IL-13 decreases susceptibility to airway epithelial SARS-CoV-2 infection but increases disease severity in vivo.

Treatments available to prevent progression of virus-induced lung diseases, including coronavirus disease 2019 (COVID-19) are of limited benefit once respiratory failure occurs. The efficacy of approved and emerging cytokine signaling-modulating antibodies is variable and is affected by disease course and patient-specific inflammation patterns. Therefore, understanding the role of inflammation on the viral infectious cycle is critical for effective use of cytokine-modulating agents. We investigated the role of the type 2 cytokine IL-13 on SARS-CoV-2 binding/entry, replication, and host response in primary HAE cells in vitro and in a model of mouse-adapted SARS-CoV-2 infection in vivo. IL-13 protected airway epithelial cells from SARS-CoV-2 infection in vitro by decreasing the abundance of ACE2-expressing ciliated cells rather than by neutralization in the airway surface liquid or by interferon-mediated antiviral effects. In contrast, IL-13 worsened disease severity in mice; the effects were mediated by eicosanoid signaling and were abolished in mice deficient in the phospholipase A2 enzyme PLA2G2D. We conclude that IL-13-induced inflammation differentially affects multiple steps of COVID-19 pathogenesis. IL-13-induced inflammation may be protective against initial SARS-CoV-2 airway epithelial infection; however, it enhances disease progression in vivo. Blockade of IL-13 and/or eicosanoid signaling may be protective against progression to severe respiratory virus-induced lung disease.

A predominately pulmonary activation of complement in a mouse model of severe COVID-19.

Evidence from in vitro studies and observational human disease data suggest the complement system plays a significant role in SARS-CoV-2 pathogenesis, although how complement dysregulation develops in patients with severe COVID-19 is unknown. Here, using a mouse-adapted SARS-CoV-2 virus (SARS2-N501YMA30) and a mouse model of severe COVID-19, we identify significant serologic and pulmonary complement activation following infection. We observed C3 activation in airway and alveolar epithelia, and in pulmonary vascular endothelia. Our evidence suggests that while the alternative pathway is the primary route of complement activation, components of both the alternative and classical pathways are produced locally by respiratory epithelial cells following infection, and increased in primary cultures of human airway epithelia in response to cytokine exposure. This locally generated complement response appears to precede and subsequently drive lung injury and inflammation. Results from this mouse model recapitulate findings in humans, which suggest sex-specific variance in complement activation, with predilection for increased C3 activity in males, a finding that may correlate with more severe disease. Our findings indicate that complement activation is a defining feature of severe COVID-19 in mice and lay the foundation for further investigation into the role of complement in COVID-19.

Adaptation of SARS-CoV-2 to ACE2H353K mice reveals new spike residues that drive mouse infection.

The Coronavirus Disease 2019 (COVID-19) pandemic continues to cause extraordinary loss of life and economic damage. Animal models of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection are needed to better understand disease pathogenesis and evaluate preventive measures and therapies. While mice are widely used to model human disease, mouse angiotensin converting enzyme 2 (ACE2) does not bind the ancestral SARS-CoV-2 spike protein to mediate viral entry. To overcome this limitation, we "humanized" mouse Ace2 using CRISPR gene editing to introduce a single amino acid substitution, H353K, predicted to facilitate S protein binding. While H353K knockin Ace2 (mACE2H353K) mice supported SARS-CoV-2 infection and replication, they exhibited minimal disease manifestations. Following 30 serial passages of ancestral SARS-CoV-2 in mACE2H353K mice, we generated and cloned a more virulent virus. A single isolate (SARS2MA-H353K) was prepared for detailed studies. In 7-11-month-old mACE2H353K mice, a 104 PFU inocula resulted in diffuse alveolar disease manifested as edema, hyaline membrane formation, and interstitial cellular infiltration/thickening. Unexpectedly, the mouse-adapted virus also infected standard BALB/c and C57BL/6 mice and caused severe disease. The mouse-adapted virus acquired five new missense mutations including two in spike (K417E, Q493K), one each in nsp4, nsp9, and M and a single nucleotide change in the 5' untranslated region. The Q493K spike mutation arose early in serial passage and is predicted to provide affinity-enhancing molecular interactions with mACE2 and further increase the stability and affinity to the receptor. This new model and mouse-adapted virus will be useful to evaluate COVID-19 disease and prophylactic and therapeutic interventions.IMPORTANCEWe developed a new mouse model with a humanized angiotensin converting enzyme 2 (ACE2) locus that preserves native regulatory elements. A single point mutation in mouse ACE2 (H353K) was sufficient to confer in vivo infection with ancestral severe acute respiratory syndrome-coronavirus-2 virus. Through in vivo serial passage, a virulent mouse-adapted strain was obtained. In aged mACE2H353K mice, the mouse-adapted strain caused diffuse alveolar disease. The mouse-adapted virus also infected standard BALB/c and C57BL/6 mice, causing severe disease. The mouse-adapted virus acquired five new missense mutations including two in spike (K417E, Q493K), one each in nsp4, nsp9, and M and a single nucleotide change in the 5' untranslated region. The Q493K spike mutation arose early in serial passage and is predicted to provide affinity-enhancing molecular interactions with mACE2 and further increase the stability and affinity to the receptor. This new model and mouse-adapted virus will be useful to evaluate COVID-19 disease and prophylactic and therapeutic interventions.

Shuttle peptide delivers base editor RNPs to rhesus monkey airway epithelial cells in vivo.

Gene editing strategies for cystic fibrosis are challenged by the complex barrier properties of airway epithelia. We previously reported that the amphiphilic S10 shuttle peptide non-covalently combined with CRISPR-associated (Cas) ribonucleoprotein (RNP) enabled editing of human and mouse airway epithelial cells. Here, we derive the S315 peptide as an improvement over S10 in delivering base editor RNP. Following intratracheal aerosol delivery of Cy5-labeled peptide in rhesus macaques, we confirm delivery throughout the respiratory tract. Subsequently, we target CCR5 with co-administration of ABE8e-Cas9 RNP and S315. We achieve editing efficiencies of up-to 5.3% in rhesus airway epithelia. Moreover, we document persistence of edited epithelia for up to 12 months in mice. Finally, delivery of ABE8e-Cas9 targeting the CFTR R553X mutation restores anion channel function in cultured human airway epithelia. These results demonstrate the therapeutic potential of base editor delivery with S315 to functionally correct the CFTR R553X mutation in respiratory epithelia.

IL-13 induced inflammation increases DPP4 abundance but does not enhance MERS-CoV replication in airway epithelia.

BACKGROUND: Chronic pulmonary conditions such as asthma and COPD increase the risk of morbidity and mortality during infection with the Middle East respiratory syndrome coronavirus (MERS-CoV). We hypothesized that individuals with such comorbidities are more susceptible to MERS-CoV infection due to increased expression of its receptor, dipeptidyl peptidase 4 (DPP4). METHODS: We modeled chronic airway disease by treating primary human airway epithelia with the Th2 cytokine IL-13, examining how this impacted DPP4 protein levels along with MERS-CoV entry and replication. RESULTS: IL-13 exposure for 3 days led to increased DPP4 protein abundance, while a 21-day treatment increased DPP4 levels and caused goblet cell metaplasia. Surprisingly, despite this increase in receptor availability, MERS-CoV entry and replication were not significantly impacted by IL-13 treatment. CONCLUSIONS: Our results suggest that increased DPP4 abundance is likely not the primary mechanism leading to increased MERS severity in the setting of Th2 inflammation. Transcriptional profiling analysis highlighted the complexity of IL-13 induced changes in airway epithelia, including altered expression of genes involved in innate immunity, antiviral responses, and maintenance of the extracellular mucus barrier. These data suggest that additional factors likely interact with DPP4 abundance to determine MERS-CoV infection outcomes.

Intersubject Variation in ACE2 Protein Expression in Human Airway Epithelia and Its Relationship to Severe Acute Respiratory Syndrome Coronavirus 2.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 ) initiates entry into airway epithelia by binding its receptor, angiotensin-converting enzyme 2 (ACE2). METHODS: To explore whether interindividual variation in ACE2 abundance contributes to variability in coronavirus disease 2019 (COVID-19) outcomes, we measured ACE2 protein abundance in primary airway epithelial cultures derived from 58 human donor lungs. RESULTS: We found no evidence for sex- or age-dependent differences in ACE2 protein expression. Furthermore, we found that variations in ACE2 abundance had minimal effects on viral replication and induction of the interferon response in airway epithelia infected with SARS-CoV-2. CONCLUSIONS: Our results highlight the relative importance of additional host factors, beyond viral receptor expression, in determining COVID-19 lung disease outcomes.

Inter-individual Variation in Receptor Expression Influences MERS-CoV Infection and Immune Responses in Airway Epithelia.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes respiratory infection in humans, with symptom severity that ranges from asymptomatic to severe pneumonia. Known risk factors for severe MERS include male sex, older age, and the presence of various comorbidities. MERS-CoV gains entry into cells by binding its receptor, dipeptidyl peptidase 4 (DPP4), on the surface of airway epithelia. We hypothesized that expression of this receptor might be an additional determinant of outcomes in different individuals during MERS-CoV infection. To learn more about the role of DPP4 in facilitating MERS-CoV infection and spread, we used ELISA and immunofluorescent staining to characterize DPP4 expression in well-differentiated primary human airway epithelia (HAE). We noted wide inter-individual variation in DPP4 abundance, varying by as much as 1000-fold between HAE donors. This variability appears to influence multiple aspects of MERS-CoV infection and pathogenesis, with greater DPP4 abundance correlating with early, robust virus replication and increased cell sloughing. We also observed increased induction of interferon and some interferon-stimulated genes in response to MERS-CoV infection in epithelia with the greatest DPP4 abundance. Overall, our results indicate that inter-individual differences in DPP4 abundance are one host factor contributing to MERS-CoV replication and host defense responses, and highlight how HAE may serve as a useful model for identifying risk factors associated with heightened susceptibility to serious respiratory pathogens.

COVID-19 treatments and pathogenesis including anosmia in K18-hACE2 mice.

The ongoing coronavirus disease 2019 (COVID-19) pandemic is associated with substantial morbidity and mortality. Although much has been learned in the first few months of the pandemic, many features of COVID-19 pathogenesis remain to be determined. For example, anosmia is a common presentation, and many patients with anosmia show no or only minor respiratory symptoms1. Studies in animals infected experimentally with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of COVID-19, provide opportunities to study aspects of the disease that are not easily investigated in human patients. Although the severity of COVID-19 ranges from asymptomatic to lethal2, most experimental infections provide insights into mild disease3. Here, using K18-hACE2 transgenic mice that were originally developed for SARS studies4, we show that infection with SARS-CoV-2 causes severe disease in the lung and, in some mice, the brain. Evidence of thrombosis and vasculitis was detected in mice with severe pneumonia. Furthermore, we show that infusion of convalescent plasma from a recovered patient with COVID-19 protected against lethal disease. Mice developed anosmia at early time points after infection. Notably, although pre-treatment with convalescent plasma prevented most signs of clinical disease, it did not prevent anosmia. Thus, K18-hACE2 mice provide a useful model for studying the pathological basis of both mild and lethal COVID-19 and for assessing therapeutic interventions.

Heterogeneous expression of the SARS-Coronavirus-2 receptor ACE2 in the human respiratory tract.

BACKGROUND: Zoonotically transmitted coronaviruses are responsible for three disease outbreaks since 2002, including the current COVID-19 pandemic, caused by SARS-CoV-2. Its efficient transmission and range of disease severity raise questions regarding the contributions of virus-receptor interactions. ACE2 is a host ectopeptidase and the receptor for SARS-CoV-2. Numerous reports describe ACE2 mRNA abundance and tissue distribution; however, mRNA abundance is not always representative of protein levels. Currently, there is limited data evaluating ACE2 protein and its correlation with other SARS-CoV-2 susceptibility factors. MATERIALS AND METHODS: We systematically examined the human upper and lower respiratory tract using single-cell RNA sequencing and immunohistochemistry to determine receptor expression and evaluated its association with risk factors for severe COVID-19. FINDINGS: Our results reveal that ACE2 protein is highest within regions of the sinonasal cavity and pulmonary alveoli, sites of presumptive viral transmission and severe disease development, respectively. In the lung parenchyma, ACE2 protein was found on the apical surface of a small subset of alveolar type II cells and colocalized with TMPRSS2, a cofactor for SARS-CoV2 entry. ACE2 protein was not increased by pulmonary risk factors for severe COVID-19. Additionally, ACE2 protein was not reduced in children, a demographic with a lower incidence of severe COVID-19. INTERPRETATION: These results offer new insights into ACE2 protein localization in the human respiratory tract and its relationship with susceptibility factors to COVID-19.

K18-hACE2 Mice for Studies of COVID-19 Treatments and Pathogenesis Including Anosmia.

The ongoing COVID-19 pandemic is associated with substantial morbidity and mortality. While much has been learned in the first months of the pandemic, many features of COVID-19 pathogenesis remain to be determined. For example, anosmia is a common presentation and many patients with this finding show no or only minor respiratory signs. Studies in animals experimentally infected with SARS-CoV-2, the cause of COVID-19, provide opportunities to study aspects of the disease not easily investigated in human patients. COVID-19 severity ranges from asymptomatic to lethal. Most experimental infections provide insights into mild disease. Here, using K18-hACE2 mice that we originally developed for SARS studies, we show that infection with SARS-CoV-2 causes severe disease in the lung, and in some mice, the brain. Evidence of thrombosis and vasculitis was detected in mice with severe pneumonia. Further, we show that infusion of convalescent plasma (CP) from a recovered COVID-19 patient provided protection against lethal disease. Mice developed anosmia at early times after infection. Notably, while treatment with CP prevented significant clinical disease, it did not prevent anosmia. Thus K18-hACE2 mice provide a useful model for studying the pathological underpinnings of both mild and lethal COVID-19 and for assessing therapeutic interventions.

Heterogeneous expression of the SARS-Coronavirus-2 receptor ACE2 in the human respiratory tract.

BACKGROUND: Zoonotically transmitted coronaviruses are responsible for three disease outbreaks since 2002, including the current COVID-19 pandemic, caused by SARS-CoV-2. Its efficient transmission and range of disease severity raise questions regarding the contributions of virus-receptor interactions. ACE2 is a host ectopeptidase and the receptor for SARS-CoV-2. Numerous reports describe ACE2 mRNA abundance and tissue distribution; however, mRNA abundance is not always representative of protein levels. Currently, there is limited data evaluating ACE2 protein and its correlation with other SARS-CoV-2 susceptibility factors. MATERIALS AND METHODS: We systematically examined the human upper and lower respiratory tract using single-cell RNA sequencing and immunohistochemistry to determine receptor expression and evaluated its association with risk factors for severe COVID-19. FINDINGS: Our results reveal that ACE2 protein is highest within regions of the sinonasal cavity and pulmonary alveoli, sites of presumptive viral transmission and severe disease development, respectively. In the lung parenchyma, ACE2 protein was found on the apical surface of a small subset of alveolar type II cells and colocalized with TMPRSS2, a cofactor for SARS-CoV2 entry. ACE2 protein was not increased by pulmonary risk factors for severe COVID-19. Additionally, ACE2 protein was not reduced in children, a demographic with a lower incidence of severe COVID-19. INTERPRETATION: These results offer new insights into ACE2 protein localization in the human respiratory tract and its relationship with susceptibility factors to COVID-19.

Single-Dose, Intranasal Immunization with Recombinant Parainfluenza Virus 5 Expressing Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Spike Protein Protects Mice from Fatal MERS-CoV Infection.

Middle East respiratory syndrome coronavirus (MERS-CoV) can cause severe and fatal acute respiratory disease in humans and remains endemic in the Middle East since first being identified in 2012. There are currently no approved vaccines or therapies available for MERS-CoV. In this study, we evaluated parainfluenza virus 5 (PIV5)-based vaccine expressing the MERS-CoV envelope spike protein (PIV5/MERS-S) in a human DPP4 knockin C57BL/6 congenic mouse model (hDPP4 KI). Following a single-dose intranasal immunization, PIV5-MERS-S induced neutralizing antibody and robust T cell responses in hDPP4 KI mice. A single intranasal administration of 104 PFU PIV5-MERS-S provided complete protection against a lethal challenge with mouse-adapted MERS-CoV (MERSMA6.1.2) and improved virus clearance in the lung. In comparison, single-dose intramuscular immunization with 106 PFU UV-inactivated MERSMA6.1.2 mixed with Imject alum provided protection to only 25% of immunized mice. Intriguingly, an influx of eosinophils was observed only in the lungs of mice immunized with inactivated MERS-CoV, suggestive of a hypersensitivity-type response. Overall, our study indicated that PIV5-MERS-S is a promising effective vaccine candidate against MERS-CoV infection.IMPORTANCE MERS-CoV causes lethal infection in humans, and there is no vaccine. Our work demonstrates that PIV5 is a promising vector for developing a MERS vaccine. Furthermore, success of PIV5-based MERS vaccine can be employed to develop a vaccine for emerging CoVs such as SARS-CoV-2, which causes COVID-19.

Engineered amphiphilic peptides enable delivery of proteins and CRISPR-associated nucleases to airway epithelia.

The delivery of biologic cargoes to airway epithelial cells is challenging due to the formidable barriers imposed by its specialized and differentiated cells. Among cargoes, recombinant proteins offer therapeutic promise but the lack of effective delivery methods limits their development. Here, we achieve protein and SpCas9 or AsCas12a ribonucleoprotein (RNP) delivery to cultured human well-differentiated airway epithelial cells and mouse lungs with engineered amphiphilic peptides. These shuttle peptides, non-covalently combined with GFP protein or CRISPR-associated nuclease (Cas) RNP, allow rapid entry into cultured human ciliated and non-ciliated epithelial cells and mouse airway epithelia. Instillation of shuttle peptides combined with SpCas9 or AsCas12a RNP achieves editing of loxP sites in airway epithelia of ROSAmT/mG mice. We observe no evidence of short-term toxicity with a widespread distribution restricted to the respiratory tract. This peptide-based technology advances potential therapeutic avenues for protein and Cas RNP delivery to refractory airway epithelial cells.

IFN-I response timing relative to virus replication determines MERS coronavirus infection outcomes.

Type 1 IFNs (IFN-I) generally protect mammalian hosts from virus infections, but in some cases, IFN-I is pathogenic. Because IFN-I is protective, it is commonly used to treat virus infections for which no specific approved drug or vaccine is available. The Middle East respiratory syndrome-coronavirus (MERS-CoV) is such an infection, yet little is known about the role of IFN-I in this setting. Here, we show that IFN-I signaling is protective during MERS-CoV infection. Blocking IFN-I signaling resulted in delayed virus clearance, enhanced neutrophil infiltration, and impaired MERS-CoV-specific T cell responses. Notably, IFN-I administration within 1 day after infection (before virus titers peak) protected mice from lethal infection, despite a decrease in IFN-stimulated gene (ISG) and inflammatory cytokine gene expression. In contrast, delayed IFN-ß treatment failed to effectively inhibit virus replication, increased infiltration and activation of monocytes, macrophages, and neutrophils in the lungs, and enhanced proinflammatory cytokine expression, resulting in fatal pneumonia in an otherwise sublethal infection. Together, these results suggest that the relative timing of the IFN-I response and maximal virus replication is key in determining outcomes, at least in infected mice. By extension, IFN-αß or combination therapy may need to be used cautiously to treat viral infections in clinical settings.

Attenuation of pulmonary ACE2 activity impairs inactivation of des-Arg9 bradykinin/BKB1R axis and facilitates LPS-induced neutrophil infiltration.

Angiotensin-converting enzyme 2 (ACE2) is a terminal carboxypeptidase with important functions in the renin-angiotensin system and plays a critical role in inflammatory lung diseases. ACE2 cleaves single-terminal residues from several bioactive peptides such as angiotensin II. However, few of its substrates in the respiratory tract have been identified, and the mechanism underlying the role of ACE2 in inflammatory lung disease has not been fully characterized. In an effort to identify biological targets of ACE2 in the lung, we tested its effects on des-Arg9 bradykinin (DABK) in airway epithelial cells on the basis of the hypothesis that DABK is a biological substrate of ACE2 in the lung and ACE2 plays an important role in the pathogenesis of acute lung inflammation partly through modulating DABK/bradykinin receptor B1 (BKB1R) axis signaling. We found that loss of ACE2 function in mouse lung in the setting of endotoxin inhalation led to activation of the DABK/BKB1R axis, release of proinflammatory chemokines such as C-X-C motif chemokine 5 (CXCL5), macrophage inflammatory protein-2 (MIP2), C-X-C motif chemokine 1 (KC), and TNF-α from airway epithelia, increased neutrophil infiltration, and exaggerated lung inflammation and injury. These results indicate that a reduction in pulmonary ACE2 activity contributes to the pathogenesis of lung inflammation, in part because of an impaired ability to inhibit DABK/BKB1R axis-mediated signaling, resulting in more prompt onset of neutrophil infiltration and more severe inflammation in the lung. Our study identifies a biological substrate of ACE2 within the airways, as well as a potential new therapeutic target for inflammatory diseases.

Mouse-adapted MERS coronavirus causes lethal lung disease in human DPP4 knockin mice.

The Middle East respiratory syndrome (MERS) emerged in Saudi Arabia in 2012, caused by a zoonotically transmitted coronavirus (CoV). Over 1,900 cases have been reported to date, with ∼36% fatality rate. Lack of autopsies from MERS cases has hindered understanding of MERS-CoV pathogenesis. A small animal model that develops progressive pulmonary manifestations when infected with MERS-CoV would advance the field. As mice are restricted to infection at the level of DPP4, the MERS-CoV receptor, we generated mice with humanized exons 10-12 of the mouse Dpp4 locus. Upon inoculation with MERS-CoV, human DPP4 knockin (KI) mice supported virus replication in the lungs, but developed no illness. After 30 serial passages through the lungs of KI mice, a mouse-adapted virus emerged (MERSMA) that grew in lungs to over 100 times higher titers than the starting virus. A plaque-purified MERSMA clone caused weight loss and fatal infection. Virus antigen was observed in airway epithelia, pneumocytes, and macrophages. Pathologic findings included diffuse alveolar damage with pulmonary edema and hyaline membrane formation associated with accumulation of activated inflammatory monocyte-macrophages and neutrophils in the lungs. Relative to the parental MERS-CoV, MERSMA viruses contained 13-22 mutations, including several within the spike (S) glycoprotein gene. S-protein mutations sensitized viruses to entry-activating serine proteases and conferred more rapid entry kinetics. Recombinant MERSMA bearing mutant S proteins were more virulent than the parental virus in hDPP4 KI mice. The hDPP4 KI mouse and the MERSMA provide tools to investigate disease causes and develop new therapies.

Novel Innate Immune Genes Regulating the Macrophage Response to Gram Positive Bacteria.

Host variation in Toll-like receptors and other innate immune signaling molecules alters infection susceptibility. However, only a portion of the variability observed in the innate immune response is accounted for by known genes in these pathways. Thus, the identification of additional genes that regulate the response to Gram positive bacteria is warranted. Bone marrow-derived macrophages (BMMs) from 43 inbred mouse strains were stimulated with lipotechoic acid (LTA), a major component of the Gram positive bacterial cell wall. Concentrations of the proinflammatory cytokines IL-6, IL-12, and TNF-α were measured. In silico whole genome association (WGA) mapping was performed using cytokine responses followed by network analysis to prioritize candidate genes. To determine which candidate genes could be responsible for regulating the LTA response, candidate genes were inhibited using RNA interference (RNAi) and were overexpressed in RAW264.7 macrophages. BMMs from Bdkrb1-deficient mice were used to assess the effect of Bdkrb1 gene deletion on the response to LTA, heat-killed Streptococcus pneumoniae, and heat-killed Staphylococcus aureus WGA mapping identified 117 loci: IL-6 analysis yielded 20 loci (average locus size = 0.133 Mb; 18 genes), IL-12 analysis produced 5 loci (0.201 Mb average; 7 genes), and TNF-α analysis yielded 92 loci (0.464 Mb average; 186 genes of which 46 were prioritized by network analysis). The follow-up small interfering RNA screen of 71 target genes identified four genes (Bdkrb1, Blnk, Fbxo17, and Nkx6-1) whose inhibition resulted in significantly reduced cytokine production following LTA stimulation. Overexpression of these four genes resulted in significantly increased cytokine production in response to LTA. Bdkrb1-deficient macrophages were less responsive to LTA and heat-killed S. aureus, validating the genetic and RNAi approach to identify novel regulators of the response to LTA. We have identified four innate immune response genes that may contribute to Gram positive bacterial susceptibility.

Newborn Cystic Fibrosis Pigs Have a Blunted Early Response to an Inflammatory Stimulus.

RATIONALE: Studies suggest that inappropriate responses to proinflammatory stimuli might contribute to inflammation in cystic fibrosis (CF) lungs. However, technical challenges have made it difficult to distinguish whether altered responses in CF airways are an intrinsic defect or a secondary effect of chronic disease in their tissue of origin. The CF pig model provides an opportunity to study the inflammatory responses of CF airways at birth, before the onset of infection and inflammation. OBJECTIVES: To test the hypothesis that acute inflammatory responses are perturbed in porcine CF airways. METHODS: We investigated the inflammatory responses of newborn CF and non-CF pig airways following a 4-hour exposure to heat-killed Staphylococcus aureus, in vivo and in vitro. MEASUREMENTS AND MAIN RESULTS: Following an in vivo S. aureus challenge, markers of inflammation were similar between CF and littermate control animals through evaluation of bronchoalveolar lavage and tissues. However, transcriptome analysis revealed genotype-dependent differences as CF pigs showed a diminished host defense response compared with their non-CF counterparts. Furthermore, CF pig airways exhibited an increase in apoptotic pathways and a suppression of ciliary and flagellar biosynthetic pathways. Similar differences were observed in cultured airway epithelia from CF and non-CF pigs exposed to the stimulus. CONCLUSIONS: Transcriptome profiling suggests that acute inflammatory responses are dysregulated in the airways of newborn CF pigs.

Airway acidification initiates host defense abnormalities in cystic fibrosis mice.

Cystic fibrosis (CF) is caused by mutations in the gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. In humans and pigs, the loss of CFTR impairs respiratory host defenses, causing airway infection. But CF mice are spared. We found that in all three species, CFTR secreted bicarbonate into airway surface liquid. In humans and pigs lacking CFTR, unchecked H(+) secretion by the nongastric H(+)/K(+) adenosine triphosphatase (ATP12A) acidified airway surface liquid, which impaired airway host defenses. In contrast, mouse airways expressed little ATP12A and secreted minimal H(+); consequently, airway surface liquid in CF and non-CF mice had similar pH. Inhibiting ATP12A reversed host defense abnormalities in human and pig airways. Conversely, expressing ATP12A in CF mouse airways acidified airway surface liquid, impaired defenses, and increased airway bacteria. These findings help explain why CF mice are protected from infection and nominate ATP12A as a potential therapeutic target for CF.

Middle East Respiratory Syndrome Coronavirus Causes Multiple Organ Damage and Lethal Disease in Mice Transgenic for Human Dipeptidyl Peptidase 4.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes life-threatening disease. Dipeptidyl peptidase 4 (DPP4) is the receptor for cell binding and entry. There is a need for small-animal models of MERS, but mice are not susceptible to MERS because murine dpp4 does not serve as a receptor. We developed transgenic mice expressing human DPP4 (hDPP4) under the control of the surfactant protein C promoter or cytokeratin 18 promoter that are susceptible to infection with MERS-CoV. Notably, mice expressing hDPP4 with the cytokeratin 18 promoter developed progressive, uniformly fatal disease following intranasal inoculation. High virus titers were present in lung and brain tissues 2 and 6 days after infection, respectively. MERS-CoV-infected lungs revealed mononuclear cell infiltration, alveolar edema, and microvascular thrombosis, with airways generally unaffected. Brain disease was observed, with the greatest involvement noted in the thalamus and brain stem. Animals immunized with a vaccine candidate were uniformly protected from lethal infection. These new mouse models of MERS-CoV should be useful for investigation of early disease mechanisms and therapeutic interventions.