Pak2-mediated phosphorylation promotes RORγt ubiquitination and inhibits colonic inflammation.

Dysregulated interleukin-17 (IL-17) expression and its downstream signaling is strongly linked to inflammatory bowel diseases (IBDs). However, the molecular mechanisms by which the function of RORγt, the transcription factor of IL-17, is regulated remains elusive. By a mass spectrometry-based approach, we identify that Pak2, a serine (S)/threonine (T) kinase, directly associates with RORγt. Pak2 recognizes a conserved KRLS motif within RORγt and phosphorylates the S-316 within this motif. Genetic deletion of Pak2 in Th17 cells reduces RORγt phosphorylation, increases IL-17 expression, and induces severe colitis upon adoptive transfer to Rag1-/- mice. Similarly, reconstitution of RORγt-S316A mutant in Rorc-/- Th17 cells enhances IL-17 expression and colitis severity. Mechanistically, we demonstrate that Pak2-mediated phosphorylation causes a conformational change resulting in exposure of the ubiquitin ligase Itch interacting PPLY motif and degradation of RORγt. Thus, we have uncovered a mechanism by which the activity of RORγt is regulated that can be exploited therapeutically.

INTRODUCTION

Interleukin-17 (IL-17) is a proinflammatory cytokine that is produced by a variety of immune cells, including the Th17 subset of helper CD4+ T cells, δ T cells, and innate lymphoid cells (Honda and Littman, 2016; Kumar et al., 2021b; Zhou and Sonnenberg, 2020). IL-17 signals through the IL-17 receptor, composed of IL-17RA and IL-17RC receptor subunits (Gaffen, 2009). The binding of IL-17 to its receptor activates the target cells, such as epithelial cells, endothelial cells, and fibroblasts, and induces the expression of CXCL1, CXCL2, and CXCL8, which attract myeloid cells such as neutrophils and promote inflammation (Gaffen, 2009). IL-17 is critically essential for host defense against fungal and bacterial infections. However, dysregulated IL-17 expression and downstream signaling are involved in several human diseases, including inflammatory bowel disease (IBD) (McGeachy et al., 2019; Patel and Kuchroo, 2015).

The orphan nuclear receptor RORγt is a transcription factor crucial for IL-17 expression (Ivanov et al., 2006; Zhang et al., 2008). RORγt consists of a ligand-independent activation function 1 helix, a DNA-binding domain, a flexible hinge domain, and a C-terminal ligand-binding domain (Kumar et al., 2021b). The zinc finger motifs within the DNA-binding domain recognize the ROR response elements within the IL-17 promoter to induce IL-17 expression (Kumar et al., 2021b). Given the critical role of IL-17 in inflammatory diseases, RORγt has emerged as an attractive target for pharmacological interventions (Beringer et al., 2016). However, a clear understanding of the regulation of RORγt is currently lacking, which is absolutely necessary to target RORγt effectively.

Crosstalk between different posttranslational modifications plays a critical role in regulating the activity of transcription factors (Hunter, 2007). We have demonstrated earlier that the E3 ligase Itch targets RORγt for ubiquitination, and Itch deficiency results in spontaneous rectal prolapse as a result of severe spontaneous colitis (Kathania et al., 2016). Here, we show how Pak2-mediated phosphorylation facilitates RORγt ubiquitination to prevent excessive Th17 response. A clear understanding of this regulatory pathway could lead to the development of safe and highly selective modulators of RORγt as a targeting strategy for inflammatory diseases driven by IL-17.

RESULTS

Pak2 interacts and phosphorylates RORγt

To gain molecular insights into the regulation of RORγt, we immunoprecipitated RORγt from the cell lysate of in vitro-generated Th17 cells and subjected it to mass spectrometry (MS) analysis. Through this approach, we identified Pak2 as a RORγt-binding protein (Figures 1A and S1A). We also precipitated RORγt from the cell lysate of CD4+ T cells sorted from colonic lamina propria lymphocytes (cLPLs) of mice treated with DSS and subjected it to MS analysis and found Pak2 as a RORγt-binding protein (Figure S1B). To validate the MS results, we transiently transfected 293T cells with plasmids encoding Myc-tagged Pak2 and FLAG-tagged RORγt. Cell lysates were immunoprecipitated with either anti-FLAG or anti-Myc antibodies. Samples immunoprecipitated with the anti-Myc antibody contained FLAG-RORγt, and vice versa (Figure S1C), suggesting that Pak2 and RORγt physically interact with each other. To determine whether this interaction occurred between endogenous proteins in primary cells, we performed immunoprecipitation assays with anti-Pak2 and anti-RORγt antibodies using the lysate of in vitro-generated Th17 cells and found an interaction between RORγt and Pak2 (Figure 1B). Similar results were obtained in CD4+ T cells sorted from cLPLs of mice treated with DSS (Figure S1D). We also performed glutathione S-transferase (GST) pull-down assays using recombinant Pak2 protein. GST-RORγt, but not GST alone, precipitated recombinant Pak2 (Figure 1C), suggesting a direct interaction.

Figure 1.

Pak2 interacts and phosphorylates RORγt

(A) Lysate of Th17 cells was precipitated using anti-RORγt antibody and subjected to high-resolution liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis. A representative LC-MS/MS spectrum corresponding to SDNGELEDKPPAPPVR of Pak2 is shown. Observed b and y ions are indicated.

(B) Lysates were prepared from Th17 cells and immunoprecipitated using anti-RORγt and anti-Pak2 antibody analyzed by western blotting with anti-RORγt and anti-Pak2 antibody.

(C) Immunoblotting of recombinant Pak2 precipitated with GST or GST-RORγt. Below are input controls for recombinant Pak2.

(D) Sequence alignment of RORγt showing the conserved KRLS motif.

(E) Pak2 was knocked down in CD4+ T cells using shRNA and cultured under Th17-inducing conditions. The cells were restimulated with anti-CD3 and anti-CD28 antibodies, and the lysate was subjected to immunoprecipitation (IP) with anti-RORγt antibody and analyzed by p-Ser specific antibody.

(F) ADP-glow kinase assay result is represented as relative luciferase units (RLUs).

(G) CD4+ T cells from Pak2CD4 were reconstituted with either wild-type or kinase-dead mutant of Pak2 and cultured under Th17 condition. Cell lysates were subjected to IP with anti-RORγt antibody and analyzed by p-Ser specific antibody.

(H) CD4+ T cells were isolated from Rorc−/− mice, reconstituted with either wild-type or RORγt-ΔS316A mutant, and cultured under Th17 condition. Cell lysates were subjected to IP with anti-V5 antibody and analyzed by p-Ser specific antibody.

Data are representative of two independent experiments. Data represent means ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.

Pak2 is a Serine/Threonine protein kinase that phosphorylates its target proteins on KRX[S/T] motif (Taglieri et al., 2014). Amino acid sequence analysis revealed a highly conserved KRLS motif in RORγt, which could be potentially phosphorylated (Figure 1D). To test if Pak2 phosphorylates RORγt, we knocked down Pak2 in in vitro-generated Th17 cells using short hairpin RNA (shRNA) lentiviral particles and found that inhibiting Pak2 reduced the phosphorylation of RORγt. As seen in Figure 1E, knocking down Pak2 reduced RORγt phosphorylation. Further, we confirmed that Pak2 phosphorylates RORγt by performing an in vitro kinase assay. As shown in Figure 1F, incubation of RORγt with wild-type Pak2, but not a Pak2 kinase-dead mutant (Pak2-ΔK278R), resulted in RORγt phosphorylation. Similarly, incubation of the RORγt-ΔS316A mutant with wild-type Pak2 exhibited a defect in phosphorylation. These data suggest that Pak2 phosphorylates RORγt at Serine 316 (S316). To further confirm these results, we reconstituted CD4+ T cells isolated from Pak2CD4 mice with either wild-type Pak2 or Pak2-ΔK278R by lentivirus transduction. We found RORγt phosphorylation in Pak2−/− cells reconstituted with wild-type Pak2 but not the Pak2-ΔK278R mutant (Figure 1G). Similarly, reconstitution of Rorc−/− CD4+ T cells with wild type, but not the RORγt-ΔS316A mutant, resulted in RORγt phosphorylation (Figure 1H). These data collectively indicate that Pak2 phosphorylates RORγt at S316 in Th17 cells.

Inhibition of Pak2 enhances IL-17 expression and colitis

To gain insights into the functional consequence of Pak2-mediated phosphorylation of RORγt, we analyzed the expression of Il17a and Il17f mRNA in in vitro-generated Th17 cells from wild-type and Pak2CD4 mice. Results of real-time PCR experiments showed that Pak2 deficiency enhances the expression of Il17a and Il17f (Figures S2A and S2B). However, no significant difference in Rorc mRNA level was observed (Figure S2C). To gain in vivo evidence for Pak2-mediated phosphorylation of RORγt in colonic inflammation, we sorted CD4+ T cells from wild-type and Pak2CD4 mice, and the cells were differentiated under Th17 polarizing conditions. The cells were then adoptively transferred into Rag1−/− mice as described before (Lee et al., 2009). The Rag1−/− mice that received Pak2−/− Th17 cells showed more body weight loss, an increased fecal occult blood (FOB) score, increased diarrhea, splenomegaly, reduced colon length, and a higher weight-to-length ratio of the colon compared with the mice that received wild-type Th17 cells (Figures 2A–2E). Colonoscopic examination showed increased inflammation in mice that received Pak2−/− Th17 cells compared with those that received wild-type Th17 cells (Figures 2F and 2G). Similarly, histo-logical analysis of H&E-stained sections showed greater infiltration of inflammatory cells, more crypt damage, and higher clinical scores in mice that received Pak2−/− Th17 cells than wild-type cells (Figures 2H and 2I). Real-time PCR analysis showed higher expression of ll17a and Il17f mRNA in the colonic mucosa of mice that received Pak2−/− Th17 cells (Figures 2M and 2N). Further, flow cytometric analysis (Figure S2D) showed an increased IL-17 expression by the adoptively transferred Pak2−/− Th17 cells in Rag1−/− mice compared with wild-type Th17 cells (Figure 2J). However, no significant changes in interferon gamma (IFN-γ) production (Figures 2K and S2E) was observed. Also, as expected, no change in IL-17 expression by innate lymphoid cells (ILCs) of host Rag1−/− mice was observed (Figure 2L). These data suggest that Pak2-mediated phosphorylation in Th17 cells plays a crucial role in colonic inflammation.

Figure 2.

Pak2 inhibition enhances IL-17 expression and Th17 cell-induced colitis

Th17 cells were generated in vitro using CD4+ T cells isolated from wild-type and Pak2CD4 mice and injected intraperitoneally into Rag1−/− mice (n = 5 animals per group).

(A) Body weight change in percentage.

(B) FOB score.

(C) Diarrhea score.

(D) Representative image of colon and spleen.

(E) Colon weight to length ratio.

(F) Representative colonoscopic images.

(G) Colonoscopic score.

(H) Representative H&E images. Scale bars, 100 μm.

(I) Histology score.

(J and K) cLPLs were stained with anti-CD4, anti-IL-17, and anti-IFN-γ antibodies and analyzed by flow cytometry.

(L) Flow cytometric analysis of IL-17 expression by ILCs in cLPLs.

(M–O) RNA isolated from cLPLs was assayed for the expression of Il17a, Il17f, and Rorc by real-time PCR.

(P) Lysates from CD4+ T cells sorted from cLPLs were analyzed by immunoblotting with anti-RORγt and anti-actin antibodies.

Data are representative of two independent experiments (n = 5). Dots indicate individual mice. Data represent means ± SD. *p < 0.05; **p < 0.01; ***p < 0.001. N.S., not significant.

To test if increased IL-17 expression by the adoptively transferred Pak2−/− Th17 cells is potentially due to elevated Rorc mRNA expression, we performed real-time PCR analysis. No change in Rorc mRNA level (Figure 2O) was noted. However, immunoblotting the lysate of cLPLs showed an increased level of RORγt protein in mice that received Pak2−/− Th17 cells compared with wild-type Th17 cells (Figure 2P). These data suggest that Pak2 regulates RORγt protein turnover without affecting RORγt transcription.

Pak2 enhances the ubiquitination of RORγt

Next, we sought to investigate how Pak2 regulates RORγt protein turnover. We have previously shown that Itch targets RORγt for ubiquitination (Kathania et al., 2016). Since phosphorylation can affect protein turnover via ubiquitination (Hunter, 2007), we hypothesized that Pak2-mediated phosphorylation promotes RORγt ubiquitination. To test this hypothesis, we performed a ubiquitination assay by co-expressing either wild-type RORγt, RORγt-ΔS316A kinase mutant, or RORγt-ΔS316D phospho-mimetic mutants along with the Itch. Our results showed an increased polyubiquitination of RORγt-ΔS316D (Figure 3A), suggesting that phosphorylation regulates ubiquitination of RORγt. To further confirm that phosphorylation promotes RORγt ubiquitination, we performed in vitro ubiquitin assays using recombinant wild-type RORγt or RORγt mutants (RORγt-ΔS316A or RORγt-ΔS316D) along with recombinant Itch. Increased ubiquitination of RORγt-ΔS316D phospho-mimetic mutant was observed compared with the RORγt-DS-A phospho-null mutant (Figure 3B). Finally, we analyzed ubiquitination of RORγt in in vitro-generated Th17 cells from Pak2CD4 mice lentivirally transduced with wild-type Pak2 or the Pak2-ΔK278R kinase-dead mutant. We found an increased ubiquitination in the cells transduced with wild-type Pak2 compared with the Pak2-ΔK278R kinase-dead mutant (Figure 3C).

Figure 3.

Pak2 enhances the ubiquitination of RORγt and regulates IL-17 expression

(A) 293T cells were co-transfected with Itch, RORγt, or RORγt mutants. Cell lysates were prepared and subjected to immunoprecipitation using GST-TUBE, followed by immunoblotting analysis with the indicated antibodies.

(B) In vitro ubiquitination of RORγt and RORγt mutants.

(C) CD4+ T cells from Pak2CD4 mice were reconstituted with either wild-type Pak2 or Pak2ΔK278R mutant and cultured under Th17 condition. Cell lysates were immunoprecipitated using GST-TUBE, followed by immunoblotting with the indicated antibodies.

(D) CD4+ T cells from Rorc−/− mice were reconstituted with wild-type RORγt or RORγt-ΔS316D mutant. Cells were treated with cycloheximide (CHX) for the indicated times. Cell lysates were analyzed by immunobloting with anti-RORγt antibody and anti-actin antibody.

(E) Th17 cells generated in vitro from Pak2CD4 mice were reconstituted with wild-type Pak2 and Pak2ΔK278R mutant. Cells were treated with CHX for the indicated times. Cell lysates were analyzed by immunoblotting with anti-RORγt antibody, anti-V5, and anti-actin antibody.

(F) Protein mRNA to ratio of wild-type RORγt and RORγtΔS316D mutant at indicated time points following stimulation with PMA/ionomycin activation and CHX.

(G) In silico analysis of mutation of S316D on RORγt.

(H) Immunoblotting of recombinant Itch precipitated with GST, GST- RORγt, or GST-RORγt mutants. Below are input controls for recombinant GST alone, GST-RORγt, GST-RORγt mutants, and Itch.

(I) CD4+ T cells from Rorc−/− mice and reconstituted with either wild-type RORγt or RORγt phospho-mutants. Cells were then cultured under Th17 conditions, and Il17a expression was analyzed by real-time PCR. Below is the western blot expression of RORγt and RORγt phospho-mutants.

Data are representative of three independent experiments. Data represent means ± SD. *p < 0.05; **p < 0.01; ***p < 0.001. N.S., not significant.

To investigate if phosphorylation-regulated ubiquitination affected RORγt protein turnover, we performed a cycloheximide (CHX) chase experiment. The primary CD4+ T cells from Rorc−/− mice were lentivirally transduced with wild-type RORγt and RORγt-ΔS316D phospho-mimetic mutants. The cells were then treated with CHX, and the level of RORγt was analyzed by immunoblotting. Our results showed a reduced abundance of RORγt protein in RORγt-ΔS316D compared with wild-type RORγt transduced cells (Figure 3D). Similarly, the CHX chase experiment in in vitro-generated Th17 cells from Pak2CD4 mice reconstituted with wild-type Pak2 or the Pak2-ΔK278R kinase-dead mutant also showed a reduced abundance of RORγt protein in wild-type Pak2 transduced cells compared with the Pak2-ΔK278R kinase-dead mutant (Figure 3E). These results strongly suggested that phosphorylation increases RORγt protein turnover. Additionally, the mRNA-to-protein ratio of RORγt in Rorc−/− CD4+ T cells reconstituted with wild-type RORγt and RORγt-ΔS316D phospho-mimetic mutant was lower compared with wild-type-RORγt (Figure 3F).

Itch is a HECT type E3 ligase that contains four WW domains (each of which contains two conserved tryptophan residues), which recognizes proline-rich PPXY (P, proline; X, any amino acid; Y, tyrosine) motifs in its substrate proteins (Venuprasad et al., 2015). We showed earlier that Itch binds to the conserved PPLY motif on RORγt via its WW domain (Kathania et al., 2016). To gain molecular insights into the mechanism by which phosphorylation at S316 promotes RORγt ubiquitination, we performed homology modeling of mouse RORγt. In silico analysis of the modeled structure showed that S316 made an H-bond(3.6 Å) with a side-chain amino group of asparagine (N) 253 of the neighboring α-helix and stabilized the ligand-binding domain (LBD) of RORγt, hence there was less accessibility of the PPLY motif (Figure 3G). We then substituted the S316 with phospho-mimetic aspartic acid residues (D) 316, and the energy of the modeled structure was minimized after the substitution mutation. The modeled structure revealed that the H-bond interaction between S316 to N253 is abolished after phosphorylation of serine, suggesting that phosphorylation may provide increased accessibility of the PPLY motif of RORγt to Itch (Figure 3G). To validate the computational analysis, we performed a GST pull-down assay using purified wild-type Itch, GST-RORγt, GST-RORγt-ΔS316A, and GST-RORγt-ΔS316D mutants. Our results showed that Itch association with RORγt-ΔS316D mutant was more prominent compared with GST-RORγt or GST-RORγt-ΔS316A mutant (Figure 3H). These results suggest that phosphorylation enhances the binding affinity of Itch to RORγt.

Phosphorylation of RORγt inhibits IL-17 expression and colonic inflammation

To confirm that Pak2-mediated phosphorylation prevents excessive IL-17-mediated inflammation, we reconstituted Rorc−/− CD4+ T cells with wild-type RORγt or RORγt-ΔS316A or RORγt-ΔS316D mutants. Cells were cultured under Th17 polarizing conditions, and expression of IL-17 was measured by real-time PCR. Our results showed an increased Il17a expression in Rorc−/− cells expressing RORγt-ΔS316A mutant compared with wild-type RORγt, whereas significantly reduced Il17a expression was observed in cells expressing RORγt-ΔS316D mutant (Figure 3I). However, no significant difference in Rorc mRNA was observed (Figure S2F).

To investigate the physiological impact of RORγt phosphorylation on the regulation of IL-17-mediated inflammation in vivo, we utilized an adoptive transfer colitis model (Lee et al., 2009). We sorted CD4+CD25− cells from Rorc−/− mice and reconstituted them with either empty vector, wild-type RORγt, or RORγt-ΔS316A or RORγt-ΔS316D mutants. The cells were differentiated under Th17 polarizing conditions for 5 days, and 5 × 105 cells were adoptively transferred into Rag1−/− mice (Figure 4A). The mice were monitored for signs of disease, including weight loss, FOB, and diarrhea. Rag1−/− mice that received Th17 cells expressing a phosphorylation-deficient mutant, RORγt-ΔS316A, exhibited greater loss of body weight, higher FOB, diarrhea scores, splenomegaly, reduced colon length, and a higher weight-to-length ratio of the colon compared with host mice that received Th17 cells expressing RORγt-ΔS316D mutant and wild-type RORγt (Figures 4B–4F). Colonoscopic examination revealed enhanced inflammation in Rag1−/− mice that received Th17 cells expressing RORγt-ΔS316A mutant compared with RORγt-ΔS316D mutant or wild-type RORγt (Figures 4G and 4H). Histological analysis of H&E-stained sections showed greater infiltration of inflammatory cells, more crypt damage, and higher clinical scores for Rag1−/− mice that received RORγt-ΔS-A mutant expressing cells compared with RORγt-ΔS-D mutant or wild-type RORγt cells (Figures 4I and 4J). Rag1−/− mice that received Th17 cells expressing RORγt-ΔS316A mutant had higher expression of ll17a and Il17f mRNA in the colonic mucosa (Figure 4K). Similarly, flow cytometric analysis of cLPLs showed increased IL-17 expression in Rag1−/− mice that received Th17 cells expressing the RORγt-ΔS316A mutant compared with cells expressing the RORγt-ΔS316D mutant or wild-type RORγt. However, there was no change in IFN-γ production by adoptively transferred cells (Figure 4L and 4M). Further expression of IL-17-induced chemokines and MMPs Cxcl1, Ccl2, Ccl7, Ccl20, Mmp1, Mmp2, Mmp3, and Mmp13 were significantly upregulated in Rag1−/− mice that received Th17 cells expressing RORγt-ΔS316A mutation (Figures 3A and 3B). To test the potential difference in Rorc gene expression in adoptively transferred Th17 cells expressing wild-type RORγt and RORγt mutants, we performed real-time PCR experiments using RNA isolated from cLPLs of Rag1−/− mice. As shown in Figure 4K, no significant changes in Rorc mRNA expression were observed. However, immunoblotting the lysates with anti-V5 antibody showed increased RORγt protein expression of the RORγt-ΔS316A mutant, suggesting a defect in turnover of RORγt protein (Figure 4N). Together, these findings suggest that Pak2-mediated phosphorylation of RORγt negatively regulates IL-17 expression and controls colonic inflammation.

Figure 4.

RORγt phosphorylation regulates colonic inflammation

(A) Schematic diagram of Th17 cell-induced colitis model (n = 5 animals per group).

(B) Body weight change in percentage.

(C) FOB score.

(D) Diarrhea score.

(E) Spleen and colon length.

(F) Colon weight-to-length ratio.

(G) Representative colonoscopic images.

(H) Colonoscopic score.

(I) Representative H&E images.

(J) Histology score.

(K) Real-time PCR analysis of Il17a, Il17f, and Rorc.

(L and M) Flow cytometric analysis of IL-17 and IFN-γ by adoptively transferred Th17 cells.

(N) Lysates from cLPLs were analyzed by immunoblotting with anti-RORγt and anti-actin antibodies.

Data are representative of two independent experiments (n = 5). Dots indicate individual mice. Data represent means ± SD. *p < 0.05; **p < 0.01; ***p < 0.001. N.S., not significant.

DISCUSSION

Genome-wide association studies have highlighted the central role of the RORγt:IL-17 pathway in several human inflammatory diseases (Duerr et al., 2006; Lock et al., 2002; Parkes et al., 2013). Accordingly, the IL-17-inducing transcription factor RORγt has emerged as an attractive target for pharmacological interventions (Beringer et al., 2016). Our results show that Pak2-mediated phosphorylation of RORγt at S316 inhibits IL-17 expression by promoting ubiquitination of RORγt. Pak2−/− Th17 cells are highly colitogenic due to elevated IL-17 expression. Similarly, transfer of Th17 cells expressing RORγt-ΔS316A phosphor mutant resulted in severe colitis, whereas the RORγt-ΔS316D mutation that mimics the constitutively phosphorylated form of the RORγt mutant resulted in attenuated colitis in Rag1−/− mice. Mechanistically, we demonstrated that S316 phosphorylation of RORγt enhances its interaction with Itch due to increased exposure of the E3 ligase Itch interacting PPLY motif. Our results could be exploited therapeutically to inhibit IL-17-mediated inflammation.

Pak2 was shown to be essential for maintaining regulatory T cell (Treg) stability and their suppressive function, and loss of Pak2, specifically in Tregs, resulted in defective Treg function (O’Hagan et al., 2017). Our data show that Pak2 plays a distinct role in Th17 cells by facilitating RORγt degradation to curb excessive IL-17-mediated inflammation. Adoptive transfer of Pak2-deficient Th17 cells into Rag1−/− mice (which are devoid of T cells and Tregs) resulted in severe colitis. T cells recovered from the lamina propria of the recipient Rag1−/− mice exhibited an increased level of RORγt protein. Similarly, adoptive transfer of Th17 cells expressing the RORγt-ΔS316A phospho-null mutant resulted in a defect in RORγt protein turnover and elevated IL-17 expression. These data strongly suggest a distinct function for Pak2 in regulating Th17 cells and Tregs.

IL-17 is produced by a variety of immune cells, including Th17 cells, ILCs, Tc17 cells, natural killer (NK) cells, and neutrophils (Kumar et al., 2021b). The cell-surface receptors involved and the intracellular signaling pathways leading to the secretion of cytokines significantly differ in these cells. However, it remains poorly understood if different immune cells’ regulation of IL-17 expression varies or adopts the same pathways. The data presented here identify a previously unknown mechanism of regulation of the RORγt-IL-17 pathway by Pak2 in Th17 cells. Our assay systems exclude the regulation of other IL-17-producing cells such as NK cells, CD8+ T cells, and ILCs. If Pak2 regulates IL-17 expression in these cells by a similar mechanism remains unknown, which requires further detailed studies, including cell-type-specific knockout mice (e.g., NK cell or ILC-specific Pak2−/− mice) and additional adoptive transfer experiments.

The upstream mechanisms that activate the Pak2-Itch pathway to trigger RORγt degradation in Th17 cells remain un clear. Pak2 is shown to be activated by Cdc42/Rac downstream of Vav following T cell receptor (TCR) stimulation (Ha et al., 2015; Taglieri et al., 2014). Therefore, it is possible that in Th17 cells, Pak2 acts as a feedback mechanism and inhibits IL-17 expression by phosphorylation of RORγt. It is also possible that IL-17R signaling activates Pak2 in Th17 cells to impede the pathogenicity of Th17 cells. Such a phenomenon has been reported recently via the induction of IL-24 (Chong et al., 2020). Additionally, Pak2 is involved in TGF-β signaling (Sato et al., 2013; Wilkes and Leof, 2006; Wilkes et al., 2003, 2005, 2009; Yan et al., 2012), which regulates Th17 development (Korn et al., 2009; Patel and Kuchroo, 2015). Therefore, it is also possible that Pak2 activated via the transforming growth factor β (TGF-β) pathway may trigger RORγt phosphorylation. Further, a detailed investigation of these pathways is essential to fully understand how RORγt protein turnover is regulated during an inflammatory response to prevent chronic inflammation.

The current strategies of targeting IL-17 using antagonistic antibodies (against IL-17 or its receptor) have shown some success in treating psoriasis and ankylosing spondylitis (Beringer et al., 2016); however, clinical trials using this approach led to mixed results in rheumatoid arthritis (RA) (Fragoulis et al., 2016; Genovese et al., 2013; Hueber et al., 2010) and failure in IBDs (Fitzpatrick, 2013; Hueber et al., 2012; Targan et al., 2016). Several recent studies have shown that RORγt function and protein stability are modified by posttranslational mechanisms, including ubiquitination, acetylation, and SUMOylation (Kumar et al., 2021b). A defect in RORγt protein degradation has been reported in tumor-infiltrated Th17 cells of patients with colorectal cancer (Sun et al., 2021). CRNDE-h protein was shown to form a complex with the PPLY region of RORγt, and binding of CRNDE-h to RORγt in patients’ Th17 cells prevented ubiquitination and proteasomal degradation of RORγt by impeding its association with the Itch protein (Sun et al., 2021). On the contrary, our results show that Pak2 facilitates the binding of Itch to RORγt and promotes RORγt degradation. A clear understanding of the mechanism that regulates RORγt degradation could lead to alternative avenues to promote RORγt degradation at the site of inflammation. Such a strategy could transiently inhibit IL-17, resulting in therapeutic inhibition of Th17-mediated inflammation without deleterious effects observed in antibody-mediated systemic blocking of IL-17.

Limitations of the study

Although our study comprehensively shows that phosphorylation promotes ubiquitination of RORγt by enhancing its interaction with Itch, additional biophysical and cryoelectron microscopy evidence is necessary for the phosphorylation-dependent conformational changes in RORγt. In our in vivo experiments, we have lentivirally expressed RORγt-ΔS316A in CD4+T cells; studies using RORγt-ΔS316A point mutation knockin mice will be necessary to determine the broader physiological relevance of our findings. Finally, in this study, we utilized only the Th17-adoptive transfer colitis model to demonstrate that phosphorylation of RORγt regulates Th17 cell-induced colon inflammation. If the Pak2-RORγt-Itch pathway operates in other Th17-dependent disease models needs to be tested.

STAR★METHODS

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact: K Venuprasad (venuprasad.poojary@utsouthwestern.edu).

Materials availability

This work did not generate any unique reagents.

Data and code availability

This paper does not report the original code. All software utilized is freely or commercially available and is listed in the key resources table. All data reported in this paper will be shared by the lead contact upon request.

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Anti-c-Myc (9E10)	Santa Cruz Biotechnology	Cat: sc-40; RRID:AB_2857941
Anti-Pak2	Santa Cruz Biotechnology	Cat: sc-133113; RRID:AB_1568809
Anti-Flag (M2)	Sigma Aldrich	Cat: F1804; RRID:AB_262044
Anti-RORγt (FKJS-9)	eBioscience	Cat:14698882; RRID:AB_1834475
Anti-Itch (32)	BD Bioscience	Cat: 611198; RRID:AB_398732
Anti-RORγt (O28-835)	BD Bioscience	Cat: 562197; RRID:AB_10894594
Anti-GST (B14)	Santa Cruz Biotechnology	Cat: sc-138; RRID:AB_627677
Anti-Ahr (B-11)	Santa Cruz Biotechnology	Cat: sc-74571; RRID:AB_2223948
Anti-β-actin(C4)	Santa Cruz Biotechnology	Cat: sc-47778; RRID:AB_626632
Anti-mouse-HRP	Amersham Bioscience	Cat: NA931; RRID:AB_772210
Anti-rabbit-HRP	Cell Signaling Technology	Cat: 7074; RRID:AB_2099233
Clean-Blot™ IP (HRP)	Themo Fisher Scientific	Cat: 21230; RRID:AB_2864363
Veriblot IP (HRP)	Abcam	Cat: 131366; RRID:AB_2892718
Anti-CD45-APC (104)	eBioscience	Cat:17045482; RRID:AB_469400
Anti-IL17-PerCP-Cy5.5 (TC11)	Biolegend	Cat:506920; RRID:AB_961384
Anti-CD4-FITC (GK1.5)	Biolegend	Cat:100406; RRID:AB_312691
Anti-IFN-γ-APC	BD Bioscience	Cat:554413;RRID:AB_398551
FC-block (2.4G2)	BD Bioscience	Cat:553142; RRID:AB_394657
Anti-mouse CD3ε	Biolegend	Cat:100223; RRID:AB_1877072
Anti-mouse IFN-γ	BioXCell	Cat:BE0054; RRID:AB_1107692
Anti-mouse CD28	BioXCell	Cat:BE0015; RRID:AB_1107624
Chemicals, peptides, and recombinant proteins
Murine IL-2	Peprotech	Cat: 212–12
Murine IL-6	Peprotech	Cat: 216–16B
Murine TGF-β1	Peprotech	Cat: 100–21
Pak2 shRNA(m) lentiviral particle	Santa Cruz Biotechnology	Cat: sc-36184-V
Live Dead Aqua	Invitrogen	Cat: L34957
Cytofix/Cytoperm	BD Bioscience	Cat: 55028
Perm/Wash Buffer	BD Bioscience	Cat: 554723
Lamina Propria Dissociation Kit (mouse)	Miltenyi Biotec	Cat:130097410
Naive CD4+ T Cell Isolation Kit (mouse)	Miltenyi Biotec	Cat:130104453
CD4+ T Cell Isolation Kit (mouse)	Miltenyi Biotec	Cat:130104454
Verso cDNA Synthesis Kit	Themo Fisher Scientific	Cat: AB1453/B
SYBR GREEN I Master Mix (2X)	Roche Diagnostic	Cat: 507203180
OneTaq 2X Master Mix	NEB	Cat: M0482L
Q5® Site-Directed Mutagenesis Kit	NEB	Cat: E0554S
QIAGEN Plasmid Plus Maxi Kit	Qiagen	Cat: 12963
QIAquick Gel Extraction Kit	Qiagen	Cat: 28706
RNeasy Micro Kit	Qiagen	Cat: 74004
RNeasy Mini Kit	Qiagen	Cat: 74104
Luciferase Assay System	Promega	Cat: E1500
LS Columns MACS Separation Columns	Miltenyi Biotec	Cat:130042401
cOmplete™, Protease Inhibitor Cocktail	Roche	Cat:4693159001
Phorbol 12-myristate 13-acetate (PMA)	Sigma Aldrich	Cat: P8139
Phorbol 12-myristate 13-acetate (PMA)	Sigma Aldrich	Cat: P8139
Ionomycin calcium salt	Sigma Aldrich	Cat: I0634
Restore™ Western Blot Stripping Buffer	Thermo Fisher Scientific	Cat: 21063
ECL Prime Detection Reagent	GE Healthcare	Cat: RPN2236
Lipofectamine™ 2000	Themo Fisher Scientific	Cat:11668019
RPMI 1640 Medium	Themo Fisher Scientific	Cat:11875093
HBSS	Themo Fisher Scientific	Cat:14175103
Golgi Stop	BD Bioscience	Cat: 554724
Golgi Plug	BD Bioscience	Cat: 555029
RNase-Free DNase Set	Qiagen	Cat: 79254
RBC Lysis Buffer (10X)	Biolegend	Cat: 420301
SYBR Safe DNA Gel Stain	Invitrogen	Cat: S33102
Protein A/G PLUS-Agarose	Santa Cruz Biotechnology	Cat: sc2003
Experimental models: Cell lines
HEK293T	ATCC	Cat: CRL-3216
Experimental models: Organisms/strains
Mouse: C57BL/6	Jackson Laboratory	Cat:000664; RRID:IMSR_JAX:000664
Mouse: Rag1−/−	Jackson Laboratory	Cat:002216; RRID:IMSR_JAX:002216
Mouse: Pak2f/f	Dr. Jonathan Chernoff
Mouse: Rorctm1Litt/J	Jackson Laboratory	Cat:007571; RRID:IMSR_JAX:007571
Mouse: CD4cre	Jackson Laboratory	Cat: 022071; RRID:IMSR_JAX:022071
Recombinant DNA
Plasmid:MIGR-RORγt	Addgene	Cat: 24069
Software and algorithms
FlowJo v10.5.3	BD Bioscience	https://www.flowjo.com
GraphPad Prism v7	GraphPad	https://www.graphpad.com
Biorender	Biorender	https://biorender.com/
PyMol	Schrodinger	https://pymol.org/2/

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Mice

C57BL/6 mice, Rag1−/− mice, Rorc mice, and CD4 mice were purchased from the Jackson Laboratory. Pak2 mice were described before (Kosoff et al., 2013). Equal number of male and female mice were used. All mice were housed in micro isolator cages in the barrier facility of the University of Texas Southwestern Medical Center. All experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of the University of Texas Southwestern Medical Center.

Th17 cell-induced colitis

Naïve CD4+ T cells were isolated from spleen and lymph nodes from wild-type, Pak2CD4 mice or Rorc−/− mice using CD4+ T cell isolation kit. Cells were then stained with antibodies against APC-CD25 and FITC-CD4 and were sorted for CD4+CD25− populations (Kumar et al., 2021a). CD4+CD25− cells from Rorc−/− mice (male and female, 7–8 week old) were lentivirally transduced with RORγt and RORγt phospho mutants (ΔS316A and ΔS316D) for 24 h. Transduced cells were then cultured under Th17 inducing conditions for 5 days. Rag1−/− mice (male and female, 7–8 week old) were injected intra-peritoneally with 5 × 105 Th17 cells and monitored for disease severity up to 8 weeks.

METHOD DETAILS

Plasmid construction and cell transfection

Myc-Itch, HA-Ub (wild-type), and Flag-mRORγt have been described (Kathania et al., 2016). Transient transfection of 293T cells was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Myc-Pak2 and Myc-Pak2-ΔK278R were created and cloned into pCDNA3.1 between NotI and BamHI sites. The sequences of all clones were verified.

Lentiviral transduction

Expression clones were created by Gateway cloning technology according to the manufacturer’s instructions (Invitrogen). Lentiviral expression clones of mouse mRORγt-ΔS316A, mRORγt-ΔS316D, Pak2, and Pak2-ΔK278R were constructed by first subcloning into pENTR-3C entry vector (Invitrogen), and then subcloned to pLenti6.2/N-Lumio/V5-DEST (Invitrogen) via Gateway cloning. The ViraPower lentiviral expression system (Invitrogen) was used for packaging and producing lentiviruses in 293FT cells. The supernatant was harvested after 72 h. CD4+ T cells were transduced using lentiviruses in the presence of 5 μg/mL polybrene (Santa Cruz), and the media was changed after 12 h.

Protein identification by liquid chromatography (LC)-tandem MS

The protein samples were processed and analyzed at the Mass Spectrometry Facility of the Department of Pathology at the University of Michigan. Gel slices (10 slices/lane) were destained with 30% methanol for 4 h. Upon reduction (10 mM DTT) and alklylation (65 mM 2-Chloroacetamide) of the cysteines, proteins were digested overnight with sequencing grade, modified trypsin (Promega). The resulting peptides were resolved on a nano-capillary reverse phase column (Acclaim PepMap C18, 2 micron, 25 cm, ThermoScientific) using a 1% acetic acid/acetonitrile gradient at 300 nL/min and directly introduced in to Orbitrap Fusion tribrid mass spectrometer (Thermo Scientific, San Jose CA). MS1 scans were acquired at 120K resolution. Data-dependent high-energy C-trap dissociation MS/MS spectra were acquired with top speed option (3 sec) following each MS1 scan (relative CE ~32%). Proteins were identified by searching the data against the Mus musculus database (24861 entries) using Proteome Discoverer (v1.4, Thermo Scientific). Search parameters included MS1 mass tolerance of 10 ppm and fragment tolerance of 0.2 Da; two missed cleavages were allowed; carbamidimethylation of cysteine was considered fixed modification and oxidation of methionine were considered a potential modification. Percolator algorithm was used for discriminating between correct and incorrect spectrum identification. The false discovery rate (FDR) was calculated at the peptide level and peptides with <1% FDR were retained. Immunoprecipitated proteins were separated by SDS-PAGE. In-gel digestion with trypsin, followed by protein identification using LC-tandem MS, was performed as described elsewhere.43 Briefly, tryptic peptides were resolved on a nano-LC column (Magic AQ C18; Michrom Bioresources, Auburn, CA) and introduced into an Orbitrap mass spectrometer (Thermo Scientific, Waltham, MA). The Orbitrap was set to collect a high-resolution MS1 (FWHM 30,000@400 m/z), followed by the data-dependent collision-induced dissociation spectra on the “top 9” ions in the linear ion trap. Spectra were searched against a human protein database (UniProt release 2011_05) using the X!Tandem/TPP software suite.44 Proteins identified with a Protein Prophet probability Z0.9 false discovery rate o2% were considered for further analysis.

Real-time PCR analysis

Total RNA was prepared using RNeasy Mini Kit (Qiagen), followed by cDNA synthesis using Verso cDNA kit (Thermo Scientific). Quantitative real-time PCR was performed on a Mastercycler Realplex2 (Eppendorf). Light Cycler 480 SYBR Green I master reaction mix (Roche) was used in a 20 μL reaction volume. The expression of individual genes was normalized to the expression of actin. Cycling conditions were: 95°C for 2 min, followed by 50 cycles of 95°C for 15 sec, 55°C for 15 sec, and 72°C for 20 sec.

Th17 cell differentiation

For in vitro experiments, naïve CD4+ T cells from the spleen and lymph nodes were differentiated into Th17 cells in plates coated with anti-CD3 (1 μg/mL) and anti-CD28 (2 μg/mL). The cells were cultured in the presence of TGF-β (5 ng/mL), IL-6 (20 ng/mL), anti-IL-4 (5 μg/mL) and anti-IFN-γ (5 μg/mL) antibodies for 5 days.

Flow cytometry

Lamina propria lymphocytes were isolated using the Lamina Propria Dissociation Kit (Miltenyi Biotec) following the manufacturer’s instructions. Cells were washed with 1X PBS and then incubated with Fc block (553142, BD Bioscience). Cells were then stained with Live-Dead aqua (L34957, Invitrogen) for live cells and further stained with combinations of antibodies. Antibodies used were Lin-PB (79724, BioLegend), CD45.2-APC (17-0454-81, eBioscience), CD4-FITC (100406, BioLegend), IL-17-PerCP-Cy5.5 (506920, BioLegend), IFN-γ-APC (1554413, BD Bioscience). Data were acquired with a FACSCanto II (BD) and analyzed with FlowJo software (Tree Star).

Immunoprecipitation and immunoblot analysis

Cell lysates were prepared from 293T cells transfected with Myc-Pak2 and Flag-RORγt plasmids after 36 h by homogenizing in NP-40 lysis buffer. Protein estimations were done using the Pierce BCA protein assay kit according to the manufacturer’s protocol. Whole-cell lysates were precleared with 20 μL of Protein A/G plus agarose beads (Santa Cruz) for 1 h at 4°C. Lysates were then incubated with 1 μg of the desired antibody overnight at 4°C followed by a further 1 h of incubation at 4°C with 25 μL of Protein A/G beads. The immunocomplexes were washed five times with lysis buffer and denatured using 4X Laemmli buffer. Further, they were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Blots were visualized using Amersham ECL plus immunoblot analysis detection system (GE, Chicago, IL) on a Biorad ChemiDoc. For reprobing, membranes were stripped by incubation in a stripping buffer (62.5 mM Tris-HCl, pH 6.7; 100 mM 2-mercaptoethanol and 2% SDS) at 55°C for 45 min and washed thoroughly before reprobing.

In vitro kinase assays

Pak2 kinase activity was assessed using ADP-Glo Kinase Assay Kit (V9101, Promega), following the manufacturer’s instructions. Recombinant RORγt protein (1 μg) was incubated with 100 ng of recombinant Pak2 kinase. The kinase reaction with a final ATP concentration of 50 μM was incubated at room temperature for 2 h. The kinase reaction was then terminated by adding ADP-Glo reagent for 40 min, followed by the addition of kinase detection reagent for 30 min incubation before reading the luminescence on a multimode microplate reader.

Ubiquitination assay

293T cells were transfected with Flag-RORγt, Myc-Itch, and various other constructs as indicated. MG132 was added 6 h before cell lysis. Cells were washed three times with PBS and lysed in NP-40 lysis buffer. Immunoprecipitation was performed using anti-Flag antibody. RORγt-associated ubiquitin was analyzed by immunoblot using antibody against HA. An in vitro ubiquitination assay was performed using the E2-Ubiquitin Conjugation Kit (Abcam, Cambridge, MA) with GST-RORγt, GST-RORγt-ΔS316A or GST-RORγt-ΔS316D protein as substrate and Itch as the E3 ligase following the manufacturer’s protocol.

TUBE pull-down assay

The pull-down of ubiquitinated proteins was performed with GST-tagged TUBE, following the manufacturer’s instructions (Life Sensors). Briefly, for Pan–Ub, cells were collected, washed twice with PBS, and lysed with TUBE buffer supplemented with protease inhibitors (Pierce) and 5 mM NEM. Clarified lysates were incubated for 2 h on equilibrated glutathione Sepharose-GST-TUBE under rotation (Mukherjee et al., 2020). The resin was washed 3 times with TBS-T (200 mM Tris-HCl pH 8.0, 0.15 M NaCl, 0.1% Tween-20) and resolved by SDS-PAGE. RORγt-associated ubiquitin was analyzed by immunoblot using antibody against HA.

Protein expression and purification

RORγt and RORγt mutant (ΔS316A and ΔS316D) were sub-cloned into pGEX-6P1-DEST vector containing GST tag at N-terminal. Further, GST–RORγt and GST–RORγt mutant proteins were individually expressed in Rosetta™(DE3) pLysS E. coli. Cleared lysates were prepared and the soluble fusion proteins were purified on glutathione Sepharose 4B GST-tagged protein purification resin (GE Healthcare Life Sciences) and tested by immunoblot using monoclonal anti-GST (Cell Signaling Technology). GST-Itch protein was purified as shown previously (Paul et al., 2018).

Homology modeling

The protein sequences of mouse RORγt were retrieved from the UniProt database (P51450-2). Homologs to the target mouse RORγt LBD domain sequence were identified by PSI-blast for PDB databank on NCBI. The three-dimensional structure of mouse RORγt LBD domain was generated by SWISS-MODEL using the PDB: 5EJV. The substitution mutation of S316 to D316 was performed in PyMol. H-bond distances between the amino acids were measured in PyMol. Further, the analysis of the modeled structure of the LBD domain of mouse RORγt by AlphaFold (AF-P51450-F1) was done in PyMol.

Colonoscopic examination

To measure colitis severity, a high-resolution murine video endoscopic system was used. For monitoring colon inflammation in vivo, mice were anesthetized with Ketamine/Xylazine, and endoscopy was performed using a mini-endoscope from Karl-Storz. Grading of colitis scores was performed according to the mouse endoscopic index of colitis severity (MEICS). In brief, MEICS was determined by perianal findings, wall transparency, intestinal bleeding, and focal lesion. Each of these four different parameters of inflammation was given a score from 0 to 3, resulting in a total MEICS ranging from 0 to 12 (Kodani et al., 2013).

QUANTIFICATION AND STATISTICAL ANALYSIS

Data are represented as mean ± SD. Differences in group survival were analyzed by the Kaplan-Meier test using Prism 8 (GraphPad Software). Statistical significance was determined by unpaired Student’s t-test and one-way ANOVA; p< 0.05 was considered statistically significant.