Zfp362 potentiates murine colonic inflammation by constraining Treg cell function rather than promoting Th17 cell differentiation

Mucosal barrier integrity and pathogen clearance is a complex process influenced by both Th17 and Treg cells. Previously, we had described the DNA methylation profile of Th17 cells and identified Zinc finger protein (Zfp)362 to be uniquely demethylated. Here, we generated Zfp362−/− mice to unravel the role of Zfp362 for Th17 cell biology. Zfp362−/− mice appeared clinically normal, showed no phenotypic alterations in the T‐cell compartment, and upon colonization with segmented filamentous bacteria, no effect of Zfp362 deficiency on Th17 cell differentiation was observed. By contrast, Zfp362 deletion resulted in increased frequencies of colonic Foxp3+ Treg cells and IL‐10+ and RORγt+ Treg cell subsets in mesenteric lymph nodes. Adoptive transfer of naïve CD4+ T cells from Zfp362−/− mice into Rag2−/− mice resulted in a significantly lower weight loss when compared with controls receiving cells from Zfp362+/+ littermates. However, this attenuated weight loss did not correlate with alterations of Th17 cells but instead was associated with an increase of effector Treg cells in mesenteric lymph nodes. Together, these results suggest that Zfp362 plays an important role in promoting colonic inflammation; however, this function is derived from constraining the effector function of Treg cells rather than directly promoting Th17 cell differentiation.


Introduction
CD4 + Th cells are crucial for the maintenance of immunological homeostasis and host defense against pathogens, and the differentiation of distinct CD4 + Th cell subsets is partly controlled by epigenetic mechanisms [1,2].Especially, selective demethylation of CpG motifs can result in the preferential and/or stable expression of genes.In a previous study, we performed the first genomewide analysis of changes in DNA methylation patterns during the differentiation of naïve CD4 + T cells (Tnaïve) into Th17 cells [3].A unique epigenetic signature consisting of seven genes (Il17a, Zfp362, Ccr6, Acsbg1, Dpp4, Rora, Dclk1) characterizing Th17 cells was defined.Just as the other signature genes, zinc finger protein (Zfp)362 was found to be exclusively demethylated only in ex vivo isolated Th17 cells but not in Tnaïve or Th1 cells.In addition, the selective DNA demethylation was associated with an increased Zfp362 expression in ex vivo isolated Th17 cells [3].Thus, the DNA methylation and mRNA expression pattern of Zfp362 is congruent with the patterns of the key functional Th17 cell markers Il17a and Ccr6.Interestingly, Zfp362 was additionally found to be specifically demethylated in γδ T cells, although to a slightly lesser extent than in Th17 cells [3].
Besides our own studies [3,4], only very limited knowledge has been accumulated about Zfp362, which belongs to the C2H2type zinc finger proteins, known to act as transcription factors and/or transcriptional regulators or co-factors.More precisely, Zfp362 is a multiple adjacent-fingered Zfp, and proteins of this group are usually incorporated into transcriptional complexes formed on nuclear hormone receptors such as RORγt (retinoic acid receptor-related orphan nuclear receptor gamma thymusspecific variant).Thus, Zfps can modulate the transcriptional activity of nuclear hormone receptors through interaction with other regulatory molecules inside the complex [5].According to a secondary structure and disorder prediction by the Phyre2 web portal [6], the C-terminal domain of the protein consists of an array of five to six C2H2-type zinc finger motifs, indicating the DNA-binding ability of Zfp362.In contrast, the N-terminal domain of Zfp362 seems to be intrinsically disordered.A diverse set of functions was ascribed to intrinsically disordered regions.Most often, intrinsically disordered regions are associated with proteinprotein interactions and undergo disorder-to-structure transitions upon binding of their partners [7].Together, this led us to hypothesize that Zfp362 might act as a transcriptional regulator influencing the differentiation and functional properties of Th17 cells.
Th17 cells, characterized by the expression of the transcription factor RORγt, play a critical role in maintaining immunological integrity and pathogen clearance, especially at mucosal surfaces [8].Principally, one can distinguish nonpathogenic and pathogenic Th17 cells [9][10][11].Nonpathogenic Th17 cells display a regulatory program to secrete immunoregulatory cytokines, such as IL-4 and IL-10, and express essential negative regulators of different pathophysiological responses such as CD5L (CD5 antigen-like) or GATA3.In contrast, pathogenic Th17 cells coexpress the Th1 lineage identification factor T-bet and secrete IFN-γ and GM-CSF (granulocyte-macrophage colony-stimulating factor).In mice, homeostatic tissue-resident Th17 cells can be promoted by commensals, such as segmented filamentous bacteria (SFB), whereas pathogenic Th17 cells are for example induced by Citrobacter rodentium [12,13].γδ T cells are another IL-17producing T-cell population, together with Th17 cells belonging to the type 3 immune cells.In fact, γδ T cells are the main source of IL-17 under steady-state conditions in mice [14,15].
In the present study, we investigate the role of Zfp362 in the differentiation and functional properties of Th17 cells.To this end, Zfp362 −/− mice were generated and studied under steadystate or inflammatory conditions.Together, our findings suggest that Zfp362 constrains the effector function of Treg cells, thereby potentiating inflammatory T-cell responses within the intestine fully independent of Th17 cell differentiation.

Zfp362-deficient mice are healthy and harbor a normal CD4 + T-cell compartment
Previously, we had demonstrated that Zfp362 is part of the epigenetic signature characterizing Th17 cells [3].In order to study the unknown role of Zfp362 for Th17 cell biology, Zfp362-deficient mice were generated.To this end, embryonic stem cells harboring the "knockout-first allele" (tm1a) construct inserted into intron 4 of the Zfp362 locus were obtained from Eucomm [16] (Supplementary Information Fig. S1A).Generated animals were backcrossed to C57BL/6J background for ten generations.All mice were genotyped by PCR before usage in any experiment to ensure the homozygous presence (Zfp362 −/− ) or absence (Zfp362 +/+ ) of the "tm1a" construct (Supplementary Information Fig. S1B).
When housed under steady-state conditions in a specific pathogen-free (SPF) environment with a low microbiota diversity [17], Zfp362 −/− mice appeared clinically normal, that is, develop, grow, and reproduce normally.As Th17 cells play a critical role in maintaining immunological integrity, especially at mucosal surfaces [8] and thus are essential components of the gastrointestinal immune system, the colon (CO) and small intestine (SI) of Zfp362 −/− mice and Zfp362 +/+ littermates were inspected by histology.Both Zfp362 −/− and Zfp362 +/+ mice showed a comparably low total score, based on signs of inflammation, epithelial erosion, hyperplasia, single cell apoptosis, and the area involved, in the CO and SI (Fig. 1A and B).

Zfp362 −/− mice colonized with SFB exhibit alterations in Treg cells without affecting Th17 cells
In order to describe the functional relevance of Zfp362 in particular for Th17 cells in vivo, we colonized Zfp362 −/− mice with SFB.These commensal bacteria can modulate the number and phenotype of Th17 cells, particularly in the SI of mice by stimulating innate and adaptive immune responses [12,13].Here, Zfp362 −/− and Zfp362 +/+ mice were housed in cages from SFB-colonized mice, that is, colonization by coprophagy, and the colonization state of the mice was verified by quantitative PCR after seven days.Zfp362 −/− and Zfp362 +/+ mice housed under SPF conditions devoid of SFB were used as additional controls.Two weeks after colonization, the CD4 + T-cell compartment of the CO, SI, CO-draining mLN, SI-draining mLN, pLN, and spleen was assessed by flow cytometry (Supplementary Information Figs.S4 and S5).As anticipated, colonization with SFB resulted in increased frequencies of Th17 cells (RORγt + Foxp3 − CD4 + ), especially in the CO, SI, and mLNs (Fig. 2A; Supplementary Information Figs.S5A  and S6A).Yet, no significant differences were observed between Zfp362 −/− and Zfp362 +/+ mice with regard to Th17 cells, with the exception of pLN, where a significantly increased frequency was found in Zfp362 −/− mice upon SFB colonization (Supplementary Information Fig. S6A).Next, the expression of IL-17A was investigated as an indicator of Th17 cell functionality [11].In line with the increased frequencies of total Th17 cells, frequencies of IL-17A + Th17 cells also expanded upon colonization with SFB (Fig. 2B; Supplementary Information Figs.S5B and S6B).When compared with Zfp362 +/+ littermates, Zfp362 −/− mice housed under SPF conditions showed a slight trend for increased IL-17A production in the CO, SI, and mLNs, which was diminished or even reverted upon colonization with SFB (Fig. 2B).In the spleen, the frequency of IL-17A + Th17 cells significantly declined in noncolonized Zfp362 −/− mice when compared with Zfp362 +/+ littermates, a phenotype that was still visible as a trend after colonization (Supplementary Information Fig. S6B).γδ T cells are another IL-17-producing T-cell population and belong together with Th17 cells to type 3 immune cells [14,15].While the frequency of total γδ T cells was neither influenced by SFB colonization nor by Zfp362 deficiency, a significant increase of IL-17A + γδ T cells was found in pLN and CO-draining mLN of SFB-colonized Zfp362 −/− mice when compared with Zfp362 +/+ littermates.This effect was not observed for IFN-γ + γδ T cells (Supplementary Information Fig. S7).
As another major effector Th cell population, Th1 cells (Tbet + Foxp3 − CD4 + ) were investigated regarding their frequencies and cytokine expression.Yet, colonization with SFB and also Zfp362 deficiency only very marginally affected total Th1 cell frequencies and IFN-γ expression in all investigated organs (Fig. 2C and D; Supplementary Information Figs.S5C,D and S6C,D).Nonclassic Th1 cells (ncTh1; RORγt + T-bet + Foxp3 − CD4 + ) are derived from Th17 cell precursors and characterized by simultaneous expression of the master transcription factors T-bet and RORγt [19].Expectedly, the frequency of these cells increased in the CO, SI, and mLNs upon colonization with SFB [20], but no major impact of Zfp362 deficiency was found (Fig. 2E; Supplementary Information Figs.S5E and S6E).compartment was investigated.Unexpectedly, Zfp362 deficiency had an impact on the frequency, cytokine expression, and subset composition of Treg cells.First, a higher frequency of colonic Treg cells was found in SFB-colonized Zfp362 −/− when compared with Zfp362 +/+ mice (Fig. 2F; Supplementary Information Figs. S5F and S6F).Second, irrespective of the SFB colonization status Zfp362 −/− mice showed a higher frequency of IL-10 + Treg cells in mLN, and a higher frequency was observed even in pLN upon SFB colonization (Fig. 2G; Supplementary Information Figs.S5G and  S6G).Finally, significantly increased frequencies of RORγt + Treg cells were found in SI-draining mLN of Zfp362 −/− when compared with Zfp362 +/+ mice, while the increase of this Treg cell subset did not reach significance in pLN (Fig. 2H; Supplementary Information Figs.S5H and S6H).Despite these alterations in the frequency, cytokine expression, and subset composition of Treg cells, the histological inspection of the CO and SI did not reveal any differences between SFB-colonized Zfp362 −/− mice and Zfp362 +/+ littermates (Supplementary Information Fig. S8).In effect, SFBcolonized mice exhibited comparable total inflammation scores as noncolonized mice.Taken together, only a minor impact of Zfp362 deficiency was found on the differentiation and function of type 3 immune cells.In contrast, Zfp362 deficiency resulted in considerable alterations within the Treg cell compartment.

Zfp362 expression is not restricted to Th17 cells
As we observed a mild effect of Zfp362 deficiency on Treg cells in vivo, we mined the existing snRNA-seq data of sorted intestinal CD44 + CD62L − CD4 + T cells for Zfp362 expression and could detect a broad distribution of Zfp362 expression across almost all identified clusters of intestinal CD4 + T cells (Supplementary Information Fig. S9A).However, the number of positive cells per cluster was too low to make any statement regarding a preferential Zfp362 expression in particular Th cell subsets.To increase the sensitivity of the detection of Zfp362 expression, we sorted Th17 (RORγt + Foxp3 − CD4 + ), RORγt − Treg cells (Foxp3 + RORγt − CD4 + ), and RORγt + Treg cells (RORγt + Foxp3 + CD4 + ) from different organs of reporter mice [21] and determined Zfp362 expression by real-time quantitative PCR.We could detect low Zfp362 expression levels in all sorted T-cell subsets from SLOs and confirm the even lower Zfp362 expression levels in subsets isolated from the CO and SI.Interestingly, in the spleen Zfp362 expression was slightly but significantly increased in RORγt − Treg cells when compared with RORγt + Treg cells and Th17 cells (Supplementary Information Fig. S9B).In conclusion, Zfp362 expression is not restricted to Th17 cells, but expressed across multiple Th cell subsets at low levels.

Zfp362 loss does neither affect Treg cell-mediated suppression nor Th cell differentiation in vitro
Having demonstrated a marginally increased Zfp362 expression in Foxp3 + Treg cells, we next aimed to investigate whether Zfp362 has an impact on the suppressive capacity of Treg cells.To that end, an in vitro suppression assay was performed by co-culturing sorted Treg cells from Zfp362 −/− mice or Zfp362 +/+ littermate controls with cell tracker-labeled congenic Tnaïve cells at different Treg cell to Tnaïve ratios.However, both Zfp362 −/− and Zfp362 +/+ Treg cells displayed an equal suppression of Tnaïve proliferation at all ratios tested (Supplementary Information Fig. S10A-C), suggesting that the suppressive capacity of Treg cells is not grossly affected by Zfp362 deficiency.
Next, we assessed if Zfp362 can modulate the differentiation of Th cell subsets and if Zfp362 deficiency would tip the balance toward the differentiation of Treg cells.We performed a series of in vitro differentiation cultures under varying conditions using Tnaïve cells from Zfp362 −/− mice or Zfp362 +/+ littermate controls.Cells were cultured under non-polarizing Th0, iTreg, Th17, and Th17 w/o IL-6 (absence of IL-6) conditions for four days, followed by flow cytometric analysis.As expected, cells cultured under Th17 conditions showed a high frequency of RORγt + and IL-17A + cells, but no differences were found between cultured cells from Zfp362 −/− mice or Zfp362 +/+ littermate controls (Fig. 3A and B; Supplementary Information Fig. S10D-F).A similar observation was made for cells cultured under Th17 w/o IL-6 conditions, resulting in lower frequencies of RORγt + and hardly any IL-17A + cells.Cultivation of cells under iTreg and Th17 w/o IL-6 conditions resulted in clearly detectable frequencies of Foxp3 + cells.Here, slightly reduced frequencies of Foxp3 + cells were found in cultures of Zfp362-deficient when compared with Zfp362 +/+ control cells (Fig. 3C; Supplementary Information Fig. S10G).In summary, Zfp362 deficiency did neither affect the suppressive function of Treg cells nor resulted in preferential Treg cell differentiation when tested in vitro.

CD4 + T cells from Zfp362 −/− mice are less colitogenic
The mild Treg cell phenotype observed in vivo (Fig. 2F-H) could not be recapitulated in vitro.To study the T-cell-specific effect of Zfp362 deficiency in vivo and to exclude that a T-cell-extrinsic effect caused the observed mild alterations within the CD4 + Tcell compartment, we made use of the adoptive transfer colitis model.Importantly, Th17 cells were shown to play a critical role in the pathogenesis of colitis [22][23][24], wherefore this model is ideally suited to study the impact of Zfp362 on Th17 cell differentiation and function in more detail.Thereto, 3 × 10 5 Tnaïve isolated from either Zfp362 −/− mice or Zfp362 +/+ littermates were injected into sex-and age-matched Rag2 −/− mice (Supplementary Information Fig. S11A and B).Rag2 −/− mice injected with PBS served as control.The body weight of the mice was monitored twice a week since weight loss is a hallmark of colitis [25].Intriguingly, mice injected with Tnaïve from Zfp362 −/− mice lost significantly less weight than mice injected with Tnaïve from Zfp362 +/+ mice (Fig. 4A), suggesting a decreased pathogenicity of Zfp362-deficient CD4 + T cells.While at the end of the experiment the length of the CO or SI relative to the weight of the organ was comparable between the two groups, indicating a similar degree of inflammation when compared with the PBS-treated control group (Fig. 4B), mice receiving Tnaïve from Zfp362 −/− mice exhibited a slightly reduced total inflammation score of the CO when compared with mice receiving Tnaïve from Zfp362 +/+ mice (Fig. 4C and D).This attenuated inflammation of the colonic tissue in mice receiving Tnaïve from Zfp362 −/− mice was most pronounced with regard to the parameter of single-cell apoptosis, which is a prime parameter of transfer colitis and often associated with elevated levels for tumor necrosis factor, inducible nitric oxide synthase, and p53 [26].Thus, the histological examination of the CO is consistent with the observed body weight changes, supporting the hypothesis that Zfp362 deficiency may result in a reduced pathogenicity of CD4 + T cells (Table S1).
In order to investigate the impact of Zfp362 deficiency on the CD4 + T-cell compartment and specifically on Th17 cells under these inflammatory conditions in more detail, single-cell suspensions from the CO, SI, CO-draining mLN, SI-draining mLN, pLN, and spleen were analyzed by flow cytometry (Supplementary Information Fig. S11C).Similar to the results obtained in the SFB colonization experiments, differences in Th17 cell frequency and expression of IL-17A between mice receiving Tnaïve from Zfp362 −/− or Zfp362 +/+ donors were neglectable (Fig. 5A and B; Supplementary Information Figs.S12A,B and S13A,B).Additionally, for Th1 cell frequency, ncTh1 cell frequency, and expression of IFN-γ no alterations were found (Fig. 5C-E; Supplementary Information Figs.S12C-E and S13C-E).Furthermore, no impact of Zfp362 deficiency on the total Treg cell frequency (Fig. 5F; Supplementary Information Figs.S12F and 13F) or T-bet + Treg cells (Fig. 5G; Supplementary Information Figs.S12G and S13G) was observed.However, interestingly, a mild increase of IL-10 + (p = 0.43) and RORγt + (p = 0.098) Treg cells and a significant increase of CD69 + CD103 + Treg cells was detected in COdraining mLN of recipient mice receiving Tnaïve from Zfp362 −/− mice compared with controls (Fig. 5H-J; Supplementary Information Figs.S12H-J and S13H-J).Together, these findings indicate that Zfp362 plays a T-cell-intrinsic role in the pathogenicity of CD4 + T cells.

Discussion
Epigenetic modifications are critical for the heritable but plastic fixation of distinct gene expression patterns.Thus, DNA methylation plays a key role in the lineage fate decision and functional properties of CD4 + T helper cells [1,2].In mammals, especially the demethylation of cytosines in CpG dinucleotides of the DNA strongly correlates with the stable expression of a gene.Intriguingly, for Treg cells, it was shown that the CpG DNA demethylation at a set of Treg cell-specific epigenetic signature genes not only A transfer colitis was induced in 8-week-old male and female Rag2 −/− mice by intraperitoneal injection of 3 × 10 5 naïve CD4 + T cells isolated from sex-and age-matched Zfp362 −/− mice or Zfp362 +/+ littermates.Rag2 −/− mice injected with PBS served as control.(A) Over the course of the experiment, animals were monitored regarding their weight twice a week.Data are depicted as mean ± 95% CI and summarized from ten independent experiments (n = 23-30 biological replicates per group in total).Symbols indicate the different experimental groups according to the legend.The significance of differences between means was assessed using an ordinary two-way ANOVA with Šidák's multiple comparison test (mean ± 95% CI; *p < 0.05 and **p < 0.01).(B) Four weeks after adoptive transfer, mice were sacrificed, and the length and weight of the colon (left) and small intestine (SI; right) was assessed and depicted in scatter plots with bars.Data are summarized from six independent experiments (n = 7-9 biological replicates per group in total).Each symbol represents an individual mouse.The significance of differences between means was assessed using Kruskal-Wallis test (mean ± SD). (C, D) Inflammation of the colon was assessed by histology.(C) Exemplary H&E stainings of colons from mice receiving Tnaïve from Zfp362 +/+ mice, Tnaïve from Zfp362 −/− mice, or PBS only.Black bar represents approx.50 μm.Invasion of inflammatory cells (i), epithelial hyperplasia (h), and sloughing of epithelia cells (e) are labeled.(D) Scatter plots with bars present the total inflammation score, and the single scores for inflammation, epithelial erosion, hyperplasia, single cell apoptosis, and the area involved.Each symbol represents an individual mouse.Data are summarized from three to five independent experiments (n = 9-14 biological replicates per group in total).The significance of differences between means was assessed using Kruskal-Wallis test with Dunn's multiple comparison test (mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001).
results in the imprinting of a unique transcriptional program but also leads to the stabilized expression of the respective molecules, which are all crucial for the suppressive activity of Treg cells [27][28][29].Recently, we described the first epigenetic signature characterizing Th17 cells consisting of seven genes (Il17a, Zfp362, Ccr6, Acsbg1, Dpp4, Rora, Dclk1) [3].Among them, Il17a as the major effector cytokine of Th17 cells essential for host defense against microbial organisms, [11] and the chemokine receptor Ccr6 critical for accurate migration of Th17 cells during homeostasis and inflammation [30] are well described regarding their functional role within Th17 cells.Also, for the transcription factor RORα [31] and the Dipeptidylpeptidase IV (Dpp4; CD26) acting as a costimulatory molecule in T-cell activation [32] a critical role for Th17 cell function was demonstrated.Yet, at present, the contri-bution of the transcriptional co-modulator Zfp362, the acyl-CoA synthetase Acsbg1, and the serine/threonine-protein kinase Dclk1 to the differentiation, maintenance, functional properties, and/or plasticity of Th17 cells are completely unknown.
Akin to Il17a and Ccr6, Zfp362 showed a strong and homogeneous demethylation at all inspected CpG motives as well as enhanced mRNA expression in Th17 cells [3].Accordingly, we attempted to reveal the role of Zfp362 for Th17 cell biology by making use of newly generated Zfp362 knock-out mice in the present study.Under steady-state conditions, Zfp362-deficient mice appeared clinically normal, that is, develop, grow, and reproduce normally.Neither the histological examination of the CO and SI, the analysis of the CD4 + T-cell compartment by high-dimensional flow cytometric analysis nor the snRNA-seq Figure 5. Upon adoptive transfer, CD4 + T cells from Zfp362 −/− mice show no major alterations within the Th17 cell compartment.A transfer colitis was induced in 8-week-old male and female Rag2 −/− mice by intraperitoneal injection of 3 × 10 5 naïve T cells isolated from sexand age-matched Zfp362 −/− mice or Zfp362 +/+ littermates.Four weeks after adoptive transfer, mice were sacrificed, and the CD4 + T-cell composition was assessed by flow cytometry.(A-J) Scatter plots with bars of indicated cell populations in colon-draining mesenteric lymph nodes (COmLN), small intestine-draining mLN (SImLN), colon (CO), and small intestine (SI).Numbers in the representative dot plots from CO indicate the frequencies of cells within the depicted gates or quadrants.Summarizing scatter plots with bars show frequencies of indicated T-cell populations.Data are summarized from five independent experiments (n = 10-12 biological replicates per group in total).Each symbol represents an individual mouse.The significance of differences between means was assessed using an ordinary two-way ANOVA with Šidák's multiple comparison test (mean ± SD; *p < 0.05).
analysis revealed any major differences between Zfp362 −/− mice and Zfp362 +/+ littermates used as controls.However, due to the homeostatic state of the animals and the housing in a SPF environment with a very low microbial diversity [17,33], the majority of CD4 + T cells are naïve, and Th17 cells are almost completely absent.Thus, to be able to study the role of Zfp362 for Th17 cells, we colonized Zfp362 −/− mice and Zfp362 +/+ littermates with SFB, a Clostridia-related spore-forming gram-positive bacterium with a strong Th17 cell-inducing capacity [12,13,34].As expected, we could reproduce a sizeable increase in the frequency of total and also IL-17A + Th17 cells in the gastrointestinal tract and mLN upon colonization with SFB.Surprisingly, Zfp362 deficiency did neither markedly impact the generation of Th17 cells nor their IL-17A expression.On the contrary, we observed a role of Zfp362 for Treg cell frequency, cytokine production, and subset composition within the gastrointestinal tract and especially within mLN.Treg cells are essential for intestinal homeostasis by protecting from inappropriate immune responses and ensuring tolerance toward the vast diversity of antigens derived from food and commensal bacteria in the gastrointestinal tract [35].The gut-draining mLN, furthermore, are important sites for the de novo induction and proliferation of Treg cells [36,37].The immunomodulatory cytokine IL-10 represents a key effector molecule for suppressive Treg cells and was shown to be required for the maintenance of intestinal homeostasis [38].A subset of IL-10 + Treg cells is constituted by RORγt + Treg cells.These cells possess a superior suppressive capacity and are essential for intestinal homeostasis [4,39].Intriguingly, RORγt + Treg cells show a strong and homogeneous demethylation at the Zfp362 locus [4], implying that Zfp362 might rather play a role in the differentiation of RORγt + IL-10 + Treg cells.
However, due to the ubiquitous deletion of Zfp362 in the applied mouse model, a T-cell-extrinsic effect on the observed alterations within the CD4 + T-cell compartment could not be excluded.Hence, to study the role of Zfp362 more specifically on CD4 + T cells, a transfer colitis model was employed.Interestingly, Rag2 −/− mice receiving Tnaïve from Zfp362 −/− mice lost significantly less weight than mice receiving Zfp362-sufficient Tnaïve.Additionally, signs of inflammation within the CO assessed by histology were slightly attenuated in mice receiving Zfp362-deficient Tnaïve when compared with mice adoptively transferred with Tnaïve from Zfp362 +/+ mice.Although Th17 and Th1 cells were reported to be the main drivers of colitis [22][23][24], no differences were observed in the frequency or respective cytokine expression of these cell populations between mice receiving Tnaïve from Zfp362 −/− or Zfp362 +/+ donors.Again, differences in the CD4 + T-cell compartment between these two experimental groups were only observed within the Treg cell population of CO-draining mLN.During experimental colitis, Treg cells accumulate and proliferate within mLN and the CO [40].Here, Zfp362 deficiency resulted in an increase of CD69 + CD103 + Treg cells.This subset of effector Treg cells was previously shown to possess an increased expression of diverse activation and effector molecules, such as KLRG1, ICOS, CTLA-4, CD39, and CD73 [41,42].Furthermore, RORγt + and IL-10 + Treg cells were slightly increased in the CO-draining mLN of mice receiving Tnaïve from Zfp362 −/− mice.Importantly, the production of IL-10 from Treg cells and also the presence of RORγt + Treg cells were shown to be critical for the resolution of colitis [40,[43][44][45].Thus, the observed attenuated body weight loss and slightly reduced total inflammation score of the CO after colitis induction with Zfp362-deficient Tnaïve might be rather due to an increased effector activity of Treg cells than an impairment of the Th17 cell compartment.Strikingly, Zfp362 was described to be a part of the "core" chromatin accessibility signature of murine ST2 (Il1rl1)-expressing tissue Treg cells [46] and human tissue-repair Treg cells [47].Additionally, Zfp362 was previously found to be upregulated on mRNA level in CCR2 + CD4 + T cells, which were demonstrated to have an immunoregulatory function and attenuate lung fibrosis development [48].
Our global analyses did not only confirm Zfp362 expression in Th17 and Treg cells but also in additional Th cell subsets with a rather low expression in the intestine and slightly higher -but still low -expression levels in Th cell subsets isolated from SLOs.However, we could not detect any impact of Zfp362 deficiency on the suppressive capacity of Tregs in vitro and in vitro Th cell differentiation cultures did not reveal a preferential Treg cell differentiation in cultured Zfp362-deficient T cells.Thus, a prominent role of Zfp362 to prevent the trans-differentiation of Th17 to Treg cells can be excluded.
Viewed as a whole, the data from the present study indicate that Zfp362, a member of the unique epigenetic signature of Th17 cells, can potentiate inflammatory T-cell responses within the intestine fully independently of Th17 cell differentiation.Unexpectedly, Treg cells appear to be more strongly influenced by the activity of this transcriptional modulator.Future studies are necessary to reveal the molecular mechanisms by which Zfp362 constrains the effector functions of Treg cells in more detail.

Mice
CD4 cre Zfp362 fl/fl mice (B6.Zfp362 tm1c(Eucomm)Hmgu -D2-Tg(CD4 cre ) 1Cwi /J) were generated by crossing mice derived from embryonic stem cells harboring the tm1a construct within the Zfp362 locus (obtained from Eucomm) [16] to a flip-recombinase expressing mouse strain.After removing the flip recombinase, mice harboring the tm1c construct were obtained.Further crossing with CD4 cre mice resulted in mice with a T-cell-specific Zfp362 deficiency (Zfp362 (CD4)−/− ).All mice used in this study (B6.Zfp362 tm1a(Eu-comm)Hmgu mice, B6.Rag tm1Cgn (Rag2 -/-) mice, B6.Zfp362 tm1c(Eucomm)Hmgu -D2-Tg(CD4 cre ) 1Cwi /J (CD4 cre Zfp362 fl/fl ), B6;FVB/N-Rorc (EGFP)1Ebe -Foxp3 (tm1Flv) , B6.Rorc(gt)-Gfp TG -Foxp3-IRES-mRFP mice (RORγt × Foxp3), and B6.Foxp3 tm1(hCD2/CD52)Shori -Ptprc a (Foxp3 hCD2 )) were bred and maintained at the central animal facility of the Helmholtz Centre for Infection Research (HZI, Braunschweig, Germany), which provides state-of-the-art laboratory animal care and service.All mice were housed in barriers under SPF conditions in isolated, ventilated cages, and handled by personnel appropriately trained as well as dedicated animal care staff to assure the highest possible hygienic standards and animal welfare in compliance with German and European animal welfare guidelines.According to the German Animal Welfare Act ( §4, section 4), sacrificing animals solely to remove organs for scientific purposes is notified to the competent authority.Animal experiments were approved by the Lower Saxony Committee on the Ethics of Animal Experiments as well as the responsible state office (Lower Saxony State Office of Consumer Protection and Food Safety) under permit number 33.19-42502-04-20/3540.This study was carried out in accordance with the principles of the Basel Declaration as well as recommendations as defined by Federation of European Laboratory Animal Science Associations and the German animal welfare body GV-SOLAS (Society for Laboratory Animal Science).For all experiments, heterozygous Zfp362 +/− mice were bred to obtain Zfp362 −/− mice and Zfp362 +/+ littermate controls.Before experimental use, all mice were genotyped by subjecting genomic DNA to PCR testing for the integrity of intron 4 (F: 5'-CAGCGACTCATGTGGAAGGT; R: 5'-GGCACTACCGAAACCGAGAA) and for the presence of the 'tm1a' construct (F: 5'-CAGCGACTCATGTGGAAGGT; R: 5'-GTGGGAAAGGGTTCGAAGTT).

Preparation of single-cell suspensions from different organs
For the preparation of single-cell suspensions from lymphoid organs, mice were sacrificed by CO 2 asphyxiation.Spleen, lymph nodes, and thymus were isolated, mechanically disrupted using a plunger, and filtered through a nylon mesh with a pore size of 100 μm.Cell suspensions were washed and splenocytes were subjected to erythrocyte lysis buffer (0.01 M potassium bicarbonate, 0.155 M ammonium chloride, and 0.1 mM EDTA at pH 7.5).After an additional washing step, all cell suspensions were filtered through nylon meshes with a pore size of 30 μm.For the preparation of single-cell suspensions from the CO, colons were opened longitudinally and washed with predigestion medium (PBS, containing 0.2% BSA (Merck) and 5 mM EDTA (Sigma-Aldrich)) to remove feces.Subsequently, colons were incubated with predigestion medium at 37°C three times for 15 min.After washing with Hank's Balanced Salt Solution (HBSS with Mg 2+ and Ca 2+ ; Gibco), predigested colons were cut into 1-2 mm pieces and digested in HBSS containing 1 mg ml −1 Collagenase D (Roche), 100 μg ml −1 DNaseI (Roche), and 25 mM HEPES (Gibco) at 37°C for 1 h.Subsequently, cells were subjected to 30%/70% Percoll (GEHealthcare) gradient centrifugation (780 g, room temperature, 20 min, acceleration and brake off).Cells in the interphase layer were harvested, washed, and resuspended in PBS containing 0.2% BSA.For the preparation of single-cell suspensions from SI, the mouse lamina propria dissociation kit (Miltenyi Biotec) using the gentleMACS TM Octo dissociator with heaters was applied according to the manufacturer's protocol.
For high-dimensional analysis of flow cytometric data, raw FCS data were pregated manually on Live/Dead − CD3 + CD8 − CD4 + cells using FlowJo® prior to file export.Exported files were analyzed through successive FlowSOM clustering and t-Distributed Stochastic Neighbor Embedding (t-SNE) representation using R (v 4.1.2).Similar event numbers for each sample per condition group and equal event numbers per condition were used for the analysis.t-SNE representations were built on the phenotypic markers RORγt, CD62L, CD25, T-bet, CD44, and Foxp3 for samples from the intestine (CO and SI), and additionally on CCR6 for samples from secondary lymphoid organs.The characteristics of each identified FlowSOM cluster were assessed by means of histograms.
For cell sorting, single-cell suspensions of indicated organs were stained with the respective antibodies and Lin − cells of interest (unless stated otherwise -Lin: CD8α, CD45R, CD11c, F4/80) were sorted by FACS utilizing a BD FACSAria-Ilu, BD FACSAria-II SORP or BD FACSAria-Fusion (all BD Biosciences).

Single-cell transcriptome analysis
Lin − CD44 + CD62L − CD4 + T cells were sorted from CO-and SI-derived single-cell suspensions of Zfp362 (CD4)−/− or Zfp362 fl/fl control mice.For sorting, single-cell suspensions from seven mice per genotype were pooled.For droplet-based snRNA-seq, libraries were prepared using single cell multiome ATAC + gene expression kit according to the manufacturer's instructions (10x Genomics), targeting 5000 nuclei per sample after counting with a Countess II Automated Cell Counter (Thermo Fisher).Seven cycles were applied to generate cDNA followed by 15 cycles for the final library generation.Generated snRNA-seq libraries were sequenced on a NovaSeq 6000 (100 cycles, pairedend).Raw gene counts were obtained by aligning reads to the mm10 genome (refdata-cellranger-arc-mm10-2020-A-2.0.0) using CellRanger software (cellranger-arc-2.0.1, 10x Genomics).Single-nucleus RNA counts of the pooled CD4 + T-cell wildtype control and Zfp362 knockout libraries were analyzed using the Seurat framework [50] (v.4.3.0) in R (v.4.1.3).To this end, the filtered cell barcode matrices from CellRanger were loaded into a Seurat object.Cells with 200 or less and 6000 or more detected unique molecular identifiers were excluded and also cells with 5% or more of the total counts mapping to mitochondrial genes.Then, the cell count matrices for each sample were normalized using scTransform [51] (v.0.3.5) with default parameters.Following the official Seurat documentation to integrate samples using a scTransform-based workflow, we applied the functions SelectInte-grationFeatures, PrepSCTIntegration, FindIntegrationAnchors, and IntegrateData with 3000 features and 30 dimensions.Dimension reduction and clustering were then performed on the integrated assay using RunPCA, FindNeighbors, FindClusters, and RunUMAP in order with 20 significant dimensions as determined by Elbow-Plot, a clustering resolution of 0.3, and all other parameters set at default.The identified clusters were then annotated manually using the literature and external databases as references [52,53].Two tiny clusters of contaminating Plasma B and epithelial cells were removed to obtain a high-quality and clean data set.For downstream cluster/cell type marker and differential gene expression analysis, raw counts from the RNA assay were first size-normalized and then log-scaled using the NormalizeData function.To obtain fold-changes and p-values for cell contrasts, we used the FindMarkers function together with the MAST [54] model bioconductor package (v1.20.0) on the normalized counts.Plots were generated using the packages ggplot2 (v3.3.6) and cowplot (v1.1.1).Absolute log-foldchanges were required to be greater than 0.1 and adjusted p-values were required to be less than the alpha level 0.01 for cluster markers and 0.05 for comparing between knockout and control cells, to consider a gene being differentially expressed.Data sets generated for this study are available from GEO under the accession number GSE229676.

Colonization with SFB
Eight-week-old Zfp362 −/− mice and Zfp362 +/+ littermate controls were colonized with SFB by coprophagy.For this purpose, mice were housed in cages containing bedding and feces from cages of already SFB-colonized mice.Seven days later, the colonization status of Zfp362 −/− mice and Zfp362 +/+ littermate controls was checked.To that end, feces samples were collected and DNA was extracted following established protocols based on the combination of mechanical disruption (bead-beating) and phenol/chloroform-based purification [55].In brief, feces samples were suspended in a solution containing 500 μL of extraction buffer (200 mM Tris, 20 mM EDTA, 200 mM NaCl, pH 8.0), 200 μl of 20% SDS, 500 μL of phenol:chloroform:isoamyl alcohol (24:24:1), and 100 μL of 0.1 mm zirconia/silica.The samples were homogenized twice using a bead beater (BioSpec) for 2 min.After DNA precipitation, crude DNA extracts were resuspended in TE buffer containing 100 μg mL −1 RNase and column-purified in order to remove PCR inhibitors (BioBasic).PCR of isolated DNA from feces samples was performed to detect SFB using specific primers (F: 5'-GACGCTGAGGCATGAGAGCAT and R: 5'-GACG-GCACGGATTGTTATTCA).Fourteen days after SFB colonization, mice were sacrificed by CO 2 asphyxiation, and either CO and SI were prepared for histology, or single-cell suspensions from small intestine, CO-draining mLNs, SI-draining mLN, pLN, and spleen were prepared for flow cytometry.

Histology
Colon and SI samples rolled up to "swiss roles" were fixed in 4% neutrally-buffered formaldehyde and embedded in paraffin according to standard histological procedures.Sections of approx.3 μm thickness were stained with hematoxylin-eosin and evaluated by light microscopy randomized and blinded to the experimental groups.The histological scoring used to microscopically evaluate the severity of inflammation (Supplementary Information Table S1) was adapted from the TJL-score developed for scoring colitis in mice by The Jackson Laboratory [56].The scores were added up to a total of up to 15 per section, and the scores of the three sections to a total of up to 45 per sample.

Transfer colitis
Naïve CD4 + T cells (Tnaïve) were sorted by fluorescenceactivated cell sorting (FACS) as CD62L high CD25 − CD4 + Lin − (Lin: CD8α, CD11c) from single-cell suspensions of pooled spleens and pLNs from 8-week-old Zfp362 −/− mice or Zfp362 +/+ littermate controls.Sorted cells were washed twice with PBS and 3 × 10 5 Tnaïve were injected intraperitoneally in a volume of 100 μl PBS into sex-and age-matched Rag2 −/− recipient mice.Rag2 −/− mice injected with only 100 μL PBS served as controls.The body weight and health status of recipient mice were monitored twice a week over the course of 4 weeks.At the end of the experiment, mice were sacrificed by CO 2 asphyxiation, and either the CO and SI were analyzed by histology, or single-cell suspensions from the CO, small intestine, CO-draining mLNs, SI-draining mLN, pLN, and spleen were prepared for flow cytometry.

Statistical analysis
The GraphPad Prism software v 9.1.2(Graph-Pad) was used to perform all statistical analyses.If not stated otherwise, data were presented as mean ± SD.The significance of differences between means was assessed using an ordinary two-way ANOVA with Šidák's multiple comparison test.A p-value below 0.05 was considered significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 1 .
Figure 1.Zfp362 knock-out mice exhibit a normal health status and T-cell composition.(A) Microscopic signs of inflammation were assessed by histology of the colon.(Left) Exemplary H&E staining of the colon from Zfp362 +/+ and Zfp362 −/− mice.Black bar represents approx.50 μm.(Right) Scatter plot with bars presenting the total inflammation score of colons from Zfp362 +/+ and Zfp362 −/− mice are shown.(B) Microscopic signs of inflammation were assessed by histology of the small intestine (SI).(Left) Exemplary hematoxylin and eosin (H&E) staining of the SI from Zfp362 +/+ and Zfp362 −/− mice.Black bar represents approx.50 μm.(Right) Scatter plots with bars presenting the total inflammation score of SIs from Zfp362 +/+ and Zfp362 −/− mice are shown.The total inflammation score was based on signs of inflammation, epithelial erosion, hyperplasia, single-cell apoptosis, and the area involved.Each symbol represents an individual mouse.Data are summarized from three to four independent experiments (n = 8-9 biological replicates per group in total).The significance of differences between means was assessed using Mann-Whitney test (mean ± SD). (C) The T-cell composition in the colon and SI of Zfp362 −/− mice and Zfp362 +/+ littermates was assessed by high-dimensional flow Finally, the Treg cell cytometry.Live/Dead − CD3 + CD8 − CD4 + T cells were manually gated in FlowJo.(Left) t-Distributed Stochastic Neighbor Embedding (t-SNE) of CD4 + T cells built on key markers (RORγt, CD62L, CD25, T-bet, CD44, and Foxp3).Colors indicate FlowSOM clusters.(Right) Average frequencies of FlowSOM clusters in scatter plots with bars.(D) The T-cell composition in mLN, pLN, and spleen of Zfp362 −/− mice and Zfp362 +/+ littermates was assessed by high-dimensional flow cytometry.(Left) t-SNE of CD4 + T cells built on key markers (CCR6, RORγt, CD62L, CD25, T-bet, CD44, and Foxp3).Colors indicate FlowSOM clusters.(Right) Average frequencies of FlowSOM clusters in scatter plots with bars.(C, D) Each symbol represents an individual mouse.The significance of differences between means was assessed using an ordinary two-way ANOVA with Šidák's multiple comparison test (mean ± SD; *p < 0.05 and **p < 0.01).Data are summarized from three independent experiments (n = 7-8 biological replicates per group in total).

Figure 4 .
Figure 4. Transfer of CD4 + T cells from Zfp362 −/− mice causes less severe colitis.A transfer colitis was induced in 8-week-old male and female Rag2 −/− mice by intraperitoneal injection of 3 × 10 5 naïve CD4 + T cells isolated from sex-and age-matched Zfp362 −/− mice or Zfp362 +/+ littermates.Rag2 −/− mice injected with PBS served as control.(A) Over the course of the experiment, animals were monitored regarding their weight twice a week.Data are depicted as mean ± 95% CI and summarized from ten independent experiments (n = 23-30 biological replicates per group in total).Symbols indicate the different experimental groups according to the legend.The significance of differences between means was assessed using an ordinary two-way ANOVA with Šidák's multiple comparison test (mean ± 95% CI; *p < 0.05 and **p < 0.01).(B) Four weeks after adoptive transfer, mice were sacrificed, and the length and weight of the colon (left) and small intestine (SI; right) was assessed and depicted in scatter plots with bars.Data are summarized from six independent experiments (n = 7-9 biological replicates per group in total).Each symbol represents an individual mouse.The significance of differences between means was assessed using Kruskal-Wallis test (mean ± SD). (C, D) Inflammation of the colon was assessed by histology.(C) Exemplary H&E stainings of colons from mice receiving Tnaïve from Zfp362 +/+ mice, Tnaïve from Zfp362 −/− mice, or PBS only.Black bar represents approx.50 μm.Invasion of inflammatory cells (i), epithelial hyperplasia (h), and sloughing of epithelia cells (e) are labeled.(D) Scatter plots with bars present the total inflammation score, and the single scores for inflammation, epithelial erosion, hyperplasia, single cell apoptosis, and the area involved.Each symbol represents an individual mouse.Data are summarized from three to five independent experiments (n = 9-14 biological replicates per group in total).The significance of differences between means was assessed using Kruskal-Wallis test with Dunn's multiple comparison test (mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001).