
==== Front
Mol Ther
Mol Ther
Molecular Therapy
1525-0016
1525-0024
American Society of Gene & Cell Therapy

S1525-0016(23)00322-2
10.1016/j.ymthe.2023.06.011
Original Article
Preventing occludin tight-junction disruption via inhibition of microRNA-193b-5p attenuates viral load and influenza-induced lung injury
Vaswani Chirag M. 12
Varkouhi Amir K. 3
Gupta Sahil 245
Ektesabi Amin M. 24
Tsoporis James N. 2
Yousef Sadiya 2
Plant Pamela J. 2
da Silva Adriana L. 67
Cen Yuchen 8
Tseng Yi-Chieh 8
Batah Sabrina S. 9
Fabro Alexandre T. 9
Advani Suzanne L. 2
Advani Andrew 2
Leong-Poi Howard 24
Marshall John C. 24
Garcia Cristiana C. 1011
Rocco Patricia R.M. 67
Albaiceta Guillermo M. 121314
Sebastian-Bolz Steffen 1
Watts Tania H. 15
Moraes Theo J. 816
Capelozzi Vera L. 18
dos Santos Claudia.C. claudia.dossantos@unityhealth.to
1241719∗
1 Department of Physiology, Temerty Faculty of Medicine, University of Toronto, Toronto, ON, Canada
2 Keenan Research Centre for Biomedical Science, St. Michael’s Hospital, Toronto, ON, Canada
3 Department of Chemistry and Environmental Science, New Jersey Institute of Technology, Newark, NJ, USA
4 Institute of Medical Sciences, Faculty of Medicine, University of Toronto, Toronto, ON, Canada
5 Faculty of Medicine, School of Medicine, The University of Queensland, Herston, QLD 4006, Australia
6 Laboratory of Pulmonary Investigation, Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
7 COVID-19 Virus Network from Ministry of Science, Technology, and Innovation, Brazilian Council for Scientific and Technological Development, and Foundation Carlos Chagas Filho Research Support of the State of Rio de Janeiro, Brazil
8 Program in Translational Medicine, SickKids Research Institute, Toronto, ON, Canada
9 Department of Pathology and Legal Medicine, Ribeirão Preto Medical School, University of São Paulo, São Paulo, Brazil
10 Laboratory of Respiratory, Exanthematic Viruses, Enterovirus and Viral Emergencies, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil
11 Integrated Research Group on Biomarkers. René Rachou Institute, FIOCRUZ Minas, Belo Horizonte, Brazil
12 Departamento de Biología Funcional, Instituto Universitario de Oncología del Principado de Asturias, Universidad de Oviedo, Oviedo, Spain
13 Unidad de Cuidados Intensivos Cardiológicos, Hospital Universitario Central de Asturias, Oviedo, Spain
14 CIBER-Enfermedades Respiratorias, Instituto de Salud Carlos III, Madrid, Spain
15 Department of Immunology, Temerty Faculty of Medicine, University of Toronto, Toronto, ON, Canada
16 Department of Pediatrics University of Toronto and Respirology, Hospital for Sick Children, Toronto, ON, Canada
17 Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, Toronto, ON, Canada
18 Department of Pathology, University of São Paulo, São Paulo, Brazil
19 Interdepartmental Division of Critical Care, St Michael’s Hospital, University of Toronto, Toronto, ON, Canada
∗ Corresponding author: Claudia. C. dos Santos, MSc, MD, Clinician-Scientist, Associate Professor of Medicine, Interdepartmental Division of Critical Care, St. Michael’s Hospital/University of Toronto, 30 Bond Street, Room 4-008, Toronto, ON M5B 1WB, Canada. claudia.dossantos@unityhealth.to
06 9 2023
19 6 2023
31 9 26812701
2 3 2023
14 6 2023
© 2023 The Author(s)
2023
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Virus-induced lung injury is associated with loss of pulmonary epithelial-endothelial tight junction integrity. While the alveolar-capillary membrane may be an indirect target of injury, viruses may interact directly and/or indirectly with miRs to augment their replication potential and evade the host antiviral defense system. Here, we expose how the influenza virus (H1N1) capitalizes on host-derived interferon-induced, microRNA (miR)-193b-5p to target occludin and compromise antiviral defenses. Lung biopsies from patients infected with H1N1 revealed increased miR-193b-5p levels, marked reduction in occludin protein, and disruption of the alveolar-capillary barrier. In C57BL/6 mice, the expression of miR-193b-5p increased, and occludin decreased, 5–6 days post-infection with influenza (PR8). Inhibition of miR-193b-5p in primary human bronchial, pulmonary microvascular, and nasal epithelial cells enhanced antiviral responses. miR-193b-deficient mice were resistant to PR8. Knockdown of occludin, both in vitro and in vivo, and overexpression of miR-193b-5p reconstituted susceptibility to viral infection. miR-193b-5p inhibitor mitigated loss of occludin, improved viral clearance, reduced lung edema, and augmented survival in infected mice. Our results elucidate how the innate immune system may be exploited by the influenza virus and how strategies that prevent loss of occludin and preserve tight junction function may limit susceptibility to virus-induced lung injury.

Graphical abstract

The temporal-dependent inhibition of microRNA-193b-5p reduces tight junction protein occludin transcript degradation, lung edema, and viral load, and augments survival in influenza-infected mice. The results elucidate how strategies that prevent loss of occludin and preserve tight junction function may limit susceptibility to virus-induced lung injury.

Keywords

acute respiratory distress syndrome
acute lung injury
influenza virus
tight junctions
occludin
microRNA
miR-193b
interferon beta
alveolar-capillary membrane
antiviral immune response
==== Body
pmcIntroduction

Inadequate immunity to the continually evolving influenza virus (IV) variants cause sporadic outbreaks that are often difficult to predict and continue to remain a public health concern.1 The 1918 pandemic claimed the lives of over 40 million people.2 The 2009 H1N1 pandemic strain resulted in an estimated 500,000 fatalities.3 IV can reach deep into the lung airspaces and cause alveolar-capillary injury, diffuse alveolar damage, exudative edema, and immune cell infiltration resulting in acute respiratory distress syndrome (ARDS)4,5; the leading cause of multi-organ failure4,6 and mortality in critically ill influenza-infected patients.7

Alveolar-capillary damage1,4 in virus-induced ARDS is associated with injury to tight junctions (TJs).8 TJs are dynamic multiprotein complexes that function as paracellular gates that restrict diffusion between compartments on the basis of size and charge.9,10,11 Beyond barrier protection, TJs transmit signals to the cell interior that regulate the cytoskeleton, gene expression, cell proliferation and differentiation, affecting various cellular processes including microbial pathogenesis.11,12,13 Various pathogens have developed elegant strategies to disrupt TJs in order to reach their receptors and alter the host’s immune responses, while others utilize TJ proteins as receptors for entry into cells.14 Disruption of TJs, including occludin (Ocln), leads to increases in paracellular permeability and polarity defects which facilitate viral or bacterial cell entry and spread.15,16 Evidence supports the role of Ocln, and associated structural proteins as targets for various classes of viruses. Ocln is a receptor for the hepatitis C virus (HCV),5,17 coxsackievirus,18 porcine epidemic diarrhea virus,19 and Toxoplasma gondii.20 Ocln may also affect virus pathogenicity by regulating viral replication as Ocln-deficient cells are resistant to HCV infection5 and Ocln-targeting antibodies prevent HCV infection in vitro21 and in vivo.22 In contrast, IVs,23 West Nile virus,24 and rotaviruses25 disrupt TJs by causing a decrease in Ocln protein expression facilitating viral entry into cells. However, the molecular mechanisms of IV-induced Ocln decrease, its effects on lung injury, and the impact of preserving Ocln in acute virus-induced lung injury remain largely unknown.

MicroRNAs (miRs) are small (∼22 nucleotides) non-coding RNAs that regulate gene expression at the post-transcriptional level.26 MicroRNAs can regulate TJ expression, modulate epithelial/endothelial barrier function and can be used directly or indirectly to augment viral replication potential.27,28 We have recently identified miR-193b-5p, as a miR that binds to the 3′ untranslated region (UTR) of the Ocln mRNA, causing decreases in Ocln in sepsis-induced lung injury.28 miR-193b has been implicated in viral infection,29,30 cell-cycle regulation,31 tumor suppression,32 fat metabolism,33 and vasculogenesis.34 Whether miR-193b-5p contributes to IV-induced lung injury remains to be determined. Here, we found that in lungs from patients who died with ARDS, expression levels of miR-193b-5p are increased, while Ocln is decreased at early, as well as late stages of IV-associated ARDS. In a murine model of H1N1 influenza (PR8), miR-193b-5p inhibition prevents loss of Ocln; enhances interferon beta (IFN-β) and interferon regulated genes (IRGs) expression, reduces viral replication, histological evidence of lung injury and mortality. Silencing of Ocln reconstituted the wild-type (WT) injurious phenotype in miR-193b-deficient mice. Taken together, our work suggests Ocln plays a fundamental role in the pathogenesis of IV infection and lung injury; and that inhibition of host-derived miR-193b-5p may enhance interferon antiviral responses, prevent Ocln TJ disruption attenuating viral load and influenza-induced lung injury.

Results

Decreased Ocln protein, TJ disruption, and miR-193b-5p expression in lung biopsies from patients with IV-induced ARDS

Analysis of differentially expressed genes from IV-infected vs. non-infected human lung tissue samples (GEO accession no. GSE163959) identified miR-193b as the most likely miRs regulating the gene expression pattern seen in lungs from IV-infected patients compared with non-infected controls (adjusted p value of 2.7 × 10−11, Figure S1A). Human lung biopsies from IV-infected patients showed distinct pathological features of ARDS and reduced Ocln protein compared with non-infected controls in pulmonary bronchial epithelial, endothelial, and alveolar epithelial cells (Figure 1A, clinical data presented in Table 1). In three representative patients, IV infection demonstrated transudative edema and granulomatous tissue (Figure 1B, top panel) and showed reduced Ocln staining in lung endothelial cells relative to non-infected control (by immunofluorescence for Ocln and colocalization with CD34, Figure 1B, middle panel). In both ARDS patients, the transmission electron microscopy images demonstrated progressive disruption of TJs over the course of infection associated with viral inclusions (Figure 1B lower panel). RNA was extracted from formalin-fixed paraffin-embedded (FFPE) biopsy lung tissues (Figure 1C) to demonstrate a 2- to 7-fold increase in miR-193b-5p (copies/μL) contained in digital droplets relative to non-infected controls (Figure 1D). Since miRs in the same gene cluster or family may be co-regulated at the transcriptional level and/or may share functional relationships by coregulating the same biological process,35 we looked at the expression of miR-365a1, a member of the miR-193b/365a genomic cluster, and found that this was not differentially expressed in our biopsy lung tissues (Figure S1B). Using miRNAscope, we probed for miR-193b-5p in situ in human lung autopsy samples, and visually identified miR-193b-5p punctae within H1N1-infected lungs; in distal bronchi, in edema filled alveoli, and around pulmonary arterioles (Figure S1C). We further determined that the expression of miR-193b-5p increased in response to recombinant IFN-β by stimulating human primary distal bronchial airway epithelial cells (BEAS2b) with log-fold increasing doses of recombinant human IFN-β; the copy number of miR-193b-5p increased 3- to 5-fold over 4 h stimulation with 1,000 IU (international units) human recombinant IFN-β (1 pg = 1.2 × 102 IU, Figure 1E). To determine if the promoter of miR-193b is IFN-β responsive, we made a construct with the 5′ UTR of miR-193b and demonstrated, using luciferase fluorescence, increased promoter activity in response to IFN-β as well as TNF-α, indicating their regulation on miR-193b (Figure 1F). Independent bronchoalveolar lavage (BAL) samples obtained from IV-infected patients, compared with critically ill non-IV-infected patients (Toronto Public Health Laboratory and Regional Ethics Committee Comité de ética de la investigación Clinica del Principado de Asturias, Spain, ref. 22/17) and were sequenced for miR gene expression (Figure 1G) and showed increases in miR-193b-5p expression. IFN-γ and TNF-α proteins were increased using ELISA on the same BALf samples (Figures 1H, 1I, and 1J, respectively).Figure 1 Tight junctions are disrupted, mi-193b-5p expression is increased, while occludin protein is decreased in lung biopsies from patients infected with influenza virus

(A) Immunohistochemistry shows marked reduction in occludin DAB staining in influenza-infected human lung tissues compared with non-IV infected control lungs in pulmonary bronchial epithelial, endothelial, and alveolar epithelial cells. (B) Photomicrographs of three representative human histological lung sections comparing non-infected lung biopsies with the IV-induced features of acute respiratory distress syndrome (ARDS) and organizing phase pneumonia. Top panel: hematoxylin and eosin (H&E) staining showing progressive histological changes: intimate association between the basal lamina and the epithelial cells (top left); diffuse alveolar damage (DAD) and acute alveolar edema (top middle); organized or proliferative changes (top right). Middle panel: immunofluorescent staining showing co-localization of occludin (yellow, white arrows) with CD34 (green, white arrows, middle left), and occludin staining reduction in areas of acute DAD (middle mid). Progressive changes associated with increase in granulation tissue deposition is associated with decrease in the staining intensity for occludin (middle right). Bottom panel: transmission electron microscopy (TEM) images demonstrated preservations in tight junctions in the absence of viral infection (bottom left). In the acute stages of viral-induced DAD, tight junction disruptions can be seen at the apical sides between endothelial and epithelial cells associated with intra-alveolar edema (bottom middle). Disruptions of tight junctions at the ultrastructure level in the acute stage of viral-induced DAD (bottom middle). Viral particles can be seen free, adjacent to peripheral apical junctions and lateral to tight junctions invading the type 2 alveolar epithelium in organizing pneumonia (bottom right). The organizing phase of infection is associated with severe tight junction disruption with evidence of H1N1 viral particles invading the type 2 alveolar epithelium (bottom right). Abbreviations: tight junctions (TJ), virus (Vi). Clinical features of patients 1–5 are presented in Table 1. (C) Formalin-fixed, paraffin-embedded lung tissues were isolated for total RNA. Whisker plots showing significant increase in miR-193b-5p levels using. (D) Digital droplet PCR (ddPCR) showing increased copies/μL of miR-193b-5p comparing non-infected vs. H1N1-infected lungs (data are presented as median ± interquartile range [IQR], n = 3–7; ∗p < 0.05, ∗∗p < 0.01, ∗∗p < 0.001, ∗∗∗∗p < 0.0001; Mann-Whitney). (E) Bar graph showing dose-dependent increase in miR-193b-5p expression in response to human recombinant IFN-β treatment (IU, international units, 4 h) in human bronchial epithelial cells (BEAS2b) using ddPCR (data are presented as median ± IQR; ∗p = 0.05, p = 0.01; Kruskal-Wallis; symbols represent results for independent experiments (n = 3–7). (F) Exogenous stimulation of the miR-193b promoter construct with recombinant IFN-β and TNF-α shows a significant increase in nanoluc/Firefly dual luciferase expression relative to promoterless backbone expression with 6 h of treatment. (G) Human bronchoalveolar lavage fluid (BALf) was collected from influenza-infected vs. non-infected patients, isolated and sequenced for microRNA measuring a significant increase in (H) miR-193b-5p expression. ELISA demonstrates significant increase in both (I) TNF-α and (J) IFN-γ (median ± IQR; ∗p < 0.05, ∗∗p < 0.01; Mann-Whitney).

Table 1 Clinical features of the patients

	Patients	
Case 1 (MN)	Case 2 (AGC)	Case 3 (CMSM)	Case 4 (LO)	Case 5 (CFNM)	
Age (years)	35	35	39	81	51	
Sex	male	female	female	female	male	
Premorbid disease	absent	absent	absent	RA	absent	
Illness (daysa)	4	7	5	5	10	
Oseltamir (daysa)	10	14	10	4	4	
Steroids (daysa)	10	9	16	12	20	
Intubation (daysa)	10	8	25	17	20	
Status	alive	alive	dead	alive	dead	
NPA	+	+	–	+	–	
Lung biopsy	+	+	+	+	+	
Lung EM	+	+	+	+	+	
EM, electron microscopy for SALI associated to influenza A; RA, rheumatoid arthritis; NPA, nasopharyngeal aspirate. Case 3 was pregnant.

a Duration of illness or treatment.

PR8 infection in mice recapitulates hallmark pathological features of human IV-induced ARDS

We established an intranasal murine model of influenza A infection with PR8 (Influenza, Strain A/Puerto Rico/8/34 [H1N1] at 107 TCID50 [50% tissue culture infectivity dose]) that recapitulated unique pathological features of human IV-induced ARDS. This model is uniformly lethal by 8 days post-infection. WT mice were infected intranasally with PR8, and lungs were analyzed at days 3, 4, 5, and 6 post-infections (Figure 2A). Macroscopically, murine lungs became progressively more congested, hemorrhagic, and hyperemic after day 3 post-infection (Figure 2B). As early as day 4 post-PR8 infection, mice exhibited prominent bronchiolar epithelial necrosis with basement membrane detachments, epithelial necrosis, and neutrophilic infiltrates consistent with severe interstitial pneumonitis. Alveolar collapse, homogeneous septal thickening with cell infiltration, and hyaline membrane resembled features of human ARDS-associated diffuse alveolar damage (Figure 2Cix, x, xi, xii). Hyperplasia of alveolar cells with viral cytopathic changes were present (Figure S2A), similar to pathological changes evident in human severe H1N1 infection (Figure 2Cv, vi, vii, viii). Histopathologic changes showed a similar degree and pattern of lung injury distribution in mice and humans (Figure S2C). Disruptions of the intercellular junctional complexes (TJ) and opening of cell-cell contacts of type II alveolar epithelial cells (yellow square) were observed in both the transmission electron micrographs of alveolar septa in mouse and human H1N1-infected lungs (Figures 2D and 2E, respectively).Figure 2 PR8 intranasal infection increases endogenous miR-193b-5p, localized in macrophages, pulmonary endothelial, and epithelial cells, associated with reduced occludin and mitochondrial antiviral signaling mRNA expression

(A) Schematic of the experiment. WT (C57/bl6 10–12 weeks) mice were infected intranasally (IN) with PR8 (Influenza, Strain A/Puerto Rico/8/34 [H1N1] at 107 TCID50) and degree of lung injury was assessed at day 0 (baseline), and 3, 4, 5, and 6 post-infection (dpi). (B) Gross pathology images of lungs harvested on days 0, 4, 5, and 6 dpi showing increasing engorgement, hyperemia, and hemorrhagic changes over time. (C) Representative photomicrographs of H&E-stained lung sections (4 μm) showing lung injury in mice infected with PR8 compared with human lungs from H1N1-induced ARDS (i, ii, iii, iv). Necrotizing bronchiolitis and diffuse alveolar damage (DAD) were evident as early as 4 dpi in mice lung parenchyma, resembling pathological features of infected human tissue (v, vi, vii, viii). Prominent alveolar collapse, with intra-alveolar edema, DAD, and homogeneous septal thickening associated with inflammation and hyaline membrane deposition (ix, x, xi, xii). (D and E) Transmission electron micrographs of alveolar septa in mouse and human H1N1-treated lungs with diminishing of electron-dense materials, indicating disruptions of intercellular junctional complexes (TJ), and opening of cell-cell contacts of type II alveolar epithelial cells (AEC-II) (yellow square) were observed. HM, hyaline membranes; AEC, alveolar epithelial cells; TJ, tight junctions; Sq, squamous metaplasia. Bar graphs showing significant changes in the expression of (F) miR-193b-5p and (G) occludin (Ocln) 5–6 days post-PR8 inoculation in mice whole lungs. (H) Western blot demonstrating reduced occludin protein expression in mice lungs 6 days post influenza infection. Bar graphs showing significant changes in the expression of (I) mitochondrial antiviral signaling protein (MAVS), (J) IFN-β, and (K) IFN-γ in PR8-infected mice lungs relative to day 0 (n = 5–7, data are presented as median ± IQR; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 Tukey’s multiple comparisons test).

Increase in miR-193b-5p decreases Ocln and MAVS in PR8-induced acute lung injury

To determine the role of miR-193b-5p in PR8-induced lung injury, C57BL/6J mice were randomized to receive PR8 or mock infection with an equal volume of saline (Figures 2A–2C). Lungs were collected daily from days 3 to 6 post-infection. Infection resulted in ≥15% weight loss and 50% mortality rate by day 6 (Figures S2A and S2B). The levels of miR-193b-5p expression increased by 2- to 8-fold by 5–6 days post-infection (Figure 2F).

We have previously shown that the sequences for human and mouse miR-193b-5p are homologous,36 and that miR-193b-5p binds to a conserved seed sequence in the 3′ UTR of Ocln resulting in its silencing.37 Here we noted that increased levels of miR-193b-5p and the loss of Ocln expression at days 6 and 6 post-infection (Figure 2E) were related with worsening lung injury. In independent experiments, increased levels of miR-193b-5p were confirmed using digital droplet PCR (Figure S3C). Both the miR-193b-3p sister strand and miR-365a1-3p were not differentially expressed in our PR8 model (Figures S3D and S3E).

In addition to Ocln, the gene encoding for mitochondrial antiviral signaling (MAVS) protein is also a computationally predicted target of miR-193b-5p (MiRTarBase v.8.0). MAVS is an adaptor for the retinoic acid-inducible gene I-like receptors that mediate the transcriptional induction of type 1 interferons such as IFN-β and other IRGs that collectively establish the antiviral host response.38 We found significant decrease in the expression of MAVS by 5–6 days post-infection (Figure 2I), but this had no impact on type 1 IFN-β gene expression. IFN-β expression increased at day 3 post-infection and continued to rise and remain elevated until mice were humanely sacrificed at day 6 post-infection (Figure 2J). Type 2 IFN, IFN-β, peaked at 4 days post-infection and remained modestly increased until day 6 post-infection (Figure 2K).

To identify which cells expressed miR-193b-5p in vivo, we performed in situ hybridization with miR-193b-5p-specific probes and co-stained for macrophages (CD64; Figure S3F), endothelial (CD34; Figure S3G), and epithelial (transcription termination factor 1 [TTF1]) (Figure S3H) cells using specific markers to demonstrate miR-193b-5p punctae within all three cell types by day 4 post-PR8 infection.

miR-193b-5p and Ocln inversely affect IV replication and IRG expression in human primary bronchial airway epithelial and pulmonary microvascular endothelial cells

Based on the in situ evidence of miR-193b-5p localization in both murine and human pulmonary epithelial and endothelial cells, miR-Ocln rescue and Ocln knockdown experiments were performed in human bronchoalveolar distal airway epithelial cells (BEAS2b) and human pulmonary microvascular endothelial cells (HPMECs).39 Treating cells with increasing PR8 multiplicity of infection (MOI) resulted in increased miR-193b-5p expression levels in both BEAS2b (Figure S3I) and HPMECs (Figure S3J) at 24 h. BEAS2b cells were transfected with miR-193b-5p mimic (MIM), inhibitor (INH), or negative control (NC), followed by IV infection at an MOI of 1. Overexpression of miR-193b-5p in BEAS2b cells transfected with MIM resulted in increased detection of viral mRNA expression at 12 and 24 h post-infection (Figure 3). Viral RNA polymerase subunit 1 (PB1) was significantly increased at 12 h post-infection, and viral hemagglutinin (HA) was increased at 12 and 24 h post infection (Figures 3A and 3B).Figure 3 miR-193b-5p mimic enhances virus proliferation and attenuates type 1 interferon responses while miR-193b-5p inhibition reduces viral gene expression and enhances antiviral responses

BEAS2b cells were infected with H1N1 (MOI = 1.0) 24 h after transfection with miR-193b-5p mimic (MIM), inhibitor (INH), or their respective negative controls (NCs). Samples were collected at 12, 24, and/or 48 h post-infection (hpi) for analysis. Real-time qPCR for miR-193b-5p were normalized to miR-191 levels, and viral mRNA hemagglutinin (HA), viral RNA polymerase (PB1), and neuraminidase (NA) normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Bar graphs showing miR-193b-5p MIM transfection results in global increases in viral mRNA transcripts (A) 12 hpi and (B) 24 hpi. miR-193b-5p INH transfection reduces viral mRNA expression sustained to (C) 12, (D) 24, and (E) 48 hpi. (F) Transfection of the miR-193b-5p MIM increases while the INH reduces viral titers at 24 h (plaque-forming units, PFU/mL). (G) Representative western blot demonstrating knockdown (KD) of Ocln using a specific siRNA against Ocln. (H) Increased PB1, HA, and NA viral transcripts after Ocln KD. Bar graphs showing decreased expression of (I) IFN-β, (J) interferon-induced protein with tetratricopeptide repeats 1 (IFIT-1), and (K) bone marrow stromal cell antigen 2 (BST2) after Ocln KD. HPMECs infected with H1N1 (MOI = 1.0) or mock infected (saline [SAL]) 24 h after transfection with INH. Bar graphs show increased expression of IFN-β and interferon-regulated genes (IRGs) (L) IFN-β, (M) interferon regulatory factor 1 (IRF-1), (N) IFIT-1, (O) BST2. Primary human nasal epithelial cells (HNECs) transduced with lentivector overexpressing the miR-193b-5p INH or negative control (NC). Three weeks (21 days) after transduction cells were infected with respiratory syncytial virus (RSV) (n = 3) and assessed after 72 h. (P) GFP fluorescent photomicrographs of NECS transduced with the LV overexpressing the miR-193b-5p INH, the empty LV, or the negative control before RSV infection, and at 72 h after RSV infection (5× and 10× magnification). (Q) Overexpression of miR-193b-5p INH significantly reduced viral load 3 days after RSV infection. (R–V) BEAS2b cells were stimulated with human recombinant IFN-β at 1,000 IU and infected with LV-miR-193b-5p INH or blank vector (empty LV) resulting in increased miR-193b-5p (R), and decreased expression of Ocln (S), MAVS (T), and IFIT-1 (U), and signal transducer and activator of transcription 1 (STAT1) (V). Data are presented as median ± IQR. Data are presented as median ± IQR; individual symbols represent results for each independent experiment (n = 3–8). Comparisons between two groups (Mann-Whitney), multiple groups (Kruskal-Wallis), and for data that were normally distributed (Kolmogorov-Smirnov): Tukey’s test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

In contrast, transfection with the miR-193b-5p INH resulted in decreased viral mRNA expression (HA and PB1) as early as 12–24 hpi (Figures 3C and 3D). However, we noticed an increase in HA and decreased PB1 transcripts by 48 hpi (Figure 3E). Viral plaque-forming units were significantly increased upon delivery of the miR-193b-5p MIM (shown in red) and decreased after treatment with the INH (shown in blue, Figure 3F). The absence of complementarity between the miR-193b sequence and the virus mRNAs sequences negates the possibility of direct mimicry of host miR by the virus.

Gene silencing of Ocln with an anti-Ocln siRNA enhances viral mRNA transcription and impairs IFN response in vitro

Since Ocln is a target of miR-193b-5p and other viruses use Ocln to mediate pathogen entry into cells, we assessed the direct effects of Ocln knockdown on IV viral mRNA expression. Decrease in Ocln protein expression was shown by western blot after transfection with Ocln siRNA compared with a scrambled siRNA in BEAS2b cells (Figure 3G) and quantitated through densitometry analysis (Figure S3K). Figure 3H shows that decreased Ocln expression was associated with increased expression of viral polymerase (PB1), HA, and neuraminidase. Silencing Ocln resulted in impaired type 1 IFN responses, increased viral load, and impairment of host antiviral response factors, including IFN-β (Figure 3I), IFN-induced protein with tetratricopeptide repeats 1 (IFIT-1) (Figure 3J), and bone marrow stromal cell antigen 2 (BST-2) (Figure 3K) compared with the mock saline controls, recapitulating the effects of miR-193-5p MIM transfection in vitro and the effects of miR-193b-5p MIM transfection in vitro.

miR-193b-5p inhibits type 1 IFN responses in vitro

HPMECs were treated with the miR-193b-5p INH or NC, followed by PR8 infection at an MOI of 1. Treatment with the INH resulted in increased expression of IFN-β (Figure 3L) and upregulation of other IFN-related genes—IFN regulatory factor 1 (Figure 3M), IFIT-1 (Figure 3N), and BST-2 (Figure 3O)—compared with NC. Taken together our data suggest miR-193b-5p downregulates type 1 interferon responses in vitro.

The action of miR-193b-5p on antivirus responses is not restricted to IV

To determine whether miR-193b-5p also plays a role in other important respiratory virus infection we infected primary human nasal epithelial cells (HNECs) with respiratory syncytial virus (RSV). Since HNECs are difficult to transfect, we generated a lentivirus vector overexpressing miR-193b-5p INH or empty vector (blank vector) that we used to determine the effect of miR-193b-5p inhibition in HNECs.40 HNECs were harvested from consenting healthy volunteers and cultured in an air-fluid interface. Cells were transduced with the empty vector or the LV-miR-193b-5p-INH and allowed to differentiate for 21 days, after which they were infected with RSV. Transduction with the LV-miR-193b-5p-IHN prevented RSV infection-induced loss of normal HNEC morphology over 72 h (Figure 3P, left hand panel) compared with empty vector (Figure 3Q, right hand panel) and reduced log-fold viral titers in vitro (Figure 3Q), suggesting the broad applicability of the miR-193b-5p target in viral injury.

Since the lentivirus could potentially impact the IFN response, we determined the effect of the LV on IRG expression by stimulating cells transduced with either the LV-miR-193b-5p INH or empty vector with human recombinant IFN-β (1,000 IU) for 24 h. Stimulation with recombinant IFN-β resulted in increased miR-193b-5p; this was unaffected by transduction with the empty vector, but was mitigated by transduction with the LV-miR-193b-5p-INH (Figure 3R).

Ocln expression levels increased significantly in cells transduced with the LV-miR-193b-5p-INH (Figure 3S). Transduction with the empty vector did not prevent recombinant IFN-β-induced decrease in MAVS expression, while LV delivery of the miR-193b-5p INH resulted in increased MAVS expression (Figure 3T). While the expression of IFIT-1 was unaffected by LV-miR-193b-5p-INH, the expression of STAT1 was markedly enhanced by miR-193b-5p inhibition (Figures 3U and 3V). These results indicate that the role of miR-193b-5p on antivirus responses is not restricted to IV and suggest a broader applicability for miR-193b-5p inhibition for preserving or augmenting antiviral responses and decreasing viral load.

Mice deficient in miR-193b confer resistance to PR8 and have reduced lung injury and improved antiviral responses

Mice with a deletion of miR-193b (miR-193b-365a1 knockout [KO]) or their WT littermates were randomized to receive intranasally 30 μL of PR8 at 107 TCID50 or equal volume saline (mock infection) and were followed for 6 days. Absence of miR-193b conferred resistance to PR8-induced morbidity, as documented by reduced change in percent loss of body weight (Figure S3L) and body temperature (Figure S3M), and an overall reduced hyperemic lung (Figure 4A). In an independent experiment, in situ hybridization for miR-193b-5p shows increased visible punctae in the lung parenchyma from WT mice appearing at 6 days post-infection (Figure 4B). miR-193b KO mice were included as a NC. RNU6 stained intensely in the lung tissues as a positive control for the assay, and the scrambled control was used to demonstrate the specificity of the assay with minimal to no visible punctates.Figure 4 miR-193b KO mice are protected against loss of occludin, tight junction disruption, lung injury, and PR8-induced mortality

(A) Whole-lung images between non-infected vs. 6 days PR8-infected WT lungs vs. miR193b KO mice showing less-prominent pulmonary hepatization. (B) In situ images showing increase in miR-193b-5p clusters (oval) and punctae (arrows) in murine lungs at 6 days post-PR8 infection vs. non-infected in WT mice relative to miR193b KO mice controls. RNU6 stained intensely as a positive control, and the scrambled control showed no visible punctates, demonstrating the specificity of the assay. (C) Mouse lung histology stained for routine H&E demonstrates extensive diffuse alveolar damage relative to miR193b KO mice. (D) Survival curve demonstrates significant survival advantage of miR-193b KO compared with WT littermates infected with PR8 (n = 5–6 per group, TCID50 of 107, log rank, ∗∗p = 0.01). Comparison of PR8-induced lung injury in miR-193b KO vs. WT littermates as demonstrated by bronchoalveolar lavage fluid (BALf). (E) Trend in decreased total cells, (F) reduced protein content, and (G) significant reduction in neutrophil infiltrates by 6 dpi. Real-time qPCR showed no difference in (H) IL-6 in lung tissues, but a significant increase (I) in occludin (Ocln), (J) mitochondrial antiviral signaling (MAVS), (K) interferon-induced transmembrane protein 1 (IFITM1), (L) interferon alpha and beta receptor subunit 1 (IFNAR1), and (M) interferon beta transcripts at 6 dpi. Individual symbols represent individual mice. Data are presented as median ± IQR (∗p = 0.05, ∗∗p = 0.01; Mann-Whitney for two groups and Kruskal-Wallis for multiple groups).

Progression of PR8-induced lung injury was markedly attenuated in miR-193b KO mice. Common histopathological features of severe human IV lower respiratory tract infection, including diffuse alveolar damage (Figure 4C) with hyaline membrane formation, patchy interstitial lymphoplasmacytic infiltrates, bronchiolitis with squamous metaplasia, and/or pulmonary congestion with various degrees of hemorrhage,7,41 were significantly reduced in miR-193b KOs (Figure S4).

Seven-day mortality was significantly decreased in miR-193b KO mice (Figure 4D) compared with WT PR8-infected mice. At day 6 post-infection, miR-193b KO mice had decreased markers of lung injury including lower BALf total cell infiltrates, protein exudation (Figures 4E and 4F), and percent polymorphonuclear (PMN) cells in alveolar spaces (Figure 4G). We did not detect any difference in the expression of inflammation-related cytokine IL-6 mRNA (Figure 4H). However, antiviral gene expression including miR-193b-5p targets Ocln (Figure 4I), MAVS (Figure 4J), as well as IRGs, including IFITM1 (Figure 4K), the IFN alpha and beta receptor subunit 1 (IFNAR1) (Figure 4L), and IFN-β (Figure 4L), were markedly increased in miR-193b KOs compared with WT PR8-infected mice.

Decreased viral inclusions, preserved Ocln protein levels, and TJ integrity in PR8-infected miR-193b KO mice

Transmission electron micrography of lungs collected at day 6 post-PR8 infection from WT and miR-193b KO mice demonstrated that intercellular junctional complexes were markedly disrupted in WT mice (Figures 5A and 5B; Figure 5K, red arrows) with marked opening of alveolar epithelial cell-cell contacts (Figures 5A and 5B; Figure 5G, black arrows). Importantly, viral particles were seen totally free in cytoplasm adjacent to disrupted TJs (Figures 5A and 5B; Figure 5C, red arrows) contrasting with well-enclosed viral particles adjacent to non-fragmented TJs in miR-193b KO lungs (Figures 5A and 5B; Figure 5D, red arrows). Semiquantitative analysis of lung injury score performed by a lung pathologist (blinded to genotype and group assignment) supported preservation of alveolar epithelial cell (Figure 5C), adherens junction integrity (Figure 5D), TJs (Figure 5E), and was associated with significant reductions in viral inclusions (Figure 5F) in miR-193b KO mice compared with WT on days 4, 5, and 6 post-infection.Figure 5 miR-193b KO mice have preserved occludin and tight junction architecture with reduced viral inclusions

Representative electron microscopy tissue images showing changes in lung ultrastructure morphology in (A) non-infected (0 dpi): (left panels) the alveolar-capillary barrier (AA and B), alveolar cell side (AE and F), and endothelial cell side (AI and J). (B) PR8 infected (6 dpi): PR8-infected lungs (left panels) viral inclusions (BC and D), epithelium TJ opening (BJ and H), and endothelial TJ opening (BK and L) WT littermate controls compared with miR-193b KO mice (N = 3). At low magnification, type I alveolar epithelial cells (AE-I) and type II AECs (AE-II) were intimately associated with each other through tight junctions (the electron-dense materials at the lateral side close to the apical surfaces, yellow circle) and adherens junctions (located adjacent to the tight junctions, black arrowheads). In PR8-infected WT lungs, a decrease in electron-dense materials indicated disruption of intercellular junctional complexes (K, red arrows) and opening of AEC cell-cell contacts (G, black arrows). In WT lungs, viral particles are totally free in the cytoplasm adjacent to disrupted TJ (C, red arrows) contrasting with well-enclosed viral particles adjacent to non-fragmented TJs in miR-93b KO lungs (D, red arrows). The sections are representative lung sections from four mice per group. Scale bars, 5 mm (low magnification) and 1 mm (high magnification). AE-I, type I alveolar epithelial cells; AE-II, type II alveolar epithelial cells; EC, endothelial cells; cap, capillaries; Vir, viral particles. Each TEM image (20 images per animal) was analyzed for alveolar epithelial cells integrity, tight junction integrity, adherens junction integrity, and viral particles at three different magnifications. Semiquantitative lung score documented significant increases in parameters for loss of (C) alveolar epithelial cell integrity, (D) adherens junction integrity, (E) tight junction integrity, and (F) significantly reduced viral inclusions in miR-193b KO mice compared with WT on 4, 5, and 6 days post-infection. Submicroscopic findings were graded according to a 5-point semiquantitative severity-based scoring system where: 0 = integrity of the submicroscopic parameters, 1 = changes in 1%–25%, 2 = changes in 26%–50%, 3 = changes in 51%–75%, and 4 = changes in 76%–100% of the examined tissue.6 The pathologist (V.L.C.) working on the light microscopy and TEM images was blinded to group assignment. Data are presented as means ± SD (n = 4–6, two-way ANOVA, Sidak’s multiple comparisons test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). (G) Immunohistochemistry for occludin shows miR-193b KO mice lungs have preserved occludin protein expression 5–6 days post-infection relative to WT. (H) Representative western blot demonstrated increased Ocln protein expression in miR-193b KO mice relative to WT littermates at 4–6 dpi.

Ocln protein expression was preserved in miR-193b KO compared with WT mice. Ocln expression was decreased in bronchial epithelial cells by immunohistochemistry in WT mice infected with PR8 (Figure 5G); immunoblotting for Ocln confirmed preservation of Ocln in PR8 infected miR-193b KO mice compared with WTs (Figure 5H). Interestingly, immunoblotting for MAVS revealed that absence of miR-193b did not protect from PR8-induced decrease in MAVS protein expression, suggesting that, while MAVS may be a computational target of miR-193b-5p, other mechanisms may be responsible for the reduction in MAVS protein expression in vivo (data not shown).

These results demonstrate that absence of miR-193b results in preserved Ocln mRNA and protein expression, reduced TJ morphological disruptions, reduced loss of compartmentalization of virus particles, improved antiviral responses, and decreased lung injury and mortality post-PR8 infection.

Ocln gene silencing reconstitutes susceptibility to PR8 infection in miR-193b KO mice

To determine if Ocln is a critical target of miR-193b-5p playing a direct role in PR8-induced acute lung injury we randomized miR-193b KO mice and WT littermates to intubation and intratracheal delivery of 1.5 nmol of an anti-Ocln siRNA 24 h before intranasal inoculation with PR8 or saline (mock infection). We monitored behavioral and physiological parameters of mouse wellbeing daily and harvested lungs for analysis 5 days post-infection (Figure 6A).Figure 6 Knocking down occludin in miR-193b KO mice reconstitutes susceptibility to PR8-induced acute lung injury

(A) Schematic of experimental design showing intratracheal delivery of siRNA against occludin (Ocln) or scrambled siRNA 1 day before intranasal infection with PR8 (TCID50 of 107). Lungs were harvested 5 dpi. (B) Gross lung pathology images showed increased pulmonary edema, hyperemia, and hemorrhagic changes in WT and miR-193b KO mice that received the siRNA against Ocln compared with miR-193b KO mice that received the scrambled siRNA. Graphs show worsening physiological parameters in WT and miR-193b KO mice that received the siRNA against Ocln (C) loss of body weight and (D) decreased body temperature. (E) Ocln knock down reduced Ocln message expression in the KO mice by about 50%; this was associated with the reduced expression of (F) IFN-β, (G) MAVS, and increased expression of (H) iNOS. Symbols represent results for individual mice; two-way ANOVA, Sidak’s multiple comparisons test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). (I) Representative lung histology sections stained with H&E showed severe lymphocytic infiltration and alveolar collapse in miR-193b KO mice that received the siRNA against Ocln compared with non-infected WT, miR-193b KO, and mice that received the scrambled siRNA. (J) Immunohistochemistry showing decreased Ocln protein expression in siRNA-Ocln-treated miR-193b KO mice associated with diffuse alveolar damage with denudation of the membrane and cellular infiltrates in the airspaces using immunohistochemistry DAB staining. (K) Representative images of cytospin showing increased (L) total cell count, (M) total neutrophil infiltrates, and (N) total protein (edema) in the bronchoalveolar lavage fluid (BALf) from mice treated with the siRNA against Ocln compared with WT and miR-193b KO treated with scrambled siRNA, recapitulating injurious WT phenotype. (O) While WT mice showed non-significant increase in viral load with the siRNA against Ocln, in miR-193b KO mice, silencing Ocln increased viral load in whole-lung tissue homogenates by 6 days post-infection relative to scrambled control treated mice (data are presented as means ± SD, n = 3–6; two-way ANOVA [Kolmogorov–Smirnov], Sidak’s multiple comparisons test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

Gross lung morphology showed that miR-193b KO lungs treated with the siRNA against Ocln were markedly hyperemic, consistent with severe pulmonary hepatization similar to WT littermate positive controls. In contrast, miR-193b KOs randomized to receive the scrambled siRNA were protected from PR8-induced lung injury (Figure 6B). Silencing Ocln in miR-193b KO mice infected with PR8 resulted in increased loss of percent body weight (<5%) and temperature (<5%) relative to scrambled control (Figures 6C and 6D) associated with increased mortality. Lung Ocln transcripts were significantly reduced in both WT and miR-193b KO mice treated with the siRNA against Ocln relative to scrambled control (Figure 6E), demonstrating successful knockdown of Ocln. Lung IFN-β levels significantly decreased in mice where we knocked down Ocln in WT and KO mice treated with the siRNA (Figure 6F). Lung MAVS expression in KO mice treated with the siRNA against Ocln was significantly decreased, with a non-significant decrease in the WT mice (Figure 6G). Severe IV infection induced activation of innate immune cells neutrophils and macrophages, which increases the expression of inducible nitric oxide synthase (iNOS).42 Lung iNOS was elevated in both genotypes that received the siRNA against Ocln relative to scrambled controls (Figure 6H), suggesting increased inflammation following Ocln knockdown.

Histological assessments of lung injury were consistent with progression of lung injury following Ocln silencing in both genotypes (Figures 6I and 6J). Analysis of BALf demonstrated increased total cell infiltration (Figures 6K and 6L), PMN cells (Figure 6M), and total protein influx (Figure 6N). While Ocln silencing in WT mice non-significantly increased viral load in whole-lung tissue homogenates relative to scrambled control, the injurious effects were significantly reconstituted in miR-193b KOs (Figure 6O). Collectively, these results demonstrate that silencing Ocln in miR-193b KO mice abrogates the protective effects of miR-193b deletion and partially reverts the lung injury phenotype back to that seen in WT controls infected with PR8. Taken together, the data underscore the importance of both miR-193b and Ocln in PR8-induced acute lung injury.

Intratracheal administration of miR-193b-5p INH mitigated loss of Ocln, reduced viral load, and improved survival in PR8-induced acute lung injury

C57/BL6 mice were randomized to receive the miR-193b-5p INH through intratracheal administration on days 4 or 5 post-PR8 infection (at 107 TCID50) or the same dose of a non-human miRNA NC. A schematic of the experiment is shown in Figure 7A. Improved survival was noted when the INH was delivered on both days 4 and 5, but maximum improvement was following delivery at day 4 post-infection (Figures 7B and 7C). Lung tissues and BALf were collected on day 6 from mice treated at days 4 or 5 to determine levels of miR-193b-5p and assess lung injury-related outcomes. Treatment with miR-193b-5p INH resulted in decreased miR-193b-5p (Figure 7D), increased Ocln mRNA (Figure 7E), and protein expression (Figures 7F and 7G) in lung tissues. In addition, this treatment resulted in decreased viral titers measured using hemagglutination assay (Figure 7H), decreased expression of the viral nuclear capsid protein as measured by western blot (Figures 7I and 7J), increased IFN-β (Figure 7K), and decreased IL-6 (Figure 7L) gene expression. Infected mice treated with the miR-193b-5p INH also demonstrated decreased levels of systemic acidosis as measured by serum pH (Figure 7M) and circulating lactate (Figure 7N).Figure 7 Inhibition of miR-193b-5p mitigates lung injury in a pre-clinical model of PR8

(A) Schematic of experimental design. C57bl6 mice (10–12 weeks) infected with H1N1 (107 TCID50) were randomized to miR-193b-5p inhibitor (INH) or negative control (NC). INH and NC were delivered intratracheally (i.t.) at 4 or 5 dpi. Lungs from mice were harvested 6 dpi for lung injury assessment; a separate set of mice were followed to determine survival. Kaplan-Meier curves demonstrated survival advantage when the miR-193b-5p INH is delivered at (B) day 4 and (C) day 5 relative to NC (non-human RNA sequence, n = 8–14, log rank Mantel-Cox; ∗p = 0.05, ∗∗p = 0.01). Semiquantitative PCR shows significant (D) decrease in miR-193b-5p and (E) increased occludin (Ocln) transcript levels 6 dpi in miR-193b-5p INH- vs. NC-treated groups. (F) Representative western blot demonstrates increased Ocln protein expression relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (G) and quantified using arbitrary units (AU) between INH and NC (H), and were quantified by hemagglutination assay (HA/mL). (I) Representative western blot and (J) quantification demonstrates reduced expression of viral nuclear protein (NP) in INH relative to NC-treated H1N1 lungs. Bar graph showing significant (K) increase in IFN-β (antiviral response) and (L) decrease in inflammatory cytokine IL-6 in H1N1-infected lungs treated with the INH compared with the NC. Decreased severity of lung injury outcomes was evident at day 6 as determined by: (M) increased pH (N) decreased serum lactate (marker of systemic dysfunction), decrease in markers of pulmonary vascular leakage, and inflammation including decreased Evans blue dye incorporation into lung tissues (O) and decreased bronchoalveolar lavage fluid (P), total protein (Q), IgM levels, and total cellular infiltrates (R). (S) Significant reductions in myeloperoxidase (MPO) activity and (T) NADPH oxidase 2 (Nox2) expression levels in miR-193b-5p INH-treated lungs. Expression levels were quantified relative to GAPDH in arbitrary units (AU). Individual symbols represent results for individual mice. Data are presented as median ± IQR, Mann-Whitney U; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

Decreased Evans blue dye incorporation in lung tissue (Figure 7O) and BALf levels of total protein (Figure 7P) and IgM (Figure 7Q) were used to demonstrate decreased exudative edema formation in mice that received the miR-193b-5p-INH compared with NC. This was associated with decreased inflammatory cell infiltration (Figure 7R), myeloperoxidase activity (Figure 7S), and NADPH oxidase 2 expression (Figure 7T). The HI perfect reagent (HPF) alone did not seem to impart significant independent effect compared with the HPF carrying the NC inhibitor (Figures S3N, S3O, and S3P), therefore our results show the comparisons of just the NC and INH. Our results show how the timeline inhibition of miR-193b-5p in a PR8 murine model, can preserve Ocln transcript and protein expression, reduce lung injury, increase IFN-β, viral clearance and improve survival in vivo.

Discussion

MicroRNAs play a pivotal role in modulating the expression of various protein coding genes simultaneously and therefore are uniquely positioned to orchestrate a coordinated antiviral response.43,44 Not surprisingly, viruses have developed a variety of different ways to sabotage this response.7,8,45 Here, we demonstrate that, during IV infection, host-derived miR-193b-5p is induced by IFN-β and, in a feedback loop, acts to limit overexpression of IFN-β, which indirectly regulates the function of TJs by regulating Ocln expression levels in epithelial and endothelial pulmonary cells in a miR-193b-5p-dependent fashion. Various therapeutic strategies to silence Ocln are available for antiviral administration and are in phase 3 clinical trials to treat HCV infections.46,47 However, strategies to preserve or modulate virus-induced disruptions of TJs and Ocln expression are not well elucidated and may be warranted for severe IV and other respiratory virus infections such as RSV. Our paper highlights the role of host-derived miR-193b-5p in the regulation of IFN-β and Ocln-dependent TJs in human cells and in mice infected with PR8. Inhibition or absence of miR-193b can mitigate H1N1- and PR8-induced cell injury and morbidity and mortality, respectively; by preserving Ocln expression and enhancing host antiviral responses.

At baseline, this miR-193b-5p is nearly undetectable; its expression in vitro can be induced by lipopolysaccharides (main component of Gram-negative bacteria), TNF-α,37 and IL-1β.48 It has been implicated in cancer metastasis,49 angiotensin-mediated inhibition of apoptosis,50 binding and regulation of histone deacetylase 7,48 and Polo-like kinase 1, a critical regulator of cell-cycle progression and apoptosis.31 Converging evidence suggests that both strands of miR-193b (-3p and -5p) play important roles in respiratory virus infections. We found that, during influenza infection, IFN-β induction of miR-193b-5p contributes to the pathogenesis of influenza through the loss of Ocln. In our models, mir-193b-5p expression levels are increased in response to recombinant IFN-β regulating the expression of other IRGs. Both IVs and other respiratory viruses such as coronaviruses (SARS/COVID) encode proteins capable of disrupting members of the membrane-associated guanylate kinases (MAGUK) family of proteins (MUPP 1, MUPP 2, MUPP 3, and Ocln). Moreover, members of at least nine different virus families use apical-junctional complex molecules as receptors or disrupt these proteins to gain access to their receptors in the paracellular space. Naturally, multiple viruses have devised specific strategies to prevent the host from defending the apical-junctional complex as a part of their pathological mechanisms. Ocln is one of these MAGUK targets, regulated by miR-193b-5p.

Here, we show that inhibition of miR-193b-5p or miR-193b deficiency resulted in preserved Ocln levels, TJ integrity, and enhanced type 1 IFN responses, resulting in reduced lung injury and mortality. In vitro delivery of an siRNA against Ocln resulted in increased viral mRNA transcripts and impairment of IRG expression, demonstrating that Ocln is required for type 1 IFN responses. Electron microscopy confirms the importance of miR-193b-5p in H1N1 pathology; viral inclusions, TJ disruption, and increased paracellular spaces were significantly more prominent in WT compared with miR-193b KO mice infected with PR8. While important in regulating Ocln and IFN-β-dependent responses, our data by no means exonerate other miR-193b-5p mechanisms of action (i.e., other target genes) in influenza-induced lung injury—this is an ongoing area of research in the lab.

Gain- and loss-of-function experiments demonstrate the importance of miR-193b-5p to the regulation of IFN responses via regulation of IRGs. Although MAVS is a putative target of miR-193b-5p, our data do not support this being an effector of miR-193b-5p-induced effects since the absence of miR-193b-5p does not preclude decreases in MAVS protein expression. In addition to MAVS, various antiviral transcripts were significantly increased in miR-193b KO mice including IFITM 1 and 3. IFIT-1 proteins mediate antiviral activity against a broad range of viruses, such as influenza, through the suppression of translation initiation and sequestering viral proteins or RNA in the cytoplasm.51,52 Emerging evidence demonstrates that IFITM1 is localized predominantly at the plasma membrane,53 whereas IFITM2 and 3 localized within the endosomal and lysosomal compartments.54 IFITM3 exhibits its antiviral nature by trapping virions, including influenza, in endocytic compartments, resulting in their degradation.51,54,55 IFITM restriction is not a global property of the cell but rather localized to late or lysosomal compartments. IFITM 1, 2, and 3 have all been proven to exhibit antiviral properties including but not limited to SARS, Middle Eastern respiratory syndrome, and influenza-related viruses.56,57 Tetherin is encoded by BST-258 and inhibits the release of influenza and other viruses from the surface of infected cells via induction of apoptosis.59 The absence of IFIT-1 is not critical in influenza restriction and, similarly, the anti-viral function of BST-2 can be partially antagonized.60 IFNAR1 has recently been shown to play a critical role in other forms of respiratory virus lung infections such as infections with SARS-coronavirus 2, as inherited IFNAR1 deficiency predisposes not only to severe COVID-19 pneumonia but also multisystem inflammatory syndrome.61

The contribution of Ocln to permeability has been demonstrated through numerous lines of investigation: (1) blocking the extracellular loops62,63 and reducing the protein content alters paracellular permeability,64 (2) overexpression or prevention of Ocln degradation enhances transepithelial resistance (TER),65 and (3) synthetic peptides mimicking the extracellular loop compete with loop-loop interactions causing increased vascular leak.66 The debate on the role of Ocln in barrier protection centers on the fact that TJ ultrastructure appears unaltered in Ocln-deficient mice, and early studies suggested normal TER and permeability in isolated tissue preparations.67 These initial studies, however, may not have fully appreciated the role of Ocln (and TJs) in cell signaling and cell-cell and cell-matrix functions.68 Moreover, recent studies show that, although TJs are morphologically intact, Ocln-deficient mice have complex histological phenotypes characterized by chronic inflammation and poor TJ integrity in several epithelial/endothelial tissues, pointing to an important role for Ocln in TJ stability, signaling, and barrier function as opposed to TJ assembly.67 Here, we did not use Ocln KO mice, primarily because current data suggest that these mice have developed alternative strategies to compensate for the absence of Ocln and therefore are not ideal for our PR8-induced acute lung injury model. Hence, we delivered an siRNA to knock down Ocln acutely in vivo. When we knock down Ocln in miR-193b KO mice we partially abrogate the protective effects of miR-193b deletion in mice—we think in part because Ocln expression may not be entirely sufficient to explain the decrease in vascular leakage in our model. While we investigated the role of miR-193b-5p and Ocln in an influenza viral injury model, the putative targets list for miR-193b-5p is enriched for genes encoding for proteins associated with cell-cell adhesion and cell junction assembly including claudins (CLDN-2, -19), CLCN5 (chloride channel Cl−/H+ exchanger), and non-integrin membrane-ECM interaction proteins (thrombospondin 1 and 2), which may also account for the therapeutic effect of miR-193b-5p inhibition on vascular leakage. We are currently investigating the pulmonary differential expression profile in infected vs. non-infected miR-193b KO mice vs. WT littermates. This may provide novel insights into the role of miR-193b in host-immune regulation in virus-induced ARDS.

MicroRNAs are recognized as attractive targets for intervention as their aberrant expression in various pathological contexts can play critical roles in the development and progression of diseases.69,70 In the context of virus-induced lung injury, miR command multiple gene targets,26 and thus delivering miR mimics or inhibitors may reduce various syndromic features of lung injury and enhance survival. Synthesized single-stranded oligonucleotides (antagomirs) as well as inhibitors are showing promise as interventions for various diseases.70 Studies have demonstrated their use in HCV,71 chronic inflammatory diseases,72 cardiac injury,73 and metabolic diseases.74 The administration of a lentivector overexpressing the miR-193b-5p INH in the presence of IFN-β rescues MAVS and Ocln, demonstrating the therapeutic potential of our inhibitor. Our findings were further supported by the overexpression of miR-193b-5p INH in a lentivector to reduce viral load and preserve TJ integrity in human primary nasal epithelial cells infected with a non-IV RSV, broadening the potential applicability of exploiting this miR in other respiratory virus disease models.

Because of their high transduction efficiency, 70% of all gene therapy clinical trials involve the use of lentiviruses (LVs). Concerns regarding high immunogenicity and the risks of insertional mutagenesis has limited the enthusiasm for translation to the clinic.75 Enhanced safety profiles and lower production costs have led to the emergence of nonviral vectors as an attractive alternative for the delivery of synthetic siRNAs19,75,76 and miRs.69,70,77 Lipid nanoparticles have gained much attention for the successful delivery of therapeutic RNAs in vaccines for SARS-COV278,79 and treatment of hereditary amyloid transthyretin-mediated amyloidosis80 and tuberculosis.81,82

In our pre-clinical in vivo study, cationic lipids containing the commercially available miR INH or the NC lipid micelles were generated upon mixture with the QIAGEN HPF. The miR INH was modified to reduce in vivo degradation (2′-O-methyl-group modified oligonucleotides). We have previously published successful delivery of our miR-193b-5p INH in an vivo model of sepsis using this methodology.83 In this article, we show that delivery of the miR-193b-5p INH on days 4 and 5 post-infection reduces the expression levels of miR-193b-5p, 24–48 h post-inhibitor delivery. Future work will build off our proof-of-concept experiments to develop therapeutically relevant carriers, such as lipid nanoparticles, for the delivery of exogenous therapeutic miRs.

In summary, we expose the pathogenic nature of miR-193b-5p and its role in working at the behest of the virus, where its timely increase assists in the reduction of the fortified barrier-Ocln enabling a viral attack to the host system. The timely delivery of the miR-193b-5p INH, mitigates the loss of TJ Ocln, reduces viral load, and enhances host antiviral responses, thus positioning this miR as a possible novel therapeutic target in influenza-induced ARDS.

Materials and methods

Cell culture

Human bronchoalveolar distal airway epithelial cells (BEAS2b) were generously gifted by Dr. Haibo Zhang (University of Toronto, Canada, Department of Anesthesia). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. HPMECs were purchased from PromoCELL (Heidelberg, Germany) and cultured in endothelial cell basal medium MV 2 (EBM-2) (CC-3202, Lonza) supplemented with endothelial cell growth medium (EGM-2MV) SingleQuot Kit supplements and growth factors (CC4176, Lonza). All in vitro experiments with HPMECs were performed with cells at passages 3 to 7.

In vitro miR gain- and loss-of-function experiments

Cells were incubated at 70%–80% confluency for 24 h with 5 nM syn-hsa-miR-193b-5p miScript mimic (MIM; MSY0004767), 50 nM anti-hsa-miR-193b-5p miScript miRNA INH (MIN0004767), 50 nM hsa-miRNA inhibitor NC(1027271), 75 nM occludin siRNA (Ocln KD; SI05054385), or 5 or 75 nM AllStar siRNA NC (1027280) (all purchased from QIAGEN), using HPF (301705, QIAGEN) or Lipofectamine RNAiMAX (13778150, Thermo Fisher Scientific) as per the manufacturer’s instructions.

In vivo knockdown of Ocln

C57Bl6 mice were intubated 1ne day before PR8 inoculation using Ambion In Vivo Pre-Designed silencing RNA (siRNA, cat. no. 4457308) targeting Ocln, and the Ambion In Vivo Negative Control no. 1 siRNA (cat. no. 4457287). SiRNA were re-suspended with the invivofectamine 3.0 Reagent (cat. no. IVF3001), with each mouse receiving 1.6 nmol of siRNA.

Influenza infection in vitro

BEAS2b, Madin-Darby canine kidney (MDCK), and HPMECs cells were infected with 1.0 MOI of Influenza A/Puerto Rico/8/34 (H1N1; PR8; Charles River Laboratories) in DMEM, α-minimum essential medium (MEM), or EBM-2, respectively. Cells were supplemented with 2% FBS and 1% penicillin-streptomycin. Samples were collected at 12, 24, or 48 h post-infection.

Animal care

All experimental procedures and protocols were approved by the Animal Care Committee of St. Michael’s Hospital, Toronto, ON, Canada (ACC906). Eight- to 12-week-old male C57Bl/6J WT mice were purchased from Jackson Laboratories. miR-193-365-1 knockout mice (miR-193 KO) were a generous gift from Dr. Lothar Hennighausen (National Institutes of Health). miR-193-365-1 KO mice (miR-193 KO were bred in-house at the Keenan Research Center vivarium. Generation of KO mice has been previously characterized, and the microbiome from different genotypes was not different.84 miR-193 KO (C57Bl/6 × 129SvEv/Tac background) were bred with C57Bl/6J mice to produce heterozygotes (miR-193 Hets). DNA from ear notches was extracted using REDExtract-N-Amp (cat. no. XNAT, Sigma-Aldrich). PCR was performed per the manufacturer’s instructions. Primer sequences are given in Figure S5. Primer pair 1 produces a 546 bp band in miR-193 KO animals while primer pair 2 produces a roughly 500 bp band in miR-193b WT animals. The presence of two bands, as resolved on a 3% agarose gel, indicates heterozygosity (Figure S5). All mice were housed on a standard 12 h light/dark cycle and had free access to food (Teklan Glocbal 18% Protein Rodent Diet) and reverse osmosis water. Infection experiments were performed in a biosafety level 2 (BSL-2) room in the vivarium. Non-infected mice were housed in the same BSL-2 facility room as the infected mice to minimize potential confoundment. All animals were monitored closely and treated humanely in accordance to the guidelines outlined in the Canadian Council of Animal Care. Animals were sacrificed by anesthetic overdose (isoflurane) at the endpoint of each study. Assessment of physiological parameters were performed by an experimenter blinded to the assigned groups, and welfare assessment of the mice was performed in accordance with the St. Michael’s Hospital Vivarium Facility. All mice were randomized to experimental groups by providing a unique ID number per mouse in experiments, and were assigned to treatment groups using a random number generator (https://www.calculatorsoup.com) in accordance to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.85 ARRIVE 2.0 guideline checklist is provided in the supplemental information.

Influenza infection in vivo

Influenza A/PR/8/34 H1N1 was grown in eggs and tissue culture infectious dose determined by infection of MDCK cells86 and mice were anesthetized with 2%–5% isoflurane. Animals were held upright during administration. PR8 (107 TCID50) in a total volume of 30 μL was administered intranasally using a pipette. Animals were then placed on a bedding-free cage on top of a heating blanket until recovery. Intratracheal instillation of oligonucleotide mixtures were performed at days 4, 5, or 6 post-infections. Formal sample size calculation based on our preliminary experiments indicates that with eight mice per group we will have a power of 0.8 (type I error of 0.5) to detect statistically significant differences in survival and BALf differential cell count between mice treated with miR-193b-5p INH vs. NC.

BALf and lung tissue harvesting

After anesthetizing the animal in 2%–5% isoflurane with continuous oxygenation, the trachea was intubated with a 20-gauge cannula, and the right main step bronchus tied off. BALf collection was performed by instilling 0.5 mL of saline into the left lung and re-aspirating the fluid three times. After cardiac puncture and exsanguination, blood was collected and immediately centrifuged for serum. The right lungs were collected, and flash frozen in liquid nitrogen for subsequent real-time qRT-PCR and protein quantification. After centrifugation, the BALf supernatant was used for protein determination using a Bradford assay. The pellet was re-suspended in PBS for total cell count using a hemocytometer and trypan blue dye. The re-suspended pellet was centrifuged onto cytospin slides, which were stained with hematoxylin and eosin with the Hemacolor Stain Set (EMD Millipore, Billerica, MA) to distinguish monocytes, neutrophils, and macrophages for subsequent cell differential analysis. Lungs were also assessed for viral load determination (plaque assay), inflammation (neutrophil count), histology, western blots, immunohistochemistry, immunoblotting, or RNA analysis.

Assessment of viral load

Standard agarose-based plaque assays were performed from the BALf. MDCK cells, an accepted tight-junction forming the epithelial cell model,65 were maintained at 37°C in a humidified 5% CO2 chamber under stationary conditions, cultured to 90% confluency. Each well of a 6-well plate is seeded at 1 × 106 cells and cultured in MEM (Gibco/Invitrogen, Carlsbad, CA) containing 10% FBS, 100 units/mL penicillin (Gibco), and 100 μg/mL streptomycin (Gibco). After two washes with serum-free DMEM (Gibco/Invitrogen), the cells are maintained in serum-free DMEM at 37°C for 1 h. Then, each well was overlaid with 200 μL of diluted BAL of serially diluted solutions of inoculum and incubated at 37°C for 1 h. After one wash in serum-free DMEM, the cells were overlaid with serum-free DMEM containing 0.6% agarose, 0.1% diethylamino ethyl-dextran (Sigma-Aldrich), and 7 μg/mL trypsin (Sigma-Aldrich). The cells were cultured at 37°C for 72 h, fixed in 10% formaldehyde, and then stained with 0.037% methylene blue and each experiment was performed in triplicate.

Tissue culture infectivity dose

Samples were dispensed into a clear, round-bottomed, 96-well plate and serially diluted (1:10) in replicates of four down the plate from row A to H, along with a saline only NC and a PR8-positive control. MDCK cells were seeded at 105 cells per well. Plates were incubated at 37°C with 5% CO2. The following day, cells were washed twice with serum-free DMEM and incubated for 4 days with DMEM supplemented with 2% FBS, 1% penicillin-streptomycin, 1% L-glutamine, 1% NEAA, and 1× TPCK-treated trypsin. Post-incubation, 0.5% red blood cell solution was added to each well and degree of agglutination was measured after 2 h at 4°C. The TCID50 was calculated based on the ReedMunch method.87

mRNA and miRNA analyses

Frozen right lungs were first homogenized using a mortar and pestle. Total RNA was then extracted from 1 mg of homogenized lung by the TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized from 1 μg of RNA using the SuperScript First-Strand Synthesis System for reverse transcription (RT) PCR (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. Real-time qPCR was performed using the ViiA 7 Real-Time PCR System (Thermo Fisher Scientific). The relative change in gene expression or miRNA level was calculated by the ΔΔCT method. Gene expression was normalized to the housekeeping gene β-actin, and miR-193b-5p (QIAGEN, cat no. 339350) level was normalized to the housekeeping miRNA miR-191-5p (QIAGEN, cat no. 339350) and U6 (QIAGEN, cat no. 339350). Fold change was then normalized to appropriate controls. Primer list is provided in Table S1.

Immunoblotting protein expression

Lung tissues were homogenized, and cells were lysed using RIPA lysis buffer in loading buffer (Bio-Rad, no. 161–0737). Proteins were resolved on a 10% SDS-PAGE gel, transferred on to nitrocellulose membranes (Amersham Biosciences), and blocked in 5% milk as previously described.88 Primary antibodies were probed overnight at 4°. Western blot bands were analyzed using Bio-Rad Gel Doc 2000. Densitometry was performed using GelQuantNet software. Antibodies used include occludin (71–1500, Invitrogen, used for tissue samples), occludin (sc-133255, Santa Cruz, used for cell samples), MAVS (no. 3993, Cell Signaling), GAPDH (no. 2118, Cell Signaling), and β-actin (sc-47778, Santa Cruz).

IFN treatment

BEAS2b cells were treated with Recombinant Human IFN-β Protein (8499-IF, R&D). The specific activity of Recombinant Human IFN-β is approximately 2.8 × 108 IU/mg, which is calibrated against human IFN-β World Health Organization International Standard (NIBSC code: 00/572). Cells were treated at log-fold increase in IFN-β. Cells were lysed with TRIzol Reagent (Thermo Fisher Scientific) and RNA was extracted 4 h post-treatment.

Lentiviral vectors overexpressing miR-193b-5p INH or NC

Third generation replication-defective and self-inactivating, VSVG-pseudotyped integrating lentivectors (LVs) were produced. Human embryonic kidney cells (HEK293T) cells were co-transfected with four plasmid vectors: (1) bicistronic LV transfer plasmid carrying the miR-193b INH or scrambled control (NC) sequence plus the gene for green fluorescent protein (GFP), high-efficiency LV packaging, and envelope plasmids (gifts from Professor Gerard Wagemaker, University of Rotterdam) including (2) LV packaging plasmid (pMDL-g/pRRE; encoding for gag and pol proteins), (3) LV packaging plasmid (pRSV-REV; encoding for rev protein), and (4) LV envelope plasmid pMD-VSVg (encoding for VSVg: vesicular stomatitis virus glycoprotein). Viral particles were harvested 48 h post-transfection by cell supernatant centrifugation, dispersed in PBS, and stored at −80°C. Functional viral titers were calculated based on transduction of 293T cells with serial dilutions of the virus and by flow cytometry to measure percentage of GFP-positive cells. GFP expression was confirmed by microscopy and cell death by lactate dehydrogenase levels in conditioned medium.

Primary HNECs

Nasal brushings were performed from consenting healthy volunteers (REB no. 10000 61106 Hospital for Sick Children Research Ethics Board) and cells were cultured in basal epithelial growth medium. After two passages, cells were plated on collagen-coated 96-well trans wells for differentiation. After 21 days of growth under air-liquid interface conditions, cells were infected. Lentivirus (LV) vectors were used to transduce these cells and deliver the miR-193b-5p INH and NC sequences. At 21 days, cells were infected with PR8 (MOI = 1). Effect of miR-193b-5p inhibition was determined at 72 h post-infection. We exploited the existing system using RSV to demonstrate our approach and methodology.40

Digital droplet PCR

RNA (from homogenized whole lung tissue, or cells) was reverse transcribed using RT-specific TaqMan primers (cat. nos. 4427975 and 4427975, Thermo Fischer Scientific) and the TaqMan MicroRNA Reverse Transcription Kit (cat. no. 4366596, Thermo Fischer Scientific). ddPCR Supermix for Probes (cat. no. 186-3010, Bio-Rad) was used as per the manufacturer’s instructions. In brief, 20 μL reaction volumes, containing 1× master mix, primers and TaqMan probes (cat. no. 4427975, Thermo Fischer Scientific), and 2 μL of cDNA were loaded on disposable droplet generator cartridges (before 12.05.2014 cat. no. 186-3008, from 12.05.2014 cat. no. 186-4008, gaskets cat. no. 186-3009, Bio-Rad). Droplets were generated with 70 μL of droplet generation oil (cat. no. 186-3005, Bio-Rad) using the QX100 system (Bio-Rad). Droplets were then transferred to a 96-well PCR plate (cat. no. 0030128.613, TwinTec, Eppendorf, Hamburg, Germany). The samples were partitioned into ∼20,000 nL sized oil droplets through a water-oil emulsion technique. Partitioned droplets were thermocycled to endpoint in a 96-well PCR plate. After PCR, the sealed plates were placed in the droplet reader from the QX100 system (Bio-Rad), and droplets were analyzed as per the manufacturer’s recommendations (Droplet Reader Oil, cat. no. 186-3004). Fluorescence amplitudes were read for all droplets in each sample well in a droplet flow cytometer.

Histology

The left lung was fixed in 10% neutral-buffered formalin and embedded in paraffin. Sections (4 μm thick) were cut longitudinally from the central zone with a microtome and stained with hematoxylin-eosin for histologic analysis. Photomicrographs at magnifications of ×25, ×100, and ×400 were obtained from eight nonoverlapping fields of view per section under a light microscope (Olympus BX51, Olympus Latin America, Brazil).

miRNAscopy in situ

In situ hybridization for miR-193b-5p was performed using miRNAscope HD Detection Reagent-RED (cat. no. 3324510, Advanced Cell Diagnostics). The manual RNU6 (positive) control probe (cat. no. 727871-S1) and scrambled (negative) control probe (cat. no. 727881-S1) in channel S1 were used for the miRNAscope HD RED assay. The RNA-Protein Co-Detection Ancillary Kit (cat. no. 323180) allowed simultaneous co-detection of the miR-193b-5p probe (cat. no. 892401- S1), and primary antibodies TTF1 (cat. no. BS-0826R), CD34 (cat. no. PA585917), and CD68 (cat. no. PA5109344) were stained with Alexa Fluor 633 (cat. no. A21070).

Dual-Luciferase assay

The 2,800 bp of genomic sequence upstream of the human miR-193b-365a1 initiation codon (Ensembl ID ENSG00000207639) with flanking XhoI and BglII sites was synthesized by Epoch Gene into a pBluescript II SK (−) backbone. The promoter was digested using XhoI and BglII in NEB Buffer 3.1 and the fragment was cloned into the pNL1.2[NlucP] (cat. no. N1011) reporter vector. HEK293T cells were co-transfected with the promoter-reporter vector and a Firefly luciferase plasmid, pGL4.54[luc2/TK] vector (Promega) using Lipofectamine 3000 (Thermo Fisher Scientific) for 24 h. The cell culture medium was replaced with fresh DMEM medium containing Hu-IFN-β (1,000 IU) and TNF-α (10 ng/mL) at 6 h post-transfection. Mock-transfected cells were not treated with Hu-IFN-β or TNF-α. Cells were lysed, and the luciferase signal was measured using a Nano-Glo Dual-Luciferase Reporter Assay System (Promega). The results are expressed as the normalized ratio of Nanoluc to Firefly luciferase intensity in miR promoter vs. backbone only control. All experiments were repeated three times.

Ethics approval

REB no. 21-228C (St. Michael’s Hospital, Toronto, Canada), REB no. 02-0118-U/05-0016-C (Toronto Public Health Laboratory, Canada), REB no. 10000 61106 (Hospital for Sick Children, Toronto, Canada), REB no. 14.048–900 (University of Sao Paolo, Brazil), and REB no. 22/17 University of Asturias, Oviedo, Spain).

FFPE human samples

Archival blocks of FFPE lung tissue from five patients who died with ARDS (two males and three females; mean age 48 years) and three control patients deceased from cancer (lung negative for malignancy: two males and one female; mean age 64 years) were obtained (CEP 14.048-900, University of Sao Paolo) and RNA was extracted from FFPE human sections using the RNeasy FFPE kit (QIAGEN, cat. no. 73504) according to the manufacturer’s instructions.

Statistics analysis

All studies were randomized and blinded. Observers assessing endpoints were blinded. Formal sample size calculations are based on mortality, levels of miR-193b, or degree of lung injury. All data were analyzed using Kruskal-Wallis one-way analysis of variance (ANOVA) or two-way ANOVA as appropriate. We used false discovery rate to correct for multiple hypothesis testing.

Supplemental information

Document S1. Figures S1–S5 and Table S1

Document S2. Article plus supplemental information

Data availability

Data available upon request from corresponding author.

Acknowledgments

We would like to thank St. Michael’s Hospital Research Core Facility Staff members (Drs. Xiaofeng Lu and Caterina Di Ciano-Oliveira) and the vivarium staff for their expertise and technical support for the experiments. We further extend gratitude to Dr. Yuexin Shan, Michael Kim, Shehla Itzari, Dr. Paul Turgeon, and Dr. Ana Paula Monteiro, for their expertise and advice in the experimental models. We also thank the patients and family who donated samples for research purposes. Graphical abstract images were created with BioRender.com. This work was supported by the 10.13039/501100000024 Canadian Institutes of Health Research (grant no. MOP-130331 to CCDS). The research reported in this publication was supported in part by the São Paulo Research Foundation (10.13039/501100001807 FAPESP ) (2018/20403-6), Carlos Chagas Filho Foundation for Supporting Research in the State of Rio de Janeiro (10.13039/501100004586 FAPERJ E-26/210.181/2020), and the 10.13039/501100003593 National Council for Scientific and Technological Development (CNPq-483005/2012-6, 401700/2020-8) and 10.13039/501100002322 CAPES /DFATD (88881.158922/2017-01).

Author contributions

Concept and design, C.M.V., A.K.V., T.H.W., T.J.M., J.N.T., and C.C.d.S.; performed experiments and original data collection, C.M.V., A.K.V., V.L.C., S.G., A.M.E., S.Y., P.J.P., A.L.d.S., Y.C., Y.-C.T., C.C.G., S.S.-B., A.T.F., S.L.A., and A.A.; data analysis and interpretation, C.M.V., V.L.C., S.G., A.M.E., P.J.P., S.L.A., A.A., and C.C.d.S.; manuscript editing and discussion, C.M.V., A.K.V., S.G., A.M.E., J.N.T., A.L.d.S., T.H.W., C.C.G., P.R.M.R., G.M.A., T.J.M., S.S.-B., A.M., S.S.B., J.C.M., and C.C.d.S.

Declaration of interests

The authors declare no competing interests.

Supplemental information can be found online at https://doi.org/10.1016/j.ymthe.2023.06.011.
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