Monocyte Production of C1q Potentiates CD8+ T Cell Function Following Respiratory Viral Infection.

Respiratory viral infections remain a leading cause of morbidity and mortality. Using a murine model of human metapneumovirus (HMPV), we identified recruitment of a C1q-expressing inflammatory monocyte population concomitant with viral clearance by adaptive immune cells. Genetic ablation of C1q led to reduced CD8+ T cell function. Production of C1q by a myeloid lineage was necessary to enhance CD8+ T cell function. Activated and dividing CD8+ T cells expressed a C1q receptor, gC1qR. Perturbation of gC1qR signaling led to altered CD8+ T cell IFN-γ production, metabolic capacity, and cell proliferation. Autopsy specimens from fatal respiratory viral infections in children demonstrated diffuse production of C1q by an interstitial population. Humans with severe COVID-19 infection also demonstrated upregulation of gC1qR on activated and rapidly dividing CD8+ T cells. Collectively, these studies implicate C1q production from monocytes as a critical regulator of CD8+ T cell function following respiratory viral infection. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

IFN-λ drives distinct lung immune landscape changes and antiviral responses in human metapneumovirus infection.

Human metapneumovirus (HMPV) is a primary cause of acute respiratory infection, yet there are no approved vaccines or antiviral therapies for HMPV. Early host responses to HMPV are poorly characterized, and further understanding could identify important antiviral pathways. Type III interferon (IFN-λ) displays potent antiviral activity against respiratory viruses and is being investigated for therapeutic use. However, its role in HMPV infection remains largely unknown. Here, we show that IFN-λ is highly upregulated during HMPV infection in vitro in human and mouse airway epithelial cells and in vivo in mice. We found through several immunological and molecular assays that type II alveolar cells are the primary producers of IFN-λ. Using mouse models, we show that IFN-λ limits lung HMPV replication and restricts virus spread from upper to lower airways but does not contribute to clinical disease. Moreover, we show that IFN-λ signaling is predominantly mediated by CD45- non-immune cells. Mice lacking IFN-λ signaling showed diminished loss of ciliated epithelial cells and decreased recruitment of lung macrophages in early HMPV infection along with higher inflammatory cytokine and interferon-stimulated gene expression, suggesting that IFN-λ may maintain immunomodulatory responses. Administration of IFN-λ for prophylaxis or post-infection treatment in mice reduced viral load without inflammation-driven weight loss or clinical disease. These data offer clinical promise for IFN-λ in HMPV treatment. IMPORTANCE: Human metapneumovirus (HMPV) is a common respiratory pathogen and often contributes to severe disease, particularly in children, immunocompromised people, and the elderly. There are currently no licensed HMPV antiviral treatments or vaccines. Here, we report novel roles of host factor IFN-λ in HMPV disease that highlight therapeutic potential. We show that IFN-λ promotes lung antiviral responses by restricting lung HMPV replication and spread from upper to lower airways but does so without inducing lung immunopathology. Our data uncover recruitment of lung macrophages, regulation of ciliated epithelial cells, and modulation of inflammatory cytokines and interferon-stimulated genes as likely contributors. Moreover, we found these roles to be distinct and non-redundant, as they are not observed with knockout of, or treatment with, type I IFN. These data elucidate unique antiviral functions of IFN-λ and suggest IFN-λ augmentation as a promising therapeutic for treating HMPV disease and promoting effective vaccine responses.

PD-1 Impairs CD8+ T Cell Granzyme B Production in Aged Mice during Acute Viral Respiratory Infection.

CD8+ T cell dysfunction contributes to severe respiratory viral infection outcomes in older adults. CD8+ T cells are the primary cell type responsible for viral clearance. With increasing age, CD8+ T cell function declines in conjunction with an accumulation of cytotoxic tissue-resident memory (TRM) CD8+ T cells. We sought to elucidate the role of PD-1 signaling on aged CD8+ T cell function and accumulation of CD8+ TRM cells during acute viral respiratory tract infection, given the importance of PD-1 regulating CD8+ T cells during acute and chronic infections. PD-1 blockade or genetic ablation in aged mice yielded improved CD8+ T cell granzyme B production comparable to that in young mice during human metapneumovirus and influenza viral infections. Syngeneic transplant and adoptive transfer strategies revealed that improved granzyme B production in aged Pdcd1-/- CD8+ T cells was primarily cell intrinsic because aged wild-type CD8+ T cells did not have increased granzyme B production when transplanted into a young host. PD-1 signaling promoted accumulation of cytotoxic CD8+ TRM cells in aged mice. PD-1 blockade of aged mice during rechallenge infection resulted in improved clinical outcomes that paralleled reduced accumulation of CD8+ TRM cells. These findings suggest that PD-1 signaling impaired CD8+ T cell granzyme B production and contributed to CD8+ TRM cell accumulation in the aged lung. These findings have implications for future research investigating PD-1 checkpoint inhibitors as a potential therapeutic option for elderly patients with severe respiratory viral infections.

Terminally exhausted CD8+ T cells contribute to age-dependent severity of respiratory virus infection.

BACKGROUND: Lower respiratory infections are a leading cause of severe morbidity and mortality among older adults. Despite ubiquitous exposure to common respiratory pathogens throughout life and near universal seropositivity, antibodies fail to effectively protect the elderly. Therefore, we hypothesized that severe respiratory illness in the elderly is due to deficient CD8+ T cell responses. RESULTS: Here, we establish an aged mouse model of human metapneumovirus infection (HMPV) wherein aged C57BL/6 mice exhibit worsened weight loss, clinical disease, lung pathology and delayed viral clearance compared to young adult mice. Aged mice generate fewer lung-infiltrating HMPV epitope-specific CD8+ T cells. Those that do expand demonstrate higher expression of PD-1 and other inhibitory receptors and are functionally impaired. Transplant of aged T cells into young mice and vice versa, as well as adoptive transfer of young versus aged CD8+ T cells into Rag1-/- recipients, recapitulates the HMPV aged phenotype, suggesting a cell-intrinsic age-associated defect. HMPV-specific aged CD8+ T cells exhibit a terminally exhausted TCF1/7- TOX+ EOMES+ phenotype. We confirmed similar terminal exhaustion of aged CD8+ T cells during influenza viral infection. CONCLUSIONS: This study identifies terminal CD8+ T cell exhaustion as a mechanism of severe disease from respiratory viral infections in the elderly.

Monocyte production of C1q potentiates CD8 + T cell effector function following respiratory viral infection.

Respiratory viral infections remain a leading cause of morbidity and mortality. Using a murine model of human metapneumovirus (HMPV), we identified recruitment of a C1q-producing inflammatory monocyte population concomitant with viral clearance by adaptive immune cells. Genetic ablation of C1q led to reduced CD8 + T cell function. Production of C1q by a myeloid lineage was sufficient to enhance CD8 + T cell function. Activated and dividing CD8 + T cells expressed a putative C1q receptor, gC1qR. Perturbation of gC1qR signaling led to altered CD8 + T cell IFN-γ production and metabolic capacity. Autopsy specimens from fatal respiratory viral infections in children demonstrated diffuse production of C1q by an interstitial population. Humans with severe COVID-19 infection also demonstrated upregulation of gC1qR on activated and rapidly dividing CD8 + T cells. Collectively, these studies implicate C1q production from monocytes as a critical regulator of CD8 + T cell function following respiratory viral infection.

Human Metapneumovirus Reinfection in Aged Mice Recapitulates Increased Disease Severity in Elderly Humans Infected with Human Metapneumovirus.

Human metapneumovirus (HMPV) is a leading cause of respiratory infection in adults >65 y. Nearly all children worldwide are seropositive for HMPV by age 5 y, but reinfections occur throughout life, and there is no licensed vaccine. Recurrent HMPV infection is mild and self-resolving in immunocompetent individuals. However, elderly individuals develop severe respiratory disease on HMPV reinfection that leads to a high risk for morbidity and mortality. In this study, we developed a mouse model to mirror HMPV reinfection in elderly humans. C57BL/6J mice were infected with HMPV at 6-7 wk old, aged in-house, and rechallenged with high-dose virus at 70 wk. Aged rechallenged mice had profound weight loss similar to primary infected mice, increased lung histopathology, and accumulated cytotoxic CD8+CD44+CD62L-CD69+CD103+ memory cells despite having undetectable lung virus titer. When aged mice 14 mo postinfection (p.i.) or young mice 5 wk p.i. were restimulated with HMPV cognate Ag to mimic epitope vaccination, aged mice had an impaired CD8+ memory response. Convalescent serum transfer from young naive or 5 wk p.i. mice into aged mice on day of infection did not protect. Aged mice vaccinated with UV-inactivated HMPV also exhibited diminished protection and poor CD8+ memory response compared with young mice. These results suggest aged individuals with HMPV reinfection have a dysregulated CD8+ memory T cell response that fails to protect and exacerbates disease. Moreover, aged mice exhibited a poor memory response to either epitope peptide or UV-inactivated vaccination, suggesting that aged CD8+ T cell dysfunction presents a barrier to effective vaccination strategies.

RAGE contributes to allergen driven severe neutrophilic airway inflammation via NLRP3 inflammasome activation in mice.

Background: Asthma is a major public healthcare burden, affecting over 300 million people worldwide. While there has been great progress in the treatment of asthma, subsets of patients who present with airway neutrophilia, often have more severe disease, and tend to be resistant to conventional corticosteroid treatments. The receptor for advanced glycation endproducts (RAGE) plays a central role in the pathogenesis of eosinophilic asthma, however, it's role in neutrophilic asthma remains largely uninvestigated. Methods: A mouse model of severe steroid resistant neutrophilic airway disease (SSRNAD) using the common fungal allergen Alternaria alternata (AA) was employed to evaluate the effects of genetic ablation of RAGE and pharmacological inhibition of the NLRP3 inflammasome on neutrophilic airway inflammation. Results: AA exposure induced robust neutrophil-dominant airway inflammation and increased BALF levels of Th1/Th17 cytokines in wild-type mice, which was significantly reduced in RAGE-/- mice. Serum levels of IgE and IgG1 were increased similarly in both wild-type and RAGE-/- mice. Pharmacological inhibition of NLRP3 blocked the effects of AA exposure and NLRP3 inflammasome activation was RAGE-dependent. Neutrophil extracellular traps were elevated in the BALF of wild-type but not RAGE-/- mice and an atypical population of SiglecF+ neutrophils were identified in the BALF. Lastly, time-course studies found that RAGE expression promoted sustained neutrophil accumulation in the BALF of mice in response to AA.

Prior respiratory syncytial virus infection reduces vaccine-mediated Th2-skewed immunity, but retains enhanced RSV F-specific CD8 T cell responses elicited by a Th1-skewing vaccine formulation.

Respiratory syncytial virus (RSV) remains the most common cause of lower respiratory tract infections in children worldwide. Development of a vaccine has been hindered due the risk of enhanced respiratory disease (ERD) following natural RSV exposure and the young age (<6 months) at which children would require protection. Risk factors linked to the development of ERD include poorly neutralizing antibody, seronegative status (never been exposed to RSV), and a Th2-type immune response. Stabilization of the more antigenic prefusion F protein (PreF) has reinvigorated hope for a protective RSV vaccine that elicits potent neutralizing antibody. While anecdotal evidence suggests that children and adults previously exposed to RSV (seropositive) are not at risk for developing vaccine associated ERD, differences in host immune responses in seropositive and seronegative individuals that may protect against ERD remain unclear. It is also unclear if vaccine formulations that skew towards Th1- versus Th2-type immune responses increase pathology or provide greater protection in seropositive individuals. Therefore, the goal of this work was to compare the host immune response to a stabilized prefusion RSV antigen formulated alone or with Th1 or Th2 skewing adjuvants in seronegative and seropositive BALB/c mice. We have developed a novel BALB/c mouse model whereby mice are first infected with RSV (seropositive) and then vaccinated during pregnancy to recapitulate maternal immunization strategies. Results of these studies show that prior RSV infection mitigates vaccine-mediated skewing by Th1- and Th2-polarizing adjuvants that was observed in seronegative animals. Moreover, vaccination with PreF plus the Th1-skewing adjuvant, Advax, increased RSV F85-93-specific CD8 T cells in both seronegative and seropositive dams. These data demonstrate the importance of utilizing seropositive animals in preclinical vaccine studies to assess both the safety and efficacy of candidate RSV vaccines.

Nitroalkene fatty acids modulate bile acid metabolism and lung function in obese asthma.

Bile acid profiles are altered in obese individuals with asthma. Thus, we sought to better understand how obesity-related systemic changes contribute to lung pathophysiology. We also test the therapeutic potential of nitro-oleic acid (NO2-OA), a regulator of metabolic and inflammatory signaling pathways, to mitigate allergen and obesity-induced lung function decline in a murine model of asthma. Bile acids were measured in the plasma of healthy subjects and individuals with asthma and serum and lung tissue of mice with and without allergic airway disease (AAD). Lung function, indices of inflammation and hepatic bile acid enzyme expression were measured in obese mice with house dust mite-induced AAD treated with vehicle or NO2-OA. Serum levels of glycocholic acid and glycoursodeoxycholic acid clinically correlate with body mass index and airway hyperreactivity whereas murine levels of ß-muricholic acid and tauro-ß-muricholic acid were significantly increased and positively correlated with impaired lung function in obese mice with AAD. NO2-OA reduced murine bile acid levels by modulating hepatic expression of bile acid synthesis enzymes, with a concomitant reduction in small airway resistance and tissue elastance. Bile acids correlate to body mass index and lung function decline and the signaling actions of nitroalkenes can limit AAD by modulating bile acid metabolism, revealing a potential pharmacologic approach to improving the current standard of care.

The perplexing role of RAGE in pulmonary fibrosis: causality or casualty?

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease in which most patients die within 3 years of diagnosis. With an unknown etiology, IPF results in progressive fibrosis of the lung parenchyma, diminishing normal lung function, which results in respiratory failure, and eventually, death. While few therapies are available to reduce disease progression, patients continue to advance toward respiratory failure, leaving lung transplantation the only viable option for survival. As incidence and mortality rates steadily increase, the need for novel therapeutics is imperative. The receptor for advanced glycation endproducts (RAGE) is most highly expressed in the lungs and plays a significant role in a number of chronic lung diseases. RAGE has long been linked to IPF; however, confounding data from both human and experimental studies have left an incomplete and perplexing story. This review examines the present understanding of the role of RAGE in human and experimental models of IPF, drawing parallels to recent advances in RAGE biology. Moreover, this review discusses the role of RAGE in lung injury response, type 2 immunity, and cellular senescence, and how such mechanisms may relate to RAGE as both a biomarker of disease progression and potential therapeutic target in IPF.The reviews of this paper are available via the supplemental material section.

Nrf2 through Aryl Hydrocarbon Receptor Regulates IL-22 Response in CD4+ T Cells.

IL-17A and IL-22 derived from Th17 cells play a significant role in mucosal immunity and inflammation. TGF-ß and IL-6 promote Th17 differentiation; however, these cytokines have multiple targets. The identification and screening of additional molecules that regulate IL-17A and IL-22 responses in certain inflammatory conditions is of great clinical significance. In this study, we show that CDDO-Im, a specific Nrf2 activator, promotes IL-17A and IL-22 responses in murine Th17 cells. In contrast, CDDO-Im inhibits IL-17A response in multiple sclerosis patient-derived PBMCs. However, Nrf2 specifically regulates IL-22 response in vivo. Nrf2 acts through the regulation of antioxidant response element (ARE) binding motifs in target genes to induce or repress transcription. Promoter analysis revealed that Il17a, Rorc, and Ahr genes have several ARE motifs. We showed that Nrf2 bound to ARE repressor (ARE-R2) of Rorc and inhibited Rorc-dependent IL-17A transactivation. The luciferase reporter assay data showed that CDDO-Im regulated Ahr promoter activity. Chromatin immunoprecipitation quantitative PCR data showed that Nrf2 bound to ARE of AhR. Finally, we confirmed that the CDDO-Im-mediated induction of IL-22 production in CD4+ T cells was abrogated in CD4-specific Ahr knockout mice (AhrCD4 ). CH-223191, a specific AhR antagonist, inhibits CDDO-Im-induced IL-22 production in CD4+ T cells, which further confirmed the AhR-dependent regulation. Collectively, our data showed that Nrf2 via AhR pathways regulated IL-22 response in CD4+ T cells.

The axis of the receptor for advanced glycation endproducts in asthma and allergic airway disease.

Asthma is a generalized term that describes a scope of distinct pathologic phenotypes of variable severity, which share a common complication of reversible airflow obstruction. Asthma is estimated to affect almost 400 million people worldwide, and nearly ten percent of asthmatics have what is considered "severe" disease. The majority of moderate to severe asthmatics present with a "type 2-high" (T2-hi) phenotypic signature, which pathologically is driven by the type 2 cytokines Interleukin-(IL)-4, IL-5, and IL-13. However, "type 2-low" (T2-lo) phenotypic signatures are often associated with more severe, steroid-refractory neutrophilic asthma. A wide range of clinical and experimental studies have found that the receptor for advanced glycation endproducts (RAGE) plays a significant role in the pathogenesis of asthma and allergic airway disease (AAD). Current experimental data indicates that RAGE is a critical mediator of the type 2 inflammatory reactions which drive the development of T2-hi AAD. However, clinical studies demonstrate that increased RAGE ligands and signaling strongly correlate with asthma severity, especially in severe neutrophilic asthma. This review presents an overview of the current understandings of RAGE in asthma pathogenesis, its role as a biomarker of disease, and future implications for mechanistic studies, and potential therapeutic intervention strategies.

Prefusion RSV F Immunization Elicits Th2-Mediated Lung Pathology in Mice When Formulated With a Th2 (but Not a Th1/Th2-Balanced) Adjuvant Despite Complete Viral Protection.

Respiratory syncytial virus (RSV) remains the most common cause of lower respiratory tract infections in children worldwide. Development of a vaccine has been hindered by the risk of developing enhanced respiratory disease (ERD) upon natural exposure to the virus. Generation of higher quality neutralizing antibodies with stabilized pre-fusion F protein antigens has been proposed as a strategy to prevent ERD. We sought to test whether there was evidence of ERD in naïve BALB/c mice immunized with an unadjuvanted, stabilized pre-fusion F protein, and challenged with RSV line 19. We further sought to determine the extent to which formulation with a Th2-biased (alum) or a more Th1/Th2-balanced (Advax-SM) adjuvant influenced cellular responses and lung pathology. When exposed to RSV, mice immunized with pre-fusion F protein alone (PreF) exhibited increased airway eosinophilia and mucus accumulation. This was further exacerbated by formulation of PreF with Alum (aluminum hydroxide). Conversely, formulation of PreF with a Th1/Th2-balanced adjuvant, Advax-SM, not only suppressed RSV viral replication, but also inhibited airway eosinophilia and mucus accumulation. This was associated with lower numbers of lung innate lymphocyte cells (ILC2s) and CD4+ T cells producing IL-5+ or IL-13+ and increased IFNγ+ CD4+ and CD8+ T cells, in addition to RSV F-specific CD8+ T cells. These data suggest that in the absence of preimmunity, stabilized PreF antigens may still be associated with aberrant Th2 responses that induce lung pathology in response to RSV infection, and can be prevented by formulation with more Th1/Th2-balanced adjuvants that enhance CD4+ and CD8+ IFNγ+ T cell responses. This may support the use of stabilized PreF antigens with Th1/Th2-balanced adjuvants like, Advax-SM, as safer alternatives to alum in RSV vaccine candidates.

STAT2 Limits Host Species Specificity of Human Metapneumovirus.

The host tropism of viral infection is determined by a variety of factors, from cell surface receptors to innate immune signaling. Many viruses encode proteins that interfere with host innate immune recognition in order to promote infection. STAT2 is divergent between species and therefore has a role in species restriction of some viruses. To understand the role of STAT2 in human metapneumovirus (HMPV) infection of human and murine tissues, we first infected STAT2-/- mice and found that HMPV could be serially passaged in STAT2-/-, but not WT, mice. We then used in vitro methods to show that HMPV inhibits expression of both STAT1 and STAT2 in human and primate cells, but not in mouse cells. Transfection of the murine form of STAT2 into STAT2-deficient human cells conferred resistance to STAT2 inhibition. Finally, we sought to understand the in vivo role of STAT2 by infecting hSTAT2 knock-in mice with HMPV, and found that mice had increased weight loss, inhibition of type I interferon signaling, and a Th2-polarized cytokine profile compared to WT mice. These results indicate that STAT2 is a target of HMPV in human infection, while the murine version of STAT2 restricts tropism of HMPV for murine cells and tissue.

The receptor for advanced glycation end products is a critical mediator of type 2 cytokine signaling in the lungs.

BACKGROUND: Asthma is estimated to effect more than 300 million persons worldwide, leading to nearly 250,000 deaths annually. The majority of patients with mild-to-severe asthma have what is deemed "type-2 high" asthma, which is driven by the prototypical type 2 cytokines IL-4, IL-5, and IL-13. Studies have indicated that the receptor for advanced glycation end products (RAGE) is a critical molecule in the pathogenesis of experimental asthma/allergic airway inflammation. More specifically, RAGE expressed on stromal cells, rather than hematopoietic cells, is critical to induction of asthma/allergic airway inflammation by driving type 2 inflammatory responses. However, the role of RAGE in directly mediating type 2 cytokine signaling has never been investigated. OBJECTIVE: The goal of this study was to test the hypothesis that RAGE mediates type 2 cytokine-induced signal transduction, airway inflammation, and mucus metaplasia in the lungs. METHODS: Wild-type (WT) and RAGE knockout (RAGE-/-) mice, were intranasally administered rIL-5/rIL-13 or rIL-4 alone, and signal transducer and activator of transcription 6 (STAT6) signaling, airway inflammation, and mucus metaplasia were assessed. A RAGE small-molecule antagonist was used to determine the effects of pharmacologically inhibiting RAGE on type 2 cytokine-induced effects. RESULTS: Administration of type 2 cytokines induced pronounced airway inflammation and mucus metaplasia in WT mice, which was nearly completely abrogated in RAGE-/- mice. In addition, treatment with a RAGE-specific antagonist diminished the effects of type 2 cytokines in WT mice and in primary human bronchial epithelial cell cultures. Genetic ablation or pharmacologic inhibition of RAGE blocks the effects of IL-13 and IL-4 by inhibiting sustained STAT6 activation and downstream target gene expression in mice and in human bronchial epithelial cells. CONCLUSIONS: This study is the first to indicate that RAGE is a critical component of type 2 cytokine signal transduction mechanisms, which is a driving force behind type 2-high asthma.

CD4+ Regulatory T Cells Exert Differential Functions during Early and Late Stages of the Immune Response to Respiratory Viruses.

Acute respiratory virus infection (ARI) induces CD8+ T cells with diminished cytokine production and functional impairment. The role of cellular mediators of immune impairment, specifically CD4+ regulatory T cells (Tregs), is incompletely understood in ARI. Tregs are known suppressors of effector T cell function, but whether they are detrimental or beneficial in ARI remains controversial. We show in this paper that Treg depletion leads to increased CD8+ T cell function and lower virus titer in mice infected with human metapneumovirus. We further demonstrate that Tregs play a temporal role in the immune response to human metapneumovirus and influenza: Treg depletion before infection pathologically reduces virus-specific CD8+ T cell numbers and delays virus clearance, whereas depletion 2 d postinoculation enhances CD8+ T cell functionality without reducing virus-specific CD8+ T cell numbers. Mechanistically, Treg depletion during immune priming led to impaired dendritic cell and CD8+ T cell migration. Further, early Treg depletion was associated with immune skewing toward a type 2 phenotype characterized by increased type 2 innate lymphoid cells and TH2 CD4+ T cells, which was not observed when Treg depletion was delayed until after inoculation. These results indicate that the presence of Tregs at inoculation is critical for efficient priming of the CD8+ T cell response to ARI, whereas later in infection, Tregs are dispensable for virus clearance.

HMGB1 promotes ductular reaction and tumorigenesis in autophagy-deficient livers.

Autophagy is important for liver homeostasis, and the deficiency leads to injury, inflammation, ductular reaction (DR), fibrosis, and tumorigenesis. It is not clear how these events are mechanistically linked to autophagy deficiency. Here, we reveal the role of high-mobility group box 1 (HMGB1) in two of these processes. First, HMGB1 was required for DR, which represents the expansion of hepatic progenitor cells (HPCs) implicated in liver repair and regeneration. DR caused by hepatotoxic diets (3,5-diethoxycarbonyl-1,4-dihydrocollidine [DDC] or choline-deficient, ethionine-supplemented [CDE]) also depended on HMGB1, indicating that HMGB1 may be generally required for DR in various injury scenarios. Second, HMGB1 promoted tumor progression in autophagy-deficient livers. Receptor for advanced glycation end product (RAGE), a receptor for HMGB1, was required in the same two processes and could mediate the proliferative effects of HMBG1 in isolated HPCs. HMGB1 was released from autophagy-deficient hepatocytes independently of cellular injury but depended on NRF2 and the inflammasome, which was activated by NRF2. Pharmacological or genetic activation of NRF2 alone, without disabling autophagy or causing injury, was sufficient to cause inflammasome-dependent HMGB1 release. In conclusion, HMGB1 release is a critical mechanism in hepatic pathogenesis under autophagy-deficient conditions and leads to HPC expansion as well as tumor progression.

SOD3 improves the tumor response to chemotherapy by stabilizing endothelial HIF-2α.

One drawback of chemotherapy is poor drug delivery to tumor cells, due in part to hyperpermeability of the tumor vasculature. Extracellular superoxide dismutase (SOD3) is an antioxidant enzyme usually repressed in the tumor milieu. Here we show that specific SOD3 re-expression in tumor-associated endothelial cells (ECs) increases doxorubicin (Doxo) delivery into and chemotherapeutic effect on tumors. Enhanced SOD3 activity fostered perivascular nitric oxide accumulation and reduced vessel leakage by inducing vascular endothelial cadherin (VEC) transcription. SOD3 reduced HIF prolyl hydroxylase domain protein activity, which increased hypoxia-inducible factor-2α (HIF-2α) stability and enhanced its binding to a specific VEC promoter region. EC-specific HIF-2α ablation prevented both the SOD3-mediated increase in VEC transcription and the enhanced Doxo effect. SOD3, VEC, and HIF-2α levels correlated positively in primary colorectal cancers, which suggests a similar interconnection of these proteins in human malignancy.