Projects & Methods
To begin addressing our goals, we developed a mouse model, inducing asthma susceptibility in young mice by exposing their mothers to diesel exhaust particles (DEPs). We chose to use DEPs because of their strong associations with childhood asthma and a growing number of epidemiological studies hinting that DEPs may have transgenerational effects. We found that exposure of female mice to DEPs caused predisposition to asthma in their offspring (Manners et al., J Allergy Clin Immunol. 2014, 134:63-72, and Lenberg et al., J Allergy Clin Immunol. 2018, 141:1118-1122). This was linked to shifts in numbers/activation status of several immune cell subsets. Among these cell subsets were natural killer (NK) cells. The association of NK cells with asthma was surprising because it did not fit into the traditional paradigm of NK cells serving as a body’s defense instrument designed to kill. Instead, in the context of maternal DEPs, NK cells promoted the type-2 immune response (Qian et al., J Clin Invest. 2020, 130: 4133-4151). As such, they played an important role in the development of asthma susceptibility. We then discovered that ‘DEP’ NK cells drove the type-2 immune response and asthma through activation of airway epithelial cells. Epithelial activation was a result of cooperation between two NK cell mediators – the granule protease granzyme B and the cytokine IL-13. The key outcome of epithelial activation was transcription of the IL-25 gene. Epithelial IL-25 activated group 2 innate lymphoid cells (ILC2s) and Th2 cells, leading to asthma. Lastly, our in vitro experiments using DEPs, human cord blood and human airway epithelial cells suggested that a similar NK cell-dependent pathway might exist in humans. One of the most intriguing findings of our study was demonstration of epithelial cell activation and induction of the IL-25 gene transcription by granzyme B. This was unexpected because the traditionally-described function of granzyme B is to induce cell death. Cell death occurs after intracellular delivery of granzyme B via cleavage and activation of pro-apoptotic caspases. We showed that extracellular granzyme B did not kill but instead it activated epithelial cells via protease-activated receptor 2 (PAR2). Intriguingly, granzyme B-triggered mechanism that we uncovered resembles some of the mechanisms that are elicited by allergens. House dust mite and pro-allergic fungi (Aspergillus and Alternaria) contain proteases and induce asthma partly via PAR2-mediated activation of the airway epithelium. Thus, to trigger asthma, NK cells activate same pathways as allergens do. Currently, we are performing experiments to define the molecular basis of pro-allergic program in ‘DEP’ NK cells, and elucidate maternal and offspring-derived signals that led to formation of this program.
Another pathway that was activated in our model was the IL1b pathway (Lenberg et al., J Allergy Clin Immunol. 2018, 141:1118-1122). Depletion of IL1b in pups of DEP-exposed mothers reduced their asthma, underscoring importance of this cytokine in the development of asthma traits in our model. We then showed that IL1b and NK cells were not linked, indicating that their effects on type-2 inflammation and asthma were independent of each other. Our goals for the IL1b project are to delineate how IL1b controls offspring asthma, and what leads to its overproduction in our model.
As mentioned earlier, maternal exposure to DEPs led to activation of several other cells and pathways in offspring. We have projects to determine what roles these cells and pathways play in asthma and what mechanisms lead to their activation.
Lastly, we are testing blood samples from diesel exhaust-exposed children with asthma for activation of cells and pathways that we discovered in our mouse model.
- Mouse models of asthma
- Mouse models of prenatally-induced predisposition to asthma
- Analysis of neonatal mice
- Dissection and analysis of embryonic organs and placentas
- Isolation of immune cell subsets from mouse tissues and human peripheral blood
- Immune cell transfer approaches
- In vitro assays to study activation of immune cells
- Multi-color flow cytometry
- Confocal microscopy
- Molecular biology, including gene cloning, site-directed mutagenesis, mammalian cell transfection, generation of recombinant retroviruses and retroviral infection, protein expression and purification
- Assays to study activation of signaling molecules