cells

Article -β Intensifies -23-Driven Pathogenicity of T Helper Cells in Neuroinflammatory Disease

Agnieshka Agasing, James L. Quinn, Gaurav Kumar and Robert C. Axtell *

Department of and Clinical , Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA; [email protected] (A.A.); [email protected] (J.L.Q.); [email protected] (G.K.) * Correspondence: [email protected]; Tel.: +1-(405)-271-2354

Abstract: Interferon (IFN)-β is a popular therapy for (MS). However, 25–40% of patients are nonresponsive to this therapy, and it worsens neuromyelitis optica (NMO), another neuroinflammatory disease. We previously identified, in both NMO patients and in mice, that IFN-β treatment had inflammatory effects in T Helper (TH) 17-induced disease through the production of the inflammatory IL-6. However, other studies have shown that IFN-β inhibits the differentiation and function of TH17 cells. In this manuscript, we identified that IFN-β had differential effects on discrete stages of TH17 development. During early TH17 development, IFN-β inhibits IL-17 production. Conversely, during late TH17 differentiation, IFN-β synergizes with IL-23 to promote a pathogenic T cell that has both TH1 and TH17 characteristics and expresses elevated levels of the potent inflammatory IL-6 and GM-CSF and the BLIMP. Together, these findings help resolve a paradox surrounding IFN-β and TH17-induced disease and  illuminate the pathways responsible for the pathophysiology of NMO and MS patients who are  IFN-β nonresponders. Citation: Agasing, A.; Quinn, J.L.; Kumar, G.; Axtell, R.C. Interferon-β Keywords: experimental autoimmune encephalomyelitis; T helper 17; interferon-beta Intensifies Interleukin-23-Driven Pathogenicity of T Helper Cells in Neuroinflammatory Disease. Cells 2021, 10, 2139. https://doi.org/ 1. Introduction 10.3390/cells10082139 IFN-β remains a popular treatment for relapsing-remitting multiple sclerosis (RRMS). However, despite the clinical efficacy of IFN-β, approximately 25–40% of RRMS patients Academic Editor: Edwin Wan respond poorly to this treatment [1], and it consistently worsens disease in patients with neuromyelitis optica (NMO), a neuroinflammatory disease often misdiagnosed as MS [2,3]. Received: 10 July 2021 These observations demonstrate that there are limitations to the therapeutic benefits of IFN-β. Accepted: 19 August 2021 β Published: 20 August 2021 IFN- is a pleiotropic cytokine that has many effects on the immune system. Although the mechanism of action of IFN-β in MS is currently unclear, some have speculated that the

Publisher’s Note: MDPI stays neutral efficacy of this therapy is achieved by inhibiting the function of T Helper (TH) 17 cells [4–6]. with regard to jurisdictional claims in However, biomarker studies of MS and NMO patients, as well as animal experiments, have published maps and institutional affil- confounded this theory. iations. In the MS population, there are clear responders and nonresponders to IFN-β therapy. The differences in response in patients have been associated with therapeutic noncom- pliance [7], the development of neutralizing antibodies [8], as well as differences in the immunological signatures of patients [9]. We previously identified that the balance of TH1 to TH17 signatures determined the efficacy of IFN-β therapy. High TH1 signatures Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. and high levels of IL-7 before initiation of therapy are associated with a good response to β This article is an open access article IFN- [10,11]. Alternatively, elevated TH17 cytokine signatures in a subset of MS patients distributed under the terms and are associated with nonresponders [9,11,12]. In NMO, a disease with a strong association conditions of the Creative Commons with TH17 signatures [13], patients have an increased frequency of relapses when treated Attribution (CC BY) license (https:// with IFN-β [2,3,14]. creativecommons.org/licenses/by/ We recently reported, in both NMO patients and in mice, that IFN-β treatment had 4.0/). inflammatory effects in T Helper (TH) 17 induced through the action of the inflammatory

Cells 2021, 10, 2139. https://doi.org/10.3390/cells10082139 https://www.mdpi.com/journal/cells Cells 2021, 10, 2139 2 of 9

cytokine IL-6 [15]. However, other studies have shown that IFN-β inhibits TH17 [4–6]. This manuscript now provides data to reconcile the conflicting reports of IFN-β on the differentiation and pathogenic function of TH17 cells in neuro-autoimmune diseases.

2. Materials and Methods 2.1. Mice Eight- to ten-week-old female C57BL/6J mice were purchased from Jackson Labora- tory, housed in the Oklahoma Medical Research Foundation animal facility and treated in compliance with the Institutional Animal Care and Use Committee (IACUC).

2.2. Culturing Encephalitogenic T Cells

Donor C57BL/6 mice were subcutaneously immunized with 150 µg MOG35–55 peptide (Genemed Synthesis Inc., San Antonio, TX, USA) emulsified in Complete Freund’s adjuvant (5 mg/mL heat-killed M. tuberculosis). This was followed by an intraperitoneal (I.P.) injection of 250 ng of Bordetella pertussis toxin (List Biological Laboratories Inc., Campbell, CA, USA) in PBS at day 0 and day 2 post-immunization. Donor mice were sacrificed day 10 post-immunization, and spleens and lymph nodes were harvested and mechanically disrupted to obtain a single-cell suspension. 2.5 × 106 cells/mL were stimulated for three days with MOG35–55 (10 µg/mL), IL-23 (10 ng/mL; R & D Systems, Minneapolis, MN, USA) and anti-IFN-γ (10 µg/mL; eBioscience, San Diego, CA, USA) in the presence or absence of IFN-β (100 U/mL; PBL, Novato, CA, USA) in complete RPMI 1640 (Gibco, Waltham, MA, USA). To block IL-6 signaling, cells were cultured with anti-IL-6 (10 µg/mL; BD Biosciences, Franklin Lakes, NJ, USA).

2.3. Adoptive Transfer EAE C57BL/6J recipient mice were I.P. injected with 5 × 106 cells and monitored daily for clinical scores. Paralysis was assessed using the following standard clinical score: (0) healthy, (1) loss of tail tone, (2) partial hind-limb paralysis, (3) complete hind-limb paralysis, (4) forelimb paralysis, and (5) moribund/dead. Transfer EAE mice were sacrificed at day 15, and spinal cords were fixed and sectioned for histological analysis using H & E and Luxol fast blue staining.

2.4. Isolation of CNS-Infiltrating Cells Mice were perfused with PBS, and spinal cords were collected and homogenized through mechanical disruption. CNS homogenates were incubated with DNAse (5 µL/mL; Sigma) and collagenase D (4 mg/mL; Roche) at 37 ◦C for 1 h, and cells were isolated using a Percoll gradient.

2.5. Flow Cytometric Analysis of Cytokine Expression Cells were stimulated with 50 ng/mL PMA (Sigma-Aldrich, St. Louis, MO, USA), 500 ng/mL ionomycin (Sigma-Aldrich), and monensin (BD Biosciences) for 4 h as de- scribed by the manufacturer’s protocol. Cells were then stained with CD4 (eBioscience) or CD19 (eBioscience), fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences) and stained for IL-17 (BioLegend), IFN-γ (BioLegend) and/or IL-6 (eBioscience). Data was collected on LSRII (BD Biosciences) and analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA).

2.6. Analysis of Cytokine Production by ELISA Culture supernatants were collected for ELISA. IL-17, IL-10, IL-6 and GM-CSF levels were assessed using anti-mouse ELISA kits (eBioscience).

2.7. RNA Isolation and Quantitative Real Time RT-PCR RNA was extracted from cells with an RNAeasy Mini Kit (QIAGEN). cDNA was generated using an iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). qRT-PCR Cells 2021, 10, 2139 3 of 9

was performed using forward and reverse primers (see Table S1), cDNA and iQ SYBR Green Supermix (Bio-Rad) on a 7900HT Fast Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). Sample reactions were carried out in triplicate. GAPDH was used as a reference , and the relative gene expression analysis was measured using the 2-∆∆Ct method [16].

2.8. Intracellular Staining of Phosphorylated STAT Proteins Cells from the spleen and lymph nodes of MOG-immunized mice were cultured as described above. After 0, 15, 30 and 60 min, cells were fixed with 4% paraformaldehyde for 10 min, centrifuged at 1500 rpm for 5 min and permeabilized on ice with 100% cold methanol for another 10 min. Cells were then washed with PBS and stained with the following antibodies: anti-CD4 (eBioscience), pSTAT1 (BD Biosciences), pSTAT3 (BD Bio- sciences), pSTAT4 (eBioscience), pSTAT5 (eBioscience) and pSTAT6 (eBioscience) for flow cytometric analysis.

2.9. In Vitro TH17 Differentiation Spleen cells from healthy mice were depleted of CD8+ T lymphocytes using CD8a (Ly-2) MicroBeads (Miltenyi Biotec) and cultured in two sequential phases. For TGF-Beta- dependent TH17 differentiation, cells were cultured (5 × 106 cells/mL) with antibodies against CD3 (eBioscience; 10 µg/mL) and CD28 (eBioscience; 0.5 µg/mL) and the cytokines IL-6 (R & D Systems; 20 ng/mL) and TGF-β (R & D Systems; 1 ng/mL) in the presence or absence of IFN-β (100 U/mL) for three days. For IL-23-dependent TH17 differentiation, the cells initially cultured with IL-6 and TGF-β (without IFN-β) were washed and recultured for an additional three days with anti-CD3 (10 µg/mL), anti-CD28 (0.5 µg/mL), IL-23 (10 ng/mL) and anti-IFN-γ (10 µg/mL) with or without IFN-β (100 U/mL).

2.10. Statistical Analysis Data are presented as means ± s.e.m., and statistical significance was determined using Mann–Whitney tests. In the case of three or more data sets, means were compared using the Kruskal–Wallis test with a Dunn’s multiple comparison correction. Differences were considered significant for p < 0.05. Statistical analyses were performed using Prism 6 (GraphPad, San Diego, CA, USA).

3. Results 3.1. IFN-β Potentiates the Pathogenicity of Myelin-Enriched TH17 Cells Previous studies have shown that IFN-β treatment worsened TH17-induced EAE [11]. To understand how IFN-β increased TH17 disease severity, we assessed the direct effects that IFN-β stimulation had on the encephalitogenic capacity of IL-23-derived TH17 cells. We cultured donor cells with MOG35–55 and IL-23 in the presence or absence of IFN-β prior to the transfer into healthy recipient mice. Mice which received IL-23- and IFN- β-stimulated cells showed exacerbated disease scores compared to recipient mice that received cells stimulated with only IL-23 (Figure1A). In agreement with the clinical scores, the histological analysis of spinal cords indicated that mice receiving IFN-β-stimulated cells had increased CNS-inflammation compared to mice receiving cells stimulated with only IL-23 (Figure1B). We next examined the production of IL-17 and IFN-γ in the CNS of recipient mice 15 days following adoptive transfer. In the spinal cords, mice receiving cells stimulated with IFN-β and IL-23 had significantly elevated numbers of IFN-γ+IL-17- and IFN-γ+IL-17+ TH cells compared to mice that received cells stimulated with only IL-23 (Figure1C–E). The differences in the numbers of IFN-γ-IL-17+ TH cells were nearly significant between both groups of mice (Figure1F). Cells 2021, 10, x FOR PEER REVIEW 4 of 9

- + Cells 2021, 10differences, 2139 in the numbers of IFN-γ IL-17 TH cells were nearly significant between both 4 of 9 groups of mice (Figure 1F).

Figure 1. IFN-β increases the pathogenicity of IL-23-stimulated, myelin-enriched TH17 cells. (A) Clinical scores of mice Figure 1. IFN-β increases the pathogenicity of IL-23-stimulated, myelin-enriched TH17 cells. (A) with EAE induced by adoptive transfer of splenocytes cultured with MOG35–55, IL-23 and anti-IFN-γ in the presence (+) or absenceClinical (−) of scores recombinant of mice mouse with IFN-EAEβ induced(n = 20–21 by mice adop pertive group transfer from threeof splenocytes independent cultured experiments). with MOG (B) Histology35– of spinal55, cord IL-23 sections and anti-IFN- from representativeγ in the presence mice from (+) the or− absenceIFN-β and (−) + of IFN- recombinantβ groups. Sections mouse were IFN- obtainedβ (n = 20 15–21 days post- transfermice and per stained group with from H & three E. Arrows independent indicate demyelinating experiments). lesions (B) inHistology the brain andof spinal spinal cord.cord (sectionsC) Representative from flow cytometryrepresentative plots of CD4+ mice T cellsfrom expressing the − IFN- IFN-β γandand + IL-17 IFN- inβthe groups. spinal cordsSections of EAE were mice, obtained 15 days 15 post-transfer. days post- Absolute numberstransfer of T helperand stained cells that with are H (D &) IFN-E. Arrowsγ+IL-17 indicate-,(E) IFN- demyelinatingγ+IL-17+ and (F lesions) IFN-γ- inIL-17 the+ brainin the and spinal spinal cords cord. of EAE mice. Error(C bars) Representative represent means flow± s.e.m. cytometry Statistical plots analysis of CD4+ was T performed cells expressing using Mann IFN-−Whitneyγ and IL-17 tests. inp valuesthe spinal < 0.05 were + consideredcords of significant EAE mice, (** indicates15 days ppost-transfer.≤ 0.01, * indicates Absolutep < 0.05). numbers of T helper cells that are (D) IFN-γ IL- 17-, (E) IFN-γ+IL-17+ and (F) IFN-γ-IL-17+ in the spinal cords of EAE mice. Error bars represent means ± s.e.m. Statistical analys3.2.is IFN- wasβ Inducesperformed Inflammatory using Mann Cytokines−Whitney in Myelin-Enrichedtests. p values < TH170.05 were Cells consid- ered significant (** indicatesIt has p ≤ been0.01, reported* indicates that p < IFN- 0.05).β inhibits IL-17 expression [4–6]. However, we detected increased numbers of IL-17+ TH cells in the CNS of recipient mice that received IFN- 3.2. IFN-β Induces Inflammatoryβ-treated cells. Cytokines Therefore, in Myelin-Enriched we next determined TH17 the Cells phenotype of the cells before they It has been reportedwere transferred that IFN-β into inhibits recipient IL-17 mice. expression Strikingly, [4–6]. we However, observed that we detected CD4+ T cells treated increased numberswith of IL-17 IFN-β+ THand cells IL-23 in had the increased CNS ofIL-17 recipient expression mice measuredthat received by FACS, IFN- qRT-PCRβ- and + treated cells. Therefore,ELISA we compared next determined to CD4 T cellsthe treatedphenotype with of only the IL-23 cells (Figure before2A–C). they Wewere also assessed the expression of other inflammatory cytokines. We observed that in IL-23-stimulated transferred into recipient mice. Strikingly, we observed that CD4+ T cells treated with IFN- conditions, IFN-β significantly elevated IL-6, although the amounts were substantially β and IL-23 had increasedlower than IL-17 IL-17 expression (Figure2D). measured T cells are by not FACS, thought qRT-PCR of as a majorand ELISA source com- of IL-6. In these pared to CD4+ T cellsculture treated conditions, with only we IL-23 observed (Figure that IFN-2A–C).β increased We also IL-6 assessed production the expres- in B-cells in a dose- sion of other inflammatorydependent cytokines. manner (Figure We2 observedE). Recently, that it has in beenIL-23-stimulated reported that GM-CSF conditions, is a key cytokine IFN-β significantlyproduced elevated byIL-6, TH although cells which the promotes amounts neuroinflammation were substantially [17]. lower Here, than we show IL- that IFN-β 17 (Figure 2D). T cellssignificantly are not thought elevates GM-CSFof as a major expression source in of IL-23-stimulated IL-6. In these conditionsculture condi- (Figure2F). tions, we observed thatIL-6 IFN- isβ a criticalincreased factor IL-6 for production TH17 differentiation in B-cells and in IL-17a dose-dependent expression [18 ]. Our data β manner (Figure 2E).demonstrating Recently, it thathas IFN- been increasedreported the that expression GM-CSF of is IL-17 a key and cytokine IL-6 led us pro- to hypothesize that IFN-β indirectly induced IL-17 through IL-6. We tested this hypothesis by inhibiting IL- duced by TH cells which promotes [17]. Here, we show that IFN-β 6 in these cultures with a neutralizing antibody. We observed that blocking IL-6 expression significantly elevateshad GM-CSF no effect expression on IL-17 expression in IL-23-stimulated (Figure2G). However, conditions we (Figure did find 2F). that IL-6 inhibition IL-6 is a criticalsignificantly factor for reducedTH17 differentiation GM-CSF expression and IL-17 (Figure expression2H). We recently [18]. Our reported data that in vivo demonstrating thatadministration IFN-β increased ofIFN- the expressionβ exacerbated of paralysisIL-17 and and IL-6 elevated led us to GM-CSF hypothesize+ TH cells in CNS that IFN-β indirectlymice induced with TH17-EAE IL-17 through [15]. Furthermore, IL-6. We tested the therapeuticthis hypothesis blockade by inhibiting of IL-6 reversed these IL-6 in these cultureseffects with of IFN-a neutralizingβ [15]. Our inan vitrotibody.data We now observed provide further that blocking evidence thatIL-6 IFN- ex-β-mediated pression had no effectinduction on IL-17 of GM-CSF expression is partially (Figure dependent 2G). However, on elevated we did IL-6 find expression. that IL-6 In summary, β inhibition significantlythese reduced results show GM-CSF that IFN- expression, with IL-23, (Figure induces 2H). a highly We recently inflammatory reported cell population. that in vivo administration of IFN-β exacerbated paralysis and elevated GM-CSF+ TH cells in CNS mice with TH17-EAE [15]. Furthermore, the therapeutic blockade of IL-6 reversed

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these effects of IFN-β [15]. Our in vitro data now provide further evidence that IFN-β- mediated induction of GM-CSF is partially dependent on elevated IL-6 expression. In summary, these results show that IFN-β, with IL-23, induces a highly inflammatory cell Cells 2021, 10, 2139 5 of 9 population.

FigureFigure 2. IFN-β 2.induces IFN-β inflammatory induces expression cytokine in IL-23-stimulated expression in TH17 IL-23-stimulated cultures. Spleen TH17 and lymph cultures. node cells of MOG-immunizedSpleen and lymph C57BL/6J node mice cells were of MOG-immunize cultured with MOGd C57BL/6J35–55, IL-23, mice and were anti-IFN- culturedγ in the with presence MOG35–55 or absence, IL- of IFN-β.(23,A) and Representative anti-IFN-γ FACS in the plots presence of CD4+ or T cellsabsence expressing of IFN- IFN-β. γ(Aand) Representative IL-17. (B) Expression FACS of plots IL-17 of transcripts CD4+ T in the culturedcells cells expressing measured by IFN- qRT-PCR.γ and (IL-17.C) Secreted (B) Expression IL-17 levels of in IL-17 the culture transcripts supernatants in the measured cultured by cells ELISA. measured (D) Secreted levels ofby IL-6 qRT-PCR. in the culture (C) Secreted supernatants IL-17 measured levels in by the ELISA. cultur (Ee )supernatants Intracellular IL-6 meas wasured measured by ELISA. in CD19 (D)+ SecretedB cells by flow cytometry.levels (F )of GM-CSF IL-6 in in the the culture culture supernatants supernatants measured measured by by ELISA. ELISA. The ( concentrationE) Intracellular of (IL-6G) IL-17 was and measured (H) GM-CSF from culturein CD19 supernatants+ B cells by from flow IFN- cytometry.β ± α IL-6 ( stimulationF) GM-CSF were in the assessed culture by supern ELISA.atants Statistical measured analysis by was ELISA. determined using MannThe concentration−Whitney tests. ofp values(G) IL-17 < 0.05 and were (H considered) GM-CSF significantfrom culture (**** supernatants indicates p < 0.0001, from ***IFN- indicatesβ ± α IL-6p <0.001, * indicatesstimulationp < 0.05). were Error assessed bars indicate by ELISA. s.e.m. Statistical analysis was determined using Mann−Whitney tests. p values < 0.05 were considered significant (**** indicates p < 0.0001, *** indicates p < 0.001, * indicates p < 0.05). Error bars3.3. indicate IFN-β s.e.m.Induces Key Transcription Factors for the Differentiation of Pathogenic T Cells We observed that IFN-β increased inflammatory cytokine production from T-helper 3.3. IFN-β Inducescells. Key Transcription Therefore, we Factors determined for th ife transcriptionDifferentiation factors of Pathogenic important T forCells the differentiation of pathogenic T-cell populations were altered by IFN-β. Transcription factors for TH17 and TH1 differentiation have been shown to promote the inflammatory potential of T-

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We observed that IFN-β increased inflammatory cytokine production from T-helper cells. Therefore, we determined if transcription factors important for the differentiation of pathogenic T-cell populations were altered by IFN-β. Transcription factors for TH17 and Cells 2021, 10,TH1 2139 differentiation have been shown to promote the inflammatory potential of T-cells 6 of 9 [19–21]. We found that IFN-β significantly upregulated the TH17-lineage transcription factors RORγt and Runx1 (Figure 3A,B), which was consistent with our observation of an increased IL-17 producticells [19on–21 by]. We IFN found-β. We that also IFN- observedβ significantly that upregulatedthe TH1-lineage the TH17-lineage transcription transcription factor Runx3 (Figurefactors 3C ROR), butγ tnot and T Runx1-bet (Figure (Figure 3A,B),D), was which significantly was consistent elevated with our by observationIFN-β. of an β Interestingly, it hasincreased been suggested IL-17 production that cells by IFN- that .have We also both observed TH1 and that TH17 the TH1-lineage phenotypes transcription factor Runx3 (Figure3C), but not T-bet (Figure3D), was significantly elevated by IFN- β. are highly pathogenic. In a recent study, Blimp-1 was identified as a crucial IL-23-induced Interestingly, it has been suggested that cells that have both TH1 and TH17 phenotypes transcription factorare that highly help pathogenic.ed drive TH17 In a recent-mediated study, Blimp-1 was identified through as the a crucialinduction IL-23-induced of GM-CSF [22]. Wetranscription observed factor that Blimp that helped-1 expression drive TH17-mediated was markedly inflammation increased through in IFN the-β- induction and IL-23-stimulatedof GM-CSF cells compared [22]. We observed to cells stimulated that Blimp-1 with expression only IL was-23 markedly(Figure 3 increasedE). IFN- in IFN-β- β signals through andthe IL-23-stimulatedJAK-STAT pathway, cells compared primarily to through cells stimulated STAT1. with However, only IL-23 IFN (Figure-β can3 E). IFN- β also activate othersignals STAT through transcription the JAK-STAT factors, pathway, including primarily STAT3 through[23]. STAT1 STAT1. signaling However, is IFN- β can required for TH1 alsodifferentiation activate other, and STAT STAT3 transcription is required factors, for includingTH17 differentiation STAT3 [23]. STAT1 [24,25 signaling]. is We found that IFNrequired-β resulted for TH1 in the differentiation, intracellular and phosphorylation STAT3 is required of forSTAT1 TH17 ( differentiationFigure 3F). [24,25]. We found that IFN-β resulted in the intracellular phosphorylation of STAT1 (Figure3F ). We found no induction of STAT3, STAT4, STAT5 or STAT6 phosphorylation by IFN-β We found no induction of STAT3, STAT4, STAT5 or STAT6 phosphorylation by IFN-β (Figure 3F). Our data(Figure demonstrate3F ). Our data that demonstrate IFN-β promotes that IFN- aβ phenotypepromotes a that phenotype has both that TH1 has both TH1 and TH17 characteristics,and TH17 a characteristics,phenotype shown a phenotype to be highly shown pathogenic to be highly [26]. pathogenic [26].

FigureFigure 3. Transcriptional 3. Transcriptional differences differences in pathogenic in pathogenic IL-23-stimulated IL-23 TH17-stimulated cells induced TH17 bycells IFN- inducedβ treatment. by IFN Spleen-β and lymphtreatment. node cells fromSpleen MOG and35–55 lymph-immunized node cells C57BL/6J from miceMOG were35–55 cultured-immunized with MOGC57BL/6J35–55, mice IL-23 andwere anti-IFN- culturedγ in the presencewith or MOG absence35–55 of, IFN-IL-23β andfor threeanti- days.IFN-γ Gene in the expression presence was or absence measured of by IFN qRT-PCR-β for three for the days. transcription Gene ex- factors; (A) RORpressionγt, (B) Runx1, was measured (C) Runx3, by (D qRT) T-bet-PCR and for (E )the Blimp-1. transcription Cells from factors; the spleen (A) RORγt, and lymph (B) nodes Runx1, of MOG-immunized(C) Runx3, + mice were(D) T stimulated-bet and ( withE) Blimp MOG-35–551. Cells, anti-IFN- from γthe, IL-23 spleen± IFN- andβ lymphfor 0, 15, nodes 30 and of 60 MOG min.- CD4immunizedT cells weremice assessedwere for phosphorylationstimulated of with (F) STAT1, MOG STAT3,35–55, anti STAT4,-IFN STAT5-γ, IL and-23 STAT6± IFN- usingβ for flow0, 15, cytometry. 30 and The60 min. results CD4 are+ representative T cells were of two to fourassessed independent for phosphorylation experiments. Statistical of (F) analysisSTAT1, wasSTAT3, determined STAT4, using STAT5 Mann and− WhitneySTAT6 using tests. pflowvalues cytome- < 0.05 were consideredtry. The significant results (**** are indicatesrepresentativep < 0.0001, of two *** indicates to four independentp < 0.001, ** indicates experiments.p < 0.01). Statistical analysis was determined using Mann−Whitney tests. p values < 0.05 were considered significant (**** indicates p < 0.0001, *** indicates3.4. p IFN-< 0.001,β Has ** indicates Differential p < Effects 0.01). on TGF-β-Dependent and IL-23-Dependent TH17 Differentiation 3.4. IFN-β Has DifferentialThe Effects data described on TGF- aboveβ-dependent contradict and reports IL-23-d showingependent that TH17 IFN- β inhibits TH17 devel- Differentiation opment. In culture, TH17 cells can develop in the presence or absence of TGF-β or IL-23 (Figure4A). TGF- β, with IL-6, initiates the differentiation of naïve T cells to TH17 cells;

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The data described above contradict reports showing that IFN-β inhibits TH17 de- velopment. In culture, TH17 cells can develop in the presence or absence of TGF-β or IL- Cells 2021, 10, 2139 7 of 9 23 (Figure 4A). TGF-β, with IL-6, initiates the differentiation of naïve T cells to TH17 cells; however, these TH17 cells are not highly inflammatory [27]. IL-23 is also involved with the differentiation of TH17 cells and is essential in driving the inflammatory functions of these cells [27,28]. Wehowever, speculated these TH17that IFN- cellsβ are may not have highly different inflammatory effects [ 27on]. these IL-23 isdiscrete also involved with the differentiation of TH17 cells and is essential in driving the inflammatory functions of pathways of TH17 differentiation. First, we assessed the effect of IFN-β on TGF-β-medi- these cells [27,28]. We speculated that IFN-β may have different effects on these discrete + ated TH17 differentiation.pathways We of differentiated TH17 differentiation. resting First, CD4 we T assessed cells with the IL-6 effect and of IFN- TGF-β onβ and TGF- β-mediated found that IFN-β significantlyTH17 differentiation. reducedWe the differentiated expression restingof IL-17 CD4 and+ T GM-CSF cells with in IL-6 these and TGF-cul- β and found tures (Figure 4B,C). thatNext, IFN- weβ assessedsignificantly the effects reduced of the IFN- expressionβ on IL-23-mediated of IL-17 and GM-CSFTH17 devel- in these cultures opment. Here we took(Figure the4 initialB,C). Next, TH17 we cultures assessed (TGF- the effectsβ and of IFN-IL-6 βwithouton IL-23-mediated IFN-β) and TH17 res- development. timulated the cells withHere weIL-23 took in thethe initial presence TH17 or cultures absence (TGF- of βIFN-andβ IL-6. In withoutthis condition, IFN-β) and we restimulated found that IFN-β significantlythe cells with increased IL-23 in thethe presence expression or absence of IL-17 of but IFN- hadβ. In no this effect condition, on GM- we found that β CSF expression (FigureIFN- 4B,C).significantly These data increased demonstrate the expression that TGF- of IL-17β and but IL-23 had noare effect important on GM-CSF expres- sion (Figure4B,C). These data demonstrate that TGF- β and IL-23 are important factors that factors that impact the function of IFN-β on TH17 development. impact the function of IFN-β on TH17 development.

Figure 4. Impact of IFN-β on cytokine production during TGF-β-dependent and IL-23-dependent TH17 differentiation. SpleenFigure and 4. lymph Impact node of cellsIFN- fromβ on healthycytokine C57BL/6J production were during depleted TGF- of CD8+β-dependent cells and and differentiated IL-23-dependent in the culturing schemeTH17 depicted differentiation. in (A). For Spleen TGF-β and-dependent lymph node TH17 cells differentiation, from healthy cells C57BL/6J were stimulated were depleted with anti-CD3, of CD8+ anti-CD28, IL-6,cells TGF- andβ ± differentiatedIFN-β. For IL-23-dependent in the culturing TH17 scheme differentiation, depicted cellsin (A were). For stimulated TGF-β-dependent with anti-CD3, TH17 anti-CD28, differ- IL-23, entiation, cells were stimulated with anti-CD3, anti-CD28, IL-6, TGF-β ± IFN-β. For IL-23-dependent anti-IFN-γ ± IFN-β. The concentrations of (B) IL-17A and (C) GM-CSF secreted were determined by ELISA following TH17 differentiation, cells were stimulated with anti-CD3, anti-CD28, IL-23, anti-IFN-γ ± IFN-β. The early and late differentiation. Statistical analysis was performed using one-way ANOVAs. p values < 0.05 were considered concentrations of (B) IL-17A and (C) GM-CSF secreted were determined by ELISA following early significant (**** indicates p < 0.0001). Error bars indicate s.e.m. and late differentiation. Statistical analysis was performed using one-way ANOVAs. p values < 0.05 were considered significant4. Discussion (**** indicates p < 0.0001). Error bars indicate s.e.m. These new data help to rectify the apparent conflicting theories on how IFN-β affects 4. Discussion TH17 function. One predominant theory behind the efficacy of IFN-β is that this therapy These new datareduces help to disease rectify by the inhibiting apparent TH17 conflicting differentiation theories and on function how IFN- [4–β6 ].affects However, MS and TH17 function. OneNMO predominant patients withtheo highry behind TH17 signaturesthe efficacy and of mice IFN- withβ is that TH17-induced this therapy EAE have exac- reduces disease by inhibitingerbated disease TH17 when different treatediation with and IFN- functionβ [2,3,9,11 [4–6].,13,14 However,,29]. Our new MS dataand provide key

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insights into how IFN-β paradoxically increases TH17 pathology. IFN-β has differential effects on discrete pathways of TH17 development. In accordance with previous reports, IFN-β inhibits IL-17 production; however, this is during TGF-β-dependent TH17 devel- opment, which does not produce a pathogenic cell population [27]. In contrast, IFN-β enhances the pathological functions of T cells during TH17 development, which is driven by IL-23 [27,28]. Our experiments define a mechanism where IFN-β with IL-23 induces a highly pathogenic T helper cell population that expresses elevated levels of Blimp1, the TH17-lineage RORγt and Runx1, and the TH1-lineage gene Runx3. These pathogenic T cells secrete elevated levels of IL-6 which contribute to the secretion of the inflammatory cytokine GM-CSF. This study provides key insights into how IFN-β drives pathology in diseases with elevated TH17 signatures. Our observations suggest that IFN-β with IL-23 can alter both the transcriptional and cytokine profiles towards an inflammatory phenotype during TH17- mediated disease. These findings provide further evidence that IFN-β may worsen in certain neuro-autoimmune diseases, such as in patients with NMO.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/cells10082139/s1, Table S1: List of Primers. Author Contributions: Conceptualization, R.C.A. and A.A.; Data curation, R.C.A., A.A., J.L.Q. and G.K.; Original draft preparation, A.A.; Review and editing, R.C.A. and A.A.; Funding acquisition, R.C.A. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by the National Multiple Sclerosis Society (RG-1602-07722) and the National Institutes of Health (R01AI137047 and R01EY027346). Institutional Review Board Statement: The study was conducted according to the guidelines of the institutional IACUC of Oklahoma Medical Research Foundation (protocol 18-21 approved on 7/17/2018). Informed Consent Statement: Not applicable. Data Availability Statement: All data from this manuscript will be provided to anyone upon request. Conflicts of Interest: R.C.A. has consulted Roche, EMD-Serono, Biogen-Idec, and is on the advisory board for Progentec Diagnostics Inc.

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