The Curcumin Analog GO-Y030 Controls the Generation and Stability of Regulatory T Cells.

Regulatory T cells (Tregs) play a crucial role in preventing antitumor immune responses in cancer tissues. Cancer tissues produce large amounts of transforming growth factor beta (TGF-ß), which promotes the generation of Foxp3+ Tregs from naïve CD4+ T cells in the local tumor microenvironment. TGF-ß activates nuclear factor kappa B (NF-κB)/p300 and SMAD signaling, which increases the number of acetylated histones at the Foxp3 locus and induces Foxp3 gene expression. TGF-ß also helps stabilize Foxp3 expression. The curcumin analog and antitumor agent, GO-Y030, prevented the TGF-ß-induced generation of Tregs by preventing p300 from accelerating NF-κB-induced Foxp3 expression. Moreover, the addition of GO-Y030 resulted in a significant reduction in the number of acetylated histones at the Foxp3 promoter and at the conserved noncoding sequence 1 regions that are generated in response to TGF-ß. In vivo tumor models demonstrated that GO-Y030-treatment prevented tumor growth and reduced the Foxp3+ Tregs population in tumor-infiltrating lymphocytes. Therefore, GO-Y030 exerts a potent anticancer effect by controlling Treg generation and stability.

Fatty acid-binding protein 3 controls contact hypersensitivity through regulating skin dermal Vγ4+ γ/δ T cell in a murine model.

BACKGROUND: Fatty acid-binding protein 3 (FABP3) is a cytosolic carrier protein of polyunsaturated fatty acids (PUFAs) and regulates cellular metabolism. However, the physiological functions of FABP3 in immune cells and how FABP3 regulates inflammatory responses remain unclear. METHODS: Contact hypersensitivity (CHS) induced by 2,4-dinitrofluorobenzene (DNFB) and fluorescein isothiocyanate was applied to the skin wild-type and Fabp3-/- mice. Skin inflammation was assessed using FACS, histological, and qPCR analyses. The development of γ/δ T cells was evaluated by a co-culture system with OP9/Dll1 cells in the presence or absence of transgene of FABP3. RESULTS: Fabp3-deficient mice exhibit a more severe phenotype of contact hypersensitivity (CHS) accompanied by infiltration of IL-17-producing Vγ4+ γ/δ T cells that critically control skin inflammation. In Fabp3-/- mice, we found a larger proportion of Vγ4+ γ/δ T cells in the skin, even though the percentage of total γ/δ T cells did not change at steady state. Similarly, juvenile Fabp3-/- mice also contained a higher amount of Vγ4+ γ/δ T cells not only in the skin but in the thymus when compared with wild-type mice. Furthermore, thymic double-negative (DN) cells expressed FABP3, and FABP3 negatively regulates the development of Vγ4+ γ/δ T cells in the thymus. CONCLUSIONS: These findings suggest that FABP3 functions as a negative regulator of skin inflammation through limiting pathogenic Vγ4+ γ/δ T-cell generation in the thymus.

GITR controls intestinal inflammation by suppressing IL-15-dependent NK cell activity.

Glucocorticoid-induced TNFR family related gene (GITR) is a member of the TNFR superfamily that is expressed on cells of the immune system. Although the protective and pathogenic roles of GITR in T cell immunity are well characterized, the role of GITR in innate immunity in the intestinal tissues has not been well clarified. In this study, using a dextran sulfate sodium (DSS)-induced colitis model in mice, we found that GITR-deficiency rendered mice more susceptible to acute intestinal inflammation and that a significantly higher number of activated natural killer (NK) cells was accumulated in the colonic lamina propria of Gitr-/- mice as compared to wild-type mice. Additionally, Rag2-/- Gitr-/- mice, which lack T cells but have NK cells, also displayed more severe colonic inflammation than Rag2-/- mice. In contrast, an anti-GITR agonistic antibody significantly alleviated colitis in Rag2-/- mice. Engagement of GITR inhibited IL-15-mediated activating signaling events in NK cells, which include cell activation and proliferation, and production of cytokines and cytotoxic granules. Taken together, our results provide the first evidence that GITR negatively controls intestinal inflammation through NK cell functions.

Costello syndrome model mice with a HrasG12S/+ mutation are susceptible to develop house dust mite-induced atopic dermatitis.

Costello syndrome is an autosomal dominant disorder that is caused by germline HRAS mutations. Patients with Costello syndrome present craniofacial abnormalities, cardiac defects, and cancer predisposition, as well as skin abnormalities, including papillomas, keratosis pilaris, and eczematous dermatitis. However, the mechanisms underlying the dermatological abnormalities remain unclear. Here, we demonstrated that knock-in mice expressing an Hras G12S mutation (HrasG12S/+ mice) are susceptible to develop atopic dermatitis (AD)-like skin lesions, including eczema, pruritus, elevated serum IgE levels, acanthosis, and the infiltration of mast cells, basophils, and type-2 innate lymphoid cells in the dermis, after stimulation with house dust mite allergens (Dermatophagoides farinae, Dfb). Reduced skin barrier function, increased proliferation of phosphorylated ERK (p-ERK)-positive epidermal cells, and increased Th2-type cytokines as well as epithelial cell-derived cytokines, including IL-33, were observed in the skin tissue of HrasG12S/+ mice compared with Hras+/+ mice. Cultured HrasG12S/+ keratinocytes exhibited increased IL-33 expression after Dfb stimulation. PD0325901, an MEK inhibitor, ameliorated AD-like symptoms in HrasG12S/+ mice, showing decreased proliferation of p-ERK-positive epidermal cells and decreased expression of IL-33. Our findings indicate that the epidermis of HrasG12S/+ mice stimulated by Dfb strongly induced IL-33 expression and type-2 innate lymphoid cells, resulting in AD-like skin lesions. These results suggest that the epidermis of HrasG12S/+ mice are prone to development of eczematous dermatitis stimulated with house dust mite allergens.

Germ-Free Conditions Modulate Host Purine Metabolism, Exacerbating Adenine-Induced Kidney Damage.

Alterations in microbiota are known to affect kidney disease conditions. We have previously shown that germ-free conditions exacerbated adenine-induced kidney damage in mice; however, the mechanism by which this occurs has not been elucidated. To explore this mechanism, we examined the influence of germ-free conditions on purine metabolism and renal immune responses involved in the kidney damage. Germ-free mice showed higher expression levels of purine-metabolizing enzymes such as xanthine dehydrogenase, which converts adenine to a nephrotoxic byproduct 2,8-dihydroxyadenine (2,8-DHA). The germ-free mice also showed increased urinary excretion of allantoin, indicating enhanced purine metabolism. Metabolome analysis demonstrated marked differences in the purine metabolite levels in the feces of germ-free mice and mice with microbiota. Furthermore, unlike the germ-free condition, antibiotic treatment did not increase the expression of purine-metabolizing enzymes or exacerbate adenine-induced kidney damage. Considering renal immune responses, the germ-free mice displayed an absence of renal IL-17A expression. However, the adenine-induced kidney damage in wild-type mice was comparable to that in IL-17A-deficient mice, suggesting that IL-17A does not play a major role in the disease condition. Our results suggest that the enhanced host purine metabolism in the germ-free mice potentially promotes the conversion of the administered adenine into 2,8-DHA, resulting in exacerbated kidney damage. This further suggests a role of the microbiota in regulating host purine metabolism.

TRAF5 Deficiency Ameliorates the Severity of Dextran Sulfate Sodium Colitis by Decreasing TRAF2 Expression in Nonhematopoietic Cells.

TNFR-associated factor 5 (TRAF5) is a cytosolic adaptor protein and functions as an inflammatory regulator. However, the in vivo function of TRAF5 remains unclear, and how TRAF5 controls inflammatory responses in the intestine is not well understood. In this study, we found that intestinal epithelial cells from Traf5-/- mice expressed a significantly lower level of NF-κB-regulated proinflammatory genes, such as Tnf, Il6, and Cxcl1, as early as day 3 after dextran sulfate sodium (DSS) exposure when compared with wild-type mice. The intestinal barrier integrity of DSS-treated Traf5-/- mice remained intact at this early time point, and Traf5-/- mice showed decreased body weight loss and longer colon length at later time points. Surprisingly, the protein level of TRAF2, but not TRAF3, was reduced in colon tissues of Traf5-/- mice after DSS, indicating the requirement of TRAF5 for TRAF2 protein stability in the inflamed colon. Experiments with bone marrow chimeras confirmed that TRAF5 deficiency in nonhematopoietic cells caused the attenuated colitis. Our in vitro experiments demonstrated that proinflammatory cytokines significantly promoted the degradation of TRAF2 protein in Traf5-/- nonhematopoietic cells in a proteasome-dependent manner. Collectively, our data suggest a novel regulatory function of TRAF5 in supporting the proinflammatory function of TRAF2 in nonhematopoietic cells, which may be important for acute inflammatory responses in the intestine.

IQ motif-containing GTPase-activating protein 1 is essential for the optimal maintenance of lung ILC2s.

Group 2 innate lymphoid cells (ILC2s) play critical roles in type 2 immunity and are crucial for pathogenesis of various types of inflammatory disease. IQ motif-containing GTPase-activating protein 1 (IQGAP1) is a ubiquitously expressed scaffold protein that is involved in multiple cellular functions such as cell survival and trafficking. While the roles for IQGAP1 in T and B lymphocytes have been uncovered, the physiological significance of IQGAP1 in innate lymphocytes remains to be elucidated. In the current study, we demonstrate that using bone marrow chimeras, the deficiency of IQGAP1 caused an impaired survival of lung ILC2s in a cell-intrinsic manner and that Iqgap1-/- mice displayed decreased accumulation of ILC2s after administration of papain and thereby reduced the pathology of the disease. Moreover, Iqgap1-/- ILC2s showed a significantly enhanced apoptosis as compared to wild-type ILC2s under both steady-state and inflammatory conditions. Together these results identify for the first time that IQGAP1 is essential for homeostasis of ILC2s in the lung.

TNF Receptor-Associated Factor 5 Limits Function of Plasmacytoid Dendritic Cells by Controlling IFN Regulatory Factor 5 Expression.

The physiological functions of TNF receptor-associated factor 5 (TRAF5) in the skin inflammation and wound healing process are not well characterized. We found that Traf5 -/- mice exhibited an accelerated skin wound healing as compared with wild-type counterparts. The augmented wound closure in Traf5 -/- mice was associated with a massive accumulation of plasmacytoid dendritic cells (pDCs) into skin wounds and an enhanced expression of genes related to wound repair at skin sites. In accordance with this result, adoptive transfer of Traf5 -/- pDCs, but not wild-type pDCs, into the injured skin area in wild-type recipient mice significantly promoted skin wound healing. The expression of skin-tropic chemokine receptor CXCR3 was significantly upregulated in Traf5-/- pDCs, and treatment with a CXCR3 inhibitor cancelled the promoted wound healing in Traf5-/- mice, suggesting a pivotal role of CXCR3 in pDC-dependent wound healing. Traf5 -/- pDCs displayed significantly higher expression of IFN regulatory factor 5 (IRF5), which correlated with greater induction of proinflammatory cytokine genes and CXCR3 protein after stimulation with TLR ligands. Consistently, transduction of exogeneous TRAF5 in Traf5-/- pDCs normalized the levels of abnormally elevated proinflammatory molecules, including IRF5 and CXCR3. Furthermore, knockdown of IRF5 also rescued the abnormal phenotypes of Traf5-/- pDCs. Therefore, the higher expression and induction of IRF5 in Traf5-/- pDCs causes proinflammatory and skin-tropic characteristics of the pDCs, which may accelerate skin wound healing responses. Collectively, our results uncover a novel role of TRAF5 in skin wound healing that is mediated by IRF5-dependent function of pDCs.

CD45+CD326+ Cells are Predictive of Poor Prognosis in Non-Small Cell Lung Cancer Patients.

PURPOSE: The epithelial-to-mesenchymal transition, the major process by which some cancer cells convert from an epithelial phenotype to a mesenchymal one, has been suggested to drive chemo-resistance and/or metastasis in patients with cancer. However, only a few studies have demonstrated the presence of CD45/CD326 doubly-positive cells (CD45/CD326 DPC) in cancer. We deployed a combination of cell surface markers to elucidate the phenotypic heterogeneity in non-small cell lung cancer (NSCLC) cells and identified a new subpopulation that is doubly-positive for epithelial and non-epithelial cell-surface markers in both NSCLC cells and patients' malignant pleural effusions. EXPERIMENTAL DESIGN: We procured a total of 39 patients' samples, solid fresh lung cancer tissues from 21 patients and malignant pleural effusion samples from 18 others, and used FACS and fluorescence microscopy to check their surface markers. We also examined the EGFR mutations in patients with known acquired EGFR mutations. RESULTS: Our data revealed that 0.4% to 17.9% of the solid tumor tissue cells and a higher percentage of malignant pleural effusion cells harbored CD45/CD326 DPC expressing both epithelial and nonepithelial surface markers. We selected 3 EGFR mutation patients and genetically confirmed that the newly identified cell population really originated from cancer cells. We also found that higher proportions of CD45/CD326 DPC are significantly associated with poor prognosis. CONCLUSIONS: In conclusion, varying percentages of CD45/CD326 DPC exist in both solid cancer tissue and malignant pleural effusion in patients with NSCLC. This CD45/CD326 doubly-positive subpopulation can be an important key to clinical management of patients with NSCLC.

TNF receptor associated factor 5 controls oncostatin M-mediated lung inflammation.

Oncostatin M (OSM) is involved in pathogenesis of several human inflammatory diseases including lung inflammation and fibrosis. Although accumulating evidence indicates that OSM mediates lung inflammation, the precise mechanism for OSM on lung inflammation still remains unclear. In this study, we found that OSM receptor was abundantly expressed on endothelial and stromal/fibroblast cells in the lung of mice. In vitro stimulation with OSM upregulated vascular cell adhesion molecule-1 (VCAM-1), which promotes eosinophil infiltration in the lung tissues, on freshly-isolated lung stromal/fibroblast cells from wild-type mice. However, these cells from TNF receptor associated factor 5 (TRAF5)-deficient mice failed to show the increase in VCAM-1 expression after OSM stimulation. Furthermore, Traf5-/- mice showed markedly attenuated lung inflammation in terms of eosinophil infiltration upon intranasal administration with OSM as compared to wild-type mice. These results indicate that TRAF5 is crucially involved in OSM-mediated lung inflammation probably by inducing lung stromal/fibroblast cell activation.

ER Stress Protein CHOP Mediates Insulin Resistance by Modulating Adipose Tissue Macrophage Polarity.

Obesity represents chronic inflammatory states promoted by pro-inflammatory M1-macrophage infiltration into white adipose tissue (WAT), thereby inducing insulin resistance. Herein, we demonstrate the importance of an ER stress protein, CHOP, in determining adipose tissue macrophage (ATM) polarity and systemic insulin sensitivity. A high-fat diet (HFD) enhances ER stress with CHOP upregulation in adipocytes. CHOP deficiency prevents HFD-induced insulin resistance and glucose intolerance with ATM M2 predomination and Th2 cytokine upregulation in WAT. Whereas ER stress suppresses Th2 cytokine expression in cultured adipocytes, CHOP knockdown inhibits this downregulation. In contrast, macrophage responsiveness to Th1/Th2 cytokines is unchanged regardless of whether CHOP is expressed. Furthermore, bone marrow transplantation experiments showed recipient CHOP to be the major determinant of ATM polarity. Thus, CHOP in adipocytes plays important roles in ATM M1 polarization by altering WAT micro-environmental conditions, including Th2 cytokine downregulation. This molecular mechanism may link adipose ER stress with systemic insulin resistance.

Mesenteric lymph nodes contribute to proinflammatory Th17-cell generation during inflammation of the small intestine in mice.

T cells of the small intestine, including Th17 cells, are critically involved in host protection from microbial infection, and also contribute to the pathogenesis of small bowel inflammatory disorders. Accumulating evidence suggests that mesenteric lymph nodes (MLNs) play important roles in gut-tropic T-cell generation, although it is still unclear if MLNs are involved in the pathogenesis of small intestine inflammation. To address this issue, we analyzed the roles of both MLNs and Peyer's patches (PPs) by evaluating MLN- or PP-deficient mice in an experimental model of small intestine inflammation, induced by CD3-specific mAb injection. Interestingly, MLNs, but not PPs, were essential for the pathogenesis of intestinal inflammation, in particular the accumulation and infiltration of CD4(+) T-cell populations, including Th17 cells, from the blood. In addition, CD4(+) T-cell accumulation was dependent on the function of the α4 ß7 integrin. Furthermore, MLN removal led to a significantly reduced number of peripheral α4 ß7 (+) CD4(+) effector memory T cells under normal conditions, suggesting that MLNs may play a role in maintaining the number of gut-tropic CD4(+) effector memory T cells circulating in the blood. Taken together, the present study highlights the important role of MLNs in contributing to the pathogenesis of small intestine inflammation.

The adaptor TRAF5 limits the differentiation of inflammatory CD4(+) T cells by antagonizing signaling via the receptor for IL-6.

The physiological functions of members of the tumor-necrosis factor (TNF) receptor (TNFR)-associated factor (TRAF) family in T cell immunity are not well understood. We found that in the presence of interleukin 6 (IL-6), naive TRAF5-deficient CD4(+) T cells showed an enhanced ability to differentiate into the TH17 subset of helper T cells. Accordingly, TH17 cell-associated experimental autoimmune encephalomyelitis (EAE) was greatly exaggerated in Traf5(-/-) mice. Although it is normally linked with TNFR signaling pathways, TRAF5 constitutively associated with a cytoplasmic region in the signal-transducing receptor gp130 that overlaps with the binding site for the transcription activator STAT3 and suppressed the recruitment and activation of STAT3 in response to IL-6. Our results identify TRAF5 as a negative regulator of the IL-6 receptor signaling pathway that limits the induction of proinflammatory CD4(+) T cells that require IL-6 for their development.

Gene therapy model of X-linked severe combined immunodeficiency using a modified foamy virus vector.

X-linked severe combined immunodeficiency (SCID-X1) is an inherited genetic immunodeficiency associated with mutations in the common cytokine receptor γ chain (γc) gene, and characterized by a complete defect of T and natural killer (NK) cells. Gene therapy for SCID-X1 using conventional retroviral (RV) vectors carrying the γc gene results in the successful reconstitution of T cell immunity. However, the high incidence of vector-mediated T cell leukemia, caused by vector insertion near or within cancer-related genes has been a serious problem. In this study, we established a gene therapy model of mouse SCID-X1 using a modified foamy virus (FV) vector expressing human γc. Analysis of vector integration in a human T cell line demonstrated that the FV vector integration sites were significantly less likely to be located within or near transcriptional start sites than RV vector integration sites. To evaluate the therapeutic efficacy, bone marrow cells from γc-knockout (γc-KO) mice were infected with the FV vector and transplanted into γc-KO mice. Transplantation of the FV-treated cells resulted in the successful reconstitution of functionally active T and B cells. These data suggest that FV vectors can be effective and may be safer than conventional RV vectors for gene therapy for SCID-X1.

Y chromosome-linked B and NK cell deficiency in mice.

There are no primary immunodeficiency diseases linked to the Y chromosome, because the Y chromosome does not contain any vital genes. We have established a novel mouse strain in which all males lack B and NK cells and have Peyer's patch defects. By 10 wk of age, 100% of the males had evident immunodeficiencies. Mating these immunodeficient males with wild-type females on two different genetic backgrounds for several generations demonstrated that the immunodeficiency is linked to the Y chromosome and is inherited in a Mendelian fashion. Although multicolor fluorescence in situ hybridization analysis showed that the Y chromosome in the mutant male mice was one third shorter than that in wild-type males, exome sequencing did not identify any significant gene mutations. The precise molecular mechanisms are still unknown. Bone marrow chimeric analyses demonstrated that an intrinsic abnormality in bone marrow hematopoietic cells causes the B and NK cell defects. Interestingly, fetal liver cells transplanted from the mutant male mice reconstituted B and NK cells in lymphocyte-deficient Il2rg(-/-) recipient mice, whereas adult bone marrow transplants did not. Transducing the EBF gene, a master transcription factor for B cell development, into mutant hematopoietic progenitor cells rescued B cell but not NK cell development both in vitro and in vivo. These Y chromosome-linked immunodeficient mice, which have preferential B and NK cell defects, may be a useful model of lymphocyte development.

Homeostatic proliferation of naive CD4+ T cells in mesenteric lymph nodes generates gut-tropic Th17 cells.

Homeostatic proliferation of naive T cells in the spleen and cutaneous lymph nodes supplies memory-phenotype T cells. The "systemic" proliferative responses divide distinctly into fast or slow cell division rates. The fast proliferation is critical for generation of effector memory T cells. Because effector memory T cells are abundant in the lamina propria of the intestinal tissue, "gut-specific" homeostatic proliferation of naive T cells may be important for generation of intestinal effector memory T cells. However, such organ-specific homeostatic proliferation of naive T cells has not yet been addressed. In this study, we examined the gut-specific homeostatic proliferation by transferring CFSE-labeled naive CD4(+) T cells into sublethally irradiated mice and separately evaluating donor cell division and differentiation in the intestine, mesenteric lymph nodes (MLNs), and other lymphoid organs. We found that the fast-proliferating cell population in the intestine and MLNs had a gut-tropic α4ß7(+) Th17 phenotype and that their production was dependent on the presence of commensal bacteria and OX40 costimulation. Mesenteric lymphadenectomy significantly reduced the Th17 cell population in the host intestine. Furthermore, FTY720 treatment induced the accumulation of α4ß7(+)IL-17A(+) fast-dividing cells in MLNs and eliminated donor cells in the intestine, suggesting that MLNs rather than intestinal tissues are essential for generating intestinal Th17 cells. These results reveal that MLNs play a central role in inducing gut-tropic Th17 cells and in maintaining CD4(+) T cell homeostasis in the small intestine.