Maf deficiency in T cells dysregulates Treg - TH17 balance leading to spontaneous colitis

Imbratta, Claire; Leblond, Marine M.; Bouzourène, Hanifa; Speiser, Daniel E.; Velin, Dominique; Verdeil, Grégory

doi:10.1038/s41598-019-42486-2

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Published: 16 April 2019

Maf deficiency in T cells dysregulates T_reg - T_H17 balance leading to spontaneous colitis

Claire Imbratta¹,
Marine M. Leblond¹,
Hanifa Bouzourène²,
Daniel E. Speiser ORCID: orcid.org/0000-0003-2031-3250¹,
Dominique Velin³ &
…
Grégory Verdeil¹

Scientific Reports volume 9, Article number: 6135 (2019) Cite this article

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Abstract

The maintenance of homeostasis in the gut is a major challenge for the immune system. Here we demonstrate that the transcription factor MAF plays a central role in T cells for the prevention of gastro-intestinal inflammation. Conditional knock out mice lacking Maf in all T cells developed spontaneous late-onset colitis, correlating with a decrease of FOXP3⁺RORγt⁺ T cells proportion, dampened IL-10 production in the colon and an increase of inflammatory T_H17 cells. Strikingly, FOXP3⁺ specific conditional knock out mice for MAF did not develop colitis and demonstrated normal levels of IL-10 in their colon, despite the incapacity of regulatory T cells lacking MAF to suppress colon inflammation in Rag1^−/− mice transferred with naïve CD4⁺ T cells. We showed that one of the cellular sources of IL-10 in the colon of these mice are T_H17 cells. Thus, MAF is critically involved in the maintenance of the gut homeostasis by regulating the balance between T_reg and T_H17 cells either at the level of their differentiation or through the modulation of their functions.

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Introduction

Maf encodes for a transcription factor belonging to the AP-1 family (MAF or c-MAF). It has been studied for many years in T cells, and more specifically in CD4⁺ T helper (T_H) cell differentiation. Its role in Il4 transcriptional regulation and T_H2 regulation was first established using a transgenic mouse system for Maf^1,2. Maf was also shown to play a prominent role in T_H17 cells in both mice and human, through the regulation of Il10 and Il23r expression^3,4 and in T_FH cells together with the transcription factor Bcl6⁵. MAF inactivation in T cells affects susceptibility to disease in a context-specific manner, depending on the T cell polarization induced by this disease with a general tendency to increase inflammatory responses over tolerance⁶. In invariant natural killer T (iNKT) cells, MAF regulates the expression of IL-17A and RORγt⁷. Interestingly, Maf was found upregulated in CD8⁺ T cells infiltrated in human and murine melanoma. This expression led to intratumoral T cell dysfunction through the regulation of genes involved in T cell exhaustion⁸.

The potential role of Maf in regulatory T cells (T_reg) is less clear. MAF cooperates with AhR in FOXP3⁻ T regulatory 1 (T_R1) cells to control Il10 expression⁹. Recently, a subset of T_reg expressing both FOXP3 and RORγt, the master transcription factor of T_H17 cells, has been characterized in more detail¹⁰. Mostly present in the gut, RORγt⁺ T_reg have enhanced suppressive activity compared to RORγt^- T_reg and maintain gut homeostasis to microbiota^11,12,13. The development of this population, described in both mice and human^11,12, is tightly linked to the presence of the microbiota but not to dietary antigens¹⁴. Transcriptomic analysis showed an enriched expression of Maf in RORγt⁺ T_reg¹³ and it was shown that the inactivation of Maf in T_reg strongly affects its function and differentiation into RORγt⁺ T_reg^15,16. Recent studies have linked MAF to the modulation of a large immunoregulatory and tissue-residency program in human T_H17 cells producing Il-10. These results are reminiscent of the role of MAF in the regulation of tolerance in the gut that is dependent on induced Treg cells¹⁷. MAF can bind in the vicinity of many genes expressed in recently activated T_H17 cell subsets. This study suggests that binding with other transcriptional partners to regulatory regions of the genome would explain the different effect of MAF on the expression of genes encoding either tolerogenic or inflammatory molecules.

In an attempt to clarify the role of Maf in T cells in vivo, we studied mice inactivated for Maf in all T cells (Maf^ΔTcells). Interestingly, these mice developed late onset colitis correlating with a decrease of RORγt⁺ T_reg and an increase of T_H17 cells in the colon and the mesenteric LNs. The disease was associated with increased production of TNFα, IFNγ, IL-1β and a lack of IL-10 in the colon of Maf^ΔTcells mice. Using an adoptive cell transfer model in Rag1^−/− mice, we showed that Maf-deficient T_reg are inefficient in preventing colitis, demonstrating the role of Maf in regulating T_reg function. Moreover, we observed that Maf^ΔTcells mice develop exacerbated T_H17 response against Helicobacter pylori, a human pathobiont colonizing the stomach mucosa, leading to the inability of the bacteria to establish a chronic state of infection in Maf^ΔTcells mice. Strikingly, mice deficient for Maf in T_reg alone (Maf^ΔTreg mice) did not develop colitis and demonstrated normal levels of IL-10 in their colon. Compared to Maf^ΔTcells, we observed that T_H17 cells of Maf^ΔTreg mice produce IL-10. Taken together, our data demonstrated that Maf is playing a major role in the maintenance of gastro-intestinal homeostasis through the regulation of functions of both T_reg and T_H17 cells. Maf regulates the differentiation of RORγt⁺ T_reg, the suppressive activities of T_reg as well as the activity of T_H17 cells from the gut.

Results

T cell specific Maf-deficient mice develop spontaneous colitis

To study the role of Maf in T cells, we generated CD4^cre Maf^fl/fl mice (Maf^ΔTcells), in which all T cells (CD4⁺ and CD8⁺) are inactivated for the expression of Maf. Interestingly, these mice spontaneously developed strong colitis, associated with a defect in weight gain starting at 16 weeks of age (Fig. 1A), concomitant with the appearance of diarrhea. These symptoms increased as the mice got older – with up to a 15% difference of body weight at 26 weeks of age between Maf^ΔTcells mice and Cre-negative littermates (Maf^f/f) (Fig. 1A). Macroscopic and microscopic examination of the gastrointestinal system revealed severe inflammation of all the parts of the colon with a clear enlargement in most of the mice (Fig. 1B,C) but no clear difference in the length of the colon (Supplementary Fig. 1A). We did not observe similar features in other organs such as liver or kidney (Supplementary Fig. 1B-D). Hematoxylin and eosin staining on histological sections of colon samples showed massive infiltration of cells in the lamina propria and submucosal spread (Fig. 1C). The scoring of histological sections revealed that the majority of mice had severe colitis (4/7 mice with the score of 5) (Fig. 1D). We found a significant increase in the number of total immune cells both in the mesenteric LN (mLN) and in the colon of Maf^ΔTcells mice compared to the one from Maf^f/f littermates (Fig. 1E). Both CD4⁺ and CD8⁺ T cells were more abundant in the colon of Maf^ΔTcells mice compared to Maf^f/f littermates (Fig. 1F). Maf^ΔTcells mice treated with antibiotics developed no or only mild colitis (Supplementary Fig. 1E,F) demonstrating the requirement of microbiota in the development of this phenotype. Thus, the absence of Maf in T cells leads to spontaneous colitis in mice.

In vitro differentiation of Maf-deficient CD4⁺ T cells in RORγt⁺ T_reg is defective

As previously described, MAF has been shown to regulate cytokine production in various CD4⁺ T_H subsets. To determine whether MAF deficiency in CD4⁺ T cells can also alter the differentiation of any of these subsets, we differentiated naïve CD4⁺ T cells with polarizing medium to obtain T_H1, T_H2, T_reg and T_H17 (FOXP3⁻ or FOXP3⁺) cells in vitro (Supplementary Fig. 2A–C). Comparison of the expression of the master transcription factors TBET (for T_H1), GATA3 (for T_H2), FOXP3 (for T_reg) and RORγt (for T_H17) between CD4⁺ T cells from Maf^ΔTcells and Maf^f/f mice did not show significant differences (Fig. 2A,B). However, in the T_H17 polarizing condition, we observed the development of two distinct populations when analyzing FOXP3 expression. For the FOXP3⁺ population, also named RORγt⁺ T_reg, there was a significant decrease in RORγt level in CD4⁺ T cells from Maf^ΔTcells mice compared to the one from Maf^f/f mice, from 35% to 10% of CD4⁺ T cells respectively (Fig. 2A,B). In these polarizing conditions, MAF levels were the highest in RORγt⁺ T_reg and T_H17, with approximately 80% of MAF- expressing cells in both subsets. MAF is also expressed in RORγt^- T_reg, though at lower level - around 8% of the cells - whereas it is almost absent in T_H1 and T_H2 (Fig. 2C,D). These in vitro experiments of differentiation suggest that MAF plays a role for the differentiation of RORγt⁺ T_reg and might impact the physiological functions of T_H17 cells.

T cell specific Maf-deficient mice are deprived of RORγt⁺ T_reg

To confirm the results obtained in vitro, we first looked at the abundance of transcripts encoding for cytokines in the total colon of aged Maf^f/f or Maf^ΔTcells mice (20 weeks or more). We found higher expression of Tnfa, Ifng and Il1b in Maf^ΔTcells mice compared to Maf^f/f littermates (Fig. 3A), in accordance with a higher infiltration of immune cells and a stronger inflammation in the colon. The transcripts encoding for Il4 were slightly less abundant in Maf^ΔTcells mice and, strikingly, Il10 expression level was much lower in colons from Maf^ΔTcells mice (Fig. 3A). We then determined the in vivo expression of MAF in the various subsets. MAF was expressed in RORγt⁺ FOXP3⁺ T_reg and RORγt⁺ FOXP3⁻ T_H17 cells- around 40% and 20%, respectively - from the colon (Fig. 3B). In the colon, around 15% of RORγt⁻ T_reg expressed MAF (Fig. 3B). The proportion of RORγt⁺ T_reg was dramatically decreased in spleens, mLN and colons of Maf^ΔTcells mice, while we found a significant increase in percentages as well as total numbers of RORγt⁻ T_reg from these organs compared to Maf^f/f mice (Fig. 3C,D and Supplementary Fig. 3A). We also observed elevated percentages and cell numbers of T_H17 cells in spleens, mLN and colons of Maf^ΔTcells mice compared to Maf^f/f mice (Fig. 3C,D and Supplementary Fig. 3A). In Maf^f/f mice, RORγt⁺ T_reg proportions increased with age to reach 10% of the total CD4⁺ T cell population in the colon at 25-weeks age mice (Fig. 3E). The defect in RORγt⁺ T_reg in Maf^ΔTcells mice is already detected at 2 weeks of age (i.e. the weaning period) compared to Maf^f/f mice (Fig. 3E). Interestingly, the level of MAF in these three populations followed the same upward trend with time (Fig. 3F). RORγt⁻ T_reg increase was already present in Maf^ΔTcells mice at the age of 2 weeks and tended to expand over time (Fig. 3E). However, the increase of T_H17 cells was only observed at an advanced age in Maf^ΔTcells mice, correlating with the development of colitis. These data indicate that the deletion of MAF in T cells alters differentiation of highly suppressive RORγt⁺ T_reg whereas it favors RORγt⁻ T_reg accumulation and later T_H17 cells expansion. Loss of the equilibrium between these subsets in the colon is associated with colitis onset.

Maf-deficient T_reg fail to prevent colitis development in vivo

The dramatic decrease in RORγt⁺ T_reg was associated with an increase in classical RORγt⁻ T_reg in Maf^ΔTcells mice (Fig. 3D). However, the development of spontaneous colitis in Maf^ΔTcells mice implies that Maf-deficient T_reg are unable to offset the decrease in highly suppressive RORγt⁺ T_reg. This would suggest that the remaining Maf-deficient T_reg are dysfunctional and thus inefficient at suppressing T_H17 cells. To test this hypothesis, we examined the capacity of wild type (WT) or Maf-deficient T_reg to suppress gut inflammation driven by the injection of naïve CD4⁺ T cells into Rag1^−/− mice¹⁸. We transferred either WT or Maf-deficient naïve CD4⁺ T cells alone or in the presence of either WT or Maf-deficient T_reg into Rag1^−/− recipient mice. Both WT and Maf-deficient naïve CD4⁺ T cells induced weight loss and cell infiltration in the colon when transferred alone (Fig. 4A,B) without any significant difference in the histologic score between the two groups (Fig. 4C). When we co-transferred WT T_reg, the weight loss and the infiltration of cells in the colon were prevented with WT and Maf-deficient naïve CD4⁺ T cells (Fig. 4A,B). Strikingly, Maf-deficient T_reg failed to prevent colitis in all transferred groups compared to WT T_reg, with a significant difference for the weight loss and the histological score (Fig. 4A–C). Histological sections of colons from recipient mice transferred with Maf-deficient T_reg displayed massive infiltration of cells in the associated lamina propria, with a disorganized architecture that was not observed in mice transferred with WT T_reg (Fig. 4B,C). We confirmed the presence of T_reg in all co-transferred groups (Fig. 4D) and the expression of MAF in WT transferred T_reg (Fig. 4E). Taken together, these results demonstrated that Maf-deficient T_reg are at least partially dysfunctional since they cannot prevent inflammation and colitis development.

T_reg specific Maf-deficient mice do not develop spontaneous colitis

We showed that both T_reg differentiation and function were largely affected in Maf^ΔTcells mice (Figs 3 and 4). To test whether the defect of MAF in Treg was sufficient to recapitulate the phenotype of colitis, we generated Foxp3^YFPcre Maf^fl/fl mice (Maf^ΔTreg), in which only T_reg are inactivated for the expression of Maf. We observed a strong decrease in percentages as well as total numbers of RORγt⁺ T_reg in the colon and in the mLN, mirrored by a slight increase in percentages of RORγt⁻ T_reg and T_H17 cells (Fig. 5A,C and Supplementary Fig. 3). We confirmed the complete deletion of Maf only in T_reg from Maf^ΔTreg mice (Fig. 5B). However, we did not observe any signs of colitis, confirmed by histological colon section and body weight measurement of these mice compared to Maf^f/f mice (Fig. 5D,E) even in aged (6–12 months) Maf^ΔTreg mice (Fig. 5F). Similarly, the number of immune cells infiltrated in the colon of Maf^f/f and Maf^ΔTreg was not different (Fig. 5G). We noticed a slight increase in cells in mLN (Fig. 5G) and no difference in the number of CD4⁺ or CD8⁺ T cells in the colon of Maf^ΔTreg mice compared to WT (Fig. 5H). This showed that Maf inactivation in T_reg is not sufficient to induce spontaneous colitis.

Maf deletion impairs IL-10 production by T_reg and T_H17 cells

To uncover why Maf^ΔTreg mice do not develop colitis, we measured transcript levels of cytokines encoding genes by quantitative RT-PCR in total colon of Maf^f/f, Maf^ΔTreg or Maf^ΔTcells mice aged of more than 20 weeks. As expected, we found higher expression of transcripts encoding for Maf in colon from Maf^ΔTreg mice compared to colon from Maf^ΔTcells mice, confirming that conventional T cells infiltrated in the colon still expressed Maf in Maf^ΔTreg mice (Fig. 6A). When looking at transcripts encoding for Il10, we found a similar pattern of expression than the one for Maf, with a higher level of transcript in Maf^f/f and Maf^ΔTreg mice compared to Maf^ΔTcells mice (Fig. 6A). To confirm that T_reg from Maf^ΔTreg are really deficient for Maf and Il10 production, T_reg from the spleen and LN of Maf^f/f, Maf^ΔTreg or Maf^ΔTcells mice were sorted using flow cytometry. We measured high levels of transcripts for Maf and Il10 in T_reg cells from Maf^f/f mice, while the levels of transcripts for these genes were low in T_reg cells from both Maf^ΔTreg and Maf^ΔTcells mice (Fig. 6B). Similarly, we confirmed that upon restimulation, T_reg from mLN and colon of Maf^ΔTreg or Maf^ΔTcells mice expressed decreased levels of IL-10 compared to Maf^f/f counterparts (Fig. 6C,D). This reduction is largely observed in RORγt⁺ T_reg but also to a lesser extent in RORγt⁻ T_reg from mLN of Maf^ΔTreg mice, suggesting a dependence for MAF for IL-10 production in T_reg. These data suggest that Maf expression in FOXP3⁻ T cells is able to compensate for sufficient amounts of Il10 production to prevent colitis development in Maf^ΔTreg mice. To determine which other T cell type could produce IL-10, we stimulated ex-vivo CD4⁺ T cells from mLN and colon of mice. We observed that IL-10 production by T_H17 cells from mLN of Maf^ΔTcells mice was decreased compared to production from Maf^f/f and Maf^ΔTreg mice (Fig. 6E). This suggests that IL-10 production by T_H17 cells in the gut is also regulated by Maf. The reduction of IL-10 expression not only by T_H17 cells, but also T_reg, could explain colitis onset observed only in mice where all the T cells are inactivated for Maf. Furthermore, we observed an increased percentage in IFNγ⁺T_H17 cells while the percentage of IL-17A⁺ T_H17 cells remained similar in Maf^ΔTcells mice compared to Maf^f/f or Maf^ΔTreg mice (Fig. 6F). Together, these data demonstrate that Maf is an essential driver of T_reg and T_H17 immunoregulatory function, especially through IL-10 regulation.

Maf ^ΔTcells mice eliminate Helicobacter pylori through exacerbated T_H17 response

Recently, Gabrysova L et al. demonstrated that T_H17 response was inhibited in Maf^ΔTcells mice in an experimental autoimmune encephalomyelitis (EAE) model⁶. This conclusion is not in line with our observation that Maf^ΔTcells mice develop a colitis (Fig. 1C) characterized by the accumulation of T_H17 cells in the colon lamina propria (Fig. 3D). In order to re-evaluate our results in a different experimental setting, we infected Maf^ΔTcells mice with the human pathobiont Helicobacter pylori. H. pylori infects the stomach mucosa and relies on T_reg and IL-10 to chronically infect its host^19,20. Moreover, it has been established that H. pylori can be cleared from the stomach mucosa by a T_H17 response triggered by vaccination²¹. Two months after infection, quantification of the bacterial burden by numeration of colony-forming unit (CFU) in the mouse stomach showed that Maf^ΔTcells mice cannot be infected by H. pylori (Fig. 7A). Remarkably, as compared to infected Maf^f/f mice, we observed increased mRNA levels encoding for Cd4, Tnfα, Inos, Il17a, Il22 and the antimicrobial peptides RegIIIβ and RegIIIγ in the stomach mucosa of Maf^ΔTcells mice (Fig. 7B–E). This pattern of increased expression, which is characteristic of a T_H17 response, is very similar to the vaccine-induced T_H17 response that clear H. pylori infection in WT mice²². In addition, we found higher Il2 production as well as higher Foxp3 level in Maf^ΔTcells mice (Supplementary Fig. 4). Collectively, these data suggest that Maf^ΔTcells mice can eliminate H. pylori infection by generating an exacerbated T_H17 inflammatory program.

Discussion

Our study shows that the deletion of Maf in all T cells (Maf^ΔTcells mice) drives spontaneous late-onset colitis (Fig. 1). To date, no study has shown that the deletion of Maf in T cells was sufficient and necessary to drive colitis. The development of the colitis is associated with the dysregulation of the T_reg-T_H17 equilibrium (Fig. 3D,E) and a large decrease in the production of IL-10 (Fig. 3A). Inactivation of Maf in T cells impaired the differentiation of RORγt⁺ T_reg (Fig. 3C) and favoured the accumulation of colitogenic T_H17 cells in the colon of the mice (Fig. 3D). The onset of the colitis is dependent on the presence of microbiota since Maf^ΔTcells mice treated with antibiotics develop only mild colitis (Supplementary Fig. 1E,F). The first visible signs of colitis developed around the age of 15 weeks (Fig. 1A). It correlates with the natural differentiation and/or accumulation of RORγt⁺ T_reg and MAF expression in these cells in the colon of WT animals (Fig. 3E,F). This RORγt T_reg population is preferentially found in the colon (Fig. 3C) and represents the main producer of IL-10 (Fig. 6D)^10,16. The role of MAF in the regulation of the RORγt T_reg population was demonstrated very recently using mice in which Maf is deleted in T_reg (Maf^ΔTreg)^15,16. In these two studies, the authors showed that the differentiation of this particular regulatory population is altered in the absence of Maf.

The low proportion of RORγt⁺ T_reg in the colon of Maf^ΔTcells mice was paradoxically associated with an increase in proportion and numbers of RORγt⁻ T_reg. We provide evidence that T_reg in Maf^ΔTcells mice are not fully functional. Indeed, compared to WT T_reg, Maf-deficient T_reg do not control the development of colitis induced by the transfer of naïve CD4⁺ T cells into Rag1^−/− mice (Fig. 4). Moreover, we observed that Maf-deficient RORγt⁻ T_reg produce lower level of IL-10 as compared to WT RORγt⁻ T_reg (Fig. 6C,D). These findings are in appearance not entirely in line with the study of Xu et al. showing that Maf -deficient nT_reg are still able to suppress colon inflammation in Rag1^−/− mice transferred with naïve CD4⁺ T cells¹⁵. However, given the nature of their experimental setting in which Maf -deficient nT_reg were isolated from H. hepaticus colonized mice and transferred in H. hepaticus infected mice, we assume that the antigen specificity of some Maf-deficient nT_reg could compensate for their partial loss of function. One other possibility would be the effect of potentially contaminating Maf-deficient T cells transferred with sorted Maf-deficient Treg. We assume that this potentially pathogenic T cells are also present in the “naïve” Maf-deficient T cells transferred in the mice. However, control Treg transferred together with “naïve” Maf-deficient T cells are still able to control colitis, which make this possibility unlikely. Altogether, it can be suggested that despite their normal differentiation, Maf-deficient RORγt⁻ T_reg are unable of preventing colitis due to their loss of function (decreased IL-10 production), combined to their low specificity to the gut microbiota in our experimental setting.

An impaired differentiation of colon RORγt⁺ T_reg was also observed in Maf^ΔTreg mice (Fig. 5A). Despite this absence, the mice did not develop colitis even at old ages (Fig. 5D–F). The absence of colitis onset is associated with a very limited accumulation of T_H17 cells (Fig. 5C) and normal expression level of IL-10 in the colon (Fig. 6A). This result is similar to what is described previously in these mice, with only a mild inflammation detected in H. hepaticus free animals aged of 6 months¹⁵.

One of the differences between Maf^ΔTreg and Maf^ΔTcells mice is that RORγt⁺ T_H17 cells do not express Maf in Maf^ΔTcells mice (Fig. 2C). It has been shown that MAF plays a prominent role in T_H17 cells through the regulation of IL-10 production^4,17. We indeed observed in vivo that RORγt⁺ T_H17 cells from Maf^ΔTcells mice do not produce IL-10 while Maf^f/f and Maf^ΔTreg counterparts do (Fig. 6E). Furthermore, we showed an increase of IFN-γ⁺ T_H17 cells from Maf^ΔTcells compared to Maf^f/f and Maf^ΔTreg mice (Fig. 6F).

Maf can affect other T cell subtypes that might participate to colitis development. A recent study on the role of MAF in iNKT cells showed that MAF inactivation in these cells leads to a decrease in IL-17a production⁷. In our system this would mean that inactivation of MAF in these cells would limit the development of colitis, which is however not the case. MAF regulates IL-4 but not IL-10 production in T_H2 cells²³. The absence of MAF leads to a decreased expression of Il-4²³, which is not in accordance with an increased pathology. T_H2 cells are a minority of the CD4 T cells that we find in the colon of the mice and do not increase among CD4 T cells in Maf^ΔTcells (Supplementary Fig. 3C). Despite the involvement of T_H2 cells in the development of ulcerative colitis in human²⁴, we think for these reasons that it is unlikely that the development of colitis after MAF inactivation is related to T_H2 cells. T_R1 cells, may also be a source of IL-10. Indeed, it was described that MAF is present in this population and regulates the expression of Il10 together with AhR⁹. We did not find any IL-10 producing RORgt- Foxp3- CD4 T cells in the colon of our mice, which limits the potential role of Tr1 in our system (Supplementary Fig. 3C). Altogether, the absence of IL-10 production by both RORγt^+/− T_reg and T_H17 cells associated with the increase production of IFN-γ by T_H17 cells in Maf^ΔTcells mice most probably lead to colitis onset.IL-10 production by T_reg is essential to maintain homeostasis at environmental surfaces by directly suppressing pathogenic T_H17 cells and T_H17/T_H1 cells²⁵. Several years ago, work led by Rudensky et al. showed that selective disruption of Il10 in T_reg leads to spontaneous colitis²⁶. However, mice lacking IL10RA²⁷ in T_reg that show reduced expression of IL-10, develop a more severe colitis, suggesting that compensatory mechanisms orchestrated by non-T_reg can participate to maintain homeostasis through regulation of T_reg function. Although restricted to the colon, the phenotype observed in our model looks similar to the colitis observed in T cell–specific IL-10 mutant mice²⁸ or Il10^−/− mice²⁹. This is in line with recent study showing that Maf is a common regulator of IL-10 in CD4⁺ T cells⁶. We were able to link this phenotype to decreased suppressive capacity of all Maf-deficient T_reg, but particularly RORγt⁺ T_reg, leading to uncontrolled T_H17-driven inflammation.

Recent reports suggested that RORγt is induced after the differentiation of naïve cells into Foxp3⁺ T_reg^13,30. How MAF is regulating the development of these cells remains an open question. MAF is induced by TGF-β and IL-6 in CD4⁺ T_H subsets and CD8⁺ T cells^3,4,8,15. Wheaton et al. proposed that IL-6 can upregulate MAF in pre-differentiated T_reg leading to RORγt acquisition. Indeed, RORγt has been identified as a direct target of MAF⁶. Observation that selective disruption of STAT3 in T_reg leads to a decrease of RORγt⁺ T_reg (11) in mice suggest that STAT3 may also be required in this process of differentiation. Thus, MAF appears to be at least in part regulated through STAT3 activating cytokines such as IL-6. However, what is exactly regulating MAF expression in the gut remains to be precisely determined.

A recent study showed that additionally to its direct regulation of Il10 expression, Maf is also an inhibitor of Il2⁶. In the Maf-deficient T cells, the increased production of IL-2 has been shown to inhibit the T_H17 cell response in the EAE induced model⁶. In our study, we observed that after infection with H. pylori, the bacteria is cleared from the stomach mucosa of Maf^ΔTcells mice but establish a chronic infection in Maf^f/f mice. We measured an increased level in transcripts coding for Il2 and Foxp3 in the stomach mucosa of H. pylori-infected Maf^ΔTcells mice (Supplementary Fig. 4), leading to the possibility that increased number of classical T_reg are recruited in the stomach mucosa of Maf^ΔTcells mice through an Il2-dependent pathway. However, we also found high amount of Il17a and Il22, indicating the recruitment of T_H17 cells (Fig. 7B,E) leading to the clearance of H. pylori from the stomach mucosa of Maf^ΔTcells mice. These results clearly demonstrate that MAF plays a major role in maintaining the T_reg -T_H17 equilibrium not only in the colon but also in the gastric mucosa. In addition, it can be suggested that the development of a vaccine directed against a pathobiont, such as H. pylori, might be facilitated by a vaccine formulation that prevent MAF expression in primed T cells.

Altogether, we established that the role of Maf was not only restricted to T_reg but also to conventional T cells, especially T_H17 cells, establishing Maf as a major regulator of the T_reg – T_H17 balance in the gastro-intestinal tract. On a broader perspective, MAF expression in colonic lymphocytes appears to be strongly associated with an anti-inflammatory type of response. However, the implication of Maf in immune responses appears to be largely location- and context-dependent⁶.

Material and Methods

Mice

Rag1^−/− and Foxp3^{YFPcre 26} mice were kindly provided by P.C Ho. Foxp3^YFPcre and CD4^cre ³¹ mice were crossed with Maf^fl/fl mice³² to generate Foxp3^YFPcre Maf^fl/fl (Maf^ΔTreg) and CD4^cre Maf^fl/fl (Maf^ΔTcells), respectively. Mice were bred and maintained in a specific-pathogen-free (SPF) environment, which excludes Helicobacter hepaticus, of the animal facility of the University of Lausanne. The animals used were cohoused and littermate controls are referred to as Maf^f/f mice. Experiments were performed in compliance with the University of Lausanne Institutional regulations and were approved by the veterinarian authorities of the Canton de Vaud (Switzerland).

Isolation of cells

Single-cell suspensions from spleens and mesenteric lymph nodes were obtained after mashing the organs through a 70 µM cell strainer. Colons were collected in calcium and magnesium free Hank’s Balanced Salt Solution (HBSS) (ThermoFisher Scientific) supplemented with 2% of Fetal Calf Serum (FCS) (HBSS 2%) on ice. Samples were further flushed with HBSS 2% and cut longitudinally into 3–4 pieces (2–3 cm). Minced tissues were treated with EDTA 1 mM (ThermoFisher Scientific) and DTT 1 µM (AppliChem) in HBSS 10% solution for 20 min under shaking at 37 °C. After intraepithelial lymphocytes (IEL) removal, cells from the lamina propria were washed twice in HBSS 10% and incubated with Collagenase D (1 mg/ml) (Roche) and complete RPMI (ThermoFisher Scientific) for 30 min under shaking at 37 °C. To isolate leucocytes, supernatants were centrifuged in density gradients 40%/70% Percoll (GE Healthcare Life Sciences) for 30 min at 2000 rpm. All isolated cells were washed in complete RPMI and filtrated before staining.

T cell isolation and transfer into Rag1 ^−/− mice

Donor cells were obtained from spleens and LN of both WT (obtained from Cre-negative littermates and C57Bl6 mice) and Maf^ΔTcells female mice. CD4⁺ T cells were enriched using EasySep Mouse CD4⁺ T Cell Isolation Kit (StemCell Technologies) according to the manufacturer’s recommendations. CD4⁺CD45RB^hiCD25^lo and CD4⁺CD45RB^loCD25^hi were isolated by sorting on a FACS Aria cell sorter (BD Biosciences). CD4⁺CD45RB^hiCD25^lo cells (0.5 × 10⁶) were intravenously injected alone or in combination with CD4⁺CD45RB^loCD25^hi cells (0.4 × 10⁶) into males and females Rag1^−/− mice from 10 to 15 weeks age. Mice were weighed and monitored weekly. 6 weeks after transfer, mice were sacrificed.

Histopathology and scoring

Colons were fixed with histological tissue fixative (Sigma), embedded in paraffin and stained with haematoxylin and eosin. Histopathological score (0–6) was calculated based on inflammatory cells infiltration (0–3) and tissues abnormalities (0–3) by a pathologist.

In vitro T cell differentiation

Naïve CD4⁺CD25^loCD62^hiCD44^lo cells were isolated by using EasySep Mouse Naïve CD4⁺ T Cell Isolation Kit (Stemcell Technologies) from spleens and LN of Maf^f/f and Maf^ΔTcells mice, and activated with plate-bound anti-CD3 (Biolegend, 5 μg/ml) and soluble anti-CD28 (Biolegend, 1 μg/ml) supplemented with mIL12 (10 ng/ml) and neutralizing antibody anti-IL-4 (clone 11B11, 10 μg/ml) (T_H1), mIL-4 (10 ng/ml) and anti-IFN-γ (clone: XMG-121,10 μg/ml) (T_H2), hIL-2 (50U/ml) and TGF-β (10 ng/ml) (T_reg), TGF-β (5 ng/ml) and IL-6 (40 ng/ml) (FOXP3⁺ and FOXP3⁻ T_H17). Cells were incubated 5 days at 37 °C, 5% CO₂.

Antibodies, intracellular staining and flow cytometry

All antibodies used are listed in Table 1. For intracellular cytokine staining, cells were restimulated with PMA (Sigma, 50 ng/ml) and Ionomycin (Sigma, 100 ng/ml) for 4 h at 37 °C in presence of Golgi Plug (BD Biosciences). After staining for viability using LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (ThermoFisher Scientific) and extracellular markers, cells were fixed and permeabilized with the FOXP3 Transcription Factor Staining Buffer Set (eBiosciences) according to manufacturer’s recommendations. Following permeabilization, cells were intracellularly stained for specific cytokine and acquired on a LSRII flow cytometer (BD). Data were analysed with FlowJo software V10.

Table 1 List of the antibodies used for flow cytometry.

Full size table

Real-time qPCR analysis

Colons were collected, dried and ground into a powder using liquid nitrogen. Total RNA was isolated using RNAeasy Mini Kit (Qiagen, #74194) according to the manufacturer’s recommendations. cDNA was retro-transcribed using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, # 4368814) and used for quantitative PCR. The KAPA SYBR® FAST qPCR Master Mix (2×) Kit (Sigma, #KK4618) was used for SYBR analysis. The housekeeping gene beta2microglobulin was used to normalize gene expression. Sequences of primers used are listed in Table 2.

Table 2 List of the primers used for quantitative RT-PCR.

Full size table

Bacteria and infection

H. pylori P49, kindly provided by Harry Kleanthous (Acambis, Cambridge, MA), is a human clinical isolate adapted to mice. H. pylori P49 expresses VacA but not CagA. H. pylori P49 was grown on Helicobacter pylori-selective agar plate (Oxoid, Basingstoke, UK) and brain heart infusion broth supplemented with 0.25% yeast extract and 10% fetal calf serum (PAA, Pasching, Austria) under microaerophilic conditions, as previously describe³³. Mice were treated on day 4 and 5 after birth with 5 × 10⁸ H. pylori P49, administered by orogastric gavage in 200 μL BHI.

Antibiotics treatment

Maf^f/f and Maf^ΔTcells pregnant females were fed with antibiotics in their drinking water consisting of 0.5 mg/ml Amoxicillin (Mepha Pharma AG) and 5 mg/ml Enrofloxacine (Bayer). Antibiotic-containing drinking water was changed once a week until analysis. Offspring were then treated until their sacrifice at 17 weeks age.

Statistical analysis

Unless otherwise stated, unpaired non-parametric Mann-Whitney t tests were used to calculate statistical significance using GraphPad Prism software. P values: *P < 0.05, **0.01 < P < 0.05, ***P < 0.001.

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Acknowledgements

We thank the group of Dr. Caroline Pot (D. Duc; S. Vigne) for technical help and Dr. Connie Gilfillan for critical reading of the manuscript. This work was supported by grants from the Swiss Cancer League (G.V: 3679-08-2015), the Max Cloëtta Foundation (G.V), the Cancer Research Institute, USA (G.V, D.E.S), by the Swiss National Foundation grant (D.V: 310030_173197) and Ludwig Cancer Research, USA.

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Department of Oncology, University of Lausanne, Lausanne, 1066, Switzerland
Claire Imbratta, Marine M. Leblond, Daniel E. Speiser & Grégory Verdeil
UNISciences, University of Lausanne, UniLabs, Lausanne, 1066, Switzerland
Hanifa Bouzourène
Service of Gastroenterology and Hepatology, Department of Medicine, Lausanne University Hospital, Lausanne, 1066, Switzerland
Dominique Velin

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Claire Imbratta
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Contributions

C.I., M.M.L., D.V. and G.V. designed, performed and analysed the data. H.B. performed the analysis for the histology. G.V., C.I., D.V. and D.E.S. wrote, reviewed and revised the paper.

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Correspondence to Grégory Verdeil.

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Imbratta, C., Leblond, M.M., Bouzourène, H. et al. Maf deficiency in T cells dysregulates T_reg - T_H17 balance leading to spontaneous colitis. Sci Rep 9, 6135 (2019). https://doi.org/10.1038/s41598-019-42486-2

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Received: 28 November 2018
Accepted: 01 April 2019
Published: 16 April 2019
DOI: https://doi.org/10.1038/s41598-019-42486-2

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