Regnase-1 D141N mutation induces CD4+ T cell-mediated lung granuloma formation via upregulation of Pim2.

Regnase-1 is an RNase that plays a critical role in negatively regulating immune responses by destabilizing inflammatory mRNAs. Dysfunction of Regnase-1 can be a major cause of various inflammatory diseases with tissue injury and immune cell infiltration into organs. This study focuses on the role of RNase activity of Regnase-1 in developing inflammatory diseases. We have constructed mice with a single point mutation at the catalytic center of Regnase-1 RNase domain, which lacks endonuclease activity. D141N mutant mice demonstrated systemic inflammation, immune cell infiltration into various organs and progressive development of lung granuloma. CD4+ T cells, mainly affected by this mutation, upregulated mTORC1 pathway and facilitated the autoimmune trait in D141N mutation. Moreover, serine/threonine kinase Pim2 contributed to lung inflammation in this mutation. Inhibition of Pim2 kinase activity ameliorated granulomatous inflammation, immune cell infiltration and proliferation in the lungs. Additionally, Pim2 inhibition reduced the expression of adhesion molecules on CD4+ T cells, suggesting a role for Pim2 in facilitating leukocyte adhesion and migration to inflamed tissues. Our findings provide new insights into the role of Regnase-1 RNase activity in controlling immune function and underscore the therapeutic relevance of targeting Pim2 to modulate abnormal immune responses.

TAK1-binding proteins (TAB)2 and TAB3 are redundantly required for TLR-induced cytokine production in macrophages.

Transforming growth factor-ß-activated kinase 1 (TAK1) plays a pivotal role in innate and adaptive immunity. TAK1 is essential for the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB pathways downstream of diverse immune receptors, including Toll-like receptors (TLRs). Upon stimulation with TLR ligands, TAK1 is activated via recruitment to lysine 63-linked polyubiquitin chain through TAK1-binding proteins (TAB) 2 and TAB3. However, the physiological importance of TAB2 and TAB3 in macrophages is still controversial. A previous study has shown that mouse bone marrow-derived macrophages (BMDMs) isolated from mice double deficient for TAB2 and TAB3 produced tumor necrosis factor (TNF)-α and interleukin (IL)-6 to the similar levels as control wild-type BMDMs in response to TLR ligands such as lipopolysaccharide (LPS) or Pam3CSK4, indicating that TAB2 and TAB3 are dispensable for TLR signaling. In this study, we revisited the role of TAB2 and TAB3 using an improved mouse model. We observed a significant impairment in the production of pro-inflammatory cytokines and chemokine in LPS- or Pam3CSK4-treated BMDMs deficient for both TAB2 and TAB3. Double deficiency of TAB2 and TAB3 resulted in the decreased activation of NF-κB and MAPK pathways as well as the slight decrease in TAK1 activation in response to LPS or Pam3CSK4. Notably, the TLR-mediated expression of inhibitor of NF-κB (IκB)ζ was severely compromised at the protein and mRNA levels in the TAB2/TAB3 double-deficient BMDMs, thereby impeding IL-6 production. Our results suggest that TAB2 and TAB3 play a redundant and indispensable role in TLR signaling pathway.

CGRP sensory neurons promote tissue healing via neutrophils and macrophages.

The immune system has a critical role in orchestrating tissue healing. As a result, regenerative strategies that control immune components have proved effective1,2. This is particularly relevant when immune dysregulation that results from conditions such as diabetes or advanced age impairs tissue healing following injury2,3. Nociceptive sensory neurons have a crucial role as immunoregulators and exert both protective and harmful effects depending on the context4-12. However, how neuro-immune interactions affect tissue repair and regeneration following acute injury is unclear. Here we show that ablation of the NaV1.8 nociceptor impairs skin wound repair and muscle regeneration after acute tissue injury. Nociceptor endings grow into injured skin and muscle tissues and signal to immune cells through the neuropeptide calcitonin gene-related peptide (CGRP) during the healing process. CGRP acts via receptor activity-modifying protein 1 (RAMP1) on neutrophils, monocytes and macrophages to inhibit recruitment, accelerate death, enhance efferocytosis and polarize macrophages towards a pro-repair phenotype. The effects of CGRP on neutrophils and macrophages are mediated via thrombospondin-1 release and its subsequent autocrine and/or paracrine effects. In mice without nociceptors and diabetic mice with peripheral neuropathies, delivery of an engineered version of CGRP accelerated wound healing and promoted muscle regeneration. Harnessing neuro-immune interactions has potential to treat non-healing tissues in which dysregulated neuro-immune interactions impair tissue healing.

Regnase-1 downregulation promotes pancreatic cancer through myeloid-derived suppressor cell-mediated evasion of anticancer immunity.

BACKGROUND: Pancreatitis is known to be an important risk factor for pancreatic ductal adenocarcinoma (PDAC). However, the exact molecular mechanisms of how inflammation promotes PDAC are still not fully understood. Regnase-1, an endoribonuclease, regulates immune responses by degrading mRNAs of inflammation-related genes. Herein, we investigated the role of Regnase-1 in PDAC. METHODS: Clinical significance of intratumor Regnase-1 expression was evaluated by immunohistochemistry in 39 surgically-resected PDAC patients. The functional role of Regnase-1 was investigated by pancreas-specific Regnase-1 knockout mice and Kras-mutant Regnase-1 knockout mice. The mechanistic studies with gene silencing, RNA immunoprecipitation sequencing (RIP-seq) and immune cell reconstitution were performed in human/mouse PDAC cell lines and a syngeneic orthotopic tumor transplantation model of KrasG12D-mutant and Trp53-deficient PDAC cells. RESULTS: Regnase-1 expression was negatively correlated with the clinical outcomes and an independent predictor of poor relapse-free and overall survival in PDAC patients. Pancreas-specific Regnase-1 deletion in mice promoteed pancreatic cancer with PMN-MDSC infiltration and shortened their survival. A syngeneic orthotopic PDAC model exhibited that Regnase-1 downregulation accelerated tumor progression via recruitment of intratumor CD11b+ MDSCs. Mechanistically, Regnase-1 directly negatively regulated a variety of chemokines/cytokines important for MDSC recruitment and activation, including CXCL1, CXCL2, CSF2, and TGFß, in pancreatic cancer cells. We subsequently showed that IL-1ß-mediated Regnase-1 downregulation recruited MDSCs to tumor sites and promoted pancreatic cancer progression via mitigation of cytotoxic T lympohocytes-mediated antitumor immunity. CONCLUSIONS: IL-1b-mediated Regnase-1 downregulation induces MDSCs and promotes pancreatic cancer through the evasion of anticancer immunity.

Breaking self-regulation of Regnase-1 promotes its own protein expression.

The RNA-binding protein (RBP) Regnase-1 is an endonuclease that regulates immune responses by modulating target mRNA stability. Regnase-1 degrades a group of inflammation-associated mRNAs, which contributes to a balanced immune response and helps prevent autoimmune diseases. Regnase-1 also cleaves its own mRNA by binding stem-loop (SL) RNA structures in its 3'UTR. To understand how this autoregulation is important for immune responses, we generated mice with a 2-bp genome deletion in the target SL of the Regnase-1 3'-untranslated region (3'UTR). Deletion of these nucleotides inhibited SL formation and limited Regnase-1-mediated mRNA degradation. Mutant mice had normal hematopoietic cell differentiation. Biochemically, mutation of the 3'UTR SL increased Regnase-1 mRNA stability and enhanced both Regnase-1 mRNA and protein levels in mouse embryonic fibroblasts (MEFs). The expression of Il6, a Regnase-1 target gene, was constitutively suppressed at steady-state in mutant MEFs. Additionally, Regnase-1 protein expression in mutant MEFs was significantly elevated compared to that in wild-type MEFs at steady state and upon proinflammatory cytokine stimulation. These data suggest a negative feedback mechanism for Regnase-1 expression and represent a unique mouse model to probe Regnase-1 overexpression in vivo.

Keratinocyte Regnase-1, a Downregulator of Skin Inflammation, Contributes to Protection against Tumor Promotion by Limiting Cyclooxygenase-2 Expression.

We previously showed that the ribonuclease Regnase-1 (Reg1) in keratinocytes plays a role in mitigating skin inflammation by downregulating proinflammatory cytokines. In this study, we explored whether Reg1 also has a protective role against skin carcinogenesis. The chemically induced two-stage carcinogenesis protocol revealed that epidermis-specific Reg1-deficient (Reg1-knockout [Reg1-cKO]) mice developed skin tumors with shorter latency and more multiplicity than control mice. In addition, repeated UVB irradiation readily provoked solar keratosis-like lesions in Reg1-cKO mice. Increased levels of cyclooxygenase 2, whose mRNA (Ptgs2) is reportedly a target of Reg1, have been known to be associated with the development of squamous cell carcinomas. Indeed, Ptgs2 mRNA levels were upregulated in the skin of Reg1-cKO mice after treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. The level of prostaglandin E2 was higher in 12-O-tetradecanoylphorbol-13-acetate‒treated Reg1-cKO mouse skin than in control mice skin. Moreover, in vivo inhibition of cyclooxygenase 2 attenuated the 12-O-tetradecanoylphorbol-13-acetate‒induced epidermal thickening in Reg1-cKO mice. Finally, REG1 knockdown in human squamous cell carcinomas lines enhanced PTGS2 mRNA levels after 12-O-tetradecanoylphorbol-13-acetate treatment. In conclusion, epidermal Reg1 plays a regulatory role not only in skin inflammation but also in tumor promotion through the downregulation of cyclooxygenase 2. Therefore, forced expression of Reg1 under inflammatory conditions may be relevant to preventing skin cancer.

Interleukin-33 facilitates liver regeneration through serotonin-involved gut-liver axis.

BACKGROUND AND AIMS: Insufficient liver regeneration causes post-hepatectomy liver failure and small-for-size syndrome. Identifying therapeutic targets to enhance hepatic regenerative capacity remains urgent. Recently, increased IL-33 was observed in patients undergoing liver resection and in mice after partial hepatectomy (PHx). The present study aims to investigate the role of IL-33 in liver regeneration after PHx and to elucidate its underlying mechanisms. APPROACH AND RESULTS: We performed PHx in IL-33 -/- , suppression of tumorigenicity 2 (ST2) -/- , and wild-type control mice, and found deficiency of IL-33 or its receptor ST2 delayed liver regeneration. The insufficient liver regeneration could be normalized in IL-33 -/- but not ST2 -/- mice by recombinant murine IL-33 administration. Furthermore, we observed an increased level of serotonin in portal blood from wild-type mice, but not IL-33 -/- or ST2 -/- mice, after PHx. ST2 deficiency specifically in enterochromaffin cells recapitulated the phenotype of delayed liver regeneration observed in ST2 -/- mice. Moreover, the impeded liver regeneration in IL-33 -/- and ST2 -/- mice was restored to normal levels by the treatment with (±)-2,5-dimethoxy-4-iodoamphetamine, which is an agonist of the 5-hydroxytrytamine receptor (HTR)2A. Notably, in vitro experiments demonstrated that serotonin/HTR2A-induced hepatocyte proliferation is dependent on p70S6K activation. CONCLUSIONS: Our study identified that IL-33 is pro-regenerative in a noninjurious model of liver resection. The underlying mechanism involved IL-33/ST2-induced increase of serotonin release from enterochromaffin cells to portal blood and subsequent HTR2A/p70S6K activation in hepatocytes by serotonin. The findings implicate the potential of targeting the IL-33/ST2/serotonin pathway to reduce the risk of post-hepatectomy liver failure and small-for-size syndrome.

The microsomal prostaglandin E synthase-1/prostaglandin E2 axis induces recovery from ischaemia via recruitment of regulatory T cells.

AIMS: Microsomal prostaglandin E synthase-1 (mPGES-1)/prostaglandin E2 (PGE2) induces angiogenesis through the prostaglandin E2 receptor (EP1-4). Among immune cells, regulatory T cells (Tregs), which inhibit immune responses, have been implicated in angiogenesis, and PGE2 is known to modulate the function and differentiation of Tregs. We hypothesized that mPGES-1/PGE2-EP signalling could contribute to recovery from ischaemic conditions by promoting the accumulation of Tregs. METHODS AND RESULTS: Wild-type (WT), mPGES-1-deficient (mPges-1-/-), and EP4 receptor-deficient (Ep4-/-) male mice, 6-8 weeks old, were used. Hindlimb ischaemia was induced by femoral artery ligation. Recovery from ischaemia was suppressed in mPges-1-/- mice and compared with WT mice. The number of accumulated forkhead box protein P3 (FoxP3)+ cells in ischaemic muscle tissue was decreased in mPges-1-/- mice compared with that in WT mice. Expression levels of transforming growth factor-ß (TGF-ß) and stromal cell derived factor-1 (SDF-1) in ischaemic tissue were also suppressed in mPges-1-/- mice. The number of accumulated FoxP3+ cells and blood flow recovery were suppressed when Tregs were depleted by injecting antibody against folate receptor 4 in WT mice but not in mPges-1-/- mice. Recovery from ischaemia was significantly suppressed in Ep4-/- mice compared with that in WT mice. Furthermore, mRNA levels of Foxp3 and Tgf-ß were suppressed in Ep4-/- mice. Moreover, the number of accumulated FoxP3+ cells in ischaemic tissue was diminished in Ep4-/- mice compared with that in Ep4+/+ mice. CONCLUSION: These findings suggested that mPGES-1/PGE2 induced neovascularization from ischaemia via EP4 by promoting the accumulation of Tregs. Highly selective EP4 agonists could be useful for the treatment of peripheral artery disease.

The Role of mPGES-1 in Promoting Granulation Tissue Angiogenesis Through Regulatory T-cell Accumulation.

BACKGROUND/AIM: Microsomal prostaglandin E synthase-1 (mPGES-1) is an enzyme, which catalyzes the final step of prostaglandin E2 (PGE2) synthesis. PGE2 in involved in wound-induced angiogenesis. Regulatory T cells (Tregs) regulate not only immune tolerance but also tissue repair and angiogenesis. We examined whether the mPGES-1/PGE2 axis contributes to wound-induced angiogenesis and granulation tissue formation through Treg accumulation. MATERIALS AND METHODS: The dorsal subcutaneous tissues of male mPGES-1-deficient (mPGES-1-/-) and C57BL/6 wild-type (WT) mice were implanted with polyurethane sponge disks. Angiogenesis was estimated by determining the wet weight of sponge tissues and the expression of proangiogenic factors including CD31, vascular endothelial growth factor (VEGF), and transforming growth factor ß (TGF-ß) in granulation tissues. RESULTS: Angiogenesis was suppressed in mPGES-1-/- mice compared with WT mice, which was associated with attenuated forkhead box P3 (Foxp3) expression and Foxp3+ Treg accumulation. The number of cells double-positive for Foxp3/TGFß and Foxp3/VEGF were lower in mPGES-1-/- mice than in WT mice. Neutralizing Tregs with antibodies (Abs) against CD25 or folate receptor 4 (FR4) inhibited the Foxp3+ Treg angiogenesis and accumulation in WT mice, but not in mPGES-1-/- mice. The topical application of PGE2 into the implanted sponge enhanced angiogenesis and accumulation of Tregs expressing TGFß and VEGF in WT and mPGES-1-/- mice. CONCLUSION: Tregs producing TGFß and VEGF accumulate in wounds and contribute to angiogenesis through mPGES-1-derived PGE2 mPGES-1 induction may control angiogenesis in skin wounds by recruiting Tregs.

B cell-intrinsic TBK1 is essential for germinal center formation during infection and vaccination in mice.

The germinal center (GC) is a site where somatic hypermutation and clonal selection are coupled for antibody affinity maturation against infections. However, how GCs are formed and regulated is incompletely understood. Here, we identified an unexpected role of Tank-binding kinase-1 (TBK1) as a crucial B cell-intrinsic factor for GC formation. Using immunization and malaria infection models, we show that TBK1-deficient B cells failed to form GC despite normal Tfh cell differentiation, although some malaria-infected B cell-specific TBK1-deficient mice could survive by GC-independent mechanisms. Mechanistically, TBK1 phosphorylation elevates in B cells during GC differentiation and regulates the balance of IRF4/BCL6 expression by limiting CD40 and BCR activation through noncanonical NF-κB and AKTT308 signaling. In the absence of TBK1, CD40 and BCR signaling synergistically enhanced IRF4 expression in Pre-GC, leading to BCL6 suppression, and therefore failed to form GCs. As a result, memory B cells generated from TBK1-deficient B cells fail to confer sterile immunity upon reinfection, suggesting that TBK1 determines B cell fate to promote long-lasting humoral immunity.

ILC2s Improve Glucose Metabolism Through the Control of Saturated Fatty Acid Absorption Within Visceral Fat.

Background and aims: Group 2 innate lymphoid cells (ILC2s) have been implicated in the regulation of metabolic homeostasis in mice. Methods: In this study, the role of ILC2s in white adipose tissue (WAT) was investigated using ST2, an IL-33 receptor that is expressed on ILC2 knockout mice. Results: The deficiency of ST2 decreased ILC2s in WAT, whereas ex-ILC2, which acquired group 1 innate lymphoid cell (ILC1)-like traits, was increased. This led to significant metabolic disorders such as visceral fat obesity, decreased browning in WAT, reduction of energy metabolism, and impaired glucose tolerance, compared to wild type (WT) mice. Those metabolic abnormalities of ST2-knockout (ST2KO) mice were not ameliorated by IL-33 administration, but impaired glucose tolerance and visceral fat obesity were significantly improved by transplantation of ILCs from the bone marrow of WT mice. The relative expression of Cd36 in WAT increased due to the deficiency of ST2, and the storage of saturated fatty acids in WAT of ST2KO mice was significantly higher than that of WT mice. Moreover, saturated fatty acids aggravated the chronic inflammation in adipocytes, promoted the differentiation of M1-like macrophages, and inhibited that of M2-like macrophages. Conclusions: Our results indicated that ILC2 regulates diet-induced obesity and chronic inflammation through the regulation of saturated fatty acid absorption in visceral adipose tissue.

Immunoregulatory Monocyte Subset Promotes Metastasis Associated With Therapeutic Intervention for Primary Tumor.

Systemic and local inflammation associated with therapeutic intervention of primary tumor occasionally promotes metastatic recurrence in mouse and human. However, it remains unclear what types of immune cells are involved in this process. Here, we found that the tissue-repair-promoting Ym1+Ly6Chi monocyte subset expanded as a result of systemic and local inflammation induced by intravenous injection of lipopolysaccharide or resection of primary tumor and promoted lung metastasis originating from circulating tumor cells (CTCs). Deletion of this subset suppressed metastasis induced by the inflammation. Furthermore, transfer of Ym1+Ly6Chi monocytes into naïve mice promoted lung metastasis in the mice. Ym1+Ly6Chi monocytes highly expressed matrix metalloproteinase-9 (MMP-9) and CXCR4. MMP-9 inhibitor and CXCR4 antagonist decreased Ym1+Ly6Chi-monocyte-promoted lung metastasis. These findings indicate that Ym1+Ly6Chi monocytes are therapeutic target cells for metastasis originating from CTCs associated with systemic and local inflammation. In addition, these findings provide a novel predictive cellular biomarker for metastatic recurrence after intervention for primary tumor.

Rhodobacter azotoformans LPS (RAP99-LPS) Is a TLR4 Agonist That Inhibits Lung Metastasis and Enhances TLR3-Mediated Chemokine Expression.

The lipopolysaccharides (LPSs) of Rhodobacter are reported to be TLR4 antagonists. Accordingly, the extract of Rhodobacter azotoformans (RAP99) is used as a health supplement for humans and animals in Japan to regulate immune responses in vivo. We previously analyzed the LPS structure of RAP99 (RAP99-LPS) and found it is different from that of E. coli-LPS but similar to lipid A from Rhodobacter sphaeroides (RSLA), a known antagonist of TLR4, with both having three C14 fatty acyl groups, two C10 fatty acyl groups, and two phosphates. Here we show that RAP99-LPS has an immune stimulatory activity and acts as a TLR4 agonist. Pretreatment of RAP99-LPS suppressed E. coli-LPS-mediated weight loss, suggesting it is an antagonist against E. coli-LPS like other LPS isolated from Rhodobacter. However, injections of RAP99-LPS caused splenomegaly and increased immune cell numbers in C57BL/6 mice but not in C3H/HeJ mice, suggesting that RAP99-LPS stimulates immune cells via TLR4. Consistently, RAP99-LPS suppressed the lung metastasis of B16F1 tumor cells and enhanced the expression of TLR3-mediated chemokines. These results suggest that RAP99-LPS is a TLR4 agonist that enhances the activation status of the immune system to promote anti-viral and anti-tumor activity in vivo.

Loss of FCHSD1 leads to amelioration of chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD/emphysema) is a life-threatening disorder and there are few effective therapies. Cigarette smoke-induced oxidative stress, airway inflammation, and apoptosis of lung cells have been reported to be involved in the pathogenesis of COPD/emphysema and lead to alveolar septal destruction. Here we show that the expression level of FCH and double SH3 domains 1 (FCHSD1) was drastically increased in mice in response to elastase instillation, an experimental model of COPD. FCHSD1 is a member of the F-BAR family with two SH3 domains. We found that Fchsd1 knockout (Fchsd1-/-) mice were protected against airspace enlargement induced by elastase. Elastase-instilled lungs of Fchsd1-/- mice showed reduced inflammation and apoptosis compared with WT mice. We also found that elastase-induced reduction of Sirtuin 1 (SIRT1) levels, a histone deacetylase reported to protect against emphysema, was attenuated in the lungs of Fchsd1-/- mice. Furthermore, FCHSD1 deficiency enhanced nuclear translocation of nuclear factor-like 2 (NRF2), a redox-sensitive transcription factor, following H2O2 stimulation. Conversely, Fchsd1 overexpression inhibited NRF2 nuclear translocation and increased the reduction of SIRT1 levels. Notably, FCHSD1 interacted with NRF2 and SNX9. Our results show that FCHSD1 forms a multicomplex with NRF2 and SNX9 in the cytosol that prevents NRF2 from translocating to the nucleus. We propose that FCHSD1 promotes initiation of emphysema development by inhibiting nuclear translocation of NRF2, which leads to down-regulation of SIRT1.

Novel insights into the pathogenesis of lung fibrosis: the RBM7-NEAT1-CXCL12-SatM axis at fibrosis onset.

Fibrosis is a life-threatening disorder with significant morbidity and mortality and is caused by excessive formation of connective tissue that can affect several important organs. Fibrosis in organ tissues is caused by an abnormal wound-healing process from repeated injuries. In our recent study using a mouse model of bleomycin-induced lung fibrosis, we examined the role of RNA-binding motif protein 7 (RBM7) on the development of lung fibrosis. RBM7 is up-regulated in the injured lung epithelium and disturbs normal epithelial cell repair and regeneration by promoting apoptosis of damaged epithelial cells. RBM7 causes the decay of nuclear-enriched abundant transcript 1 (NEAT1), which results in apoptosis of lung epithelial cells. These apoptotic cells then produce C-X-C motif chemokine ligand 12 (CXCL12), which leads to the recruitment of a fibrosis-promoting monocyte population called segregated-nucleus-containing atypical monocytes (SatM) to the damaged area, followed by the initiation and promotion of lung fibrosis. Here, we review recent insights into the cross-talk between lung parenchymal cells and hematopoietic cells during the development of pulmonary fibrosis.

Loss of IL-33 enhances elastase-induced and cigarette smoke extract-induced emphysema in mice.

BACKGROUND: IL-33, which is known to induce type 2 immune responses via group 2 innate lymphoid cells, has been reported to contribute to neutrophilic airway inflammation in chronic obstructive pulmonary disease. However, its role in the pathogenesis of emphysema remains unclear. METHODS: We determined the role of interleukin (IL)-33 in the development of emphysema using porcine pancreas elastase (PPE) and cigarette smoke extract (CSE) in mice. First, IL-33-/- mice and wild-type (WT) mice were given PPE intratracheally. The numbers of inflammatory cells, and the levels of cytokines and chemokines in the bronchoalveolar lavage (BAL) fluid and lung homogenates, were analyzed; quantitative morphometry of lung sections was also performed. Second, mice received CSE by intratracheal instillation. Quantitative morphometry of lung sections was then performed again. RESULTS: Intratracheal instillation of PPE induced emphysematous changes and increased IL-33 levels in the lungs. Compared to WT mice, IL-33-/- mice showed significantly greater PPE-induced emphysematous changes. No differences were observed between IL-33-/- and WT mice in the numbers of macrophages or neutrophils in BAL fluid. The levels of hepatocyte growth factor were lower in the BAL fluid of PPE-treated IL-33-/- mice than WT mice. IL-33-/- mice also showed significantly greater emphysematous changes in the lungs, compared to WT mice, following intratracheal instillation of CSE. CONCLUSION: These observations suggest that loss of IL-33 promotes the development of emphysema and may be potentially harmful to patients with COPD.

Eosinophils attenuate hepatic ischemia-reperfusion injury in mice through ST2-dependent IL-13 production.

Eosinophils are a myeloid cell subpopulation that mediates type 2 T helper cell immune responses. Unexpectedly, we identified a rapid accumulation of eosinophils in 22 human liver grafts after hepatic transplantation. In contrast, no eosinophils were detectable in healthy liver tissues before transplantation. Studies with two genetic mouse models of eosinophil deficiency and a mouse model of antibody-mediated eosinophil depletion revealed exacerbated liver injury after hepatic ischemia and reperfusion. Adoptive transfer of bone marrow-derived eosinophils normalized liver injury of eosinophil-deficient mice and reduced hepatic ischemia and reperfusion injury in wild-type mice. Mechanistic studies combining genetic and adoptive transfer approaches identified a critical role of suppression of tumorigenicity (ST2)-dependent production of interleukin-13 by eosinophils in the hepatoprotection against ischemia-reperfusion-induced injury. Together, these data provide insight into a mechanism of eosinophil-mediated liver protection that could serve as a therapeutic target to improve outcomes of patients undergoing liver transplantation.

Neutrophil Gelatinase-Associated Lipocalin Protects Acinar Cells From Cerulein-Induced Damage During Acute Pancreatitis.

OBJECTIVES: Elevated neutrophil gelatinase-associated lipocalin (NGAL) is a promising marker for severe acute pancreatitis (SAP) and multiple organ failure, suggesting systemic and local contributions during pancreatitis. We investigated the role of NGAL locally on acinar cell biology. METHODS: Western blot, reverse transcriptase-polymerase chain reaction, and immunohistochemistry analysis were performed to analyze the levels of NGAL receptors, apoptotic and regeneration markers, and 4-hydroxynonenal (4HNE) levels, 3-[4,5-Dimethylthiazole-2-yl]-2, 5-diphenyltetrazolium bromide assay, and annexin V/propidium iodide staining were used to evaluate cell viability, and effect on endothelial cells was accessed by endothelial permeability assay. RESULTS: Cerulein treatment at 20 µM for 12 hours significantly reduced acinar cell viability by 40%, which was rescued by NGAL at 800 and 1600 ng/mL concentrations, observed during mild and SAP, respectively. Mechanistically, NGAL significantly reduced the levels of reactive oxygen species and 4HNE adduct formation in a 24p3R-dependent manner and upregulated the expression of acinar cell regeneration markers, like CDK-2, CDK-4, and C-myc. However, SAP levels of NGAL significantly increased endothelial permeability and downregulated the levels of ZO-1, and cerulein treatment in NGAL knockout mice showed increased levels of 4HNE adducts. CONCLUSIONS: Neutrophil gelatinase-associated lipocalin rescues intracellular reactive oxygen species during pancreatitis and promotes survival and regeneration of acinar cells.

JMJD3 acts in tandem with KLF4 to facilitate reprogramming to pluripotency.

The interplay between the Yamanaka factors (OCT4, SOX2, KLF4 and c-MYC) and transcriptional/epigenetic co-regulators in somatic cell reprogramming is incompletely understood. Here, we demonstrate that the histone H3 lysine 27 trimethylation (H3K27me3) demethylase JMJD3 plays conflicting roles in mouse reprogramming. On one side, JMJD3 induces the pro-senescence factor Ink4a and degrades the pluripotency regulator PHF20 in a reprogramming factor-independent manner. On the other side, JMJD3 is specifically recruited by KLF4 to reduce H3K27me3 at both enhancers and promoters of epithelial and pluripotency genes. JMJD3 also promotes enhancer-promoter looping through the cohesin loading factor NIPBL and ultimately transcriptional elongation. This competition of forces can be shifted towards improved reprogramming by using early passage fibroblasts or boosting JMJD3's catalytic activity with vitamin C. Our work, thus, establishes a multifaceted role for JMJD3, placing it as a key partner of KLF4 and a scaffold that assists chromatin interactions and activates gene transcription.

Hepatic lipocalin 2 promotes liver fibrosis and portal hypertension.

Advanced fibrosis and portal hypertension influence short-term mortality. Lipocalin 2 (LCN2) regulates infection response and increases in liver injury. We explored the role of intrahepatic LCN2 in human alcoholic hepatitis (AH) with advanced fibrosis and portal hypertension and in experimental mouse fibrosis. We found hepatic LCN2 expression and serum LCN2 level markedly increased and correlated with disease severity and portal hypertension in patients with AH. In control human livers, LCN2 expressed exclusively in mononuclear cells, while its expression was markedly induced in AH livers, not only in mononuclear cells but also notably in hepatocytes. Lcn2-/- mice were protected from liver fibrosis caused by either ethanol or CCl4 exposure. Microarray analysis revealed downregulation of matrisome, cell cycle and immune related gene sets in Lcn2-/- mice exposed to CCl4, along with decrease in Timp1 and Edn1 expression. Hepatic expression of COL1A1, TIMP1 and key EDN1 system components were elevated in AH patients and correlated with hepatic LCN2 expression. In vitro, recombinant LCN2 induced COL1A1 expression. Overexpression of LCN2 increased HIF1A that in turn mediated EDN1 upregulation. LCN2 contributes to liver fibrosis and portal hypertension in AH and could represent a new therapeutic target.