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. 2019 May 8;14(5):e0216056.
doi: 10.1371/journal.pone.0216056. eCollection 2019.

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

Janelle M Veazey  1 , Timothy J Chapman  2 , Timothy R Smyth  3 , Sara E Hillman  2 , Sophia I Eliseeva  2 , Steve N Georas  1   2   3
Affiliations

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

Janelle M Veazey et al. PLoS One. .

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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. MDA5, not TLR3, promotes neutrophil accumulation and CXCL1 production after polyI:C challenge.
Wild type C57BL/6 mice, TLR3-/-, or MDA5-/- mice were administered normal saline vehicle (Veh) or 10–20 μg polyI:C (pIC) for three days. Twenty-four hours later, mice were sacrificed and bronchoalveolar lavage (BAL) was performed. BAL neutrophil percentages were determined using cytospin (A), and levels of CXCL1 were measured in cell-free supernatants by ELISA (B). Data are mean ± SD pooled from three independent experiments using both male and female mice. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons post-test analysis. * indicates p<0.05 when comparing Veh vs, polyI:C treated mice of a given genotype. # indicates p<0.05 when comparing wild-type vs. gene-targeted mice. ** and ## indicate p<0.01. *** and ### indicate p<0.005. n.s. = not-significant (p>0.05).
Fig 2
Fig 2. BAL levels of IL-6, INF-λ and MIP-1α are differentially affected by TLR3 and MDA5 deficiency.
Wild type C57BL/6 mice, TLR3-/-, or MDA5-/- mice were administered normal saline vehicle (Veh) or 10–20 μg polyI:C (pIC) for three days. Twenty-four hours later, mice were sacrificed and bronchoalveolar lavage (BAL) was performed. BAL levels of IL-6 (A), IFN-lambda (B), and MIP-1α (C) were measured in cell-free supernatants by ELISA. Data are mean ± SD pooled from three independent experiments using both male and female mice. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons post-test analysis. Statistically significant results (p<0.05) are indicated as in the legend to Fig 1.
Fig 3
Fig 3. PolyI:C induces "inside/out" pulmonary leak.
C57BL/6, TLR3-/- or MDA5-/- mice were challenged with saline vehicle (Veh) or 10 μg polyI:C (pIC) for three days. Twenty-four hours after the final challenge, BAL fluids were collected and analyzed for (A) albumin levels by ELISA, and (B) total protein content by Bradford assay. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons post-test analysis. Statistically significant results (p<0.05) are indicated as in the legend to Fig 1.
Fig 4
Fig 4. Validating a method to measure "outside-in" leak in vivo.
C57BL/6 wild-type mice were challenged with saline vehicle or 10 μg polyI:C (pIC) for three days. Twenty-three hours after the final challenge, 0.2 mg 4kDa FITC-dextran was instilled oropharyngeally into the airway. One hour later, mice were euthanized and BAL fluids, BAL cell pellets, blood and lung tissue were collected. FITC-dextran levels were determined by fluorimetry using a standard curve to calculate total microgram amounts. Data shown are mean±SD from (A) BAL fluids, (B) serum, (C) BAL cell pellets, (D) lung tissue supernatants, and (E) lung cell pellets. BALF, serum, lung homogenate, and lysate from lung cell pellet were analyzed for. Data are mean ± SD pooled from two independent experiments (male and female). Asterisks indicate **p<0.01 and *p<0.05 using unpaired Student’s t-test.
Fig 5
Fig 5. TLR3, not MDA5, regulates polyI:C-induced epithelial barrier dysfunction.
Transepithelial flux of inhaled FITC-dextran was measured in wild type C57B/6 mice, TLR3-deficient, and MDA5-deficient mice using methods described in Fig 3. Data were normalized to saline challenged wild-type mice (set at 100%). PolyI:C induces rapid clearance of inhaled FITC-dextran out of the airspaces (A) and its accumulation in serum (B), indicative of reduced epithelial barrier integrity. PolyI:C-induced epithelial barrier function is attenuated in TLR3-deficient (but not MDA5-deficient) mice. Data are mean ± SD pooled from four independent experiments using male and female mice. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons post-test analysis. Statistically significant results (p<0.05) are indicated as in the legend to Fig 1.
Fig 6
Fig 6. Inside/out barrier function assayed measured after FITC-dextran i.p. injection is also TLR3-dependent.
Wild-type and gene-targeted mice were challenged with vehicle (Veh) or inhaled polyI:C (pIC) for three days. FITC-dextran (4 kDa, 5 mg) was injected intra-peritoneally (i.p.), and 1 hour later mice were sacrificed and BAL fluids were collected and analyzed for neutrophil percentages (A), total protein content (B), or relative fluorescence (C, normalized to saline challenged wild-type mice). Data are mean ± SD pooled from one experiment, representative of two. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons post-test analysis. Statistically significant results (p<0.05) are indicated as in the legend to Fig 1.
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