The Utility of Peptide Ligand-Functionalized Liposomes for Subcutaneous Drug Delivery for Arthritis Therapy.

Liposomes and other types of nanoparticles are increasingly being explored for drug delivery in a variety of diseases. There is an impetus in the field to exploit different types of ligands to functionalize nanoparticles to guide them to the diseased site. Most of this work has been conducted in the cancer field, with relatively much less information from autoimmune diseases, such as rheumatoid arthritis (RA). Furthermore, in RA, many drugs are self-administered by patients subcutaneously (SC). In this context, we have examined the attributes of liposomes functionalized with a novel joint-homing peptide (denoted ART-1) for arthritis therapy using the SC route. This peptide was previously identified following phage peptide library screening in the rat adjuvant arthritis (AA) model. Our results show a distinct effect of this peptide ligand on increasing the zeta potential of liposomes. Furthermore, liposomes injected SC into arthritic rats showed preferential homing to arthritic joints, following a migration profile in vivo similar to that of intravenously injected liposomes, except for a less steep decline after the peak. Finally, liposomal dexamethasone administered SC was more effective than the unpackaged (free) drug in suppressing arthritis progression in rats. We suggest that with suitable modifications, this SC liposomal treatment modality can be adapted for human RA therapy.

The Anti-Inflammatory and Immunomodulatory Activities of Natural Products to Control Autoimmune Inflammation.

Inflammation is an integral part of autoimmune diseases, which are caused by dysregulation of the immune system. This dysregulation involves an imbalance between pro-inflammatory versus anti-inflammatory mediators. These mediators include various cytokines and chemokines; defined subsets of T helper/T regulatory cells, M1/M2 macrophages, activating/tolerogenic dendritic cells, and antibody-producing/regulatory B cells. Despite the availability of many anti-inflammatory/immunomodulatory drugs, the severe adverse reactions associated with their long-term use and often their high costs are impediments in effectively controlling the disease process. Accordingly, suitable alternatives are being sought for these conventional drugs. Natural products offer promising adjuncts/alternatives in this regard. The availability of specific compounds isolated from dietary/medicinal plant extracts have permitted rigorous studies on their disease-modulating activities and the mechanisms involved therein. Here, we describe the basic characteristics, mechanisms of action, and preventive/therapeutic applications of 5 well-characterized natural product compounds (Resveratrol, Curcumin, Boswellic acids, Epigallocatechin-3-gallate, and Triptolide). These compounds have been tested extensively in animal models of autoimmunity as well as in limited clinical trials in patients having the corresponding diseases. We have focused our description on predominantly T cell-mediated diseases, such as rheumatoid arthritis, multiple sclerosis, Type 1 diabetes, ulcerative colitis, and psoriasis.

Microbiota-Derived Metabolites, Indole-3-aldehyde and Indole-3-acetic Acid, Differentially Modulate Innate Cytokines and Stromal Remodeling Processes Associated with Autoimmune Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of the synovial joints. Inflammation, new blood vessel formation (angiogenesis) and bone resorption (osteoclastogenesis) are three key processes involved in the joint damage and deformities of arthritis. Various gut microbiota-derived metabolites are implicated in RA pathogenesis. However, there is barely any information about the impact of two such metabolites, indole-3-aldehyde (IAld) and indole-3-acetic acid (I3AA), on arthritis-related processes. We conducted a comparative analysis of IAld and I3AA using established cell-based models to understand how they might influence RA pathogenesis. Although structurally similar, the bioactivities of these two metabolites were profoundly different. IAld but not I3AA, inhibited the expression of pro-inflammatory cytokines (IL-1ß and IL-6) in RAW 264.7 (RAW) cells stimulated with heat-killed M. tuberculosis sonicate (Mtb) and lipopolysaccharide (LPS). IAld also exhibited pro-angiogenic activity and pro-osteoclastogenic activity. In contrast, I3AA exhibited anti-angiogenic activity on endothelial cell tube formation but had no effect on osteoclastogenesis. Both IAld and I3AA have been proposed as aryl hydrocarbon receptor (AhR) agonists. Use of CH-223191, an inhibitor of the AhR, suppressed the anti-angiogenic activity of I3AA but failed to mitigate the effects of IAld. Further investigation of the anti-inflammatory activities of IAld and I3AA in LPS-treated RAW cells indicated that inhibition of MyD88-dependent activation of NF-κB and MAPK pathways was not likely involved. Our results suggest that the relative bioavailability of these indole derivatives may differentially impact RA progression and possibly other diseases that share similar cellular processes.

A novel CNS-homing peptide for targeting neuroinflammatory lesions in experimental autoimmune encephalomyelitis.

Using phage peptide library screening, we identified peptide-encoding phages that selectively home to the inflamed central nervous system (CNS) of mice with experimental autoimmune encephalomyelitis (EAE), a model of human multiple sclerosis (MS). A phage peptide display library encoding cyclic 9-amino-acid random peptides was first screened ex-vivo for binding to the CNS tissue of EAE mice, followed by in vivo screening in the diseased mice. Phage insert sequences that were present at a higher frequency in the CNS of EAE mice than in the normal (control) mice were identified by DNA sequencing. One of the phages selected in this manner, denoted as MS-1, was shown to selectively recognize CNS tissue in EAE mice. Individually cloned phages with this insert preferentially homed to EAE CNS after an intravenous injection. Similarly, systemically-administered fluorescence-labeled synthetic MS-1 peptide showed selective accumulation in the spinal cord of EAE mice. We suggest that peptide MS-1 might be useful for targeted drug delivery to CNS in EAE/MS.

Common innate pathways to autoimmune disease.

Until recently, autoimmune disease research has primarily been focused on elucidating the role of the adaptive immune system. In the past decade or so, the role of the innate immune system in the pathogenesis of autoimmunity has increasingly been realized. Recent findings have elucidated paradigm-shifting concepts, for example, the implications of "trained immunity" and a dysbiotic microbiome in the susceptibility of predisposed individuals to clinical autoimmunity. In addition, the application of modern technologies such as the quantum dot (Qdot) system and 'Omics' (e.g., genomics, proteomics, and metabolomics) data-processing tools has proven fruitful in revisiting mechanisms underlying autoimmune pathogenesis and in identifying novel therapeutic targets. This review highlights recent findings discussed at the American Autoimmune Related Disease Association (AARDA) 2019 colloquium. The findings covering autoimmune diseases and autoinflammatory diseases illustrate how new developments in common innate immune pathways can contribute to the better understanding and management of these immune-mediated disorders.

Viewing Autoimmune Pathogenesis from the Perspective of Antigen Processing and Determinant Hierarchy.

Autoimmunity results from the breakdown of immune tolerance to defined target self antigens. Like any foreign antigen, a self antigen is continuously processed by antigen-presenting cells (APCs) and its epitopes are displayed by the major histocompatibility complex on the cell surface (dominant epitopes). However, this self antigen fails to induce a T cell response as the T cells against its dominant epitopes have been purged in the thymus during negative selection. In contrast, the T cells against poorly processed (cryptic) self epitopes escape tolerance induction in the thymus and make it to the periphery. Such T cells are generally harmless as their cognate epitopes in the periphery are not presented efficiently. But, under conditions of inflammation and immune activation, previously cryptic epitopes can be revealed on the APC surface for activation of ambient T cells. This can initiate autoimmunity in individuals who are susceptible owing to their genetic and environmental constellation. Subsequent waves of enhanced processing of other epitopes on the same or different self antigens then cause "diversification" or "spreading" of the initial T cell response, resulting in propagation of autoimmunity. However, depending on the disease process and the self antigen involved, "epitope spreading" may instead contribute to natural regression of autoimmunity. This landmark conceptual framework developed by Eli Sercarz and his team ties together determinant hierarchy, selection of epitope-specific T cells, and the induction/progression of autoimmunity. I am extremely fortunate to have worked with Eli and to have been a part of this fascinating research endeavor.

Peptide-targeted liposomal delivery of dexamethasone for arthritis therapy.

Aim: Rheumatoid arthritis is an autoimmune disease affecting the joints. Antiarthritic drugs are given systemically, thereby exposing various healthy organs to these drugs, resulting in adverse reactions. Accordingly, there is an urgent need for targeted drug delivery methods for inflamed joints. Materials & methods: We developed a liposomal drug delivery system using a novel peptide ligand (CKPFDRALC) named ART-2, which homes to the inflamed joints when injected intravenously to rats with adjuvant-induced arthritis. Results: The ART-2-coated liposomes encapsulating an antiarthritic drug, dexamethasone (DEX), were more effective in inhibiting arthritis progression than control-DEX liposomes or free DEX, despite a comparable safety profile. Conclusion: Peptide-targeted therapy has advantages over conventional drug delivery and can be adapted for rheumatoid arthritis therapy.

Celastrol suppresses experimental autoimmune encephalomyelitis via MAPK/SGK1-regulated mediators of autoimmune pathology.

OBJECTIVE AND DESIGN: Multiple sclerosis (MS) is a debilitating autoimmune disease involving immune dysregulation of the pathogenic T helper 17 (Th17) versus protective T regulatory (Treg) cell subsets, besides other cellular aberrations. Studies on the mechanisms underlying these changes have unraveled the involvement of mitogen-activated protein kinase (MAPK) pathway in the disease process. We describe here a gene expression- and bioinformatics-based study showing that celastrol, a natural triterpenoid, acting via MAPK pathway regulates the downstream genes encoding serum/glucocorticoid regulated kinase 1 (SGK1), which plays a vital role in Th17/Treg differentiation, and brain-derived neurotrophic factor (BDNF), which is a neurotrophic factor, thereby offering protection against experimental autoimmune encephalomyelitis (EAE) in mice. METHODS: We first tested the gene expression profile of splenocytes of EAE mice in response to the disease-related antigen, myelin oligodendrocyte glycoprotein (MOG), and then examined the effect of celastrol on that profile. RESULTS: Interestingly, celastrol reversed the expression of many MOG-induced genes involved in inflammation and immune pathology. The MAPK pathway involving p38MAPK and ERK was identified as one of the mediators of celastrol action. It involved suppression of SGK1 but upregulation of BDNF, which then contributed to protection against EAE. CONCLUSION: Our results not only provide novel insights into disease pathogenesis, but also offer promising therapeutic targets for MS.

Modulation of autoimmune arthritis by environmental 'hygiene' and commensal microbiota.

Observations in patients with autoimmune diseases and studies in animal models of autoimmunity have revealed that external environmental factors including exposure to microbes and the state of the host gut microbiota can influence susceptibility to autoimmunity and subsequent disease development. Mechanisms underlying these outcomes continue to be elucidated. These include deviation of the cytokine response and imbalance between pathogenic versus regulatory T cell subsets. Furthermore, specific commensal organisms are associated with enhanced severity of arthritis in susceptible individuals, while exposure to certain microbes or helminths can afford protection against this disease. In addition, the role of metabolites (e.g., short-chain fatty acids, tryptophan catabolites), produced either by the microbes themselves or from their action on dietary products, in modulation of arthritis is increasingly being realized. In this context, re-setting of the microbial dysbiosis in RA using prebiotics, probiotics, or fecal microbial transplant is emerging as a promising approach for the prevention and treatment of arthritis. It is hoped that advances in defining the interplay between gut microbiota, dietary products, and bioactive metabolites would help in the development of therapeutic regimen customized for the needs of individual patients in the near future.

The miRNA Expression Profile of Experimental Autoimmune Encephalomyelitis Reveals Novel Potential Disease Biomarkers.

Multiple sclerosis (MS) is a debilitating autoimmune disease affecting over 2.3 million people worldwide, and it is characterized by inflammation and demyelination of nerve cells. The currently available biomarkers for the diagnosis and management of MS have inherent limitations, therefore, additional new biomarkers are needed. We studied the microRNA (miRNA) profile of splenocytes of mice having experimental autoimmune encephalomyelitis (EAE), a model of human MS. A miRNA-microarray analysis revealed increased expression of nine miRNAs (let-7e, miR-23b, miR-31, miR-99b, miR-125a, miR-146b, miR-155, miR-193b, and miR-221) following EAE development. Interestingly, serum levels of miR-99b, miR-125a, and miR-146b were significantly higher in EAE mice compared to normal mice. Bioinformatics analysis revealed the experimentally validated as well as predicted gene targets of specific miRNAs that are important for disease progression in MS. Specifically, we observed inverse correlation in the levels of miR-99b versus LIF, and between miR-125a versus BDNF and LIF. Our results suggest that above-mentioned miRNAs may play a crucial role in the pathogenesis of MS, and that miR-99b, miR-125a, and miR-146b in particular may serve as useful biomarkers for disease activity.

NF-KappaB Pathway Is Involved in Bone Marrow Stromal Cell-Produced Pain Relief.

Bone marrow stromal cells (BMSCs) produce long-lasting attenuation of pain hypersensitivity. This effect involves BMSC's ability to interact with the immune system and activation of the endogenous opioid receptors in the pain modulatory circuitry. The nuclear factor kappa B (NF-κB) protein complex is a key transcription factor that regulates gene expression involved in immunity. We tested the hypothesis that the NF-κB signaling plays a role in BMSC-induced pain relief. We focused on the rostral ventromedial medulla (RVM), a key structure in the descending pain modulatory pathway, that has been shown to play an important role in BMSC-produced antihyperalgesia. In Sprague-Dawley rats with a ligation injury of the masseter muscle tendon (TL), BMSCs (1.5 M/rat) from donor rats were infused i.v. at 1 week post-TL. P65 exhibited predominant neuronal localization in the RVM with scattered distribution in glial cells. At 1 week, but not 8 weeks after BMSC infusion, western blot and immunostaining showed that p65 of NF-κB was significantly increased in the RVM. Given that chemokine signaling is critical to BMSCs' pain-relieving effect, we further evaluated a role of chemokine signaling in p65 upregulation. Prior to infusion of BMSCs, we transduced BMSCs with Ccl4 shRNA, incubated BMSCs with RS 102895, a CCR2b antagonist, or maraviroc, a CCR5 antagonist. The antagonism of chemokines significantly reduced BMSC-induced upregulation of p65, suggesting that upregulation of p65 was related to BMSCs' pain-relieving effect. We then tested the effect of a selective NF-κB activation inhibitor, BAY 11-7082. The mechanical hyperalgesia of the rat was assessed with the von Frey method. In the pre-treatment experiment, BAY 11-7082 (2.5 and 25 pmol) was injected into the RVM at 2 h prior to BMSC infusion. Pretreatment with BAY 11-7082 attenuated BMSCs' antihyperalgesia, but post-treatment at 5 weeks post-BMSC was not effective. On the contrary, in TL rats receiving BAY 11-7082 without BMSCs, TL-induced hyperalgesia was attenuated, consistent with dual roles of NF-κB in pain hypersensitivity and BMSC-produced pain relief. These results indicate that the NF-κB signaling pathway in the descending circuitry is involved in initiation of BMSC-produced behavioral antihyperalgesia.

The Micro-RNA Expression Profiles of Autoimmune Arthritis Reveal Novel Biomarkers of the Disease and Therapeutic Response.

Rheumatoid arthritis (RA) is a chronic autoimmune disease of the joints affecting about 0.3⁻1% of the population in different countries. About 50⁻60 percent of RA patients respond to presently used drugs. Moreover, the current biomarkers for RA have inherent limitations. Consequently, there is a need for additional, new biomarkers for monitoring disease activity and responsiveness to therapy of RA patients. We examined the micro-RNA (miRNA) profile of immune (lymphoid) cells of arthritic Lewis rats and arthritic rats treated with celastrol, a natural triterpenoid. Experimental and bioinformatics analyses revealed 8 miRNAs (miR-22, miR-27a, miR-96, miR-142, miR-223, miR-296, miR-298, and miR-451) and their target genes in functional pathways important for RA pathogenesis. Interestingly, 6 of them (miR-22, miR-27a, miR-96, miR-142, miR-223, and miR-296) were further modulated by celastrol treatment. Interestingly, serum levels of miR-142, miR-155, and miR-223 were higher in arthritic versus control rats, whereas miR-212 showed increased expression in celastrol-treated rats compared with arthritic rats or control rats. This is the first study on comprehensive miRNA expression profiling in the adjuvant-induced arthritis (AA) model and it also has revealed new miRNA targets for celastrol in arthritis. We suggest that subsets of the above miRNAs may serve as novel biomarkers of disease activity and therapeutic response in arthritis.

Natural Products for the Treatment of Autoimmune Arthritis: Their Mechanisms of Action, Targeted Delivery, and Interplay with the Host Microbiome.

Rheumatoid arthritis (RA) is a chronic, debilitating illness characterized by painful swelling of the joints, inflammation of the synovial lining of the joints, and damage to cartilage and bone. Several anti-inflammatory and disease-modifying drugs are available for RA therapy. However, the prolonged use of these drugs is associated with severe side effects. Furthermore, these drugs are effective only in a proportion of RA patients. Hence, there is a need to search for new therapeutic agents that are effective yet safe. Interestingly, a variety of herbs and other natural products offer a vast resource for such anti-arthritic agents. We discuss here the basic features of RA pathogenesis; the commonly used animal models of RA; the mainstream drugs used for RA; the use of well-characterized natural products possessing anti-arthritic activity; the application of nanoparticles for efficient delivery of such products; and the interplay between dietary products and the host microbiome for maintenance of health and disease induction. We believe that with several advances in the past decade in the characterization and functional studies of natural products, the stage is set for widespread clinical testing and/or use of these products for the treatment of RA and other diseases.

Peptide-directed liposomal delivery improves the therapeutic index of an immunomodulatory cytokine in controlling autoimmune arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of the synovial tissue of the joints. Inadequately controlled disease may cause severe joint damage and deformity. Currently, the anti-arthritic drugs are given systemically, and therefore, they are widely distributed to other organs that are not the intended therapeutic targets. Accordingly, using a particular dose/regimen of a drug to achieve an effective local concentration of the drug in arthritic joints may lead to expected adverse effects involving other organs. Thus, improved methods of drug delivery are needed for arthritis therapy. One attractive approach is the targeting of a systemically administered drug to the inflamed joints. We describe here a prototypic drug delivery system using a novel peptide ligand denoted as ART-1. We previously reported ART-1 (=ADK) as a peptide that preferentially homes to the inflamed joints of arthritic rats and binds to synovial endothelial cells. We tested the ART-1-coated liposomes encapsulating a fluorescent compound for binding to activated endothelial cells in vitro and homing to arthritic joints in vivo, compared to control liposomes lacking the ART-1 coating. Similar liposomes but encapsulating an immunomodulatory cytokine interleukin-27 (ART-1-IL-27 liposomes) were tested for their anti-arthritic activity compared with control liposomes. ART-1-displaying liposomes showed better binding to endothelial cells as well as in vivo homing to arthritic joints compared to control liposomes. Furthermore, ART-1-IL-27 liposomes, when intravenously injected to arthritic rats after the onset of arthritis, were more effective in suppressing disease progression than control-IL-27 liposomes lacking ART-1 or free IL-27 at an equivalent dose of IL-27. In addition, ART-1-directed liposomal IL-27 had a better safety profile than undirected liposomal IL-27 or free IL-27, thereby offering an improved therapeutic index for IL-27 therapy. These results provide a proof-of concept for the use of a novel joint-homing peptide for targeted delivery of drugs including biologics or small molecule compounds to arthritic joints with enhanced efficacy and reduced systemic exposure. This targeted therapy platform may be suitable for use in RA patients.

In vivo immune interactions of multipotent stromal cells underlie their long-lasting pain-relieving effect.

Systemic infusion of bone marrow stromal cells (BMSCs), a major type of multipotent stromal cells, produces pain relief (antihyperalgesia) that lasts for months. However, studies have shown that the majority of BMSCs are trapped in the lungs immediately after intravenous infusion and their survival time in the host is inconsistent with their lengthy antihyperalgesia. Here we show that long-lasting antihyperalgesia produced by BMSCs required their chemotactic factors such as CCL4 and CCR2, the integrations with the monocytes/macrophages population, and BMSC-induced monocyte CXCL1. The activation of central mu-opioid receptors related to CXCL1-CXCR2 signaling plays an important role in BMSC-produced antihyperalgesia. Our findings suggest that the maintenance of antihypergesia can be achieved by immune regulation without actual engraftment of BMSCs. In the capacity of therapeutic use of BMSCs other than structural repair and replacement, more attention should be directed to their role as immune modulators and subsequent alterations in the immune system.

Immunomodulation of autoimmune arthritis by pro-inflammatory cytokines.

Pro-inflammatory cytokines promote autoimmune inflammation and tissue damage, while anti-inflammatory cytokines help resolve inflammation and facilitate tissue repair. Over the past few decades, this general feature of cytokine-mediated events has offered a broad framework to comprehend the pathogenesis of autoimmune and other immune-mediated diseases, and to successfully develop therapeutic approaches for diseases such as rheumatoid arthritis (RA). Anti-tumor necrosis factor-α (TNF-α) therapy is a testimony in support of this endeavor. However, many patients with RA fail to respond to this or other biologics, and some patients may suffer unexpected aggravation of arthritic inflammation or other autoimmune effects. These observations combined with rapid advancements in immunology in regard to newer cytokines and T cell subsets have enforced a re-evaluation of the perceived pathogenic attribute of the pro-inflammatory cytokines. Studies conducted by others and us in experimental models of arthritis involving direct administration of IFN-γ or TNF-α; in vivo neutralization of the cytokine; the use of animals deficient in the cytokine or its receptor; and the impact of the cytokine or anti-cytokine therapy on defined T cell subsets have revealed paradoxical anti-inflammatory and immunoregulatory attributes of these two cytokines. Similar studies in other models of autoimmunity as well as limited studies in arthritis patients have also unveiled the disease-protective effects of these pro-inflammatory cytokines. A major mechanism in this regard is the altered balance between the pathogenic T helper 17 (Th17) and protective T regulatory (Treg) cells in favor of the latter. However, it is essential to consider that this aspect of the pro-inflammatory cytokines is context-dependent such that the dose and timing of intervention, the experimental model of the disease under study, and the differences in individual responsiveness can influence the final outcomes. Nevertheless, the realization that pro-inflammatory cytokines can also be immunoregulatory offers a new perspective in fully understanding the pathogenesis of autoimmune diseases and in designing better therapies for controlling them.

Celastrol and Its Role in Controlling Chronic Diseases.

Celastrol, a triterpenoid derived from traditional Chinese medicinal plants, has anti-inflammatory, antioxidant, and anticancer activities. Celastrol has shown preventive/therapeutic effects in experimental models of several chronic diseases. These include, chronic inflammatory and autoimmune diseases (e.g., rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, inflammatory bowel disease, and psoriasis), neurodegenerative disorders (e.g., Alzheimer's disease, Parkinson's disease, and Amyotrophic lateral sclerosis), atherosclerosis, obesity, Type 2 diabetes, and cancer. Celastrol modulates intricate cellular pathways and networks associated with disease pathology, and it interrupts or redirects the aberrant cellular and molecular events so as to limit disease progression and facilitate recovery, where feasible. The major cell signaling pathways modulated by celastrol include the NF-kB pathway, MAPK pathway, JAK/STAT pathway, PI3K/Akt/mTOR pathway, and antioxidant defense mechanisms. Furthermore, celastrol modulates cell proliferation, apoptosis, proteasome activity, heat-shock protein response, innate and adaptive immune responses, angiogenesis, and bone remodeling. Current understanding of the mechanisms of action of celastrol and information about its disease-modulating activities in experimental models have set the stage for testing celastrol in clinical studies as a therapeutic agent for several chronic human diseases.

Celastrol modulates inflammation through inhibition of the catalytic activity of mediators of arachidonic acid pathway: Secretory phospholipase A2 group IIA, 5-lipoxygenase and cyclooxygenase-2.

Elevated production of arachidonic acid (AA)-derived pro-inflammatory eicosanoids due to the concerted action of secretory phospholipase A2 group IIA (sPLA2IIA), 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2) is a common feature of many inflammatory disorders. Hence, modulation of the bioactivity of these 3 enzymes is an important strategy to control inflammation. However, the failure of drugs specific for an individual enzyme (sPLA2IIA-, 5-LOX- or COX-2) and the success of 5-LOX/COX-2 dual inhibitors in effectively controlling inflammation in clinical trials prompted us to evaluate a common inhibitor for sPLA2IIA, 5-LOX and COX-2 enzymes. Celastrol, a quinone methide triterpene, was selected in this regard through molecular docking studies. We provide the first evidence for celastrol's ability to inhibit the catalytic activity of sPLA2IIA, 5-LOX and COX-2 enzymes. Celastrol significantly inhibited the catalytic activity of sPLA2IIA (IC50=6µM) in vitro, which is independent of substrate and calcium concentration. In addition, celastrol inhibited the catalytic activities of 5-LOX (IC50=5µM) and COX-2 (IC50=20µM) in vitro; sPLA2IIA-induced edema and carrageenan-induced edema in mice; and lipopolysaccharide-stimulated production of PGE2 in human neutrophils. Thus, celastrol modulates inflammatory responses by targeting multiple enzymes of AA pathway.