Zuojia Chen

FOXP3 and RORγt: Transcriptional regulation of Treg and Th17

FOXP3(+)CD4(+)CD25(+) Regulatory T (Treg) cells and IL-17 producing helper T cells (Th17) are critical subsets of T cells which play essential roles in immune homeostasis. The Forkhead family transcription factor FOXP3 is predominantly expressed in Treg cells, where the FOXP3 ensemble is essential for Treg cell development and function. As FOXP3 is to Treg cells, the orphan retinoic acid nuclear receptor (ROR) family transcription factor RORγt is essential for Th17 development and function. In this review, we summarize recent progress of our understanding towards the molecular mechanisms underlying the differentiation and function of FOXP3(+) Treg cells and RORγt expressing Th17 cells. These may provide new insights into therapeutic intervention and targeting of human immune-deficient diseases.

PIM1 kinase phosphorylates the human transcription factor FOXP3 at serine 422 to negatively regulate its activity under inflammation.

Background: FOXP3 is a key transcription factor for the development and function of Tregs. Results: PIM1-mediated phosphorylation of FOXP3 at serine 422 decreased its DNA binding activity. Conclusion: PIM1 negatively regulates FOXP3-mediated transcriptional regulation and the suppressive activity of Tregs. Significance: PIM1 is a newly identified negative regulator of the immunosuppressive activity of Tregs. Previous reports have suggested that human CD4+ CD25hiFOXP3+ T regulatory cells (Tregs) have functional plasticity and may differentiate into effector T cells under inflammation. The molecular mechanisms underlying these findings remain unclear. Here we identified the residue serine 422 of human FOXP3 as a phosphorylation site that regulates its function, which is not present in murine Foxp3. PIM1 kinase, which is highly expressed in human Tregs, was found to be able to interact with and to phosphorylate human FOXP3 at serine 422. T cell receptor (TCR) signaling inhibits PIM1 induction, whereas IL-6 promotes PIM1 expression in in vitro expanded human Tregs. PIM1 negatively regulates FOXP3 chromatin binding activity by specifically phosphorylating FOXP3 at Ser422. Our data also suggest that phosphorylation of FOXP3 at the Ser418 site could prevent FOXP3 phosphorylation at Ser422 mediated by PIM1. Knockdown of PIM1 in in vitro expanded human Tregs promoted FOXP3-induced target gene expression, including CD25, CTLA4, and glucocorticoid-induced tumor necrosis factor receptor (GITR), or weakened FOXP3-suppressed IL-2 gene expression and enhanced the immunosuppressive activity of Tregs. Furthermore, PIM1-specific inhibitor boosted FOXP3 DNA binding activity in in vitro expanded primary Tregs and also enhanced their suppressive activity toward the proliferation of T effector cells. Taken together, our findings suggest that PIM1 could be a new potential therapeutic target in the prevention and treatment of human-specific autoimmune diseases because of its ability to modulate the immunosuppressive activity of human Tregs.

The E3 Deubiquitinase USP17 Is a Positive Regulator of Retinoic Acid-related Orphan Nuclear Receptor γt (RORγt) in Th17 Cells

Background: RORγt is the master transcription factor in Th17 cells. Results: USP17 stabilizes RORγt via deubiquitination, and USP17 levels are up-regulated in systemic lupus erythematosus. Conclusion: USP17 is a positive regulator of RORγt. Significance: USP17 could be a potential drug target to modulate RORγt-mediated autoimmune diseases such as systemic lupus erythematosus. Stable retinoic acid-related orphan nuclear receptor γt (RORγt) expression is pivotal for the development and function of Th17 cells. Here we demonstrate that expression of the transcription factor RORγt can be regulated through deubiquitination, which prevents proteasome-mediated degradation. We establish that USP17 stabilizes RORγt protein expression by reducing RORγt polyubiquitination at its Lys-360 residue. In contrast, knockdown of endogenous USP17 in Th17 cells resulted in decreased RORγt protein levels and down-regulation of Th17-related genes. Furthermore, USP17 expression was up-regulated in CD4+ T cells from systemic lupus erythematosus patients. Our data reveal a molecular mechanism in which RORγt expression in Th17 cells can be positively regulated by USP17, thereby modulating Th17 cell functions.

Cutting Edge: Ubiquitin-Specific Protease 4 Promotes Th17 Cell Function under Inflammation by Deubiquitinating and Stabilizing RORγt

RORγt is a key transcription factor that controls the development and function of inflammatory Th17. The mechanisms that regulate RORγt stability remain unclear. We report that Th17 cells highly express the deubiquitinase ubiquitin-specific protease (USP)4, which is essential for maintaining RORγt and Th17 cell function. Inhibition of the catalytic activity of USP4 with vialinin A, a compound derived from Chinese traditional medicine, dampened Th17 differentiation. USP4 interacted and deubiquitinated K48-linked polyubiquitination of RORγt, thereby promoting RORγt function and IL-17A transcription. Interestingly, TGF-β plus IL-6 enhanced USP4-mediated deubiquitination of RORγt. Moreover, USP4 and IL-17 mRNA, but not RORγt mRNA, were significantly elevated in CD4+ T cells from patients with rheumatic heart disease. Thus, USP4 could be a novel therapeutic target for the treatment of Th17-modulated autoimmune diseases.

Negative Regulation of Interferon-induced Transmembrane Protein 3 by SET7-mediated Lysine Monomethylation

Background: IFITM3 is a general antiviral host restriction factor against RNA viruses. Results: SET7-mediated monomethylation of IFITM3 at Lys-88 negatively affected its antiviral activity toward vesicular stomatitis virus (VSV) and influenza A virus (IAV) infection. Conclusion: The monomethylation of antiviral host restriction factors may perturb their function. Significance: Targeting the SET7 pathway could provide new antiviral therapeutic strategies. Although lysine methylation is classically known to regulate histone function, its role in modulating antiviral restriction factor activity remains uncharacterized. Interferon-induced transmembrane protein 3 (IFITM3) was found monomethylated on its lysine 88 residue (IFITM3-K88me1) to reduce its antiviral activity, mediated by the lysine methyltransferase SET7. Vesicular stomatitis virus and influenza A virus infection increased IFITM3-K88me1 levels by promoting the interaction between IFITM3 and SET7, suggesting that this pathway could be hijacked to support infection; conversely, IFN-α reduced IFITM3-K88me1 levels. These findings may have important implications in the design of therapeutics targeting protein methylation against infectious diseases.

Inflammation negatively regulates FOXP3 and regulatory T-cell function via DBC1.

Significance Treg cells suppress excessive and aberrant immune responses. Impaired function or homeostasis of Treg cells would induce severe autoimmune and inflammatory diseases. Forkhead box P3 (FOXP3), as a master regulator of Treg cells, forms a large complex with other binding factors to modulate Treg-cell function subtly in pathological and physiological conditions. We identified that Deleted in breast cancer 1 (DBC1) is an essential subunit of the FOXP3 complex in human CD4+ Treg cells. Our results show that the inflammatory cytokines TNF-α or IL-6 trigger FOXP3 degradation, whereas downregulation of DBC1 expression prevents FOXP3 degradation and maintains Treg-cell function under inflammatory stimuli in vitro and in vivo. These findings unveil a previously unidentified pathway for therapeutically modulating FOXP3+ Treg-cell stability under inflammation. Forkhead box P3 (FOXP3)-positive Treg cells are crucial for maintaining immune homeostasis. FOXP3 cooperates with its binding partners to elicit Treg cells’ signature and function, but the molecular mechanisms underlying the modulation of the FOXP3 complex remain unclear. Here we report that Deleted in breast cancer 1 (DBC1) is a key subunit of the FOXP3 complex. We found that DBC1 interacts physically with FOXP3, and depletion of DBC1 attenuates FOXP3 degradation in inflammatory conditions. Treg cells from Dbc1-deficient mice were more resistant to inflammation-mediated abrogation of Foxp3 expression and function and delayed the onset and severity of experimental autoimmune encephalomyelitis and colitis in mice. These findings establish a previously unidentified mechanism regulating FOXP3 stability during inflammation and reveal a pathway for potential therapeutic modulation and intervention in inflammatory diseases.

Reciprocal regulation of RORγt acetylation and function by p300 and HDAC1

T helper 17 (Th17) cells not only play critical roles in protecting against bacterial and fungal infections but are also involved in the pathogenesis of autoimmune diseases. The retinoic acid-related orphan receptor (RORγt) is a key transcription factor involved in Th17 cell differentiation through direct transcriptional activation of interleukin 17(A) (IL-17). How RORγt itself is regulated remains unclear. Here, we report that p300, which has histone acetyltransferase (HAT) activity, interacts with and acetylates RORγt at its K81 residue. Knockdown of p300 downregulates RORγt protein and RORγt-mediated gene expression in Th17 cells. In addition, p300 can promote RORγt-mediated transcriptional activation. Interestingly, the histone deacetylase (HDAC) HDAC1 can also interact with RORγt and reduce its acetylation level. In summary, our data reveal previously unappreciated posttranslational regulation of RORγt, uncovering the underlying mechanism by which the histone acetyltransferase p300 and the histone deacetylase HDAC1 reciprocally regulate the RORγt-mediated transcriptional activation of IL-17.

Deubiquitination and stabilization of T-bet by USP10.

The T-box transcriptional factor T-bet is crucial in the development, differentiation and function of Th1 cells. It drives Th1 immune response primarily through promoting expression of Th1 hallmark cytokine IFN-γ. Although T-bet was found associated with many immune-mediated diseases such as asthma and systemic sclerosis, little is known about the regulation of T-bet stability and function. Here we identified USP10, a carboxyl-terminal ubiquitin-processing protease, could interact with T-bet in the nucleus. Overexpression of USP10 directly inhibited T-bet ubiquitination and increased the expression of T-bet. We further confirmed Quercetin, a reported inhibitor of T-bet, could target USP10. Quercetin treatment downregulated USP10 and promoted T-bet degradation in a proteasome dependent way. Moreover, we found USP10 expression was upregulated in asthmatic patient PBMC, suggesting USP10 may maintain high level of T-bet and IFN-γ to fight against Th2-dominated inflammation.

Poly(ADP-ribosyl)ation of FOXP3 Protein Mediated by PARP-1 Protein Regulates the Function of Regulatory T Cells

Background: PARP-1 has importance in the immune system. Results: Poly(ADP-ribosyl)ation of FOXP3 mediated by PARP-1 destabilizes FOXP3 and negatively regulates the suppressive activity of Treg cells. Conclusion: PARP-1 negatively regulates Treg function via FOXP3 poly(ADP-ribosyl)ation. Significance: This study helps the development of PARP-1 inhibitors to prevent autoimmune diseases. Poly(ADP-ribose) polymerase 1 (PARP-1) is an ADP-ribosylating enzyme participating in diverse cellular functions. The roles of PARP-1 in the immune system, however, have not been well understood. Here we find that PARP-1 interacts with FOXP3 and induces its poly(ADP-ribosyl)ation. By using PARP-1 inhibitors, we show that reduced poly(ADP-ribosyl)ation of FOXP3 results in not only FOXP3 stabilization and increased FOXP3 downstream genes but also enhanced suppressive function of regulatory T cells. Our results suggest that PARP-1 negatively regulates the suppressive function of Treg cells at the posttranslational level via FOXP3 poly(ADP-ribosyl)ation. This finding has implications for developing PARP-1 inhibitors as potential agents for the prevention and treatment of autoimmune diseases.

USP22 is a positive regulator of NFATc2 on promoting IL2 expression

Nuclear factor of activated T cells (NFAT) is an important regulator of T cell activation. However, the molecular mechanism whereby NFATc2 regulates IL2 transcription is not fully understood. In this study, we showed that ubiquitin‐specific protease 22 (USP22), known as a cancer stem cell marker, specifically interacted with and deubiquitinated NFATc2. USP22 stabilized NFATc2 protein levels, which required its deubiquitinase activity. Consistent with these observations, depletion of USP22 in T cells reduced the expression of IL2, which is a cytokine that signifies T effector cell activation. Our findings thus unveil a previously uncharacterized positive regulator of NFATc2, suggesting that targeting the deubiquitinase activity of USP22 could have therapeutic benefit to control IL2 expression and T cell function.