Mukundan Attur

Superinduction of cyclooxygenase-2 activity in human osteoarthritis-affected cartilage. Influence of nitric oxide.

Cartilage specimens from osteoarthritis (OA)-affected patients spontaneously released PGE2 at 48 h in ex vivo culture at levels at least 50-fold higher than in normal cartilage and 18-fold higher than in normal cartilage + cytokines + endotoxin. The superinduction of PGE2 production coincides with the upregulation of cyclooxygenase-2 (COX-2) in OA-affected cartilage. Production of both nitric oxide (NO) and PGE2 by OA cartilage explants is regulated at the level of transcription and translation. Dexamethasone inhibited only the spontaneously released PGE2 production, and not NO, in OA-affected cartilage. The NO synthase inhibitor HN(G)-monomethyl-L-arginine monoacetate inhibited OA cartilage NO production by > 90%, but augmented significantly (twofold) the spontaneous production of PGE2 in the same explants. Similarly, addition of exogenous NO donors to OA cartilage significantly inhibited PGE2 production. Cytokine + endotoxin stimulation of OA explants increased PGE2 production above the spontaneous release. Addition of L-NMMA further augmented cytokine-induced PGE2 production by at least fourfold. Inhibition of PGE2 by COX-2 inhibitors (dexamethasone or indomethacin) or addition of exogenous PGE2 did not significantly affect the spontaneous NO production. These data indicate that human OA-affected cartilage in ex vivo conditions shows (a) superinduction of PGE2 due to upregulation of COX-2, and (b) spontaneous release of NO that acts as an autacoid to attenuate the production of the COX-2 products such as PGE2. These studies, together with others, also suggest that PGE2 may be differentially regulated in normal and OA-affected chondrocytes.

Periodontal Disease and the Oral Microbiota in New-Onset Rheumatoid Arthritis

OBJECTIVE To profile the abundance and diversity of subgingival oral microbiota in patients with never-treated, new-onset rheumatoid arthritis (RA). METHODS Periodontal disease (PD) status, clinical activity, and sociodemographic factors were determined in patients with new-onset RA, patients with chronic RA, and healthy subjects. Multiplexed-454 pyrosequencing was used to compare the composition of subgingival microbiota and establish correlations between the presence/abundance of bacteria and disease phenotypes. Anti-Porphyromonas gingivalis antibody testing was performed to assess prior exposure to the bacterial pathogen P gingivalis. RESULTS The more advanced forms of periodontitis were already present at disease onset in patients with new-onset RA. The subgingival microbiota observed in patients with new-onset RA was distinct from that found in healthy controls. In most cases, however, these microbial differences could be attributed to the severity of PD and were not inherent to RA. The presence and abundance of P gingivalis were also directly associated with the severity of PD and were not unique to RA. The presence of P gingivalis was not correlated with anti-citrullinated protein antibody (ACPA) titers. Overall exposure to P gingivalis was similar between patients with new-onset RA and controls, observed in 78% of patients and 83% of controls. The presence and abundance of Anaeroglobus geminatus correlated with the presence of ACPAs/rheumatoid factor. Prevotella and Leptotrichia species were the only characteristic taxa observed in patients with new-onset RA irrespective of PD status. CONCLUSION Patients with new-onset RA exhibited a high prevalence of PD at disease onset, despite their young age and paucity of smoking history. The subgingival microbiota profile in patients with new-onset RA was similar to that in patients with chronic RA and healthy subjects whose PD was of comparable severity. Although colonization with P gingivalis correlated with the severity of PD, overall exposure to P gingivalis was similar among the groups. The role of A geminatus and Prevotella/Leptotrichia species in this process merits further study.

Protein Kinase C-θ Mediates Negative Feedback on Regulatory T Cell Function

Yin-Yang T Cell Signaling Immune responses are kept in check by CD4+ regulatory T cells (Treg) that suppress other immune cells, including CD4+ effector T cells (Teff). Treg and Teff cells have many signaling components in common, yet triggering through their T cell receptors (TCRs) leads to very different outcomes. Zanin-Zhorov et al. (p. 372, published online 25 March) compared the recruitment of signaling molecules to the immunological synapse after TCR triggering in Treg and Teff cells. Although Treg cells do form synapses, signaling molecules that promote Teff activation, such as protein kinase C-θ (PKC-θ), were not recruited. Inhibition or depletion of PKC-θ in Treg cells led to suppressive activity against Teff cells, whereas costimulation enhanced PKC-θ recruitment and less suppression. Together, this suggests that PKC-θ is inflammatory in both Treg and Teff cells; however, by excluding it from the synapse, Treg cells are able to maintain suppression in the face of TCR signaling. Suppressive T cells repurpose inflammatory signaling pathways to promote their suppressive functions. T cell receptor (TCR)–dependent regulatory T cell (Treg) activity controls effector T cell (Teff) function and is inhibited by the inflammatory cytokine tumor necrosis factor–α (TNF-α). Protein kinase C-θ (PKC-θ) recruitment to the immunological synapse is required for full Teff activation. In contrast, PKC-θ was sequestered away from the Treg immunological synapse. Furthermore, PKC-θ blockade enhanced Treg function, demonstrating PKC-θ inhibits Treg-mediated suppression. Inhibition of PKC-θ protected Treg from inactivation by TNF-α, restored activity of defective Treg from rheumatoid arthritis patients, and enhanced protection of mice from inflammatory colitis. Treg freed of PKC-θ–mediated inhibition can function in the presence of inflammatory cytokines and thus have therapeutic potential in control of inflammatory diseases.

Nitric Oxide Synthase/COX Cross-Talk: Nitric Oxide Activates COX-1 But Inhibits COX-2-Derived Prostaglandin Production

It is recognized that there is molecular cross-talk between the inflammatory mediators NO and PGs that may regulate tissue homeostasis and contribute to pathophysiological processes. However, the literature is divided with respect to whether NO activates or inhibits PG production. In this study, we sought to determine whether conflicting observations could be accounted for by divergent effects of NO on the two cyclooxygenase (COX) isoforms. Exposure of resting macrophages to NO (30 μM) enhanced PGE2 release by 4.5-fold. This enhancement was inhibited by indomethacin but not by the COX-2 selective inhibitor NS398. To separate the activation of phospholipase A2 and COX, we performed experiments using fibroblasts derived from COX-1-deficient or COX-2-deficient mice. These cells exhibit increased basal PG production, which is due to a constitutively stimulated cytosolic phospholipase A2 and enhanced basal expression of the remaining COX isozyme. The exposure of COX- 2-deficient cells to exogenous NO (10 μM) resulted in a 2.4-fold increase of PGE2 release above controls. Further studies indicated that NO stimulated PGE2 release in COX-2-deficient cells, without altering COX-1 mRNA or protein expression. In contrast, NO inhibited COX-2-derived PGE2 production in both LPS-stimulated macrophages and COX-1 knockout cells. This inhibition was associated with both decreased expression and nitration of COX-2. Thus, these studies demonstrate divergent effects of NO on the COX isoforms. The regulation of PGE production by NO is therefore complex and will depend on the local environment in which these pleiotropic mediators are produced.

Decreased bacterial diversity characterizes the altered gut microbiota in patients with psoriatic arthritis, resembling dysbiosis in inflammatory bowel disease.

To characterize the diversity and taxonomic relative abundance of the gut microbiota in patients with never‐treated, recent‐onset psoriatic arthritis (PsA).

Nitric oxide synthase and cyclooxygenases: distribution, regulation, and intervention in arthritis.

Nitric oxide (NO) and prostaglandin E2 (PGE2) are two pleiotropic inflammatory mediators overproduced in arthritis-affected joints. The inducible isoform of nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) are found both in the synovial tissue and in the cartilage. Their expression is regulated by catabolic cytokines, such as interleukin-1beta and tumor necrosis factor-alpha. These inflammatory mediators play a profound role in the pathogenic processes that arise in the pannus of rheumatoid arthritis and also interfere with cartilage homeostasis in osteoarthritis. Several drugs, including nonsteroidal anti-inflammatory drugs, immunosuppressive agents, and tetracyclines, attenuate the activity of NO and PGE2. These pleiotropic mediators are targets for pharmacologic intervention and gene therapy.

Prostaglandin E2 Exerts Catabolic Effects in Osteoarthritis Cartilage: Evidence for Signaling via the EP4 Receptor

Elevated levels of PGE2 have been reported in synovial fluid and cartilage from patients with osteoarthritis (OA). However, the functions of PGE2 in cartilage metabolism have not previously been studied in detail. To do so, we cultured cartilage explants, obtained from patients undergoing knee replacement surgery for advanced OA, with PGE2 (0.1–10 μM). PGE2 inhibited proteoglycan synthesis in a dose-dependent manner (maximum 25% inhibition (p < 0.01)). PGE2 also induced collagen degradation, in a manner inhibitable by the matrix metalloproteinase (MMP) inhibitor ilomastat. PGE2 inhibited spontaneous MMP-1, but augmented MMP-13 secretion by OA cartilage explant cultures. PCR analysis of OA chondrocytes treated with PGE2 with or without IL-1 revealed that IL-1-induced MMP-13 expression was augmented by PGE2 and significantly inhibited by the cycolooygenase 2 selective inhibitor celecoxib. Conversely, MMP-1 expression was inhibited by PGE2, while celecoxib enhanced both spontaneous and IL-1-induced expression. IL-1 induction of aggrecanase 5 (ADAMTS-5), but not ADAMTS-4, was also enhanced by PGE2 (10 μM) and reversed by celecoxib (2 μM). Quantitative PCR screening of nondiseased and end-stage human knee OA articular cartilage specimens revealed that the PGE2 receptor EP4 was up-regulated in OA cartilage. Moreover, blocking the EP4 receptor (EP4 antagonist, AH23848) mimicked celecoxib by inhibiting MMP-13, ADAMST-5 expression, and proteoglycan degradation. These results suggest that PGE2 inhibits proteoglycan synthesis and stimulates matrix degradation in OA chondrocytes via the EP4 receptor. Targeting EP4, rather than cyclooxygenase 2, could represent a future strategy for OA disease modification.

Current concepts in the pathogenesis of osteoarthritis

Osteoarthritis (OA) is a degenerative joint disease that progressively causes loss of joint function and is the leading source of physical disability and impaired quality of life in industrialized nations. The burden of disease dramatically impacts health care usage and leads to total joint replacement in approximately a half-million Americans alone each year e and such consequences on society worldwide are expected to rise in coming decades with the continued expanding and aging population. There are no current interventions proven to restore cartilage or curtail the disease processes. Thus, OA often ultimately results in joint destruction, chronic pain, disability, depression and social isolation. Multiple etiologic risk factors and pathophysiologic processes all contribute to the progressive nature of the disease e and serve as targets of behavioral and pharmacologic interventions. Risk factors, such as age, gender, trauma, overuse, genetics and obesity each make contributions to initiate the process of injury in different components of the joint; then the effector biochemical processes involving the cartilage, bone, and synovium eventually intertwine and collectively damage all three components as well (Fig. 1). These effects on the tissues of all three joint compartments manifest as articular cartilage breakdown, osteophyte formation, subchondral sclerosis, bone marrow lesions and alterations of the synovium on both morphologic and biochemical levels often causing episodic synovitis. Thus, the molecular and cytokine-based events that drive joint damage in inflammatory arthritides have gradually emerged as pathogenic paradigms in OA, and will be highly relevant to the development of future OA therapeutics. With increasing appreciation of the contribution of all three joint compartments to disease progression, current research in OA pathogenesis, biomarkers and treatment has broadened immensely in recent years. In this review, we will focus on emerging pathogenic concepts that will hopefully help advance the search for effective disease-modifying osteoarthritis drugs (DMOADs).

Prospects for disease modification in osteoarthritis

Osteoarthritis (OA) can be a progressive, disabling disease, leading to diminished quality of life, and, for over 500,000 individuals annually in the US, total joint replacement. The etiology of OA will vary among individuals, with potential roles for systemic factors (such as genetics and obesity) as well as for local biomechanical factors (such as muscle weakness, joint laxity and traumatic injury). Joint deterioration occurs over extended periods of time, and the diverse molecular mechanisms that mediate pathogenic events of early, mid and late disease are not yet fully understood. The success of biologic therapies in the treatment of rheumatoid arthritis has demonstrated that the blockade of a single dominant cytokine or regulatory molecule can prevent cartilage destruction in a complex disease, and has raised expectations that mechanism-based treatments could also be developed for patients with OA. In this review, we will address the biological mechanisms that mediate structural damage in OA and examine current targets that are candidates for disease modification. The challenges to drug development and the obstacles to disease modification strategies will also be addressed.

The role of microRNA in rheumatoid arthritis and other autoimmune diseases.

MicroRNAs (miRNAs) represent a class of non-coding RNA molecules playing pivotal roles in cellular and developmental processes. miRNAs modulate the expression of multiple target genes at the post-transcriptional level and are predicted to affect up to one-third of all human protein-encoding genes. Recently, miRNA involvement in the adaptive and innate immune systems has been recognized. Rheumatoid arthritis serves an example of a chronic inflammatory disorder in which miRNAs modulate the inflammatory process in the joints, with the potential to serve as biomarkers for both the inflammatory process and the potential for therapeutic response. This review discusses the investigations that led to miRNA discovery, miRNA biogenesis and mode of action, and the diverse roles of miRNAs in modulating the immune and inflammatory responses. We conclude with a discussion of the implications of miRNA biology in rheumatoid arthritis and other autoimmune disorders.