Activation of TLR9 signaling suppresses the immunomodulating functions of CD55lo fibroblastic reticular cells during bacterial peritonitis.

Fibroblastic reticular cells (FRCs) are a subpopulation of stromal cells modulating the immune environments in health and disease. We have previously shown that activation of TLR9 signaling in FRC in fat-associated lymphoid clusters (FALC) regulate peritoneal immunity via suppressing immune cell recruitment and peritoneal resident macrophage (PRM) retention. However, FRCs are heterogeneous across tissues and organs. The functions of each FRC subset and the regulation of TLR9 in distinct FRC subsets are unknown. Here, we confirmed that specific deletion of TLR9 in FRC improved bacterial clearance and survival during peritoneal infection. Furthermore, using single-cell RNA sequencing, we found two subsets of FRCs (CD55hi and CD55lo) in the mesenteric FALC. The CD55hi FRCs were enriched in gene expression related to extracellular matrix formation. The CD55lo FRCs were enriched in gene expression related to immune response. Interestingly, we found that TLR9 is dominantly expressed in the CD55lo subset. Activation of TLR9 signaling suppressed proliferation, cytokine production, and retinoid metabolism in the CD55lo FRC, but not CD55hi FRC. Notably, we found that adoptive transfer of Tlr9 -/-CD55lo FRC from mesenteric FALC more effectively improved the survival during peritonitis compared with WT-FRC or Tlr9 -/-CD55hi FRC. Furthermore, we identified CD55hi and CD55lo subsets in human adipose tissue-derived FRC and confirmed the suppressive effect of TLR9 on the proliferation and cytokine production in the CD55lo subset. Therefore, inhibition of TLR9 in the CD55lo FRCs from adipose tissue could be a useful strategy to improve the therapeutic efficacy of FRC-based therapy for peritonitis.

Fibroblastic reticular cell-derived exosomes are a promising therapeutic approach for septic acute kidney injury.

Sepsis-induced acute kidney injury (S-AKI) is highly lethal, and effective drugs for treatment are scarce. Previously, we reported the robust therapeutic efficacy of fibroblastic reticular cells (FRCs) in sepsis. Here, we demonstrate the ability of FRC-derived exosomes (FRC-Exos) to improve C57BL/6 mouse kidney function following cecal ligation and puncture-induced sepsis. In vivo imaging confirmed that FRC-Exos homed to injured kidneys. RNA-Seq analysis of FRC-Exo-treated primary kidney tubular cells (PKTCs) revealed that FRC-Exos influenced PKTC fate in the presence of lipopolysaccharide (LPS). FRC-Exos promoted kinase PINK1-dependent mitophagy and inhibited NLRP3 inflammasome activation in LPS-stimulated PKTCs. To dissect the mechanism underlying the protective role of Exos in S-AKI, we examined the proteins within Exos by mass spectrometry and found that CD5L was the most upregulated protein in FRC-Exos compared to macrophage-derived Exos. Recombinant CD5L treatment in vitro attenuated kidney cell swelling and surface bubble formation after LPS stimulation. FRCs were infected with a CD5L lentivirus to increase CD5L levels in FRC-Exos, which were then modified in vitro with the kidney tubular cell targeting peptide LTH, a peptide that binds to the biomarker protein kidney injury molecule-1 expressed on injured tubule cells, to enhance binding specificity. Compared with an equivalent dose of recombinant CD5L, the modified CD5L-enriched FRC-Exos selectively bound PKTCs, promoted kinase PINK-ubiquitin ligase Parkin-mediated mitophagy, inhibiting pyroptosis and improved kidney function by hindering NLRP3 inflammasome activation, thereby improving the sepsis survival rate. Thus, strategies to modify FRC-Exos could be a new avenue in developing therapeutics against kidney injury.

Intraperitoneal injection of class A TLR9 agonist enhances anti-PD-1 immunotherapy in colorectal peritoneal metastases.

Peritoneal metastases are associated with a low response rate to immune checkpoint blockade (ICB) therapy. The numbers of peritoneal resident macrophages (PRMs) are reversely correlated with the response rate to ICB therapy. We have previously shown that TLR9 in fibroblastic reticular cells (FRCs) plays a critical role in regulating peritoneal immune cell recruitment. However, the role of TLR9 in FRCs in regulating PRMs is unclear. Here, we demonstrated that the class A TLR9 agonist, ODN1585, markedly enhanced the efficacy of anti-PD-1 therapy in mouse models of colorectal peritoneal metastases. ODN1585 injected i.p. reduced the numbers of Tim4+ PRMs and enhanced CD8+ T cell antitumor immunity. Mechanistically, treatment of ODN1585 suppressed the expression of genes required for retinoid metabolism in FRCs, and this was associated with reduced expression of the PRM lineage-defining transcription factor GATA6. Selective deletion of TLR9 in FRCs diminished the benefit of ODN1585 in anti-PD-1 therapy in reducing peritoneal metastases. The crosstalk between PRMs and FRCs may be utilized to develop new strategies to improve the efficacy of ICB therapy for peritoneal metastases.

Corrigendum to "Release of Danger Signals during Ischemic Storage of the Liver: A Potential Marker of Organ Damage?"

[This corrects the article DOI: 10.1155/2010/436145.].

GATA6+ Peritoneal Resident Macrophage: The Immune Custodian in the Peritoneal Cavity.

Peritoneal resident macrophages (PRMs) have been a prominent topic in the research field of immunology due to their critical roles in immune surveillance in the peritoneal cavity. PRMs initially develop from embryonic progenitor cells and are replenished by bone marrow origin monocytes during inflammation and aging. Furthermore, PRMs have been shown to crosstalk with other cells in the peritoneal cavity to control the immune response during infection, injury, and tumorigenesis. With the advance in genetic studies, GATA-binding factor 6 (GATA6) has been identified as a lineage determining transcription factor of PRMs controlling the phenotypic and functional features of PRMs. Here, we review recent advances in the developmental origin, the phenotypic identity, and functions of PRMs, emphasizing the role of GATA6 in the pathobiology of PRMs in host defense, tissue repairing, and peritoneal tumorigenesis.

Neutrophils Extracellular Traps Inhibition Improves PD-1 Blockade Immunotherapy in Colorectal Cancer.

Immune checkpoint inhibitors can improve the prognosis of patients with advanced malignancy; however, only a small subset of advanced colorectal cancer patients in microsatellite-instability-high or mismatch-repair-deficient colorectal cancer can benefit from immunotherapy. Unfortunately, the mechanism behind this ineffectiveness is unclear. The tumor microenvironment plays a critical role in cancer immunity, and may contribute to the inhibition of immune checkpoint inhibitors and other novel immunotherapies in patients with advanced cancer. Herein, we demonstrate that the DNase I enzyme plays a pivotal role in the degradation of NETs, significantly dampening the resistance to anti-PD-1 blockade in a mouse colorectal cancer model by attenuating tumor growth. Remarkably, DNase I decreases tumor-associated neutrophils and the formation of MC38 tumor cell-induced neutrophil extracellular trap formation in vivo. Mechanistically, the inhibition of neutrophil extracellular traps with DNase I results in the reversal of anti-PD-1 blockade resistance through increasing CD8+ T cell infiltration and cytotoxicity. These findings signify a novel approach to targeting the tumor microenvironment using DNase I alone or in combination with immune checkpoint inhibitors.

Mechanical Ventilation Exacerbates Poly (I:C) Induced Acute Lung Injury: Central Role for Caspase-11 and Gut-Lung Axis.

Background: The mechanisms by which moderate tidal volume ventilation (MTV) exacerbates preexisting lung injury are unclear. We hypothesized that systemic endotoxemia via the gut-lung axis would lead to non-canonical and canonical inflammasome activation and pyroptosis in a two-hit model involving polyinosinic-polycytidylic acid (Poly(I:C)), a synthetic analog of dsRNA and MTV and that this would associate with acute lung injury (ALI). Methods: Anesthetized mice were administered Poly(I:C) intratracheally and then 6 h later, they were mechanically ventilated for 4 h with otherwise non-injurious MTV (10ml/kg). Changes in intestinal and alveolar capillary permeability were measured. Further documentation of ALI was assessed by evans blue albumin permeability, protein and IL-1 family concentration in bronchoalveolar lavage fluid (BALF) or plasma, and histopathology in cohorts of wildtype (WT), whole body genetically ablated caspase-11 (caspase-11-/-), caspase-1/caspase-11 double knockout (caspase-1/11-/-), gasdermin D (GSDMD)-/-, nucleotide-binding domain leucine-rich repeat-containing protein 3 (NLRP3)-/- and advanced glycosylation end product-specific receptor (RAGE) -/- mice. Results: Non-injurious MTV exacerbated the mild lung injury associated with Poly(I:C) administration. This included the disruption of alveolar-capillary barrier and increased levels of interleukin (IL)-6, high mobility group proteins 1 (HMGB-1), IL-1ß in BALF and IL-18 in plasma. Combined (Poly(I:C)-MTV) injury was associated with increase in gastrointestinal permeability and endotoxin in plasma and BALF. Poly(I:C)-MTV injury was sensitive to caspase-11 deletion with no further contribution of caspase-1 except for maturation and release of IL-18 (that itself was sensitive to deletion of NLRP3). Combined injury led to large increases in caspase-1 and caspase-11. Genetic ablation of GSDMD attenuated alveolar-capillary disruption and release of cytokines in combined injury model. Conclusions: The previously noted exacerbation of mild Poly(I:C)-induced ALI by otherwise non-injurious MTV is associated with an increase in gut permeability resulting in systemic endotoxemia. The gut-lung axis resulted in activation of pulmonary non-canonical (cytosolic mediated caspase-11 activation) and canonical (caspase-1) inflammasome (NLRP3) mediated ALI in this two-hit model resulting in GSDMD sensitive alveolar capillary barrier disruption, pyroptosis (alveolar macrophages) and cytokine maturation and release (IL-1ß; IL-18). Pharmacologic strategies aimed at disrupting communication between gut and lung, inhibition of inflammasomes or GSDMD in pyroptosis may be useful in ALI.

Pre-operative exercise therapy triggers anti-inflammatory trained immunity of Kupffer cells through metabolic reprogramming.

Pre-operative exercise therapy improves outcomes for many patients who undergo surgery. Despite the well-known effects on tolerance to systemic perturbation, the mechanisms by which pre-operative exercise protects the organ that is operated on from inflammatory injury are unclear. Here, we show that four-week aerobic pre-operative exercise significantly attenuates liver injury and inflammation from ischaemia and reperfusion in mice. Remarkably, these beneficial effects last for seven more days after completing pre-operative exercising. We find that exercise specifically drives Kupffer cells toward an anti-inflammatory phenotype with trained immunity via metabolic reprogramming. Mechanistically, exercise-induced HMGB1 release enhances itaconate metabolism in the tricarboxylic acid cycle that impacts Kupffer cells in an NRF2-dependent manner. Therefore, these metabolites and cellular/molecular targets can be investigated as potential exercise-mimicking pharmaceutical candidates to protect against liver injury during surgery.

Identification of ILC2 in the Lung Using Flow Cytometry.

Group 2 innate lymphoid cells (ILC2), despite their scarcity, are the dominant innate lymphoid cell population in the lung, orchestrating innate immunity and adaptive immunity (Germain and Huang. Curr Opin Immunol 56:76-81, 2019; Krabbendam et al. Immunol Rev 286:74-85, 2018; Mindt et al. Front Immunol 9:840, 2018) . Recent studies reveal that ILC2 play critical roles in inflammation-associated lung injury during sepsis (Lai et al. Cell Death Dis 9:369, 2018; Xu et al. Immunol Cell Biol 96:935-947). Therefore, studies aiming to understand the pathobiology of ILC2 may reveal new therapeutic strategies for sepsis. However, the identification of ILC2 requires multiple surface and intracellular markers. This makes the detection of ILC2 in the lung challenging. Here we describe a method to detect ILC2 in the mouse lung using flow cytometry.

Single-Cell Transcriptomics Reveals Compartment-Specific Differences in Immune Responses and Contributions for Complement Factor 3 in Hemorrhagic Shock Plus Tissue Trauma.

ABSTRACT: Hemorrhagic shock with tissue trauma (HS/T) leads to the activation of a system-wide immune-inflammatory response that involves all organs and body compartments. Recent advances in single-cell analysis permit the simultaneous assessment of transcriptomic patterns in a large number of cells making it feasible to survey the landscape of immune cell responses across numerous anatomic sites. Here, we used single-cell RNA sequencing of leukocytes from the blood, liver, and spleen to identify the major shifts in gene expression by cell type and compartment in a mouse HS/T model. At 6 h, dramatic changes in gene expression were observed across multiple-cell types and in all compartments in wild-type mice. Monocytes from circulation and liver exhibited a significant upregulation of genes associated with chemotaxis and migration and a simultaneous suppression of genes associated with interferon signaling and antigen presentation. In contrast, liver conventional DC exhibited a unique pattern compared with other myeloid cells that included a pronounced increase in major histocompatibility complex class II (MHCII) gene expression. The dominant pattern across all compartments for B and T cells was a suppression of genes associated with cell activation and signaling after HS/T. Using complement factor 3 (C3) knockout mice we unveiled a role for C3 in the suppression of monocyte Major Histocompatibility Complex class II expression and activation of gene expression associated with migration, phagocytosis and cytokine upregulation, and an unexpected role in promoting interferon-signaling in a subset of B and T cells across all three compartments after HS/T. This transcriptomic landscape study of immune cells provides new insights into the host immune response to trauma, as well as a rich resource for further investigation of trauma-induced immune responses and complement in driving interferon signaling.

Maresin 1 protects the liver against ischemia/reperfusion injury via the ALXR/Akt signaling pathway.

BACKGROUND: Hepatic ischemia/reperfusion (I/R) injury can be a major complication following liver surgery contributing to post-operative liver dysfunction. Maresin 1 (MaR1), a pro-resolving lipid mediator, has been shown to suppress I/R injury. However, the mechanisms that account for the protective effects of MaR1 in I/R injury remain unknown. METHODS: WT (C57BL/6J) mice were subjected to partial hepatic warm ischemia for 60mins followed by reperfusion. Mice were treated with MaR1 (5-20 ng/mouse), Boc2 (Lipoxin A4 receptor antagonist), LY294002 (Akt inhibitor) or corresponding controls just prior to liver I/R or at the beginning of reperfusion. Blood and liver samples were collected at 6 h post-reperfusion. Serum aminotransferase, histopathologic changes, inflammatory cytokines, and oxidative stress were analyzed to evaluate liver injury. Signaling pathways were also investigated in vitro using primary mouse hepatocyte (HC) cultures to identify underlying mechanisms for MaR1 in liver I/R injury. RESULTS: MaR1 treatment significantly reduced ALT and AST levels, diminished necrotic areas, suppressed inflammatory responses, attenuated oxidative stress and decreased hepatocyte apoptosis in liver after I/R. Akt signaling was significantly increased in the MaR1-treated liver I/R group compared with controls. The protective effect of MaR1 was abrogated by pretreatment with Boc2, which together with MaR1-induced Akt activation. MaR1-mediated liver protection was reversed by inhibition of Akt. CONCLUSIONS: MaR1 protects the liver against hepatic I/R injury via an ALXR/Akt signaling pathway. MaR1 may represent a novel therapeutic agent to mitigate the detrimental effects of I/R-induced liver injury.

Mechanical Ventilation with Moderate Tidal Volume Exacerbates Extrapulmonary Sepsis-Induced Lung Injury via IL33-WISP1 Signaling Pathway.

ABSTRACT: IL-33 and WNT1-inducible secreted protein (WISP1) play central roles in acute lung injury (ALI) induced by mechanical ventilation with moderate tidal volume (MTV) in the setting of sepsis. Here, we sought to determine the inter-relationship between IL-33 and WISP1 and the associated signaling pathways in this process.We used a two-hit model of cecal ligation puncture (CLP) followed by MTV ventilation (4 h 10 mL/kg) in wild-type, IL-33-/- or ST2-/- mice or wild-type mice treated with intratracheal antibodies to WISP1. Macrophages (Raw 264.7 and alveolar macrophages from wild-type or ST2-/- mice) were used to identify specific signaling components.CLP + MTV resulted in ALI that was partially sensitive to genetic ablation of IL-33 or ST2 or antibody neutralization of WISP1. Genetic ablation of IL-33 or ST2 significantly prevented ALI after CLP + MTV and reduced levels of WISP1 in the circulation and bronchoalveolar lung fluid. rIL-33 increased WISP1 in alveolar macrophages in an ST2, PI3K/AKT, and ERK dependent manner. This WISP1 upregulation and WNT ß-catenin activation were sensitive to inhibition of the ß-catenin/TCF/CBP/P300 nuclear pathway.We show that IL-33 drives WISP1 upregulation and ALI during MTV in CLP sepsis. The identification of this relationship and the associated signaling pathways reveals a number of possible therapeutic targets to prevent ALI in ventilated sepsis patients.

A road map from single-cell transcriptome to patient classification for the immune response to trauma.

Immune dysfunction is an important factor driving mortality and adverse outcomes after trauma but remains poorly understood, especially at the cellular level. To deconvolute the trauma-induced immune response, we applied single-cell RNA sequencing to circulating and bone marrow mononuclear cells in injured mice and circulating mononuclear cells in trauma patients. In mice, the greatest changes in gene expression were seen in monocytes across both compartments. After systemic injury, the gene expression pattern of monocytes markedly deviated from steady state with corresponding changes in critical transcription factors, which can be traced back to myeloid progenitors. These changes were largely recapitulated in the human single-cell analysis. We generalized the major changes in human CD14+ monocytes into 6 signatures, which further defined 2 trauma patient subtypes (SG1 vs. SG2) identified in the whole-blood leukocyte transcriptome in the initial 12 hours after injury. Compared with SG2, SG1 patients exhibited delayed recovery, more severe organ dysfunction, and a higher incidence of infection and noninfectious complications. The 2 patient subtypes were also recapitulated in burn and sepsis patients, revealing a shared pattern of immune response across critical illness. Our data will be broadly useful to further explore the immune response to inflammatory diseases and critical illness.

Platelet-Monocyte Aggregates: Understanding Mechanisms and Functions in Sepsis.

ABSTRACT: Platelets have been shown to play an important immunomodulatory role in the pathogenesis of various diseases through their interactions with other immune and nonimmune cells. Sepsis is a major cause of death in the United States, and many of the mechanisms driving sepsis pathology are still unresolved. Monocytes have recently received increasing attention in sepsis pathogenesis, and multiple studies have associated increased levels of platelet-monocyte aggregates observed early in sepsis with clinical outcomes in sepsis patients. These findings suggest platelet-monocyte aggregates may be an important prognostic indicator. However, the mechanisms leading to platelet interaction and aggregation with monocytes, and the effects of aggregation during sepsis are still poorly defined. There are few studies that have really investigated functions of platelets and monocytes together, despite a large body of research showing separate functions of platelets and monocytes in inflammation and immune responses during sepsis. The goal of this review is to provide insights into what we do know about mechanisms and biological meanings of platelet-monocyte interactions, as well as some of the technical challenges and limitations involved in studying this important potential mechanism in sepsis pathogenesis. Improving our understanding of platelet and monocyte biology in sepsis may result in identification of novel targets that can be used to positively affect outcomes in sepsis.

Hepatocyte high-mobility group box 1 protects against steatosis and cellular stress during high fat diet feeding.

BACKGROUND: Circulating high-mobility group box 1 (HMGB1) plays important roles in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Intracellular HMGB1 is critical for the biology of hepatocytes. However, the intracellular role of HMGB1 in hepatocellular steatosis is unknown. Therefore, we aimed to investigate the role of hepatocyte-specific HMGB1 (HC-HMGB1) in development of hepatic steatosis. METHODS: Wild type (WT) C57BL/6 and HC-HMGB1-/- mice were fed high-fat diet (HFD) or low-fat diet (LFD) for up to 16 weeks. RESULTS: As expected, HMGB1 translocated from nuclear into cytoplasm and released into circulation after HFD treatment. HC-HMGB1 deficiency significantly reduced circulating HMGB1, suggesting that hepatocyte is a major source of circulating HMGB1 during NAFLD. Unexpectedly, HC-HMGB1 deficiency promoted rapid weight gain with enhanced hepatic fat deposition compared with WT at as early as 4 weeks after HFD treatment. Furthermore, there was no difference between WT and HC-HMGB1-/- mice in glucose tolerance, energy expenditure, liver damage or systemic inflammation. Interestingly, hepatic gene expression related to free fatty acid (FFA) ß-oxidation was significantly down-regulated in HC-HMGB1-/- mice compared with WT, and endoplasmic reticulum (ER) stress markers were significantly higher in livers of HC-HMGB1-/- mice. In vitro experiments using primary mouse hepatocytes showed absence of HMGB1 increased FFA-induced intracellular lipid accumulation, accompanied by increased ER-stress, significant downregulation of FFA ß-oxidation, and reduced oxidative phosphorylation. CONCLUSIONS: Our findings suggest that hepatocyte HMGB1 protects against dysregulated lipid metabolism via maintenance of ß-oxidation and prevention of ER stress. This represents a novel mechanism for HMGB1-regulation of hepatocellular steatosis, and suggests that stabilizing HMGB1 in hepatocytes may be effective strategies for prevention and treatment of NAFLD.

Notch signaling protects CD4 T cells from STING-mediated apoptosis during acute systemic inflammation.

Dysregulation of T cell apoptosis contributes to the pathogenesis of acute systemic inflammation-induced immunosuppression, as seen in sepsis and trauma. However, the regulatory mechanisms of T cell apoptosis are unclear. Activation of stimulator of interferon genes (STING) has been shown to induce T cell apoptosis. Notch was previously identified as the top negative regulator of STING in macrophages through a kinase inhibitor library screening. However, how Notch signaling regulates STING activation in T cells is unknown. Here, using a γ-secretase inhibitor to block Notch signaling, we found that Notch protected CD4 T cells from STING-mediated apoptosis during endotoxemia. Mechanistically, Notch intracellular domain (NICD) interacted with STING at the cyclic dinucleotide (CDN) binding domain and competed with CDN to inhibit STING activation. In conclusion, our data reveal a previously unidentified role of Notch in negative regulation of STING-mediated apoptosis in CD4 T cells.

LPS Induces Active HMGB1 Release From Hepatocytes Into Exosomes Through the Coordinated Activities of TLR4 and Caspase-11/GSDMD Signaling.

High-mobility group box-1 (HMGB1), a ubiquitous nuclear protein, acts as a late mediator of lethality when released extracellularly during sepsis. The major source of circulating HMGB1 in sepsis is hepatocytes. However, the mechanism of HMGB1 release of hepatocytes during sepsis is not very clear. We have previously shown that bacterial endotoxin [lipopolysaccharide (LPS)] sensing pathways, including Toll-like receptor (TLR)4 and caspase-11, regulate hepatocyte HMGB1 release in response to LPS. Here, we report the novel function of caspase-11 and gasdermin D (GsdmD) in LPS-induced active HMGB1 released from hepatocytes. HMGB1 release during endotoxemia was caspase-11/GsdmD dependent via an active way in vivo and in vitro. Caspase-11/GsdmD was responsible for HMGB1 translocation from nucleus to the cytoplasm via calcium changing-induced phosphorylation of calcium-calmodulin kinase kinase (camkk)ß during endotoxemia. Cleaved GsdmD accumulated on the endoplasmic reticulum, suggesting this may lead to calcium leak and intracellular calcium increase. Furthermore, we investigated that exosome was an important pathway for HMGB1 release from hepatocytes; this process was dependent on TLR4, independent of caspase-11 and GsdmD in vivo and in vitro. These findings provide a novel mechanism that TLR4 signaling results in an increase in caspase-11 expression, as well as increased exosome release, while caspase-11/GsdmD activation/cleavage leads to accumulation of HMGB1 in the cytoplasm through a process associated with the release of calcium from the endoplasmic reticulum and camkkß activation.

Immune-Responsive Gene 1/Itaconate Activates Nuclear Factor Erythroid 2-Related Factor 2 in Hepatocytes to Protect Against Liver Ischemia-Reperfusion Injury.

BACKGROUND AND AIMS: Itaconate, a metabolite of the tricarboxylic acid cycle, plays anti-inflammatory roles in macrophages during endotoxemia. The mechanisms underlying its anti-inflammatory roles have been shown to be mediated by the modulation of oxidative stress, an important mechanism of hepatic ischemia-reperfusion (I/R) injury. However, the role of itaconate in liver I/R injury is unknown. APPROACH AND RESULTS: We found that deletion of immune-responsive gene 1 (IRG1), encoding for the enzyme producing itaconate, exacerbated liver injury and systemic inflammation. Furthermore, bone marrow adoptive transfer experiments indicated that deletion of IRG1 in both hematopoietic and nonhematopoietic compartments contributes to the protection mediated by IRG1 after I/R. Interestingly, the expression of IRG1 was up-regulated in hepatocytes after I/R and hypoxia/reoxygenation-induced oxidative stress. Modulation of the IRG1 expression levels in hepatocytes regulated hepatocyte cell death. Importantly, addition of 4-octyl itaconate significantly improved liver injury and hepatocyte cell death after I/R. Furthermore, our data indicated that nuclear factor erythroid 2-related factor 2 (Nrf2) is required for the protective effect of IRG1 on mouse and human hepatocytes against oxidative stress-induced injury. Our studies document the important role of IRG1 in the acute setting of sterile injury induced by I/R. Specifically, we provide evidence that the IRG1/itaconate pathway activates Nrf2-mediated antioxidative response in hepatocytes to protect liver from I/R injury. CONCLUSIONS: Our data expand on the importance of IRG1/itaconate in nonimmune cells and identify itaconate as a potential therapeutic strategy for this unfavorable postsurgical complication.

TSLP protects against liver I/R injury via activation of the PI3K/Akt pathway.

Thymic stromal lymphopoietin (TSLP) is a cytokine mainly released by epithelial cells that plays important roles in inflammation, autoimmune disease, and cancer. While TSLP is expressed in the liver at high levels, the role of TSLP in liver ischemia/reperfusion (I/R) injury remains unknown. Experiments were carried out to determine the role of TSLP in liver I/R injury. Wild-type (WT) and TSLP receptor-knockout (TSLPR-/-) mice were subjected to liver partial warm I/R injury. Liver injury was assessed by measuring serum alanine aminotransferase (ALT) level, necrotic areas by liver histology, hepatocyte death, and local hepatic inflammatory responses. Signal pathways were explored in vivo and in vitro to identify possible mechanisms for TSLP in I/R injury. TSLP and TSLPR protein expression increased during liver I/R in vivo and following hepatocyte hypoxia/reoxygenation in vitro. Deletion of TSLPR or neutralization of TSLP with anti-TSLP antibody exacerbated liver injury in terms of serum ALT levels as well as necrotic areas in liver histology. Administration of exogenous recombinant mouse TSLP to WT mice significantly reduced liver damage compared with controls, but failed to prevent I/R injury in TSLPR-/- mice. TSLP induced autophagy in hepatocytes during liver I/R injury. Mechanistically, Akt was activated in WT mice during liver I/R injury. The opposite results were observed in TSLPR-/- mice. In addition, TSLP could directly induce Akt activation in hepatocytes independent of nonparenchymal cells in vitro. Furthermore, the Akt agonist, insulin-like growth factor-1 (IGF-1), prevented I/R injury in TSLPR-/- mice and an Akt inhibitor, LY294002, blocked the protective effects of TSLP in WT mice subjected to I/R. Our data indicate that TSLP protects against liver I/R injury via activation of the PI3K/Akt pathway. Through this pathway, TSLP induces autophagy in hepatocytes. Thus, TSLP is a potent inhibitor of stress-induced hepatocyte necrosis.