INDUCIBLE EPITHELIAL RESISTANCE AGAINST ACUTE VIRAL PNEUMONIA AND CHRONIC ASTHMA

Viral pneumonia remains a global health threat despite worldwide vaccination and therapeutic programs. The influenza pandemic of 1918 and SARS-CoV2 pandemic of 2019-2020 are cautionary reminders demanding the need for novel treatment strategies. Moreover, in addition to causing acute disease, respiratory virus infections are often complicated by chronic lung pathologies, such as asthma induction, progression, and exacerbation. We have reported that mice treated with a combination of inhaled Toll-like receptor (TLR) 2/6 and TLR 9 agonists (Pam2-ODN) to stimulate innate immunity are broadly protected against respiratory pathogens, but the mechanisms underlying this protection remain incompletely elucidated. Here, we show in a lethal paramyxovirus model that Pam2-ODN-enhanced survival is associated with robust virus inactivation by reactive oxygen species (ROS), which occurs prior to internalization by lung epithelial cells. We also found that mortality in sham-treated mice temporally corresponded with CD8+ T cell-enriched lung inflammation that peaks on days 11-12 after viral challenge, when the viral burden has waned to a scarcely detectable level. Pam2-ODN treatment blocked this injurious inflammation by reducing the viral burden. Alternatively, depleting CD8+ T cells 8 days after viral challenge also decreased mortality. Notably, Pam2-ODN treatment of mice lacking CD8+ T cells at any point of SeV infection showed a similar degree of protection demonstrating Pam2-ODN mediated protection independent of CD8+ T cell response. Further, Pam2-ODN treatment protected SeV challenged mice from progressing to asthma-like disease by initiating acute anti-viral effects. Interestingly, mice aerosolized with Pam2-ODN after viral clearance displayed reduced eosinophilia that was associated with reduced lung epithelial IL-33 production. Findings from this study reveal opportunities for targeted immunomodulation to protect susceptible individuals from mortality of respiratory virus infections and preventing progression to chronic asthma

Epithelial immunomodulation by aerosolized Toll-like receptor agonists prevents allergic inflammation in airway mucosa in mice

Allergic asthma is a chronic inflammatory respiratory disease associated with eosinophilic infiltration, increased mucus production, airway hyperresponsiveness, and airway remodeling. Epidemiologic data reveal that the prevalence of allergic sensitization and associated diseases has increased in the twentieth century. This has been hypothesized to be partly due to reduced contact with microbial organisms (the hygiene hypothesis) in industrialized society. Airway epithelial cells, once considered a static physical barrier between the body and the external world, are now widely recognized as immunologically active cells that can initiate, maintain, and restrain inflammatory responses, such as those that mediate allergic disease. Airway epithelial cells can sense allergens via expression of myriad Toll-like receptors (TLRs) and other pattern-recognition receptors. We sought to determine whether the innate immune response stimulated by a combination of Pam2CSK4 (“Pam2”, TLR2/6 ligand) and a class C oligodeoxynucleotide ODN362 (“ODN”, TLR9 ligand), when delivered together by aerosol (“Pam2ODN”), can modulate the allergic immune response to allergens. Treatment with Pam2ODN 7 days before sensitization to House Dust Mite (HDM) extract resulted in a strong reduction in eosinophilic and lymphocytic inflammation. This Pam2ODN immunomodulatory effect was also seen using Ovalbumin (OVA) and A. oryzae (Ao) mouse models. The immunomodulatory effect was observed as much as 30 days before sensitization to HDM, but ineffective just 2 days after sensitization, suggesting that Pam2ODN immunomodulation lowers the allergic responsiveness of the lung, and reduces the likelihood of inappropriate sensitization to aeroallergens. Furthermore, Pam2 and ODN cooperated synergistically suggesting that this treatment is superior to any single agonist in the setting of allergen immunotherapy

Inducible lung epithelial resistance requires multisource reactive oxygen species generation to protect against bacterial infections.

Pneumonia remains a global health threat, in part due to expanding categories of susceptible individuals and increasing prevalence of antibiotic resistant pathogens. However, therapeutic stimulation of the lungs' mucosal defenses by inhaled exposure to a synergistic combination of Toll-like receptor (TLR) agonists known as Pam2-ODN promotes mouse survival of pneumonia caused by a wide array of pathogens. This inducible resistance to pneumonia relies on intact lung epithelial TLR signaling, and inducible protection against viral pathogens has recently been shown to require increased production of epithelial reactive oxygen species (ROS) from multiple epithelial ROS generators. To determine whether similar mechanisms contribute to inducible antibacterial responses, the current work investigates the role of ROS in therapeutically-stimulated protection against Pseudomonas aerugnosa challenges. Inhaled Pam2-ODN treatment one day before infection prevented hemorrhagic lung cytotoxicity and mouse death in a manner that correlated with reduction in bacterial burden. The bacterial killing effect of Pam2-ODN was recapitulated in isolated mouse and human lung epithelial cells, and the protection correlated with inducible epithelial generation of ROS. Scavenging or targeted blockade of ROS production from either dual oxidase or mitochondrial sources resulted in near complete loss of Pam2-ODN-induced bacterial killing, whereas deficiency of induced antimicrobial peptides had little effect. These findings support a central role for multisource epithelial ROS in inducible resistance against a bacterial pathogen and provide mechanistic insights into means to protect vulnerable patients against lethal infections

Epithelial Immunomodulation by Aerosolized Toll like Receptor Agonists Attenuate Allergic Responsiveness in Mice

https://openworks.mdanderson.org/sumexp21/1031/thumbnail.jp

Use of a Guinea Pig-Specific Transcriptome Array for Evaluation of Protective Immunity against Genital Chlamydial Infection following Intranasal Vaccination in Guinea Pigs

<div><p>Guinea pigs have been used as a second animal model to validate putative anti-chlamydial vaccine candidates tested in mice. However, the lack of guinea pig-specific reagents has limited the utility of this animal model in <i>Chlamydia sp</i>. vaccine studies. Using a novel guinea pig-specific transcriptome array, we determined correlates of protection in guinea pigs vaccinated with <i>Chlamydia caviae</i> (<i>C. caviae</i>) via the intranasal route, previously reported by us and others to provide robust antigen specific immunity against subsequent intravaginal challenge. <i>C. caviae</i> vaccinated guinea pigs resolved genital infection by day 3 post challenge. In contrast, mock vaccinated animals continued to shed viable <i>Chlamydia</i> up to day 18 post challenge. Importantly, at day 80 post challenge, vaccinated guinea pigs experienced significantly reduced genital pathology - a sequelae of genital chlamydial infections, in comparison to mock vaccinated guinea pigs. Sera from vaccinated guinea pigs displayed antigen specific IgG responses and increased IgG1 and IgG2 titers capable of neutralizing GPIC <i>in vitro</i>. Th1-cellular/inflammatory immune genes and Th2-humoral associated genes were also found to be elevated in vaccinated guinea pigs at day 3 post-challenge and correlated with early clearance of the bacterium. Overall, this study provides the first evidence of guinea pig-specific genes involved in anti-chlamydial vaccination and illustrates the enhancement of the utility of this animal model in chlamydial pathogenesis.</p></div

Fold-change between groups from qPCR assays of selected immune response genes from the guinea pig lower genital tract.

<p>Arrows indicate direction of fold change.</p><p>* indicates <i>p</i><0.05; ** indicates <i>p</i><0.01.</p>a<p>PBS/C: mock-vaccinated and challenged, EB/C: EB-vaccinated and challenged, Naïve: non-vaccinated and not challenged.</p><p>Fold-change between groups from qPCR assays of selected immune response genes from the guinea pig lower genital tract.</p

Comparative Heatmap Depiction of Differential Gene Expression using RT-PCR array screening.

<p>Three groups of guinea pigs were used for the comparative study: non vaccinated and non challenged (naïve), mock vaccinated but challenged (PBS/C), and <i>C. caviae</i> EB vaccinated and challenged (EB/C) groups. Each group contained three animals. The tissues (upper and lower genital tracts, U and L, respectively) from the respective groups of animals were collected at days 3 and 9 after challenge. Red shading indicates an increase in expression, while blue shading indicates suppression of expression, of the gene indicated on the right side of the panel. Lighter shades including white indicate similar levels of expression. Functional gene clustering is indicated by the brackets on the left showing 3 major groups consisting of innate, Th2 and Th1/inflammatory related genes.</p

Effect of <i>C. caviae</i> Vaccination on Histopathological Lesions in the Genital tract from Guinea pigs following Chlamydial Challenge.

<p>The genital tracts from each humanely euthanized guinea pig were removed at day 80 post <i>C. caviae</i> challenge fixed and embedded and then sectioned, and analyzed microscopically after H&E staining. (A) Representative photomicrographs of histological sections from uterine tissues are shown for each group of challenged guinea pigs with <i>C. caviae</i> EB vaccination (EB, n = 3) or mock vaccinated (Mock, n = 3). The superimposed images are magnifications of the regions of the indicated boxes to show details of inflammatory cell infiltration (c and f) and hemorrhage (a and d). Original magnification of the images (b and e) is ×200, while a, c, d and f are ×400. The light blue dash lines mark superficial layer exfoliation of the endometrial epithelium of the uterus (b), whereas the light blue solid lines indicate the intact endometrial epithelium of the uterus (e). Histopathological injury scores were calculated from five distinct morphological parameters (inflammatory cell infiltration, superficial layer exfoliation, edema, congestion and hemorrhage) in the uterus (B). Scores were calculated by examination of 5 consecutive sections (2 mm-interval) in every animal. Graphs expressed as mean ± SD, and compared using paired <i>t</i>- test. The asterisk indicates statistically significant differences (* <i>p</i><0.05) between the <i>C. caviae</i> group and the mock group for the respective parameter.</p

<i>C. caviae</i> Immune Serum and Chlamydial Infectivity.

<p>Sera obtained from guinea pigs (n = 5 per group) 30 days post vaccination with <i>C. caviae</i> or mock (PBS) or 15 days after challenge (+ challenge) were heat-inactivated (HK) at 56°C for 30 min or left untreated and diluted 1∶25 in DMEM before addition into 96 well plate. Sera from each animal was incubated with <i>C. caviae</i> (2×10⁴ IFU) for 1 hr in a shaking incubator, then sera/bacterial mixtures were added to HeLa cells (0.01 MOI) for bacterial enumeration. Bacterial numbers for each group were presented as a box-and-whisker plot. * <i>p</i><0.05.</p