Patched 1 and C-C Motif Chemokine Receptor 6 Distinguish Heterogeneous T Helper 17 Subsets in Colitic Lamina Propria

ABSTRACT T helper 17 (Th17) cells contribute to the pathogenesis of inflammatory bowel diseases (IBD). However, their heterogeneity and regulatory mechanisms in IBD are not completely disclosed. A mouse colitis model was established. Th17 cells were enriched from the mesenteric lymph nodes (mLN) and lamina propria (LP). The phenotypes and functions of Th17 subsets were analyzed by flow cytometry, Immunoblotting, and real-time RT-PCR. The contributions of the Th17 subsets to colitis pathogenesis were evaluated by histology, ELISA, and flow cytometry after adoptive transfer. Smoothened (SMO), GLI family zinc finger 1 (Gli1), and GLI family zinc finger 3 (Gli3) were markedly up-regulated while Patched 1 (PTCH1) was down-regulated in LP Th17 cells in colitic lamina propria. Based on the expression of PTCH1 and C-C motif chemokine receptor 6 (CCR6), LP Th17 cells were divided into a PTCH1lowCCR6low Th17 subset and a PTCH1highCCR6high Th17 subset. The former expressed higher T-bet, IFN-γ, TNF-α, IL-1β, and GM-CSF but lower IL-17A, IL-22, IL-17F, and Gli3 than the latter. The PTCH1highCCR6high Th17 subset was more resistant to polarization towards T helper 1 (Th1) than the PTCH1lowCCR6low Th17 subset. Moreover, the PTCH1highCCR6high Th17 subset was more competent to maintain Th17 identity. The PTCH1highCCR6high Th17 subset induced less severe colitis than the PTCH1lowCCR6low Th17 subset. PTCH1highCCR6high Th17 cells are Th17 cells whereas PTCH1lowCCR6low Th17 cells are Th1-like Th17 cells. Our study deepens the understanding of Th17 heterogeneity and plasticity in colitis.


Introduction
Patients with inflammatory bowel diseases (IBD) are afflicted with persistent inflammation in the gastrointestinal tract (Bouma and Strober 2003;Silva et al. 2016). A variety of inflammatory cell types positively or negatively modulate the inflammatory cascades in the bowel (Brandtzaeg et al. 2006;Imam et al. 2018;Steinbach andPlevy 2014). 2018). Under the instructions from antigen-presenting cells and microenvironmental factors, CD4+ T lymphocytes can differentiate into distinct effector subsets, including T helper 1 (Th1), T helper 2 (Th2), regulatory T cells (Tregs), and T helper 17 cells (Th17). Th17 cells infiltrate the intestine of IBD patients to exert controversial effects in different animal models of IBD (Hou and Bishu 2020). However, recent research suggests that Th17 cells are heterogeneous, consisting of subpopulations with phenotypic and functional differences (Bystrom et al. 2019). Moreover, Th17 cells exhibit a considerable degree of plasticity, meaning that they can turn into other T lymphocyte subsets including Th1 cells and Tregs (Guery and Hugues 2015;Omenetti and Pizarro 2015). However, the heterogeneity and plasticity of Th17 cells in IBD especially in the gastrointestinal immune system are not thoroughly characterized.
The Hedgehog signaling is an evolutionarily conserved pathway playing critical roles in tissue development and homeostasis (Briscoe and Therond 2013). Hedgehog proteins, including Sonic hedgehog (SHH), Indian hedgehog (IHH), and Desert hedgehog (DHH), prompt diverse cellular reactions such as survival, proliferation, and differentiation (Briscoe and Therond 2013). In canonical Hedgehog signaling, hedgehog proteins bind to the receptor Patched 1 (PTCH1), resulting in derepression of Smoothened (SMO) and subsequent dissociation of Gli proteins (Gli1, Gli2, and Gli3) from an inhibitory complex comprising Kinesin Family Member 7 (Kif7) and Suppressor of fused protein (SUFU). Gli proteins are then processed into transcriptional factors and enter the nucleus to trigger the expression of target genes such as Ptch1 and Gli1. The impact of the Hedgehog signaling to mature T lymphocytes remains poorly understood, although previous studies suggest the involvement of the Hedgehog signaling in the activation of and cytokine expression by CD4+ T cells (Stewart et al. 2002), Th2 differentiation (Yanez et al. 2019), and cytotoxic T lymphocyte function (de la Roche ATR et al. 2013). Previous studies have suggested that the Hedgehog signaling drives differentiation and effector function of Th17 cells and is associated with the occurrence and progression of IBD Lees et al. 2008;Xie et al. 2021).
In this study, we characterized the expression of the key Hedgehog signaling components in Th17 cells in a mouse colitis model. Novel Th17 subsets, based on the expression PTCH1 and CCR6, were found in the colonic lamina propria (LP) of colitic mice. These Th17 subsets exhibited distinct cytokine profiles, plasticity, and pathogenicity. Our data indicate that the Hedgehog signaling might differentially affect different colitic Th17 subsets.

Colitis model
The study was approved by Wuhan University Animal Care and Use Committee and conducted under the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. Male homozygous IL-17A-EGFP mice (C57BL/6-Il17at tm1 /Bcgen, 8-week-old) were purchased from the Beijing Biocytogen Co., Ltd. This strain has an IRES-EGFP-SV40-polyA cassette being placed after the stop codon of the Il17a gene so IL-17A and EGFP are co-expressed. Rag1 knockout (C57BL/6J-Rag1 em1cyagen , 8-week-old) mice were purchased from Cyagen. To induce acute colitis, mice were fed with water containing 3% dextran sulfate sodium (DSS, Sigma-Aldrich) for 7 days.

Enrichment of immune cells from mesenteric lymph nodes (mLns) and lamina propria (LP)
Mice were sacrificed using carbon dioxide. Mesenteric lymph nodes were harvested and pressed through 70-µm cell strainers to prepare single-cell suspensions. Colons were collected and minced into small fragments, followed by digestion for 30 minutes at 37°C in RPMI 1640 medium supplemented with 3 mg/ml collagenase VIII, 2 mM ethylenediaminetetraacetic acid, 1 mM dithiothreitol, 5 mM HEPES, 200 U/ml DNase I, and 20% fetal calf serum. The tissues were then filtered through 70-µm cell strainers. The resultant single-cell suspensions were centrifuged at 250×g for 5 minutes and resuspended in Tris-NH 4 Cl lysis buffer for 2 minutes at room temperature to remove red blood cells. Cells were then resuspended in PBS before further testing. All chemicals and reagents were purchased from Sigma-Aldrich.

Real-time RT-PCR
The Arcturus PicoPure RNA Isolation Kit was used for RNA extraction based on the supplier's protocol. The RevertAid First Strand cDNA Synthesis Kit was used for cDNA synthesis. cDNA samples were blended with the SYBR™ Select Master Mix and PCR was run on a StepOne™ Real-Time PCR System (Applied Biosystems). All reagents were purchased from ThermoFisher. Supplementary Table 1 summarizes the primer sequences. The transcript levels of genes of interest were first normalized to the transcript level of β-actin and then computed by the 2 −ΔΔCt formula.
To evaluate naïve CD4+ T cell differentiation to Th1 cells, splenic CD4+ T cells were enriched from normal C57BL/6J mice using the Dynabeads™ Untouched™ Mouse CD4 Cells Kit (11416D, Invitrogen) following the vendor's manual. The purity of the sorted CD4+ T cells reached about 95%. CD4+ T cells were stimulated for 5 days in the same Th1-skewing condition as stated above. Four hours before the end of culture, cells were stimulated for 4 hours with 20 ng/ml phorbol 12-myristate 13-acetate (Sigma-Aldrich), 1 μg/ml ionomycin (Sigma-Aldrich), and 2 μg/ml brefeldin A (Invitrogen). Cells were then subjected to intracellular staining with the FITC anti-IL-17A antibody and APC anti-IFN-γ antibody.
To test the intrinsic ability to maintain Th17 identity, 100 µl of Th17 cells were seeded into each well of a 96-well culture plate without any stimuli. Recombinant mouse SHH was added at a final concentration of 500 ng/ml. Cells were incubated for 24 hours before IL-17A(EGFP) was measured by FACS.

Adoptive transfer
IL-17A-EGFP mice were treated with 3% DSS as described above. On day 14 after DSS treatment, Th17 subsets were sorted from the LP. Rag1 knockout (Rag1 −/− ) mice were fed with 1% DSS for four days. After that, 100 μl of PBS containing 1 × 10 5 sorted Th17 cells were retro-orbitally injected into each Rag1 −/− mouse each day for two consecutive days. Eight days after the last injection, the mice were euthanized for further assays.

ELISA
All chemicals were obtained from Sigma-Aldrich. Mouse colons were put in a digestion buffer (PBS supplemented with 20 U aprotinin A, 0.05% Tween-20, 0.1 mM benzethonium chloride, 10 mM ethylenediaminetetraacetic acid, and 0.1 mM phenylmethylsulfonyl fluoride). In general, 50 μl of digestion buffer was used for 1 mg of tissue. The colons were then homogenized on ice in tissue grinders. The homogenates were centrifuged at 12,000 rpm for 5 minutes and the supernatants were harvested for cytokine quantification using the LEGEND MAX™ mouse TNF-α ELISA kit (BioLegend), LEGEND MAX™ mouse IL-6 ELISA kit (BioLegend), and mouse IL-1 beta/IL-1F2 quantikine ELISA kit (R&D Systems), respectively.

Hematoxylin and eosin staining
Colon paraffin sections (5-µm thickness) were prepared and subjected to staining with hematoxylin and eosin following the standard protocol. The severity of inflammation was scored from 0 to 4: 0: no inflammation; 1: minimal inflammation; 2: visible but low infiltrating leukocytes; 3: significant infiltrating leukocytes, an increase in the vasculature, and colon wall thickening; 4: transmural infiltrates, goblet loss, and colon wall thickening.

Statistics
Data were shown as mean ± standard deviation. Each experiment was independently repeated 2 to 4 times. The unpaired two-tailed Student's t-test or one-way ANOVA with Tukey's test was used to test the statistical significance. A P < 0.05 is regarded as significant.

The expression of Hedgehog signaling components is changed in LP Th17 cells in colitis
We induced acute colitis in male IL-17A-EGFP transgenic mice with DSS. On day 0, day 7, and day 14 after DSS administration, immune cells were enriched from mLNs and LP ( Supplementary Fig. S1). CD3+CD4+ T lymphocytes were observed in these immune cells and IL-17A(EGFP)+ Th17 cells were found within CD3+CD4+ T lymphocytes (Figure 1(a,b)). The frequency and absolute number of Th17 cells progressively increased in both mLNs and LP (Figure 1(c,d)). The key components of the Hedgehog signaling in Th17 cells were evaluated by real-time RT-PCR. As exhibited in Figure 1(e), PTCH1 expression was not significantly changed in mLN Th17 cells but was down-regulated in LP Th17 cells on day 7 and day 14. PTCH2 expression was not profoundly altered in Th17 cells in either mLNs or LP (Figure 1(f)). SMO expression was not significantly changed in mLN Th17 cells but was significantly up-regulated in LP Th17 cells on day 7 and day 14, with the highest expression on day 7 (Figure 1(g)). Similarly, Gli1 expression was unchanged in mLN Th17 cells but was promoted in LP Th17 cells on day 7 and day 14 (Figure 1(h)). Gli3, however, was significantly increased in both mLN Th17 cells and LP Th17 cells on day 7 and day 14, with the highest level in day-7 LP Th17 cells (Figure 1(i)). No Gli2 amplification was observed (Data not shown). The Immunoblotting assay confirmed increased Gli1 expression in LP Th17 cells compared with mLN Th17 cells ( Supplementary Fig. S2). No inhibitory phosphorylation at Thr1074 was observed on Gli1 in both mLN and LP Th17 cells ( Supplementary Fig. S2).

Differential expression of PTCH1 and CCR6 mark LP Th17 subsets in colitis
Because PTCH1 mRNA was down-regulated in LP Th 17 cells, we checked its surface staining along with CCR6 (a known Th17 surface marker). As displayed in Fig. 2(a,b), mLN Th17 cells were predominantly PTCH1 high CCR6 high at each time point. Day-0 LP Th17 cells were also PTCH1 high CCR6 high . Interestingly, a PTCH1 low CCR6 low Th17 subset emerged in day-7 Th17 cells and day-14 Th17 cells, accounting for approximately 20% and 30% of LP Th17 cells, respectively (Figure 2(a,b)). The frequency of PTCH1 high CCR6 high LP Th17 cells was progressively decreased on day 7 and day 14 (Figure 2(a,b)). The absolute numbers of both subsets increased in the LP on day 7 and day 14 (Figure 2(c)). Assessment of IL-17A (EGFP) intensity suggested that the PTCH1 low CCR6 low Th17 subset expressed lower IL-17A than the PTCH1 high CCR6 high Th17 subset (Figure 2(d,e)). In addition, similarly differential IL-22 expression in the LP Th17 subsets was observed on day 7 but not on day 14 (Figure 2(f)). The expression of IL-17F and C-C motif chemokine ligand 20 (CCL20), which are also Th17 signature gene products, was also lower in the PTCH1 low CCR6 low subset relative to the PTCH1 high CCR6 high subset (Figure 2(g) and Supplementary Fig. S3A). However, equivalent IL-23 R (another Th17 surface marker) expression was found on the two Th17 subsets (Supplementary Fig. S3B).
Because only LP Th17 cells showed significant heterogeneity, we then focused on LP Th17 cells in the following assays. We characterized the mRNA levels of transcription factors key to the differentiation of Th1, Treg, and Th17 cells. The PTCH1 low CCR6 low subset expressed more T-bet than the PTCH1 high CCR6 high subset, whereas Foxp3 and RORγt were comparably expressed in the two subsets (Figure 3(a-c)). To further identify the subsets, we evaluated the expression of IL-17A and IFN-γ. As shown in Figure 3(d,e), around 12%-15% of PTCH1 low CCR6 low cells were IL-17A+IFN-γ+, whereas less than 3% of PTCH1 high CCR6 high cells were IL-17A+IFN-γ+, suggesting that the PTCH1 low CCR6 low subset expressed more IFN-γ than the PTCH1 high CCR6 high subset. To test the expression of other pro-inflammatory cytokines, the transcript levels of TNF-α, IL-1β, and GM-CSF were quantified in the two subsets. As shown in Figure 3(f-h), the PTCH1 low CCR6 low subset expressed higher TNF-α, IL-1β, and GM-CSF than the PTCH1 high CCR6 high subset.

Gli3 is differentially expressed in the two Th17 subsets
We then asked if the activation status of the canonical Hedgehog signaling was distinct in these Th17 subsets. To this end, the mRNAs of SMO, Gli1, and Gli3 were quantified. As demonstrated in Figure 4(a), SMO expression was equivalent in the two subsets and was lower on day 14 than day 7. Gli1 expression was also comparable in the two subsets (Figure 4(b)). Gli3 expression, however, was higher in the PTCH1 high CCR6 high subset relative to the PTCH1 low CCR6 low subset (Figure 4(c)). Moreover, Gli3 expression in each subset was lower on day 14 than day 7 (Figure 4(c)). The changes in Gli3 expression were confirmed by Immunoblotting (Figure 4(d,e)). Hence, the PTCH1 high CCR6 high subset might have stronger activation of the Hedgehog signaling, as evidenced by the higher Gli3 expression. Consistent with the intracellular cytokine staining results, the PTCH1 low CCR6 low subset possessed more IFN-γ mRNA than the PTCH1 high CCR6 high subset (Figure 4(f)).
-γ-cells and IFN-γ+ cells were significantly reduced, suggesting that the SHH suppressed the generation of Th1-like Th17 cells. Notably, in the presence of SHH, the PTCH1 high CCR6 high subset still contained more IL-17A+IFN-γ-cells and fewer IFN-γ+ cells than the PTCH1 low CCR6 low subset ( Figure 5(a,b)). Moreover, freshly sorted PTCH1 low CCR6 low cells expressed higher IL-1β and TNF-α than PTCH1 high CCR6 high cells ( Figure 5(c,d)). Under the Th1-skewing condition without SHH, both subsets upregulated IL-1β and TNF-α, but the PTCH1 low CCR6 low subset still expressed higher IL-1β and TNF-α than the PTCH1 high CCR6 high subset ( Figure 5(c,d)). SHH significantly reduced IL-1β and TNF-α in both subsets under the Th1-skewing condition ( Figure 5(c,d)). The PTCH1 low CCR6 low subset still expressed higher IL-1β and TNF-α than the PTCH1 high CCR6 high subset in the presence of SHH (Figure 5(c,d)). To analyze the effect of SHH on CD4+ T cell differentiation into Th1 cells, splenic CD4+ T cells were enriched from normal wild-type mice and cultured under the Th1 differentiation condition with or without SHH for 5 days. As shown in Supplementary Fig. S4, SHH moderately inhibited the generation of IFN-γ-expressing Th1 cells but did not affect IL-17A expression.
To check the stability of Th17 identity, the two subsets were cultured for 24 hours in the presence or absence of SHH without any other stimuli. When SHH was absent, IL-17A (EGFP)+ cells were decreased in both subsets compared with freshly sorted Th17 subsets. However, the PTCH1 high CCR6 high subset had more IL-17A(EGFP)+ cells than the PTCH1 low CCR6 low subset ( Figure 5(e,f)). SHH partially prevented the decrease of IL-17A (EGFP)+ cells in both subsets, and the PTCH1 high CCR6 high subset also had more IL-17A (EGFP)+ cells than the PTCH1 low CCR6 low subset in the presence of SHH ( Figure 5(e,f)). Therefore, compared with the PTCH1 low CCR6 low subset, the PTCH1 high CCR6 high subset was more competent to maintain Th17 identity.

PTCH1 high CCR6 high Th17 cells are less pathogenic than PTCH1 low CCR6 low Th17 cells in inducing colitis
To measure the pathogenicity of the two subsets, Rag1 −/− mice were given 1% DSS for four days and then received injections of the PTCH1 high CCR6 high subset or the PTCH1 low CCR6 low subset for two days (Supplementary Fig. S5). Eight days after the last transfer (i.e. 14 days after the start of DSS treatment), the colons were collected for evaluation. H&E staining indicated significant tissue damage and a high histological inflammation score in mice receiving the PTCH1 low CCR6 low subset (Figure 6(a,b)). In contrast, in mice receiving the PTCH1 high CCR6 high subset, less tissue damage featuring a lower histological inflammation score was found (Figure 6(a,b)). Consistently, mice receiving the PTCH1 low CCR6 low subset progressively and significantly lost body weight, whereas mice receiving the PTCH1 high CCR6 high subset lost less body weight on day 11 and 13 ( Figure 6(c)). Additionally, the expression of pro-inflammatory cytokines, such as TNFα, IL-1β, and IL-6, was higher in the colons of mice receiving the PTCH1 low CCR6 low subset, in comparison to mice receiving the PTCH1 high CCR6 high subset (Figure 6(d)). Eight days after the adoptive transfer, CD3+ T lymphocytes were retrieved from recipients' LP ( Figure 6(e)). Since Rag1 −/− mice lack mature T lymphocytes, all CD3+ T lymphocytes were donor-derived, i.e. exogenous Th17 subsets. The frequency of donor-derived PTCH1 hi CCR6 hi Th17 cells was higher than the frequency of donor-derived PTCH1 low CCR6 low Th17 cells (Figure 6(f)). The expression of IL-17A and IFN-γ in these Th17 subsets was quantified by FACS. Donor-derived PTCH1 high CCR6 high subset and donor-derived PTCH1 low CCR6 low subset expressed comparable IL-17A (Figure 6(g,h)). However, donor-derived PTCH1 high CCR6 high subset expressed fewer IFN-γ than donorderived PTCH1 low CCR6 low subset (Figure 6(g,h)). Interestingly, the PTCH1 high CCR6 high subset expressed more Ki67 than the PTCH1 low CCR6 low subset ( Figure 6(i,j)). There were no significant differences in apoptosis and necrosis between the two subsets in vivo ( Supplementary Fig. S6). Taken together, the PTCH1 high CCR6 high subset was less pathogenic than the PTCH1 low CCR6 low subset.

Discussion
The present study first unveiled the expression of important Hedgehog signaling components in Th17 cells in colitis development. The frequency of Th17 cells was boosted in both mLN and LP CD4+ T lymphocytes after colitis induction, indicating that Th17 response was initiated and promoted during the development of colitis. The transcripts of PTCH1, SMO, and Gli1 were not significantly altered in mLN Th17 cells after the onset of colitis, suggesting that the differentiation and activation of Th17 cells do not influence the transcription of these molecules. In contrast, the changes in PTCH1, SMO, and Gli1 in LP Th17 cells suggest that LP-specific factors, perhaps inflammatory mediators, enhance the transcription of these molecules. Interestingly, Gli3 was increased in both mLN Th17 cells and LP Th17 cells after the onset of colitis, signifying the activation of the hedgehog signaling in colitis-responsive Th17 cells. Therefore, our study might support the conclusion of the latest study showing that the Hedgehog signaling particularly Gli3 controls Th17 differentiation . Because Gli1 itself is the target gene of the Hedgehog signaling (Carballo et al. 2018), the remarkable increase in Gli1 implies the activation of the Hedgehog signaling in LP Th17 cells.
Another important discovery made by this study is the revelation of heterogeneous LP Th17 subsets in colitis according to the expression of PTCH1 and CCR6. Interestingly, the PTCH1 low CCR6 low subset only existed in LP Th17 cells after the onset of colitis, suggesting that this subset originated from PTCH1 high CCR6 high Th17 cells through the downregulation of CCR6 and PTCH1. What caused the down-regulation of CCR6 and PTCH1 remains unidentified. Since PTCH1 low CCR6 low Th17 cells expressed lower IL-17A and IL-22 relative to PTCH1 high CCR6 high Th17 cells while maintaining RORγt expression, perhaps the PTCH1 low CCR6 low subset was undergoing polarization toward other types of T lymphocyte subsets. Indeed, the PTCH1 low CCR6 low subset expressed higher T-bet and IFN-γ than the PTCH1 high CCR6 high subset, possibly reflecting Th1 polarization. IFN-γexpressing Th17 cells, termed Th1/Th17 or Th1-like Th17 cells, have been implicated in the pathogenesis of inflammatory disorders and autoimmunity including IBD (Kamali et al. 2019;Kotake et al. 2017). It has been reported that under the influence of IL-12, IL-23, and IL-1β when TGFβ is absent, Th17 cells lose stability and express IFN-γ (Omenetti and Pizarro 2015). Therefore, the strong immune and inflammatory milieu in LP might skew PTCH1 high CCR6 high Th17 cells towards PTCH1 low CCR6 low Th1/Th17 cells. To our knowledge, our study is the first to report the correlation between Th17 heterogeneity/plasticity and PTCH1 expression. Although no significant changes in PTCH2 transcript were found, it would be interesting to see whether PTCH2 is differentially expressed on LP Th17 cells if a PTCH2 antibody is suitable for FACS analysis.
The Hedgehog signaling has profound effects on cell fate decisions during tissue development and homeostasis via modulating tissue patterning, cell division, and differentiation (Skoda et al. 2018). We found that Gli3 was differentially expressed in the two Th17 subsets. Surprisingly, inconsistent with the differential PTCH1 expression, Gli1 expression was equivalent in the two Th17 subsets. This poses the question of whether PTCH1 expression correlates to the Hedgehog signaling activation. If extracellular Hedgehog proteins are present in inflamed colons, the activation of the Hedgehog signaling should increase the expression of both PTCH1 and Gli1. Therefore, some unknown factor(s) favors Gli3 expression rather than Gli1 expression upon the activation of the Hedgehog signaling in LP Th17 cells. Further investigations are needed to reveal the factor(s).
A previous study has demonstrated that both IHH and SHH are secreted by intestinal epithelium (Kolterud et al. 2009). In DSS-induced colitis, IHH was expressed in the whole colon but mildly down-regulated (Lee et al. 2016). In patients with Crohn's disease, Hedgehog ligands were generally expressed at the superficial epithelium (Buongusto et al. 2017). However, another study indicates strong SHH expression throughout the epithelium from the base to the lumen in both colonic Crohn's disease and ulcerative colitis (Nielsen et al. 2004). Moreover, in ulcerative colitis, CD4+ inflammatory cells also express SHH (Nielsen et al. 2004). Because both CD4+ T cells and macrophages express CD4, they likely produce SHH in the colitic microenvironment. Therefore, we think IHH is expressed by colonic epithelial cells while SHH is produced by colonic epithelial cells and CD4+ inflammatory cells in colitis.
The in vitro culture assays show that: 1) The PTCH1 high CCR6 high subset was more resistant to Th1 polarization than the PTCH1 low CCR6 low subset; 2) SHH-induced hedgehog signaling inhibited the generation of Th1-like Th17 cells. The results strongly suggest that the Hedgehog signaling plays a crucial role in maintaining Th17 identity and inhibiting Th17 plasticity. However, the mechanism by which SHH does so has yet to be found. It is broadly accepted that Th1 differentiation depends on T-bet expression (Saravia et al. 2019) and so does Th1-like Th17 polarization (Harbour et al. 2015;Krausgruber et al. 2016). Furthermore, Runx1 and Runx3 are also required for the generation of Th1-like Th17 cells (Wang et al. 2014). Our ongoing study is checking whether SHH could interfere with the expression of T-bet, Runx1, and Runx3. The adoptive transfer assay showed that the PTCH1 high CCR6 high subset was less pathogenic than the PTCH1 low CCR6 low subset, implying that the latter contributes more significantly to colitis development. Therefore, blockade of the generation of Th1-like Th17 cells might be a future therapeutic strategy to alleviate colitis severity.
Taken together, this study reveals Th17 subsets based on PTCH1 expression and the Hedgehog signaling for the first time. It deepens the understanding of Th17 heterogeneity in colitis and sheds light on the role of the Hedgehog signaling in regulating Th17 subsets.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This study was supported by the Wuhan Clinical Research Foundation (Grant# WX14C22) and Wuhan Municipal Medical Research Foundation (Grant# WX20A07)

Data availability
The data are available from the corresponding author upon reasonable request.