Emory University School of Medicine
Emory University School of Medicine
Department of Pediatrics

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Department of Pediatrics

Pathophysiology of CF-related diabetes – Innate immunity in the airway

Goal

CF is the second most common lethal genetic disease among Americans, arising from mutations in the gene encoding CFTR. CF-related diabetes (CFRD), the most common co-morbidity[1], begins to develop early in childhood and the pathological consequences are severe including significantly increased frequency of acute pulmonary exacerbations and increased rate of decline in lung function compared to CF without diabetes, resulting in poorer outcomes[2-4]. Recent [5] and prior work in the McCarty lab and Koval lab, and others, identified multiple defects in CF airway epithelial cells related to glucose handling [6] and expression and function of tight junction (TJ) proteins [7] that are responsible for the barrier function of the airway monolayer. We most recently showed that expression of mutant CFTR in airway epithelial cells strongly impacts the localization of multiple important TJ proteins, that this is worsened under conditions of hyperglycemia, and only partly recovered by treatment with CFTR correctors (see below) [5]. New preliminary data show that the actin cytoskeleton is disrupted in CF cells, which can alter TJ protein assembly and function, consistent with previous reports [8, 9]. However, these prior studies do not address the impact of impaired TJ function or actin cytoskeleton disruption on polymorphonuclear neutrophils (PMNs) that are recruited to the CF airway in response to muco-obstruction and infection. It is the damage produced by PMNs that leads to most of the morbidity and mortality in CF.

To enter the airway in response to pathogens, PMNs must exit the vasculature, cross the interstitium, and migrate across the airway epithelium (Fig. 1). However, mechanistic studies of how mutant CFTR in airway epithelial cells controls the rate and extent of PMN Trans-Epithelial Migration (TEpM) and impacts PMN function are lacking. The Tirouvanziam lab identified a new paradigm for how PMNs turn pathogenic early on upon exposure to the CF airway milieu (the “GRIM” fate) [10-16]. Notably, on their way to the lumen, PMNs release small amounts of proteases that cause cumulative, progressive destruction along their migratory path in the lamina propria [17-19], which ultimately causes bronchiectasis [19] [20-23]. Our data show that PMNs cause worse damage during TEpM across primary CF airway monolayers than non-CF controls. Hence, the rate of TEpM and activity in the sub-apical space are likely to strongly induce lung injury in CF, leading to loss of lung function and death.

The overall goal of this project is to identify the mechanisms regulating interactions between PMNs and the airway epithelium [11] that occur during TEpM, how these are altered in the CF airway, and how they are further altered under conditions mimicking CFRD. We hypothesize that the loss of CFTR in the apical plasma membrane of airway epithelial cells destabilizes the actin-dependent TJ assembly complex, leading to shifts in the association of specific TJ proteins with the plasma membrane at sites of cell-cell contact, and that this induces alterations in interactions between transmigrating PMNs and epithelial cells themselves. We also hypothesize that cell-cell interactions are further defective in the context of hyperglycemic conditioning of the epithelium, and that this leads to both retention of PMNs in the sub-apical compartment and their partial activation to adopt the GRIM phenotype, thereby leading to greater destruction of the airway epithelium. [24-27]

Methods

Flow cytometry, gene expression (RNA-Seq in both epithelial cells and neutrophils), transepithelial current measurements, immunofluorescence, neutrophil transmigration assays, bacterial killing assays, metabolic activity assays, novel assay for impact of neutrophil transmigration on airway barrier function, spatial transcriptomics, spatial metabolomics (Fig. 2).

Collaborators

Rabin Tirouvanziam, PhD, Dept. of Pediatrics, Emory. Mike Koval, PhD, Dept. of Medicine, Emory.

Funding

CF Foundation, grant MCCART21G0; CFF grant under review; NIH R01 planned



Fig. 1. Confocal images of PMNs transmigrating through a primary non-CF airway monolayer. Red: ZO-1; Blue: DAPI; and Green: PMNs. (A, B) orthogonal views. Arrows: PMNs transmigrating.



Fig. 2. Graphical representation of techniques employed by the lab to study CFRD in vitro, including neutrophil transmigration. Created with Biorender.



References

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