Exercise improves the outcome of anticancer treatment with ultrasound-hyperthermia-enhanced nanochemotherapy and autophagy inhibitor.

It has been shown that exercise has a direct impact on tumor growth along with functional improvement. Previous studies have shown that exercise decreases the risk of cancer recurrence across various types of cancer. It was indicated that exercise stimulates the immune system to fight cancer. Previous study demonstrated that pulsed-wave ultrasound hyperthermia (pUH) combined with PEGylated liposomal doxorubicin (PLD) and chloroquine (CQ) inhibits 4T1 tumors growth and delays their recurrence. In this study, we investigated if the combinatorial treatment with high-intensity interval training (HIIT) combined with pUH-enhanced PLD delivery and CQ improved the outcome. The mouse experiment composed of three groups, HIIT+PLD+pUH+CQ group, PLD+pUH+CQ group, and the control group. HIIT+PLD+pUH+CQ group received 6 weeks of HIIT (15 min per day, 5 days per week) before 4T1 tumor implantation. Seven days later, they received therapy with PLD (10 mg/kg) + pUH (3 MHz, 50% duty cycle, 0.65 W/cm2, 15min) + CQ (50 mg/kg daily). Results showed that HIIT+PLD+pUH+CQ significantly reduced the tumor volumes and brought about longer survival of tumor-bearing mice than PLD+pUH+CQ did. Blood cell components were analyzed and showed that neutrophil and reticulocytes decreased while lymphocytes increased after exercise.

Adenylate Kinase 4 Promotes Inflammatory Gene Expression via Hif1α and AMPK in Macrophages.

Macrophages comprise the front line of defense against various pathogens. Classically activated macrophages (M1), induced by IFN-γ and LPS, highly express inflammatory cytokines and contribute to inflammatory processes. By contrast, alternatively activated macrophages (M2) are induced by IL-4 and IL-13, produce IL-10, and display anti-inflammatory activity. Adenylate kinase 4 (Ak4), an enzyme that transfers phosphate group among ATP/GTP, AMP, and ADP, is a key modulator of ATP and maintains the homeostasis of cellular nucleotides which is essential for cell functions. However, its role in regulating the function of macrophages is not fully understood. Here we report that Ak4 expression is induced in M1 but not M2 macrophages. Suppressing the expression of Ak4 in M1 macrophages with shRNA or siRNA enhances ATP production and decreases ROS production, bactericidal ability and glycolysis in M1 cells. Moreover, Ak4 regulates the expression of inflammation genes, including Il1b, Il6, Tnfa, Nos2, Nox2, and Hif1a, in M1 macrophages. We further demonstrate that Ak4 inhibits the activation of AMPK and forms a positive feedback loop with Hif1α to promote the expression of inflammation-related genes in M1 cells. Furthermore, RNA-seq analysis demonstrates that Ak4 also regulates other biological processes in addition to the expression of inflammation-related genes in M1 cells. Interestingly, Ak4 does not regulate M1/M2 polarization. Taken together, our study uncovers a potential mechanism linking energy consumption and inflammation in macrophages.

Combination Therapy of Pulsed-Wave Ultrasound Hyperthermia and Immunostimulant OK-432 Enhances Systemic Antitumor Immunity for Cancer Treatment.

PURPOSE: In this study, we hypothesized that systemic antitumor immunity might be enhanced by combining pulsed-wave ultrasound hyperthermia (pUSHT) with OK-432 and that the induced antitumor immunity could confer protection against tumorigenesis. These hypotheses were tested in bilateral and rechallenged tumor models. METHODS AND MATERIALS: Bilateral and rechallenged tumor models were applied in the studies. In the bilateral tumor model, BALB/c mice were inoculated in both flanks with CT26-luc tumor cells. The tumors in the right flank were treated with 4 courses of pUSHT with or without OK-432. In the rechallenged tumor model, tumor cells were implanted into the right flank. Once formed, the tumors were treated with pUSHT with OK-432, followed by surgical resection. New tumor cells were then implanted into the contralateral flank. The antitumor response was evaluated via infiltrated immune cells and the severity of necrosis/apoptosis in tumors. RESULTS: In the bilateral tumor model, the tumor growth rate and growth activity of both treated (100% reduction) and untreated tumors (90.5% reduction) were significantly inhibited with the combination treatment compared with the sham control group, and the systemic antitumor effect was prolonged. The survival rate was significantly enhanced (sham control, 8 days; OK plus pUSHT, >20 days). IFNγ+ CD4 (treated tumor, 8.6-fold; untreated tumor, 4-fold), IFNγ+ CD8 (treated tumor, 6.7-fold; untreated tumor, 2.6-fold), and T cell and NK cell (treated tumor, 4-fold; untreated tumor, 2.5-fold) infiltration was increased in the combination group compared with the control group. In the rechallenged tumor model, new tumors failed to form with the combination treatment. CONCLUSION: This experimental study combining pUSHT and OK-432 explored a new therapeutic strategy for controlling colon cancer metastasis. The results show that the combination treatment may produce an effective antitumor immune response.

Pulsed-wave Ultrasound Hyperthermia Enhanced Nanodrug Delivery Combined with Chloroquine Exerts Effective Antitumor Response and Postpones Recurrence.

Autophagy is found to serve as a surviving mechanism for cancer cells. Inhibiting autophagy has been considered as an adjuvant anti-cancer strategy. In this study, we investigated the anti-tumor effect of combining pulsed-wave ultrasound hyperthermia (pUH) enhanced PEGylated liposomal doxorubicin (PLD) delivery with an autophagy inhibitor chloroquine (CQ). BALB/c mice bearing subcutaneous 4T1 tumor received intravenous injection of PLD (10 mg/kg) plus 15-minute on-tumor pUH on Day 5 after tumor implantation and were then fed with CQ (50 mg/kg daily) thereafter. Prolonged suppression of tumor growth was attained with PLD + pUH + CQ treatment, whereas in PLD + pUH group tumors quickly recurred after an initial inhibition. Treatment with CQ monotherapy had no benefit compared to the control group. Immunohistochemical staining and Western blotting showed that autophagy of cancer cells was blocked for the mice receiving CQ. It indicates that PLD + pUH + CQ is a promising strategy to treat cancer for a long-term inhibition.

Development of a PD-L1-Expressing Orthotopic Liver Cancer Model: Implications for Immunotherapy for Hepatocellular Carcinoma.

BACKGROUND: Anti-programmed cell death-1(anti-PD1) treatment has shown promising antitumor efficacy in patients with advanced hepatocellular carcinoma (HCC). This study sought to explore the functional significance of programmed death ligand-1 (PD-L1) expression in tumor cells in the tumor microenvironment. METHODS: The mouse liver cancer cell line BNL-MEA was transfected with PD-L1 plasmids and stable clones expressing PD-L1 were selected. An orthotopic HCC model was generated by implanting the cells into the subcapsular space of BALB/c mice. Cell growth features were measured by proliferation assay, colony formation, flow cytometry (in vitro), ultrasonography, and animal survival (in vivo). The changes in T-cell function were examined by cytokine assay, expression of T-cell related genes, and flow cytometry. The efficacy of anti-PD1 therapy was compared between the parental and PD-L1-expressing tumors. RESULTS: PD-L1 expression did not affect growth characteristics of BNL-MEA cells but downregulated the expression of genes related to T-cell activation in the tumor microenvironment. Co-culture of PD-L1-expressing BNL-MEA cells with CD8+ T cells reduced T-cell proliferation and expression of cytokines IFNγ and TNFα. Tumors with PD-L1 expression showed better response to anti-PD1 therapy and depletion of CD8+ T cells abolished the antitumor effect. The difference in treatment response between parental and PD-L1-expressing tumors disappeared when a combination of anti-PD1 and sorafenib was given. CONCLUSIONS: PD-L1 expression in HCC cells may inhibit T-cell function in the liver tumor microenvironment. Anti-PD1 therapy appeared more effective in PD-L1-expressing than nonexpressing tumors, but the difference was diminished by the addition of sorafenib.

SUMO-defective c-Maf preferentially transactivates Il21 to exacerbate autoimmune diabetes.

SUMOylation is involved in the development of several inflammatory diseases, but the physiological significance of SUMO-modulated c-Maf in autoimmune diabetes is not completely understood. Here, we report that an age-dependent attenuation of c-Maf SUMOylation in CD4+ T cells is positively correlated with the IL-21-mediated diabetogenesis in NOD mice. Using 2 strains of T cell-specific transgenic NOD mice overexpressing wild-type c-Maf (Tg-WTc) or SUMOylation site-mutated c-Maf (Tg-KRc), we demonstrated that Tg-KRc mice developed diabetes more rapidly than Tg-WTc mice in a CD4+ T cell-autonomous manner. Moreover, SUMO-defective c-Maf preferentially transactivated Il21 to promote the development of CD4+ T cells with an extrafollicular helper T cell phenotype and expand the numbers of granzyme B-producing effector/memory CD8+ T cells. Furthermore, SUMO-defective c-Maf selectively inhibited recruitment of Daxx/HDAC2 to the Il21 promoter and enhanced histone acetylation mediated by CREB-binding protein (CBP) and p300. Using pharmacological interference with CBP/p300, we illustrated that CBP30 treatment ameliorated c-Maf-mediated/IL-21-based diabetogenesis. Taken together, our results show that the SUMOylation status of c-Maf has a stronger regulatory effect on IL-21 than the level of c-Maf expression, through an epigenetic mechanism. These findings provide new insights into how SUMOylation modulates the pathogenesis of autoimmune diabetes in a T cell-restricted manner and on the basis of a single transcription factor.

Exosomal TGF-ß1 is correlated with lymphatic metastasis of gastric cancers.

Exosomes are nano-vesicles transporting bioactive material between cells. This study explored the prognostic association of exosomal TGF-ß1 with lymph node (LN) metastasis of gastric cancer (GC). TGF-ß1 expressions in the exosomes isolated from the gastroepiploic veins of 61 GC patients analyzed by ELISA. The regulatory T (Treg) cells in celiac LNs of gastric cancer analyzed by immunohistochemistry. Exosomal TGF-ß1 expression and the ratio of Treg cells in draining LNs were both significantly associated with pathological stages and LN metastasis of gastric cancer. Besides, the exosomal TGF-ß1 expression and Treg proportion in LN were also significantly correlated in gastric cancer patients. Recombinant TGF-ß1 and exosomes isolated from GC patients were used to induce FOXP3+ Treg cells from naïve T cells in vitro. Compared to the control, recombinant TGF-ß1 induced more CD25 (41%), FOXP3 (19%) and CTLA-4 (47%), while reduced CD45RA expression by 38% in primary naïve T cell cultures (p<0.01). Exosomes treatment induced more CD25 and 45% higher CTLA-4 expression, and increased 29% higher of CD45RA-negative cells than recombinant TGF-ß1 did (p<0.01). Adding TGF-ß1 neutralizing antibody partially abrogated the effects of exosomes on Treg induction. Our study showed exosomal TGF-ß1 related to lymph node metastasis and the ratio of Treg cells in lymph nodes of gastric cancers. Exosomes from gastric cancer patients could induce Treg cells formation through the effect of TGF-ß1.

Effective gene expression in the rat dorsal root ganglia with a non-viral vector delivered via spinal nerve injection.

Delivering gene constructs into the dorsal root ganglia (DRG) is a powerful but challenging therapeutic strategy for sensory disorders affecting the DRG and their peripheral processes. The current delivery methods of direct intra-DRG injection and intrathecal injection have several disadvantages, including potential injury to DRG neurons and low transfection efficiency, respectively. This study aimed to develop a spinal nerve injection strategy to deliver polyethylenimine mixed with plasmid (PEI/DNA polyplexes) containing green fluorescent protein (GFP). Using this spinal nerve injection approach, PEI/DNA polyplexes were delivered to DRG neurons without nerve injury. Within one week of the delivery, GFP expression was detected in 82.8% ± 1.70% of DRG neurons, comparable to the levels obtained by intra-DRG injection (81.3% ± 5.1%, p = 0.82) but much higher than those obtained by intrathecal injection. The degree of GFP expression by neurofilament(+) and peripherin(+) DRG neurons was similar. The safety of this approach was documented by the absence of injury marker expression, including activation transcription factor 3 and ionized calcium binding adaptor molecule 1 for neurons and glia, respectively, as well as the absence of behavioral changes. These results demonstrated the efficacy and safety of delivering PEI/DNA polyplexes to DRG neurons via spinal nerve injection.

Tumour suppressor death-associated protein kinase targets cytoplasmic HIF-1α for Th17 suppression.

Death-associated protein kinase (DAPK) is a tumour suppressor. Here we show that DAPK also inhibits T helper 17 (Th17) and prevents Th17-mediated pathology in a mouse model of autoimmunity. We demonstrate that DAPK specifically downregulates hypoxia-inducible factor 1α (HIF-1α). In contrast to the predominant nuclear localization of HIF-1α in many cell types, HIF-1α is located in both the cytoplasm and nucleus in T cells, allowing for a cytosolic DAPK-HIF-1α interaction. DAPK also binds prolyl hydroxylase domain protein 2 (PHD2) and increases HIF-1α-PHD2 association. DAPK thereby promotes the proline hydroxylation and proteasome degradation of HIF-1α. Consequently, DAPK deficiency leads to excess HIF-1α accumulation, enhanced IL-17 expression and exacerbated experimental autoimmune encephalomyelitis. Additional knockout of HIF-1α restores the normal differentiation of Dapk(-/-) Th17 cells and prevents experimental autoimmune encephalomyelitis development. Our results reveal a mechanism involving DAPK-mediated degradation of cytoplasmic HIF-1α, and suggest that raising DAPK levels could be used for treatment of Th17-associated inflammatory diseases.

Reciprocal regulation of C-Maf tyrosine phosphorylation by Tec and Ptpn22.

C-Maf plays an important role in regulating cytokine production in TH cells. Its transactivation of IL-4 is optimized by phosphorylation at Tyr21, Tyr92, and Tyr131. However, the molecular mechanism regulating its tyrosine phosphorylation remains unknown. In this study, we demonstrate that Tec kinase family member Tec, but not Rlk or Itk, is a tyrosine kinase of c-Maf and that Tec enhances c-Maf-dependent IL-4 promoter activity. This effect of Tec is counteracted by Ptpn22, which physically interacts with and facilitates tyrosine dephosphorylation of c-Maf thereby attenuating its transcriptional activity. We further show that phosphorylation of Tyr21/92/131 of c-Maf is also critical for its recruitment to the IL-21 promoter and optimal production of this cytokine by TH17 cells. Thus, manipulating tyrosine phosphorylation of c-Maf through its kinases and phosphatases can have significant impact on TH cell-mediated immune responses.

Inability to resolve specific infection generates innate immunodeficiency syndrome in Xiap-/- mice.

Emerging evidence indicates that innate immunodeficiency syndromes are linked to mutations in innate receptors and to specific infections. X-linked lymphoproliferative syndrome type-2 (XLP-2) is associated with deficiency in X-linked inhibitor of apoptosis protein (XIAP), with poorly understood molecular mechanisms. Here we showed that XIAP deficiency selectively impaired B-cell chronic lymphocytic leukemia/lymphoma 10 (BCL10)-mediated innate responses to dectin-1 ligands but did not affect responses to various Toll-like receptor agonists. Consequently, Xiap(-/-) mice became highly vulnerable on Candida albicans infection. The compromised early innate responses led to the persistent presence of C albicans and inflammatory cytokines in Xiap(-/-) mice. Furthermore, priming of Xiap(-/-) mice with the dectin-1 ligand curdlan alone resulted in XLP-2-like syndromes. Restoration of dectin-1-induced Rac1 activation and phagocytosis by resolvin D1, but not up-regulation of nuclear factor-κB, rescued Xiap(-/-) mice from C albicans lethal infection. Therefore, development of XLP-2 in XIAP-deficient patients could be partly due to sustained inflammation as a consequence of defective BCL10-dependent innate immunity toward specific pathogens. Importantly, our results suggest the potential therapeutic value of resolvin D1 in the treatment of XLP-2 and innate immunodeficiency syndromes.

Dengue viral protease interaction with NF-κB inhibitor α/ß results in endothelial cell apoptosis and hemorrhage development.

Hemorrhagic manifestations occur frequently accompanying a wide range of dengue disease syndromes. Much work has focused on the contribution of immune factors to the pathogenesis of hemorrhage, but how dengue virus (DENV) participates in the pathogenic process has never been explored. Although there is no consensus that apoptosis is the basis of vascular permeability in human dengue infections, we showed in dengue hemorrhage mouse model that endothelial cell apoptosis is important to hemorrhage development in mice. To explore the molecular basis of the contribution of DENV to endothelial cell death, we show in this study that DENV protease interacts with cellular IκBα and IκBß and cleaves them. By inducing IκBα and IκBß cleavage and IκB kinase activation, DENV protease activates NF-κB, which results in endothelial cell death. Intradermal inoculation of DENV protease packaged in adenovirus-associated virus-9 induces endothelial cell death and dermal hemorrhage in mice. Although the H51 activity site is not involved in the interaction between DENV protease and IκB-α/ß, the enzymatic activity is critical to the ability of DENV protease to induce IκBα and IκBß cleavage and trigger hemorrhage development. Moreover, overexpression of IκBα or IκBß protects endothelial cells from DENV-induced apoptosis. In this study, we show that DENV protease participates in the pathogenesis of dengue hemorrhage and discover IκBα and IκBß to be the new cellular targets that are cleaved by DENV protease.

Ets-1 facilitates nuclear entry of NFAT proteins and their recruitment to the IL-2 promoter.

E26 transformation-specific sequence 1 (Ets-1), the prototype of the ETS family of transcription factors, is critical for the expression of IL-2 by murine Th cells; however, its mechanism of action is still unclear. Here we show that Ets-1 is also essential for optimal production of IL-2 by primary human Th cells. Although Ets-1 negatively regulates the expression of Blimp1, a known suppressor of IL-2 expression, ablation of B lymphocyte-induced maturation protein 1 (Blimp1) does not rescue the expression of IL-2 by Ets-1-deficient Th cells. Instead, Ets-1 physically and functionally interacts with the nuclear factor of activated T-cells (NFAT) and is required for the recruitment of NFAT to the IL-2 promoter. In addition, Ets-1 is located in both the nucleus and cytoplasm of resting Th cells. Nuclear Ets-1 quickly exits the nucleus in response to calcium-dependent signals and competes with NFAT proteins for binding to protein components of noncoding RNA repressor of NFAT complex (NRON), which serves as a cytoplasmic trap for phosphorylated NFAT proteins. This nuclear exit of Ets-1 precedes rapid nuclear entry of NFAT and Ets-1 deficiency results in impaired nuclear entry, but not dephosphorylation, of NFAT proteins. Thus, Ets-1 promotes the expression of IL-2 by modulating the activity of NFAT.

PTPN22 modulates macrophage polarization and susceptibility to dextran sulfate sodium-induced colitis.

PTPN22, a protein tyrosine phosphatase expressed mainly in hematopoietic cells, has been linked to many autoimmune diseases. A C-to-T single nucleotide polymorphism (SNP) at position 1858 of human PTPN22 cDNA decreases the risk of Crohn's disease. However, the function of PTPN22 and the mechanism by which this SNP reduces the risk of Crohn's disease are poorly understood. We find that PTPN22 is expressed in macrophages. It suppresses M1 macrophage polarization and reciprocally promotes the expression of M2-associated genes. PTPN22-deficient mice develop severe colitis induced by dextran sulfate sodium, and their intestinal macrophages express higher levels of M1 genes but lower levels of M2-associated genes. Furthermore, the protective T allele of the C1858T SNP is associated with attenuated expression of inflammatory cytokines and a higher level of PTPN22 in human M1 macrophages. This T allele-associated aberrant expression of PTPN22 is partly attributed to an autoinhibition mechanism, in which PTPN22 suppresses its own expression in M1 but not M2 macrophages. Our data not only demonstrate a critical role of PTPN22 in regulating macrophage polarization but also provide a molecular explanation for the protective effect of the C1858T SNP in Crohn's disease.

Galectin-3 modulates Th17 responses by regulating dendritic cell cytokines.

Galectin-3 is a ß-galactoside-binding animal lectin with diverse functions, including regulation of T helper (Th) 1 and Th2 responses. Current data indicate that galectin-3 expressed in dendritic cells (DCs) may be contributory. Th17 cells have emerged as critical inducers of tissue inflammation in autoimmune disease and important mediators of host defense against fungal pathogens, although little is known about galectin-3 involvement in Th17 development. We investigated the role of galectin-3 in the induction of Th17 immunity in galectin-3-deficient (gal3(-/-)) and gal3(+/+) mouse bone marrow-derived DCs. We demonstrate that intracellular galectin-3 negatively regulates Th17 polarization in response to the dectin-1 agonist curdlan (a ß-glucan present on the cell wall of fungal species) and lipopolysaccharide, agents that prime DCs for Th17 differentiation. On activation of dectin-1, gal3(-/-) DCs secreted higher levels of the Th17-axis cytokine IL-23 compared with gal3(+/+) DCs and contained higher levels of activated c-Rel, an NF-κB subunit that promotes IL-23 expression. Levels of active Raf-1, a kinase that participates in downstream inhibition of c-Rel binding to the IL23A promoter, were impaired in gal3(-/-) DCs. Modulation of Th17 by galectin-3 in DCs also occurred in vivo because adoptive transfer of gal3(-/-) DCs exposed to Candida albicans conferred higher Th17 responses and protection against fungal infection. We conclude that galectin-3 suppresses Th17 responses by regulating DC cytokine production.

Galectin-3 negatively regulates dendritic cell production of IL-23/IL-17-axis cytokines in infection by Histoplasma capsulatum.

Galectin-3 (gal3) is known for its immunoregulatory functions in infectious, autoimmune, and inflammatory diseases. However, little is known about its regulatory role in the host's IL-17A response to infection. Using a mouse model of histoplasmosis in which both Th1 and Th17 responses contribute to fungal clearance, we investigated how gal3 regulates IL-17A responses. Our study showed that Histoplasma infection induced gal3(-/-) dendritic cells to produce significantly higher levels of IL-23, TGF-ß1, and IL-1ß than did gal3(+/+) cells. Infected by the same inoculum of Histoplasma, gal3(-/-) mice had lower fungal burden and produced higher levels of IL-23/IL-17-axis cytokines and lower levels of IL-12 and IFN-γ. Additionally, there was an increase in Th17 cells and a reduction in Th1 cells in infected gal3(-/-) mice. In vitro Th1/Th17-skewing experiments excluded the intrinsic effect of gal3 on Th cell differentiation. Although neutrophils from both gal3(+/+) and gal3(-/-) mice produced IL-17A upon IL-23 stimulation, their contribution to IL-17A production was greater in gal3(-/-) mice than in gal3(+/+) mice. Compared with gal3(+/+) dendritic cells, adoptive transfer of gal3(-/-) dendritic cells resulted in production of significantly higher levels of IL-17-axis cytokines and reduced fungal burden. It appears that reduced fungal burden and preferential IL-17A response in gal3(-/-) mice by both Th17 cells and neutrophils were the result of preferential production of IL-23/IL-17-axis cytokines by dendritic cells. Our study showed that gal3 negatively regulates IL-17A responses through inhibition of IL-23/IL-17-axis cytokine production by dendritic cells.

14-3-3θ is a binding partner of rat Eag1 potassium channels.

The ether-à-go-go (Eag) potassium (K(+)) channel belongs to the superfamily of voltage-gated K(+) channel. In mammals, the expression of Eag channels is neuron-specific but their neurophysiological role remains obscure. We have applied the yeast two-hybrid screening system to identify rat Eag1 (rEag1)-interacting proteins from a rat brain cDNA library. One of the clones we identified was 14-3-3θ, which belongs to a family of small acidic protein abundantly expressed in the brain. Data from in vitro yeast two-hybrid and GST pull-down assays suggested that the direct association with 14-3-3θ was mediated by both the N- and the C-termini of rEag1. Co-precipitation of the two proteins was confirmed in both heterologous HEK293T cells and native hippocampal neurons. Electrophysiological studies showed that over-expression of 14-3-3θ led to a sizable suppression of rEag1 K(+) currents with no apparent alteration of the steady-state voltage dependence and gating kinetics. Furthermore, co-expression with 14-3-3θ failed to affect the total protein level, membrane trafficking, and single channel conductance of rEag1, implying that 14-3-3θ binding may render a fraction of the channel locked in a non-conducting state. Together these data suggest that 14-3-3θ is a binding partner of rEag1 and may modulate the functional expression of the K(+) channel in neurons.

Tyrosine phosphorylation of c-Maf enhances the expression of IL-4 gene.

Maf proteins are involved in a variety of biological processes, such as oncogenesis, lens development, and differentiation. In immune system, c-Maf transactivates IL-4 promoter, and ectopic expression of c-Maf skews primary T cell response toward the Th2 pathway. Numerous transcription factors are subjected to posttranslational modification. In this study, to our knowledge, we show for the first time that c-Maf is subjective to tyrosine phosphorylation in Th cells and that the level of its tyrosine phosphorylation positively correlates with IL-4 expression by peripheral Th cells, but is negatively associated with the severity of disease in NOD mice. c-Maf undergoes tyrosine phosphorylation at Tyr(21), Tyr(92), and Tyr(131) residues in Th2 cells. Furthermore, tyrosine phosphorylation at these three residues is critical for the recruitment of c-Maf to IL-4 promoter and IL-4 production in Th cells. Taken together, this study sheds new light on the role of posttranslational modification of c-Maf in IL-4 production and Th cell-mediated autoimmune diseases.

PTPN22.6, a dominant negative isoform of PTPN22 and potential biomarker of rheumatoid arthritis.

PTPN22 is a tyrosine phosphatase and functions as a damper of TCR signals. A C-to-T single nucleotide polymorphism (SNP) located at position 1858 of human PTPN22 cDNA and converting an arginine (R620) to tryptophan (W620) confers the highest risk of rheumatoid arthritis among non-HLA genetic variations that are known to be associated with this disease. The effect of the R-to-W conversion on the phosphatase activity of PTPN22 protein and the impact of the minor T allele of the C1858T SNP on the activation of T cells has remained controversial. In addition, how the overall activity of PTPN22 is regulated and how the R-to-W conversion contributes to rheumatoid arthritis is still poorly understood. Here we report the identification of an alternative splice form of human PTPN22, namely PTPN22.6. It lacks the nearly entire phosphatase domain and can function as a dominant negative isoform of the full length PTPN22. Although conversion of R620 to W620 in the context of PTPN22.1 attenuated T cell activation, expression of the tryptophan variant of PTPN22.6 reciprocally led to hyperactivation of human T cells. More importantly, the level of PTPN22.6 in peripheral blood correlates with disease activity of rheumatoid arthritis. Our data depict a model that can reconcile the conflicting observations on the functional impact of the C1858T SNP and also suggest that PTPN22.6 is a novel biomarker of rheumatoid arthritis.

SUMOylation attenuates c-Maf-dependent IL-4 expression.

The function of transcription factors can be critically regulated by SUMOylation. c-Maf, the cellular counterpart of v-maf oncogene, is a potent transactivator of the IL-4 gene in Th2 cells. We found in a yeast two-hybrid screen that c-Maf can interact with Ubc9 and PIAS1, two key enzymes of the SUMOylation pathway. In this study, we report that c-Maf co-localized with these two SUMO (small ubiquitin-like modifier) ligases in the nucleus and that c-Maf can be SUMOylated in vitro and also in primary Th2 cells. We also demonstrated that lysine-33 is the dominant, if not the only, SUMO acceptor site of c-Maf. SUMOylation of c-Maf attenuated its transcriptional activity. Reciprocally, a SUMOylation resistant c-Maf was more potent than WT-c-Maf in driving IL-4 production in c-Maf-deficient Th2 cells. Furthermore, we showed that ablation of the SUMO site did not alter the subcellular localization or the stability of c-Maf protein but instead enhanced its recruitment to the Il4-promoter. We conclude that SUMOylation at lysine-33 is a functionally critical post-translational modification event of c-Maf in Th cells.