Heme sequestration by hemophilin from Haemophilus haemolyticus reduces respiratory tract colonization and infection with non-typeable Haemophilus influenzae.

Iron acquisition is a key feature dictating the success of pathogen colonization and infection. Pathogens scavenging iron from the host must contend with other members of the microbiome similarly competing for the limited pool of bioavailable iron, often in the form of heme. In this study, we identify a beneficial role for the heme-binding protein hemophilin (Hpl) produced by the non-pathogenic bacterium Haemophilus haemolyticus against its close relative, the opportunistic respiratory tract pathogen non-typeable Haemophilus influenzae (NTHi). Using a mouse model, we found that pre-exposure to H. haemolyticus significantly reduced NTHi colonization of the upper airway and impaired NTHi infection of the lungs in an Hpl-dependent manner. Further, treatment with recombinant Hpl was sufficient to decrease airway burdens of NTHi without exacerbating lung immunopathology or systemic inflammation. Instead, mucosal production of the neutrophil chemokine CXCL2, lung myeloperoxidase, and serum pro-inflammatory cytokines IL-6 and TNFα were lower in Hpl-treated mice. Mechanistically, H. haemolyticus suppressed NTHi growth and adherence to human respiratory tract epithelial cells through the expression of Hpl, and recombinant Hpl could recapitulate these effects. Together, these findings indicate that heme sequestration by non-pathogenic, Hpl-producing H. haemolyticus is protective against NTHi colonization and infection. IMPORTANCE: The microbiome provides a critical layer of protection against infection with bacterial pathogens. This protection is accomplished through a variety of mechanisms, including interference with pathogen growth and adherence to host cells. In terms of immune defense, another way to prevent pathogens from establishing infections is by limiting the availability of nutrients, referred to as nutritional immunity. Restricting pathogen access to iron is a central component of this approach. Here, we uncovered an example where these two strategies intersect to impede infection with the respiratory tract bacterial pathogen Haemophilus influenzae. Specifically, we find that a non-pathogenic (commensal) bacterium closely related to H. influenzae called Haemophilus haemolyticus improves protection against H. influenzae by limiting the ability of this pathogen to access iron. These findings suggest that beneficial members of the microbiome improve protection against pathogen infection by effectively contributing to host nutritional immunity.

Neutrophil responsiveness to IL-10 impairs clearance of Streptococcus pneumoniae from the lungs.

The early immune response to bacterial pneumonia requires a careful balance between pathogen clearance and tissue damage. The anti-inflammatory cytokine interleukin (IL)-10 is critical for restraining otherwise lethal pulmonary inflammation. However, pathogen-induced IL-10 is associated with bacterial persistence in the lungs. In this study, we used mice with myeloid cell specific deletion of IL-10R to investigate the cellular targets of IL-10 immune suppression during infection with Streptococcus pneumoniae, the most common bacterial cause of pneumonia. Our findings suggest that IL-10 restricts the neutrophil response to S. pneumoniae, as neutrophil recruitment to the lungs was elevated in myeloid IL-10 receptor (IL-10R)-deficient mice and neutrophils in the lungs of these mice were more effective at killing S. pneumoniae. Improved killing of S. pneumoniae was associated with increased production of reactive oxygen species and serine protease activity in IL-10R-deficient neutrophils. Similarly, IL-10 suppressed the ability of human neutrophils to kill S. pneumoniae. Burdens of S. pneumoniae were lower in myeloid IL-10R-deficient mice compared with wild-type mice, and adoptive transfer of IL-10R-deficient neutrophils into wild-type mice significantly improved pathogen clearance. Despite the potential for neutrophils to contribute to tissue damage, lung pathology scores were similar between genotypes. This contrasts with total IL-10 deficiency, which is associated with increased immunopathology during S. pneumoniae infection. Together, these findings identify neutrophils as a critical target of S. pneumoniae-induced immune suppression and highlight myeloid IL-10R abrogation as a mechanism to selectively reduce pathogen burdens without exacerbating pulmonary damage.

Lack of VMP1 impairs hepatic lipoprotein secretion and promotes non-alcoholic steatohepatitis.

BACKGROUND & AIMS: Vacuole membrane protein 1 (VMP1) is an endoplasmic reticulum (ER) transmembrane protein that regulates the formation of autophagosomes and lipid droplets. Recent evidence suggests that VMP1 plays a critical role in lipoprotein secretion in zebra fish and cultured cells. However, the pathophysiological roles and mechanisms by which VMP1 regulates lipoprotein secretion and lipid accumulation in non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are unknown. METHODS: Liver-specific and hepatocyte-specific Vmp1 knockout mice as well as Vmp1 knock-in mice were generated by crossing Vmp1flox or Vmp1KI mice with albumin-Cre mice or by injecting AAV8-TBG-cre, respectively. Lipid and energy metabolism in these mice were characterized by metabolomic and transcriptome analyses. Mice with hepatic overexpression of VMP1 who were fed a NASH diet were also characterized. RESULTS: Hepatocyte-specific deletion of Vmp1 severely impaired VLDL secretion resulting in massive hepatic steatosis, hepatocyte death, inflammation and fibrosis, which are hallmarks of NASH. Mechanistically, loss of Vmp1 led to decreased hepatic levels of phosphatidylcholine and phosphatidylethanolamine as well as to changes in phospholipid composition. Deletion of Vmp1 in mouse liver also led to the accumulation of neutral lipids in the ER bilayer and impaired mitochondrial beta-oxidation. Overexpression of VMP1 ameliorated steatosis in diet-induced NASH by improving VLDL secretion. Importantly, we also showed that decreased liver VMP1 is associated with NAFLD/NASH in humans. CONCLUSIONS: Our results provide novel insights on the role of VMP1 in regulating hepatic phospholipid synthesis and lipoprotein secretion in the pathogenesis of NAFLD/NASH. LAY SUMMARY: Non-alcoholic fatty liver disease and its more severe form, non-alcoholic steatohepatitis, are associated with a build-up of fat in the liver (steatosis). However, the exact mechanisms that underly steatosis in patients are not completely understood. Herein, the authors identified that the lack of a protein called VMP1 impairs the secretion and metabolism of fats in the liver and could therefore contribute to the development and progression of non-alcoholic fatty liver disease.

Hepatocytic p62 suppresses ductular reaction and tumorigenesis in mouse livers with mTORC1 activation and defective autophagy.

BACKGROUND & AIMS: Either activation of mTORC1 due to loss of Tsc1 (tuberous sclerosis complex 1) or defective hepatic autophagy due to loss of Atg5 leads to spontaneous liver tumorigenesis in mice. The purpose of this study was to investigate the mechanisms by which autophagy contributes to the hepatic metabolic changes and tumorigenesis mediated by mTORC1 activation. METHODS: Atg5 Flox/Flox (Atg5F/F) and Tsc1F/F mice were crossed with albumin-Cre mice to generate liver-specific Atg5 knockout (L-Atg5 KO), L-Tsc1 KO and L-Atg5/Tsc1 double KO (DKO) mice. These mice were crossed with p62/Sqstm1F/F (p62) and whole body Nrf2 KO mice to generate L-Atg5/Tsc1/p62 and L-Atg5/Tsc1-Nrf2 triple KO mice. These mice were housed for various periods up to 12 months, and blood and liver tissues were harvested for biochemical and histological analysis RESULTS: Deletion of Atg5 in L-Tsc1 KO mice inhibited liver tumorigenesis but increased mortality and was accompanied by drastically enhanced hepatic ductular reaction (DR), hepatocyte degeneration and metabolic reprogramming. Deletion of p62 reversed DR, hepatocyte degeneration and metabolic reprogramming as well as the mortality of L-Atg5/Tsc1 DKO mice, but unexpectedly promoted liver tumorigenesis via activation of a group of oncogenic signaling pathways. Nrf2 ablation markedly improved DR with increased hepatocyte population and improved metabolic reprogramming and survival of the L-Atg5/Tsc1 DKO mice without tumor formation. Decreased p62 and increased mTOR activity were also observed in a subset of human hepatocellular carcinomas. CONCLUSIONS: These results reveal previously undescribed functions of hepatic p62 in suppressing tumorigenesis and regulating liver cell repopulation and metabolic reprogramming resulting from persistent mTORC1 activation and defective autophagy. LAY SUMMARY: Metabolic liver disease and viral hepatitis are common chronic liver diseases and risk factors of hepatocellular carcinoma, which are often associated with impaired hepatic autophagy and increased mTOR activation. Using multiple genetically engineered mouse models of defective hepatic autophagy and persistent mTOR activation, we dissected the complex mechanisms behind this observation. Our results uncovered an unexpected novel tumor suppressor function of p62/Sqstm1, which regulated liver cell repopulation, ductular reaction and metabolic reprogramming in liver tumorigenesis.

Liver-specific deletion of mechanistic target of rapamycin does not protect against acetaminophen-induced liver injury in mice.

BACKGROUND: Acetaminophen (APAP) overdose can cause liver injury and liver failure, which is one of the most common causes of drug-induced liver injury in the United States. Pharmacological activation of autophagy by inhibiting mechanistic target of rapamycin (mTOR) protects against APAP-induced liver injury likely via autophagic removal of APAP-adducts and damaged mitochondria. In the present study, we aimed to investigate the role of genetic ablation of mTOR pathways in mouse liver in APAP-induced liver injury and liver repair/regeneration. METHODS: Albumin-Cre (Alb-Cre) mice, mTORf/f and Raptorf/f mice (C57BL/6J background) were crossbred to produce liver-specific mTOR knockout (L-mTOR KO, Alb Cre+/-, mTORf/f) and liver-specific Raptor KO (L-Raptor, Alb Cre+/-, Raptor f/f) mice. Alb-Cre littermates were used as wild-type (WT) mice. These mice were treated with APAP for various time points for up to 48 h. Liver injury, cell proliferation, autophagy and mTOR activation were determined. RESULTS: We found that genetic deletion of neither Raptor, an important adaptor protein in mTOR complex 1, nor mTOR, in the mouse liver significantly protected against APAP-induced liver injury despite increased hepatic autophagic flux. Genetic deletion of Raptor or mTOR in mouse livers did not affect APAP metabolism and APAP-induced c-Jun N-terminal kinase (JNK) activation, but slightly improved mouse survival likely due to increased hepatocyte proliferation. CONCLUSIONS: Our results indicate that genetic ablation of mTOR in mouse livers does not protect against APAP-induced liver injury but may slightly improve liver regeneration and mouse survival after APAP overdose.

Autophagy in liver diseases: A review.

The liver is a highly dynamic metabolic organ that plays critical roles in plasma protein synthesis, gluconeogenesis and glycogen storage, cholesterol metabolism and bile acid synthesis as well as drug/xenobiotic metabolism and detoxification. Research from the past decades indicate that autophagy, the cellular catabolic process mediated by lysosomes, plays an important role in maintaining cellular and metabolic homeostasis in the liver. Hepatic autophagy fluctuates with hormonal cues and the availability of nutrients that respond to fed and fasting states as well as circadian activities. Dysfunction of autophagy in liver parenchymal and non-parenchymal cells can lead to various liver diseases including non-alcoholic fatty liver diseases, alcohol associated liver disease, drug-induced liver injury, cholestasis, viral hepatitis and hepatocellular carcinoma. Therefore, targeting autophagy may be a potential strategy for treating these various liver diseases. In this review, we will discuss the current progress on the understanding of autophagy in liver physiology. We will also discuss several forms of selective autophagy in the liver and the molecular signaling pathways in regulating autophagy of different cell types and their implications in various liver diseases.

Autophagy and liver cancer.

Autophagy is a highly conserved catabolic process that degrades cytosolic proteins and organelles via formation of autophagosomes that fuse with lysosomes to form autolysosomes, whereby autophagic cargos are degraded. Numerous studies have demonstrated that autophagy plays a critical role in the regulation of liver physiology and homeostasis, and impaired autophagy leads to the pathogenesis of various liver diseases such as viral hepatitis, alcohol associated liver diseases (AALD), non-alcoholic fatty liver diseases (NAFLD), and liver cancer. Recent evidence indicates that autophagy may play a dual role in liver cancer: inhibiting early tumor initiation while promoting progression and malignancy of already formed liver tumors. In this review, we summarized the progress of current understanding of how hepatic viral infection, alcohol consumption and diet-induced fatty liver diseases impair hepatic autophagy. We also discussed how impaired autophagy promotes liver tumorigenesis, and paradoxically how autophagy is required to promote the malignancy and progression of liver cancer. Understanding the molecular mechanisms underlying how autophagy differentially affects liver cancer development and progression may help to design better therapeutic strategies for prevention and treatment of liver cancer.