Pro-Inflammatory Th17 Cells and Glucocorticoid Resistance: Implications for Chronic Inflammatory Diseases and Their Treatment
Th17 have heterogeneous phenotypes and functions, and can have pro- or anti-inflammatory activities. Pathogenic Th17 cells have been associated to several inflammatory and autoimmune diseases. Besides IL-17A and IL-17F, they also produce GM-CSF and IFN-γ, and express high levels of IL-23R. These cells are refractory to glucocorticoid (GC)-mediated T cell suppression, with different degree of resistance in different diseases. GC resistance is mediated by multiple mechanisms that need to be elucidated. Different subsets of Th17 cells with distinct GC sensitivity probably exist, and GC treatment for a long period could be responsible for the depletion of GC-sensitive T cells and the expansion of GC resistant subsets, thus worsening the condition of patients affected by inflammatory diseases.
CD4+ T helper (Th) lymphocytes are crucial players in immune responses. These cells are classified in distinct subtypes according to the different cytokines they produce and the different transcription factors they express. The characteristics of the antigen and of the microenvironment within the site of inflammation decide which type of T cell subset is activated.
Th1 and Th2 cells are the first two subsets identified in 1986 by Mosman et al. . Th1 cells, characterized by the expression of T-bet and the production of IFN-γ, are mainly involved in the immune responses against intracellular bacteria; Th2 cells, characterized by the expression of GATA-3 and the production of IL-4, IL-5 and IL-13, drive immune responses during helminth infections and allergies .
Further studies have demonstrated that Th lymphocyte populations are not limited to these two subsets, as other subtypes exist. In 2005 it was demonstrated that a distinct T cell subset is responsible for the production of the cytokine IL-17 (also indicated as IL-17A), and it was named Th17 . These cells also produce the cytokines IL-17F, IL-22 and GM-CSF, and are characterized by the expression of the transcription factor RORγt , and of the chemokine receptor CCR6 . IL-17 promotes recruitment and activation of neutrophils and macrophages in the site of inflammation and is fundamental for the activation of efficacious host immune responses against intracellular bacteria and fungi.
However, Th17 have been also associated to the pathogenesis of several inflammatory and autoimmune diseases [6-8]. Patients with multiple sclerosis have high concentrations of IL-17 in their serum and cerebrospinal fluid. Furthermore, it has been demonstrated that Th17 cells have a greater ability when compared to other T cell subsets to pass across the blood brain barrier and to infiltrate the parenchyma of the central nervous system . Th17 cells have also been implicated in rheumatoid arthritis (RA) and in inflammatory bowel disease (IBD). Experimental studies have demonstrated the presence of IL-17 in inflamed joints of animal models of RA , and clinical studies have evidenced increased percentages of Th17 in patients affected by RA , and by IBD . Due to their pathogenetic role in different chronic inflammatory diseases, Th17 cells represent a key target of therapeutic approaches.
Recent studies performed in animal models have demonstrated that the characteristics and the role in human physiopathology of Th17 are more complex than they were considered when this cell subset was identified. Th17 have heterogeneous phenotypes and functions, and can have pro- or anti-inflammatory activities depending on the stimuli they receive .
Figure 1. Phenotypic characteristics of pro- and anti-inflammatory Th17 cells. Protective anti-inflammatory Th17 cells, in addition to the transcription factor RORγT, also express the transcription factor FoxP3 (expressed also by the anti-inflammatory Treg cells), and produce the anti-inflammatory cytokine IL-10. The pathogenic pro-inflammatory Th17 cells express high levels of IL-23 receptor and integrin avβ3 on their surface, produce the pro-inflammatory cytokines granulocyte-macrophage colony-stimulating factor and IFN-γ, and express the Th1 transcription factor T-bet.
In particular, the pathogenic pro-inflammatory Th17 cells express high levels of IL-23 receptor and integrin avβ3 on their surface, produce the pro-inflammatory cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and IFN-γ and express the Th1 transcription factor T-bet, while the protective Th17 cells produce the anti-inflammatory cytokine IL-10 [13,14].The molecular microenvironment in which Th17 mature is able to influence the differentiation program of these cells, determining the development of a protective or a pathogenic immune response.
Stimulation of T cells with the cytokines TGF-β and IL-6 generates Th17 which produce IL-17A, IL-17F, IL-21 and IL-10, express the transcription factors RORγt and FoxP3, and show immunoregulatory functions being able to suppress effector T cells . These cells are not able to induce autoimmunity when transferred in vivo, and are considered protective . The presence of IL-23 promotes the differentiation of pathogenic Th17 cells that, if transferred in vivo, trigger type I diabetes in NOD mice or experimental autoimmune encephalomyelitis (EAE) in the mouse model of multiple sclerosis [15,16]. These data together with the observation that IL-23 and IL-23R knockout mice are resistant to the development of autoimmunity  strongly indicate a key role of IL-23 in promoting the differentiation of pathogenic Th17 cells able to induce autoimmunity .
Different studies have clearly demonstrated the involvement in autoimmune diseases of pathogenic Th17 cells. Besides IL-17A and IL-17F, they also produce GM-CSF and IFN-γ, and express high levels of IL-23R. In contrast, non-pathogenic Th17 produce IL-17 and IL-10, and show anti-inflammatory activities. An expansion of pro-inflammatory Th17 cells, also indicated as Th17.1, has been observed in CNS from mice with EAE and in patients with multiple sclerosis [18,19]. IFN-γ producing Th17 cells have also been detected in the synovial fluid from children affected by juvenile idiopathic arthritis , and they have been implicated in the severity of graft versus host disease after allogeneic hematopoietic stem cell transplantation .
These pathogenic cells concomitantly express the transcription factors RORγt and T-bet, and the pro-inflammatory chemokine receptors CCR6 and CXCR3. Their development is sustained by the cytokine IL-23 , and inhibited by IL-21 . Due to the emerging role played by Th17.1 cells in the pathogenic mechanisms characterizing various chronic inflammatory autoimmune diseases, this T cell subset is becoming an interesting therapeutic target.
Th17 and glucocorticoid resistance
Glucocorticoids (GCs) are drugs commonly used to treat different inflammatory conditions including asthma, rheumatoid arthritis, multiple sclerosis and Crohn’s disease. A major problem is represented by the existence of steroid-resistant individuals who need very high doses of GCs to control the disease. Studies in animal models and in humans strongly suggest the involvement of a steroid-resistant Th17 phenotype .
Figure 2. Differences in molecular patterns characterizing Th17 cells with distinct glucocorticoid (GC) sensitivity. Pro-inflammatory GC-sensitiveTh17 cells stably express on the surface the molecule multi-drug resistance type 1 (MDR1), and are refractory to GC-mediated T cell suppression. These cells also express high levels of BCL-2 and RORγt, which confer resistance to GC-induced apoptosis, and of STAT3 that inhibits glucocorticoid receptor functions. Furthermore, data on the ability of anti-IL-6R mAbs to restore GC sensitivity in Th17 cells, strongly suggest a role of high IL-6 levels in the promotion of Th17 GC resistance. Differently, the GC-sensitive Th17 cells do not express MDR1 and are characterized by the expression of the protein glucocorticoid-induced leucine zipper (GILZ) that, by inhibiting the Th17 transcriptional program essential for Th17 cell differentiation, is a direct mediator of GC inhibitory activity of Th17 inflammation.
In a recent study, Ramesh and colleagues  demonstrated that pro-inflammatory Th17 cells, producing both IL-17 and IFN-g, stably express on the surface the molecule multi-drug resistance type 1 (MDR1), an ATP-dependent multi-drug transporter, also known as P-glycoprotein, associated to tumor resistance to chemotherapy [25; see also Important new features and glucocorticoid-resistance of pro-inflammatory Th17 Cells]. The same authors demonstrate that patients affected by Crohn’s disease have an expansion in their gut of MDR1+ IFN-g-producing Th17, and that these cells are refractory to GC-mediated T cell suppression. Nevertheless, blocking MDR1 with the selective inhibitor elacridar did not restore Th17 sensitivity to GC treatment.
GCs mediate their activity through the glucocorticoid receptor (GR). Intracellular levels of GR and GR isoform composition determine cell responsiveness to GC [26,27]. Of interest, different Th subsets have distinct GC sensitivity, as recently reviewed by Banuelos and Lu . Th1 cells are susceptible to GC-induced apoptosis and cytokine suppression, Th2 are sensitive only to cytokine suppression, while Th17 are resistant to both GC-mediated activities.
This gradient of GC sensitivity among Th subsets is reflected by different levels of GC resistance in inflammatory diseases [29,30]. Banuelos and Lu put the interesting questions whether autoimmune diseases can be categorized into endotypes characterized by distinct GC sensitivity, and whether long-term GC therapy determines the shift from a GC-sensitive to a GC-resistant Th subset.
In a more recent review Banuelos and colleagues focus the attention on the different GC sensitivity/resistance of Th17 cells observed in different inflammatory diseases . For example, Th17 are sensitive to GC treatment in psoriasis while they are resistant in Crohn’s disease [32,24]. The authors suggest that differences in molecular patterns characterizing human chronic inflammatory diseases strongly influence GC sensitivity of Th17.
Various factors have been implicated in GC resistance. In a murine model of asthma and in humans with mild allergies Banuelos et al. have demonstrated that Th17 resistance to GC-induced apoptosis is associated to high levels of the anti-apoptotic factor BCL-2 expressed in these cells . Furthermore, the transcription factor RORγt, which characterizes Th17, is able to counteract GC-induced apoptosis , and STAT3, another transcription factor expressed by these cells, inhibits GR functions . Studies in multiple sclerosis have suggested a role for the cytokine IL-6 in GC resistance of Th17. GCs inhibit the production of this pro-inflammatory cytokine in multiple sclerosis, and it has been observed that high levels of IL-6 are associated to the presence of Th17 resistant to GC treatment . Of interest, the use of anti-IL-6R mAbs enhances the ability of GCs to inhibit Th17 response . All these findings strongly suggest that Th17 cells are intrinsically resistant to GC suppression.
Nevertheless, recent evidence demonstrate that in certain diseases Th17 cells are sensitive to GC inhibitory activity . For example, GCs are efficacious in reducing plaques and associated symptoms in psoriasis, a disease characterized by a strong Th17-mediated inflammation. The protein glucocorticoid-induced leucine zipper (GILZ) has been identified as a direct mediator of GC inhibitory activity of Th17 inflammation in psoriasis. This protein inhibits the Th17 transcriptional program directly binding to the promoters of the genes BATF, STAT3, IRF4, and RORct, which are essential for Th17 cell differentiation. As GCs are able to increase GILZ levels in Th17 cells, the decreased Th17 cell number and activity observed in psoriasis has been at least in part attributed to GILZ .
All these data indicate that GC resistance is mediated by multiple mechanisms, which, at least in part, need to be elucidated. As Banuelos and colleagues conclude , different subsets of Th17 cells with distinct GC sensitivity probably exist, and long-term GC treatment could deplete GC-sensitive T cells and promote the expansion of GC resistant subsets, thus sustaining the inflammatory process. GC resistance represents a fundamental topic to identify new therapeutic approaches able to contrast more efficiently the various chronic inflammatory diseases. In a recent study, by the use of a murine model of autoimmunity and human T cells in vitro, Schewitz-Bowers and colleagues  have demonstrated that GC-resistant Th17 are sensitive to the calcineurin inhibitor cyclosporine A, sustaining the usefulness of alternative therapeutic approaches with reduced effects in nonimmune tissues for the treatment of GC-resistant inflammatory diseases.
Elisabetta Profumo – Department. of Cardiovascular, Dysmetabolic and Aging-associated Diseases, Istituto Superiore di Sanità, Rome, Italy; E-mail: firstname.lastname@example.org; Ph: +39-06-49902760; Fax: +39-06-49902886
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