Cytokine signatures of end organ injury in COVID-19.

Increasing evidence has shown that Coronavirus disease 19 (COVID-19) severity is driven by a dysregulated immunologic response. We aimed to assess the differences in inflammatory cytokines in COVID-19 patients compared to contemporaneously hospitalized controls and then analyze the relationship between these cytokines and the development of Acute Respiratory Distress Syndrome (ARDS), Acute Kidney Injury (AKI) and mortality. In this cohort study of hospitalized patients, done between March third, 2020 and April first, 2020 at a quaternary referral center in New York City we included adult hospitalized patients with COVID-19 and negative controls. Serum specimens were obtained on the first, second, and third hospital day and cytokines were measured by Luminex. Autopsies of nine cohort patients were examined. We identified 90 COVID-19 patients and 51 controls. Analysis of 48 inflammatory cytokines revealed upregulation of macrophage induced chemokines, T-cell related interleukines and stromal cell producing cytokines in COVID-19 patients compared to the controls. Moreover, distinctive cytokine signatures predicted the development of ARDS, AKI and mortality in COVID-19 patients. Specifically, macrophage-associated cytokines predicted ARDS, T cell immunity related cytokines predicted AKI and mortality was associated with cytokines of activated immune pathways, of which IL-13 was universally correlated with ARDS, AKI and mortality. Histopathological examination of the autopsies showed diffuse alveolar damage with significant mononuclear inflammatory cell infiltration. Additionally, the kidneys demonstrated glomerular sclerosis, tubulointerstitial lymphocyte infiltration and cortical and medullary atrophy. These patterns of cytokine expression offer insight into the pathogenesis of COVID-19 disease, its severity, and subsequent lung and kidney injury suggesting more targeted treatment strategies.

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.

Targeted antioxidants as therapeutics for treatment of pneumonia in the elderly.

Community acquired pneumonia is a leading cause of mortality in the United States. Along with predisposing comorbid health status, age is an independent risk factor for determining the outcome of pneumonia. Research over the last few decades has contributed to better understanding the underlying immunodysregulation and imbalanced redox homeostasis tied to this aged population group that increases susceptibility to a wide range of pathologies. Major approaches include targeting oxidative stress by reducing ROS generation at its main sources of production which includes the mitochondrion. Mitochondria-targeted antioxidants have a number of molecular strategies that include targeting the biophysical properties of mitochondria, mitochondrial localization of catalytic enzymes, and mitigating mitochondrial membrane potential. Results of several antioxidant studies both in vitro and in vivo have demonstrated promising potential as a therapeutic in the treatment of pneumonia in the elderly. More human studies will need to be conducted to evaluate its efficacy in this clinical setting.

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.

Aging and Lung Disease.

People worldwide are living longer, and it is estimated that by 2050, the proportion of the world's population over 60 years of age will nearly double. Natural lung aging is associated with molecular and physiological changes that cause alterations in lung function, diminished pulmonary remodeling and regenerative capacity, and increased susceptibility to acute and chronic lung diseases. As the aging population rapidly grows, it is essential to examine how alterations in cellular function and cell-to-cell interactions of pulmonary resident cells and systemic immune cells contribute to a higher risk of increased susceptibility to infection and development of chronic diseases, such as chronic obstructive pulmonary disease and interstitial pulmonary fibrosis. This review provides an overview of physiological, structural, and cellular changes in the aging lung and immune system that facilitate the development and progression of disease.

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.

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.

Age-enhanced endoplasmic reticulum stress contributes to increased Atg9A inhibition of STING-mediated IFN-ß production during Streptococcus pneumoniae infection.

Pneumococcal infections remain a leading cause of death in persons ≥ 65 y of age. Recent reports have illustrated detrimental changes in the endoplasmic reticulum stress response or unfolded protein response in aging and age-related diseases; however, the relationship between aging, the unfolded protein response, and innate immune responses to Streptococcus pneumoniae has not been fully elucidated. Our results illustrate that stimulator of IFN genes-mediated production of IFN-ß during S. pneumoniae infection is decreased in aged hosts. Enhanced endoplasmic reticulum stress in response to S. pneumoniae augmented inositol-requiring protein 1/X-box binding protein 1-mediated production of autophagy-related gene 9 (Atg9a). Knockdown of Atg9a or treatment with gemcitabine HCl resulted in enhanced stimulator of IFN genes-mediated production of IFN-ß by aged macrophages. Consecutive treatments with gemcitabine during in vivo S. pneumoniae infection decreased morbidity and mortality in aged hosts, which was associated with decreased Atg9a expression, increased IFN-ß production, and improved bacterial clearance from lung tissue. Taken together, data presented in this study provide new evidence as to why older persons are more susceptible to S. pneumoniae, and provide a possible mechanism to enhance these responses, thereby decreasing morbidity and mortality in this population.

Impaired NLRP3 inflammasome function in elderly mice during influenza infection is rescued by treatment with nigericin.

The NLRP3 inflammasome is activated in the lung during influenza viral infection; however, the impact of aging on inflammasome function during influenza infection has not been examined. In this study, we show that elderly mice infected with a mouse-adapted strain of influenza produced lower levels of IL-1ß during in vitro and in vivo infection. Dendritic cells from elderly mice exhibited decreased expression of ASC, NLRP3, and capase-1 but increased expression of pro-IL-1ß, pro-IL-18, and pro-IL-33 compared with dendritic cells from young infected mice. Treatment with nigericin during influenza infection augmented IL-1ß production, increased caspase-1 activity, and decreased morbidity and mortality in elderly mice. Our study demonstrates for the first time, to our knowledge, that during influenza viral infection, elderly mice have impaired NLRP3 inflammasome activity and that treatment with nigericin rescues NLRP3 activation in elderly hosts.

Efficacy of a vaccine that links viral epitopes to flagellin in protecting aged mice from influenza viral infection.

Influenza vaccines are less effective in older people than younger people. This impaired ability to protect older people from influenza viral lung infection has important implications as older people suffer a higher morbidity and mortality from influenza viral lung infection than younger people. Therefore, the development of novel effective vaccines that induce protection from influenza viral infections in older people are urgently needed. We had previously shown that direct linking the TLR5 activator, flagellin, to viral peptides induces effective immunity to viral antigens in young mice and people, respectively. In this study, we tested the efficacy of this vaccine platform with the hemagglutinin peptide of the influenza A strain virus (vaccine denoted as STF2.HA1-2) in protecting aged mice from subsequent influenza viral lung infection. We found that a 3.0 µg dose of the vaccine was effective in reducing mortality and increasing clinical well-being during influenza viral lung infection in aged mice. However, this effect was inferior to the response induced in young mice. Defects in the adaptive immune system but not the innate immune system were associated with this reduced effectiveness of the vaccine with aging. Our results indicate that the STF2.HA1-2 vaccine is effective in protecting aged hosts from influenza lung infection, although defects in the adaptive immune system with aging may limit the effectiveness of this vaccine in older people.

Aging promotes neutrophil-induced mortality by augmenting IL-17 production during viral infection.

Morbidity and mortality associated with viral infections increase with age, although the underlying mechanisms are unclear. Here, we investigated whether aging alters inflammatory responses during systemic viral infection and thereby contributes to virus-induced death. We found that infection of aged mice with systemic herpes viruses led to rapid increases in serum IL-17, neutrophil activation, and mortality due to hepatocyte necrosis. In contrast, all young mice survived infection, displaying weaker IL-17 induction and neutrophil activation. Natural killer T (NKT) cells isolated from the livers of aged mice produced more IL-17 than did young cells, and adoptively transferred aged NKT cells induced liver injury in young mice impaired in viral control. Importantly, IL-17 neutralization or neutrophil depletion during viral infection reduced liver damage and prevented death of aged mice. These results demonstrate that, during systemic viral infection, aging alters the host-pathogen interaction to overproduce IL-17, contributing to liver injury and death.

Aging impairs IFN regulatory factor 7 up-regulation in plasmacytoid dendritic cells during TLR9 activation.

Plasmacytoid dendritic cells (pDCs) are innate sensors that produce IFN-alpha in response to viral infections. Determining how aging alters the cellular and molecular function of these cells may provide an explanation of increased susceptibility of older people to viral infections. Hence, we examined whether aging critically impairs pDC function during infection with HSV-2, a viral pathogen that activates TLR9. We found that impaired IFN-alpha production by aged murine pDCs led to impaired viral clearance with aging. Upon TLR9 activation, aged pDCs displayed defective up-regulation of IFN-regulatory factor 7, a key adaptor in the type I IFN pathway, as compared with younger counterparts. Aged pDCs had more oxidative stress, and reducing oxidative stress in aged pDCs partly recovered the age-induced IFN-alpha defect during TLR9 activation. In sum, aging impairs the type I IFN pathway in pDCs, and this alteration may contribute to the increased susceptibility of older people to certain viral infections.

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.