Miz1 represses type I interferon production and limits viral clearance during influenza A virus infection.

Type I interferons (IFNs) are critical for the antiviral immune response, and fine-tuning type I IFN production is critical to effectively clearing viruses without causing harmful immunopathology. We showed that the transcription factor Miz1 epigenetically repressed the expression of genes encoding type I IFNs in mouse lung epithelial cells by recruiting histone deacetylase 1 (HDAC1) to the promoters of Ifna and Ifnb. Loss of function of Miz1 resulted in augmented production of these type I IFNs during influenza A virus (IAV) infection, leading to improved viral clearance in vitro and in vivo. IAV infection induced Miz1 accumulation by promoting the cullin-4B (CUL4B)-mediated ubiquitylation and degradation of the E3 ubiquitin ligase Mule (Mcl-1 ubiquitin ligase E3; also known as Huwe1 or Arf-BP1), which targets Miz1 for degradation. As a result, Miz1 accumulation limited type I IFN production and favored viral replication. This study reveals a previously unrecognized function of Miz1 in regulating antiviral defense and a potential mechanism for influenza viruses to evade host immune defense.

Miz1 promotes KRAS-driven lung tumorigenesis by repressing the protocadherin Pcdh10.

Targeting KRAS-mutated non-small-cell lung cancer (NSCLC) remains clinically challenging. Here we show that loss of function of Miz1 inhibits lung tumorigenesis in a mouse model of oncogenic KRAS-driven lung cancer. In vitro, knockout or silencing of Miz1 decreases cell proliferation, clonogenicity, migration, invasion, or anchorage-independent growth in mutant (MT) KRAS murine or human NSCLC cells but has unremarkable impact on non-tumorigenic cells or wild-type (WT) KRAS human NSCLC cells. RNA-sequencing reveals Protocadherin-10 (Pcdh10) as the top upregulated gene by Miz1 knockout in MT KRAS murine lung tumor cells. Chromatin immunoprecipitation shows Miz1 binding on the Pcdh10 promoter in MT KRAS lung tumor cells but not non-tumorigenic cells. Importantly, silencing of Pcdh10 rescues cell proliferation and clonogenicity in Miz1 knockout/knockdown MT KRAS murine or human tumor cells, and rescues allograft tumor growth of Miz1 knockout tumor cells in vivo. Miz1 is upregulated in MT KRAS lung tumor tissues compared with adjacent non-involved tissues in mice. Consistent with this, Miz1 is upregulated while Pcdh10 is downregulated in human lung adenocarcinomas (LUAD) compared with normal tissues, and high Miz1 levels or low Pcdh10 levels are associated with poor survival in lung cancer patients. Furthermore, the Miz1 signature is associated with worse survival in MT but not WT KRAS LUAD, and Pcdh10 is downregulated in MT compared to WT KRAS LUAD. Taken together, our studies implicate the Miz1/Pcdh10 axis in oncogenic KRAS-driven lung tumorigenesis.

Suppression of Allergic Asthma by Loss of Function of Miz1-mediated Th1 Skewing.

Asthma is the most prevalent chronic respiratory disease worldwide. There is currently no cure, and it remains an important cause of morbidity and mortality. Here we report that lung-specific loss of function of the transcription factor Miz1 (c-Myc-interacting zinc finger protein-1) upregulates the pro-T-helper cell type 1 cytokine IL-12. Upregulation of IL-12 in turn stimulates a Th1 response, thereby counteracting T-helper cell type 2 response and preventing the allergic response in mouse models of house dust mite- and OVA (ovalbumin)-induced asthma. Using transgenic mice expressing Cre under a cell-specific promoter, we demonstrate that Miz1 acts in lung epithelial cells and dendritic cells in asthma. Chromatin immunoprecipitation coupled with high-throughput DNA sequencing or quantitative PCR reveals the binding of Miz1 on the Il12 promoter indicating direct repression of IL-12 by Miz1. In addition, HDAC1 (histone deacetylase 1) is recruited to the Il12 promoter in a Miz1-depdenent manner, suggesting epigenetic repression of Il12 by Miz1. Furthermore, Miz1 is upregulated in the lungs of asthmatic mice. Our data together suggest that Miz1 is upregulated during asthma, which in turn promotes asthma pathogenesis by preventing Th1 skewing through the transcriptional repression of IL-12.

Age-related Differences in the Nasal Mucosal Immune Response to SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 180 million people since the onset of the pandemic. Despite similar viral load and infectivity rates between children and adults, children rarely develop severe illness. Differences in the host response to the virus at the primary infection site are among the mechanisms proposed to account for this disparity. Our objective was to investigate the host response to SARS-CoV-2 in the nasal mucosa in children and adults and compare it with the host response to respiratory syncytial virus (RSV) and influenza virus. We analyzed clinical outcomes and gene expression in the nasal mucosa of 36 children with SARS-CoV-2, 24 children with RSV, 9 children with influenza virus, 16 adults with SARS-CoV-2, and 7 healthy pediatric and 13 healthy adult controls. In both children and adults, infection with SARS-CoV-2 led to an IFN response in the nasal mucosa. The magnitude of the IFN response correlated with the abundance of viral reads, not the severity of illness, and was comparable between children and adults infected with SARS-CoV-2 and children with severe RSV infection. Expression of ACE2 and TMPRSS2 did not correlate with age or presence of viral infection. SARS-CoV-2-infected adults had increased expression of genes involved in neutrophil activation and T-cell receptor signaling pathways compared with SARS-CoV-2-infected children, despite similar severity of illness and viral reads. Age-related differences in the immune response to SARS-CoV-2 may place adults at increased risk of developing severe illness.

miR-221-5p-Mediated Downregulation of JNK2 Aggravates Acute Lung Injury.

Sepsis and acute lung injury (ALI) are linked to mitochondrial dysfunction; however, the underlying mechanism remains elusive. We previously reported that c-Jun N-terminal protein kinase 2 (JNK2) promotes stress-induced mitophagy by targeting small mitochondrial alternative reading frame (smARF) for ubiquitin-mediated proteasomal degradation, thereby preventing mitochondrial dysfunction and restraining inflammasome activation. Here we report that loss of JNK2 exacerbates lung inflammation and injury during sepsis and ALI in mice. JNK2 is downregulated in mice with endotoxic shock or ALI, concomitantly correlated inversely with disease severity. Small RNA sequencing revealed that miR-221-5p, which contains seed sequence matching to JNK2 mRNA 3' untranslated region (3'UTR), is upregulated in response to lipopolysaccharide, with dynamically inverse correlation with JNK2 mRNA levels. miR-221-5p targets the 3'UTR of JNK2 mRNA leading to its downregulation. Accordingly, miR-221-5p exacerbates lung inflammation and injury during sepsis in mice by targeting JNK2. Importantly, in patients with pneumonia in medical intensive care unit, JNK2 mRNA levels in alveolar macrophages flow sorted from non-bronchoscopic broncholaveolar lavage (BAL) fluid were inversely correlated strongly and significantly with the percentage of neutrophils, neutrophil and white blood cell counts in BAL fluid. Our data suggest that miR-221-5p targets JNK2 and thereby aggravates lung inflammation and injury during sepsis.

Immune response to SARS-CoV-2 in the nasal mucosa in children and adults.

RATIONALE: Despite similar viral load and infectivity rates between children and adults infected with SARS-CoV-2, children rarely develop severe illness. Differences in the host response to the virus at the primary infection site are among the proposed mechanisms. OBJECTIVES: To investigate the host response to SARS-CoV-2, respiratory syncytial virus (RSV), and influenza virus (IV) in the nasal mucosa in children and adults. METHODS: Clinical outcomes and gene expression in the nasal mucosa were analyzed in 36 children hospitalized with SARS-CoV-2 infection, 24 children with RSV infection, 9 children with IV infection, 16 adults with mild to moderate SARS-CoV-2 infection, and 7 healthy pediatric and 13 healthy adult controls. RESULTS: In both children and adults, infection with SARS-CoV-2 leads to an interferon response in the nasal mucosa. The magnitude of the interferon response correlated with the abundance of viral reads and was comparable between symptomatic children and adults infected with SARS-CoV-2 and symptomatic children infected with RSV and IV. Cell type deconvolution identified an increased abundance of immune cells in the samples from children and adults with a viral infection. Expression of ACE2 and TMPRSS2 - key entry factors for SARS-CoV-2 - did not correlate with age or presence or absence of viral infection. CONCLUSIONS: Our findings support the hypothesis that differences in the immune response to SARS-CoV-2 determine disease severity, independent of viral load and interferon response at the primary infection primary site.

Epithelial cell-specific loss of function of Miz1 causes a spontaneous COPD-like phenotype and up-regulates Ace2 expression in mice.

Cigarette smoking, the leading cause of chronic obstructive pulmonary disease (COPD), has been implicated as a risk factor for severe disease in patients infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we show that mice with lung epithelial cell-specific loss of function of Miz1, which we identified as a negative regulator of nuclear factor κB (NF-κB) signaling, spontaneously develop progressive age-related changes resembling COPD. Furthermore, loss of Miz1 up-regulates the expression of Ace2, the receptor for SARS-CoV-2. Concomitant partial loss of NF-κB/RelA prevented the development of COPD-like phenotype in Miz1-deficient mice. Miz1 protein levels are reduced in the lungs from patients with COPD, and in the lungs of mice exposed to chronic cigarette smoke. Our data suggest that Miz1 down-regulation-induced sustained activation of NF-κB-dependent inflammation in the lung epithelium is sufficient to induce progressive lung and airway destruction that recapitulates features of COPD, with implications for COVID-19.

JNK2 up-regulates hypoxia-inducible factors and contributes to hypoxia-induced erythropoiesis and pulmonary hypertension.

The hypoxic response is a stress response triggered by low oxygen tension. Hypoxia-inducible factors (HIFs) play a prominent role in the pathobiology of hypoxia-associated conditions, including pulmonary hypertension (PH) and polycythemia. The c-Jun N-terminal protein kinase (JNK), a stress-activated protein kinase that consists of two ubiquitously expressed isoforms, JNK1 and JNK2, and a tissue-specific isoform, JNK3, has been shown to be activated by hypoxia. However, the physiological role of JNK1 and JNK2 in the hypoxic response remains elusive. Here, using genetic knockout cells and/or mice, we show that JNK2, but not JNK1, up-regulates the expression of HIF-1α and HIF-2α and contributes to hypoxia-induced PH and polycythemia. Knockout or silencing of JNK2, but not JNK1, prevented the accumulation of HIF-1α in hypoxia-treated cells. Loss of JNK2 resulted in a decrease in HIF-1α and HIF-2α mRNA levels under resting conditions and in response to hypoxia. Consequently, hypoxia-treated Jnk2-/- mice had reduced erythropoiesis and were less prone to polycythemia because of decreased expression of the HIF target gene erythropoietin (Epo). Jnk2-/- mice were also protected from hypoxia-induced PH, as indicated by lower right ventricular systolic pressure, a process that depends on HIF. Taken together, our results suggest that JNK2 is a positive regulator of HIFs and therefore may contribute to HIF-dependent pathologies.

The kinase Jnk2 promotes stress-induced mitophagy by targeting the small mitochondrial form of the tumor suppressor ARF for degradation.

Mitophagy is essential for cellular homeostasis, but how mitophagy is regulated is largely unknown. Here we found that the kinase Jnk2 was required for stress-induced mitophagy. Jnk2 promoted ubiquitination and proteasomal degradation of the small mitochondrial form of the tumor suppressor ARF (smARF). Loss of Jnk2 led to the accumulation of smARF, which induced excessive autophagy that resulted in lysosomal degradation of the mitophagy adaptor p62 at steady state. Depletion of p62 prevented Jnk2-deficient cells from mounting mitophagy upon stress. Jnk2-deficient mice displayed defective mitophagy, which resulted in tissue damage under hypoxic stress, as well as hyperactivation of inflammasomes and increased mortality in sepsis. Our findings define a unique mechanism of maintaining immunological homeostasis that protects the host from tissue damage and mortality.

Suppression of inflammation and acute lung injury by Miz1 via repression of C/EBP-δ.

Inflammation is essential for host defense but can cause tissue damage and organ failure if unchecked. How the inflammation is resolved remains elusive. Here we report that the transcription factor Miz1 was required for terminating lipopolysaccharide (LPS)-induced inflammation. Genetic disruption of the Miz1 POZ domain, which is essential for the transactivation or repression activity of Miz1, resulted in hyperinflammation, lung injury and greater mortality in LPS-treated mice but a lower bacterial load and mortality in mice with Pseudomonas aeruginosa pneumonia. Loss of the Miz1 POZ domain prolonged the expression of proinflammatory cytokines. After stimulation, Miz1 was phosphorylated at Ser178, which was required for recruitment of the histone deacetylase HDAC1 to repress transcription of the gene encoding C/EBP-δ, an amplifier of inflammation. Our data provide a long-sought mechanism underlying the resolution of LPS-induced inflammation.