The Nr4a family regulates intrahepatic Treg proliferation and liver fibrosis in MASLD models.

Nonalcoholic steatohepatitis (NASH) is a chronic progressive liver disease and highly prevalent worldwide. NASH is characterized by hepatic steatosis, inflammation, fibrosis and liver damage, which eventually results in liver dysfunction due to cirrhosis or hepatocellular carcinoma. However, the cellular and molecular mechanisms underlying NASH progression remain largely unknown. Here, we found an increase of Nr4a family of orphan nuclear receptors expression in intrahepatic T cells from mice with diet-induced NASH. Loss of Nr4a1 and Nr4a2 in T cell (dKO) ameliorated liver cell death and fibrosis, thereby mitigating liver dysfunction in NASH mice. dKO resulted in reduction of infiltrated macrophages and Th1/Th17 cells, whereas massive accumulation of T regulatory (Treg) cells in the liver of NASH mice. Combined single-cell RNA transcriptomic and TCR sequencing analysis revealed that intrahepatic dKO Tregs exhibited enhanced TIGIT and IL10 expression and were clonally expanded during NASH progression. Mechanistically, we found that dKO Tregs expressed high levels of Batf which promotes Treg cell proliferation and function upon TCR stimulation. Collectively, our findings not only provide an insight into the impact of intrahepatic Treg cells on NASH pathogenesis, but also suggest a therapeutic potential of targeting of Nr4a family to treat the disease.

SOCS, SPRED, and NR4a: Negative regulators of cytokine signaling and transcription in immune tolerance.

Cytokines are important intercellular communication tools for immunity. Most cytokines utilize the JAK-STAT and Ras-ERK pathways to promote gene transcription and proliferation; however, this signaling is tightly regulated. The suppressor of cytokine signaling (SOCS) family and SPRED family are a representative negative regulators of the JAK-STAT pathway and the Ras-ERK pathway, respectively. The SOCS family regulates the differentiation and function of CD4+ T cells, CD8+ T cells, and regulatory T cells, and is involved in immune tolerance, anergy, and exhaustion. SPRED family proteins have been shown to inactivate Ras by recruiting the Ras-GTPase neurofibromatosis type 1 (NF1) protein. Human genetic analysis has shown that SOCS family members are strongly associated with autoimmune diseases, allergies, and tumorigenesis, and SPRED1 is involved in NF1-like syndromes and tumors. We also identified the NR4a family of nuclear receptors as a key transcription factor for immune tolerance that suppresses cytokine expression and induces various immuno-regulatory molecules including SOCS1.

Immunomodulation by Inflammation during Liver and Gastrointestinal Tumorigenesis and Aging.

Chronic inflammation is thought to promote tumorigenesis and metastasis by several mechanisms, such as affecting tumor cells directly, establishing a tumor-supporting microenvironment, enhancing tumor angiogenesis, and suppressing antitumor immunity. In this review, we discuss the recent advances in our understanding of how inflammation induces the immunosuppressive tumor microenvironment, such as increasing the level of pro-inflammatory cytokines, chemokines, and immunosuppressive molecules, inducing immune checkpoint molecules and cytotoxic T-cell exhaustion, and accumulating regulatory T (Treg) cells and myeloid-derived suppressor cells (MDSCs). The suppression of antitumor immunity by inflammation is especially examined in the liver and colorectal cancer. In addition, chronic inflammation is induced during aging and causes age-related diseases, including cancer, by affecting immunity. Therefore, we also discuss the age-related diseases regulated by inflammation, especially in the liver and colon.

The E3 ligase VHL promotes follicular helper T cell differentiation via glycolytic-epigenetic control.

Follicular helper T (Tfh) cells are essential for germinal center formation and effective humoral immunity, which undergo different stages of development to become fully polarized. However, the detailed mechanisms of their regulation remain unsolved. Here we found that the E3 ubiquitin ligase VHL was required for Tfh cell development and function upon acute virus infection or antigen immunization. VHL acted through the hypoxia-inducible factor 1α (HIF-1α)-dependent glycolysis pathway to positively regulate early Tfh cell initiation. The enhanced glycolytic activity due to VHL deficiency was involved in the epigenetic regulation of ICOS expression, a critical molecule for Tfh development. By using an RNA interference screen, we identified the glycolytic enzyme GAPDH as the key target for the reduced ICOS expression via m6A modification. Our results thus demonstrated that the VHL-HIF-1α axis played an important role during the initiation of Tfh cell development through glycolytic-epigenetic reprogramming.

Immune regulation by protein ubiquitination: roles of the E3 ligases VHL and Itch.

Protein ubiquitination is an important means of post-translational modification which plays an essential role in the regulation of various aspects of leukocyte development and function. The specificity of ubiquitin tagging to a protein substrate is determined by E3 ubiquitin ligases via defined E3-substrate interactions. In this review, we will focus on two E3 ligases, VHL and Itch, to discuss the latest progress in understanding their roles in the differentiation and function of CD4+ T helper cell subsets, the stability of regulatory T cells, effector function of CD8+ T cells, as well as the development and maturation of innate lymphoid cells. The biological implications of these E3 ubiquitin ligases will be highlighted in the context of normal and dysregulated immune responses including the control of homeostasis, inflammation, auto-immune responses and anti-tumor immunity. Further elucidation of the ubiquitin system in immune cells will help in the design of new therapeutic interventions for human immunological diseases and cancer.

The E3 ligase VHL controls alveolar macrophage function via metabolic-epigenetic regulation.

Metabolic pathways such as glycolysis or oxidative phosphorylation play a key role in regulating macrophage function during inflammation and tissue repair. However, how exactly the VHL-HIF-glycolysis axis is involved in the function of tissue-resident macrophages remains unclear. Here we demonstrate that loss of VHL in myeloid cells resulted in attenuated pulmonary type 2 and fibrotic responses, accompanied by reduced eosinophil infiltration, decreased IL-5 and IL-13 concentrations, and ameliorated fiber deposition upon challenge. VHL deficiency uplifted glycolytic metabolism, decreased respiratory capacity, and reduced osteopontin expression in alveolar macrophages, which impaired the function of type 2 innate lymphoid cells but was significantly reversed by HIF1α inhibition or ablation. The up-regulated glycolysis altered the epigenetic modification of osteopontin gene, with the metabolic intermediate 3-phosphoglyceric acid as a key checkpoint controller. Thus, our results indicate that VHL acts as a crucial regulatory factor in lung inflammation and fibrosis by regulating alveolar macrophages.

The E3 ligases Itch and WWP2 cooperate to limit TH2 differentiation by enhancing signaling through the TCR.

The mechanisms by which the sensitivity of naive CD4+ T cells to stimulation by the cognate antigen via the T cell antigen receptor (TCR) determines their differentiation into distinct helper T cell subsets remain elusive. Here we demonstrate functional collaboration of the ubiquitin E3 ligases Itch and WWP2 in regulating the strength of the TCR signal. Mice lacking both Itch and WWP2 in T cells showed spontaneous autoimmunity and lung inflammation. CD4+ T cells deficient in Itch and WWP2 exhibited hypo-responsiveness to TCR stimulation and a bias toward differentiation into the TH2 subset of helper T cells. Itch and WWP2 formed a complex and cooperated to enhance TCR-proximal signaling by catalyzing the conjugation of atypical ubiquitin chains to the phosphatase SHP-1 and reducing the association of SHP-1 with the tyrosine kinase Lck. These findings indicate that targeted ubiquitination regulates the strength of the TCR signal and differentiation toward the TH2 lineage.

The E3 ligase Itch in immune regulation and beyond.

Itch or itchy E3 ubiquitin ligase was initially discovered by genetic studies on the mouse coat color changes, and its deletion results in an itchy phenotype with constant skin scratching and multi-organ inflammation. It is a member of the homologous to E6-associated protein C-terminus (HECT)-type family of E3 ligases, with the protein-interacting WW-domains for the recruitment of substrate and the HECT domain for the transfer of ubiquitin to the substrate. Since its discovery, numerous studies have demonstrated that Itch is involved in the control of many aspects of immune responses including T-cell activation and tolerance and T-helper cell differentiation. Itch is also implicated in other biological contexts such as tumorigenesis, development, and stress responses. Many signaling pathways are regulated by Itch-promoted ubiquitylation of diverse target proteins. Itch is also involved in human diseases. Here, we discuss the major progress in understanding the biological significance of Itch-promoted protein ubiquitylation in the immune and other systems and in Itch-mediated regulation of signal transduction.

The ubiquitin system in immune regulation.

The ubiquitin system plays a pivotal role in the regulation of immune responses. This system includes a large family of E3 ubiquitin ligases of over 700 proteins and about 100 deubiquitinating enzymes, with the majority of their biological functions remaining unknown. Over the last decade, through a combination of genetic, biochemical, and molecular approaches, tremendous progress has been made in our understanding of how the process of protein ubiquitination and its reversal deubiquitination controls the basic aspect of the immune system including lymphocyte development, differentiation, activation, and tolerance induction and regulates the pathophysiological abnormalities such as autoimmunity, allergy, and malignant formation. In this review, we selected some of the published literature to discuss the roles of protein-ubiquitin conjugation and deubiquitination in T-cell activation and anergy, regulatory T-cell and T-helper cell differentiation, regulation of NF-κB signaling, and hematopoiesis in both normal and dysregulated conditions. A comprehensive understanding of the relationship between the ubiquitin system and immunity will provide insight into the molecular mechanisms of immune regulation and at the same time will advance new therapeutic intervention for human immunological diseases.

Haploinsufficiency of SAMD9L, an endosome fusion facilitator, causes myeloid malignancies in mice mimicking human diseases with monosomy 7.

Monosomy 7 and interstitial deletion of 7q (-7/7q-) are well-recognized nonrandom chromosomal abnormalities frequently found among patients with myelodysplastic syndromes (MDSs) and myeloid leukemias. We previously identified candidate myeloid tumor suppressor genes (SAMD9, SAMD9-like = SAMD9L, and Miki) in the 7q21.3 subband. We established SAMD9L-deficient mice and found that SAMD9L(+/-) mice as well as SAMD9L(-/-) mice develop myeloid diseases resembling human diseases associated with -7/7q-. SAMD9L-deficient hematopoietic stem cells showed enhanced colony formation potential and in vivo reconstitution ability. SAMD9L localizes in early endosomes. SAMD9L-deficient cells showed delays in homotypic endosome fusion, resulting in persistence of ligand-bound cytokine receptors. These findings suggest that haploinsufficiency of SAMD9L and/or SAMD9 gene(s) contributes to myeloid transformation.

Poly-ADP ribosylation of Miki by tankyrase-1 promotes centrosome maturation.

During prometaphase, dense microtubule nucleation sites at centrosomes form robust spindles that align chromosomes promptly. Failure of centrosome maturation leaves chromosomes scattered, as seen routinely in cancer cells, including myelodysplastic syndrome (MDS). We previously reported that the Miki (LOC253012) gene is frequently deleted in MDS patients, and that low levels of Miki are associated with abnormal mitosis. Here we demonstrate that Miki localizes to the Golgi apparatus and is poly(ADP-ribosyl)ated by tankyrase-1 during late G2 and prophase. PARsylated Miki then translocates to mitotic centrosomes and anchors CG-NAP, a large scaffold protein of the γ-tubulin ring complex. Due to impairment of microtubule aster formation, cells in which tankyrase-1, Miki, or CG-NAP expression is downregulated all show prometaphase disturbances, including scattered and lagging chromosomes. Our data suggest that PARsylation of Miki by tankyrase-1 is a key initial event promoting prometaphase.

The dynactin complex maintains the integrity of metaphasic centrosomes to ensure transition to anaphase.

The dynactin complex is required for activation of the dynein motor complex, which plays a critical role in various cell functions including mitosis. During metaphase, the dynein-dynactin complex removes spindle checkpoint proteins from kinetochores to facilitate the transition to anaphase. Three components (p150(Glued), dynamitin, and p24) compose a key portion of the dynactin complex, termed the projecting arm. To investigate the roles of the dynactin complex in mitosis, we used RNA interference to down-regulate p24 and p150(Glued) in human cells. In response to p24 down-regulation, we observed cells with delayed metaphase in which chromosomes frequently align abnormally to resemble a "figure eight," resulting in cell death. We attribute the figure eight chromosome alignment to impaired metaphasic centrosomes that lack spindle tension. Like p24, RNA interference of p150(Glued) also induces prometaphase and metaphase delays; however, most of these cells eventually enter anaphase and complete mitosis. Our findings suggest that although both p24 and p150(Glued) components of the dynactin complex contribute to mitotic progression, p24 also appears to play a role in metaphase centrosome integrity, helping to ensure the transition to anaphase.

Up-regulation of survivin by the E2A-HLF chimera is indispensable for the survival of t(17;19)-positive leukemia cells.

The E2A-HLF fusion transcription factor generated by t(17;19)(q22;p13) translocation is found in a small subset of pro-B cell acute lymphoblastic leukemias (ALLs) and promotes leukemogenesis by substituting for the antiapoptotic function of cytokines. Here we show that t(17;19)+ ALL cells express Survivin at high levels and that a dominant negative mutant of E2A-HLF suppresses Survivin expression. Forced expression of E2A-HLF in t(17;19)(-) leukemia cells up-regulated Survivin expression, suggesting that Survivin is a downstream target of E2A-HLF. Analysis using a counterflow centrifugal elutriator revealed that t(17;19)+ ALL cells express Survivin throughout the cell cycle. Reporter assays revealed that E2A-HLF induces survivin expression at the transcriptional level likely through indirect down-regulation of a cell cycle-dependent cis element in the promoter region. Down-regulation of Survivin function by a dominant negative mutant of Survivin or reduction of Survivin expression induced massive apoptosis throughout the cell cycle in t(17;19)+ cells mainly through caspase-independent pathways involving translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus. AIF knockdown conferred resistance to apoptosis caused by down-regulation of Survivin function. These data indicated that reversal of AIF translocation by Survivin, which is induced by E2A-HLF throughout the cell cycle, is one of the key mechanisms in the protection of t(17;19)+ leukemia cells from apoptosis.

A major lipid raft protein raftlin modulates T cell receptor signaling and enhances th17-mediated autoimmune responses.

The membrane microdomains known as lipid rafts have been shown to act as platforms for the initiation of various receptor signals. Through proteomic analysis, we have identified a novel protein termed Raftlin (raft-linking protein) as a major protein in lipid rafts. To determine the physiological and immunological functions of Raftlin in mammals, we generated Raftlin-deficient mice, as well as Raftlin-transgenic (Tg) mice. Although Raftlin was originally identified in B cells, we observe no severe abnormalities in the B cells of these mice, presumably due to a high expression of Raftlin-homologue (Raftlin-2). T cells, in contrast, expressed a substantial amount of Raftlin but no Raftlin-2. In Raftlin-deficient mice, T cell-dependent Ab production was reduced, and experimental autoimmune encephalomyelitis, a Th17-dependent autoimmune disease model, was ameliorated. In Raftlin-Tg mice, in contrast, Ab production was enhanced and experimental autoimmune encephalomyelitis was more severe. Cytokine production, especially that of IL-17, was reduced in Raftlin-deficient T cells, while it was enhanced in Raftlin-Tg T cells. We found that these changes were associated with the strength of the TCR-mediated signals. Importantly, localization of Lck protein in the lipid rafts was enhanced by Raftlin overexpression and reduced by Raftlin deficiency. These data indicate that Raftlin modulates TCR signals and is necessary for the fine-tuning of T cell-mediated immune responses.

Identification of a common microdeletion cluster in 7q21.3 subband among patients with myeloid leukemia and myelodysplastic syndrome.

Monosomy 7 and interstitial deletions in the long arm of chromosome 7 (-7/7q-) is a common nonrandom chromosomal abnormality found frequently in myeloid disorders including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and juvenile myelomonocytic leukemia (JMML). Using a short probe-based microarray comparative genomic hybridization (mCGH) technology, we identified a common microdeletion cluster in 7q21.3 subband, which is adjacent to 'hot deletion region' thus far identified by conventional methods. This common microdeletion cluster contains three poorly characterized genes; Samd9, Samd9L, and a putative gene LOC253012, which we named Miki. Gene copy number assessment of three genes by real-time PCR revealed heterozygous deletion of these three genes in adult patients with AML and MDS at high frequency, in addition to JMML patients. Miki locates to mitotic spindles and centrosomes and downregulation of Miki by RNA interference induced abnormalities in mitosis and nuclear morphology, similar to myelodysplasia. In addition, a recent report indicated Samd9 as a tumor suppressor. These findings indicate the usefulness of the short probe-based CGH to detect microdeletions. The three genes located to 7q21.3 would be candidates for myeloid tumor-suppressor genes on 7q.

Peptidoglycan and lipopolysaccharide activate PLCgamma2, leading to enhanced cytokine production in macrophages and dendritic cells.

In macrophages and monocytes, microbial components trigger the production of pro-inflammatory cytokine through Toll-like receptors (TLRs). Although major TLR signaling pathways are mediated by serine/threonine kinases, including TAK1, IKK and MAP kinases, tyrosine phosphorylation of intracellular proteins by TLR ligands has been suggested in a number of reports. Here, we demonstrated that peptidoglycan (PGN) of a Gram-positive bacterial cell wall component, a TLR2 ligand and lipopoysaccharide (LPS) of a Gram-positive bacterial component, a TLR4 ligand induced tyrosine phosphorylation of phospholipase Cgamma-2 (PLCgamma2), leading to intracellular free Ca2+ mobilization in bone marrow-derived macrophages (BMMphi) and bone marrow-derived dendritic cells (BMDC). PGN- and LPS-induced Ca2+ mobilization was not observed in BMDC from PLCgamma2 knockout mice. Thus, PLCgamma2 is essential for TLR2 and TLR4-mediated Ca2+ flux. In PLCgamma2-knockdown cells, PGN-induced IkappaB-alpha phosphorylation and p38 activation were reduced. Moreover, PLCgamma2 was necessary for the full production of TNF-alpha and IL-6. These data indicate that the PLCgamma2 pathway plays an important role in bacterial ligands-induced activation of macrophages and dendritic cells.

A novel Zinc finger protein, ZCCHC11, interacts with TIFA and modulates TLR signaling.

Toll-like receptors (TLRs) play an important role as a sensor of microbial pathogens in the innate immune response. TLRs transmit signals through the recruitment of adaptor proteins including tumor necrosis factor-associated factor 6 (TRAF6), which mediates the activation of IkappaB kinase (IKK). TIFA (TRAF-interacting protein with a forkhead-associated (FHA) domain) has been shown to bind to TRAF6 and activate IKK by promoting the oligomerization and ubiquitin-ligase activity of TRAF6. FHA domains preferentially bind to phospho-threonine residues in their targets. Here, we identified a novel zinc finger protein, ZCCHC11, that interacts with TIFA from phosphoproteins of a macrophage cell line, RAW 264.7, by using affinity purification with GST-TIFA and mass spectrometric analysis. By a search of the EST database, we found a 200kDa full-length form (ZCCHC11L). ZCCHC11L was mostly located to the nucleus, but translocated into the cytoplasm in response to LPS and bound to TIFA. Overexpression and knockdown by siRNA indicated that ZCCHC11 functions as a negative regulator of TLR-mediated NF-kappaB activation. The N-terminal region (ZCCHC11S) including C2H2-type [corrected] Zn-finger motif was sufficient for suppression of NF-kappaB. We propose that ZCCHC11 is a unique TLR signal regulator, which interacts with TIFA after LPS treatment and suppresses the TRAF6-dependent activation of NF-kappaB.

FLN29, a novel interferon- and LPS-inducible gene acting as a negative regulator of toll-like receptor signaling.

Lipopolysaccharide (LPS) activates macrophages through toll-like receptor (TLR) 4. Although the mechanism of the TLR signaling pathway has been well documented, the mechanism of the negative regulation in response to LPS, particularly LPS tolerance, is still poorly understood. In this study we identified and characterized a novel interferon- and LPS-inducible gene, FLN29, which contains a TRAF6-related zinc finger motif and TRAF family member-associated NF-kappaB activator-related sequences. The induction of FLN29 was dependent on STAT1. The forced expression of FLN29 in macrophage-like RAW cells resulted in the suppression of TLR-mediated NF-kappaB and mitogen-activated protein kinase activation, while a reduced expression of FLN29 by small interfering RNA partly cancelled the down-regulation of LPS signaling. Furthermore, we demonstrated that NF-kappaB activation induced by TRAF6 and TAB2 was impaired by co-expression of FLN29, suggesting FLN29 may regulate the downstream of TRAF6. Taken together, FLN29 is a new negative feedback regulator of TLR signaling.

Modulation of TLR signalling by the C-terminal Src kinase (Csk) in macrophages.

In macrophages and monocytes, lipopolysaccharide (LPS) triggers the production of pro-inflammatory cytokine through Toll-like receptor (TLR) 4. Although major TLR signalling pathways are mediated by serine or threonine kinases including IKK, TAK1, p38 and JNKs, a number of reports suggested that tyrosine phosphorylation of intracellular proteins is involved in LPS signalling. Here, we identified several tyrosine-phosphorylated proteins using mass spectrometric analysis in response to LPS stimulation. Among these proteins, we characterized C-terminal Src kinase (Csk), which negatively regulates Src-like kinases in RAW 264.7 cells using RNAi knockdown technology. Unexpectedly, LPS-induced CD40 activation and the secretion of pro-inflammatory cytokine such as IL-6 and TNF-alpha, was down-regulated in Csk knockdown cells. Furthermore, overall cellular tyrosine phosphorylation and TLR4-mediated activation of IkappaB-alpha, Erk and p38 but not of JNK, were also down-regulated in Csk knockdown cells. The protein expression levels of a tyrosine kinase, Fgr, were reduced in Csk knockdown cells, suggesting that Csk is a critical regulator of TLR4-mediated signalling by modifying the levels of Src-like kinases.