Meihong Deng

Lipopolysaccharide Clearance, Bacterial Clearance, and Systemic Inflammatory Responses Are Regulated by Cell Type–Specific Functions of TLR4 during Sepsis

The morbidity associated with bacterial sepsis is the result of host immune responses to pathogens, which are dependent on pathogen recognition by pattern recognition receptors, such as TLR4. TLR4 is expressed on a range of cell types, yet the mechanisms by which cell-specific functions of TLR4 lead to an integrated sepsis response are poorly understood. To address this, we generated mice in which TLR4 was specifically deleted from myeloid cells (LysMTLR4KO) or hepatocytes (HCTLR4KO) and then determined survival, bacterial counts, host inflammatory responses, and organ injury in a model of cecal ligation and puncture (CLP), with or without antibiotics. LysM-TLR4 was required for phagocytosis and efficient bacterial clearance in the absence of antibiotics. Survival, the magnitude of the systemic and local inflammatory responses, and liver damage were associated with bacterial levels. HCTLR4 was required for efficient LPS clearance from the circulation, and deletion of HCTLR4 was associated with enhanced macrophage phagocytosis, lower bacterial levels, and improved survival in CLP without antibiotics. Antibiotic administration during CLP revealed an important role for hepatocyte LPS clearance in limiting sepsis-induced inflammation and organ injury. Our work defines cell type–selective roles for TLR4 in coordinating complex immune responses to bacterial sepsis and suggests that future strategies for modulating microbial molecule recognition should account for varying roles of pattern recognition receptors in multiple cell populations.

Lipopolysaccharide Stimulates p62-Dependent Autophagy-Like Aggregate Clearance in Hepatocytes

Impairment of autophagy has been associated with liver injury. TLR4-stimulation by LPS upregulates autophagy in hepatocytes, although the signaling pathways involved remain elusive. The objective of this study was to determine the signaling pathway leading to LPS-stimulated autophagy in hepatocytes. Cell lysates from livers of wild type (WT; C57BL/6) mice given LPS (5 mg/kg-IP) and hepatocytes from WT, TLR4ko, and MyD88ko mice treated with LPS (100 ng/mL) up to 24 h were collected. LC3II, p62/SQSTM1, Nrf2, and beclin1 levels were determined by immunoblot, immunofluorescence, and qPCR. Autophagy-like activation was measured by GFP-LC3-puncta formation and LC3II-expression. Beclin1, Nrf2, p62, MyD88, and TIRAP were knocked-down using siRNA. LC3II-expression increased in both liver and hepatocytes after LPS and was dependent on TLR4. Beclin1 expression did not increase after LPS in hepatocytes and beclin1-knockdown did not affect LC3II levels. In hepatocytes given LPS, expression of p62 increased and p62 colocalized with LC3. p62-knockdown prevented LC3II puncta formation. LPS-induced LC3II/p62-puncta also required MyD88/TIRAP signaling and localization of both Nrf2 and NFκB transcription factors to the nucleus to upregulate p62-expression. Therefore, TLR4-activation by LPS in hepatocytes induces a p62-mediated, not beclin1-mediated, autophagy-like clearance pathway that is hepatoprotective by clearing aggregate-prone or misfolded proteins from the cytosol and preserving energy homeostasis under stress.

Shedding of the tumor necrosis factor (TNF) receptor from the surface of hepatocytes during sepsis limits inflammation through cGMP signaling

Drugs used to treat erectile dysfunction may be repurposed to inhibit inflammation and treat patients with sepsis. Taming TNF during sepsis The proinflammatory cytokine tumor necrosis factor (TNF) is required for an effective immune response to invading pathogens; however, excessive TNF signaling through its receptor TNFR leads to cell death and tissue damage, such as occurs during sepsis. The proteolytic shedding of TNFR from the cell surface decreases TNF signaling in cells and reduces TNF concentrations in the plasma. Deng et al. showed that treating mouse or human hepatocytes with the bacterial product LPS resulted in increased shedding of TNFR by the protease TACE, an event dependent on the second messenger cGMP. Treating mice that had sepsis with drugs that increased the plasma concentration of cGMP decreased inflammation and increased survival, suggesting that a similar strategy might provide a therapy against sepsis in humans. Proteolytic cleavage of the tumor necrosis factor (TNF) receptor (TNFR) from the cell surface contributes to anti-inflammatory responses and may be beneficial in reducing the excessive inflammation associated with multiple organ failure and mortality during sepsis. Using a clinically relevant mouse model of polymicrobial abdominal sepsis, we found that the production of inducible nitric oxide synthase (iNOS) in hepatocytes led to the cyclic guanosine monophosphate (cGMP)–dependent activation of the protease TACE (TNF-converting enzyme) and the shedding of TNFR. Furthermore, treating mice with a cGMP analog after the induction of sepsis increased TNFR shedding and decreased systemic inflammation. Similarly, increasing the abundance of cGMP with a clinically approved phosphodiesterase 5 inhibitor (sildenafil) also decreased markers of systemic inflammation, protected against organ injury, and increased circulating amounts of TNFR1 in mice with sepsis. We further confirmed that a similar iNOS-cGMP-TACE pathway was required for TNFR1 shedding by human hepatocytes in response to the bacterial product lipopolysaccharide. Our data suggest that increasing the bioavailability of cGMP might be beneficial in ameliorating the inflammation associated with sepsis.

Oestrogen sulfotransferase ablation sensitizes mice to sepsis

Sepsis is the hosts deleterious systemic inflammatory response to microbial infections. Here we report an essential role for the estrogen sulfotransferase (EST or SULT1E1), a conjugating enzyme that sulfonates and deactivates estrogens, in sepsis response. Both the cecal ligation and puncture (CLP) and lipopolysacharide (LPS) models of sepsis induce the expression of EST and compromise the activity of estrogen, an anti-inflammatory hormone. Surprisingly, EST ablation sensitizes mice to sepsis-induced death. Mechanistically, EST ablation attenuates sepsis-induced inflammatory responses due to compromised estrogen deactivation, leading to increased sepsis lethality. In contrast, transgenic overexpression of EST promotes estrogen deactivation and sensitizes mice to CLP-induced inflammatory response. The induction of EST by sepsis is NF-κB dependent and EST is a NF-κB target gene. The reciprocal regulation of inflammation and EST may represent a yet to be explored mechanism of endocrine regulation of inflammation, which has an impact on the clinical outcome of sepsis.

Toll-like Receptor 4 Signaling on Dendritic Cells Suppresses Polymorphonuclear Leukocyte CXCR2 Expression and Trafficking via Interleukin 10 During Intra-abdominal Sepsis.

BACKGROUND Toll-like receptor 4 (TLR4) is a critical receptor involved in the sensing of gram-negative bacterial infection. However, the roles of TLR4 in sepsis are cell-type specific. Dendritic cells (DCs) are known to play a central role in microbial detection, alerting the immune system to the presence of infection and coordinating adaptive immune response. The goal of this study was to investigate the impact of DC-specific TLR4 signaling on host defense against intra-abdominal polymicrobial sepsis. METHODS C57BL/6, global Tlr4 knockout, cell-specific knockout control, and CD11c-specific Tlr4(-/-) mice underwent cecal ligation and puncture (CLP). RESULTS Specific deletion of TLR4 on DCs in mice improved survival and enhanced bacterial clearance. Deletion of TLR4 on DCs was associated with lower levels of circulating interleukin 10 (IL-10), higher polymorphonuclear leukocyte (PMN) accumulation in the peritoneal cavity, and higher expression of chemokine (C-X-C motif) receptor 2 (CXCR2) on PMNs after CLP. In vitro studies of DC and neutrophil cocultures confirmed that TLR4-dependent secretion of IL-10 from DCs regulated neutrophil CXCR2 expression. CONCLUSIONS Our data shed light on a previously unrecognized role for TLR4 signaling on DCs in driving IL-10 secretion during sepsis and, through this pathway, regulates PMN recruitment via suppression of CXCR2 expression.

cGAS-mediated autophagy protects the liver from ischemia/reperfusion injury independent of STING

Liver ischemia-reperfusion (I/R) injury occurs through induction of oxidative stress and release of damage-associated molecular patterns (DAMPs), including cytosolic DNA released from dysfunctional mitochondria or from the nucleus. Cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) is a cytosolic DNA sensor known to trigger stimulator of interferon genes (STING) and downstream type 1 interferon (IFN-I) pathways, which are pivotal innate immune system responses to pathogen. However, little is known about the role of cGAS/STING in liver I/R injury. We subjected C57BL/6 (WT), cGAS knockout (cGAS-/-), and STING-deficient (STINGgt/gt) mice to warm liver I/R injury and that found cGAS-/- mice had significantly increased liver injury compared with WT or STINGgt/gt mice, suggesting a protective effect of cGAS independent of STING. Liver I/R upregulated cGAS in vivo and also in vitro in hepatocytes subjected to anoxia/reoxygenation (A/R). We confirmed a previously published finding that hepatocytes do not express STING under normoxic conditions or after A/R. Hepatocytes and liver from cGAS-/- mice had increased cell death and reduced induction of autophagy under hypoxic conditions as well as increased apoptosis. Protection could be restored in cGAS-/- hepatocytes by overexpression of cGAS or by pretreatment of mice with autophagy inducer rapamycin. Our findings indicate a novel protective role for cGAS in the regulation of autophagy during liver I/R injury that occurs independently of STING. NEW & NOTEWORTHY Our studies are the first to document the important role of cGAS in the acute setting of sterile injury induced by I/R. Specifically, we provide evidence that cGAS protects liver from I/R injury in a STING-independent manner.

The Endotoxin Delivery Protein HMGB1 Mediates Caspase-11-Dependent Lethality in Sepsis

Graphical Abstract Figure. No caption available. SUMMARY Caspase‐11, a cytosolic endotoxin (lipopolysaccharide: LPS) receptor, mediates pyroptosis, a lytic form of cell death. Caspase‐11‐dependent pyroptosis mediates lethality in endotoxemia, but it is unclear how LPS is delivered into the cytosol for the activation of caspase‐11. Here we discovered that hepatocyte‐released high mobility group box 1 (HMGB1) was required for caspase‐11‐dependent pyroptosis and lethality in endotoxemia and bacterial sepsis. Mechanistically, hepatocyte‐released HMGB1 bound LPS and targeted its internalization into the lysosomes of macrophages and endothelial cells via the receptor for advanced glycation end‐products (RAGE). Subsequently, HMGB1 permeabilized the phospholipid bilayer in the acidic environment of lysosomes. This resulted in LPS leakage into the cytosol and caspase‐11 activation. Depletion of hepatocyte HMGB1, inhibition of hepatocyte HMGB1 release, neutralizing extracellular HMGB1, or RAGE deficiency prevented caspase‐11‐dependent pyroptosis and death in endotoxemia and bacterial sepsis. These findings indicate that HMGB1 interacts with LPS to mediate caspase‐11‐dependent pyroptosis in lethal sepsis. HIGHLIGHTSHepatocyte‐released HMGB1 mediates caspase‐11‐dependent lethality in sepsisHMGB1‐LPS complexes are taken up by macrophages and endothelial cells via RAGEHMGB1 destabilizes phagolysosomes for the transfer of LPS to cytosolic caspase‐11Neutralizing extracellular HMGB1 inhibits caspase‐11 activation in sepsis &NA; Caspase‐11, a cytosolic LPS receptor, mediates endotoxic shock. Deng and colleagues demonstrate that hepatocyte‐released HMGB1 mediates caspase‐11‐dependent pyroptosis and lethality in sepsis by delivering extracellular LPS into the cytosol of macrophages and endothelial cells, where LPS activates caspase‐11.

Cyclic stretch induced IL-33 production through HMGB1/TLR-4 signaling pathway in murine respiratory epithelial cells

Interleukin 33 (IL-33), an inflammatory and mechanically responsive cytokine, is an important component of a TLR4-dependent innate immune process in mucosal epithelium. Although TLR4 also plays a role in sensing biomechanical stretch, a pathway of stretch-induced TLR4-dependent IL-33 biosynthesis has not been revealed. In the current study, we show that short term (6 h) cyclic stretch (CS) of cultured murine respiratory epithelial cells (MLE-12) increased intracellular IL-33 expression in a TLR4 dependent fashion. There was no detectable IL-33 in conditioned media in this interval. CS, however, increased release of the notable alarmin, HMGB1, and a neutralizing antibody (2G7) to HMGB1 completely abolished the CS mediated increase in IL-33. rHMGB1 increased IL-33 synthesis and this was partially abrogated by silencing TLR4 suggesting additional receptors for HMGB1 are involved in its regulation of IL-33. Collectively, these data reveal a HMGB1/TLR4/IL-33 pathway in the response of respiratory epithelium to mechanical stretch.

iNOS promotes CD24+CD133+ liver cancer stem cell phenotype through a TACE/ADAM17-dependent Notch signaling pathway

Significance CD24+CD133+ liver cancer stem cells (LCSCs) express higher levels of the inducible nitric oxide synthase (iNOS) and possess self-renewal and tumor growth properties. iNOS is associated with more aggressive hepatocellular carcinoma (HCC), leading to the upregulation of Notch1 signaling. The activation of Notch1 by iNOS/NO is dependent on cGMP/PKG-mediated activation of TACE and upregulation of iRhom-2. The expression of iNOS, CD24, and CD133 correlates with the expression of activated TACE and Notch signaling in more aggressive human HCC. These findings have implications for understanding how LCSCs are regulated in the setting of chronic inflammation, where signals to upregulate iNOS are often present. Targeting iNOS could have therapeutic benefit in HCC. The inducible nitric oxide synthase (iNOS) is associated with more aggressive solid tumors, including hepatocellular carcinoma (HCC). Notch signaling in cancer stem cells promotes cancer progression and requires Notch cleavage by ADAM (a disintegrin and metalloprotease) proteases. We hypothesized that iNOS/NO promotes Notch1 activation through TACE/ADAM17 activation in liver cancer stem cells (LCSCs), leading to a more aggressive cancer phenotype. Expression of the stem cell markers CD24 and CD133 in the tumors of patients with HCC was associated with greater iNOS expression and worse outcomes. The expression of iNOS in CD24+CD133+ LCSCs, but not CD24−CD133− LCSCs, promoted Notch1 signaling and stemness characteristics in vitro and in vivo, as well as accelerating HCC initiation and tumor formation in the mouse xenograft tumor model. iNOS/NO led to Notch1 signaling through a pathway involving the soluble guanylyl cyclase/cGMP/PKG-dependent activation of TACE/ADAM17 and up-regulation of iRhom2 in LCSCs. In patients with HCC, higher TACE/ADAM17 expression and Notch1 activation correlated with poor prognosis. These findings link iNOS to Notch1 signaling in CD24+CD133+ LCSCs through the activation of TACE/ADAM17 and identify a mechanism for how iNOS contributes to progression of CD24+CD133+ HCC.

Interleukin-33 contributes to ILC2 activation and early inflammation-associated lung injury during abdominal sepsis

Sepsis is defined as infection with organ dysfunction due to a dysregulated immune response. The lung is one of the most vulnerable organs at the onset of sepsis. Interleukin (IL)‐33 can be released by injured epithelial and endothelial cells in the lung and regulate immune activation and infiltration. Therefore, we postulated that IL‐33 would contribute to the immune response in the lung during sepsis. Using the cecal ligation and puncture (CLP) sepsis model, we show here that IL‐33 contributes significantly to both sepsis‐induced inflammation in the lung and systemic inflammatory response in the early phase of sepsis. Despite the higher intra‐peritoneal bacterial burden, the absence of IL‐33 resulted in less infiltration of neutrophils and monocytes into the lungs in association with lower circulating, lung and liver cytokine levels as well as reduced lung injury at 6 h after sepsis. IL‐33 was required for the upregulation of IL‐5 in type 2 Innate Lymphoid Cells (ILC2), while IL‐5 neutralization suppressed neutrophil and monocyte infiltration in the lungs during CLP sepsis. This reduction in leukocyte infiltration in IL‐33‐deficient mice was reversed by administration of recombinant IL‐5. These results indicate that IL‐33 plays a major role in the local inflammatory changes in the lung, in part, by regulating IL‐5 and this axis contributes to lung injury early after the onset of sepsis.