Rajesh Rajaiah

Heat-shock proteins can promote as well as regulate autoimmunity

Heat-shock proteins (Hsps) are among the most highly conserved and immunogenic proteins shared by microbial agents and mammals. Under physiological conditions, the ubiquitously distributed Hsps maintain the integrity and function of other cellular proteins when cells are exposed to stressful stimuli. However, owing to their conserved nature and stress inducibility, Hsps may become targets of immune response. The T cells and/or antibodies induced by a microbial Hsp may crossreact with the corresponding mammalian Hsp (molecular mimicry) and trigger an autoimmune response, which if unchecked can lead to immune pathology and clinical manifestations. Furthermore, enhanced expression of Hsp under stress can unveil previously hidden antigenic determinants that can initiate and perpetuate autoimmune reactivity. Also, the innate immune mechanisms activated by an Hsp can reinforce and even direct the type of adaptive immune response to that protein. Hsps have been implicated in the induction and propagation of autoimmunity in several diseases, including rheumatoid arthritis, atherosclerosis and type 1 diabetes. However, Hsps possess immunoregulatory attributes as well and therefore, are being exploited for immunomodulation of various immune-mediated disorders.

Green Tea Protects Rats against Autoimmune Arthritis by Modulating Disease-Related Immune Events

Green tea, a product of the dried leaves of Camellia sinensis, is the most widely consumed beverage in the world. The polyphenolic compounds from green tea (PGT) possess antiinflammatory properties. We investigated whether PGT can afford protection against autoimmune arthritis and also examined the immunological basis of this effect using the rat adjuvant arthritis (AA) model of human rheumatoid arthritis (RA). AA can be induced in Lewis rats (RT.1(l)) by immunization with heat-killed Mycobacterium tuberculosis H37Ra (Mtb), and arthritic rats raise a T cell response to the mycobacterial heat-shock protein 65 (Bhsp65). Rats consumed green tea (2-12 g/L) in drinking water for 1-3 wk and then were injected with Mtb to induce disease. Thereafter, they were observed regularly and graded for signs of arthritis. Subgroups of these rats were killed at defined time points and their draining lymph node cells were harvested and tested for T cell proliferative and cytokine responses. Furthermore, the sera collected from these rats were tested for anti-Bhsp65 antibodies. Feeding 8 g/L PGT to Lewis rats for 9 d significantly reduced the severity of arthritis compared with the water-fed controls. Interestingly, PGT-fed rats had a lower concentration of the proinflammatory cytokine interleukin (IL)-17 but a greater concentration of the immunoregulatory cytokine IL-10 than controls. PGT feeding also suppressed the anti-Bhsp65 antibody response. Thus, green tea induced changes in arthritis-related immune responses. We suggest further systematic exploration of dietary supplementation with PGT as an adjunct nutritional strategy for the management of RA.

Celastrus-derived Celastrol suppresses autoimmune arthritis by modulating antigen-induced cellular and humoral effector responses

Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovial inflammation and articular damage. Proinflammatory cytokines, antibodies, and matrix-degrading enzymes orchestrate the pathogenic events in autoimmune arthritis. Accordingly, these mediators of inflammation are the targets of several anti-arthritic drugs. However, the prolonged use of such drugs is associated with severe adverse reactions. This limitation has necessitated the search for less toxic natural plant products that possess anti-arthritic activity. Furthermore, it is imperative that the mechanism of action of such products be explored before they can be recommended for further preclinical testing. Using the rat adjuvant-induced arthritis model of human RA, we demonstrate that celastrol derived from Celastrus has potent anti-arthritic activity. This suppression of arthritis is mediated via modulation of the key proinflammatory cytokines (IL-17, IL-6, and IFN-γ) in response to the disease-related antigens, of the IL-6/IL-17-related transcription factor STAT3, of antibodies directed against cyclic citrullinated peptides and Bhsp65, and of the activity of matrix metalloproteinase-9 and phospho-ERK. Most of the clinical and mechanistic attributes of celastrol are similar to those of Celastrus extract. Several studies have addressed the antitumor activity of celastrol. Our study highlights the anti-arthritic activity of Celastrus-derived celastrol and the underlying mechanisms. These results provide a strong rationale for further testing and validation of the use of celastrol and the natural plant extract from Celastrus as an adjunct (with conventional drugs) or alternative modality for the treatment of RA.

Interleukin-27 and Interferon-γ Are Involved in Regulation of Autoimmune Arthritis

Inflammation underlying immune pathology and tissue damage involves an intricate interplay between multiple immunological and biochemical mediators. Cytokines represent the key immune mediators that trigger a cascade of reactions that drive processes such as angiogenesis and proteolytic damage to tissues. IL-17 has now been shown to be a pivotal cytokine in many autoimmune diseases, supplanting the traditional Th1-Th2 paradigm. Also, the dual role of proinflammatory IFN-γ has unraveled new complexities in the cytokine biology of such disorders. A major hurdle in fully understanding the effector pathways in these disorders is the lack of information regarding the temporal kinetics of the cytokines during the course of the disease, as well as the interplay among the key cytokines. Using an experimental model of arthritic inflammation, we demonstrate that the temporal expression of cytokines during the incubation phase is a critical determinant of disease susceptibility. The susceptible rats raised a vigorous IL-17 response early, followed by IFN-γ and IL-27 response in that sequence, whereas the resistant rats displayed an early and concurrent response to these three cytokines. Accordingly, treatment with exogenous IFN-γ/IL-27 successfully controlled arthritic inflammation and inhibited the defined mediators of inflammation, angiogenesis, cell survival, apoptosis, and tissue damage. Furthermore, IFN-γ enhanced IL-27 secretion, revealing a cooperative interplay between the two cytokines. Our results offer a novel immunobiochemical perspective on the pathogenesis of autoimmune arthritis and its therapeutic control.

Nicotine-induced differential modulation of autoimmune arthritis in the Lewis rat involves changes in interleukin-17 and anti-cyclic citrullinated peptide antibodies.

OBJECTIVE Rheumatoid arthritis (RA) is a debilitating autoimmune disease, and smoking is an important environmental factor in a subset of RA patients. A role of the cholinergic antiinflammatory pathway in autoimmune inflammation is increasingly being realized. Nicotine is a major component of cigarette smoke, and it stimulates the α7 nicotinic acetylcholine receptors. Therefore, defining the mechanisms underlying the immunomodulatory effects of nicotine on arthritis is of high relevance. The purpose of this study was to address this issue using the rat adjuvant-induced arthritis (AIA) model of human RA. METHODS Lewis rats were immunized subcutaneously with heat-killed Mycobacterium tuberculosis H37Ra for disease induction. Rats were treated with nicotine intraperitoneally either before (pretreatment) or after (posttreatment) the onset of AIA. Control rats received the vehicle (buffer) in place of nicotine. The severity of arthritis was assessed and graded. The draining lymph node cells were tested for T cell proliferative and cytokine responses against the disease-related antigen mycobacterial heat-shock protein 65. The sera were tested for anti-cyclic citrullinated peptide (anti-CCP) antibodies and anti-mycobacterial Hsp65 antibodies. RESULTS Nicotine pretreatment aggravated the arthritis, whereas nicotine posttreatment suppressed the disease. This altered severity of AIA directly correlated with the levels of the anti-CCP antibodies, of the Th1/Th17 cytokines, and of the corresponding dendritic cell-derived cytokines. The majority of these effects on cellular responses could be replicated in vitro. CONCLUSION Nicotine-induced modulation of AIA involves specific alterations in the disease-related cellular and humoral immune responses in AIA. These results are of significance in advancing our understanding of the pathogenesis of RA.

Immunomodulation of Autoimmune Arthritis by Herbal CAM.

Rheumatoid arthritis (RA) is a debilitating autoimmune disease of global prevalence. The disease is characterized by synovial inflammation leading to cartilage and bone damage. Most of the conventional drugs used for the treatment of RA have severe adverse reactions and are quite expensive. Over the years, increasing proportion of patients with RA and other immune disorders are resorting to complementary and alternative medicine (CAM) for their health needs. Natural plant products comprise one of the most popular CAM for inflammatory and immune disorders. These herbal CAM belong to diverse traditional systems of medicine, including traditional Chinese medicine, Kampo, and Ayurvedic medicine. In this paper, we have outlined the major immunological pathways involved in the induction and regulation of autoimmune arthritis and described various herbal CAM that can effectively modulate these immune pathways. Most of the information about the mechanisms of action of herbal products in the experimental models of RA is relevant to arthritis patients as well. The study of immunological pathways coupled with the emerging application of genomics and proteomics in CAM research is likely to provide novel insights into the mechanisms of action of different CAM modalities.

Dissociation of Endotoxin Tolerance and Differentiation of Alternatively Activated Macrophages

Endotoxin tolerance is a complex phenomenon characterized primarily by decreased production of proinflammatory cytokines, chemokines, and other inflammatory mediators, whereas the expression of other genes are induced or unchanged. Endotoxin tolerance is induced by prior exposure of murine macrophages/human monocytes, experimental animals, or people to TLR ligands. Although recent studies reported a possible relationship between endotoxin tolerance and differentiation of alternatively activated macrophages (AA-MΦs or M2), we show in this study that LPS pretreatment of IL-4Rα−/− and STAT6−/− macrophages, which fail to develop into AA-MΦs, resulted in tolerance of proinflammatory cytokines, as well as molecules and chemokines previously associated with AA-MΦs (e.g., arginase-1, mannose receptor, CCL2, CCL17, and CCL22). In contrast to LPS, wild-type (WT) MΦs pretreated with IL-4, the prototype inducer of AA-MΦs, did not induce endotoxin tolerance with respect to proinflammatory cytokines, AA-MΦ–associated chemokines, negative regulators, NF-κB binding and subunit composition, and MAPKs; conversely, IL-13−/− macrophages were tolerized equivalently to WT MΦs by LPS pretreatment. Further, IL-4Rα deficiency did not affect the reversal of endotoxin tolerance exerted by the histone deacetylase inhibitor trichostatin A. Like WT mice, 100% of LPS-tolerized IL-4Rα–deficient mice survived LPS + d-galactosamine–induced lethal toxicity and exhibited decreased serum levels of proinflammatory cytokines and AA-MΦ–associated chemokines induced by LPS challenge compared with nontolerized mice. These data indicate that the signaling pathways leading to endotoxin tolerance and differentiation of AA-MΦs are dissociable.

CD14 dependence of TLR4 endocytosis and TRIF signaling displays ligand specificity and is dissociable in endotoxin tolerance.

Significance MyD88-dependent signaling is cluster of differentiation 14 (CD14)-dependent only at low LPS concentrations, whereas activation of the TIR-domain–containing adapter-inducing IFN-β (TRIF) pathway requires CD14 at all LPS concentrations, leading to internalization of the Toll-like receptor 4 (TLR4) complex into endosomes whereupon TRIF is recruited. Using alternative TLR4 agonists, or macrophages rendered tolerant to LPS, we dissociate TLR4 complex internalization from CD14 and TRIF-dependent signaling. In response to LPS, CD14 contributes to the formation of a TLR4/MD2 complex dimer that, in turn, promotes endocytosis and IRF3 activation. Dimerization of Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD2) heterodimers is critical for both MyD88- and TIR-domain–containing adapter-inducing IFN-β (TRIF)-mediated signaling pathways. Recently, Zanoni et al. [(2011) Cell 147(4):868–880] reported that cluster of differentiation 14 (CD14) is required for LPS-/Escherichia coli- induced TLR4 internalization into endosomes and activation of TRIF-mediated signaling in macrophages. We confirmed their findings with LPS but report here that CD14 is not required for receptor endocytosis and downstream signaling mediated by TLR4/MD2 agonistic antibody (UT12) and synthetic small-molecule TLR4 ligands (1Z105) in murine macrophages. CD14 deficiency completely ablated the LPS-induced TBK1/IRF3 signaling axis that mediates production of IFN-β in murine macrophages without affecting MyD88-mediated signaling, including NF-κB, MAPK activation, and TNF-α and IL-6 production. However, neither the MyD88- nor TRIF-signaling pathways and their associated cytokine profiles were altered in the absence of CD14 in UT12- or 1Z105-treated murine macrophages. Eritoran (E5564), a lipid A antagonist that binds the MD2 “pocket,” completely blocked LPS- and 1Z105-driven, but not UT12-induced, TLR4 dimerization and endocytosis. Furthermore, TLR4 endocytosis is induced in macrophages tolerized by exposure to either LPS or UT12 and is independent of CD14. These data indicate that TLR4 receptor endocytosis and the TRIF-signaling pathway are dissociable and that TLR4 internalization in macrophages can be induced by UT12, 1Z105, and during endotoxin tolerance in the absence of CD14.

Exogenous tumour necrosis factor α induces suppression of autoimmune arthritis

IntroductionOur previous studies showed that arthritic Lewis (LEW) rats produced the highest levels of tumour necrosis factor (TNF)α in the recovery phase of adjuvant arthritis (AA), suggesting a correlation between high TNFα levels and reduced severity of arthritis. To further explore this correlation, we compared the TNFα secretion profile of the AA-resistant Wistar Kyoto (WKY) rats with that of LEW rats, determined the effect of exogenous TNFα on the course of AA in LEW rats, and examined various mechanisms involved in TNFα-induced disease modulation.MethodsA cohort each of LEW and WKY rats was immunised subcutaneously with heat-killed Mycobacterium tuberculosis H37Ra (Mtb). At different time points thereafter, subgroups of rats were killed and their draining lymph node cells were tested for cytokine production. Another group of LEW rats was injected with TNFα intraperitoneally daily for a total of 10 injections, 3 before and 6 after Mtb challenge, and then observed for signs of AA. In parallel, TNFα-treated rats were examined for changes in other cytokines, in CD4+CD25+ T cell frequency, and in indoleamine 2,3-dioxygenase (IDO) mRNA expression levels.ResultsLEW rats displayed a TNFα secretion profile that was opposite to that of the WKY rats. Furthermore, TNFα treatment significantly downmodulated the severity of AA in LEW rats, and decreased the interferon (IFN)-γ secretion in response to the pathogenic determinant of the disease-related antigen. No significant alterations were observed in other parameters tested.ConclusionThe role of endogenous TNFα in the induction and propagation of arthritis is well established. However, exogenous TNFα can downmodulate the course of AA, displaying an immunoregulatory functional attribute of this cytokine.

Suppression of ongoing experimental arthritis by a Chinese herbal formula (Huo-Luo-Xiao-Ling Dan) involves changes in antigen-induced immunological and biochemical mediators of inflammation

Rheumatoid arthritis (RA) is one of the major autoimmune diseases of global prevalence. The use of the anti-inflammatory drugs for the treatment of RA is associated with severe adverse reactions and toxicity. This limitation has necessitated the search for novel therapeutic products. We report here a traditional Chinese medicine-based herbal formula, Huo luo xiao ling dan (HLXL), which has potent antiarthritic activity as validated in the rat adjuvant-induced arthritis (AA) model. HLXL (2.3 g/Kg) was fed to Lewis (RT.11) rats daily by gavage beginning at the onset of arthritis and then continued through the observation period. HLXL inhibited the severity of ongoing AA. This suppression of arthritis was associated with significant alterations in the T cell proliferative and cytokine responses as well as the antibody response against the disease-related antigen, mycobacterial heat-shock protein 65 (Bhsp65). There was a reduction in the level of the proinflammatory cytokines IL-17 and IL-1β but enhancement of the anti-inflammatory cytokine IL-10 level. In addition, there was inhibition of both the anti-Bhsp65 antibody response and the serum level of nitric oxide. Thus, HLXL is a promising CAM modality for further testing in RA patients.