International Journal of Molecular Sciences

Article Semaphorin4A-Plexin D1 Axis Induces Th2 and Th17 While Represses Th1 Skewing in an Autocrine Manner

1,2, 3,4, 1,2 1,2 Tiago Carvalheiro † , Carlos Rafael-Vidal †, Beatriz Malvar-Fernandez , Ana P. Lopes , Jose M. Pego-Reigosa 3,4, Timothy R. D. J. Radstake 1,2 and Samuel Garcia 1,2,3,4,* 1 Department of Rheumatology & Clinical Immunology, University Medical Center Utrecht, University of Utrecht, 3508 GA Utrecht, The Netherlands; [email protected] (T.C.); [email protected] (B.M.-F.); [email protected] (A.P.L.); [email protected] (T.R.D.J.R.) 2 Center for Translational Immunology, University Medical Center Utrecht, University of Utrecht, 3508 GA Utrecht, The Netherlands 3 Rheumatology & Immuno-mediated Diseases Research Group (IRIDIS), Galicia Sur Health Research Institute (IIS Galicia Sur), SERGAS-UVIGO, 36312 Vigo, Spain; [email protected] (C.R.-V.); [email protected] (J.M.P.-R.) 4 Rheumatology Department, University Hospital Complex of Vigo, 36312 Vigo, Spain * Correspondence: [email protected]; Tel.: +34-986-515-463 These authors equally contributed to this work. †  Received: 31 August 2020; Accepted: 19 September 2020; Published: 22 September 2020 

Abstract: (Sema)4A is a transmembrane glycoprotein that is elevated in several autoimmune diseases such as systemic sclerosis, rheumatoid arthritis and multiple sclerosis. Sema4A has a key role in the regulation of Thelper Th1 and Th2 differentiation and we recently demonstrated that CD4+ activation induces the expression of Sema4A. However, the autocrine role of Sema4A on Th cell differentiation remains unknown. Naïve Th cells from healthy controls were cell sorted and differentiated into Th1, Th2 and Th17 in the presence or absence of a neutralizing against the Sema4A PlexinD1. expression was determined by quantitative PCR and protein expression by ELISA and flow cytometry. We found that the expression of Sema4A is induced during Th1, Th2 and Th17 differentiation. PlexinD1 neutralization induced the differentiation of Th1 cells, while reduced the Th2 and Th17 skewing. These effects were associated with an upregulation of the T-bet by Th1 cells, and to downregulation of GATA3 and RORγt in Th2 cells and Th17 cells, respectively. Finally, PlexinD1 neutralization regulates the systemic sclerosis patients serum-induced production by CD4+ T cells. Therefore, the autocrine Sema4A-PlexinD1 signaling acts as a negative regulator of Th1 skewing but is a key mediator on Th2 and Th17 differentiation, suggesting that dysregulation of this axis might be implicated in the pathogenesis of CD4+ T cell-mediated diseases.

Keywords: semaphorin4A; plexinD1; CD4+ T cell differentiation; T helper cells; systemic sclerosis

1. Introduction T helper (Th) cells are essential regulators of the adaptive immune responses. There are three major subsets of Th cells, which can be identified based on their cytokine production and immune function. Th1 cells are characterized by the secretion of interferon-γ (IFN-γ) and are involved in the host defense against intracellular pathogens; Th2 cells usually secret interleukin (IL)-4, IL-5 and IL-13 and are critical for mediating immune responses against extracellular parasites; finally Th17 cells mainly produce IL-17, IL-21 and IL-22, and are pivotal in the defense against extracellular bacteria and fungi [1–3]. Dysregulation of Th has been implicated in several diseases.

Int. J. Mol. Sci. 2020, 21, 6965; doi:10.3390/ijms21186965 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 6965 2 of 11

Hyperactivation of Th1 and Th17 cells confers susceptibility to autoimmune diseases and is associated Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 2 of 11 with the pathology of multiple sclerosis (MS), inflammatory bowel disease, systemic sclerosis (SSc), psoriasis,bacteria rheumatoid and fungi [1,2,3]. arthritis Dysregulation (RA) and spondiloarthropaties of Th homeostasis has [ 4been–7]. implicated On the other in several hand, diseases. Th2 cells are implicatedHyperactivation in the pathogenesis of Th1 and of Th17 asthmatic cells andconfers allergic susceptibility diseases [to8 ,9autoimmune]. diseases and is Semaphorinassociated with (Sema) the pathology 4A is a transmembraneof multiple sclero glycoproteinsis (MS), inflammatory belonging bowel to the disease, semaphorin systemic family, whichsclerosis is a large (SSc), group psoriasis, of proteins rheumatoid initially arthritis described (RA) and as spondiloarthropaties axonal guidance molecules [4,5,6,7]. On [10 the]. Sema4A other is mainlyhand, produced Th2 cells byare implicated dendritic in cells, the pathogenesis monocytes of and asthmatic and allergic [11 diseases], although [8,9]. it can be also expressedSemaphorin by activated (Sema) CD4 4A+ T is cells a transmembrane and germinal glyc centeroprotein B cells belonging [12–14 ].to Sema4Athe semaphorin binds family, to different which is a large group of proteins initially described as axonal guidance molecules [10]. Sema4A is receptors expressed on CD4+ T cells, such as PlexinB1, PlexinB2, PlexinD1, -1 (NRP-1), mainly produced by dendritic cells, monocytes and macrophages [11], although it can be also Immunoglobulin-likeexpressed by activated transcript CD4+ T 4cells (ILT)4 and andgerminal Tim-2, center although B cells the[12,13,14]. last has Sema4A been reportedbinds to different in mice and has noreceptors human expressed orthologous on CD4 [12–+ 18T ].cells, Several such studiesas PlexinB1, have PlexinB2, shown thatPlexinD1, Sema4A Neuropilin-1 expression (NRP-1), is elevated in diseasesImmunoglobulin-like in which Th transcript cells play 4 an(ILT)4 essential and Tim-2, role, although including the asthma,last has been RA, reported MS and in SSc mice [11 and–13 ,19]. Furthermore,has no human functional orthologous studies have[12,13,14,15,16,17,18]. demonstrated thatSeveral Sema4A studies is a keyhave moleculeshown that in the Sema4A regulation of naïveexpression CD4+ isT elevated cell diff inerentiation, diseases in althoughwhich Th ce dillsff erentplay an e ffessentialects in mouserole, including and human asthma, CD4 RA,+ MST cells haveand been SSc reported. [11,12,13,19]. In mice, Furthermore, Sema4A isfunctional involved studies in T cell have priming demonstrated and Th1 that skewing, Sema4A both is a processes key + mediatedmolecule by Tim-2in the [regulation14,20]. Besides of naïve that, CD4 Sema4AT cell differentiation, deficiency induces although Th2 different skewing effects and in enhances mouse the + severityand ofhuman allergic CD4 responsesT cells have [21 been–23 ].reported. Finally, In Sema4A mice, Sema4A induced is involved the expression in T cell priming of IL-17 and by Th1 CD4 + T skewing, both processes mediated by Tim-2 [14,20]. Besides that, Sema4A deficiency induces Th2 cells from autoimmune encephalomyelitis mice [11,24]. In human CD4+ T cells, exogenous Sema4A skewing and enhances the severity of allergic responses [21,22,23]. Finally, Sema4A induced the inducesexpression ILT-4 mediated of IL-17 by Th2 CD4 diff+ erentiationT cells from and, autoimmune on the contrary encephalomyelitis to mice studies, mice [11,24]. inhibits In Th1 human skewing. + Moreover,CD4+ T we cells, also exogenous found that Sema4A Sema4A induces secreted ILT-4 mediated by activated Th2 differentiation CD4 T cells and, of SSc on the patients contrary is ableto to inducemice the studies, expression inhibits of Th17 Th1 cytokinesskewing. Moreover, in a PlexinB2, we also PlexinD1 found andthat NRP-1Sema4A dependent secreted by manner activated [12 ,13]. Altogether,CD4+ T cells of these SSc patients data demonstrate is able to induce that the exogenous expression Sema4A of Th17 is involved in a in PlexinB2, human PlexinD1 naïve Th cell differentiation.and NRP-1 dependent However, manner whether [12,13]. autocrine Sema4A is involved on this process and the functional consequencesAltogether, of its inhibitionthese data demonstrate are still unknown. that exogenous Sema4A is involved in human naïve Th cell differentiation. However, whether autocrine Sema4A is involved on this process and the functional 2. Resultsconsequences of its inhibition are still unknown.

2.1. Sema4A2. Results Is Induced During CD4+ T Cell Differentiation

+ Although2.1. Sema4A previous is Induced reports During CD4 have+ T shown Cell Differentiation that Sema4A is not expressed in naïve CD4 T and + only induced after CD4 T cells activation [12,13], its expression during Th cell di+ fferentiation is Although previous reports have shown that+ Sema4A is not expressed in naïve CD4 T and only still unknown.induced after Indeed, CD4+ unstimulated T cells activation naïve [12,13], CD4 itsT cellsexpression did not during express Th SEMA4A. cell differentiation However, is upon still Th1, Th2 andunknown. Th17 diIndeed,fferentiation unstimulated SEMA4A naïve expression CD4+ T cells is did upregulated not express at SEMA4A. day 3, although However, di uponfferences Th1, were only significantTh2 and Th17 in differentiation the Th2 subset. SEMA4A Despite expression the lower is geneupregulated expression at day levels 3, although compared differences to day were 3, at day 7 weonly still observedsignificant an in upregulationthe Th2 subset. of Despite SEMA4A the (Figurelower gene1A). expression In addition, levels we alsocompared determined to day the3, at e ffect of Thday diff 7erentiation we still observed on the an secretion upregulation of Sema4A. of SEMA4A At day (Figure 3, the 1A). Sema4A In addition, levels we were also alreadydetermined elevated the in the threeeffect Th of cellTh differentiation subsets compared on the to secretion the non-stimulated of Sema4A. At naïve day CD43, the+ TSema4A cells, even levels though were already it was only + significantelevated in in the the Th17 three subset. Th cell subsets At day co 7,mpared the secretion to the non-stimulated of Sema4A was naïve significantly CD4 T cells, higher even though in thethree it was only significant in the Th17 subset. At day 7, the secretion of Sema4A was significantly higher differentiated Th subsets compared to the non-differentiated naïve T cells (Figure1B). in the three differentiated Th subsets compared to the non-differentiated naïve T cells (Figure 1B).

Figure 1. Sema4A is induced during T helper cell differentiation. Sema4A mRNA (A) and protein (B) Figure 1. Sema4A is induced during T helper cell differentiation. Sema4A mRNA (A) and protein (B) levels in unstimulated naïve CD4+ T cells or in Th1, Th2 or Th17 differentiated cells for 3 days (n = 4) levels in unstimulated naïve CD4+ T cells or in Th1, Th2 or Th17 differentiated cells for 3 days (n = 4) and 7 days (n = 6). Means and SEM are shown. * p < 0.05 and **** p < 0.0001 compared to the medium at day 3. ### p < 0.001 and #### p < 0.0001 compared to the medium at day 7. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 11

and 7 days (n = 6). Means and SEM are shown. * p < 0.05 and **** p < 0.0001 compared to the medium at day 3. ### p < 0.001 and #### p < 0.0001 compared to the medium at day 7. Int. J. Mol. Sci. 2020, 21, 6965 3 of 11 2.2. Autocrine Sema4A–plexinD1 Signaling Induces Th2 and Th17 Differentiation and Suppresses Th1 Skewing 2.2. Autocrine Sema4A–plexinD1 Signaling Induces Th2 and Th17 Differentiation and Suppresses Th1 SkewingWe have previously shown that neutralization of autocrine/paracrine Sema4A signaling reduced the expression of Th17 cytokines by the total CD4+ T cells [13], thus we next analyzed the We have previously shown that neutralization of autocrine/paracrine Sema4A signaling reduced effect of the inhibition of this signaling during Th helper cell differentiation. For this purpose we the expression of Th17 cytokines by the total CD4+ T cells [13], thus we next analyzed the effect of used an anti-PlexinD1 neutralizing antibody, as PlexinD1 is the most expressed Sema4A receptor in the inhibition of this signaling during Th helper cell differentiation. For this purpose we used an naïve CD4+ T cells [13]. Compared to the respective isotype control, the neutralization of PlexinD1 anti-PlexinD1 neutralizing antibody, as PlexinD1 is the most expressed Sema4A receptor in naïve induced the expression of IFNG in Th1-diferentiated cells, while it reduced the expression of Th2 CD4+ T cells [13]. Compared to the respective isotype control, the neutralization of PlexinD1 induced cytokines (IL4 and IL5), and Th17 cytokines (IL17 and IL22) cytokines by Th2 and Th17 the expression of IFNG in Th1-diferentiated cells, while it reduced the expression of Th2 cytokines differentiated cells, respectively. TNF expression was upregulated in the three cell subsets but the (IL4 and IL5), and Th17 cytokines (IL17 and IL22) cytokines by Th2 and Th17 differentiated cells, expression was not modulated by the PlexinD1 neutralization (Figure 2). Consistently, we also respectively. TNF expression was upregulated in the three cell subsets but the expression was not found these effects earlier in the differentiation process, as at day 3 PlexinD1 blocking upregulated modulated by the PlexinD1 neutralization (Figure2). Consistently, we also found these e ffects earlier IFNG levels and reduced IL4 and IL17 expression in the respective Th subsets, Th1, Th2 and Th17 in the differentiation process, as at day 3 PlexinD1 blocking upregulated IFNG levels and reduced IL4 (Figure S1). and IL17 expression in the respective Th subsets, Th1, Th2 and Th17 (Figure S1).

IFNG IL4 IL17 IgG 0.054 * α-PlexinD1 # ## 25 # 0.07 0.20 0.06 20 # 0.15 0.05 # 15 0.04 0.10 x 1000 x 10 0.03

Ct 0.02 0.05 Δ 5 - 0.01 2 0 0.00 0.00 Med Th1 Th2 Th17 Med Th1 Th2 Th17 Med Th1 Th2 Th17 TNF IL5 IL22 * 1.8 # # 0.07 0.35 # # # 0.30 1.5 # ## 0.06 0.25 1.2 0.05 0.04 0.20 0.9 0.15 x 1000 0.03 0.6 0.10 Ct 0.02 Δ - 0.3 0.01 0.05 2 0.0 0.00 0.00 Med Th1 Th2 Th17 Med Th1 Th2 Th17 Med Th1 Th2 Th17 Figure 2. Sema4A-PlexinD1 axis inhibits Th1 and promotes Th2 and Th17 differentiation. Cytokines mRNAFigure 2. expression Sema4A-PlexinD1 in naïve CD4axis +inhibitsT cells Th1 diff erentiatedand promotes either Th2 in and medium Th17 differentiation. (Med), Th1, Th2 Cytokines or Th17 dimRNAfferentiation expression cocktails in naïve in the CD4 presence+ T cells of andifferentiated anti-PlexinD1 either antibody in medium or its respective(Med), Th1, isotype Th2 or control Th17 fordifferentiation 7 days (n = 6).cocktails Data is in presented the presence as connected of an anti-PlexinD1 dots. * p < 0.05.antibody # p < or0.05 its andrespective ## p < 0.01isotype compared control tofor the 7 days medium. (n = 6). Data is presented as connected dots. * p < 0.05. # p < 0.05 and ## p < 0.01 compared to the medium. We next confirmed these results at the protein level; on Th1 cells, PlexinD1 neutralization significantlyWe next increased confirmed the these secretion results of IFNat theγ and protein showed level; a trendon Th1 towards cells, PlexinD1 a higher productionneutralization of thissignificantly cytokine increased after restimulation the secretion with of IFN PMAγ /andionomycin. showed a On trend the towards other hand, a higher neutralization production ofof this the Sema4A-PlexinD1cytokine after restimulation signaling significantly with PMA/ionomyci reduced then. secretion On the andother the hand, production neutralization of IL-4 and of IL-17 the bySema4A-PlexinD1 Th2 and Th17 cells, signaling respectively signific (Figureantly reduced3A,B and the Supplementary secretion and the Figure production S2). We of also IL-4 observed and IL-17 a reductionby Th2 and on Th17 IL-22 cells, production respectively by Th17 (Figure cells, 3A,B but di andfferences Supplementary were not significantFigure S2). (Figure We also3B). observed a reductionTo rule on out IL-22 the possibilityproduction of by unspecific Th17 cells, effects but duedifferences to cell viability were not and significant/or proliferation, (Figure we 3B). analyzed the viability of a naïve CD4+ T cell and proliferation during differentiation. At day 3, non-differentiated naïve T cells did not proliferate, but in the Th1, Th2 and Th17 subsets, the proliferation rate was nearly 100%, independent of PlexinD1 neutralization (Figure S3A). Overall, cell viability was similar through all the culture conditions either at day 3 and day 7, without effect of the PlexinD1 neutralization. Nevertheless, the overall cell viability at day 7 was slightly lower compared with day 3 (Figure S3B).

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Figure 3.FigureSema4A-PlexinD1 3. Sema4A-PlexinD1 axis axis inhibits inhibits Th1 Th1 andand promotes Th2 Th2 and and Th17 Th17 differentiation. differentiation. Cytokine Cytokine + secretionsecretion (A) and (A intracellular) and intracellular cytokine cytokine production production (B)) in in naïve naïve CD4 CD4+ T cellsTcells differentiated differentiated with Th1, with Th1, Th2 or Th17Th2 or di Th17fferentiation differentiation cocktails cocktails in in the the presence presence of of an an anti-PlexinD1 anti-PlexinD1 antibody antibody or its orrespective its respective isotypeisotype control control for 7 daysfor 7 days (n = (n6). = 6). Data Data is is presented presented as as connected connected dots. dots. * p < * 0.05p < and0.05 ** and p < 0.01. ** p < 0.01.

2.3. PlexinD1To Neutralization rule out the possibility Regulates of TBX21, unspecific GATA3 effects and due RORC to cell Expressionviability and/or proliferation, we analyzed the viability of a naïve CD4+ T cell and proliferation during differentiation. At day 3, Wenon-differentiated next analyzed whether naïve T PlexinD1cells did not neutralization proliferate, but also in modulates the Th1, Th2 the and expression Th17 subsets, of T-bet, the GATA3 and RORproliferationγt, the master rate was transcription nearly 100%, factorsindependent involved of PlexinD1 in Th neutralization cell differentiation (Figure [S3A).3]. The Overall, expression levels ofcell TBX21, viability the was gene similar encoding through all T-bet, the culture was increased conditions oneither Th1-di at dayff erentiated3 and day 7, cellswithout at effect day 3 and 7 of differentiationof the PlexinD1 in the neutralization. presence of Nevertheless, the anti-PlexinD1 the overall antibody. cell viability On theat day contrary, 7 was slightly GATA3 lower expression compared with day 3 (Figure S3B). was reduced on Th2-differentiated cells at both day 3 and 7, although differences were not significant. Similarly,2.3. PlexinD1PlexinD1 Neutralization neutralization Regulates significantly TBX21, GATA3 reduced and RORC the expression Expression of RORC, the gene that γ encodes RORWe nextt, in analyzed Th17 cells whether at dayPlexinD1 3 and neutralizati 7 (Figureon 4alsoA,B). modulates Therefore, the expression our data of suggest T-bet, that Sema4A-PlexinD1GATA3 and ROR axisγ impactst, the master the transcription expression factors of master involved transcription in Th cell differentiation factors involved [3]. The in the Th cell diInt.fferentiationexpression J. Mol. Sci. 2020 levels processes., 21, xof FOR TBX21, PEER the REVIEW gene encoding T-bet, was increased on Th1-differentiated cells at day 5 of 11 3 and 7 of differentiation in the presence of the anti-PlexinD1 antibody. On the contrary, GATA3 expression was reduced on Th2-differentiated cells at both day 3 and 7, although differences were not significant. Similarly, PlexinD1 neutralization significantly reduced the expression of RORC, the gene that encodes RORγt, in Th17 cells at day 3 and 7 (Figure 4A,B). Therefore, our data suggest that Sema4A-PlexinD1 axis impacts the expression of master transcription factors involved in the Th cell differentiation processes.

Figure 4. Sema4A-PlexinD1 axis regulates the expression of transcription factors involved in T helper cell Figure 4. Sema4A-PlexinD1 axis regulates the expression of transcription factors involved in T helper differentiation. (A,B) TBX21, GATA3 and RORC expression in naïve CD4+ T cells differentiated either cell differentiation. (A,B) TBX21, GATA3 and RORC expression in naïve CD4+ T cells differentiated either in medium or Th1, Th2 or Th17 differentiation cocktails in the presence of an anti-PlexinD1 antibody or its respective isotype control for 7 days (A, n = 6) and 3 days (B, n = 4). Data is presented as connected dots. * p < 0.05 and ** p < 0.01. # p < 0.05 and ## p < 0.01 compared to the medium.

2.4. Sema4A-PlexinD1 Blockade Reduces PlexinB2 Expression During Th17 Cell Differentiation As Sema4A has multiple receptors [10], we also determined whether blockade of the Sema4A-PlexinD1 axis modulates the expression of Sem4A receptors. The neutralization of PlexinD1 did not affect the mRNA expression of PlexinD1, PlexinB1, NRP-1 or ILT-4 in any of the subsets analyzed at day 7 (Figure 5A). However, PlexinD1 blockage led to a significant reduction in PLEXINB2 expression in Th17-diferentiated cells at both day 3 and 7 (Figure 5B,C) and to a lower percentage of PlexinB2 positive cells at day 3 of Th17 differentiation (Figure 5D and Figure S4).

Figure 5. Sema4A-PlexinD1 axis induces the expression of PlexinB2 in Th17 cells. (A) mRNA expression of Sema4A receptors in naïve CD4+ T cells differentiated in medium or Th1, Th2 or Th17

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in medium or Th1, Th2 or Th17 differentiation cocktails in the presence of an anti-PlexinD1 antibody Figure 4. Sema4A-PlexinD1 axis regulates the expression of transcription factors involved in T helper or itscell respective differentiation. isotype (A,B control) TBX21, for GATA3 7 days and (A ,RORCn = 6) expression and 3 days in naïve (B, n CD4= 4).+ T cells Data differentiated is presented as connectedeither dots.in medium * p < 0.05or Th1, and Th2 ** p or< Th170.01. differentiati # p < 0.05 andon cocktails ## p < 0.01 in the compared presence toof thean anti-PlexinD1 medium. antibody or its respective isotype control for 7 days (A, n = 6) and 3 days (B, n = 4). Data is presented 2.4. Sema4A-PlexinD1as connected dots. Blockade * p < 0.05 Reduces and ** p PlexinB2 < 0.01. # p Expression< 0.05 and ## during p < 0.01 Th17compared Cell to Di thefferentiation medium. As2.4. Sema4ASema4A-PlexinD1 has multiple Blockade receptors Reduces PlexinB2 [10], we Expression also determined During Th17 whetherCell Differentiation blockade of the Sema4A-PlexinD1As Sema4A axis has modulates multiple the receptors expression [10], of we Sem4A also receptors.determined The whether neutralization blockade ofof PlexinD1 the did notSema4A-PlexinD1 affect the mRNA axis modulates expression the of expression PlexinD1, of PlexinB1,Sem4A receptors. NRP-1 The or neutralization ILT-4 in any of of PlexinD1 the subsets analyzeddid not at day affect 7 (Figure the mRNA5A). However,expression PlexinD1of PlexinD1, blockage PlexinB1, led NRP-1 to a significant or ILT-4 in reduction any of the in subsets PLEXINB2 expressionanalyzed in Th17-diferentiatedat day 7 (Figure 5A). cells However, at both dayPlexin 3 andD1 blockage 7 (Figure led5B,C) to anda significant to a lower reduction percentage in of PlexinB2PLEXINB2 positive expression cells at day in Th17-diferentiated 3 of Th17 differentiation cells at both (Figure day 53D and and 7 Figure(Figure S4).5B,C) and to a lower percentage of PlexinB2 positive cells at day 3 of Th17 differentiation (Figure 5D and Figure S4).

FigureFigure 5. Sema4A-PlexinD1 5. Sema4A-PlexinD1 axis inducesaxis induces the expression the expression of PlexinB2 of PlexinB2 in Th17 in cells.Th17 (Acells.) mRNA (A) mRNA expression of Sema4Aexpression receptors of Sema4A in naïve receptors CD4+ inT cellsnaïve di CD4fferentiated+ T cells differentiated in medium orin Th1,medium Th2 or or Th1, Th17 Th2 di fforerentiation Th17 cocktails in the presence of an anti-PlexinD1 antibody or its respective isotype control for 7 days

(n = 6). (B–D) PlexinB2 mRNA (B,C) and protein (D) expression in naive CD4+ T cells differentiated in medium or Th1, Th2 or Th17 differentiation cocktails in the presence of an anti-PlexinD1 antibody or its respective isotype control for 7 days (B, n = 6) and 3 days (C,D, n = 4). Data are presented as connected dots. * p < 0.05. # p < 0.05 compared to the medium.

2.5. PlexinD1 Neutralization Regulates CD4+ T Cells Cytokine Production Induced by Systemic Sclerosis Patient Serum Previously we have shown that Sema4A is elevated in the serum of SSc patients and it induces the production of Th17 cytokines [13]. In addition, both IL-17 and IL-4 cytokines are elevated in SSc patients and have been shown to play an important role in SSc pathogenesis [25–29]. Thus, in order to confirm the functional role of the Sema4A-PlexinD1 axis on a CD4+ T cell-mediated disease, we investigated the role of PlexinD1 neutralization on the CD4+ T cells cytokine production induced by the serum of SSc patients. In CD4+ T cells activated in the presence of SSc-serum, the neutralization of PlexinD1 significantly induced the secretion of IFN-γ, while the secretion of IL-17 was significantly reduced (Figure6). The secretion of IL-4 despite the low production and undetectable in some donors, shows a trend towards a reduction after the neutralization of PlexinD1 (Figure6). Altogether, these data Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 6 of 11

differentiation cocktails in the presence of an anti-PlexinD1 antibody or its respective isotype control for 7 days (n = 6). (B–D) PlexinB2 mRNA (B,C) and protein (D) expression in naive CD4+ T cells differentiated in medium or Th1, Th2 or Th17 differentiation cocktails in the presence of an anti-PlexinD1 antibody or its respective isotype control for 7 days (B, n = 6) and 3 days (C,D, n = 4). Data are presented as connected dots. * p < 0.05. # p < 0.05 compared to the medium.

2.5. PlexinD1 Neutralization Regulates CD4+ T Cells Cytokine Production Induced by Systemic Sclerosis Patient Serum Previously we have shown that Sema4A is elevated in the serum of SSc patients and it induces the production of Th17 cytokines [13]. In addition, both IL-17 and IL-4 cytokines are elevated in SSc patients and have been shown to play an important role in SSc pathogenesis [25,26,27,28,29]. Thus, in order to confirm the functional role of the Sema4A-PlexinD1 axis on a CD4+ T cell-mediated disease, we investigated the role of PlexinD1 neutralization on the CD4+ T cells cytokine production induced by the serum of SSc patients. In CD4+ T cells activated in the presence of SSc-serum, the Int.neutralization J. Mol. Sci. 2020 of, 21 PlexinD1, 6965 significantly induced the secretion of IFN-γ, while the secretion of6 IL-17 of 11 was significantly reduced (Figure 6). The secretion of IL-4 despite the low production and undetectable in some donors, shows a trend towards a reduction after the neutralization of PlexinD1 suggest(Figure that6). Sema4A-PlexinD1Altogether, these signalingdata suggest is involved that Sema4A-PlexinD1 in the elevated levels signaling of IL-17 is and involved IL-4 observed in the inelevated these patients. levels of IL-17 and IL-4 observed in these patients.

IFN-γ IL-4 IL-17 0.08 1750 * 25 1200 * ## ## ### ## 1500 20 1250 900 1000 15 600 750 10

pg/mL 500 300 5 250 0 0 0 Medium IgG αPlexinD1 Medium IgG αPlexinD1 Medium IgG αPlexinD1 SSc serum SSc serum SSc serum

Figure 6. Sema4A-PlexinD1 axis regulates CD4+ T cytokine production induced by systemic sclerosis Figure 6. Sema4A-PlexinD1 axis regulates CD4+ T cytokine production induced by systemic sclerosis patient serum. IFN-γ, IL-4 and IL-17 secretion by activated CD4+ T cells, pretreated for 1 h with an patient serum. IFN-γ, IL-4 and IL-17 secretion by activated CD4+ T cells, pretreated for 1 h with an anti-PlexinD1 antibody or its respective isotype control (IgG) and incubated with the serum of SSc anti-PlexinD1 antibody or its respective isotype control (IgG) and incubated with the serum of SSc patients patients (20% v/v) for 5 days (n = 8). Data are presented as connected dots. * p < 0.05. ## p < 0.01 and (20% v/v) for 5 days (n = 8). Data are presented as connected dots. * p < 0.05. ## p < 0.01 and ### p < 0.001 ### p < 0.001 compared to medium. compared to medium. 3. Discussion 3. Discussion Here, we demonstrated the role of autocrine Sema4A-PlexinD1 axis on CD4+ T cell differentiation and itsHere, contribution we demonstrated to T cell-mediated the diseases.role of autocrine Previous reports Sema4A-PlexinD1 have shown that axis Sema4A on CD4 is expressed+ T cell bydifferentiation activated CD4 and+ T its cells contribution and memory to T Th2 cell-mediated cells [12,13 ].diseases. Here we Previous first show reports that thehave expression shown that of Sema4ASema4A wasis expressed induced duringby activated the CD4 CD4+ +T T di cellsfferentiation and memory and, Th2 together cells [12,13]. with the Here fact thatwe first Sema4A showwas that involvedthe expression in T cell of activationSema4A was and induced differentiation, during the suggest CD4+ thatT differentiation autocrine Sema4A and, together may be with involved the fact in thesethat Sema4A processes. was involved in T cell activation and differentiation, suggest that autocrine Sema4A maySecondly, be involved we in demonstrated these processes. that the autocrine Sema4A-PlexinD1 axis was a negative regulator of humanSecondly, Th1 we diff demonstratederentiation and that played the autocrine a crucial Sema4A-PlexinD1 role in Th2 skewing. axis was These a negative findings regulator are in accordanceof human withTh1 thedifferentiation work of Lu Nand et al. played [12], in a which crucia exogenousl role in Sema4ATh2 skewing. administration These findings repressed are Th1 in diaccordancefferentiation with and the induced work Th2of Lu skewing. N et al. In[12], addition, in which we exogenous unprecedently Sema4A showed administration that Sema4A-PlexinD1 repressed axisTh1 isdifferentiation also involved onand Th17 induced differentiation, Th2 skewing. which is In in lineaddition, with previous we unprecedently works, which showed demonstrate that thatSema4A-PlexinD1 Sema4A induces axis IL-17 is also production involved in on both Th17 mouse differ andentiation, human which CD4+ isT in cells line [11 with,13,24 previous]. Importantly, works, thewhich effect demonstrate of the Sema4A-PlexinD1 that Sema4Aaxis induces on T helperIL-17 production cell differentiation in both ismouse supported and human by the regulation CD4+ T cells of the[11,13,24]. master transcriptionImportantly, factorsthe effect T-bet, of GATA3the Sema4A-P and RORC.lexinD1 Similarly axis on to ourT helper previous cell workdifferentiation [13], but on is thesupported contrary by to the the regulation work of Lu of N the et al.master [12], transcription the autocrine factors Sema4A-PlexinD1 T-bet, GATA3 signaling and RORC. did not Similarly affect theto CD4our +previousT cell proliferation. work [13], Thesebut on di thefferences contrary may to be the attributed work of to theLu stimulationN et al. [12], with the exogenous autocrine + + Sema4A, the use of CD4 T cells from buffy coats and the different manner of CD4 T cell activation, as these authors used an anti-CD3 antibody at a suboptimal concentration and did not activated the CD28 signaling pathway. Altogether, these data suggest that the Sema4A-PlexinD1 axis might be implicated in the maintenance of the homeostatic levels of Th1, Th2 and Th17 cells and therefore in the proper host defense against intracellular and extracellular pathogens [1,2]. This role of Sema4A-PlexinD1 was observed in combination with key cytokines involved on Th differentiation, such as IL12 (Th1), IL-4 (Th2) and IL-23 (Th17) [4,5,9]. However, whether Sema4A is capable of modulating Th differentiation in the absence of these cytokines or is a costimulatory pathway implicated in Th skewing needs to be further elucidated. Sema4A binds to different receptors in a cell type and context dependent manner [10,30]. Here we found that PlexinD1 is involved in the differentiation of three CD4+ T subsets. To our knowledge PlexinD1 is the only Sema4A receptor involved in the inhibition of Th1 differentiation, while in the Th2 skewing both PlexinD1 and ILT-4 are implicated [12]. The neutralization of Sema4A-PlexinD1 axis downregulated the expression of PlexinB2 in Th17 cells, and as PlexinB2 is involved in the secretion of Th17 cytokines by activated CD4+ T cells [13], the Sema4A-PlexinB2 axis seems also to be implicated Int. J. Mol. Sci. 2020, 21, 6965 7 of 11 in the Th17 skewing. Hence, Sema4A plays a central role in CD4+ T cell differentiation through the involvement of different receptors. Nevertheless, the role of other Sema4A receptors involved in T cell function, such as NRP-1, PlexinB1 or PlexinB2 needs to be elucidated. Lastly, we translated our findings to a CD4+ T cell-mediated disease and we demonstrated that the neutralization of PlexinD1 suppressed the CD4+ T cell secretion of IL-17 and IL-4 induced by the serum of SSc patients. Although PlexinD1 can bind to other semaphorin family members, mainly Sema3E [10,31], but also Sema3C, Sema3D and Sema3G [32–34], Sema4A is the only known PlexinD1 ligand able to induce T cell activation. This fact, together with the elevated Sema4A expression observed in SSc patients [13], suggest that the Sema4A-PlexinD1 axis is responsible, at least in part, for the elevated IL-4 and IL-17 levels observed in these patients. As Sema4A expression is also elevated in other CD4+ T cell-mediated diseases including, asthma, RA and MS [11,12,19,24], it is tempting to speculate that Sema4A-PlexinD1 signaling is involved in the dysregulation of Th cell homeostasis observed in these diseases and targeting this axis might be a beneficial therapeutic approach. As binding of PlexinD1 to its different ligands is not only involved in T cell differentiation, but also plays a key role in vascular, cardiac, skeletal and neuronal development and homeostasis [35–40], we consider that neutralizing Sema4A might be a better therapeutic option than blocking PlexinD1. A limitation of our study is that we could not test this possibility, due to the lack of commercially available anti-Sema4A neutralizing . Therefore, further studies using other methodologies such as gene silencing or deletion are needed to elucidate the pathological role of Sema4A on CD4+ T cell-mediated diseases.

4. Materials and Methods

4.1. Naïve CD4+ T Cell Isolation Peripheral blood mononuclear cells (PBMCs) from healthy controls were isolated by the Ficoll gradient (GE Healthcare, Zwijndrecht, The Netherlands). Cells were further isolated using the CD4+ T Cell Isolation Kit on an autoMACS Pro Separator, according to the manufacturer’s instructions (Miltenyi Biotec, Leiden, The Netherlands). To isolate the naïve subpopulation, total CD4+ T cells were stained with anti-CD4 APC-eF780 (eBioscience, Nieuwegein, The Netherlands), anti-CD27 BV510 (Biolegend, Amsterdam, The Netherlands), anti-CD25 PE, anti-CD127 Alexa Fluor-647 and anti-CD45RO PE/Cy7 (BD Biosciences, Vianen, The Netherlands) antibodies and purified on a BD + FACSAria™ III cell sorter (BD Biosciences, Vianen, The Netherlands). Naïve CD4 CD25−CD27 + CD45RO− T cells were defined according to the gating strategy shown in Figure S5. Purity was consistently > 99%.

4.2. Naïve CD4+ T Cell Differentiation Naïve CD4+ T cells were preincubated for 1 h with the neutralizing antibody anti-PlexinD1 or its respective isotype control, goat IgG (both 2.5 µg/mL, R&D systems, Abingdon, United Kingdom) in RPMI-GlutaMAX (Thermo Fisher Scientific, Nieuwegein, The Netherlands) supplemented with 10% fetal bovine serum (FBS, Biowest, Amsterdam, The Netherlands), 10.000 I.E. penicillin-streptomycin (Thermo Fisher Scientific, Nieuwegein, The Netherlands) and 1:500 Primocin (Invivogen, Toulouse France). Next, cells were activated with Dynabeads Human T-Activator CD3/CD28 (Thermo Fisher Scientific, Nieuwegein, The Netherlands) at a bead-to-cell ratio of 1:5 and differentiated either to Th1, Th2 or Th17 for 7 days with the specific T helper cell differentiation cocktails (Table S1). At day 3, half of the medium was replaced with fresh medium containing the differentiation cocktails and the anti-PlexinD1 antibody or the isotype control. For the intracellular cytokine staining, 1 µg/mL of phorbol 12-myristate 13-acetate (PMA), 50 ng/mL of ionomycin (both from Sigma Aldrich, Zwijndrecht, The Netherlands) and 1 µg/mL of GolgiStop (BD Biosciences, Vianen, The Netherlands) were added for the final 4 h of stimulation. Int. J. Mol. Sci. 2020, 21, 6965 8 of 11

4.3. CD4+ T Cell Stimulation Total CD4+ T cells were pretreated for 1 h with the anti-PlexinD1 antibody or its respective isotype control IgG (both 2.5 µg/mL) and then activated with Dynabeads Human T-Activator CD3/CD28 (Thermo Fisher Scientific, Nieuwegein, The Netherlands) at a bead-to-cell ratio of 1:5 and incubated with the serum of SSc patients (20% v/v), for 5 days. All included patients fulfilled the ACR/EULAR 2013 classification criteria for SSc [41]. All patients provided informed written consent approved by the local institutional medical ethics review boards prior to inclusion in this study (NL47151.041.13, on 29 November 2013). Samples and clinical information were treated anonymously immediately after collection. Demographics and clinical characteristics of the included patients are detailed in Table S2.

4.4. Flow Cytometry CD4+ T cells were stained with Fixable Viability Dye (eBioscience, Nieuwegein, The Netherlands), and antibodies for PlexinB2 APC and its respective isotype control (both from R&D systems, Abingdon, United Kingdom). Alternatively, cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience, Nieuwegein, The Netherlands), and stained for IL-17A FITC, IL-22 APC, IFNγ PerCP-Cy5.5 (all from eBioscience, Nieuwegein, The Netherlands), IL-4 BV711, IL-13 PE and TNF BV421 (all from BD Biosciences, Vianen, The Netherlands). For proliferation analysis, CD4+ T cells were labeled with CellTrace Violet (1.5 µM, Thermo Fisher Scientific, Nieuwegein, The Netherlands) prior to culture. Samples were acquired on a BD LSR Fortessa (BD Biosciences), or on a BD FACSCanto (BD Biosciences, Vianen, The Netherlands) using the BD FACSDiva software (BD Biosciences, Vianen, The Netherlands). FlowJo software (BD Biosciences, Vianen, The Netherlands) was used for data analyses. All flow cytometry data is presented as the percentage of positive cells.

4.5. Cytokine Measurement Sema4A (Biomatik LLC, Wilmington, DE, USA), IL-17A, IFN-γ (eBioscience, Nieuwegein, The Netherlands) and IL-4 (R&D systems, Abingdon, United Kingdom) were measured by ELISA in cell-free supernatants, according to the manufacturer’s instructions.

4.6. RT-PCR and Quantitative qPCR RNA was isolated using the RNeasy micro Kit and RNase-Free DNase Set (Qiagen, Venlo, The Netherlands). Total RNA was reverse-transcribed using an iScript cDNA Synthesis kit (Biorad, Veenendaal, The Netherlands). qPCR reactions were performed using SYBR green (Applied Biosystem, Nieuwerkerk aan Den Ijssel, The Netherlands) with a StepOne Plus Real-Time PCR system (Applied Biosystems, Nieuwerkerk aan Den Ijssel, The Netherlands). cDNA was amplified using specific primers (all from Integrated DNA Technologies, Inc. (IDT), Leuven, Belgium, see Table S3). Relative levels of gene expression were normalized to the B2M housekeeping gene. The relative quantity of mRNA was calculated using the formula 2 ∆Ct 1000. − × 4.7. Statistical Analysis Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software, Inc., San Diego, CA, USA). Potential differences between experimental groups were analyzed by a parametric ANOVA test. p < 0.05 was considered statistically significant.

5. Conclusions The Sema4A-PlexinD1 axis is an important regulator of T helper cell differentiation and therefore regulating Sema4A levels might be a potential therapeutic approach in CD4+ T cell-mediated diseases. Int. J. Mol. Sci. 2020, 21, 6965 9 of 11

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/18/ 6965/s1. Author Contributions: Conceptualization, T.C., A.P.L., T.R.D.J.R. and S.G.; methodology, T.C., B.M.-F., A.P.L. and S.G.; formal analysis T.C., C.R.-V., B.M.-F., A.P.L., J.M.P-R., T.R.D.J.R. and S.G.; Writing—Original draft preparation, C.R.-V., and S.G.; Writing—Review and editing, T.C., C.R.-V., B.M.-F., A.P.L., J.M.P.-R., T.R.D.J.R. and S.G.; funding acquisition, T.R.D.J.R. and S.G. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Dutch Arthritis Association (project grant NR18-1-301 to S. García). C.R-V. is supported by a predoctoral fellowship from Xunta de Galicia (IN606A-2020/043). T.C. and A.P.L. were supported by grants from the Portuguese National Funding Agency for Science, Research and Technology: Fundação para a Ciência e a Tecnologia (SFRH/BD/93526/2013 and SFRH/BD/116082/2016, respectively). S.G. is supported by the Miguel Servet program (CP19/00005) from the Instituto de Salud Carlos III (ISCIII) and the European Social Fund (“Investing in your future”). Acknowledgments: The authors would like to thank Michel Olde Nordkamp for technical support. The graphical abstract figure was created with BioRender.com. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

Sema4A Semaphorin4A Th T helper IFN-γ Interferon-γ IL Interleukin ILT4 Immunoglobulin-like transcript 4 SSc Systemic sclerosis NRP-1 Neuropilin-1

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