Celastrol, a Chinese herbal compound, controls autoimmune inflammation by altering the balance of pathogenic and regulatory T cells in the target organ

Abstract

Inflammation is an integral component of autoimmune arthritis. The balance of pathogenic T helper 17 (Th17) and protective T regulatory (Treg) cells can influence disease severity, and its resetting offers an attractive approach to control autoimmunity. We determined the frequency of Th17 and Treg in the joints of rats with adjuvant arthritis (AA), a model of rheumatoid arthritis (RA). We also investigated the impact of Celastrol, a bioactive compound from the traditional Chinese medicine Celastrus that can suppress AA, on Th17/Treg balance in the joints. Celastrol treatment reduced Th17 cells but increased Treg in the joints, and it inhibited Th17 differentiation but promoted Treg differentiation in vitro by blocking the activation of pSTAT3. Furthermore, Celastrol limited the production of Th17-differentiating cytokines and chemokines (CCL3, CCL5). Thus, Celastrol suppressed arthritis in part by altering Th17/Treg ratio in inflamed joints, and it should be tested as a potential adjunct/alternative for RA therapy.

Keywords: Celastrol;; Chinese medicine;; Experimental arthritis;; Immune regulation; Rheumatoid arthritis;; T helper 17 cells;; T regulatory cells;.

Figure 1. Celastrol treatment downregulates the frequency of IL-17-producing T cells, but upregulates Treg in the SIC of arthritic joints

On d 18 after Mtb immunization of rats, SIC were collected from the joints of arthritic rats treated with Celastrol or vehicle (n= 8 each), and the cells analyzed by flow cytometry for IL-17-/IFN-γ -expressing CD4⁺ (A) and CD8⁺ (B) T cells. In each section (A/B), the left panel shows a representative profile, whereas the right panel shows the average (mean ± SEM) values of the group. Also depicted is the relative proportion of IL-17-producing only, IFN-γ-producing only, and IL-17-IFN-γ double producing cells among CD4 (C) and CD8 (D) T cells from SIC. Sections ‘C’ and ‘D’ correspond to sections ‘A’ and ‘B’, respectively. The frequency of Treg cells in the SIC (n= 8) was also determined (E). The Th17/Treg ratio (n = 8) (left panel) and Tc17/Treg ratio (n = 8) (right panel) are also shown (F). (Where indicated, the error bars represent SEM.)

Figure 2. Celastrol treatment downregulates the frequency of Th17, but has little effect on Th1 and Treg cells in the draining lymph nodes

The draining lymph node cells were harvested on d 18 after disease induction from rats treated with Celastrol or vehicle (n = 3 rats per group). These cells were analyzed by flow cytometry for the frequency of CD4+ T cells expressing IL-17 (A), IFN-γ (B), or Foxp3 (C). In each row, the left panel shows a representative profile, whereas the right panel shows the average (mean ± SEM) values of the group.

Figure 3. Celastrol inhibits the in vitro differentiation of Th17, but augments that of Treg

(A) Naive CD4⁺ T cells were isolated from mouse splenocytes that had been polyclonally stimulated for 3 d with plate-bound anti-CD3 and soluble anti-CD28 in the presence or absence of different cytokines and varying concentrations of Celastrol as follows: TGF-β and IL-6 and anti-IL-2 antibody (bottom panel); IL-6 and TGFβ (middle panel); or IL-2 and TGFβ (top panel), as described in detail under Materials and Methods. In each case, flow cytometric analysis of intracellular staining of IL-17 and Foxp3 was performed. A representative profile for each experiment is shown in each section. (B) Intracellular staining for RORγt was performed in cells shown in the bottom panel of section ‘A’. A representative profile (left panel) and average (mean + SEM, n= 3) values of the group (right panel) are shown.

Figure 4. Celastrol inhibits IL-6 receptor signaling via reducing STAT3 signaling

Mouse CD4⁺ T cells were treated with Celastrol (300 nM) for 8 h and then activated with IL-6 (2 ng/mL) for the indicated duration (minutes). (A) Cell lysates were collected and pSTAT3 and pSTAT5 were tested using a western blot assay. Total STAT3 and total STAT5 were used as protein-loading controls. The gel was then subjected to densitometry and the values were normalized to appropriate controls. (B) Total RNA isolated from cells treated as described above was tested for STAT3-induced gene SOCS3 by qRT-PCR (mean ± SEM, n = 3).

Figure 5. Celastrol reduces the production of IL-6 and IL-1β by SAC and SIC

SAC and SIC from Mtb-immunized arthritic rats treated with Celastrol or the vehicle were harvested on day 18 of AA. (A) Cells were restimulated in vitro for 24 h with or without Mtb sonicate (10 µg/ml). Thereafter, cell-free culture supernatants were collected, and the levels of IL-1β and IL-6 were measured by a Multiplex assay. (B–C) SAC from untreated arthritic rats were restimulated in vitro for 6 h with Mtb sonicate in the presence or absence of Celastrol (100 or 300 nM) (n = 3 each) (B). Similarly, SAC from arthritic rats treated with Celastrol or the vehicle were restimulated in vitro for 6 h with or without Mtb sonicate (n = 3) (C). In each case (B/C), the total RNA prepared from cells was processed by quantitative RT-PCR to measure IL-1β and IL-6 mRNA expression (top panel), and values were normalized to the respective hypoxanthine-guanine phosphoribosyltransferase mRNA levels and expressed as relative message. In parallel (bottom panel), cell lysates were prepared from SAC and the levels of pro-IL-1β (p31) and IL-1β (p17) were analyzed by western blot. (D) NF-kB inhibition. HEK 293T cells were transfected with an expression construct containing Firefly luciferase and a promoter containing three copies of the NF-kB binding site (NF-kB luciferase). Cells were pretreated with indicated concentrations of Celastrol for 2 h followed by stimulation with TNF-α for 6 h. Luciferase activity was read and the results presented as percent inhibition over the medium control (mean ± SEM, n = 3). (E) SAC from an untreated arthritic rat were treated with Mtb sonicate as in ‘C’ but with or without an inflammasome inhibitor (Glyben, also known as Glibenclamide), a protease inhibitors (yVAD: caspase-1 inhibitor; MMP-9 inhibitor; AEBSF: serine protease inhibitor), or Celastrol for 24 h. The culture supernatants were analyzed for pro-IL-1β and mature IL-1β by western blot.

Figure 6. Celastrol limits the inflammatory cell infiltration into the joints and the chemotactic migration of cells in vitro, and inhibits antigen-induced T cell proliferation

(A) Neutrophils (CD45+ CD11b/c+ Rat Granulocyte Marker+) and T cells (CD3+) were isolated from SIC from arthritic joints and enumerated by flow cytometry. The Th1, Th17, and Treg cells were detected by intracellular staining for IFN-γ, IL-17, and Foxp3, respectively. Data from 4–5 independent experiments (n = 3 rats per group) are presented. (B) Total SIC from arthritic rats treated with Celastrol or vehicle were stimulated ex vivo with Mtb sonicate. Chemokines in cell supernatants were measured by Multiplex assay. (C) LNC derived from untreated arthritic rats were allowed to migrate using a Transwell membrane in the presence of CCL3 or CCL5 (50 ng/mL) and Celastrol at the indicated concentrations. Cells treated with vehicle served as controls for Celastrol-treated cells (n = 3). (Where indicated, the error bars represent SEM.). (D) Celastrol limits T cell proliferative responses to arthritis-related antigens in lymph nodes. Ex vivo T cell recall responses to arthritis-related antigens were measured using LNC of Celastrol-/vehicle-treated rats. The draining LNC from these rats were harvested 18 d after Mtb-immunization and tested in a proliferation assay using the indicated recall antigens (mean ± SEM, n = 3). (Keyhole limpet hemocyanin, KLH; Mycobacterium tuberculosis H37Ra, Mtb; Mycobacterial heat shock protein 65, Bhsp65; and Rat heat shock protein 65, Rhsp65.).