Opposite effects of miR-155 in the initial and later stages of lipopolysaccharide (LPS)-induced inflammatory response.

Although microRNA-155 (miR-155) is considered a pro-inflammatory mediator, cumulative evidence indicates that it also has anti-inflammatory effects in macrophages and dendritic cells. In this study, we identified the dramatic expression changes of more than half of potential miR-155-targeted genes upon lipopolysaccharide (LPS) stimulation; 223 genes were down-regulated and 85 genes were up-regulated, including suppressor of cytokine signaling 1 (SOCS1) and transforming growth factor-ß-activated kinase 1-binding protein 2 (TAB2), two well-known genes involved in miR-155-mediated regulation of the Toll-like receptor 4 (TLR4) signaling pathway. We also found that miR-155 acted as an anti-inflammatory mediator in the initial stage of LPS-induced inflammatory response mainly through repressing TAB2 protein translation, and as a pro-inflammatory mediator by down-regulating SOCS1 in the later stage. Meanwhile, overexpression of TAB2 3' untranslated region (UTR) in macrophages promoted the development of endotoxin tolerance by competing for binding with miR-155, which resulted in an elevated expression level of SOCS1 protein. These findings provide new insights for understanding the regulatory mechanisms in fine-tuning of LPS-induced innate immune response.

Effects of Granulocyte Colony-Stimulating Factor on Proliferation and Apoptosis of B Cells in Bone Marrow of Healthy Donors.

BACKGROUND AND OBJECTIVE: The aim of this study was to investigate the effects of granulocyte colony-stimulating factor (G-CSF) on the proliferation and apoptosis of bone marrow (BM) B cells from healthy donors and its mechanism. MATERIALS AND METHODS: The proliferation ability and apoptosis of BM cells from healthy donors before and after in vivo G-CSF application were determined by multiparameter flow cytometry. The gene expression of B cells was detected by RNA-Seq. In vitro experiments were performed to investigate the effects of G-CSF on the proliferation and apoptosis of BM B cells through which gene. RESULTS: Treating healthy donors with G-CSF significantly decreased proliferation and increased apoptosis of BM B cells. The proliferation of CD19+CD27- B cell subgroup and CD19+CD24hiCD38hi B cell subset were also decreased. G-CSF also significantly altered proapoptotic genes, cell cycle arrest genes, and DNA replication and cell cycle genes, especially significantly increased SOCS1 expression of BM B cells. In vitro experiments showed that SOCS1 overexpression did not affect B cell proliferation ability and apoptosis. CONCLUSIONS: Our results suggest that extensive effects of G-CSF on BM B cells, such as inhibiting proliferation, inducing apoptosis, and altering a series of gene expression.

Pneumolysin binds to the mannose receptor C type 1 (MRC-1) leading to anti-inflammatory responses and enhanced pneumococcal survival.

Streptococcus pneumoniae (the pneumococcus) is a major cause of mortality and morbidity globally, and the leading cause of death in children under 5 years old. The pneumococcal cytolysin pneumolysin (PLY) is a major virulence determinant known to induce pore-dependent pro-inflammatory responses. These inflammatory responses are driven by PLY-host cell membrane cholesterol interactions, but binding to a host cell receptor has not been previously demonstrated. Here, we discovered a receptor for PLY, whereby pro-inflammatory cytokine responses and Toll-like receptor signalling are inhibited following PLY binding to the mannose receptor C type 1 (MRC-1) in human dendritic cells and mouse alveolar macrophages. The cytokine suppressor SOCS1 is also upregulated. Moreover, PLY-MRC-1 interactions mediate pneumococcal internalization into non-lysosomal compartments and polarize naive T cells into an interferon-γlow, interleukin-4high and FoxP3+ immunoregulatory phenotype. In mice, PLY-expressing pneumococci colocalize with MRC-1 in alveolar macrophages, induce lower pro-inflammatory cytokine responses and reduce neutrophil infiltration compared with a PLY mutant. In vivo, reduced bacterial loads occur in the airways of MRC-1-deficient mice and in mice in which MRC-1 is inhibited using blocking antibodies. In conclusion, we show that pneumococci use PLY-MRC-1 interactions to downregulate inflammation and enhance bacterial survival in the airways. These findings have important implications for future vaccine design.

Clinico-biological significance of suppressor of cytokine signaling 1 expression in acute myeloid leukemia.

Suppressor of cytokine signaling 1 (SOCS1) protein, which encodes a member of signal transducers and activators of transcription-induced inhibitors, takes part in a negative regulation of cytokine signaling. The mechanism of SOCS1 in tumor carcinogenesis is complex and there have been no studies concerning the clinic-biologic implication of SOCS1 expression in acute myeloid leukemia (AML). Here, we first identified that higher bone marrow (BM) SOCS1 expression was closely associated with older age, FLT3-ITD, NPM1 and DNMT3A mutations, but negatively correlated with CEBPA mutation in patients with de novo AML. Compared to patients with lower SOCS1 expression, those with higher expression had lower complete remission rates and shorter overall survival. Further, higher expression of SOCS1 in the BM was an independent unfavorable prognostic factor irrespective of age, white blood cell, cytogenetics and gene mutations. Next, we generated zebrafish model overexpressing SOCS1 by spi1 promoter, which showed kidney marrow from adult SOCS1 zebrafish had increased myelopoiesis, myeloid progenitors and the kidney or spleen structure were effaced and distorted, mimicking leukemia phenotype. The SOCS1/FLT3-ITD double transgenic fish could further facilitate the leukemic process. The results indicate SOCS1 plays an important role in AML and its higher expression serves as a new biomarker to risk-stratify AML patients.

Agomelatine, a MT1/MT2 melatonergic receptor agonist with serotonin 5-HT2C receptor antagonistic properties, suppresses Prevotella intermedia lipopolysaccharide-induced production of proinflammatory mediators in murine macrophages.

OBJECTIVE: This study was performed in an attempt to examine the influence of agomelatine in mitigating the generation of proinflammatory mediators in RAW264.7 murine macrophages exposed to lipopolysaccharide (LPS) obtained from Prevotella intermedia, a gram-negative anaerobic bacterium that is related with various types of periodontal diseases, and the molecular mechanisms behind its effects. DESIGN: LPS from P. intermedia strain ATCC 25611 was prepared employing the conventional phenol-water procedure. Conditioned culture media were analyzed for the levels of nitric oxide (NO), interleukin-1ß (IL-1ß) and IL-6. Real-time PCR analysis was carried out to determine the mRNA levels of inducible NO synthase (iNOS), IL-1ß, IL-6 and SOCS1. Protein expression levels were evaluated by immunoblot test. NF-κB-dependent SEAP reporter assay was performed using a reporter cell line. DNA-binding activities of NF-κB subunits were analyzed utilizing the ELISA-based kits. RESULTS: Agomelatine was found to down-regulate significantly the generation of iNOS-derived NO, IL-1ß and IL-6 as well as the expression of their mRNAs in cells activated with P. intermedia LPS. Agomelatine decreased NF-κB-dependent SEAP release caused by P. intermedia LPS. Agomelatine did not inhibit NF-κB transcription induced by LPS at the level of IκB-α degradation. Instead, LPS-induced nuclear translocation and DNA binding of NF-κB p50 subunit was blocked by agomelatine. P. intermedia LPS-elicited activation of STAT1 and STAT3 was reduced notably by co-treatment with agomelatine. Agomelatine showed a tendency to enhance mRNA level of SOCS1 in LPS-activated cells as well. CONCLUSIONS: Agomelatine merits further evaluation to reveal its usefulness on the host modulation of periodontal disease.

SOCS3 inhibits the pathological effects of IL-22 in non-melanoma skin tumor-derived keratinocytes.

Basal cell carcinomas (BCC) and squamous-cell carcinomas (SCC) are common malignancies in humans, caused by neoplastic transformation of keratinocytes of the basal or suprabasal layers of epidermis, respectively. Tumor-infiltrating lymphocytes (TILs) are frequently found in BCC and SCC, and functionally promote epithelial carcinogenesis. TILs secreting IL-22, in particular, participate to BCC and SCC growth by inducing keratinocyte proliferation and migration, as well as the expression of inflammatory, anti-apoptotic and pro-angiogenic genes.In this study, we identified SOCS3 as a valid candidate to be manipulated for suppressing tumorigenic functions in BCC and SCC. We found that SOCS3 and SOCS1 expression was reduced in vivo, in tumor lesions of BCC and SCC, as compared to other skin inflammatory conditions such as psoriasis, despite the high number of IL-22-secreting TILs. Moreover, IL-22 was not able to induce in vitro the transcriptional expression of SOCS3 in BCC-or SCC-derived keratinocytes, contrarily to healthy cells. Aimed at rescuing SOCS3 activity in these tumor contexts, a SOCS3-derived peptide, named KIR-ESS, was synthesized, and its ability in suppressing IL-22-induced responses was evaluated in healthy and transformed keratinocytes. We found that KIR-ESS peptide efficiently suppressed the IL-22 molecular signaling in keratinocytes, by acting on STAT3 and Erk1/2 cascade, as well as on the expression of STAT3-dependent downstream genes. Interestingly, after treatment with peptide, both healthy and transformed keratinocytes could no longer aberrantly proliferate and migrate in response to IL-22. Finally, treatment of athymic nude mice bearing SCC xenografts with KIR-ESS peptide concomitantly reduced tumor growth and activated STAT3 levels. As a whole, these data provides the rationale for the use in BCC and SCC skin tumors of SOCS3 mimetics, being able to inhibit the deleterious effects of IL-22 in these contexts.

SOCS1 Regulates Apoptosis and Inflammation by Inhibiting IL-4 Signaling in IL-1ß-Stimulated Human Osteoarthritic Chondrocytes.

Recently, Suppressor of Cytokine Signaling 1 (SOCS1) was identified as a potential therapeutic target for osteoarthritis (OA) treatment. However, the mechanisms and signaling pathways of SOCS1 in the regulation of OA development are unclear. The purpose of the current study was to investigate whether interleukin- (IL-) 4 was involved in regulatory mechanism of SOCS1 in human osteoarthritic chondrocytes. First, IL-1ß was used to stimulate human osteoarthritic chondrocytes isolated from the articular cartilage of OA patients undergoing total knee replacement. The protein and mRNA expression levels of SOCS1 were upregulated in IL-1ß-stimulated human osteoarthritic chondrocytes compared with control cells. The knockdown of SOCS1 increased cell viability and inhibited cell apoptosis. It was also found that IL-4 expression was increased by SOCS1 silencing. Additionally, knockdown of IL-4 reduced cell viability and increased cell apoptosis of osteoarthritic chondrocytes transfected with SOCS1 siRNA. Moreover, the decreased expression of inflammatory factors induced by SOCS1 was enhanced by IL-4 knockdown. In conclusion, IL-4 signaling plays a crucial role in the regulatory functions of SOCS1 in apoptosis and inflammation in human osteoarthritic chondrocytes. These findings provide a potential therapeutic target for the clinical treatment of OA.

Thymus-Derived Regulatory T Cell Development Is Regulated by C-Type Lectin-Mediated BIC/MicroRNA 155 Expression.

Thymus-derived regulatory T (tTreg) cells are key to preventing autoimmune diseases, but the mechanisms involved in their development remain unsolved. Here, we show that the C-type lectin receptor CD69 controls tTreg cell development and peripheral Treg cell homeostasis through the regulation of BIC/microRNA 155 (miR-155) and its target, suppressor of cytokine signaling 1 (SOCS-1). Using Foxp3-mRFP/cd69+/- or Foxp3-mRFP/cd69-/- reporter mice and short hairpin RNA (shRNA)-mediated silencing and miR-155 transfection approaches, we found that CD69 deficiency impaired the signal transducer and activator of transcription 5 (STAT5) pathway in Foxp3+ cells. This results in BIC/miR-155 inhibition, increased SOCS-1 expression, and severely impaired tTreg cell development in embryos, adults, and Rag2-/- γc-/- hematopoietic chimeras reconstituted with cd69-/- stem cells. Accordingly, mirn155-/- mice have an impaired development of CD69+ tTreg cells and overexpression of the miR-155-induced CD69 pathway, suggesting that both molecules might be concomitantly activated in a positive-feedback loop. Moreover, in vitro-inducible CD25+ Treg (iTreg) cell development is inhibited in Il2rγ-/-/cd69-/- mice. Our data highlight the contribution of CD69 as a nonredundant key regulator of BIC/miR-155-dependent Treg cell development and homeostasis.

Resveratrol upregulates SOCS1 production by lipopolysaccharide-stimulated RAW264.7 macrophages by inhibiting miR-155.

Resveratrol is a polyphenolic compound extracted from grapes and the Chinese herb, Polygonum cuspidatum. In the present study, in order to elucidate the molecular mechanisms of action of resveratrol in host immune cells, we examined the effects of resveratrol on the inflammatory response in lipopolysaccharide (LPS)­stimulated RAW264.7 murine macrophages. The cells were treated with resveratrol prior to stimulation with LPS (1 µg/ml). Resveratrol downregulated the expression of inflammatory markers, such as tumor necrosis factor (TNF)-α and interleukin (IL)­6, induced by LPS, and inhibited the phosphorylation of mitogen-activated protein kinases (MAPKs) and signal transducer and activator of transcription (STAT)1/STAT3. Resveratrol also upregulated the production of suppressor of cytokine signaling 1 (SOCS1; a STAT inhibitor) and suppressed the expression of miR­155, which plays an essential role in the innate and adaptive immune response. Given the elevated levels of SOCS1 in LPS-induced inflammation, our results suggest that resveratrol exerts anti-inflammatory effects due to the upregulation of SOCS1, which is a potential target of miR­155, as well as of miR­155 mimics and inhibitors. These findings suggest the benefits of resveratrol, which are derived from its regulation of SOCS1 expression via the inhibition of miR­155, and indicate that resveratrol may be developed as a useful agent for the treatment of inflammatory diseases.

Suppressor of Cytokine Signaling (SOCS)1 Regulates Interleukin-4 (IL-4)-activated Insulin Receptor Substrate (IRS)-2 Tyrosine Phosphorylation in Monocytes and Macrophages via the Proteasome.

Allergic asthma is a chronic lung disease initiated and driven by Th2 cytokines IL-4/-13. In macrophages, IL-4/-13 bind IL-4 receptors, which signal through insulin receptor substrate (IRS)-2, inducing M2 macrophage differentiation. M2 macrophages correlate with disease severity and poor lung function, although the mechanisms that regulate M2 polarization are not understood. Following IL-4 exposure, suppressor of cytokine signaling (SOCS)1 is highly induced in human monocytes. We found that siRNA knockdown of SOCS1 prolonged IRS-2 tyrosine phosphorylation and enhanced M2 differentiation, although siRNA knockdown of SOCS3 did not affect either. By co-immunoprecipitation, we found that SOCS1 complexes with IRS-2 at baseline, and this association increased after IL-4 stimulation. Because SOCS1 is an E3 ubiquitin ligase, we examined the effect of proteasome inhibitors on IL-4-induced IRS-2 phosphorylation. Proteasomal inhibition prolonged IRS-2 tyrosine phosphorylation, increased ubiquitination of IRS-2, and enhanced M2 gene expression. siRNA knockdown of SOCS1 inhibited ubiquitin accumulation on IRS-2, although siRNA knockdown of SOCS3 had no effect on ubiquitination of IRS-2. Monocytes from healthy and allergic individuals revealed that SOCS1 is induced by IL-4 in healthy monocytes but not allergic cells, whereas SOCS3 is highly induced in allergic monocytes. Healthy monocytes displayed greater ubiquitination of IRS-2 and lower M2 polarization than allergic monocytes in response to IL-4 stimulation. Here, we identify SOCS1 as a key negative regulator of IL-4-induced IRS-2 signaling and M2 differentiation. Our findings provide novel insight into how dysregulated expression of SOCS increases IL-4 responses in allergic monocytes, and this may represent a new therapeutic avenue for managing allergic disease.

HCV-induced miR146a controls SOCS1/STAT3 and cytokine expression in monocytes to promote regulatory T-cell development.

Host innate and adaptive immune responses must be tightly regulated by an intricate balance between positive and negative signals to ensure their appropriate onset and termination while fighting pathogens and avoiding autoimmunity; persistent pathogens may usurp these regulatory machineries to dampen host immune responses for their persistence in vivo. Here, we demonstrate that miR146a is up-regulated in monocytes from hepatitis C virus (HCV)-infected individuals compared to control subjects. Interestingly, miR146a expression in monocytes without HCV infection increased, whereas its level in monocytes with HCV infection decreased, following Toll-like receptor (TLR) stimulation. This miR146a induction by HCV infection and differential response to TLR stimulation were recapitulated in vitro in monocytes co-cultured with hepatocytes with or without HCV infection. Importantly, inhibition of miR146a in monocytes from HCV-infected patients led to a decrease in IL-23, IL-10 and TGF-ß expressions through the induction of suppressor of cytokine signalling 1 (SOCS1) and the inhibition of signal transducer and activator transcription 3 (STAT3), and this subsequently resulted in a decrease in regulatory T cells (Tregs) accumulated during HCV infection. These results suggest that miR146a may regulate SOCS1/STAT3 and cytokine signalling in monocytes, directing T-cell differentiation and balancing immune clearance and immune injury during chronic viral infection.

Overexpression of Bcl-2, SOCS 1, 3 and Cdh 1, 2 are associated with the early neoplasic changes in modified 4-nitroquinoline 1-oxide-induced murine oral cancer model.

BACKGROUND: The objective was to assess histopathological changes and the expression of proliferating cell nuclear antigen (PCNA), Bcl-2, suppressor of cytokine signaling (SOCS) 1 and 3, Vimentin, TWIST1, and Cdh 1 and 2 in early stages of experimental oral carcinogenesis process using a shorter period of exposure to 4-nitroquinoline oxide (4-NQO) model. METHODS: In this study, 20 rats were divided into control group (n = 10), sacrificed on the first day of the experiment, and experimental group (n = 10) treated with 50 ppm of 4-NQO solution dissolved in drinking water for 8 and 12 weeks. The histological sections were stained with H&E or subjected to immunohistochemistry for detecting PCNA, Bcl-2, SOCS 1 and 3, and STAT 3. Some specimens were used for verification of Vimentin expression, Cdh 1, Cdh 2, and TWIST1 by RT-qPCR. RESULTS: At both 8 and 12 weeks, morphological changes occurred mainly in the posterior portion of the tongue and were limited to the epithelial tissue, including moderate to severe dysplasia at 8 weeks, and severe dysplasia with exacerbation of atypical cells at 12 weeks. Expression of SOCS 1 and 3 increased from 8 to 12 weeks (P < 0.05), whereas STAT 3 expression was reduced mainly at 12 weeks (P < 0.05) in comparison with the control group. The expression of all epithelial-mesenchymal transition markers (EMT) was increased after 12 weeks, reaching statistical significance (P < 0.05) for Cdh 1 and 2. CONCLUSIONS: Together, the results suggested that overexpression of Bcl-2, SOCS 1 and 3, and Cdh 1 and 2 is associated with the early neoplasic changes in modified 4-nitroquinoline 1-oxide-induced murine oral cancer model.

MicroRNA-155-5p is associated with oral squamous cell carcinoma metastasis and poor prognosis.

BACKGROUND: Abnormal miRNA expression was recently implicated in the metastasis of oral squamous cell carcinoma (OSCC) and with a poor prognosis. The initiation of the invasion-metastasis cascade involves epithelial-mesenchymal transition (EMT). Our aim was to clarify how miRNA, especially miR-155-5p misexpression contributes to OSCC metastasis through EMT. METHODS: We collected tumor samples from 73 subjects with OSCC. The samples were analyzed by quantitative reverse-transcription polymerase chain reaction (qRT-PCR), and correlations between miR-155-5p levels and clinical characteristics were investigated. OSCC cell lines were analyzed by miRNA microarray and by transfection with a miR-155-5p mimic or inhibitor, followed by proliferation and wound-healing migration assays. qRT-PCR analyses of EMT makers in cells transfected with miR-155-5p inhibitor were performed. RESULTS: We found high miR-155-5p expression in tissue samples from subjects with OSCC that had metastasized to cervical lymph nodes. HSC-3 cells also strongly expressed miR-155-5p. The epithelial marker E-cadherin was strongly expressed in HSC-3 cells transfected with miR-155-5p inhibitor, and we observed elevated SOCS1 and decreased STAT3 expression in these cells. CONCLUSIONS: Our results suggest that miR-155-5p causes OSCC to metastasize, and could serve as a novel therapeutic target for OSCC.