Is TGFβ as an anti-inflammatory cytokine required for differentiation of inflammatory T_H17 cells?

Abstract

T-Helper 17 (T_H17) cells are a CD4⁺ T_H subset that plays a critical role in the pathophysiology of inflammatory disorders, especially chronic forms. It seems that the derivation of T_H17 cells from their precursors take place in inflammatory microenvironment. The role of transforming growth factor (TGF)-β as an anti-inflammatory cytokine in T_H17 cell differentiation is controversial. To address some of the discrepancies that exist among different studies, this study was undertaken to more clarify the TGFβ role in human T_H17 cell differentiation. Here, CD4⁺ T-cells were isolated from peripheral blood samples and cultured in X-VIVO 15 serum-free medium. Purified cells were then treated with different combinations of polarizing cytokines (interleukin [IL]1-β, -6, and -23, with or without TGFβ), neutralizing anti-interferon (IFN)-γ and anti-IL-4 antibodies and polyclonal stimulators anti-CD3 and -CD28 antibodies, and then analyzed for IL-17, IFNγ, Foxp3, and CD25 expression by flow cytometry and for release of IL-17, -21, -22, and -10 into culture media by ELISA. The effects of selective inhibition of TGFβ signaling pathway on T_H17 cell polarization were also determined by using small molecules SB-431542 and A83-01. The current study found that a combination of pro-inflammatory cytokines, including IL-1β, -6, and -23, but not TGFβ, could be used as a cytokine combination to induce development of human T_H17 cells. It was also shown that TGFβ acted as a negative regulator in this regard and also led to reduced IL-17 and IL-22 production while inducing Foxp3 expression. Indeed, blocking of TGFβ signaling pathways by selective inhibitors up-regulated T_H17 cell differentiation. From the data here, we concluded that TGFβ down-regulates human T_H17 cell differentiation and that a presence of pro-inflammatory cytokines (along with IFNγ and IL-4 neutralizing antibodies) is sufficient for optimal differentiation of human T_H17 cells.

Keywords:

• A83-01
• TH17 cell differentiation
• Foxp3
• pro-inflammatory cytokines
• SB-431542
• TGFβ
• TGFβ signaling
• TH17 cell
• TH17/1 cell
• Treg cell

Introduction

Traditionally, helper CD4⁺ T (T_H) cells have been categorized into various subsets. The T-Helper 17 (T_H17) subset is identified on the basis of an ability to produce interleukin-17 (IL-17) (also called IL-17A), IL-17F, -21, -22, -23, -6, and characterized by expression of retinoic acid-related orphan receptor (RORC) (the human ortholog of mouse ROR-γt) and signal transducer and activator of transcription-3 (STAT-3) (Annunziato et al. 2007; Korn et al. 2009). As all T_H17 cell-derived cytokines have pro-inflammatory properties, it is not surprising T_H17 cells are implicated in a wide range of inflammatory disorders including experimental autoimmune encephalomyelitis, multiple sclerosis, autoimmune arthritis, and inflammatory bowel disease (Furuzawa-Carballeda et al. 2007).

Several studies have shown an association between severity in inflammatory conditions and T_H17 cell responses in humans and mice (Furuzawa-Carballeda et al. 2007; Maddur et al. 2012). Thus, identification of factors responsible for T_H17 cell differentiation is of great interest due to the importance of these cells in health and disease. Since the discovery of T_H17 cells, efforts have been made to identify factors that polarize T_H17 cells. Some studies have showed that a combination of IL-6 and transforming growth factor (TGF)-β was crucial for optimal differentiation of CD4⁺ T-cells to T_H17 cells in mice (Mangan et al. 2006; Veldhoen et al. 2006). Most of the information about differentiation of T_H17 cells has been derived from experimental mouse models; comparably little is known about key polarizing factors needed for human T_H17 cell development (Streeck et al. 2008). In the human scenario, it was shown that pro-inflammatory cytokines IL-1β, IL-6, and IL-23, as well as TGFβ were all essential for T_H17 cell polarization (Manel et al. 2008; O’Garra et al. 2008; Volpe et al. 2008). However, some studies reported that pro-inflammatory cytokines but not TGFβ was needed for development of this cell type (Acosta-Rodriguez et al. 2007; Ghoreschi et al. 2010). Other studies have also reported dose-dependent effects of TGFβ on T_H17 cell differentiation (Hakemi et al. 2011; Ghaedi et al. 2015). Clearly, studies are somewhat conflicting with regard to roles of cytokines in T_H17 cell generation and the uncertainty as to whether or not TGFβ has any role to play, has not yet been fully resolved.

Cytokines are critical messengers that control the differentiation and directing of T_H cells. TGFβ is believed to promote anti-inflammatory conditions by inducing the expansion of CD4⁺ Foxp3⁺ T_reg (T-regulatory) cells that are essential for immunological tolerance (Shevach et al. 2008). TGFβ signaling is also essential for regulating the expansion, activation, and effector function of mature CD4⁺ T-cells (Letterio 2005). The mechanism of TGFβ signaling is now understood in better detail. Specifically, TGFβ signals through a receptor complex comprised of a Type I and Type II receptor, both serine/threonine kinases. The TGFβ dimers bind to a Type II receptor that recruits, phosphorylates, and activates the Type I receptor. This finally leads to SMAD (Sma- and Mad-related protein) complex accumulation in the nucleus that, in turn, act as transcription factors in the regulation of target gene expression (Shi & Massague 2003).

Small molecule inhibitors have proven extremely useful to investigate signal transduction pathways. The inhibitors SB-431542 and A83-01 are known potent specific inhibitors of TGFβ superfamily Type I receptors (Inman et al. 2002). There are some reports that SB-431542 can reduce Foxp3 expression and inhibit T_reg cell-mediated suppression (Eddahri et al. 2006; Oida et al. 2006). Another study noted that T-cells treated with TGFβ RI kinase inhibitor SB-431542 retained a low amount of T_H17 cell polarization (Hasan et al. 2015). Indeed, to our knowledge, there has been no report about effects of A83-01 in this regard, although A83-01 is known to be more potent in inhibiting TGFβ signaling than SB-431542 (Tojo et al. 2005). By blocking TGFβ signaling pathway (using SB-431542 and A83-01), the role of TGFβ in development of human T_H17 cells can be determined more accurately.

As noted above, there is some controversy regarding the role of TGFβ in T_H17 cell differentiation. It seems these cells are derived under the influence of inflammatory microenvironments and inflammatory cytokines. Because TGFβ is a known anti-inflammatory protein, its presence and participation in T_H17 cell differentiation in inflammatory conditions is a subject of debate as well. Thus, the purpose of the current study was to evaluate the effects of TGFβ on human T_H17 cell differentiation and to identify cytokine combination that could optimize human T_H17 cell polarization. As part of these efforts, the selective inhibitors of TGFβ superfamily Type I receptors (SB-431542 and A83-01) were employed.

Material and methods

Cell isolation and differentiation of human T_H17 cells

Peripheral blood mononuclear cells (PBMC) were obtained from the peripheral blood of healthy human volunteers following obtaining a written informed consent from each subject. The informed consent was prepared and approved by the Review Board at Royan Institute (Tehran, Iran). PMBC were isolated by density gradient centrifugation using Ficoll-Histopaque solution.

From each sample, CD4⁺ T-cells were isolated by magnetic activated cell sorting (MACS) using human CD4 MicroBeads (Miltenyi Biotec, London, UK), according to the manufacturer instructions. The purity of separated CD4⁺ T-cells was ≥96% as determined by flow cytometry. The cells were cultured in X-VIVO 15 Chemically-Defined Serum-free Hematopoietic Cell Medium (Lonza, Allendale, NJ) in 24-well plates at a density of 3 × 10⁵ cells/well and were treated with different combinations of recombinant polarizing cytokines including human IL-1β (20 ng/ml), human IL-6 (40 ng/ml), human IL-23 (20 ng/ml) (all recombinant cytokines from Peprotech, London, UK). Neutralizing antibodies (purified NA/LE Mouse anti-human IFNγ [5 μg/ml; BD Pharmingen, Oxford, UK] and anti-IL-4 [5 μg/ml; R&D Systems, Abingdon, UK]) with T-cell expanders (anti-human CD3 and anti-CD28 Functional Grade [each at 2 μg/ml; eBioscience; Hatfield, UK]) were also used in the cultures. As controls, cells in some wells were only treated by the T-cell expanders (i.e. the cytokine-untreated cells).

For determining the role of TGFβ in human T_H17 cell polarization, in some experiments different concentrations of TGFβ1 (10, 20, 40, 80, or 160 ng/ml) were added to cultured cells as well. In addition, the SB-431542 (10 μM; Sigma, St. Louis, MO) and A83-01 (3 μM, Sigma), were added to culture medium in some culture wells, as selective inhibitors of TGFβ signaling pathways, to more clarify the role of TGFβ in induction of T_H17 cells. On Days 3 and 6 of culture, all above cytokines and antibodies were refurbished through replacement of culture medium. On Day 7 of culture, the cells were re-stimulated with anti-CD3 and anti-CD28 in the presence of the transport inhibitor Brefeldin A (BFA) (5 μg/ml; Sigma) for 12 hr. The cells were then harvested for flow cytometric analysis of intracellular IL-17, IFNγ, CD25, and Foxp3 and the culture supernatants were collected for cytokine analysis.

Flow cytometry and intracellular staining

For each control and test sample, single cell suspension of 0.5–1.0 × 10⁶ cells were washed with BD Perm/Wash Buffer (BD, London, UK). After washing, 200 μl BD Cyto-fix/Cytoperm solution was added to each cell pellet and the cells were incubated for 20–30 min at 4 °C. The cells were then washed twice with BD Perm/Wash Buffer and incubated in phosphate-buffered saline (PBS, pH 7.4) containing 5% bovine serum albumin (BSA, Sigma) for 10–15 min at room temperature. After washing with BD Perm/Wash Buffer, the cells were aliquoted into dedicated tubes and then treated with conjugated antibody at manufacturer-recommended titers (i.e. incubated 30–45 min at 4 °C in the dark). PE (phycoerythrin)-labeled mouse anti-human antibodies against intracellular cytokines IL-17A (eBioscience), IFNγ (BD Bioscience), Foxp3 (Biolegend, San Diego, CA), and CD25 (BD Bioscience), as well as PE-conjugated mouse IgG1κ Isotype Control (BD Bioscience) were used in dedicated tubes for cell staining. All samples were analyzed with Flow cytometer (BD FACSCalibur^TM) and Flow-Jo software. A minimum of 30 000 events/sample was acquired.

Analysis of cytokine production by ELISA

Levels of IL-17, IL-21, IL-22, and IL-10 in culture supernatants were measured using the appropriate commercial ELISA Ready-SET-Go kits (eBioscience), according to manufacturer instructions. The level of sensitivity of the kits was 4 pg IL-17/ml, 8 pg IL-21/ml, 8 pg IL-22/ml, and 2 pg IL-10/ml.

Statistical analysis

All results are expressed as means ± SEM. Statistical analysis was performed using a one-way analysis of variance (ANOVA). All calculations were performed using Prism Software (v.6.0, GraphPad, San Diego, CA). A p-value < 0.05 was considered significant. All experiments were repeated on at least five PBMC samples prepared from different healthy adult donors.

Results

IL-17-producing T_H cell generation requires pro-inflammatory cytokines but not TGFβ

To define cytokine requirements for the induction of human T_H17 cell development, differentiation of isolated CD4⁺ T-cells with pro-inflammatory cytokines (IL-1β, IL-6, IL-23), neutralizing anti-IFNγ and anti-IL-4 antibodies (for inhibition of T_H1 and T_H2 differentiation, respectively), and polyclonal stimulation of T-cells through anti-CD3 and -CD28 antibodies was performed. Figure 1 presents images of proliferated CD4⁺ T-cell colonies during any T_H17 cell polarization. To assess the role of TGFβ in induction of human T_H17 cell differentiation, this study evaluated T_H17 cell polarization in the absence/presence of TGFβ (10–160 ng/ml in range). The results showed that TGFβ acted as a negative regulator for human T_H17 cell development and that a presence of pro-inflammatory IL-1β, IL-6, and IL-23 was sufficient for induction of T_H17 cells and increased production of IL-17. The percentages of cells expressing intracellular IL-17 decreased from 29.41 [±0.84]% to ≈15% in the presence of TGFβ. Nevertheless, TGFβ could not completely suppress IL-17 expression and T_H17 differentiation (Figure 2(A,B)).

Figure 1. Colonies of proliferating CD4⁺ T-cells during in vitro human T_H17 cell differentiation. For stimulation and expansion of the T-cells, polyclonal stimulators (including anti-CD3 and anti-CD28 antibodies) were used (see “Methods” section). Representative micro-images are shown with different magnifications.

Figure 2. IL-1β, IL-6, and IL-23 – but not TGFβ – are required for optimal in vitro differentiation of human CD4⁺ T_H17 cells. (A) Flow cytometric dot-plot analysis of intracellular IL-17 expression by CD4⁺ T-cells. Representative data of five independent experiments (i.e. PBMC from five different donors) are shown. (B) Columns represent mean percentage (±SEM) of T-cells expressing intracellular IL-17 in each culture condition. ns: Non-significant (*p < 0.05).

In a second set of experiments, to explore the role of TGFβ signaling on T_H17 development in vitro, effects from inhibition of TGFβ receptor signaling pathways by addition of SB-431542 and A83-01 were evaluated. The results indicated that blocking TGFβ signaling could restore T_H17 cell differentiation and production of IL-17 (Figure 2(A,B)). Collectively, these data demonstrated that TGFβ and TGFβ signaling were not required for, and were down-regulators of, in vitro development of human T_H17 cells.

Human T_H17 cells produce IFNγ (T_H17/1 cells) in a manner regulated by TGFβ

A majority of IL-17-secreting T_H cells also produce IFNγ; these T_H17 cells are known as T_H17/1 cells and produce both IL-17 and IFNγ (Zielinski et al. 2012; Duhen & Campbell 2014). The current experiments revealed that a certain percentage (10.29 [±0.75]%) of the human T_H17 cells differentiated under the influence of IL-1β, IL-6, and IL-23, and produced IFNγ along with IL-17 (IL-17 + IFNγ + T_H cells). The results also indicated that TGFβ modulated secretion of IFNγ (Figure 3(A,B)) and reduced T_H17 cell differentiation. Addition of SB-431542 and A83-01 as TGFβ signal pathway inhibitors restored IFNγ production by the T_H17 cells.

Figure 3. IFNγ production by human T-cells differentiated in presence of IL-1β, IL-6, and IL-23 – with or without TGFβ as well. (A) Flow cytometric analysis of intracellular expression of IFNγ by T_H17 cells derived from CD4⁺ T-cells. (B) Mean expression levels (±SEM) from five independent experiments (i.e. PBMC from five different donors) using various culture conditions (i.e. different TGFβ concentration and inhibitors of TGFβ signaling pathways) are shown. ns: Non-significant (*p < 0.05).

TGFβ shifts human T_H17 cell polarization to Foxp3⁺ cells

During differentiation of CD4⁺ T_H cells to IL-17-producing cells, a presence of polarizing factors is essential. In the case of TGFβ, its presence up-regulates expression of Foxp3 transcription factor, a lineage specification factor for T_reg cells. The present results showed that pro-inflammatory IL-1β, IL-6, and IL-23 were required for T_H17 cell development but did not induce Foxp3 expression; in contrast, with an increasing presence of TGFβ (10–160 ng/ml), expression of Foxp3 increased – an outcome that indicated there was T_reg cell differentiation instead of T_H17 cell development (Figure 4(A,B)). Both SB-431542 and A83-01 repressed Foxp3 induction.

Figure 4. TGFβ effects on Foxp3 expression by CD4⁺ T-cells differentiated under T_H17 cell-promoting conditions. (A) Intracellular staining of Foxp3. (B) Columns are mean (±SEM) Foxp3 expression levels in five independent experiments (i.e. PBMC from five different donors) (*p < 0.05).

To further investigate TGFβ effects on T_H cells during differentiation, CD25 expression on the cells was measured. As seen in Figure 5, TGFβ suppressed CD25 expression and increasing TGFβ concentrations led to an overall decline in cell CD25 expression. These data showed TGFβ could suppress T_H17 differentiation, in part, through reduced T-cell activation. By blocking TGFβ signaling using SB-431542 and A83-01, expression of CD25 was re-enhanced to >70% of its expression in the absence of TGFβ (Figure 5(A,B)).

Figure 5. TGFβ effects on in vitro T_H17 cell differentiation/activation of T-cells. (A) Dot-plots show expression of CD25 by differentiated T-cells. (B) Column bars are means ± SEM of five independent experiments (i.e. PBMC from five different donors) (*p < 0.05).

T_H17 cell-derived cytokines modulated by TGFβ

These experiments also measured production of cytokines associated with T_H17 cell differentiation, e.g. IL-17, IL-21, IL-22, and IL-10, in the cultures. The results showed that a combination of IL-1β, IL-6 and IL-23 in the presence of anti-IFNγ and anti-IL-4 antibodies and polyclonal stimulators (anti-CD3 + anti-CD28) were necessary to induce human T_H17 cells that produced >200 pg/ml IL-17 and 240 pg/ml IL-22 (T_H17 cell hallmark cytokines) and low amounts of IL-21 and IL-10. TGFβ significantly suppressed production of IL-17, IL-21, and IL-22 (Figure 6). Production of the same cytokines after blocking of TGFβ signaling pathways (via A83-01 and SB-431542) was also analyzed. Notably, all T_H17 cell-associated cytokines were up-regulated; this outcome emphasized the fact TGFβ could inhibit optimal T_H17 cell differentiation.

Figure 6. T_H17 cell-derived cytokines; modulation by TGFβ and blocking of TGFβ signaling. Cytokine levels in supernatants of CD4⁺ T-cells differentiated toward T_H17 states in the presence or absence of TGFβ and/or inhibitors of signaling pathways. Data showed means ± SEM from five independent experiments (i.e. PBMC from five different donors). ND: not detected (*p < 0.05).

Discussion

The current study was designed to examine the critical factors involved in regulation of human T_H17 cell differentiation, especially the role of TGFβ and related signaling in derivation of T_H17 cells in vitro. The present results indicated that pro-inflammatory cytokines (e.g. IL-1β, IL-6, IL-23) were crucial for this differentiation – independent of any TGFβ or associated signaling. Moreover, TGFβ treatments induced Foxp3 expression and reduced IL-17, IL-22 and IFNγ production – indicating a negative role for TGFβ in this differentiation process (Figure 7).

Figure 7. Schematic of human T_H17 cell differentiation by pro-inflammatory cytokines independently of TGFβ and its associated signaling. In presence of IL-1β, IL-6, and IL-23 as pro-inflammatory cytokines, T_H17 cells that secrete IL-17, IL-22, and IFNγ developed, whereas TGFβ suppressed production of IL-17, IL-22, and IFNγ and enhanced expression of Foxp3 on the differentiated cells. Selective TGFβ signaling inhibitors restored T_H17 cell differentiation.

Some studies have illustrated that TGFβ + IL-6 were critical for T_H17 cell development in mice (Bettelli et al. 2006; Mangan et al. 2006; Veldhoen et al. 2006). In contrast, the role of TGFβ in human T_H17 cell differentiation is still unclear and controversial (Table 1). The present findings were consistent with studies reporting TGFβ had a suppressive function on development of the T_H17 lineage, through reduction of IL-17 production (Acosta Rodriguez et al. 2007; Evans et al. 2007; Wilson et al. 2007). The present study also found that in vitro production of IL-17 and IL-22 was optimal in the presence of IL-1β, IL-6, and IL-23 – but only in the absence of TGFβ, and that TGFβ-related signaling was deleterious for the induction of IL-17-producing T_H cells.

Table 1. Reported role of TGFβ in human T_H17 cell differentiation.

Authors	Significant findings
Manel et al. (2008)	TGFβ, IL-1β, and IL-6, IL-21 or IL-23 are required for TH17 cell polarization.
Volpe et al. (2008)	TGFβ, IL-1β, IL-6, and IL-23 are all essential for TH17 cell differentiation.
Ghaedi et al. (2015)	Low concentrations of TGFβ increase IL-17 expression, but higher concentrations of TGFβ suppress TH17 cell differentiation in the presence of IL-1β, IL23, and IL-2.
Yang et al. (2008)	IL-1β and IL-6 induce IL-17 secretion, but TGFβ and IL-21 uniquely promote the differentiation of TH17 cells accompanied by expression of RORC.
Hakemi et al. (2011)	Absence of TGFβ: induced IL-17+ and IFNγ+ TH17 cells. Presence of TGFβ: induced IL-17+ and IFNγ− TH17 cells.
Santarlasci et al. (2009)	TGFβ suppresses T-bet and IFNγ expression but does not affect the expression of IL-17 and ROR-γt.
Evans et al. (2007)	TGFβ suppresses the generation of TH17 cells and inhibits IL-17 and IFNγ expression.
Acosta Rodriguez et al. (2007)	IL-1β and IL-6 but not TGFβ are essential for TH17 cell differentiation. TGFβ suppresses the production of IL-17 and IFNγ.
Wilson et al. (2007)	IL-1β and IL-23 drive the differentiation of TH17 cells. TGFβ suppress the expression of IL-17, IL-22, and IFNγ.
Chen et al. (2007)	IL-6 and IL-23, without TGFβ are needed for TH17 differentiation. TGFβ up-regulate the RORC expression but do not induce TH17 cell derivation.
van Beelen et al. (2007)	IL-1β and IL-23 induce development of TH17 cells through IL-17 and IFNγ production, but TGFβ and IL-6 cannot induce human TH17 cells polarization.

Contradictive studies – based on effects of TGFβ on human T_H17 cell derivation – are presented as well.

TGFβ suppresses the immune system by inhibiting the function of immune inflammatory cells and promoting the differentiation and function of T_reg cells (Shevach et al. 2008). It also seems that TGFβ plays a role in the interplay between T_H17 and T_reg cells (Awasthi et al. 2008) and can control the plasticity of T_H17/T_reg cell lineages (Lee et al. 2009; Kleinewietfeld & Hafler, 2013). The present results indicated that TGFβ promoted expression of Foxp3 and may have induced Foxp3⁺ T_reg cell populations even in the presence of T_H17-promoting cytokines. Similar with these data, there are reports that TGFβ induced T_reg cells and inhibited T_H17 cell differentiation (Shevach et al. 2008; Zhou et al. 2008). The current study also showed that TGFβ decreased cell CD25 expression. Classen et al. (2007) also reported that loss of TGFβ signaling was important for T-cell proliferation and suggested TGFβ was a prominent factor in keeping human CD4⁺ T-cells in a resting state.

Some studies have indicated there is a subtype of human T_H17 lymphocytes that is able to produce simultaneously both IL-17 and IFNγ and co-express T-bet and RORC – a hybrid (T_H17/T_H1) phenotype, i.e. T_H17/1 cells (Annunziato & Romagnani 2010; Boniface et al. 2010; Duhen et al. 2013). Peters et al. (2011) indicated that T_H17 populations contain cells ranging from ‘classical’ more regulated to ‘alternative’ more pathogenic T_H17 cells. The latter types are major cells involved in the pathogenicity of autoimmunity and inflammatory reactions; these cells produce IL-17, IL-22, and IFNγ, along with low amounts of IL-21. On the other hand, the classic T_H17 cell produces IL-17, IL-21, and IL-10, with a low amount of IL-22. In our experiences, we have obtained T_H17 lineages more similar to the pathogenic phenotype. When TGFβ plus pro-inflammatory cytokines were used, expression of both IL-17 and IFNγ decreased. Consistent with the present results, Annunziato et al. (2009) and Wilson et al. (2007) reported that use of a combination of IL-1β with IL-23 in the absence of TGFβ promoted differentiation of T_H17/1 cells that co-expressed IL-17, IFNγ, and IL-22.

There is also debate about TGFβ intracellular signaling pathways that are involved in this process (Hirahara et al. 2010). The current results demonstrated that both SB-431542 and A83-01, as selective inhibitors of TGFβ signaling pathways, restored the production of IL-17 and IL-22 from differentiating T_H17 cells. Indeed in the presence of TGFβ, selective inhibition of its signaling pathways up-regulates T_H17 cell differentiation. There is a study reported that T-cells treated with SB-431542 did not differentiate to T_H17 cells (Hasan et al. 2015). In this regard, Mohammed et al. (2010) also showed that SB-431542 caused a block in any increase in IL-17 secretion. To date, there have been no reports for an effect of A83-01 in changing potential T_H17 cell polarization. Though suggestive of a role of TGFβ-related signaling in many of these outcomes, those experiments were done with mice where TGFβ is already reported to be a prominent factor in their T_H17 cell development.

In the present study, expression of Foxp3 (demonstrating T_reg cell polarization) was inhibited by SB-431542 or A83-01 molecules even when TGFβ was present. Some other studies have also shown that SB-431542 abolished T_reg cell-mediated immune suppression (Eddahri et al. 2006; Oida et al. 2006). The present results were also consistent with those of Zaccone et al. (2011) and Olkhanud et al. (2011) who showed that inhibition of TGFβ signaling (using SB-431542) dramatically reduced Foxp3 expression – while expression of IFNγ that was reduced by TGFβ was now up-regulated after a blocking of TGFβ signaling. While some investigators have found A83-01 was more potent than SB-431542 in inhibiting TGFβ signaling (Tojo et al. 2005), the present study found no significant differences between the agents with regard to impact on T_H17 cell development. Therefore, while the data here showed that inhibition of TGFβ activity by blocking TGFβ signaling pathways did not restrict human T_H17 cell derivation, these results may pertain only to the set of conditions established in this in vitro experiment.

Conclusions

The present findings indicated to us that IL-1β, IL-6, and IL-23 – as pro-inflammatory cytokines – were likely required for human T_H17 cell derivation and that these effects seemed to be independent of TGFβ and related signaling pathways. We also concluded that a presence of TGFβ and related signaling were moreover negative factors for in vitro human T_H17 development and in fact, seemed to serve to promote T_reg cell differentiation.

Disclosure statement

The authors declare no conflicts of interest. The authors alone are responsible for the content of this manuscript.

Funding

This study was done as one part of PhD research project and financially was supported by a research grant (Grant No. 1065782) from Tarbiat Modares University and a research grant (Grant No. 91000101) from Royan Institute.