IFN-β Inhibits Human Th17 Cell Differentiation¹

Multiple sclerosis is an inflammatory demyelinating CNS disease, in which the autoimmune response is presumably directed against CNS myelin proteins. One of the hallmarks of the disease process is that the otherwise impermeable blood-brain barrier permits peripherally activated inflammatory cells to migrate into the CNS. During the episodes of disease activity, autoreactive T cells migrate into the CNS perivascular areas, where they upon reactivation with abundant myelin Ags lead to demyelination and axonal degeneration. The chronic inflammatory response leads to the neuronal conduction deficits associated with neurological symptoms and eventually to the loss of functional neuronal tissue and astrocytic gliosis, in which sclerotic plaques replace the functional neuronal tissue. Suppression of the inflammatory response, which leads to the prevention of CNS lesion formation, has been an effective therapeutic approach leading to the inhibition of disability progression in a large proportion of relapsing-remitting (RR)³ multiple sclerosis (MS) patients. IFN-β-1a has been used over the past 15 years as a first-line treatment for RR MS. Its efficacy has been documented in large placebo-controlled clinical trials by a significant decrease in the clinical relapse rate and new CNS lesion formation documented by the magnetic resonance imaging (1). Despite its established therapeutic efficacy, IFN-β’s complex mechanisms of action are not yet completely elucidated. Previous in vitro studies have suggested that the beneficial effects of IFN-β-1a in MS are due, at least in part, to an antiviral effect, the inhibition of APC maturation and T cell proliferation, the regulation of IL-12 and IL-10 cytokine transcription (2), and the blocking of cell transmigration through the blood-brain barrier (3). However, recent advances in our understanding of the development of the autoimmune response may allow us to identify novel mechanisms of action for this effective therapy.

The Th17 cell lineage is thought to play a critical role in the development of the autoimmune response in rheumatoid arthritis, psoriasis, juvenile diabetes, and autoimmune uveitis (4, 5). Several studies have documented the accumulation of Th17 cells in CNS MS lesions, suggesting that Th17 cells play a central role in the immunopathogenesis of MS (6, 7, 8). Lock et al. (7) have detected elevated IL-17 mRNA levels in active MS brain lesions in comparison to normal-appearing white matter and this was confirmed at the protein level by Tzartos et al. (8). Our recent study has identified an accumulation of Th17 cells in MS lesions, as evidenced by the increased expression of IL-17, retinoic acid-related orphan nuclear hormone receptor c (RORc) transcription factor, and multiple cytokines that mediate Th17 cell expansion (9). IL-17 gene expression is higher in the blood and cerebrospinal fluid mononuclear cells derived from MS patients than in those from healthy controls (HCs) (6). Similar to the other T cell lineages, the differentiation of Th17 cells is orchestrated by cytokines secreted by APCs. Although TGF-β and IL-6 induce Th17 cell differentiation in animal studies, this cytokine combination did not lead to the differentiation of human Th17 cells, which has required one of the following combinations: IL-6 and IL-23 (10), IL-1β and IL-6 (11), IL-1β and IL-23 (12, 13), TGF-β and IL-23 (14), or TGF-β and IL-21 (15). In contrast to mice, several human studies have reported that TGF-β even inhibits IL-17 synthesis (11). IL-23, which in mice only expands differentiated Th17 cells, is found to be a critical cytokine for Th17 cell differentiation in human studies (12, 13, 14, 16). Recently, Manel et al. (16) have demonstrated that a combination of all of these cytokines leads to the most effective differentiation of human Th17 cells.

In this study, we identified that IFN-β-1a inhibited the expression of IL-1β and IL-23, while it induced the expression of IL-12p35 and IL-27p28 in the dendritic cells (DCs) derived from MS patients and HCs. Our DC supernatant transfer experiments confirmed that the IFN-β-1a-induced changes in DC cytokine secretion suppress the Th17 markers RORc, IL-17A, and IL-23R. Furthermore, IFN-β-1a exhibited a direct effect on naive CD4⁺CD45RA⁺ T cells derived from both MS patients and HCs and inhibited their Th17 differentiation, as evidenced by the inhibition of RORc, IL-17A, and IL-23R expression.

Peripheral blood samples were collected upon obtaining institutional review board-approved informed consent from 54 MS patients and 40 HCs. The patients were not treated with immunomodulatory therapy at the time of blood collection and their treatment-free period was as reported previously from our laboratory (10, 17).

CD14⁺ monocytes were separated from PBMCs by negative selection using an EasyStep Negative Selection Monocyte Enrichment Kit (StemCell Technologies). DCs were generated from monocytes plated at 10⁶ cells/ml in low adhesion plates (Corning) in RPMI 1640 complete medium supplemented with 10% FBS, 2 mM glutamine, and 100 U/ml penicillin G, and streptomycin (Invitrogen) in the presence of GM-CSF (1000 IU/ml) and IL-4 (500 IU/ml; R&D Systems). At day 6, the cells were stained for the expression of CD11c⁺ (BD Pharmingen) and DC purity (>95%) was confirmed by flow cytometry. LPS-matured DCs were cultured in the absence or presence of IFN-β-1a for 6 h before the RNA isolation with an RNeasy kit (Qiagen).

CD4⁺CD5RA⁺ T cells were separated from the PBMCs using magnetic beads negative selection (>95% purity; Miltenyi Biotec) and stimulated at 2 × 10⁶ cells/ml with immobilized anti-CD3 (1 μg/ml, clone UCHT; R&D Systems) and anti-CD28 mAb (5 μg/ml, clone 28.2; BD Pharmingen) in Th17-polarizing conditions (IL-6 at 50 ng/ml and IL-23 at 10 ng/ml; R&D Systems) (10) in the absence or presence of IFN-β-1a (1000 IU/ml; R&D Systems). On day 3, the cells were collected for gene expression assays. The remaining cell cultures were expanded after 5 days with IL-2 (100 U/ml) for an additional 7 days and restimulated with plate-immobilized anti-CD3 and anti-CD28 mAb. The supernatants (SNs) were collected at 48 h for cytokine measurement by ELISA, and the cells were used for surface marker staining or lysed for RORc, GATA-3, and T-bet Western blot analysis. For Western blot analysis of STAT1 and STAT3 phosphorylation, CD4⁺CD45RA⁺ cells differentiated for 12 days in Th17-polarizing conditions were restimulated with anti-CD3 and anti-CD28 mAb in the absence or presence of IFN-β-1a for 1 and 6 h.

The DCs were matured with LPS (100 ng/ml; Sigma-Aldrich) in the absence or presence of IFN-β-1a for 48 h before supernatant collection. IFN-β in the SNs was neutralized with 4000 IU/ml anti-IFN-β Abs (goat anti-human polyclonal Ab; R&D Systems) 2 h before SN transfer. IL-1β and IL-23 (R&D Systems) were added to the SN at 10 and 20 ng/ml, respectively. IL-12p35 and IL-27p28 were neutralized with blocking Abs (polyclonal goat anti-human IgG Abs; R&D Systems catalog no. AF2526) at 10 μg/ml. Anti-CD3 plus anti-CD28 mAb-activated CD4⁺CD45RA⁺ T cells were cultured with 250 μl of SN for 3 days before RNA harvesting for the gene expression assays.

Total RNA isolated from DCs and CD4⁺CD45RA⁺ cells was reverse-transcribed to cDNA using an iSCRIPT cDNA synthesis kit (Bio-Rad). qRT-PCR was performed on an Applied Biosystems PRISM 7700 Sequence System. The primers for the 18S RNA, IL-1β, IL-23p19, suppressor of cytokine signaling 3 (SOCS3), IL-12p35, IL-27p28, RORc, IL-17A, IL-23R, and IL-10 were purchased from Applied Biosystems. Each sample was analyzed in triplicate. Relative gene expression was normalized against 18S RNA.

Immature and LPS-matured DCs were plated at 10⁶ cells per condition in the absence or presence of IFN-β-1a for 1 and 6 h before protein extraction, as indicated for the STAT1, STAT3, and SOCS3 blotting experiments. For detection of STAT1 phosphorylation, the DCs were pretreated with 20 μM fludarabine (FLUD; Sigma-Aldrich) for 1 h in OptiMEM medium and then matured with LPS in the absence or presence of IFN-β-1a for 1 h.

The CD4⁺CD45RA⁺ cells were cultured at 2 × 10⁶ cells per condition with IL-6 and IL-23 in the presence or absence of IFN-β-1a for 5 days, expanded with IL-2 for an additional 7 days, and restimulated with immobilized anti-CD3 plus anti-CD28 mAb for 48 h for RORc, T-bet, and GATA-3 blotting. For Western blot analysis of STAT1 and STAT3 phosphorylation, CD4⁺CD45RA⁺ cells differentiated for 12 days in Th17-polarizing conditions were restimulated with anti-CD3 and anti-CD28 mAb in the absence or presence of IFN-β-1a for 1 and 6 h. The cells were lysed with radioimmunoprecipitation assay buffer containing 2.5 mM sodium pyrophosphate, 1 mM NA₃VO₄, and 1 mM phenylmethylsulfonyl fluoride (Santa Cruz Biotechnology). The cell lysates were resolved with 5–15% gradient SDS-PAGE (Bio-Rad) and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% milk in TBS (20 mM Tris and 500 mM NaCl) and 0.1% Tween 20 at room temperature for 1 h followed by overnight incubation at 4°C with primary Abs against pSTAT1 (rabbit polyclonal Ab; Cell Signaling Technology) and total STAT1 (clone 9H2; Cell Signaling Technology), pSTAT3 (rabbit polyclonal Ab; Cell Signaling Technology) and total STAT3 (rabbit polyclonal Ab; Cell Signaling Technology), SOSC3 (rabbit polyclonal Ab; Santa Cruz Biotechnology); RORc (rabbit polyclonal Ab; Abcam), β-actin (clone Ac-74; Sigma-Aldrich), T-bet (clone 4B10; Santa Cruz Biotechnology), and GATA-3 (clone HG3-32; Santa Cruz Biotechnology). Secondary HRP-conjugated Ab was added at a dilution of 1/2000 for 1 h and the protein bands were detected with an ECL Detection System (Santa Cruz Biotechnology).

Supernatants from the CD4⁺CD45RA⁺ cell cultures were collected and stored at −80°C until the cytokine measurements. IL-17A and IL-10 secretion was measured with ELISA kits from Antigenix America and BD Pharmingen according to the manufacturers’ protocol.

IL-23R and CCR6 surface expression was measured by flow cytometry after CD4⁺CD45RA⁺ cell culture for 12 days in Th17-polarizing conditions in the absence or presence of IFN-β-1a and restimulation with anti-CD3 and anti-CD28 Abs for 48 h. The Th17-differentiated cells were stained with IL-23R-PE (clone 218213; R&D Systems), CCR6-PE Abs (clone 11AG; BD Pharmingen), and PE-conjugated mouse anti-human IgG isotype control (R&D Systems) and analyzed by FACSCalibur (BD Biosciences).

Statistical analyses of the qRT-PCR, ELISA, and FACS results were performed using a Wilcoxon-matched pairs signed ranks test (GraphPad, InStat), followed by a Benjamini and Hochberg correction for multiple comparisons. Adjusted p < 0.05 were considered significant.

Statistical analyses of gene expression in the CD4⁺CD45RA⁺ cells cultured with SNs from IFN-β-1a-treated DCs in the absence or presence of the indicated cytokines and blocking Abs were performed using a repeated measures ANOVA Friedman test with Dunnett’s posttest (GraphPad, InStat). A p < 0.05 was considered significant.

First, we investigated the effect of IFN-β-1a on the cytokines that promote Th17 differentiation, IL-1β and IL-23p19. IFN-β-1a significantly down-regulated IL-1β and IL-23p19 gene expression in DCs derived from the 10 MS patients (−2.0- and −2.9-fold, respectively) and from HCs (−3.0- and −4.7-fold, respectively) in comparison to the untreated samples (Fig. 1,A). Recent studies from our laboratory have also detected that IFN-β-1a inhibits the secretion of IL-1β and IL-23 by monocyte-derived DCs from RR MS patients (18). To further characterize the mechanisms involved in the IFN-β-1a-induced changes in DC cytokine gene expression, we demonstrated that IFN-β-1a induces STAT1 and STAT3 phosphorylation in DCs derived from MS patients (Fig. 1,B). Since STAT3 activation induces SOCS3 expression (19), we examined to what extent IFN-β-1a induces SOCS3 gene and protein expression. Our results revealed a significant up-regulation in SOCS3 gene expression upon IFN-β-1a treatment of the DCs derived from the 10 MS patients (1.6-fold) and HCs (1.7-fold). The IFN-β-1a-induced SOCS3 up-regulation was confirmed at the protein level by using Western blotting (Fig. 1 C).

We next examined the effect of IFN-β-1a on DC expression of cytokines that inhibit Th17 cell differentiation, IL-12p35 and IL-27p28. IFN-β-1a significantly induced IL-12p35 gene expression in the DCs from 10 MS patients (11.7-fold), while the increase in HCs (1.3-fold) did not reach statistical significance. IFN-β-1a also induced IL-27p28 gene expression in both MS patients and HCs (527.5- and 335.7-fold) compared with untreated samples (Fig. 2,A). We next sought to characterize the signaling events that lead to the IFN-β-1a-mediated IL-12p35 and IL-27p28 gene transcription and demonstrated that, consistent with previous reports (20), IFN-β-1a induces STAT1 phosphorylation (Fig. 1,B). We further confirmed the role of IFN-β-1a-induced STAT1 phosphorylation in the regulation of IL-27p28 gene transcription (5) by using the STAT1-specific inhibitor FLUD. LPS-matured DCs were cultured with IFN-β-1a or IFN-β-1a plus FLUD and analyzed for STAT1 phosphorylation. We demonstrated that the IFN-β-1a-induced IL-27p28 expression was mediated via STAT1 phosphorylation, since IL-27 gene expression decreased in the presence of the STAT1-specific inhibitor FLUD (Fig. 2,B). Fig. 2 B shows one representative of three similar Western blots and average results of three qRT-PCR experiments performed in triplicate.

Because previous studies have reported that IL-1β and IL-23 induce and IL-12 and IL-27 inhibit Th17 cell differentiation (12, 13), we proposed that the IFN-β-1a-induced changes in cytokine gene expression in DCs might collectively inhibit Th17 cell differentiation. To test this hypothesis, naive CD4⁺CD5RA⁺ T cells derived from 11 MS patients were stimulated with anti-CD3 and anti-CD28 Abs and cultured with the SNs from DCs matured in the absence or presence of IFN-β-1a. Before the SN transfer, the IFN-β-1a added to the DC cultures was blocked with anti-IFN-β-neutralizing Abs as described in Materials and Methods. The SNs from IFN-β-1a-treated DCs inhibited Th17 cell differentiation, as evidenced by the significantly decreased expression of the Th17 genes RORc (−6.2-fold), IL-17A (−15.2-fold), and IL-23R (−3.1-fold) in comparison to the SNs from untreated DCs (Fig. 3,A). The inhibitory effects of the SNs from DCs treated with IFN-β-1a on the Th17 cell differentiation were reversed by the addition of IL-1β, IL-23, anti-IL-27 mAbs, or a combination thereof, as evidenced by an increased/reversed IL-17A gene expression (Fig. 3,B). Fig. 3 B presents the results of eight experiments performed in triplicate. A repeated measures ANOVA Friedman test was used to test the significance of difference between gene expression in CD4⁺CD45RA⁺ cells cultured with SNs from IFN-β-1a-treated DCs and SNs supplemented with the indicated cytokines and/or blocking Abs. Asterisks indicate p < 0.05. In summary, our results demonstrate that IFN-β-1a modifies the expression of multiple cytokines in DCs, which collectively inhibit the Th17 cells’ differentiation.

A direct effect of IFN-β-1a on Th17 cell differentiation was investigated by treating activated CD4⁺CD45RA⁺ T cells cultured in the presence of the Th17-polarizing cytokines IL-6 and IL-23. IFN-β-1a treatment inhibited RORc gene expression in MS patients (−2.2-fold) and HCs (−2.3-fold) (Fig. 4,A), while the gene expressions of T-bet and GATA-3 were not changed (data not shown). These results were confirmed at the protein level by Western blotting. Fig. 4 B presents one representative of three similar blots.

Since RORc expression in humans, in contrast to mice, does not directly induce IL-17 cytokine secretion (21), we investigated the effect of IFN-β-1a on IL-17A production. IFN-β-1a inhibited IL-17A gene expression in the MS patients and HCs (−1.5- and −2.2-fold, respectively), as well as IL-17A protein secretion (−3.4- and −2.3-fold, respectively; Fig. 5,A). In contrast, IFN-β-1a up-regulated the IL-10 gene expression and the protein secretion in the T cells derived from the MS patients (3.7- and 1.5-fold, respectively) and the HCs (2.0- and 1.6-fold, respectively; Fig. 5 B). The gene expression and cytokine secretion of IFN-γ and IL-4 did not change (data not shown). In line with previous reports (22, 23), IFN-β-1a induced IL-12Rβ2 gene expression in the CD4⁺CD45RA⁺ T cells from MS patients, while the increase in HCs did not reach statistical significance (data not shown).

Studies of the mechanisms involved in the IFN-β-1a-medated inhibition of Th17 cell differentiation reveal that IFN-β-1a treatment of CD4⁺CD45RA⁺ cells cultured in the Th17-polarizing condition induces STAT1 and STAT3 phosphorylation (Fig. 5 C), which are implicated in the suppression of IL-17 and the induction of IL-10, respectively (24, 25), according to the previous animal studies.

We next examined the effect of IFN-β-1a on the Th17 cell markers IL-23R and CCR6 (13). IFN-β-1a significantly decreased the gene expression of IL-23R in the MS patients (−2.8-fold) and the HCs (−5.8-fold) (Fig. 6,A). The expression of IL-23R and CCR6 was confirmed at the protein level by flow cytometry. The cells treated with IFN-β-1a had a significantly decreased surface expression of IL-23R and CCR6 in both MS patients (−2.1- and −1.9-fold, respectively) and HCs (−2.1- and −1.7-fold, respectively). Fig. 6 B presents the results for IL-23R and CCR6 expression on the CD4⁺CD45RA⁺ cells derived from 10 MS patients and 10 HCs, as well as the representative staining for each surface marker.

Recent studies in mice deficient for IFN-β (26), type I IFN receptor (IFNAR) (27), and the Toll-IL-1R domain-containing adaptor-inducing IFN-β molecule involved in its downstream signaling (28) have demonstrated an increased susceptibility to experimental autoimmune encephalomyelitis (EAE) due to the lack of endogenous IFN-β signaling. These findings point to the role of endogenous IFN-β in the regulation of the adaptive immune response through the suppression of Th17 cells.

Our study has demonstrated that IFN-β-1a treatment decreases IL-1β gene expression in the DCs derived from MS patients and HCs. Moreover, the effect of IFN-β-1a-induced change in this cytokine production on Th17 cell differentiation was confirmed by adding back IL-1β to the SNs derived from IFN-β-1a-treated DCs, which reversed the SN suppressive effect on the Th17 cell differentiation (Fig. 3 B). The reversal of the IFN-β-1a-treated DC SN inhibitory effect on the Th17 cell differentiation, which was achieved by adding IL-1β, IL-23, and by IL-27 neutralization, indicates that multiple cytokines play complementary roles in Th17 cell differentiation.

Studies reporting increased levels of IL-23p19 protein in acute MS brain lesions and a higher production of IL-23 by DCs from MS patients in comparison to those from HCs (29) are in agreement with animal studies demonstrating that neutralization of IL-23 or the lack of IL-23p19 gene expression completely ameliorated EAE (30, 31). We have demonstrated in this study that IFN-β-1a decreases IL-23p19 gene expression in DCs from both MS patients and HCs. The functional effect of the inhibited IL-23 production on Th17 cell differentiation was confirmed by SN transfer experiments, since adding back IL-23 reversed the suppression of the IFN-β-treated DC SNs on the IL-17A gene expression in the differentiated Th17 cells. Our findings are in accordance with a recent human study that reported decreased levels of IL-23p19 mRNA in the PBMCs from MS patients treated with IFN-β-1a (32).

Next, we sought to identify the molecular mechanisms of IFN-β-1a-mediated inhibition of IL-1β and IL-23p19 production. IFN-β-1a induced a rapid phosphorylation of STAT3 in DCs, which acts as a transcription factor for SOCS3 (19, 33). In addition to the reported capacity of IFN-β-1a to induce SOCS3 (34), previous studies have reported that SOCS3 inhibits IL-1β (33, 35) and IL-23 (10, 36). Based on our results, we propose that IFN-β-1a suppressed IL-1β and IL-23p19 gene expression through the induction of SOCS3.

Although multiple earlier studies proposed a central proinflammatory role for IL-12 in the development of the autoimmune response and studies of IFN-β mechanisms of action have reported that IFN-β suppresses IL-12p40 and IL-12p70 secretion (2), Cua et al. (31) have reported that IL-23 but not IL-12 is critical for the induction of EAE, since mice deficient for IL-12p35 and IL-12Rβ2 were highly susceptible to EAE (37). In line with the recently proposed immunoregulatory role of IL-12 in the control of the autoimmune response, Annunziato et al (13) reported that IL-12 suppresses RORc expression in human Th17 cell clones. Regarding the effect of IFN-β, Gautier et al. (38) have reported that IFNAR⁻/⁻ mice had a decreased IL-12p35 accumulation, suggesting that endogenous IFN-β signaling mediates IL-12p35 expression. Consistent with these results, we report for the first time that in MS patients IFN-β-1a increases the expression of IL-12p35, an IL-12-specific component that binds to p40 to form a functional p70 heterodimer. While in agreement with previous reports we confirm that IFN-β-1a induces IL-12Rβ2 expression on T cells (22, 23), IL-12 did not play a critical role in the suppression of Th17 cell differentiation in our system, since IL-12 blockade did not reverse the inhibitory effect of SNs from IFN-β-1a-treated DCs on the Th17 cell differentiation (data not shown).

Although the inhibitory effects of IL-27 on the Th17 cell differentiation (5, 25, 39, 40) have been established in mice, the role of IL-27 in the regulation of human Th17 cell differentiation was only recently implicated in the immunomodulatory mechanisms of simvastatin and IFN-β-1a in patients with MS (10, 18). Nagai et al. (41) have reported that IFN-β treatment of human DCs induces IL-27 gene expression, but its functional effect on the Th17 cell differentiation was not addressed. A recent animal study (28) proposed a pivotal role for IL-27 in the endogenous IFN-β-mediated suppression of EAE. In IFNAR^−/− and TRIF^−/− mice, an increased susceptibility to EAE was associated with reduced IL-27 production, while the administration of IL-27 suppressed the clinical disease activity, suggesting that the IFN-β-induced IL-27 expression is a critical mechanism for the immunoregulatory effect of IFN-β. We have demonstrated in this study that IFN-β-1a induces the expression of IL-27p28 in human DCs and that their transferred SNs inhibit Th17 cell differentiation, as assessed by the decreased IL-17A gene expression. To study the mechanisms involved in the IFN-β-1a-induced IL-27 gene expression, we examined whether the phosphorylation of STAT1, a transcription factor for IL-27 (5) that is induced by IFN-β-1a (20), mediates the IFN-β-1a-induced IL-27 gene expression. Our results confirmed that IFN-β-1a-induced IL-27 expression is mediated by the STAT1 phosphorylation, since it was inhibited by the STAT1-specific inhibitor FLUD.

Furthermore, we report that IFN-β-1a treatment of naive CD4⁺CD45RA⁺ T cells in Th17-polarizing conditions leads to a decrease in the gene expression of the Th17 master regulatory transcription factor RORc, the cytokine IL-17A, and the Th17 cell surface marker IL-23R. The IFN-β-1a-mediated suppression of Th17 cell differentiation is also supported by decreased expression of the Th17 signature surface markers IL-23R and CCR6. IFN-β therapy-induced expression of IL-10 has been regarded as a hallmark of the therapeutic effect of IFN-β-1a in MS patients (2). In agreement with previous studies, we demonstrated that IFN-β-1a treatment of naive CD4⁺CD45RA⁺ T cells in Th17-polarizing conditions induces IL-10 gene expression and cytokine secretion.

Studies of the mechanisms involved in IFN-β-1a’s direct inhibition of Th17 cell differentiation revealed that IFN-β-1a induces STAT1 and STAT3 phosphorylation in CD4⁺CD45RA⁺ cells cultured in Th17-polarizing conditions. According to the reports from animal studies (25), we propose that in the human system STAT1 phosphorylation directly inhibits Th17 cell differentiation, while STAT3 phosphorylation mediates the induction of IL-10, which subsequently inhibits Th17 cell expansion (24, 25, 39).

In summary, our study has demonstrated that the therapeutic effect of IFN-β-1a in MS is associated with changes in the DC expression of cytokines that orchestrate Th17 cell differentiation. Furthermore, IFN-β-1a directly suppresses the differentiation of Th17 cells, as evidenced by down-regulation of RORc, IL-17A, and IL23R and induction of the IL-10 expression. Our study proposes a novel IFN-β-1a’s mechanism of action, which specifically targets the Th17 cell lineage and the development of the autoimmune response in MS.

After we had submitted our revised manuscript, Durelli et al. (42) reported that IFN-β-1a treatment decreases the frequency of IL-17-producing CD4⁺ cells in MS patients. Their results support our study, which further elucidates the IFN-β-1a mechanisms of action in DCs, previously identified (27) target for the IFN-β immunoregulatory effect against the Th17 cell-mediated autoimmune response.

We thank Danielle Speer, Leslie Ingram, and Selena Spears for help with blood sample collection.

The authors have no financial conflict of interest.

The authors have no financial conflict of interest.