL-6 production compared to wild type and esat-6 complemented Mtb strains. Administration of ESAT-6 but not CFP10 induced STAT3 phosphorylation and IL-6 expression in the mouse lungs, consistent with expression of ESAT-6, IL-6 and phosphorylated-STAT3 in Mtb-infected mouse lungs. We conclude that ESAT-6 stimulates macrophage IL-6 production through STAT3 activation.

Novel Resolvin D2 Receptor Axis in Infectious Inflammation.

Resolution of acute inflammation is an active process governed by specialized proresolving mediators, including resolvin (Rv)D2, that activates a cell surface G protein-coupled receptor, GPR18/DRV2. In this study, we investigated RvD2-DRV2-dependent resolution mechanisms using DRV2-deficient mice (DRV2-knockout [KO]). In polymicrobial sepsis initiated by cecal ligation and puncture, RvD2 (∼2.7 nmol/mouse) significantly increased survival (>50%) of wild-type mice and reduced hypothermia and bacterial titers compared with vehicle-treated cecal ligation and puncture mice that succumbed at 48 h. Protection by RvD2 was abolished in DRV2-KO mice. Mass spectrometry-based lipid mediator metabololipidomics demonstrated that DRV2-KO infectious exudates gave higher proinflammatory leukotriene B4 and procoagulating thromboxane B2, as well as lower specialized proresolving mediators, including RvD1 and RvD3, compared with wild-type. RvD2-DRV2-initiated intracellular signals were investigated using mass cytometry (cytometry by time-of-flight), which demonstrated that RvD2 enhanced phosphorylation of CREB, ERK1/2, and STAT3 in WT but not DRV2-KO macrophages. Monitored by real-time imaging, RvD2-DRV2 interaction significantly enhanced phagocytosis of live Escherichia coli, an action dependent on protein kinase A and STAT3 in macrophages. Taken together, we identified an RvD2/DRV2 axis that activates intracellular signaling pathways that increase phagocytosis-mediated bacterial clearance, survival, and organ protection. Moreover, these results provide evidence for RvD2-DRV2 and their downstream pathways in pathophysiology of infectious inflammation.

Abstract 13212: The Role of Macrophage STAT3 Signaling in Pathogenesis of Aortic Dissection

Aortic dissection (AD) is a fatal disease caused by the disruption of the intimomedial layer. Recent studies showed that IL-6, a STAT3-activating cytokine, and macrophages play important roles in AD. However, it is unknown whether or how activation of macrophage STAT3 is involved in AD pathogenesis. To answer this question, we examined both mouse AD model and human AD tissue. We used macrophage-specific knockout of SOCS3 (mSOCS3-KO), a negative regulator of STAT3, to specifically sensitize macrophages to STAT3 activation, and compared them with wild type (WT) mice. We created a mouse model of aortic stiffening by periaortic CaCl2 treatment of abdominal aorta, and infused them with angiotensin II (Ca+AngII), which augmented hemodynamic stress to aortic walls. Both WT and mSOCS3-KO showed microscopic injuries in aorta with equal frequencies (40%) 1 week after Ca+AngII. Whereas WT showed fibrotic repair of the injury, mSOCS3-KO failed to show such reparative response and developed large AD in 6 weeks. We analyzed the aorta 1 week after the Ca+AngII stimulation, focusing on the changes in aorta preceding AD development. Transcriptome analysis showed higher activation of cell cycle followed by higher proinflammatory response in mSOCS3-KO than in WT aorta before AD development. Ki67 staining and BrdU uptake study confirmed the proliferative response of inflammatory cells in aorta. Flowcytometric analysis showed preferential differentiation of mSOCS3-KO macrophages to proinflammatory M1 with reduction in reparative M2 population (M1:M2 = 2:1), while WT macrophages differentiated equally to M1 and M2. Immunofluorescence study of human AD tissue showed the infiltration of CD68-positive macrophages in adventitia and outer media at the site of dissection and surrounding intact aortic walls. The infiltrating cells showed high phospho- (active) STAT3 and Ki67. Interestingly, STAT3 was less active in tissue with fibrotic changes, suggesting the negative association of STAT3 activation and tissue repair. Therefore, macrophage STAT3 activation resulted in higher proliferation and M1 differentiation, which is likely to promote AD development. Over activation of STAT3 in macrophages may be a therapeutic target to mitigate the destructive process in AD.

STAT1 signaling is essential for protection against Cryptococcus neoformans infection in mice.

Nonprotective immune responses to highly virulent Cryptococcus neoformans strains, such as H99, are associated with Th2-type cytokine production, alternatively activated macrophages, and inability of the host to clear the fungus. In contrast, experimental studies show that protective immune responses against cryptococcosis are associated with Th1-type cytokine production and classical macrophage activation. The protective response induced during C. neoformans strain H99γ (C. neoformans strain H99 engineered to produce murine IFN-γ) infection correlates with enhanced phosphorylation of the transcription factor STAT1 in macrophages; however, the role of STAT1 in protective immunity to C. neoformans is unknown. The current studies examined the effect of STAT1 deletion in murine models of protective immunity to C. neoformans. Survival and fungal burden were evaluated in wild-type and STAT1 knockout (KO) mice infected with either strain H99γ or C. neoformans strain 52D (unmodified clinical isolate). Both strains H99γ and 52D were rapidly cleared from the lungs, did not disseminate to the CNS, or cause mortality in the wild-type mice. Conversely, STAT1 KO mice infected with H99γ or 52D had significantly increased pulmonary fungal burden, CNS dissemination, and 90-100% mortality. STAT1 deletion resulted in a shift from Th1 to Th2 cytokine bias, pronounced lung inflammation, and defective classical macrophage activation. Pulmonary macrophages from STAT1 KO mice exhibited defects in NO production correlating with inefficient inhibition of fungal proliferation. These studies demonstrate that STAT1 signaling is essential not only for regulation of immune polarization but also for the classical activation of macrophages that occurs during protective anticryptococcal immune responses.

Disruption of Mammalian Target of Rapamycin Complex 1 in Macrophages Decreases Chemokine Gene Expression and Atherosclerosis

The mammalian target of rapamycin complex 1 inhibitor, rapamycin, has been shown to decrease atherosclerosis, even while increasing plasma low-density lipoprotein levels. This suggests an antiatherogenic effect possibly mediated by the modulation of inflammatory responses in atherosclerotic plaques. Our aim was to assess the role of macrophage mammalian target of rapamycin complex 1 in atherogenesis. We transplanted bone marrow from mice in which a key mammalian target of rapamycin complex 1 adaptor, regulatory-associated protein of mTOR, was deleted in macrophages by Cre/loxP recombination (Mac-Rap(KO) mice) into Ldlr(-/-) mice and then fed them the Western-type diet. Atherosclerotic lesions from Mac-Rap(KO) mice showed decreased infiltration of macrophages, lesion size, and chemokine gene expression compared with control mice. Treatment of macrophages with minimally modified low-density lipoprotein resulted in increased levels of chemokine mRNAs and signal transducer and activator of transcription (STAT) 3 phosphorylation; these effects were reduced in Mac-Rap(KO) macrophages. Although wild-type and Mac-Rap(KO) macrophages showed similar STAT3 phosphorylation on Tyr705, Mac-Rap(KO) macrophages showed decreased STAT3Ser727 phosphorylation in response to minimally modified low-density lipoprotein treatment and decreased Ccl2 promoter binding of STAT3. The results demonstrate cross-talk between nutritionally induced mammalian target of rapamycin complex 1 signaling and minimally modified low-density lipoprotein-mediated inflammatory signaling via combinatorial phosphorylation of STAT3 in macrophages, leading to increased STAT3 activity on the chemokine (C-C motif) ligand 2 (monocyte chemoattractant protein 1) promoter with proatherogenic consequences.

Myeloid Clusters Are Associated with a Pro-Metastatic Environment and Poor Prognosis in Smoking-Related Early Stage Non-Small Cell Lung Cancer

BackgroundThis study aimed to understand the role of myeloid cell clusters in uninvolved regional lymph nodes from early stage non-small cell lung cancer patients.MethodsUninvolved regional lymph node sections from 67 patients with stage I–III resected non-small cell lung cancer were immunostained to detect myeloid clusters, STAT3 activity and occult metastasis. Anthracosis intensity, myeloid cluster infiltration associated with anthracosis and pSTAT3 level were scored and correlated with patient survival. Multivariate Cox regression analysis was performed with prognostic variables. Human macrophages were used for in vitro nicotine treatment.ResultsCD68+ myeloid clusters associated with anthracosis and with an immunosuppressive and metastasis-promoting phenotype and elevated overall STAT3 activity were observed in uninvolved lymph nodes. In patients with a smoking history, myeloid cluster score significantly correlated with anthracosis intensity and pSTAT3 level (P<0.01). Nicotine activated STAT3 in macrophages in long-term culture. CD68+ myeloid clusters correlated and colocalized with occult metastasis. Myeloid cluster score was an independent prognostic factor (P = 0.049) and was associated with survival by Kaplan-Maier estimate in patients with a history of smoking (P = 0.055). The combination of myeloid cluster score with either lymph node stage or pSTAT3 level defined two populations with a significant difference in survival (P = 0.024 and P = 0.004, respectively).ConclusionsMyeloid clusters facilitate a pro-metastatic microenvironment in uninvolved regional lymph nodes and associate with occult metastasis in early stage non-small cell lung cancer. Myeloid cluster score is an independent prognostic factor for survival in patients with a history of smoking, and may present a novel method to inform therapy choices in the adjuvant setting. Further validation studies are warranted.

Activated Adventitial Pulmonary Artery Fibroblasts Drive Macrophages Toward a Distinct M2-like Phenotype through STAT3 Signaling in Pulmonary Hypertension (120.36)

Abstract We have previously documented that in the adventitia of distal pulmonary arteries (dPA) in calves with pulmonary hypertension (PH) macrophages that express CD163 (marker of alternative activation, AAM) accumulate in proximity to adventitial fibroblasts (adFibs) in areas of remodeling. AAMs have been implicated in promoting remodeling in PH in response to STAT6 signaling. Our recently published data suggest that in the dPA, adFibs express an epigenetically stable pro-inflammatory phenotype capable of activating macrophages. We hypothesized that adFibs activate AAMs through STAT3 but not STAT6. adFibs were isolated and cultured from the dPA of hypertensive (PH-fibs) and control (CO-fibs) calves and used to generate conditioned media (CM). Bovine, murine and rat bone marrow derived macrophages (BMDMs) were incubated in CM for 16hrs followed by qRT-PCR analysis. In response to PH-fibs CM, bovine BMDMs expressed Cd163, Cd206, and IL4ra consistent with AAMs. Furthermore, in response to PH-Fib but not CO-Fib CM both murine and rat BMDMs expressed Cd206, Cd204, Irf4, Klf, jmjd3; STAT3-dependent genes Socs3, Cebp/b, Pim1, Nfatc2, Il4ra, Arg1, but not STAT6-dependent genes Chi3I3 and Retnla1; phospho-STAT3 but not phospho-STAT6; and mRNA for Arg1 (previously thought to be STAT6 dependent) in murine Stat6-/- BMDMs. These results define the activated dPA adFibs as a key locus of paracrine signaling that triggers STAT3 in macrophages, which then express a distinct AAM phenotype.

Suppression of SOCS3 in macrophages prevents cancer metastasis by modifying macrophage phase and MCP2/CCL8 induction

Inflammation has been demonstrated to play important roles in tumorigenesis, tumor progression, and metastasis. STAT3 has been shown to be frequently activated in a variety of human cancer cells and STAT3 signaling promotes the growth and survival of tumor cells. However, the role of STAT3 of myeloid cells associated with tumors is currently unknown. Suppressor of cytokine signaling-3 (SOCS3) has been shown to be a negative regulator of STAT3. In this study, we used macrophage specific SOCS3 conditional knockout (cKO) mice to investigate the effect of the hyperactivation of STAT3 in macrophages on tumor development and metastasis. In a subcutaneous transplantation model of B16F10 melanoma cells, although tumor sizes were not significantly different, SOCS3-cKO mice survived longer than wild-type (WT) mice did. SOCS3-cKO mice exhibited fewer lung and liver metastatic tumor nodules than WT mice when B16F10 was challenged intravenously. SOCS3 −/− macrophages stimulated with tumor lysates in vitro exhibited prolonged STAT3 phosphorylation and produced less amount of TNFα and IL-6, and higher amount of MCP2/CCL8 than WT macrophages. MCP/CCL8 was induced via STAT3 and exhibited anti-tumor metastatic effect in WT mice. These data suggest that hyperactivation of STAT3 in myeloid cells simultaneously exerted an anti-inflammatory as well as anti-tumor effects. Thus, the targeted inhibition of SOCS3 activity in macrophages may be therapeutic for the suppression of tumor metastasis.

Inhibitory effect of Physalis alkekengi L. var. franchetii extract and its chloroform fraction on LPS or LPS/IFN-γ-stimulated inflammatory response in peritoneal macrophages

The aerial parts of the plant Physalis alkekengi L. var. franchetii (PA) are traditionally used in folk medicine to treat cough, middle ear infection, sore throat, abscesses, and urinary problems. However, the cellular mechanisms underlying PA's possible anti-inflammatory effects are unknown. We examined the effect of a methanol extract of PA on the production of nitric oxide (NO), inducible NO synthase (iNOS), cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-α, and interleukin (IL)-6. We also examined the extract's effect on the activity of IκBα, mitogen-activated protein kinases (MAPKs) and STAT1 in macrophages stimulated by lipopolysaccharide (LPS) or LPS/interferon-γ (IFN-γ). We further fractioned the extract into chloroform and water to investigate which fraction possessed anti-inflammatory activity. We found that the PA methanol extract significantly reduced the production of NO, iNOS, COX-2, TNF-α, and IL-6. The extract also inhibited LPS-induced IκBα degradation and MAPK activation as well as LPS/IFN-γ-induced STAT1 activation, effects observed at a higher concentration than that required to suppress iNOS. The chloroform fraction possessed the anti-inflammatory activity of PA by inhibiting the expression of iNOS, TNF-α and IL-6 through downregulation of IκBα degradation and MAPK activation. Our findings indicate that the PA methanol extract contains different anti-inflammatory compounds, some of which suppressed iNOS expression and some of which inhibited IκBα degradation and MAPK activity. Further research is warranted to identify these anti-inflammatory components of PA and validate its use in animal studies.

Involvement of M2‐polarized macrophages in the ascites from advanced epithelial ovarian carcinoma in tumor progression via Stat3 activation

Ascites macrophages in advanced epithelial ovarian cancer (AdEOC) are involved in cancer metastasis and progression by modifying the tumor microenvironment. However, the precise mechanisms of cell-to-cell interaction between macrophages and tumor cells are still unclear. This study focused on the activation of signal transducer and activator of transcription 3 (Stat3) which is a critical signal transduction molecule at a point of convergence for numerous oncogenic signaling pathways as well as controlling the M2-poralization of macrophages. AdEOC ascites, in which high concentration of interleukin (IL)-6, IL-10, growth-related oncogene-alpha and vascular endothelial growth factor were detected, stimulated the proliferation of SKOV3 cells, a human ovarian cancer cell line. The simultaneous blocking of IL-6 and IL-10 by neutralizing antibodies suppressed ascites-induced tumor cell proliferation. Stat3 activation in SKOV3 cells was induced by co-culture with macrophages especially with macrophage colony stimulating factor-primed M2 macrophages but lesser extent with granulocyte-macrophage colony stimulating factor-primed immature macrophages. Cyclin-D1 expression in SKOV3 cells was also significantly induced by co-culture with macrophages. Blocking of Stat3 in macrophages by small interfering RNA inhibited the production of IL-6 and IL-10 by macrophages, and suppressed Stat3 activation and cyclin-D1 induction in co-cultured SKOV3 cells. Stat3 activation in SKOV3 cells was abrogated by simultaneous neutralization of IL-6 and IL-10. These results indicate that Stat3 activation by IL-6 and IL-10 plays an important role in cell-to-cell interaction between tumor cells and macrophages in the ascites of AdEOC.

The benzoxathiolone LYR‐71 down‐regulates interferon‐γ‐inducible pro‐inflammatory genes by uncoupling tyrosine phosphorylation of STAT‐1 in macrophages

Benzoxathiolone derivatives have shown anti-inflammatory and immunomodulatory potential in acne and psoriatic disorders. However, little is known about the molecular basis for these pharmacological effects. In this study, we decided to investigate the anti-inflammatory actions of a benzoxathiolone derivative LYR-71, 6-methyl-2-propylimino-6,7-dihydro-5H-benzo[1,3]oxathiol-4-one, in interferon (IFN)-gamma-activated macrophages. RAW 264.7 macrophages or primary macrophages, derived from bone marrow of C3H/HeJ mice, were stimulated with IFN-gamma in the presence of LYR-71. Nitric oxide (NO) or chemokine production was measured by Griess reaction or enzyme-linked immunosorbent assay. RAW 264.7 cells were used to examine the molecular mechanisms of LYR-71 in modulating IFN-gamma-induced inflammatory responses. LYR-71 down-regulated IFN-gamma-induced transcription of inducible NO synthase, IFN-gamma-inducible protein-10 and the monokine induced by IFN-gamma genes in macrophages. This effect was mediated by uncoupling tyrosine phosphorylation of the signal transducer and activator of transcription (STAT)-1 in response to IFN-gamma. LYR-71 directly inhibited the in vitro catalytic activity of Janus kinase (JAK)-2. Further, the inhibitory actions of LYR-71 on IFN-gamma-induced STAT-1 phosphorylation and NO production were consistently abolished in the presence of peroxyvanadate, implying another target dependent on protein tyrosine phosphatase. Taken together, LYR-71 could restrain IFN-gamma-induced inflammatory responses through uncoupling the tyrosine phosphorylation of STAT-1, an activation index of JAK-STAT-1 signalling, in macrophages. These results may provide a molecular mechanism underlying anti-inflammatory actions shown by benzoxathiolone derivatives.