Sex-Specific Alterations of the Lung Transcriptome at Birth in Mouse Offspring Prenatally Exposed to Vanilla-Flavored E-Cigarette Aerosols and Enhanced Susceptibility to Asthma.

Currently, approximately 8 million adult Americans use electronic cigarettes (e-cigs) daily, including women of childbearing age. It is known that more than 10% of women smoke during their pregnancy, and recent surveys show that rates of maternal vaping are similar to rates of maternal cigarette smoking. However, the effects of inhaling e-cig aerosol on the health of fetuses remain unknown. The objective of the present study was to increase our understanding of the molecular effects caused by in utero exposures to e-cig aerosols on developing mouse lungs and, later in life, on the offspring's susceptibility to developing asthma. METHODS: Pregnant mice were exposed throughout gestation to either filtered air or vanilla-flavored e-cig aerosols containing 18 mg/mL of nicotine. Male and female exposed mouse offspring were sacrificed at birth, and then the lung transcriptome was evaluated. Additionally, once sub-groups of male offspring mice reached 4 weeks of age, they were challenged with house dust mites (HDMs) for 3 weeks to assess asthmatic responses. RESULTS: The lung transcriptomic responses of the mouse offspring at birth showed that in utero vanilla-flavored e-cig aerosol exposure significantly regulated 88 genes in males (62 genes were up-regulated and 26 genes were down-regulated), and 65 genes were significantly regulated in females (17 genes were up-regulated and 48 genes were down-regulated). Gene network analyses revealed that in utero e-cig aerosol exposure affected canonical pathways associated with CD28 signaling in T helper cells, the role of NFAT in the regulation of immune responses, and phospholipase C signaling in males, whereas the dysregulated genes in the female offspring were associated with NRF2-mediated oxidative stress responses. Moreover, we found that in utero exposures to vanilla-flavored e-cig aerosol exacerbated HDM-induced asthma in 7-week-old male mouse offspring compared to respective in utero air + HDM controls. CONCLUSIONS: Overall, these data demonstrate that in utero e-cig aerosol exposure alters the developing mouse lung transcriptome at birth in a sex-specific manner and provide evidence that the inhalation of e-cig aerosols is detrimental to the respiratory health of offspring by increasing the offspring' susceptibility to developing lung diseases later in life.

Ferrets as a model for tuberculosis transmission.

Even with the COVID-19 pandemic, tuberculosis remains a leading cause of human death due to a single infectious agent. Until successfully treated, infected individuals may continue to transmit Mycobacterium tuberculosis bacilli to contacts. As with other respiratory pathogens, such as SARS-CoV-2, modeling the process of person-to-person transmission will inform efforts to develop vaccines and therapies that specifically impede disease transmission. The ferret (Mustela furo), a relatively inexpensive, small animal has been successfully employed to model transmissibility, pathogenicity, and tropism of influenza and other respiratory disease agents. Ferrets can become naturally infected with Mycobacterium bovis and are closely related to badgers, well known in Great Britain and elsewhere as a natural transmission vehicle for bovine tuberculosis. Herein, we report results of a study demonstrating that within 7 weeks of intratracheal infection with a high dose (>5 x 103 CFU) of M. tuberculosis bacilli, ferrets develop clinical signs and pathological features similar to acute disease reported in larger animals, and ferrets infected with very-high doses (>5 x 104 CFU) develop severe signs within two to four weeks, with loss of body weight as high as 30%. Natural transmission of this pathogen was also examined. Acutely-infected ferrets transmitted M. tuberculosis bacilli to co-housed naïve sentinels; most of the sentinels tested positive for M. tuberculosis in nasal washes, while several developed variable disease symptomologies similar to those reported for humans exposed to an active tuberculosis patient in a closed setting. Transmission was more efficient when the transmitting animal had a well-established acute infection. The findings support further assessment of this model system for tuberculosis transmission including the testing of prevention measures and vaccine efficacy.

Nrf2 Activation Protects Against Organic Dust and Hydrogen Sulfide Exposure Induced Epithelial Barrier Loss and K. pneumoniae Invasion.

Agriculture workers report various respiratory symptoms owing to occupational exposure to organic dust (OD) and various gases. Previously, we demonstrated that pre-exposure to hydrogen sulfide (H2S) alters the host response to OD and induces oxidative stress. Nrf2 is a master-regulator of host antioxidant response and exposures to toxicants is known to reduce Nrf2 activity. The OD exposure-induced lung inflammation is known to increase susceptibility to a secondary microbial infection. We tested the hypothesis that repeated exposure to OD or H2S leads to loss of Nrf2, loss of epithelial cell integrity and that activation of Nrf2 rescues this epithelial barrier dysfunction. Primary normal human bronchial epithelial (NHBE) cells or mouse precision cut-lung slices (PCLS) were treated with media, swine confinement facility organic dust extract (ODE) or H2S or ODE+H2S for one or five days. Cells were also pretreated with vehicle control (DMSO) or RTA-408, a Nrf2 activator. Acute exposure to H2S and ODE+H2S altered the cell morphology, decreased the viability as per the MTT assay, and reduced the Nrf2 expression as well as increased the keap1 levels in NHBE cells. Repeated exposure to ODE or H2S or ODE+H2S induced oxidative stress and cytokine production, decreased tight junction protein occludin and cytoskeletal protein ezrin expression, disrupted epithelial integrity and resulted in increased Klebsiella pneumoniae invasion. RTA-408 (pharmacological activator of Nrf2) activated Nrf2 by decreasing keap1 levels and reduced ODE+H2S-induced changes including reversing loss of barrier integrity, inflammatory cytokine production and microbial invasion in PCLS but not in NHBE cell model. We conclude that Nrf2 activation has a partial protective function against ODE and H2S.

Transcriptomic and ultrastructural evidence indicate that anti-HMGB1 antibodies rescue organic dust-induced mitochondrial dysfunction.

Exposure to organic dust (OD) in agriculture is known to cause respiratory symptoms including loss of lung function. OD exposure activates multiple signaling pathways since it contains a variety of microbial products and particulate matter. Previously, we have shown how OD exposure leads to the secretion of HMGB1 and HMGB1-RAGE signaling, and how this can be a possible therapeutic target to reduce inflammation. Cellular mitochondria are indispensable for homeostasis and are emerging targets to curtail inflammation. Recently, we have also observed that OD exposure induces mitochondrial dysfunction characterized by loss of structural integrity and deficits in bioenergetics. However, the role of HMGB1 in OD-induced mitochondrial dysfunction in human bronchial epithelial (NHBE) cells remains elusive. Therefore, we aimed to study whether decreased levels of intracellular HMGB1 or antibody-mediated neutralization of secreted HMGB1 would rescue mitochondrial dysfunction. Single and repeated ODE exposure showed an elongated mitochondrial network and cristolysis whereas HMGB1 neutralization or the lack thereof promotes mitochondrial biogenesis evidenced by increased mitochondrial fragmentation, increased DRP1 expression, decreased MFN2 expression, and increased PGC1α expression. Repeated 5-day ODE exposure significantly downregulated transcripts encoding mitochondrial respiration and metabolism (ATP synthase, NADUF, and UQCR) as well as glucose uptake. This was reversed by the antibody-mediated neutralization of HMGB1. Our results support our hypothesis that, in NHBE cells, neutralization of ODE-induced HMGB1 secretion rescues OD-induced mitochondrial dysfunction.

The autotransporter protein BatA is a protective antigen against lethal aerosol infection with Burkholderia mallei and Burkholderia pseudomallei.

BACKGROUND: Burkholderia mallei and Burkholderia pseudomallei are the causative agents of glanders and melioidosis, respectively. There is no vaccine to protect against these highly-pathogenic and intrinsically antibiotic-resistant bacteria, and there is concern regarding their use as biological warfare agents. For these reasons, B. mallei and B. pseudomallei are classified as Tier 1 organisms by the U.S. Federal Select Agent Program and the availability of effective countermeasures represents a critical unmet need. METHODS: Vaccines (subunit and vectored) containing the surface-exposed passenger domain of the conserved Burkholderia autotransporter protein BatA were administered to BALB/c mice and the vaccinated animals were challenged with lethal doses of wild-type B. mallei and B. pseudomallei strains via the aerosol route. Mice were monitored for signs of illness for a period of up to 40 days post-challenge and tissues from surviving animals were analyzed for bacterial burden at study end-points. RESULTS: A single dose of recombinant Parainfluenza Virus 5 (PIV5) expressing BatA provided 74% and 60% survival in mice infected with B. mallei and B. pseudomallei, respectively. Vaccination with PIV5-BatA also resulted in complete bacterial clearance from the lungs and spleen of 78% and 44% of animals surviving lethal challenge with B. pseudomallei, respectively. In contrast, all control animals vaccinated with a PIV5 construct expressing an irrelevant antigen and infected with B. pseudomallei were colonized in those tissues. CONCLUSION: Our study indicates that the autotransporter BatA is a valuable target for developing countermeasures against B. mallei and B. pseudomallei and demonstrates the utility of the PIV5 viral vaccine delivery platform to elicit cross-protective immunity against the organisms.

Use of Immunohistochemistry to Demonstrate In Vivo Expression of the Burkholderia mallei Virulence Factor BpaB During Experimental Glanders.

Burkholderia mallei causes the highly contagious and debilitating zoonosis glanders, which infects via inhalation or percutaneous inoculation and often culminates in life-threatening pneumonia and sepsis. In humans, glanders is difficult to diagnose and requires prolonged antibiotic therapy with low success rates. No vaccine exists to protect against B. mallei, and there is concern regarding its use as a bioweapon. The authors previously identified the protein BpaB as a potential target for devising therapies due to its role in adherence to host cells and the formation of biofilms in vitro and its contribution to pathogenicity in a mouse model of glanders. In the present study, the authors developed an immunostaining approach to probe tissues of experimentally infected animals and demonstrated that BpaB is produced exclusively in vivo by wild-type B. mallei in target organs from mice and marmosets. They detected the expression of BpaB by B. mallei both extracellularly and within macrophages, neutrophils, and epithelial cells in respiratory tissues (7/10 marmoset; 2/2 mouse). The authors also noted the intracellular expression of BpaB by B. mallei in macrophages in the regional lymph nodes of mice (2/2 tissues) and MALT of marmosets (4/5 tissues). It is interesting that B. mallei bacteria infecting distal organs did not express BpaB (2/2 mice; 3/3 marmosets), suggesting that the protein is not necessary for bacterial fitness in these anatomic locations. These findings underscore the value of BpaB as a target for developing medical countermeasures and provide insight into its role in pathogenesis.

Antibodies against In Vivo-Expressed Antigens Are Sufficient To Protect against Lethal Aerosol Infection with Burkholderia mallei and Burkholderia pseudomallei.

Burkholderia mallei, a facultative intracellular bacterium and tier 1 biothreat, causes the fatal zoonotic disease glanders. The organism possesses multiple genes encoding autotransporter proteins, which represent important virulence factors and targets for developing countermeasures in pathogenic Gram-negative bacteria. In the present study, we investigated one of these autotransporters, BatA, and demonstrate that it displays lipolytic activity, aids in intracellular survival, is expressed in vivo, elicits production of antibodies during infection, and contributes to pathogenicity in a mouse aerosol challenge model. A mutation in the batA gene of wild-type strain ATCC 23344 was found to be particularly attenuating, as BALB/c mice infected with the equivalent of 80 median lethal doses cleared the organism. This finding prompted us to test the hypothesis that vaccination with the batA mutant strain elicits protective immunity against subsequent infection with wild-type bacteria. We discovered that not only does vaccination provide high levels of protection against lethal aerosol challenge with B. mallei ATCC 23344, it also protects against infection with multiple isolates of the closely related organism and causative agent of melioidosis, Burkholderia pseudomallei Passive-transfer experiments also revealed that the protective immunity afforded by vaccination with the batA mutant strain is predominantly mediated by IgG antibodies binding to antigens expressed exclusively in vivo Collectively, our data demonstrate that BatA is a target for developing medical countermeasures and that vaccination with a mutant lacking expression of the protein provides a platform to gain insights regarding mechanisms of protective immunity against B. mallei and B. pseudomallei, including antigen discovery.

Use of the common marmoset to study Burkholderia mallei infection.

Burkholderia mallei is a host-adapted bacterium that does not persist outside of its equine reservoir. The organism causes the zoonosis glanders, which is endemic in Asia, Africa, the Middle East and South America. Infection by B. mallei typically occurs via the respiratory or percutaneous route, and the most common manifestations are life-threatening pneumonia and bacteremia. Glanders is difficult to diagnose and requires prolonged antibiotic therapy with low success rates. There is no vaccine to protect against B. mallei and there is concern regarding its use as a biothreat agent. Thus, experiments were performed to establish a non-human primate model of intranasal infection to study the organism and develop countermeasures. Groups of marmosets (Callithrix jacchus) were inoculated intranasally with B. mallei strain ATCC 23344 and monitored for clinical signs of illness for up to 13 days. We discovered that 83% of marmosets inoculated with doses of 2.5 X 10(4) to 2.5 X 10(5) bacteria developed acute lethal infection within 3-4 days. Signs of disease were severe and included lethargy, inappetence, conjunctivitis, mucopurulent and hemorrhagic nasal discharges, and increased respiratory effort with abdominal lifts. Burkholderia mallei was cultured from the lungs, spleen and liver of these animals, and pathologic examination of tissues revealed lesions characteristic of glanders. Challenge experiments also revealed that 91% of animals infected with doses ranging from 25 to 2.5 X 10(3) bacteria exhibited mild non-specific signs of illness and were culture negative. One marmoset inoculated with 2.5 X 10(3) organisms developed moderate signs of disease and reached humane end-points 8 days post-infection. The liver and spleen of this animal were colonized with the agent and pathological analysis of tissues showed nasal, splenic and hepatic lesions. Taken together, these data indicate that the marmoset is a suitable model to study respiratory infection by B. mallei.

In ovo and in vitro susceptibility of American alligators (Alligator mississippiensis) to avian influenza virus infection.

Avian influenza has emerged as one of the most ubiquitous viruses within our biosphere. Wild aquatic birds are believed to be the primary reservoir of all influenza viruses; however, the spillover of H5N1 highly pathogenic avian influenza (HPAI) and the recent swine-origin pandemic H1N1 viruses have sparked increased interest in identifying and understanding which and how many species can be infected. Moreover, novel influenza virus sequences were recently isolated from New World bats. Crocodilians have a slow rate of molecular evolution and are the sister group to birds; thus they are a logical reptilian group to explore susceptibility to influenza virus infection and they provide a link between birds and mammals. A primary American alligator (Alligator mississippiensis) cell line, and embryos, were infected with four, low pathogenic avian influenza (LPAI) strains to assess susceptibility to infection. Embryonated alligator eggs supported virus replication, as evidenced by the influenza virus M gene and infectious virus detected in allantoic fluid and by virus antigen staining in embryo tissues. Primary alligator cells were also inoculated with the LPAI viruses and showed susceptibility based upon antigen staining; however, the requirement for trypsin to support replication in cell culture limited replication. To assess influenza virus replication in culture, primary alligator cells were inoculated with H1N1 human influenza or H5N1 HPAI viruses that replicate independent of trypsin. Both viruses replicated efficiently in culture, even at the 30 C temperature preferred by the alligator cells. This research demonstrates the ability of wild-type influenza viruses to infect and replicate within two crocodilian substrates and suggests the need for further research to assess crocodilians as a species potentially susceptible to influenza virus infection.