RAGE contributes to allergen driven severe neutrophilic airway inflammation via NLRP3 inflammasome activation in mice

BackgroundAsthma is a major public healthcare burden, affecting over 300 million people worldwide. While there has been great progress in the treatment of asthma, subsets of patients who present with airway neutrophilia, often have more severe disease, and tend to be resistant to conventional corticosteroid treatments. The receptor for advanced glycation endproducts (RAGE) plays a central role in the pathogenesis of eosinophilic asthma, however, it’s role in neutrophilic asthma remains largely uninvestigated.MethodsA mouse model of severe steroid resistant neutrophilic airway disease (SSRNAD) using the common fungal allergen Alternaria alternata (AA) was employed to evaluate the effects of genetic ablation of RAGE and pharmacological inhibition of the NLRP3 inflammasome on neutrophilic airway inflammation.ResultsAA exposure induced robust neutrophil-dominant airway inflammation and increased BALF levels of Th1/Th17 cytokines in wild-type mice, which was significantly reduced in RAGE-/- mice. Serum levels of IgE and IgG1 were increased similarly in both wild-type and RAGE-/- mice. Pharmacological inhibition of NLRP3 blocked the effects of AA exposure and NLRP3 inflammasome activation was RAGE-dependent. Neutrophil extracellular traps were elevated in the BALF of wild-type but not RAGE-/- mice and an atypical population of SiglecF+ neutrophils were identified in the BALF. Lastly, time-course studies found that RAGE expression promoted sustained neutrophil accumulation in the BALF of mice in response to AA

The Safety and Efficacy of Early-Life RSV Vaccination Approaches: The Double-Edged Sword of Host Immunity

Respiratory syncytial virus (RSV) is the leading cause of childhood hospitalizations due to bronchiolitis in children under 5 years of age. Furthermore, hospitalization as a result of RSV-mediated bronchiolitis is associated with the subsequent development of wheezing and asthma. Despite the unquestionable need, development of a safe and effective RSV vaccine for young children has been met with significant barriers. Toward this goal, our lab investigated the safety and efficacy of two early-life vaccination approaches – maternal RSV vaccination and direct vaccination of RSV-experienced young mice. These vaccine candidates, which used the RSV pre-fusion F (PreF) protein, were evaluated on their ability to prevent RSV replication, overcome the type 2 immune bias of neonates, and elicit enhanced respiratory disease (ERD). We hypothesized that each vaccination approach would generate high titers of RSV-neutralizing antibody, thereby conferring protection from viral replication, while also failing to elicit ERD following natural RSV exposure. In our model of direct vaccination, we demonstrated that RSV-experienced young mice were capable of mounting protective anti-RSV immunity following RSV re-exposure. As expected, formulation of PreF with Th1- vs Th2-skewing adjuvants altered cellular immunity, with use of a Th2-skewing adjuvant eliciting strong type 2 immunity characterized by eosinophilia, ILC2s, and Th2 cells. However, neither vaccination group were protected from the type 2 immunopathology associated with early-life RSV infection and subsequent RSV exposure. In our model of maternal RSV vaccination, we demonstrated that passive transfer of high titers of RSV-neutralizing maternal antibody (matAb) was associated with complete protection from neonatal RSV infection. Unexpectedly, primary RSV exposure in the presence of matAb led to type 2 innate immune cell skewing and Th2 cell activation with associated immunopathology. Moreover, we also demonstrated that RSV re-exposure of maternally vaccinated offspring led to considerable type 2 inflammation, characterized by hyperresponsive ILC2s, eosinophilia, and a Th2 cell phenotype that orchestrated the associated type 2 immunopathology. Collectively, the unfavorable safety outcomes elicited following RSV re-exposure in both models of early-life RSV vaccination further underscores the importance of assessing immunity beyond antibody-mediated viral neutralization and highlights the difficulty in properly balancing early-life immune responses to RSV

Immune Cells Activating Biotin-Decorated PLGA Protein Carrier

Nanoparticle formulations have long been proposed as subunit vaccine carriers owing to their ability to entrap proteins and codeliver adjuvants. Poly(lactic-co-glycolic acid) (PLGA) remains one of the most studied polymers for controlled release and nanoparticle drug delivery, and numerous studies exist proposing PLGA particles as subunit vaccine carriers. In this work we report using PLGA nanoparticles modified with biotin (bNPs) to deliver proteins via adsorption and stimulate professional antigen-presenting cells (APCs). We present evidence showing bNPs are capable of retaining proteins through the biotin-avidin interaction. Surface accessible biotin bound both biotinylated catalase (bCAT) through avidin and streptavidin horseradish peroxidase (HRP). Analysis of the HRP found that activity on the bNPs was preserved once captured on the surface of bNP. Further, bNPs were found to have self-adjuvant properties, evidenced by bNP induced IL-1β, IL-18, and IL-12 production in vitro in APCs, thereby licensing the cells to generate Th1-type helper T cell responses. Cytokine production was reduced in avidin precoated bNPs (but not with other proteins), suggesting that the proinflammatory response is due in part to exposed biotin on the surface of bNPs. bNPs injected subcutaneously were localized to draining lymph nodes detectable after 28 days and were internalized by bronchoalveolar lavage dendritic cells and macrophages in mice in a dose-dependent manner when delivered intranasally. Taken together, these data provide evidence that bNPs should be explored further as potential adjuvanting carriers for subunit vaccines

DataSheet_3_Prior respiratory syncytial virus infection reduces vaccine-mediated Th2-skewed immunity, but retains enhanced RSV F-specific CD8 T cell responses elicited by a Th1-skewing vaccine formulation.pdf

Respiratory syncytial virus (RSV) remains the most common cause of lower respiratory tract infections in children worldwide. Development of a vaccine has been hindered due the risk of enhanced respiratory disease (ERD) following natural RSV exposure and the young age (<6 months) at which children would require protection. Risk factors linked to the development of ERD include poorly neutralizing antibody, seronegative status (never been exposed to RSV), and a Th2-type immune response. Stabilization of the more antigenic prefusion F protein (PreF) has reinvigorated hope for a protective RSV vaccine that elicits potent neutralizing antibody. While anecdotal evidence suggests that children and adults previously exposed to RSV (seropositive) are not at risk for developing vaccine associated ERD, differences in host immune responses in seropositive and seronegative individuals that may protect against ERD remain unclear. It is also unclear if vaccine formulations that skew towards Th1- versus Th2-type immune responses increase pathology or provide greater protection in seropositive individuals. Therefore, the goal of this work was to compare the host immune response to a stabilized prefusion RSV antigen formulated alone or with Th1 or Th2 skewing adjuvants in seronegative and seropositive BALB/c mice. We have developed a novel BALB/c mouse model whereby mice are first infected with RSV (seropositive) and then vaccinated during pregnancy to recapitulate maternal immunization strategies. Results of these studies show that prior RSV infection mitigates vaccine-mediated skewing by Th1- and Th2-polarizing adjuvants that was observed in seronegative animals. Moreover, vaccination with PreF plus the Th1-skewing adjuvant, Advax, increased RSV F85-93-specific CD8 T cells in both seronegative and seropositive dams. These data demonstrate the importance of utilizing seropositive animals in preclinical vaccine studies to assess both the safety and efficacy of candidate RSV vaccines.</p

DataSheet_1_Prior respiratory syncytial virus infection reduces vaccine-mediated Th2-skewed immunity, but retains enhanced RSV F-specific CD8 T cell responses elicited by a Th1-skewing vaccine formulation.pdf

Respiratory syncytial virus (RSV) remains the most common cause of lower respiratory tract infections in children worldwide. Development of a vaccine has been hindered due the risk of enhanced respiratory disease (ERD) following natural RSV exposure and the young age (<6 months) at which children would require protection. Risk factors linked to the development of ERD include poorly neutralizing antibody, seronegative status (never been exposed to RSV), and a Th2-type immune response. Stabilization of the more antigenic prefusion F protein (PreF) has reinvigorated hope for a protective RSV vaccine that elicits potent neutralizing antibody. While anecdotal evidence suggests that children and adults previously exposed to RSV (seropositive) are not at risk for developing vaccine associated ERD, differences in host immune responses in seropositive and seronegative individuals that may protect against ERD remain unclear. It is also unclear if vaccine formulations that skew towards Th1- versus Th2-type immune responses increase pathology or provide greater protection in seropositive individuals. Therefore, the goal of this work was to compare the host immune response to a stabilized prefusion RSV antigen formulated alone or with Th1 or Th2 skewing adjuvants in seronegative and seropositive BALB/c mice. We have developed a novel BALB/c mouse model whereby mice are first infected with RSV (seropositive) and then vaccinated during pregnancy to recapitulate maternal immunization strategies. Results of these studies show that prior RSV infection mitigates vaccine-mediated skewing by Th1- and Th2-polarizing adjuvants that was observed in seronegative animals. Moreover, vaccination with PreF plus the Th1-skewing adjuvant, Advax, increased RSV F85-93-specific CD8 T cells in both seronegative and seropositive dams. These data demonstrate the importance of utilizing seropositive animals in preclinical vaccine studies to assess both the safety and efficacy of candidate RSV vaccines.</p

DataSheet_1_Exacerbated lung inflammation following secondary RSV exposure is CD4+ T cell-dependent and is not mitigated in infant BALB/c mice born to PreF-vaccinated dams.pdf

Respiratory syncytial virus (RSV) is the leading cause of childhood hospitalizations due to bronchiolitis in children under 5 years of age. Moreover, severe RSV disease requiring hospitalization is associated with the subsequent development of wheezing and asthma. Due to the young age in which viral protection is needed and risk of vaccine enhanced disease following direct infant vaccination, current approaches aim to protect young children through maternal immunization strategies that boost neutralizing maternal antibody (matAb) levels. However, there is a scarcity of studies investigating the influence of maternal immunization on secondary immune responses to RSV in the offspring or whether the subsequent development of wheezing and asthma is mitigated. Toward this goal, our lab developed a murine model of maternal RSV vaccination and repeat RSV exposure to evaluate the changes in immune response and development of exacerbated lung inflammation on secondary RSV exposure in mice born to immunized dams. Despite complete protection following primary RSV exposure, offspring born to pre-fusion F (PreF)-vaccinated dams had exaggerated secondary ILC2 and Th2 responses, characterized by enhanced production of IL-4, IL-5, and IL-13. These enhanced type 2 cellular responses were associated with exaggerated airway eosinophilia and mucus hyperproduction upon re-exposure to RSV. Importantly, depletion of CD4+ T cells led to complete amelioration of the observed type 2 pathology on secondary RSV exposure. These unanticipated results highlight the need for additional studies that look beyond primary protection to better understand how maternal immunization shapes subsequent immune responses to repeat RSV exposure.</p

Interferon signaling drives epithelial metabolic reprogramming to promote secondary bacterial infection.

Clinical studies report that viral infections promote acute or chronic bacterial infections at multiple host sites. These viral-bacterial co-infections are widely linked to more severe clinical outcomes. In experimental models in vitro and in vivo, virus-induced interferon responses can augment host susceptibility to secondary bacterial infection. Here, we used a cell-based screen to assess 389 interferon-stimulated genes (ISGs) for their ability to induce chronic Pseudomonas aeruginosa infection. We identified and validated five ISGs that were sufficient to promote bacterial infection. Furthermore, we dissected the mechanism of action of hexokinase 2 (HK2), a gene involved in the induction of aerobic glycolysis, commonly known as the Warburg effect. We report that HK2 upregulation mediates the induction of Warburg effect and secretion of L-lactate, which enhances chronic P. aeruginosa infection. These findings elucidate how the antiviral immune response renders the host susceptible to secondary bacterial infection, revealing potential strategies for viral-bacterial co-infection treatment