The Role of Glutathione Reductase in Influenza Infection.

Influenza infection induces lung epithelial cell injury via programmed cell death. Glutathione, a potent antioxidant, has been reported to be associated with influenza infection. We hypothesized that lung epithelial cell death during influenza infection is regulated by glutathione metabolism. Eight-week-old male and female BALB/c mice were infected with influenza (PR8: A/PR/8/34 [H1N1]) via intranasal instillation. Metabolomic analyses were performed on whole lung lysate after influenza infection. For in vitro analysis, Beas-2B cells were infected with influenza. RNA was extracted, and QuantiTect Primer Assay was used to assess gene expression. Glutathione concentrations were assessed by colorimetric assay. Influenza infection resulted in increased inflammation and epithelial cell injury in our murine model, leading to increased morbidity and mortality. In both our in vivo and in vitro models, influenza infection was found to induce apoptosis and necroptosis. Influenza infection led to decreased glutathione metabolism and reduced glutathione reductase activity in lung epithelial cells. Genetic inhibition of glutathione reductase suppressed apoptosis and necroptosis of lung epithelial cells. Pharmacologic inhibition of glutathione reductase reduced airway inflammation, lung injury, and cell death in our murine influenza model. Our results demonstrate that glutathione reductase activity is suppressed during influenza. Glutathione reductase inhibition prevents epithelial cell death and morbidity in our murine influenza model. Our results suggest that glutathione reductase-dependent glutathione metabolism may play an important role in the host response to viral infection by regulating lung epithelial cell death.

Association of circulating cell-free double-stranded DNA and metabolic derangements in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease with unclear aetiology and poorly understood pathophysiology. Although plasma levels of circulating cell-free DNA (ccf-DNA) and metabolomic changes have been reported in IPF, the associations between ccf-DNA levels and metabolic derangements in lung fibrosis are unclear. Here, we demonstrate that ccf-double-stranded DNA (dsDNA) is increased in patients with IPF with rapid progression of disease compared with slow progressors and healthy controls and that ccf-dsDNA associates with amino acid metabolism, energy metabolism and lipid metabolism pathways in patients with IPF.

Antiviral Gene Expression in Young and Aged Murine Lung during H1N1 and H3N2.

Influenza is a respiratory virus that alone or in combination with secondary bacterial pathogens can contribute to the development of acute pneumonia in persons >65 years of age. Host innate immune antiviral signaling early in response to influenza is essential to inhibit early viral replication and guide the initiation of adaptive immune responses. Using young adult (3 months) and aged adult mice infected with mouse adapted H1N1 or H3N2, the results of our study illustrate dysregulated and/or diminished activation of key signaling pathways in aged lung contribute to increased lung inflammation and morbidity. Specifically, within the first seven days of infection, there were significant changes in genes associated with TLR and RIG-I signaling detected in aged murine lung in response to H1N1 or H3N2. Taken together, the results of our study expand our current understanding of age-associated changes in antiviral signaling in the lung.

RIPK3 Activates MLKL-mediated Necroptosis and Inflammasome Signaling during Streptococcus Infection.

Community-acquired pneumonia is the most common type of pneumonia and remains a leading cause of morbidity and mortality worldwide. Although many different pathogens can contribute to pneumonia, Streptococcus pneumoniae is one of the common bacterial pathogens that underlie community-acquired pneumonia. RIPK3 (receptor-interacting protein kinase 3) is widely recognized as a key modulator of inflammation and cell death. To elucidate a potential role of RIPK3 in pneumonia, we examined plasma from healthy control subjects and patients positive for streptococcal pneumonia. In human studies, RIPK3 protein concentrations were significantly elevated and were identified as a potential plasma marker of pneumococcal pneumonia. To expand these findings, we used an in vivo murine model of pneumococcal pneumonia to demonstrate that RIPK3 deficiency leads to reduced bacterial clearance, severe pathological damage, and high mortality. Our results illustrated that RIPK3 forms a complex with RIPK1, MLKL (mixed-lineage kinase domain-like protein), and MCU (mitochondrial calcium uniporter) to induce mitochondrial calcium uptake and mitochondrial reactive oxygen species(mROS) production during S. pneumoniae infection. In macrophages, RIPK3 initiated necroptosis via the mROS-mediated mitochondrial permeability transition pore opening and NLRP3 inflammasome activation via the mROS-AKT pathway to protect against S. pneumoniae. In conclusion, our study demonstrated a mechanism by which RIPK3-initiated necroptosis is essential for host defense against S. pneumoniae.

Hepcidin Is Essential for Alveolar Macrophage Function and Is Disrupted by Smoke in a Murine Chronic Obstructive Pulmonary Disease Model.

Chronic obstructive pulmonary disease (COPD) is a debilitating lung disease associated with cigarette smoking. Alterations in local lung and systemic iron regulation are associated with disease progression and pathogenesis. Hepcidin, an iron regulatory peptide hormone, is altered in subjects with COPD; however, the molecular role of hepcidin in COPD pathogenesis remains to be determined. In this study, using a murine model of smoke-induced COPD, we demonstrate that lung and circulating hepcidin levels are inhibited by cigarette smoke. We show that cigarette smoke exposure increases erythropoietin and bone marrow-derived erythroferrone and leads to expanded but inefficient erythropoiesis in murine bone marrow and an increase in ferroportin on alveolar macrophages (AMs). AMs from smokers and subjects with COPD display increased expression of ferroportin as well as hepcidin. Notably, murine AMs exposed to smoke fail to increase hepcidin in response to Gram-negative or Gram-positive infection. Loss of hepcidin in vivo results in blunted functional responses of AMs and exaggerated responses to Streptococcus pneumoniae infection.

DROSHA-Dependent miRNA and AIM2 Inflammasome Activation in Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease. Chronic lung inflammation is linked to the pathogenesis of IPF. DROSHA, a class 2 ribonuclease III enzyme, has an important role in the biogenesis of microRNA (miRNA). The function of miRNAs has been identified in the regulation of the target gene or protein related to inflammatory responses via degradation of mRNA or inhibition of translation. The absent-in-melanoma-2 (AIM2) inflammasome is critical for inflammatory responses against cytosolic double stranded DNA (dsDNA) from pathogen-associated molecular patterns (PAMPs) and self-DNA from danger-associated molecular patterns (DAMPs). The AIM2 inflammasome senses double strand DNA (dsDNA) and interacts with the adaptor apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), which recruits pro-caspase-1 and regulates the maturation and secretion of interleukin (IL)-1ß and IL-18. A recent study showed that inflammasome activation contributes to lung inflammation and fibrogenesis during IPF. In the current review, we discuss recent advances in our understanding of the DROSHA-miRNA-AIM2 inflammasome axis in the pathogenesis of IPF.

GLUT1-dependent glycolysis regulates exacerbation of fibrosis via AIM2 inflammasome activation.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a rapidly progressive, fatal lung disease that affects older adults. One of the detrimental natural histories of IPF is acute exacerbation of IPF (AE-IPF), of which bacterial infection is reported to play an important role. However, the mechanism by which bacterial infection modulates the fibrotic response remains unclear. OBJECTIVES: Altered glucose metabolism has been implicated in the pathogenesis of fibrotic lung diseases. We have previously demonstrated that glucose transporter 1 (GLUT1)-dependent glycolysis regulates fibrogenesis in a murine fibrosis model. To expand on these findings, we hypothesised that GLUT1-dependent glycolysis regulates acute exacerbation of lung fibrogenesis during bacterial infection via AIM2 inflammasome activation. RESULTS: In our current study, using a murine model of Streptococcus pneumoniae (S. pneumoniae) infection, we investigated the potential role of GLUT1 on mediating fibrotic responses to an acute exacerbation during bleomycin-induced fibrosis. The results of our current study illustrate that GLUT1 deficiency ameliorates S. pneumoniae-mediated exacerbation of lung fibrosis (wild type (WT)/phosphate buffered saline (PBS), n=3; WT/S. pneumoniae, n=3; WT/Bleomycin, n=5 ; WT/Bleomycin+S. pneumoniae, n=7; LysM-Cre-Glut1fl/f /PBS, n=3; LysM-Cre-Glut1fl/fl /S. pneumoniae, n=3; LysM-Cre-Glut1fl/fl /Bleomycin, n=6; LysM-Cre-Glut1fl/fl /Bleomycin+S. pneumoniae, n=9, p=0.041). Further, the AIM2 inflammasome, a multiprotein complex essential for sensing cytosolic bacterial DNA as a danger signal, is an important regulator of this GLUT1-mediated fibrosis and genetic deficiency of AIM2 reduced bleomycin-induced fibrosis after S. pneumoniae infection (WT/PBS, n=6; WT/Bleomycin+S. pneumoniae, n=15; Aim2-/-/PBS, n=6, Aim2-/-/Bleomycin+S. pneumoniae, n=11, p=0.034). GLUT1 deficiency reduced expression and function of the AIM2 inflammasome, and AIM2-deficient mice showed substantial reduction of lung fibrosis after S. pneumoniae infection. CONCLUSION: Our results demonstrate that GLUT1-dependent glycolysis promotes exacerbation of lung fibrogenesis during S. pneumoniae infection via AIM2 inflammasome activation.

DROSHA-Dependent AIM2 Inflammasome Activation Contributes to Lung Inflammation during Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) has been linked to chronic lung inflammation. Drosha ribonuclease III (DROSHA), a class 2 ribonuclease III enzyme, plays a key role in microRNA (miRNA) biogenesis. However, the mechanisms by which DROSHA affects the lung inflammation during idiopathic pulmonary fibrosis (IPF) remain unclear. Here, we demonstrate that DROSHA regulates the absent in melanoma 2 (AIM2) inflammasome activation during idiopathic pulmonary fibrosis (IPF). Both DROSHA and AIM2 protein expression were elevated in alveolar macrophages of patients with IPF. We also found that DROSHA and AIM2 protein expression were increased in alveolar macrophages of lung tissues in a mouse model of bleomycin-induced pulmonary fibrosis. DROSHA deficiency suppressed AIM2 inflammasome-dependent caspase-1 activation and interleukin (IL)-1ß and IL-18 secretion in primary mouse alveolar macrophages and bone marrow-derived macrophages (BMDMs). Transduction of microRNA (miRNA) increased the formation of the adaptor apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) specks, which is required for AIM2 inflammasome activation in BMDMs. Our results suggest that DROSHA promotes AIM2 inflammasome activation-dependent lung inflammation during IPF.

Mitochondrial Dysfunction in Aged Macrophages and Lung during Primary Streptococcus pneumoniae Infection is Improved with Pirfenidone.

Pneumococcal infections remain a leading cause of death in older adults, with the most serious cases occurring in persons ≥65 years of age. There is an urgent need to investigate molecular pathways underlying these impairments and devise new therapeutics to modulate innate immunity. The goal of our current study is to understand the impact of chronological aging on mitochondrial function in response to Streptococcus pneumoniae, a causative agent of bacterial pneumonia. Using chronologically aged murine models, our findings demonstrate that decreased ATP production is associated with dysregulated mitochondrial complex expression, enhanced oxidative stress, diminished antioxidant responses, and decreased numbers of healthy mitochondria in aged adult macrophages and lung in response to S. pneumoniae. Pre-treatment of aged macrophages with pirfenidone, an anti-fibrotic drug with antioxidant and anti-inflammatory properties, improved mitochondrial function and decreased cellular oxidative stress responses. In vivo administration of pirfenidone decreased superoxide formation, increased healthy mitochondria number, improved ATP production, and decreased inflammatory cell recruitment and pulmonary oedema in aged mouse lung during infection. Taken together, our data shed light on the susceptibility of older persons to S. pneumoniae and provide a possible therapeutic to improve mitochondrial responses in this population.

NLRP3 inflammasome activation in aged macrophages is diminished during Streptococcus pneumoniae infection.

Pneumococcal infections are the eigth leading cause of death in the United States, and it is estimated that older patients (≥65 yr of age) account for the most serious cases. The goal of our current study is to understand the impact of biological aging on innate immune responses to Streptococcus pneumoniae, a causative agent of bacterial pneumonia. With the use of in vitro and in vivo aged murine models, our findings demonstrate that age-enhanced unfolded protein responses (UPRs) contribute to diminished inflammasome assembly and activation during S. pneumoniae infection. Pretreatment of aged mice with endoplasmic reticulum chaperone and the stress-reducing agent tauroursodeoxycholic acid (TUDCA) decreased mortality in aged hosts that was associated with increased NLRP3 inflammasome activation, improved pathogen clearance, and decreased pneumonitis during infection. Taken together, our data provide new evidence as to why older persons are more susceptible to S. pneumoniae and provide a possible therapeutic target to decrease morbidity and mortality in this population.

Glucose Transporter 1-Dependent Glycolysis Is Increased during Aging-Related Lung Fibrosis, and Phloretin Inhibits Lung Fibrosis.

Aging is associated with metabolic diseases such as type 2 diabetes mellitus, cardiovascular disease, cancer, and neurodegeneration. Aging contributes to common processes including metabolic dysfunction, DNA damage, and reactive oxygen species generation. Although glycolysis has been linked to cell growth and proliferation, the mechanisms by which the activation of glycolysis by aging regulates fibrogenesis in the lung remain unclear. The objective of this study was to determine if glucose transporter 1 (GLUT1)-induced glycolysis regulates age-dependent fibrogenesis of the lung. Mouse and human lung tissues were analyzed for GLUT1 and glycolytic markers using immunoblotting. Glycolytic function was measured using a Seahorse apparatus. To study the effect of GLUT1, genetic inhibition of GLUT1 was performed by short hairpin RNA transduction, and phloretin was used for pharmacologic inhibition of GLUT1. GLUT1-dependent glycolysis is activated in aged lung. Genetic and pharmacologic inhibition of GLUT1 suppressed the protein expression of α-smooth muscle actin, a key cytoskeletal component of activated fibroblasts, in mouse primary lung fibroblast cells. Moreover, we demonstrated that the activation of AMP-activated protein kinase, which is regulated by GLUT1-dependent glycolysis, represents a critical metabolic pathway for fibroblast activation. Furthermore, we demonstrated that phloretin, a potent inhibitor of GLUT1, significantly inhibited bleomycin-induced lung fibrosis in vivo. These results suggest that GLUT1-dependent glycolysis regulates fibrogenesis in aged lung and that inhibition of GLUT1 provides a potential target of therapy of age-related lung fibrosis.

NOX4-dependent fatty acid oxidation promotes NLRP3 inflammasome activation in macrophages.

Altered metabolism has been implicated in the pathogenesis of inflammatory diseases. NADPH oxidase 4 (NOX4), a source of cellular superoxide anions, has multiple biological functions that may be of importance in inflammation and in the pathogenesis of human metabolic diseases, including diabetes. However, the mechanisms by which NOX4-dependent metabolic regulation affect the innate immune response remain unclear. Here we show that deficiency of NOX4 resulted in reduced expression of carnitine palmitoyltransferase 1A (CPT1A), which is a key mitochondrial enzyme in the fatty acid oxidation (FAO) pathway. The reduced FAO resulted in less activation of the nucleotide-binding domain, leucine-rich-repeat-containing receptor (NLR), pyrin-domain-containing 3 (NLRP3) inflammasome in human and mouse macrophages. In contrast, NOX4 deficiency did not inhibit the activation of the NLR family, CARD-domain-containing 4 (NLRC4), the NLRP1 or the absent in melanoma 2 (AIM2) inflammasomes. We also found that inhibition of FAO by etomoxir treatment suppressed NLRP3 inflammasome activation. Furthermore, Nox4-deficient mice showed substantial reduction in caspase-1 activation and in interleukin (IL)-1ß and IL-18 production, and there was improved survival in a mouse model of NLRP3-mediated Streptococcus pneumoniae infection. The pharmacologic inhibition of NOX4 by either GKT137831, which is currently in phase 2 clinical trials, or VAS-2870 attenuated NLRP3 inflammasome activation. Our results suggest that NOX4-mediated FAO promotes NLRP3 inflammasome activation.

Age-Dependent Susceptibility to Pulmonary Fibrosis Is Associated with NLRP3 Inflammasome Activation.

Aging has been implicated in the development of pulmonary fibrosis, which has seen a sharp increase in incidence in those older than 50 years. Recent studies demonstrate a role for the nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3) inflammasome and its regulated cytokines in experimental lung fibrosis. In this study, we tested the hypothesis that age-related NLRP3 inflammasome activation is an important predisposing factor in the development of pulmonary fibrosis. Briefly, young and aged wild-type and NLRP3(-/-) mice were subjected to bleomycin-induced lung injury. Pulmonary fibrosis was determined by histology and hydroxyproline accumulation. Bone marrow and alveolar macrophages were isolated from these mice. NLRP3 inflammasome activation was assessed by co-immunoprecipitation experiments. IL-1ß and IL-18 production was measured by ELISA. The current study demonstrated that aged wild-type mice developed more lung fibrosis and exhibited increased morbidity and mortality after bleomycin-induced lung injury, when compared with young mice. Bleomycin-exposed aged NLRP3(-/-) mice had reduced fibrosis compared with their wild-type age-matched counterparts. Bone marrow-derived and alveolar macrophages from aged mice displayed higher levels of NLRP3 inflammasome activation and caspase-1-dependent IL-1ß and IL-18 production, which was associated with altered mitochondrial function and increased production of reactive oxygen species. Our study demonstrated that age-dependent increases in alveolar macrophage mitochondrial reactive oxygen species production and NLRP3 inflammasome activation contribute to the development of experimental fibrosis.

Human metapneumovirus inhibits the IL-6-induced JAK/STAT3 signalling cascade in airway epithelium.

The host cytokine IL-6 plays an important role in host defence and prevention of lung injury from various pathogens, making IL-6 an important mediator in the host's susceptibility to respiratory infections. The cellular response to IL-6 is mediated through a Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) signal transduction pathway. Human metapneumovirus (hMPV) is an important causative agent of viral respiratory infections known to inhibit the IFN-mediated activation of STAT1. However, little is known about the interactions between this virus and other STAT signalling cascades. Herein, we showed that hMPV can attenuate the IL-6-mediated JAK/STAT3 signalling cascade in lung epithelial cells. HMPV inhibited a key event in this pathway by impeding the phosphorylation and nuclear translocation of STAT3 in A549 cells and in primary normal human bronchial epithelial cells. Further studies established that hMPV interrupted the IL-6-induced JAK/STAT pathway early in the signal transduction pathway by blocking the phosphorylation of JAK2. By antagonizing the IL-6-mediated JAK/STAT3 pathway, hMPV perturbed the expression of IL-6-inducible genes important for apoptosis, cell differentiation and growth. Infection with hMPV also differentially regulated the effects of IL-6 on apoptosis. Thus, hMPV regulation of these genes could usurp the protective roles of IL-6, and these data provide insight into an important element of viral pathogenesis.

Granzyme C supports efficient CTL-mediated killing late in primary alloimmune responses.

It is well established that granzymes A and B play a role in CTL killing of target cells by the perforin-dependent granule exocytosis pathway. The functions of multiple additional granzymes expressed in CTL are less well defined. In the present studies, CTL generated from mice deficient in dipeptidyl peptidase 1 (DPP1) were used to investigate the contribution of granzyme C to CTL killing of allogeneic target cells. DPP1 is required for activation of granzymes A and B by proteolytic removal of their N-terminal dipeptide prodomains while a significant portion of granzyme C is processed normally in the absence of DPP1. Cytotoxicity of DPP1(-/-) CTL generated in early (5-day) MLC in vitro and in peritoneal exudate cells 5 days after initial allogeneic sensitization in vivo was significantly impaired compared with wild-type CTL. Following 3 days of restimulation with fresh allogeneic stimulators however, cytotoxicity of these DPP1(-/-) effector cells was comparable to that of wild-type CTL. Killing mediated by DPP1(-/-) CTL following restimulation was rapid, perforin dependent, Fas independent and associated with early mitochondrial injury, phosphatidyl serine externalization, and DNA degradation, implicating a granzyme-dependent apoptotic pathway. The increased cytotoxicity of DPP1(-/-) CTL following restimulation coincided with increased expression of granzyme C. Moreover, small interfering RNA inhibition of granzyme C expression during restimulation significantly decreased cytotoxicity of DPP1(-/-) but not wild-type CTL. These results indicate that during late primary alloimmune responses, granzyme C can support CTL-mediated killing by the granule exocytosis pathway in the absence of functional granzymes A or B.

Intrahepatic lymphocyte expression of dipeptidyl peptidase I-processed granzyme B and perforin induces hepatocyte expression of serine proteinase inhibitor 6 (Serpinb9/SPI-6).

Human proteinase inhibitor 9 (PI-9/serpinB9) and the murine ortholog, serine proteinase inhibitor 6 (SPI-6/serpinb9) are members of a family of intracellular serine proteinase inhibitors (serpins). PI-9 and SPI-6 expression in immune-privileged cells, APCs, and CTLs protects these cells against the actions of granzyme B, and when expressed in tumor cells or virally infected hepatocytes, confers resistance to killing by CTL and NK cells. The present studies were designed to assess the existence of any correlation between granzyme B activity in intrahepatic lymphocytes and induction of hepatic SPI-6 expression. To this end, SPI-6, PI-9, and serpinB9 homolog expression was examined in response to IFN-alpha treatment and during in vivo adenoviral infection of the liver. SPI-6 mRNA expression increased 10- to 100-fold in the liver after IFN-alpha stimulation and during the course of viral infection, whereas no significant up-regulation of SPI-8 and <5-fold increases in other PI-9/serpinB9 homolog mRNAs was observed. Increased SPI-6 gene expression during viral infection correlated with influxes of NK cells and CTL. Moreover, IFN-alpha-induced up-regulation of hepatocyte SPI-6 mRNA expression was not observed in NK cell-depleted mice. Additional experiments using genetically altered mice either deficient in perforin or unable to process or express granzyme B indicated that SPI-6 is selectively up-regulated in hepatocytes in response to infiltration of the liver by NK cells that express perforin and enzymatically active granzyme B.

The role of serpinb9/serine protease inhibitor 6 in preventing granzyme B-dependent hepatotoxicity.

UNLABELLED: Virally infected hepatocytes are resistant to cytotoxic lymphocyte killing by perforin-dependent and granzyme-dependent effector mechanisms. The present studies were designed to examine the role of serine protease inhibitor 6 (SPI-6) in limiting granzyme B-dependent cytotoxic effector mechanisms in the liver. SPI-6-specific small interfering RNA (siRNA) administration to C57Bl/6J (B6) mice elicited transient alanine aminotransferase (ALT) elevations that were not observed in either granzyme B-deficient B6 (B6.gzmb(-/-)) or natural killer (NK) cell-depleted B6 mice. When SPI-6 expression was abolished by siRNA administration at the time of infection with a recombinant, replication-deficient adenovirus [E1-deleted adenovirus encoding beta-galactosidase (AdCMV-LacZ)], earlier and dramatically increased, and earlier ALT elevations were observed in wild-type B6 but not in B6.gzmb(-/-) or NK cell-depleted mice. When a 3-fold higher dose of AdCMV-LacZ was administered to B6 mice, the coadministration of SPI-6 siRNA resulted in the early onset of lethal, acute liver failure. Of note, the accelerated clearance of AdCMV-LacZ was observed in recipients of SPI-6 siRNA. CONCLUSION: These results indicate that the regulated expression of SPI-6 in hepatocytes during viral infection or following noninfectious causes of liver injury protects hepatocytes against excessively vigorous granzyme B-dependent killing but may also delay immune clearance of virally infected hepatocytes.