Search

Type I interferons (IFNs) consist of a group of structurally similar cytokines that play an integral role in regulating the immune response to combat lung infections. In certain models type I IFNs have also been associated with suppression of Th2-skewed immune and inflammatory responses. Transient pulmonary overexpression of the gp130 cytokine Oncostatin M (OSM) by Adenovirus vector (AdOSM) induces a robust Th2-skewed cytokine/inflammatory profile in C57Bl/6 murine lungs. In this study we assessed type I IFN function in OSM-mediated inflammation

Supplementary Figures S1-S3 from Oncostatin M Induces Bone Loss and Sensitizes Rat Osteosarcoma to the Antitumor Effect of Midostaurin In vivo

Purpose: In cultures, the cytokine oncostatin M (OSM) reduces the growth and induces differentiation of osteoblasts and osteosarcoma cells into glial/osteocytic cells. Moreover, OSM sensitizes these cells to apoptosis driven by various death inducers such as the kinase inhibitor staurosporine. Here, we asked whether OSM would have similar effects in vivo.Experimental Design: Adenoviral gene transfer of OSM (AdOSM) was done in naive and osteosarcoma-bearing rats, alone or in combination with Midostaurin (PKC412), a derivative of staurosporine currently used in cancer clinical trials. Bone variables were analyzed by micro-computed tomography scanner, by histology, and by the levels of various serum bone markers. Osteosarcoma progression was analyzed by the development of the primary bone tumor, evolution of pulmonary metastasis, histology (necrosis and fibrosis), and animal survival.Results: In naive rats, AdOSM reduced serum osteoblastic and osteoclastic markers in correlation with a reduced trabecular bone volume. In an osteosarcoma rat model, the combination of AdOSM with PKC412 reduced the progression of the primary bone tumor, pulmonary metastatic dissemination, and increased overall survival, whereas these agents alone had no antitumor effect. Increased tumor necrosis and tissue repair (fibrosis) were observed with this combination.Conclusion: These in vivo experiments confirm that systemic OSM overexpression alters osteoblast/osteosarcoma activity. Because OSM sensitizes rat osteosarcoma to apoptosis/necrosis, the use of kinase inhibitors such as Midostaurin in association with OSM could represent new adjuvant treatments for this aggressive malignancy.

BackgroundCigarette smokers are at increased risk of acquiring influenza, developing severe disease and requiring hospitalisation/intensive care unit admission following infection. However, immune mechanisms underlying this predisposition are incompletely understood, and therapeutic strategies for influenza are limited.MethodsWe used a mouse model of concurrent cigarette smoke exposure and H1N1 influenza infection, colony-stimulating factor (CSF)3 supplementation/receptor (CSF3R) blockade and single-cell RNA sequencing (scRNAseq) to investigate this relationship.ResultsCigarette smoke exposure exacerbated features of viral pneumonia such as oedema, hypoxaemia and pulmonary neutrophilia. Smoke-exposed infected mice demonstrated an increase in viral (v)RNA, but not replication-competent viral particles, relative to infection-only controls. Interstitial rather than airspace neutrophilia positively predicted morbidity in smoke-exposed infected mice. Screening of pulmonary cytokines using a novel dysregulation score identified an exacerbated expression of CSF3 and interleukin-6 in the context of smoke exposure and influenza. Recombinant (r)CSF3 supplementation during influenza aggravated morbidity, hypothermia and oedema, while anti-CSF3R treatment of smoke-exposed infected mice improved alveolar–capillary barrier function. scRNAseq delineated a shift in the distribution of Csf3+ cells towards neutrophils in the context of cigarette smoke and influenza. However, although smoke-exposed lungs were enriched for infected, highly activated neutrophils, gene signatures of these cells largely reflected an exacerbated form of typical influenza with select unique regulatory features.ConclusionThis work provides novel insight into the mechanisms by which cigarette smoke exacerbates influenza infection, unveiling potential therapeutic targets (e.g. excess vRNA accumulation, oedematous CSF3R signalling) for use in this context, and potential limitations for clinical rCSF3 therapy during viral infectious disease.

The ability to treat severe viral infections is limited by our understanding of the mechanisms behind virus-induced immunopathology. While the role of type I interferons (IFNs) in early control of viral replication is clear, less is known about how IFNs can regulate the development of immunopathology and affect disease outcomes. Here, we report that absence of type I IFN receptor (IFNAR) is associated with extensive immunopathology following mucosal viral infection. This pathology occurred independent of viral load or type II immunity but required the presence of macrophages and IL-6. The depletion of macrophages and inhibition of IL-6 signaling significantly abrogated immunopathology. Tissue destruction was mediated by macrophage-derived matrix metalloproteinases (MMPs), as MMP inhibition by doxycycline and Ro 28–2653 reduced the severity of tissue pathology. Analysis of post-mortem COVID-19 patient lungs also displayed significant upregulation of the expression of MMPs and accumulation of macrophages. Overall, we demonstrate that IFNs inhibit macrophage-mediated MMP production to prevent virus-induced immunopathology and uncover MMPs as a therapeutic target towards viral infections.

Some severe asthmatic patients experience frequent bacterial respiratory tract infections, which contribute significantly to their disease burden, and often are attributed to their use of systemic corticosteroids and comorbid bronchiectasis. We report a case of a 58-year-old woman who had prednisone-dependent asthma and exacerbations with intense mixed eosinophilic and neutrophilic bronchitis. Autosomal dominant hyper-IgE syndrome, which is a primary immunodeficiency characterized by elevated IgE, eosinophilia, and recurrent infections, caused by a novel pathogenic mutation in STAT3 was identified as the cause of her airway disease. We believe that this is the first report of the demonstration of an IL-5 driven eosinophilia that is associated with a STAT3 mutation that was treated successfully with an anti-IL5 biological.

The interleukin-1 family member IL-33 participates in both innate and adaptive T helper-2 immune cell responses in models of lung disease. The IL-6-type cytokine Oncostatin M (OSM) elevates lung inflammation, Th2-skewed cytokines, alternatively activated (M2) macrophages, and eosinophils in C57Bl/6 mice in vivo. Since OSM induces IL-33 expression, we here test the IL-33 function in OSM-mediated lung inflammation using IL-33-/- mice. Adenoviral OSM (AdOSM) markedly induced IL-33 mRNA and protein levels in wild-type animals while IL-33 was undetectable in IL-33-/- animals. AdOSM treatment showed recruitment of neutrophils, eosinophils, and elevated inflammatory chemokines (KC, eotaxin-1, MIP1a, and MIP1b), Th2 cytokines (IL-4/IL-5), and arginase-1 (M2 macrophage marker) whereas these responses were markedly diminished in IL-33-/- mice. AdOSM-induced IL-33 was unaffected by IL-6-/- deficiency. AdOSM also induced IL-33R+ ILC2 cells in the lung, while IL-6 (AdIL-6) overexpression did not. Flow-sorted ILC2 responded in vitro to IL-33 (but not OSM or IL-6 stimulation). Matrix remodelling genes col3A1, MMP-13, and TIMP-1 were also decreased in IL-33-/- mice. In vitro, IL-33 upregulated expression of OSM in the RAW264.7 macrophage cell line and in bone marrow-derived macrophages. Taken together, IL-33 is a critical mediator of OSM-driven, Th2-skewed, and M2-like responses in mouse lung inflammation and contributes in part through activation of ILC2 cells.

Although recent evidence has shown that IL‐6 is involved in enhanced alternative activation of macrophages toward a profibrotic phenotype, the mechanisms leading to their increased secretory capacity are not fully understood. Here, we investigated the effect of IL‐6 on endoplasmic reticulum (ER) expansion and alternative activation of macrophages in vitro. An essential mediator in this ER expansion process is the IRE1 pathway, which possesses a kinase and endoribonuclease domain to cleave XBP1 into a spliced bioactive molecule. To investigate the IRE1‐XBP1 expansion pathway, IL‐4/IL‐13 and IL‐4/IL‐13/IL‐6‐mediated alternative programming of murine bone marrow‐derived and human THP1 macrophages were assessed by arginase activity in cell lysates, CD206 and arginase‐1 expression by flow cytometry, and secreted CCL18 by ELISA, respectively. Ultrastructural intracellular morphology and ER biogenesis were examined by transmission electron microscopy and immunofluorescence. Transcription profiling of 128 genes were assessed by NanoString and Pharmacological inhibition of the IRE1‐XBP1 arm was achieved using STF‐083010 and was verified by RT‐PCR. The addition of IL‐6 to the conventional alternative programming cocktail IL‐4/IL‐13 resulted in increased ER and mitochondrial expansion, profibrotic profiles and unfolded protein response‐mediated induction of molecular chaperones. IRE1‐XBP1 inhibition substantially reduced the IL‐6‐mediated hyperpolarization and normalized the above effects. In conclusion, the addition of IL‐6 enhances ER expansion and the profibrotic capacity of IL‐4/IL‐13‐mediated activation of macrophages. Therapeutic strategies targeting IL‐6 or the IRE1‐XBP1 axis may be beneficial to prevent the profibrotic capacity of macrophages., IL‐6 is known to increase the polarization of alternatively activated macrophages. Here, we show that this hyperpolarization is associated with a dramatic increase in the endoplasmic reticulum, an organelle required for folding of secreted proteins. We show further that the inhibition of a known activator of the Unfolded Protein Response, IRE1, prevents both ER expansion and alternative activation of macrophages, suggesting that targeting the process of ER expansion in alternatively activated macrophages may be considered to prevent the known profibrotic activity of alternatively activated macrophages.

Chronic inflammation can be caused by adaptive immune responses in autoimmune and allergic conditions, driven by a T lymphocyte subset balance (TH1, TH2, Th17, Th22, and/or Treg) and skewed cellular profiles in an antigen-specific manner. However, several chronic inflammatory diseases have no clearly defined adaptive immune mechanisms that drive chronicity. These conditions include those that affect the lung such as nonatopic asthma or idiopathic pulmonary fibrosis comprising significant health problems. The remodeling of extracellular matrix (ECM) causes organ dysfunction, and it is largely generated by fibroblasts as the major cell controlling net ECM. As such, these are potential targets of treatment approaches in the context of ECM pathology. Fibroblast phenotypes contribute to ECM and inflammatory cell accumulation, and they are integrated into chronic disease mechanisms including cancer. Evidence suggests that innate cytokine responses may be critical in nonallergic/nonautoimmune disease, and they enable environmental agent exposure mechanisms that are independent of adaptive immunity. Innate immune cytokines derived from macrophage subsets (M1/M2) and innate lymphoid cell (ILC) subsets can directly regulate fibroblast function. We also suggest that STAT3-activating gp130 cytokines can sensitize fibroblasts to the innate cytokine milieu to drive phenotypes and exacerbate existing adaptive responses. Here, we review evidence exploring innate cytokine regulation of fibroblast behavior.

Oncostatin M (OSM), as one of the gp130/IL-6 family of cytokines, interacts with receptor complexes that include the gp130 signaling molecule and OSM receptor β OSMRβ chain subunits. OSMRβ chains are expressed relatively highly across a broad array of connective tissue (CT) cells of the lung, such as fibroblasts, smooth muscle cells, and epithelial cells, thus enabling robust responses to OSM, compared to other gp130 cytokines, in the regulation of extracellular matrix (ECM) remodeling and inflammation. OSMRβ chain expression in lung monocyte/macrophage populations is low, whereas other receptor subunits, such as that for IL-6, are present, enabling responses to IL-6. OSM is produced by macrophages and neutrophils, but not CT cells, indicating a dichotomy of OSM roles in macrophage verses CT cells in lung inflammatory disease. ECM remodeling and inflammation are components of a number of chronic lung diseases that show elevated levels of OSM. OSM-induced products of CT cells, such as MCP-1, IL-6, and PGE2 can modulate macrophage function, including the expression of OSM itself, indicating feedback loops that characterize Macrophage and CT cell interaction.

IL-33 modulates both innate and adaptive immune responses at tissue sites including lung and may play critical roles in inflammatory lung disease. Although IL-33 expression can be altered upon NF-Kappa B activation, here we examine regulation by Oncostatin M, a gp130 cytokine family member, in mouse lung tissue. Responses were assessed in BALB/c mouse lung at day 7 of transient overexpression using endotracheally administered adenovirus encoding OSM (AdOSM) or empty vector (AdDel70). Whole lung extracts showed induction of IL-33 mRNA (>20-fold) and protein (10-fold increase in immunoblots) by AdOSM relative to AdDel70. Immunohistochemistry for IL-33 indicated a marked induction of nuclear staining in alveolar epithelial cellsin vivo. Oncostatin M stimulated IL-33 mRNA and IL-33 full length protein in C10 mouse type 2 alveolar epithelial cells in culture in time-dependent and dose-dependent fashion, whereas IL-6, LIF, IL-31, IL-4, or IL-13 did not, and TGFβrepressed IL-33. IL-33 induction was associated with activation of STAT3, and pharmacological inhibition of STAT3 ameliorated IL-33 levels. These results indicate Oncostatin M as a potent inducer of IL-33 in mouse lung epithelial cells and suggest that an OSM/IL-33 axis may participate in innate immunity and inflammatory conditions in lung.

Resistin-like molecule alpha (RELM&alpha, ) and YM-1 are secreted proteins implicated in murine models of alternatively activated macrophage (AA/M2) accumulation and Th2-skewed inflammation. Since the gp130 cytokine Oncostatin M (OSM) induces a Th2-like cytokine and AA/M2 skewed inflammation in mouse lung, we here investigated regulation of RELM&alpha, and YM-1. Transient pulmonary overexpression of OSM by Adenovirus vector (AdOSM) markedly induced RELM&alpha, and YM-1 protein expression in total lung. In situ hybridization showed that RELM&alpha, mRNA was highly induced in airway epithelial cells (AEC) and was co-expressed with CD68 mRNA in some but not all CD68+ cells in parenchyma. IL-6 overexpression (a comparator gp130 cytokine) induced RELM&alpha, but at significantly lower levels. IL-6 (assessing IL-6&minus, /&minus, mice) was not required, nor was STAT6 (IL-4/13 canonical signalling) for AdOSM-induction of RELM&alpha, in AEC. AEC responded directly to OSM in vitro as assessed by pSTAT3 activation. RELM&alpha, deficient mice showed similar inflammatory cell infiltration and cytokine responses to wt in response to AdOSM, but showed less accumulation of CD206+ AA/M2 macrophages, reduced induction of extracellular matrix gene mRNAs for COL1A1, COL3A1, MMP13, and TIMP1, and reduced parenchymal alpha smooth muscle actin. Thus, RELM&alpha, is regulated by OSM in AEC and contributes to extracellular matrix remodelling in mouse lung.

To evaluate cellular response to oncostatin M (OSM) in comparison to interleukin (IL)-31, we analyzed monocyte chemoattractant protein 1 (MCP-1) as a readout for OSM responses with and without IL-4, IL-13, anti-OSM receptor β monoclonal antibody KPL-716, and anti-IL-31 receptor α antibody in human epidermal keratinocytes and human dermal fibroblasts in vitro. In human epidermal keratinocytes, OSM significantly induced STAT3 or STAT1 phosphorylation and synergized with IL-13 or IL-4 in elevating MCP-1. In human dermal fibroblasts, OSM results were similar, and leukemia inhibitory factor or IL-31 minimally activated STAT3 but not MCP-1. OSM significantly stimulated mRNA for type II IL-4 receptor and type II OSM receptor. KPL-716, not anti-IL-31Rα, significantly attenuated MCP-1 response to OSM and OSM + IL-4 in human epidermal keratinocytes and human dermal fibroblasts. OSM, not leukemia inhibitory factor or IL-31, synergized with IL-4 and IL-13 in human epidermal keratinocytes and human dermal fibroblasts, suggesting therapeutic potential of KPL-716 in inflammatory dermatologic diseases distinct from IL-31 inhibition.

Despite effective new treatments for Hepatitis C virus (HCV) infection, development of drug resistance, safety concerns and cost are remaining challenges. More importantly, there is no vaccine available against hepatitis C infection. Recent data suggest that there is a strong correlation between spontaneous HCV clearance and human NK cell function, particularly IFN-γ production. Further, IL-15 has innate antiviral activity and is also one of the main factors that activates NK cells to produce IFN-γ. To examine whether IL-15 and IFN-γ have direct antiviral activity against HCV, Huh7.5 cells were treated with either IFN-γ or IL-15 prior to HCV infection. Our data demonstrate that IFN-γ and IL-15 block HCV replication in vitro. Additionally, we show that IL-15 and IFN-γ do not induce anti-HCV effects through the type I interferon signaling pathway or nitric oxide (NO) production. Instead, IL-15 and IFN-γ provide protection against HCV via the ERK pathway. Treatment of Huh7.5 cells with a MEK/ERK inhibitor abrogated the anti-HCV effects of IL-15 and IFN-γ and overexpression of ERK1 prevented HCV replication compared to control transfection. Our in vitro data support the hypothesis that early production of IL-15 and activation of NK cells in the liver lead to control of HCV replication.

Oncostatin M (OSM) is an IL-6/LIF family cytokine that influences mesenchymal progenitor differentiation; however, the mechanisms of this activity have not been fully elucidated. Using uncommitted murine adipose tissue-derived mesenchymal progenitors, we have examined mechanisms of OSM-induced osteogenesis. Murine OSM (mOSM) induced osteogenic differentiation to a greater degree than interleukin (IL)-6 and other members of the gp130 cytokine family, promoting extracellular matrix mineralization as indicated by Alizarin Red S staining. mOSM also increased expression of osteogenesis-associated gene products BMP4, BMP7, Runx-2, and osteocalcin as assessed by immunoblotting and real-time quantitative PCR. Additionally, protein kinase C (PKC) delta activity was upregulated in response to OSM stimulation, and to a greater degree than IL-6. Knockdown of PKCdelta expression by use of RNA interference (RNAi) reduced OSM-mediated osteogenic differentiation and decreased expression of Runx-2. These findings suggest that OSM differentially promotes osteogenesis in non-committed mesenchymal progenitors relative to other gp130 cytokines. This activity correlates with selective activation of PKCdelta in OSM-treated cells, indicating that OSM-induced osteogenesis and upregulation of osteogenic gene products require activity of PKCdelta.

Although recent evidence indicates that gp130 cytokines, Oncostatin M (OSM) and IL-6 are involved in alternative programming of macrophages, their role in lung fibrogenesis is poorly understood. Here, we investigated the effect of transient adenoviral overexpression of OSM or IL-6 in mice during bleomycin-induced lung fibrosis. Lung fibrosis and M2-like macrophage accumulation were assessed by immunohistochemistry, western blotting, gene expression and flow cytometry. Ex-vivo isolated alveolar and bone marrow-derived macrophages were examined for M2-like programming and signalling. Airway physiology measurements at day 21 demonstrated that overexpression of OSM or IL-6 exacerbated bleomycin-induced lung elastance, consistent with histopathological assessment of extracellular matrix and myofibroblast accumulation. Flow cytometry analysis at day 7 showed increased numbers of M2-like macrophages in lungs of mice exposed to bleomycin and OSM or IL-6. These macrophages expressed the IL-6Rα, but were deficient for OSMRβ, suggesting that IL-6, but not OSM, may directly induce alternative macrophage activation. In conclusion, the gp130 cytokines IL-6 and OSM contribute to the accumulation of profibrotic macrophages and enhancement of bleomycin-induced lung fibrosis. This study suggests that therapeutic strategies targeting these cytokines or their receptors may be beneficial to prevent the accumulation of M2-like macrophages and the progression of fibrotic lung disease.

Arginase-1 (Arg-1)-expressing M2-like macrophages are associated with Th2-skewed immune responses, allergic airway pathology, ectopic B16 melanoma cancer growth in murine models, and can be induced by Oncostatin M (OSM) transient overexpression in vivo. Here, we compare OSM to the gp130-cytokine IL-6 in mediating macrophage polarization, and find that IL-6 overexpression alone (Ad vector, AdIL-6) did not induce Arg-1 protein in mouse lungs at day 7, nor ectopic melanoma tumor growth at day 14, in contrast to overexpression of OSM (AdOSM). AdOSM elevated levels of IL-4, IL-5 and IL-13 in bronchoalveolar lavage fluid, whereas AdIL-6 did not. Bone marrow-derived macrophages respond with Arg-1 enzymatic activity to M2 stimuli (IL-4/IL-13), which was further elevated in combination with IL-6 stimulation; however, OSM or LIF had no detectable activity in vitro. Arg-1 mRNA expression induced by AdOSM was attenuated in IL-6-/- and STAT6-/- mice, suggesting requirements for both IL-6 and IL-4/IL-13 signaling in vivo. Ectopic B16 tumor burden was also reduced in IL-6-/- mice. Thus, OSM induces Arg-1+ macrophage accumulation indirectly through elevation of Th2 cytokines and IL-6 in vivo, whereas IL-6 acts directly on macrophages but requires a Th2 microenvironment, demonstrating distinct roles for OSM and IL-6 in M2 macrophage polarization.

The synergistic effect of IL-18/IL-15/IL-12 stimulation potently activates NK cells, inducing high levels of IFN-γ production. As a result of this potent stimulatory effect, NK cell pre-activation with IL-18/IL-15/IL-12 is being developed as a cancer immunotherapy. Ex vivo expansion of NK cells enables the efficient generation of large numbers of NK cells for wide-scale and repeated therapeutic use, and is thus an important source of NK cells for clinical application. However, the effects of IL-18/IL-15/IL-12 stimulation on ex vivo expanded NK cells have not yet been assessed. Thus, the present study assessed the effects of IL-18/IL-15/IL-12 stimulation on NK cells expanded ex vivo using K562-based artificial antigen presenting cells expressing membrane-bound IL-21. We report that ex vivo expanded NK cells stimulated with IL-18/IL-15/IL-12 produce high levels of IFN-γ and TNFα, have potent cytotoxicity, and maintain prolonged IFN-γ production following removal of stimulation. IL-18/IL-15/IL-12 stimulation induces a phenotypically unique IFN-γ-producing population with reduced CD16 expression and greater CD25 expression as compared to stimulated IFN-γ- NK cells and unstimulated NK cells. We elucidate that the mechanism of synergy for induction and maintenance of IFN-γ production is not due to a further enhancement of STAT4 activation compared to stimulation with IL-12 alone. Furthermore, we demonstrate that the synergistic increase in IFN-γ is not solely under translational regulation, as elevated levels of IFN-γ mRNA contribute to the synergistic increase in IFN-γ. Overall, this study characterizes the response of ex vivo expanded NK cells to IL-18/IL-15/IL-12 stimulation and supports the use of ex vivo expanded NK cells as a feasible and efficient source of IL-18/IL-15/IL-12 pre-activated NK cells for adoptive transfer in cancer immunotherapies.

Adverse health outcomes in pulmonary fibrosis are associated with extracellular matrix (ECM) accumulation. Although transforming growth factor-β (TGF-β) has been reported to be an important regulator of fibrosis pathogenesis, TGF-β-independent pathways may also be involved. Here, we investigated responses of putative relatively fibrosis-resistant BALB/c mice to transient pulmonary overexpression of oncostatin M (OSM) using an adenovirus vector encoding OSM (AdOSM) and compared responses with the relatively fibrosis-prone C57Bl/6 strain. Interestingly, BALB/c mice showed similar ECM accumulation and collagen 1A1 and 3A1 mRNA elevation to C57Bl/6 mice 7 days after endotracheal administration of AdOSM. TGF-β1 mRNA levels and pSMAD2 signal were not regulated in either strain in total lung extracts. In contrast to C57Bl/6 mice, BALB/c mice lacked eosinophil, Th2 cytokine, and pro-inflammatory cytokine elevation in the broncholveolar space. OSM overexpression induced STAT3 activation and SMAD1/5/8 signaling suppression in lung from both mice strains, which was associated with a downregulation of BMPR2 and BMP ligands, and increased expression of the BMP antagonist gremlin. Although we also observed STAT3 activation and SMAD1/5/8 signaling suppression in mouse lung fibroblast cultures in vitro upon OSM stimulation, immunohistochemistry analyses indicated that the AdOSM-induced pSMAD1/5/8 signal suppression was primarily localized to the airway epithelium. Other gp130 cytokines including IL-6, LIF, CT-1, but not IL-31, also induced STAT3 activation and SMAD1/5/8 signaling suppression in C10 mouse lung epithelial cells and BEAS 2B bronchial epithelial cells, and we found that pharmacological inhibition of STAT3 activation reversed OSM-induced SMAD1/5/8 signaling suppression in vitro. The results demonstrate that OSM induces ECM accumulation in fibrosis-resistant BALB/c mouse lung in the absence of Th2 inflammation or TGF-β signaling, and highlight a dichotomy of STAT3 activation versus SMAD1 suppression in this process.

Inducible BALT (iBALT) is associated with immune responses to respiratory infections as well as with local pathology derived from chronic inflammatory lung diseases. In this study, we assessed the role of oncostatin M (OSM) in B cell activation and iBALT formation in mouse lungs. We found that C57BL/6 mice responded to an endotracheally administered adenovirus vector expressing mouse OSM, with marked iBALT formation, increased cytokine (IL-4, IL-5, IL-6, IL-10, TNF-α, and IL-12), and chemokine (CXCL13, CCL20, CCL21, eotaxin-2, KC, and MCP-1) production as well as inflammatory cell accumulation in the airways. B cells, T cells, and dendritic cells were also recruited to the lung, where many displayed an activated phenotype. Mice treated with control adenovirus vector (Addl70) were not affected. Interestingly, IL-6 was required for inflammatory responses in the airways and for the expression of most cytokines and chemokines. However, iBALT formation and lymphocyte recruitment to the lung tissue occurred independently of IL-6 and STAT6 as assessed in gene-deficient mice. Collectively, these results support the ability of OSM to induce B cell activation and iBALT formation independently of IL-6 and highlight a role for IL-6 downstream of OSM in the induction of pulmonary inflammation.

Background Monocyte recruitment leads to accumulation of macrophage foam cells and contributes to atherosclerotic lesion growth. Recent studies have reported that lesion‐resident macrophages can proliferate and represent a major cellular component during lesion development. This study was designed to assess whether the rate of macrophage proliferation changes during well‐established stages of lesion growth and to characterize other populations of proliferating cells within these lesions. Methods and Results Using murine models of atherosclerosis ( Apoe −/− and LDL r −/− mice) and human coronary artery lesions, in situ proliferation of lesion‐resident cells at different stages of growth was assessed by staining for Ki67 and bromodeoxyuridine (BrdU). In early lesions, close to half of all actively growing macrophages were proliferating in situ. BrdU pulse labeling allowed for accurate identification of in situ proliferating macrophages compared to those derived from monocyte recruitment. Local macrophage proliferation declined as lesions advanced. Interestingly, intimal inflammatory cell infiltrates containing proliferating T lymphocytes were identified during the active phase of lesion growth and correlated with apoptotic cell death. Inflammatory cell infiltrates were completely resolved in advanced lesions and replaced with the necrotic core. Conclusions Our findings indicate that atherosclerotic lesions contain locally proliferating macrophages primarily during early and intermediate stages of lesion growth. Furthermore, T‐lymphocyte‐enriched inflammatory cell infiltrates represent a novel subset of proliferating cells within the atherosclerotic lesion that correlate with apoptosis and precede the necrotic core. These findings have novel implications in understanding the pathogenesis of atherosclerosis and may implicate proliferating T lymphocytes as a contributing factor to lesion progression and stability.

International audience; Oncostatin M (OSM) has been reported to be overexpressed in psoriasis skin lesions and to exert proinflammatory effects in vitro on human keratinocytes. Here, we report the proinflammatory role of OSM in vivo in a mouse model of skin inflammation induced by intradermal injection of murine OSM-encoding adenovirus (AdOSM) and compare with that induced by IL-6 injection. Here, we show that OSM potently regulates the expression of genes involved in skin inflammation and epidermal differentiation in murine primary keratinocytes. In vivo, intradermal injection of AdOSM in mouse ears provoked robust skin inflammation with epidermal thickening and keratinocyte proliferation, while minimal effect was observed after AdIL-6 injection. OSM overexpression in the skin increased the expression of the S100A8/9 antimicrobial peptides, CXCL3, CCL2, CCL5, CCL20, and Th1/Th2 cytokines, in correlation with neutrophil and macrophage infiltration. In contrast , OSM downregulated the expression of epidermal differentiation genes, such as cytokeratin-10 or filaggrin. Collectively, these results support the proinflammatory role of OSM when it is overexpressed in the skin. However, OSM expression was not required in the murine model of psoriasis induced by topical application of imiquimod, as demonstrated by the inflammatory phenotype of OSM-deficient mice or wild-type mice treated with anti-OSM antibodies.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease that is unresponsive to current therapies and characterized by excessive collagen deposition and subsequent fibrosis. While inflammatory cytokines, including interleukin (IL)-6, are elevated in IPF, the molecular mechanisms that underlie this disease are incompletely understood, although the development of fibrosis is believed to depend on canonical transforming growth factor (TGF)-β signalling. We examined bleomycin-induced inflammation and fibrosis in mice carrying a mutation in the shared IL-6 family receptor gp130. Using genetic complementation, we directly correlate the extent of IL-6-mediated, excessive Stat3 activity with inflammatory infiltrates in the lung and the severity of fibrosis in corresponding gp130(757F) mice. The extent of fibrosis was attenuated in B lymphocyte-deficient gp130(757F);µMT(-/-) compound mutant mice, but fibrosis still occurred in their Smad3(-/-) counterparts consistent with the capacity of excessive Stat3 activity to induce collagen 1α1 gene transcription independently of canonical TGF-β/Smad3 signalling. These findings are of therapeutic relevance, since we confirmed abundant STAT3 activation in fibrotic lungs from IPF patients and showed that genetic reduction of Stat3 protected mice from bleomycin-induced lung fibrosis.

Bone resorption by osteoclasts and bone formation by osteoblasts are tightly coupled processes implicating factors in TNF, bone morphogenetic protein, and Wnt families. In osteoimmunology, macrophages were described as another critical cell population regulating bone formation by osteoblasts but the coupling factors were not identified. Using a high-throughput approach, we identified here Oncostatin M (OSM), a cytokine of the IL-6 family, as a major coupling factor produced by activated circulating CD14+ or bone marrow CD11b+ monocytes/macrophages that induce osteoblast differentiation and matrix mineralization from human mesenchymal stem cells while inhibiting adipogenesis. Upon activation of toll-like receptors (TLRs) by lipopolysaccharide or endogenous ligands, OSM was produced in classically activated inflammatory M1 and not M2 macrophages, through a cyclooxygenase-2 and prostaglandin-E2 regulatory loop. Stimulation of osteogenesis by activated monocytes/macrophages was prevented using neutralizing antibodies or siRNA to OSM, OSM receptor subunits gp130 and OSMR, or to the downstream transcription factor STAT3. The induced osteoblast differentiation program culminated with enhanced expression of CCAAT-enhancer-binding protein δ, Cbfa1, and alkaline phosphatase. Overexpression of OSM in the tibia of mice has led to new bone apposition with no sign of bone resorption. Two other cytokines have also a potent role in bone formation induced by monocytes/macrophages and activation of TLRs: IL-6 and leukemia inhibitory factor. We propose that during bone inflammation, infection, or injury, the IL-6 family signaling network activated by macrophages and TLR ligands stimulates bone formation that is largely uncoupled from bone resorption and is thus an important target for anabolic bone therapies. Disclosure of potential conflicts of interest is found at the end of this article.

Oncostatin M (OSM), a pleiotropic cytokine of the gp130 cytokine family, has been implicated in chronic allergic inflammatory and fibrotic disease states associated with tissue eosinophilia. Mouse (m)OSM induces airway eosinophilic inflammation and interstitial pulmonary fibrosis in vivo and regulates STAT6 activation in vitro. To determine the requirement of STAT6 in OSM-induced effects in vivo, we examined wild-type (WT) and STAT6-knockout (STAT6−/−) C57BL/6 mouse lung responses to transient ectopic overexpression of mOSM using an adenoviral vector (AdmOSM). Intratracheal AdmOSM elicited persistent eosinophilic lung inflammation that was abolished in STAT6−/− mice. AdmOSM also induced pronounced pulmonary remodeling characterized by goblet cell hyperplasia and parenchymal interstitial fibrosis. Goblet cell hyperplasia was STAT6 dependent; however, parenchymal interstitial fibrosis was not. OSM also induced airway hyperresponsiveness in WT mice that was abolished in STAT6−/− mice. OSM stimulated an inflammatory signature in the lungs of WT mice that demonstrated STAT6-dependent regulation of Th2 cytokines (IL-4, IL-13), chemokines (eotaxin-1/2, MCP-1, keratinocyte chemoattractant), and extracellular matrix modulators (tissue inhibitor of matrix metalloproteinase-1, matrix metalloproteinase-13), but STAT6-independent regulation of IL-4Rα, total lung collagen, collagen-1A1, -1A2 mRNA, and parenchymal collagen and α smooth muscle actin accumulation. Thus, overexpression of mOSM induces STAT6-dependent pulmonary eosinophilia, mucous/goblet cell hyperplasia, and airway hyperresponsiveness but STAT6-independent mechanisms of lung tissue extracellular matrix accumulation. These results also suggest that eosinophil or neutrophil accumulation in mouse lungs is not required for OSM-induced lung parenchymal collagen deposition and that OSM may have unique roles in the pathogenesis of allergic and fibrotic lung disease.

The cytokine Oncostatin M (OSM) is cytostatic, pro-apoptotic and induces differentiation of osteosarcoma cells into osteocytes, suggesting new adjuvant treatment for these bone-forming sarcomas. However, OSM systemic over-expression could lead to adverse side effects such as generalized inflammation, neoangiogenesis and osteolysis. We determine here the effect of OSM on chondrosarcoma, another primary bone sarcoma characterized by the production of cartilage matrix and altered bone remodelling. Chondrosarcomas are resistant to conventional chemotherapy and radiotherapy, and wide surgical excision remains the only available treatment. We found that OSM blocked the cell cycle in four of five chondrosarcoma cell lines, independently of p53 and presumably through the JAK3/STAT1 pathway. In two tested cell lines, OSM induced a hypertrophic chondrocyte differentiation, with an induced Cbfa1/SOX9 ratio and induced Coll10, matrix metalloproteinase 13 (MMP13) and RANKL expression. Adenoviral gene transfer of OSM (AdOSM) in the Swarm rat chondrosarcoma (SRC) model indicated that local intra-tumoral OSM over-expression reduces chondrosarcoma development not only with reduced tumor proliferation and enhanced apoptosis but also with enhanced RANKL expression, osteoclast formation and reduced bone volumes. Flu-like symptoms were induced by the AdOSM, but there was no effect on tumor angiogenesis. Therefore, OSM could be considered as a new adjuvant anti-cancer agent for chondrosarcomas. A local application of this cytokine is presumably needed to overcome the poor vascularization of these tumors and to limit the deleterious effect on other tissues. Its side effect on bone remodeling could be managed with anti-resorption agents, thus offering potential new lines of therapeutic interventions.

An alarming global rise in the prevalence of obesity and its contribution to the development of chronic diseases is a serious health concern. Recently, obesity has been described as a chronic low-grade inflammatory condition, influenced by both adipose tissue and immune cells suggesting proinflammatory cytokines may play a role in its etiology. Here we examined the effects of interleukin-15 (IL-15) on adipose tissue and its association with obesity. Over expression of IL-15 (IL-15tg) was associated with lean body condition whereas lack of IL-15 (IL-15(-/-)) results in significant increase in weight gain without altering appetite. Interestingly, there were no differences in proinflammatory cytokines such as IL-6 and tumor necrosis factor-alpha (TNF-alpha) in serum between the three strains of mice. In addition, there were significant numbers of natural killer (NK) cells in fat tissues from IL-15tg and B6 compared to IL-15(-/-) mice. IL-15 treatment results in significant weight loss in IL-15(-/-) knockout and diet-induced obese mice independent of food intake. Fat pad cross-sections show decreased pad size with over expression of IL-15 is due to adipocyte shrinkage. IL-15 induces weight loss without altering food consumption by affecting lipid deposition in adipocytes. Treatment of differentiated human adipocytes with recombinant human IL-15 protein resulted in decreased lipid deposition. In addition, obese patients had significantly lower serum IL-15 levels when compared to normal weight individuals. These results clearly suggest that IL-15 may be involved in adipose tissue regulation and linked to obesity.

Circulating interleukin-6 (IL-6) is frequently used to study surgical injury and inflammation. Measurement properties of serum IL-6 were examined following lumbar decompression surgery (LDS), including time course, sensitivity, and validity for detecting muscle trauma in comparison to the muscle cytoplasmic protein creatine kinase (CK).Seven women and seven men had serial blood samples taken in the preoperative waiting areas, immediately after surgery, at 6, 12, 24, 48 h, 4 d, and at 6 to 7 d. Lumbar surgeries were single level, decompression, with laminotomy.Time to peak serum IL-6 varied across individuals (range 6 to 48 h). However, the higher of two samples drawn within the sensitive time window (6 to 24 h) had a strong correlation with peak IL-6 (r = 0.99, P0.001). There was a moderate correlation between the rise in serum IL-6 and rise in serum CK, r = 0.56, P0.05. T-tests revealed that group mean IL-6 was significantly elevated at only one serial time point (6 h), whereas group mean CK was significantly elevated at three serial time points (6, 12, 24 h) and approached significant elevation as late as 48 h (P = 0.07). Women had lower CK concentrations at 6 and 24 h but gender differences on IL-6 were not statistically significant.The serum IL-6 response to LDS injury can be captured in a practical manner, despite individual variability in time course. Inclusion of CK measurement may improve sensitivity to the muscle trauma component of an overall injury.

Oncostatin M (OSM) is an IL-6/LIF cytokine family member whose role has been identified in a range of biological activities in vitro, including upregulation of inflammatory gene expression and regulation of connective tissue metabolism. Previously, we identified murine OSM (mOSM) as an inducer of the eosinophil-chemoattractant protein eotaxin-1, both in vitro using fibroblast cell lines and in vivo from mouse lung tissues. Using the NIH 3T3 cell line, we demonstrate the requirement of PI3′K activation for mOSM induction of eotaxin-1 through inhibition of both mOSM-stimulated mRNA and protein expression using the PI3′K antagonist LY294002. By assessment of phosphorylation of the downstream mediator Akt, we show mOSM to be differentially capable of activating PI3′K relative to the related gp130-utilizing cytokine IL-6. Assessment of eotaxin-1 gene expression utilizing PKB/Akt mutant-transfected NIH 3T3 cell lines demonstrated Akt is not involved in upstream regulation of eotaxin-1 through mOSM, indicating an alternate kinase pathway downstream of PI3′K may be involved. We demonstrate that mOSM stimulation of expression of eotaxin-1 is reduced by PD98059, a MAPK kinase inhibitor selective for MEK1. Both LY294002 and PD98059 attenuated mOSM-induced phosphorylation of ERK1/2 MAP kinase and also reduced binding of an AP-1 responsive promoter element, a transcriptional complex known to be MAPK-sensitive. Further, LY294002 pretreatment reduced mOSM-stimulated expression of the downstream AP-1 co-factor JunB, while PD98059 reduced levels of JunB as well as c-Fos. These results provide evidence for a previously unidentified signaling mechanism utilized by mOSM for the induction of eotaxin-1.

Recombinant Adenovirus type 5 constructs containing IL-6 cDNA can be used to infect cells in vitro and obtain a high level of IL-6 expression and secretion into culture media. Furthermore, Ad5-IL-6 viruses can also be used to infect Balb/c mice or Sprague-Dawley rats and obtain a high level of IL-6 expression that is sustained over a period of 3-5 days. Intratracheal infection was accompanied by dramatic increases in virus-encoded IL-6 mRNA levels in rat lung tissue, raised levels of IL-6 detected in bronchoalveolar lavage fluids and in serum, and IL-6-dependent sequelae such as liver acute phase responses. This occurs in a tissue-specific manner, depending on routes of infection by the virus. Rat lungs showed a prominent expansion (10 fold in numbers) of all classes of lymphocytes, including B cells, T helper cells (CD4+) and CTL (CD8+) at day 7 after infection which resolved significantly by day 12. Thus the associated biological effects of viral vector mediated IL-6 over-expression was also transient in nature. Other tissues can be infected with Ad5 and thus can also be induced to express selected genes in a transient fashion. We are currently examining the potential for Ad recombinant cytokine vectors in therapy for cancer and for bone marrow reconstitution after transplantation. Thus the use of recombinant Ad5 vectors may have a broad application in the study of cytokine function and possibly in future therapy as a transient gene transfer approach.

Objective To identify the genes up-regulated by interleukin-1 (IL-1) in combination with oncostatin M (OSM) in chondrocytes that may be involved in mechanisms of cartilage repair and degradation. Methods Gene microarray and real-time polymerase chain reaction (PCR) experiments were performed using RNA from SW1353 chondrocytes and primary human articular chondrocytes. Sections prepared from murine joints, injected with adenovirus vectors overexpressing IL-1 and/or OSM, were analyzed by immunohistochemistry for selected proteins. Results The combination of IL-1 and OSM markedly up-regulated the expression of various genes, including matrix metalloproteinases (MMPs), cytokines, chemokines, extracellular matrix components, and genes involved in signal transduction. Real-time PCR confirmed a synergistic induction of several MMPs, activin A, pentraxin 3 (PTX-3), and IL-8. The in vivo findings further indicated that stimulation with IL-1 plus OSM induced protein expression of activin A, PTX-3, and KC (the murine homolog of IL-8), as compared with the changes induced by individual cytokine treatment and unstimulated controls. Conclusion The results confirm that the potent proinflammatory cytokine combination of IL-1 plus OSM synergistically and coordinately up-regulates many genes and several MMPs. Moreover, chondrocytes exhibit a potential repair response following this procatabolic stimulus such that the repair mechanisms are ultimately overwhelmed by degradative processes in the cartilage. This gene-profiling study provides insight into the complex processes that mediate joint disease in the inflammatory arthritides through the coordinated expression of multiple genes.

Oncostatin M (OSM) regulates expression of various genes in connective tissue (CT) cells, including tissue inhibitor of metalloproteinases-1 (TIMP-1). In mouse fibroblast cell lines MLg, NIH 3T3 and primary mouse lung fibroblasts (MLF), murine OSM (muOSM) stimulated high TIMP-1 mRNA expression in comparison to leukemia inhibitory factor (LIF), epidermal growth factor (EGF), interleukin (IL)-1beta and transforming growth factor (TGF)beta. In cell signaling, muOSM induced strong phosphorylation of extracellular-signal regulated protein kinase (Erk) 1/2, p38 and Akt in addition to phosphorylation of signal transducer and activator of transcription (STAT) 1, STAT3 and STAT5 within 15 min. LIF and TGFbeta had no such effects. EGF stimulated comparable or lower Erk1/2, p38 and Akt phosphorylation while IL-1beta induced p38 phosphorylation in the fibroblast cell lines. The Erk1/2 inhibitor PD98059 and the p38 inhibitor SB203580 inhibited TIMP-1 mRNA response to muOSM, whereas the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 enhanced the TIMP-1 mRNA response in NIH 3T3 and MLg cells. PD98059 and SB203580, but not LY294002, also inhibited fold induction of a chloramphenicol acetyltransferase (CAT) reporter gene driven by a minimal TIMP-1 promoter that contained a proximal activator protein-1 (AP-1) site. Co-transfection with JunB or c-Jun expression vector in NIH 3T3 cells caused marked transactivation of the TIMP-1 promoter/CAT reporter gene. muOSM caused a rapid increase of JunB and c-Jun protein in NIH 3T3 cells. PD98059 partially inhibited the increase of JunB, but not c-Jun, whereas SB203580 did not induce detectable changes in expression of either AP-1 factor in response to muOSM. These results demonstrate that Erk1/2 and p38 contribute to the elevation of muOSM induced TIMP-1 expression, but PI3K does not, and suggest that Erk1/2 does so by enhancing JunB expression.

Objective To determine the effects of the proinflammatory cytokine combination of oncostatin M (OSM) and tumor necrosis factor α (TNFα) on cartilage destruction in both in vitro and in vivo model systems. Methods The release of collagen and proteoglycan was assessed in bovine cartilage explant cultures, while messenger RNA (mRNA) from bovine chondrocytes was analyzed by Northern blotting. Immunohistochemistry was performed on sections prepared from murine joints following injection of adenovirus vectors encoding murine OSM and/or murine TNFα. Results The combination of OSM + TNFα induced significant collagen release from bovine cartilage, accompanied by high levels of active collagenolytic activity. Northern blot analysis indicated that this cytokine combination synergistically induced matrix metalloproteinase 1 (MMP-1), MMP-3, and MMP-13 mRNA. The in vivo data clearly indicated that OSM + TNFα overexpression increased MMP levels and decreased levels of tissue inhibitor of metalloproteinases 1 (TIMP-1). Specifically, OSM + TNFα induced marked synovial hyperplasia, inflammation, and cartilage and bone destruction with a concomitant increase in MMP expression in both cartilage and synovium and decreased TIMP-1 expression in the articular cartilage. These effects were markedly greater than those seen with either cytokine alone. Conclusion This study demonstrates that OSM + TNFα represents a potent proinflammatory cytokine combination that markedly induces MMP production in both cartilage and synovium, thus promoting joint destruction.

Oncostatin M (OSM) is an interleukin (IL)-6 family cytokine that we have previously shown can synergize with a number of proinflammatory cytokines to promote the release of collagen from cartilage in explant culture. However, the effects of this potent cytokine combination in vivo are not known. Using adenoviral gene transfer, we have overexpressed murine IL-1 (AdmIL-1) and murine OSM (AdmOSM) intraarticularly in the knees of C57BL/6 mice. Histological analyses indicated marked synovial hyperplasia and inflammatory cell infiltration for both AdmIL-1 and AdmOSM but not in control joints. This inflammation was even more pronounced for the AdmIL-1+AdmOSM combination with evidence of cartilage and bone destruction. Significant loss of both proteoglycan and collagen was also seen for this combination, and immunohistochemistry revealed an increased expression of matrix metalloproteinases (MMPs) with decreased tissue inhibitor of metalloproteinases (TIMPs) in both articular cartilage and synovium. Similar expression profiles for MMPs/TIMPs were found in IL-1+OSM-stimulated human articular chondrocytes. Taken together, these data confirm that, in vivo, OSM can exacerbate the effects of IL-1 resulting in inflammation and tissue destruction characteristic of that seen in rheumatoid arthritis. We provide further evidence to implicate the up-regulation of MMPs as a key factor in joint pathology.