Carnosol Alleviates Collagen-Induced Arthritis by Inhibiting Th17-Mediated Immunity and Favoring Suppressive Activity of Regulatory T Cells

Current approaches are incurable for rheumatoid arthritis (RA). Regulatory T (Treg) cells and T helper cells (Th1 and Th17) are crucial in controlling the process of RA, which is characterized by inflammatory cell infiltration and bone destruction. Carnosol is an orthodiphenolic diterpene that has been extensively applied in traditional medicine for the treatment of multiple autoimmune and inflammatory diseases. Herein, we indicate that administration of carnosol dramatically alleviated the severity of collagen-induced arthritis (CIA) model with a decreased clinical score and inflammation reduction. Cellular mechanistically, carnosol inhibits the Th17 cell differentiation and maintains Treg cell suppressive function in vitro and in vivo. Meanwhile, it also restrains Treg cells from transdifferentiation into Th17 cells under inflammatory milieu. Furthermore, carnosol modulates the function of Th17 and Treg cells possibly via limiting IL-6R (CD126) expression. Collectively, our results suggest that carnosol can alleviate the severity of CIA via hiding Th17 cell differentiation and maintain the stability of Treg cells. Administration of carnosol can be applied as a potential therapy for patients with RA.


Introduction
Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by inflammatory cell infiltration and bone destruction [1,2]. Effector CD4 + T (Teff) cells, such as regulatory T (Treg) cells and other T helper cells (Th1 and Th17), are crucial in controlling the process of RA [3,4]. Importantly, the Th17/Treg imbalance contributes to the pathogenesis and progression of RA [5,6]. Therefore, targeting Th17 cells or combined with immune suppression can exert a therapeutic effect on RA [7]. Currently, the main treatments for RA are nonsteroidal anti-inflammatory drugs (NSAIDs); however, about 30%-50% of RA patients do not response to the treatment well [8]. Therefore, developing novel candidates is needed to meet the huge demand.
From the perspective of clinical translation, carnosol has the potential to treat various types of diseases. It has been documented that carnosol was capable in treating LPSinduced septic shock in mice by suppressing inflammasome activation [14]. Additionally, it was effective in treating numerous types of cancers, such as lung cancer [15], colon cancer [16], breast cancer, pancreatic cancer [17], prostate cancer [18], leukemia [19], and brain cancer [20]. Also, carnosol can protect against renal ischemia-reperfusion injury [21] and spinal cord injury [22]. Importantly, carnosol was reported to suppress AIDs, such as ovalbumin-induced allergic asthma [23] and experimental autoimmune encephalomyelitis (EAE) [24]. Li et al. firstly reported that carnosol and rosmanol synergistically alleviate murine arthritis via inhibiting TLR4/NF-κB/MAPK pathway [25]. Given the essential role of Th17 and Treg cell subsets in arthritis, we are wondering whether carnosol can modulate the differentiation and/or function of these cells that contributes to the therapeutic effects on CIA.
In this study, we observed that the administration of carnosol dramatically alleviated the severity of CIA model with a decreased clinic score and joint inflammation. Remarkably, the frequency of Th17 cells was markedly reduced, which was positively correlated with the duration of joint inflammation. Besides, carnosol can also maintain the suppressive capacity of Treg cells in CIA model. In vitro, we confirmed that carnosol inhibits the IL-6R (CD126) expression that can suppress Th17 cell differentiation and promote Treg cell differentiation. Collectively, our results suggest that carnosol can alleviate the severity of CIA via hiding Th17 cell differentiation and maintain the stability of Treg cells; administration of carnosol can be applied as a potential therapy for patients with RA.

Carnosol Alleviates the Development of CIA by
Suppressing Lymphocyte Infiltration. To investigate the therapeutic effect of carnosol in the context of autoimmune arthritis, we developed CIA model as previously described [26]. Carnosol was intraperitoneally administrated into the mice every other day after immunization for 14 continuous times at a dose of 50 mg/kg. We observed a significant attenuation in arthritis clinical scores and an obvious delay in CIA onset compared to DMSO administration (p < 0:01) (Figure 1(a)). Also, both the lymphocyte numbers in spleen and draining lymph nodes (dLN) were much decreased in carnosol-treated mice, compared with the DMSO group (p < 0:01) (Figure 1(b)). Accordingly, joint H&E staining was applied. Histological changes in the whole ankle joints demonstrated a significant decrease in synovitis, pannus formation, and destruction of bone and cartilage after treatment with carnosol (p < 0:05) (Figures 1(c) and 1(d)). Together, these results demonstrate that administration of carnosol has a robust therapeutic effect in murine experimental arthritis mice.

Carnosol
Reduces the Frequency of Th17 Cells and Local Joint Inflammation in CIA Model. As mentioned above, T helper cells are the most important proinflammatory response involved in the pathology of RA/CIA. We hypothesized that carnosol could have an impact on these effector cells. Next, we analyzed the frequencies of Th1, Th17, and Treg cells in carnosol-and DMSO-treated mice individually. As expected, the analysis clearly demonstrated that the number of Th17 cells was markedly declined in the dLN of CIA mice on day 54 compared to model or DMSO group (p < 0:01) (Figures 2(a) and 2(c)). Although the frequency of Th1 cells in carnosol treatment group was not statistically significant, it also showed a decreasing trend to some extent (Figures 2(a) and 2(c)). Meanwhile, our results revealed that carnosol was also able to induce Treg responses, which the frequency showed a slight increase, but no difference between model and vehicle group (Figures 2(b) and 2(c)). As is well known, Th17 cells play a driving role in the development of RA by altering the ratio of Treg/Th17 cells [27]. We therefore regressed the correlation between inflammatory scores (Figure 1(d)) and Th17 cell frequency (Figure 2(a)). Consistent with others, we also confirmed that Th17 frequency was positively correlated to inflammatory score in CIA mice ( Figure 2(d)). Collectively, these results indicate that carnosol alleviates CIA via suppressing the differentiation of Th17 cells.
Furthermore, we investigated the proinflammatory cytokines and Foxp3 levels in local joints using qRT-PCR analysis. The results revealed that, consistent with the Th17 frequency changing, the mRNA level of IL-17A was significantly decreased in carnosol-treated joint tissues in comparison to model or vehicle group (p < 0:05) ( Figure S1A, left panel). Although Th1 cells in carnosol-treated CIA mice were not statistically decreased, the mRNA level of IFN-γ is lowered with statistical significance in local joint (p < 0:05) ( Figure S1A, middle panel). However, Foxp3 mRNA in carnosol group was not significantly changed ( Figure S1A, right panel). Interestingly, the level of IL-6 mRNA in carnosol-treated CIA joint tissues was significantly decreased (p < 0:01) ( Figure S1B), implying that carnosol could inhibit joint erosion by suppressing IL-6 secretion. Thus, carnosol not only suppresses the differentiation of Th17 cells but also hides the transcriptional level of proinflammatory cytokines, such as IL-17A, IFN-γ, and IL-6, which contribute to the prevention of CIA progress.

Carnosol Stabilizes Treg Cell Suppressive Activity in CIA
Model. In RA, the stable function of Treg cells plays a pivotal role in maintaining immune microenvironment homeostasis [28]. Therefore, we explored the Treg cell stability in vivo using the established ex vivo experiment system. dLN CD4 + CD25 + Treg cells were sorted from carnosol-and DMSO-treated CIA mice individually. The purity was confirmed by flow cytometry (Figure 3(a)). As we had expected, CD4 + CD25 + IL-17A + Treg cells in carnosoltreated group were statistically lower than those on the corresponding groups after being stimulated with recombinant murine IL-6 (rmIL-6) for 3 days (p < 0:05) (Figures 3(b) and 3(c)). These results indicate that carnosol prevents Treg cells from converting to Th17 cells, which may sustain their suppressive function in CIA inflammatory environment. In addition to stability, we also focused on whether carnosol treatment affects the Treg cell immunosuppression. Hence, dLN CD4 + CD25 + Treg cells were harvested from each group on day 54 after CII immunization, and then, a standard in vitro functional assay was performed in the presence of rmIL-6 (Figures 3(d) and 3(e)). The results showed that Treg cells from carnosol-treated CIA mice maintained their suppressive function, which showed a decreased CFSE proliferation, while Treg cells from model-derived or DMSO-treated CIA mice notably lost their suppressive capacity.

Carnosol Diversely Modulates Th17 and Treg Cell
Differentiation. To further confirm the exact function of carnosol on Th17 and induced Treg cell (iTreg) differentiation, we used the established polarizing conditions, respectively. Splenic naïve CD4 + T cells were sorted and treated with carnosol at different concentrations. IL-17A or Foxp3 . Surprisingly, carnosol can also promote iTreg cell differentiation at the concentration of 10 μM (Figures 4(a) and 4(c)). As we know, Treg cells are heterogeneous and consist of at least two types, tTregs and iTregs [29]. iTregs are resistant to IL-6-driven Th17 cell conversion and maintain the functional capacity in the inflammatory condition [30]. IL-6 -IL-6R (CD126) signaling plays a vital role on CIA/RA progress [31]. Whether carnosol regulates IL-6 signaling in Th17 or iTreg cell differentiation process remains unclear. Given the undetected of IL-6 on T cell subsets, we found that the CD126 expression was quite lower in iTreg and Th17 cells pretreated with carnosol than that pretreated with DMSO control (Figures 4(b) and 4(d)). In addition, we also observed that the tTregs primed with carnosol are also expressed lower CD126 than that on the corresponding cells in the presence of IL-6 (Figures 4(e) and 4(f)). Signal transducer and activator of transcription 3 (STAT3) activities play a crucial role in the   differentiation of Th17 cells, which is also one of the key downstream molecule of IL-6 signaling [32]. Therefore, under Th17-polarizing condition, we verified that the phosphorylated STAT3 (p-STAT3) level was significantly decreased in the carnosol-treated group. Similarly, p-STAT3 was also diminished in carnosol-treated tTreg cells (Figures 4(g) and 4(h)). Collectively, these results reveal that carnosol inhibits Th17 cell differentiation with a dose-dependent manner but expedites iTreg differentiation at a relatively high concentration. IL-6R (CD126)-mediated inhibition of STAT3 activity provides a novel explanation for carnosol's role on Th17 and Treg cell differentiation.

Discussion
Carnosol, the product of oxidative degradation of carnosic acid, has previously been shown to exhibit antiinflammatory activity and has the potential to treat a variety of inflammatory diseases. Although it has already been shown that carnosol inhibits Th17 cell differentiation and downregulates multiple transcription factors, including NF-κB as well as STAT in EAE model [24], to our knowledge, this is the first study to show that carnosol treatment not only leads to an inhibition in Th17 differentiation but also favors the suppressive activity of Treg cells, contributing to the alleviation of CIA.
The potent arthritogenic effect of Th17 cells mainly lies in the pleiotropic function of IL-17A, which is majorly produced by Th17 cells and acts on the synovial microenvironment: (i) IL-17A acts on the synovial fibroblasts, causing the latter to enhance the production of inflammatory cytokines, such as TNF-α, IL-1β, and IL-6, (ii) IL-17A recruits macrophages and neutrophils to the site of inflammation in order to cause cell death-mediated inflammation, and (iii) IL-17A accelerates osteoclastogenesis, which leads to bone erosion and cartilage destruction [33]. In addition, IL-22 and IL-21, produced by Th17 cells, alter the glycosylation of autoantibodies and provide them with inflammatory properties. Thus, Th17 cells are potent mediators of arthritis, which coordinate tissue inflammation, cartilage damage, and bone erosion [33]. Mechanistically, IL-6 is crucial to the Th17 cell differentiation. Soluble IL-6 exerts its effects by binding to a receptor complex formed by the ligand-binding IL-6Ra chain (CD126). IL-6 binds to IL-6R, which can then trigger downstream signal transduction and gene expression. The combination of IL-6 and TGF-β induces the retinoid-related orphan receptor (ROR) γt, which are the key transcription factors in determining the differentiation of the Th17 lineage [34]. In this study, we showed that carnosol inhibits Th17 differentiation mainly via IL-6-IL-6R signaling.

BioMed Research International
Although there are many types of Treg, thymus-derived Treg (tTreg) and TGF-β-induced Treg (iTreg) are the most predominant Treg cell subsets [28]. tTreg cells are heterogeneous and unstable, which can be converted into other T helper cells in the inflammatory milieu. IL-6 secretion by CIA joints drove the conversion of CD25 + Foxp3 + tTreg cells into pathogenic Th17 cells, and in turn, IL-17A production by the formerly Foxp3 + Th17 cells induced more IL-6 from the inflamed synovial fibroblasts, which establishes a positive feedback loop in the arthritic joints [35]. We and others pro-vided more evidence that tTregs were significantly prevented but were less satisfactory in treating autoimmune arthritis because of their plasticity and conversion to Th1, Th17, or T follicular cells under inflammatory conditions [28]. Quite different from the tTreg cells, iTregs are resistant to inflammatory cytokine-driven Th17 cell conversion in the presence of IL-6 and IL-1β or even in high salt milieu [28]. Interestingly, we investigated that the effect of carnosol on CIA was also dependent on downregulating inflammatory Th17 cells and enhancing the immunosuppressive function of (e, f) CD4 + CD25 + tTreg cells were stimulated with carnosol or DMSO in the presence of IL-6 for 3 days, and CD126 expressions were tested by FACS. (g, h) Naïve CD4 + T cells were cultured under Th17-polarizing condition and treated with carnosol (10 μM), or CD4 + CD25 + tTreg cells were stimulated with carnosol (10 μM) in the presence of IL-6 for 3 days. Cells were then analyzed for STAT3 expression by western blot. Data are presented as mean ± SEM of 3 independent experiments. ( * p < 0:05, * * p < 0:01, and * * * p < 0:001; ns means not significant). One-way ANOVA followed by Tukey's multiple comparison test. 6 BioMed Research International CD4 + Foxp3 + Treg cells in draining lymph nodes. The frequency of Th17 cell reduction was positively correlated with the degree of arthritis remission. In addition, we also demonstrated that Treg cells from carnosol-treated CIA mice are more resistant to become Th17-like cells, even when stimulated with IL-1β and IL-6. Meanwhile, these Treg cells exhibited a markedly enhanced inhibitory activity in suppressing T cell proliferation, compared to the vehicletreated group. Notably, carnosol can also expedite iTreg differentiation in vitro, indicating that it may exert its protective effects by inducing more stable iTreg cells. IL-6-CD126-STAT3 signaling is essential for the progression of arthritis [32]. Previous findings confirmed that CD126 + Treg population is unstable and has compromised immunosuppressive capacity under the inflammatory condition [31].
Herein, we observed that under Th17-or iTreg-polarizing condition, carnosol reduces the CD126 expression on naïve T cells. In other words, carnosol possibly inhibits the initial IL-6R signaling activity, thus leading to lower th17 induction and more differentiation of iTreg cells. One of the downstream molecules of IL6-CD126 signaling is STAT3, which is phosphorylated and that translocate to the cell nucleus, where they act as transcription activators. Finally, we show that carnosol suppressed STAT3 phosphorylation, in response to the ligand IL-6, that blocks Th17 differentiation and favored Treg cell suppressive activity. In summary, this study demonstrates that carnosol treatment significantly delayed CIA disease onset and decreased the severity of arthritis by downregulating inflammatory Th17 cells while enhancing tTreg cell suppressive capacity. A molecular mechanism of carnosol on CIA arthritis appears to be via an inhibiting effect on Th17 cell differentiation by diminishing STAT3 phosphorylation and favoring Treg cell immunosuppressive function through inhibition of IL-6-IL-6R (CD126) signaling, indicating that administration of carnosol can be applied as a potential therapy for patients with RA.

Material and Methods
4.1. Mice. Male C57BL/6 (B6) and DBA1/J mice were purchased from Lanzhou Veterinary Research Institute. All mice were maintained in a single specific pathogen-free (SPF) room in the animal facility of the Medical College of Lanzhou University. All mice used in this study were 6 to 8 weeks old. All animal experiments were performed with the approval of the Institutional Animal Care and Use Committee of Lanzhou University and according to the approved institutional guidelines and regulations. All protocols were approved by the ethics committee of the First Hospital of Lanzhou University.

4.2.
Induction and Treatment of CIA. CIA model was developed as previously described [36]. Briefly, bovine type II collagen (CII, 2 mg/ml) was emulsified with complete Freund's adjuvant (CFA) (with heat-denatured mycobacterium 8 mg/ ml, Chondrex, LLC, Seattle, WA) at a ratio of 1 : 1. And then, the emulsified mixture was injected intradermally into the tail of DBA1/J mice. Mice were divided into different groups randomly, with or without a single intravenous injection of carnosol (MCE, Cat. No. HY-N0643) or DMSO every other day after immunization for 7 continuous times at a dose of 50 mg/kg. Mice were sacrificed, and data were collected at day 51. The clinical score of arthritis was graded on a scale of 0-4 scales as follows: grade 0, no erythema and no swelling; grade 1, slight erythema and swelling; grade 2, moderate edema and swelling; grade 3, severe swelling and significant edema; and grade 4, severe swelling and significant edema with joint rigidity. 4.4. Naïve CD4 + T Cell Isolation. Naïve CD4 + T cells were prepared as previously described [35]. Single-cell suspensions were obtained from the spleen of C57BL/6 (B6) mice. Splenic erythrocytes were eliminated with red blood cell lysis buffer (Sigma-Aldrich). Total T cells were enriched with nylon wool, and then, naïve CD4 + T cells were purified from these T cells via magnetic cell sorting by automagnetic cell sorter (MACS) (Miltenyi Biotec, Germany). In brief, total cells labeled with biotin anti-mouse CD8, CD25, B220, CD11b, CD11C, and CD49b antibodies and anti-biotin microbeads were subjected to a depletion followed by a positive selection with CD62L microbeads by auto MACS. The purity of naïve CD4 + T cells was determined as CD4 + CD25 -CD62L + with flow cytometry, and the purity of >98% naïve CD4 + T cells was used.

Histopathology.
For knee joints in CIA, both hind limbs from the CIA mice were dissected and incubated in 10% buffered formalin. Then, the specimens were sectioned and stained with H&E. Two pathologists blindly evaluated the global histological changes according to the infiltration of inflammatory cells and the thickness. For general assessment of histopathology, the paraffin-embedded tissues were sectioned (5 μM), stained with hematoxylin and eosin (H&E), and mounted onto microscope slides for analysis. The histopathologic score was evaluated by a 1-4 scale regarding the degree of cell infiltration, synovial hyperplasia, and cartilage destruction: (1) hyperplasia of the synovial membrane and presence of inflammation infiltration, (2) pannus and cartilage erosion, (3) major erosion of cartilage and subchondral bone, and (4) loss of joint integrity and ankyloses.
4.10. Statistics. Statistical analysis was performed using GraphPad Prism 8(GraphPad Software). Data were analyzed by Student's t test in the case of the two groups, and one-way ANOVA analysis was performed for the three groups in mice studies. Data are presented if not indicated elsewhere as mean ± SEM. A value of p < 0:05 was considered to be statistically significant ( * p < 0:05, * * p < 0:01, and * * * p < 0:001; ns means not significant).

Data Availability
The underlying data related to this paper were made available through contacting Yang Luo (yangluo68@sina.com).

Ethical Approval
The research was approved by the ethics committee of the First Hospital of Lanzhou University.

Consent
Consent for publication has been obtained from all authors.