Carl D. Richards

IL-4 gene therapy for collagen arthritis suppresses synovial IL-17 and osteoprotegerin ligand and prevents bone erosion

Bone destruction is the most difficult target in the treatment of rheumatoid arthritis (RA). Here, we report that local overexpression of IL-4, introduced by a recombinant human type 5 adenovirus vector (Ad5E1mIL-4) prevents joint damage and bone erosion in the knees of mice with collagen arthritis (CIA). No difference was noted in the course of CIA in the injected knee joints between Ad5E1mIL-4 and the control vector, but radiographic analysis revealed impressive reduction of joint erosion and more compact bone structure in the Ad5E1mIL-4 group. Although severe inflammation persisted in treated mice, Ad5E1mIL-4 prevented bone erosion and diminished tartrate-resistant acid phosphatase (TRAP) activity, indicating that local IL-4 inhibits the formation of osteoclast-like cells. Messenger RNA levels of IL-17, IL-12, and cathepsin K in the synovial tissue were suppressed, as were IL-6 and IL-12 protein production. Osteoprotegerin ligand (OPGL) expression was markedly suppressed by local IL-4, but no loss of OPG expression was noted with Ad5E1mIL-4 treatment. Finally, in in vitro studies, bone samples of patients with arthritis revealed consistent suppression by IL-4 of type I collagen breakdown. IL-4 also enhanced synthesis of type I procollagen, suggesting that it promoted tissue repair. These findings may have significant implications for the prevention of bone erosion in arthritis.

Induction of Osteogenesis in Mesenchymal Stem Cells by Activated Monocytes/Macrophages Depends on Oncostatin M Signaling†‡§

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. STEM CELLS 2012; 30:762–772

Murine Oncostatin M Stimulates Mouse Synovial Fibroblasts in Vitro and Induces Inflammation and Destruction in Mouse Joints in Vivo

Oncostatin M (OSM) is a multifunctional cytokine, a member of the interleukin-6/leukemia inhibitory factor (IL-6/LIF) family, that can regulate a number of connective-tissue cell types in vitro including cartilage and synovial tissue-derived fibroblasts, however its role in joint inflammation in vivo is not clear. We have analyzed murine OSM (muOSM) activity in vitro and in vivo in mouse joint tissue, to determine the potential role of this cytokine in local joint inflammation and pathology. The effects of muOSM and other IL-6/LIF cytokines on mouse synovial fibroblast cultures were assessed in vitro and showed induction of monocyte chemotactic protein-1, interleukin-6, and tissue inhibitor metalloproteinase-1, as well as enhancement of colony growth in soft agarose culture. Other IL-6/LIF cytokines including IL-6, LIF, or cardiotrophin-1, did not have such effects when tested at relatively high concentrations (20 ng/ml). To assess effects of muOSM in articular joints in vivo, we used recombinant adenovirus expressing muOSM cDNA (AdmuOSM) and injected purified recombinant virus (10(6) to 10(8) pfu) intra-articularly into the knees of various mouse strains. Histological analysis revealed dramatic alterations in the synovium but not in synovium of knees treated with the control virus Ad-dl70 or knees treated with Adm-IL-6 encoding biologically active murine IL-6. AdmuOSM effects were characterized by increases in the synovial cell proliferation, infiltration of mononuclear cells, and increases in extracellular matrix deposition that were evident at day 4, but much more marked at days 7, 14, and 21 after administration. The synovium took on characteristics similar to pannus and appeared to contact and invade cartilage. Collectively, these results provide good evidence that OSM regulates synovial fibroblast function differently than other IL-6-type cytokines, and can induce a proliferative invasive phenotype of synovium in vivo in mice on overexpression. We suggest that OSM may contribute to pathology in arthritis.

Intra-articular IL-10 gene transfer regulates the expression of collagen-induced arthritis (CIA) in the knee and ipsilateral paw

We studied the effects of local IL‐10 application, introduced by a recombinant human type 5 adenovirus vector, in the mouse knee joint during the early phase of CIA. One intra‐articular injection with the IL‐10‐expressing virus (Ad5E1mIL‐10) caused substantial over‐expression of IL‐10 in the mouse knee joint, using virus dosages which did not induce distracting inflammation. High expression of IL‐10 was noted for a few days, being maximal at day 1. One intra‐articular injection of Ad5E1mIL‐10 in the knee joints of collagen type II (CII)‐immunized mice, before onset of CIA was noted, reduced the incidence of collagen arthritis in that knee. Of high interest, the protective effect of local IL‐10 expression by Ad5E1mIL‐10 was not restricted to the knee joint alone. The arthritis incidence in the ipsilateral paw was highly suppressed. In contrast, local IL‐10 over‐expression was not effective when treatment was started after onset of CIA. Further analysis in the acute streptococcal cell wall‐induced arthritis model revealed that local IL‐10 over‐expression markedly suppressed the production of tumour necrosis factor‐alpha (TNF‐α) and IL‐1α, but had no significant effect on IL‐1β and IL‐12 production in the inflamed synovium. These data indicate that local over‐expression of IL‐10 in the knee joint of mice regulates the expression of collagen arthritis, probably through down‐regulation of TNF‐α.

Reduced preference for sucrose in autoimmune mice: a possible role of interleukin-6.

In a continuous one-bottle sucrose intake test, 4-month-old autoimmune MRL-Ipr mice show a shift to the right along the X-axis of the concentration-intake function, compared to congenic MRL +/+ controls. Using a brief (60-min) and a continuous (48-h) two-bottle test, the present report examines potential factors that could account for the reduced responsiveness to a palatable stimulus. Study 1 examines whether preference for sucrose is associated with age, changes in food/water intake, or impaired renal function. Reduced preference for sucrose was observed in 5-6-week-old MRL-Ipr males, although food/water intake or blood creatinine levels did not differ from control values. Immunosuppressive treatment abolished this deficit, suggesting a role of immune factor(s). Study 2 tests the hypothesis that chronic upregulation of the neuroactive cytokine interleukin-6 (IL-6), reported to occur from 3 weeks of age in young MRL-Ipr mice, reduces preference for sucrose. Sustained administration of IL-6 was produced by infecting healthy MRL +/+, C3H.SW and Balb/C mice with adenovirus vector carrying cDNA for murine IL-6. This resulted in high serum IL-6 levels over 5 days, a rapid decline in preference for sucrose and low blood glucose levels. The results from Study 1 indicate that impaired sensitivity to sucrose in MRL-Ipr mice can be detected before autoimmune disease is florid in MRL-Ipr mice. The results from Study 2 are consistent with altered motivation/emotional states after infection, and point to sustained IL-6 production as an early mechanism in behavioral alterations during chronic autoimmune/inflammatory conditions.

Adenoviral gene transfer of interleukin-1 in combination with oncostatin M induces significant joint damage in a murine model.

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.

Interleukin-15 contributes to the regulation of murine adipose tissue and human adipocytes.

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‐α (TNF‐α) 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.

Genetic partitioning of interleukin-6 signalling in mice dissociates Stat3 from Smad3-mediated lung fibrosis

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 gp130757F mice. The extent of fibrosis was attenuated in B lymphocyte‐deficient gp130757F;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.

Anchorage-independent colony growth of pulmonary fibroblasts derived from fibrotic human lung tissue.

Fibroblast heterogeneity is known to exist in chronically inflamed tissue such as pulmonary fibrosis (IPF) and scleroderma. We have previously shown differences in proliferation rates in primary lines and cloned lines of fibroblasts derived from IPF tissue compared with normal lung. In this study, we report that cell lines derived from fibrotic tissue demonstrate anchorage-independent growth in soft agarose culture whereas normal lung fibroblast lines do not. We also show that fibroblast lines derived from neonatal lung tissue form colonies at about the same frequency as the fibrotic cells. Colonies from both fibrotic and neonatal lines were shown to be positive for vimentin, laminin, fibronectin, fibronectin receptor, beta-actin, and tropomyosin by immunohistochemistry but were negative for desmin, keratin, Factor VIII, alpha-smooth muscle cell actin, and tenascin. Treatment with cytokines TGF-beta and PDGF or with corticosteroid modified the colony-forming capacity of fibrotic and neonatal cell lines, however, none of these treatments induced normal lung cell lines to form colonies. The presence of cells in adult fibrotic tissue with growth characteristics similar to those exhibited by neonatal cells is further evidence of fibroblast heterogeneity and suggests newly differentiated fibroblasts may be prevalent in fibrotic tissue and contribute directly to the matrix disorder seen in this disease.

Oncostatin M Regulates Eotaxin Expression in Fibroblasts and Eosinophilic Inflammation in C57BL/6 Mice

Oncostatin M (OSM) is a member of the IL-6/LIF (or gp130) cytokine family, and its potential role in inflammation is supported by a number of activities identified in vitro. In this study, we investigate the action of murine OSM on expression of the CC chemokine eotaxin by fibroblasts in vitro and on mouse lung tissue in vivo. Recombinant murine OSM stimulated eotaxin protein production and mRNA levels in the NIH 3T3 fibroblast cell line. IL-6 could regulate a small induction of eotaxin in NIH 3T3 cells, but other IL-6/LIF cytokines (LIF, cardiotrophin-1 (CT-1)) had no effect. Cell signaling studies showed that murine OSM, LIF, IL-6, and CT-1 stimulated the tyrosine phosphorylation of STAT-3, suggesting STAT-3 activation is not sufficient for eotaxin induction in NIH 3T3 cells. OSM induced ERK-1,2 and p38 mitogen-activated protein kinase phosphorylation in NIH 3T3 cells, and inhibitors of ERK (PD98059) or p38 (SB203580) could partially reduce OSM-induced eotaxin production, suggesting partial dependence on mitogen-activated protein kinase signaling. OSM (but not LIF, IL-6, or CT-1) also induced eotaxin release by mouse lung fibroblast cultures derived from C57BL/6 mice. Overexpression of murine OSM in lungs of C57BL/6 mice using an adenovirus vector encoding murine OSM resulted in a vigorous inflammatory response by day 7 after intranasal administration, including marked extracellular matrix accumulation and eosinophil infiltration. Elevated levels of eotaxin mRNA in whole lung were detected at days 4 and 5. These data strongly support a role of OSM in lung inflammatory responses that involve eosinophil infiltration.