Anti‐inflammatory and immune‐modulatory impacts of berberine on activation of autoreactive T cells in autoimmune inflammation

Abstract Autoreactive inflammatory CD4+ T cells, such as T helper (Th)1 and Th17 subtypes, have been found to associate with the pathogenesis of autoimmune disorders. On the other hand, CD4+ Foxp3+ T regulatory (Treg) cells are crucial for the immune tolerance and have a critical role in the suppression of the excessive immune and inflammatory response promoted by these Th cells. In contrast, dendritic cells (DCs) and macrophages are immune cells that through their inflammatory functions promote autoreactive T‐cell responses in autoimmune conditions. In recent years, there has been increasing attention to exploring effective immunomodulatory or anti‐inflammatory agents from the herbal collection of traditional medicine. Berberine, an isoquinoline alkaloid, is one of the main active ingredients extracted from medicinal herbs and has been shown to exert various biological and pharmacological effects that are suggested to be mainly attributed to its anti‐inflammatory and immunomodulatory properties. Several lines of experimental study have recently investigated the therapeutic potential of berberine for treating autoimmune conditions in animal models of human autoimmune diseases. Here, we aimed to seek mechanisms underlying immunomodulatory and anti‐inflammatory effects of berberine on autoreactive inflammatory responses in autoimmune conditions. Reported data reveal that berberine can directly suppress functions and differentiation of pro‐inflammatory Th1 and Th17 cells, and indirectly decrease Th cell‐mediated inflammation through modulating or suppressing other cells assisting autoreactive inflammation, such as Tregs, DCs and macrophages.


| Autoreactive Th1 and T17 cells
Autoreactive CD4 + T cells, such as T helper (Th)1 and Th17 subtypes, have been found to involve in the pathogenesis of several autoimmune disorders, including multiple sclerosis (MS), inflammatory bowel disease (IBD) and rheumatoid arthritis (RA). Th1 cells, which predominantly produce interferon gamma (IFNγ), participate in the elimination of intracellular pathogens and are involved in cell-mediated and delayed-type hypersensitivity responses.
There are several lines of evidence that Th1 cells producing IFNγ are closely correlated with the clinical severity of autoimmune diseases and can independently transfer diseases into naïve mice. In mice with experimental autoimmune encephalomyelitis (EAE) as a model of human MS, IFNγ levels within the central nervous system (CNS) have been found to associate with disease severity, with high levels detected at the peak of disease that fall as the disease spontaneously subsides into remission. 1,2 Infiltrating CD4 + T cells were found as the source of this IFNγ, 3 and the adoptive transfer of IFNγ-producing T-cell lines has been demonstrated to promote autoimmune pathologies. 2,4 The role of Th1 in autoimmune disorders is further confirmed by findings that mice lacking the Th1 lineage-specific transcription factors, T-bet and signal transducer and activator of transcription 4 (STAT4), are protected from the disease. 5 The pathogenic role of Th1 cells has been also declared in other models of autoimmunity, such as adjuvant-induced arthritis (AIA) as a model of human RA, 6 experimental autoimmune uveitis (EAU) 7,8 and experimental autoimmune myocarditis (EAM). 9,10 In parallel to Th1, some reports show that Th17 cells, a subtype of interleukin (IL)-17 secreting CD4 + Th cells, and their relevant cytokines play important role in the severity and progression of several autoimmune diseases. The pathogenic role of Th17 cells in autoimmune diseases has emerged from studies that indicate IL-17 expression is elevated at the inflamed sites in patients with RA, MS, uveitis, and psoriasis. [11][12][13][14][15][16] IL-17 is a pro-inflammatory cytokine that affects various cell types, including endothelial cells, fibroblasts, keratinocytes, epithelial cells, and macrophages, and promotes the generation of several cytokines including IL-6, IL-1, tumour necrosis factor alpha (TNF-α), transforming growth factor beta (TGF-β), granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage CSF (GM-CSF), and many chemokines such as macrophage inflammatory protein 2 (MIP-2), Cytokine-induced neutrophil chemoattractant (CINC) and monocyte chemoattractant protein 1 (MCP-1), as well as prostaglandins like PGE2. [17][18][19][20][21] A crucial result of such effects is the promotion and recruitment of neutrophils to the inflamed sites. 18,22 Moreover, IL-17 is found to induce the generation of matrix metalloproteinases (MMPs) that act to degrade target tissue during the inflammation. 18 In addition to IL-17, Th17 cells secret IL-6, TNF-α, IL-21 and IL-22 cytokines that are attributed to the destructive pro-inflammatory function of these cells. 18,23,24 Of note, the impact of the Th17 response on autoimmunity has been evaluated within the various experimental models. Th17/IL-17 deficient mice were found to has low sensitivity to AIA and EAE, 25 and treatment with IL-17R antagonist or IL-17 neutralizing antibody ameliorated the severity of AIA, EAE and EAU. 22 It is further supported by findings that show when Th17 regulatory factors, such as IL-6 and retinoic acid-related orphan receptor gamma t (RORγt), were knocked out in experimental animals, tissue infiltrating Th17 cells were significantly decreased and autoimmune inflammation was attenuated. 26 On the other hand, CD4 + Foxp3 + T regulatory (Treg) cells are crucial for immune tolerance and have a critical role in the suppression of the excessive immune and inflammatory response promoted by autoreactive Th cells. 27,28 Foxp3 + Treg cells can inhibit Th1 and Th17 differentiation and function. The nuclear transcription factor Foxp3, known as a specific marker for Treg cells, plays an essential role in the development and function of Treg cells, 29 which can suppress differentiation of Th1/Th17 cells by antagonizing the function of the transcription factors RORγt and ROR. 30 However, IL-6 overcame this suppressive effect of Foxp3 on Th17 differentiation. 30 Excessive IL-6 induces Th17, but suppresses differentiation of Treg cells, shifting the balance of Tregs towards inflammatory Th17 cells in patients with autoimmune disorders. Treg/Th17 imbalance and diminished numbers of Foxp3 + Treg cells in patients with various autoimmune diseases are associated with disease severity and activity. 31 On the contrary, DCs and macrophages, as discussed in the following sections, are immune cells that through their inflammatory functions promote autoreactive T-cell responses in autoimmune conditions. 32,33

| Berberine: a natural compound possessing immunomodulatory and anti-inflammatory properties
In recent years, there has been increasing attention to exploring effective immunomodulatory or anti-inflammatory agents from the herbal collection of traditional medicine. Herbal medicines introduce a rich source of natural compounds for the identification of new therapeutic agents having novel mechanisms of action and for providing valuable insight into new targets involved in the inflammatory process. Among medicinal plants and herbs, numerous plants of the genera Berberis and Coptis have been widely employed in traditional medicine to treat patients with abdominal pain, diarrhoea or gastroenteritis. 34 Berberine, an isoquinoline alkaloid, is one of the main bioactive ingredients in these herbs (Table 1) and has been shown to exhibit anti-inflammatory, antioxidation, anti-atherosclerotic, antimicrobial, antidiabetic, antitumour and neuroprotective effects. [35][36][37][38][39][40][41][42] Such pleiotropic biological and pharmacological properties of berberine have been suggested to be mainly attributed to its anti-inflammatory and immunomodulatory properties. 35,43 Berberine has been found to modulate and/ or suppress inflammation through suppressing the production of TNF-α, IL-6 and MCP-1, down-regulating the expression of cyclooxygenase-2 (COX-2), reducing generation of PGE2 and formation of exudates, and inhibiting the expression of MMP-2 and MMP-9 through nuclear factor-kB (NF-kB) and mitogen-activated protein kinase (MAPK) signalling cascades. 35 Table 2).

| SUPPRE SS IVE EFFEC TS OF B ERB ERINE ON AUTORE AC TIVE TH1/ TH17 CELL S
Anomalous autoreactive responses of CD4 + T helper cells, such as An early study on mice model of autoimmune tubulointerstitial nephritis showed that berberine could reduce increased levels of Th1 cells, which was associated with an improvement in renal function. 62 Experimental autoimmune neuritis (EAN) is a model of human Guillain-Barre syndrome characterized by infiltration of the peripheral nervous system by autoreactive T cells promoting demyelination and axon damage. [63][64][65] Berberine treatment was shown to ameliorate EAN severity by inhibiting the proliferation of CD4 + T cells and down-regulating Th1 (TNF-α) cytokine. 66 Results from an ex vivo study on human CD4 + T cells isolated from patients with ocular Behcet's disease 67 and Vogt-Koyanagi-Harada disease 68 indicate that berberine can suppress Th17 responses through reducing the frequency of IL-17 producing CD4 + T cells and inhibiting IL-17 production. It is further confirmed by an in vivo study on AIA rats that showed berberine administration could significantly reduce the blood levels of Th17 population and the serum levels of IL-17, which was accompanied by decreased expression of IL-17 in synovium and Th17 transcription factor RORγt in the spleen. 69 The further experimental study revealed that berberine treatment significantly attenuated the excessive response of Th1/Th17 cells through reducing elevated levels of Th1/Th17 cells and their cytokines IL-17/IFNγ in rats with EAM, which was along with marked reduction in the impaired cardiac function and the pathophysiological severity. 70 An in vitro study on naïve T cells isolated from the spleen of AIA rats indicated that berberine treatment could significantly decrease differentiation and survival of Th17 cells, in a con- and expression of T-bet in differentiating Th1 cells (Figure 1). [70][71][72] In sum, berberine can directly inhibit differentiation and function of Th1/Th17 cells and thereby decrease inflammation-mediated severity and progression of autoimmunity disease. As discussed in the following sections, berberine can also suppress inflammatory responses of T cells through indirect effect via affecting function of Treg cells, DCs, and macrophages.

| B ENEFI CIAL MODUL ATORY EFFEC TS OF B ERB ERINE ON TREG/ TH17 BAL AN CE
Treg cells are anti-inflammatory cells that secrete inhibitory cytokines including IL-10, IL-35 and TGF-β and, thereby, suppress TA B L E 2 Effects of berberine on cytokine production in various autoimmune diseases    sponses. 98 Berberine has been shown to exert anti-apoptotic effects on DCs in in vitro and in vivo models of RA. 99 Berberine could time-and dose-dependently induce apoptosis in murine bone marrow(BM)-derived DCs. 99 Freshly isolated BM cells were found to be insensitive to berberine, and the susceptibility to berberine-promoted apoptosis was increased during DC differentiation, in which mature IL-12-producing DCs showed higher sensitivity to berberine than immature DCs. Thus, berberine can selectively trigger apoptosis in mature DCs and whereby restrict DC maturation and shorten their lifespan. 99 As mature DCs play the crucial roles in pathogenic inflammation and immune responses in autoimmune diseases, berberine-induced apoptosis in mature DCs provides a major mechanism of immunomodulation that can be accounted, at least in part, for its immunosuppressive impacts observed in animal models of autoimmune diseases. Although the exact intracellular mechanisms underlying selective pro-apoptotic effect in DCs remain unknown, it has been shown that the production of reactive oxygen species (ROS) and mitochondrial depolarization, as well as caspase 3 activation, are involved in berberine-mediated apoptosis induction. 99 In accordance with the aforementioned in vitro findings, berberine was indicated to markedly reduce the ratio of mature to immature DCs in spleens, confirming its selective pro-apoptotic effect in mature DCs in vivo. 99 In this regard, berberine treatment could cause a considerable loss of DCs and an elevation in the apoptosis of DCs within spleens and lymph nodes in AIA mice, which was accompanied by the antiarthritic and immunosuppressive effects in these mice. 99 In sum, berberine has the potential to decrease survival and inflammatory functions of autoreactive APCs, mainly DCs, through inducing apoptosis and inhibiting co-stimulatory molecules and inflammatory cytokine secretion, which is accompanied with reducing F I G U R E 2 Mechanism underlying improving effects of berberine on Treg/Th17 balance. Berberine can shift differentiation of naïve CD4 + T cells into CD4 + Foxp3 + Treg cells through activating AhR transcription factor and up-regulating CYP1A1 Th1/Th17 population and ameliorating severity and progression of autoimmune disorders.

| MODUL ATORY EFFEC TS OF B ERB ERINE ON INFL AMMATORY MACROPHAG E S
Macrophages are the major innate immune cells present in almost every tissue or organ system and mainly act as phagocytic cells that engulf and digest cellular debris resulted from apoptosis, foreign substances, microbes and pathogens. 100 In addition to acting as profes- Th17 cells (191)(192)(193)(194). In berberine-administrated mice with colitis, levels of such pro-inflammatory cytokines in the colon and sera were significantly decreased, which was accompanied by a reduction in colonic macrophages and percentages of IL-6 + , IL-1β + and TNF-α + secreting macrophages among splenocytes. 73 responses; derive resistance against intracellular pathogens and tumours; and promote tissue destruction, and alternative M2 subsets, which generate anti-inflammatory cytokines and contribute to tissue repair and remodelling as well as tumour progression. 105,106 Of note, it was indicated that anti-inflammatory M2 macrophages become the more dominant macrophage population after berberine treatment in colitis mice. 73 In conclusion, the inhibitory effect of berberine on inflammatory macrophages can be considered as another mechanism through which ameliorates autoreactive T-cell responses in autoimmune disorders.

| B ERB ERINE-MED IATED AT TENUATING DEMYELINATION AND AUTOIMMUNE INFL AMMATI ON IN THE CENTR AL NERVOUS SYS TEM
As berberine can cross the blood-brain barrier (BBB), evaluating the beneficial effects of berberine on neurodegenerative diseases has attracted extensive attention. 107 were markedly alleviated in berberine-treated EAE mice. 109,110 Elevated permeability of BBB is mainly responsible for the infiltration of leucocytes into CNS and plays a key role in the initiation and progression of MS and EAE, 111 whereas preventing BBB alterations limits the severity and progression of the disease. 112,113 Interestingly, berberine-mediated reduction of leucocyte infiltration and CNS inflammation in treated EAE mice was indicated to be due to reduced BBE permeability. 109 Of note, BBB permeability is known to be elevated by MMPs, 114 Freund's complete adjuvant. 64,133 The hallmark of EAN is PNS infiltration by inflammatory cells, particularly Th1 cells and macrophages, which secrete pro-inflammatory cytokines such as TNF-α at local sites of inflammation.
Interestingly, berberine treatment was shown to significantly ameliorate EAN by suppressing both cellular and humoural immunity that are implicated in GBS/EAN. 66 In berberine-treated EAN rats, clinical symptoms, including flaccid or dragging tail and hind limb paraparesis, were detected to be significantly alleviated. 66 The ame- cells. 66 Mitogen-activated protein kinase (MAPK) signalling, a regulator of TNF-α production, is known to be suppressed by berberine and can be a possible mechanism for the inhibitory effect of berberine on TNF-α secretion by Th1 cells in EAN. 66 Besides, growing evidence shows that IL-10, commonly known as an anti-inflammatory cytokine, plays a key role in both the initiation and progression of autoimmune diseases, through activating proliferation and antibody production of B cells. [144][145][146] In GBS and EAN, IL-10 secretion is elevated and positively associated with axonal nerve damage and antiganglioside antibodies. 146 This can explain the protective effects of berberine against neuropathy in EAN mice; however, underlying mechanisms remain largely unknown. 66

| AMELI OR ATING EFFEC TS OF B ERB ERINE ON O CUL AR MANIFE S TATI ON S AND AUTOIMMUNE INFL AMMATI ON OF U VEITIS
Uveitis is a blinding intraocular inflammatory disorder caused by an autoimmune response implicated the uveal layers, the retina and vitreous. 147

CO N FLI C T O F I NTE R E S T
We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

F I G U R E 3
The direct and indirect inhibitory effects of berberine on autoreactive Th17 and Th1 cells. Through the direct route, berberine suppresses differentiation of CD4 cells into Th17 and Th1 cells and inhibits activation of these cells through decreasing expression of TNF and IL17 cytokines via inhibiting STAT3 and RORgt. Through the indirect route, berberine suppresses the activation of both Th17 and Th1 cells via modulating the activity of macrophages and DCs via suppressing the production of inflammatory cytokine. Further suppressive effect of berberine on Th17 and Th1 cells is achieved through its promoting impact on Treg differentiation and activation via inducing activity of AhR transcription factor, CYP1A1 and Foxp3. Green and red arrows reflect promoting and inhibiting effects of berberine, respectively (lead); Project administration (supporting); Supervision (equal); Validation (equal).

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analysed in this study.