Distinct roles for MDA5 and TLR3 in the acute response to inhaled double-stranded RNA

Abstract

The airway epithelial barrier is critical for preventing pathogen invasion and translocation of inhaled particles into the lung. Epithelial cells also serve an important sentinel role after infection and release various pro-inflammatory mediators that recruit and activate immune cells. Airway epithelial barrier disruption has been implicated in a growing number of respiratory diseases including viral infections. It is thought that when a pathogen breaks the barrier and gains access to the host tissue, pro-inflammatory mediators increase, which further disrupts the barrier and initiates a vicious cycle of leak. However, it is difficult to study airway barrier integrity in vivo , and little is known about relationship between epithelial barrier function and airway inflammation. Current assays of pulmonary barrier integrity quantify the leak of macromolecules from the vasculature into the airspaces (or “inside/out” leak). However, it is also important to measure the ease with which inhaled particles, allergens, or pathogens can enter the subepithelial tissues (or “outside/in” leak). We challenged mice with inhaled double stranded RNA (dsRNA) and explored the relationship between inside/out and outside/in barrier function and airway inflammation. Using wild-type and gene-targeted mice, we studied the roles of the dsRNA sensors Toll Like Receptor 3 (TLR3) and Melanoma Differentiation-Associated protein 5 (MDA5). Here we report that after acute challenge with inhaled dsRNA, airway barrier dysfunction occurs in a TLR3-dependent manner, whereas leukocyte accumulation is largely MDA5-dependent. We conclude that airway barrier dysfunction and inflammation are regulated by different mechanisms at early time points after exposure to inhaled dsRNA.

Inhibiting Protein Kinase D Promotes Airway Epithelial Barrier Integrity in Mouse Models of Influenza A Virus Infection

“…Figure 1A shows that CRT significantly and dose-dependently reduced the amount of total protein and albumin translocation into the airspace after polyI:C challenge, which are markers of inside/out leak. Similarly, Figure 1B shows that CRT treatment significantly prevented the rapid loss of 4kDa FITC-dextran out of the airspaces, which is a direct reflection of epithelial barrier function and a marker of outside/in leak ( 15 ). CRT did not inhibit the basal levels of BAL albumin detected in PBS challenged control mice (104 ± 52 vs. 131 ± 87 pg/ml, n=4, PBS vs. PBS plus CRT, p>0.05).…”

“…We first validated that polyI:C dose-dependently increased intracellular PKD activity as determined by Western blot analysis of phosphorylated PKD substrates ( 17 ), and that this was blocked by CRT ( Supplemental Figure 1 ). We then asked if CRT treatment could limit airway barrier disruption caused by dsRNA in vivo using a polyI:C inhalation challenge protocol ( 15 ). Figure 1A shows that CRT significantly and dose-dependently reduced the amount of total protein and albumin translocation into the airspace after polyI:C challenge, which are markers of inside/out leak.…”

“…As CRT treatment limits both barrier disruption and neutrophil influx, it was possible that CRT promoted barrier integrity indirectly by limiting airway inflammation, or directly by acting on airway epithelial cells. To try and distinguish between these possibilities (which are not mutually exclusive), we revisited our previously published model using wild-type, and TLR3- and MDA5-gene-targeted mice ( 15 ). In this model, we found that barrier integrity is mediated by TLR3, while acute neutrophil accumulation (but not barrier disruption) was attenuated in MDA5 knock-out mice.…”

“…Similarly, the partial protection from barrier disruption in the CC10-Cre x PKD3-floxed mice compared to CRT-treated mice suggests that other PKD isoform(s) and/or cell types contribute to regulation barrier integrity in vivo . These potential isoform-specific differences become especially intriguing in light of our recent finding that acute challenge with polyI:C promotes barrier disruption via TLR3, and cytokine production/airway inflammation via MDA5 signaling ( 15 ). It is therefore possible that PKD3 may mediate barrier disruption downstream of TLR3, while PKD1/2 may contribute to cytokine production downstream MDA5.…”

“…To assess outside-in leak, C57BL/6 mice were administered CRT and polyI:C as above. On day 3, 0.2mg 4kDa FITC-dextran was administered o.p 1 h prior to harvest, as previously reported ( 15 ). FITC-dextran levels in BAL fluid and serum were analyzed.…”