Objective Osteoarthritis (OA) is a degenerative disease resulting in severe joint cartilage destruction and disability. While the mechanisms underlying the development and progression of OA are poorly understood, gene mutations have been identified within cartilage-related signaling molecules implicating impaired cell signaling in OA and joint disease. The Notch pathway has recently been identified as a crucial regulator of growth plate cartilage development and components are expressed in joint tissues. Therefore, we set out to investigate a novel role for Notch signaling in joint cartilage development, maintenance, and the pathogenesis of joint disease. Methods We performed the first mouse genetic studies in which the core Notch signaling component, RBPjκ, was tissue-specifically deleted within joints. The Prx1Cre transgene removed Rbpjκ floxed alleles in mesenchymal joint precursor cells, while the Col2CreERT2 transgene specifically deleted Rbpjκ in postnatal chondrocytes. Articular chondrocyte cultures were also utilized to examine Notch regulation of gene expression. Results Loss of Notch signaling in mesenchymal joint precursor cells does not affect embryonic joint development, but rather results in an early, progressive OA-like pathology. Additionally, partial loss of Notch signaling in postnatal cartilage results in progressive joint cartilage degeneration and an age-related OA-like pathology. Inhibition of Notch signaling alters expression of the ECM-related factors: COL2A1, PRG4, COL10A1, MMP13, and ADAMTSs. Conclusions These data have identified the RBPjκ-dependent Notch pathway as: 1) a novel pathway involved in joint maintenance and articular cartilage homeostasis, 2) a critical regulator of articular cartilage ECM-related molecules, and 3) a potentially important therapeutic target for OA-like joint disease.

During Theiler's murine encephalomyelitis virus (TMEV) infection of macrophages, it is thought that high interleukin-6 (IL-6) levels contribute to the demyelinating disease found in chronically infected SJL/J mice but absent in B10.S mice capable of clearing the infection. Therefore, IL-6 expression was measured in TMEV-susceptible SJL/J and TMEV-resistant B10.S macrophages during their infection with TMEV DA strain or responses to lipopolysaccharide (LPS) or poly(I · C). Unexpectedly, IL-6 production was greater in B10.S macrophages than SJL/J macrophages during the first 24 h after stimulation with TMEV, LPS, or poly(I · C). Further experiments showed that in B10.S, SJL/J, and RAW264.7 macrophage cells, IL-6 expression was dependent on extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) and enhanced by exogenous IL-12. In SJL/J and RAW264.7 macrophages, exogenous IL-6 resulted in decreased TMEV replication, earlier activation of STAT1 and STAT3, production of nitric oxide, and earlier upregulation of several antiviral genes downstream of STAT1. However, neither inhibition of IL-6-induced nitric oxide nor knockdown of STAT1 diminished the early antiviral effect of exogenous IL-6. In addition, neutralization of endogenous IL-6 from SJL/J macrophages with Fab antibodies did not exacerbate early TMEV infection. Therefore, endogenous IL-6 expression after TMEV infection is dependent on ERK MAPK, enhanced by IL-12, but too slow to decrease viral replication during early infection. In contrast, exogenous IL-6 enhances macrophage control of TMEV infection through preemptive antiviral nitric oxide production and antiviral STAT1 activation. These results indicate that immediate-early production of IL-6 could protect macrophages from TMEV infection.

Interferon Response Factor 3 (IRF3) induces several NK-cell activating factors, is activated by poly-I:C, an experimental cancer therapeutic, but is suppressed during many viral infections. IRF3 Knockout (KO) mice exhibited enhanced B16 melanoma growth, impaired intratumoral NK cell infiltration, but not an impaired poly-I:C therapeutic effect due to direct suppression of B16 growth. IRF3 was responsible for poly-I:C decrease in TIM-3 expression by intratumoral dendritic cells, induction of NK-cell Granzyme B and IFN-γ, and induction of macrophage IL-12, IL-15, IL-6, and IRF3–dependent NK-activating molecule (INAM). Thus, IRF3 is a key factor controlling melanoma growth through NK-cell activities, especially during poly-I:C therapy.

Understanding Nitric Oxide (NO) in innate anti-viral immunity and immune-mediated pathology is hampered by incomplete details of its transcriptional and signaling factors. We found in macrophages that IRF3, ERK MAP-kinases, and PKR are essential to NO production in response to RNA-virus mimic, poly I:C, a TLR3 agonist. ERK's role in NO induction may be through phosphorylation of serine-171 of IRF3 and expression of NO-inducing cytokines, IL-6 and IFN-β. However, these cytokines induced less NO in IRF3 knockout or knockdown macrophages. These findings show that ERK and IRF3 coordinate induction of NO by macrophages in response to stimulation of TLR3.

The polymorphic commensal fungus Candida albicans causes life-threatening disease via bloodstream and intra-abdominal infections in immunocompromised and transplant patients. Although host immune evasion is a common strategy used by successful human fungal pathogens, C. albicans provokes recognition by host immune cells less capable of destroying it. To accomplish this, C. albicans white cells secrete a low-molecular-weight chemoattractive stimulant(s) of macrophages, a phagocyte that they are able to survive within and eventually escape from. C. albicans opaque cells do not secrete this chemoattractive stimulant(s). We report here a physiological mechanism that contributes to the differences in the interaction of C. albicans white and opaque cells with macrophages. E,E-Farnesol, which is secreted by white cells only, is a potent stimulator of macrophage chemokinesis, whose activity is enhanced by yeast cell wall components and aromatic alcohols. E,E-farnesol results in up to an 8.5-fold increase in macrophage migration in vitro and promotes a 3-fold increase in the peritoneal infiltration of macrophages in vivo. Therefore, modulation of farnesol secretion to stimulate host immune recognition by macrophages may help explain why this commensal is such a successful pathogen. Candida albicans is a serious fungal pathogen for humans (1). C. albicans is a dimorphic fungus and it has long been recognized that the ability to interconvert between the yeast and filamentous forms is essential for its pathogenicity. Thus, when we discovered that E,E-farnesol (referred to here as farnesol) is a quorum sensing molecule (QSM) for C. albicans which acts by blocking the conversion of yeasts to mycelia (2), we thought that farnesol would be an attractive lead compound in the design of novel antifungal drugs. However, this goal was not realized when we showed that farnesol itself acted as a virulence factor for C. albicans in a mouse intravenous infection model (3). These observations presented a dilemma: how can farnesol act as a virulence factor when part of its mode of action is to block the yeast-mycelium morphogenesis, which is necessary for its pathogenicity? We resolve this conundrum here by showing that farnesol is also a potent stimulator of macrophage migration both in vitro and in vivo.C. albicans is one of the best studied examples of an opportunistic fungal pathogen. It is a normal member of the human microbiota where it is found as a commensal primarily in the gastrointestinal and genitourinary tracts and on the skin of healthy individuals without causing significant disease. In healthy individuals, C. albicans commensals are controlled but tolerated while C. albicans pathogens are eliminated (reviewed in reference 4). The presence of innate immune phagocytes, which include both macrophages and neutrophils, is critical for early detection and elimination of C. albicans that have made the transition from commensal to an opportunistic pathogen (5-9; reviewed in reference 10). Thus, when normal immune responses are com...