WNT1-inducible signaling pathway protein 1 contributes to ventilator-induced lung injury

Abstract

Although strides have been made to reduce ventilator-induced lung injury (VILI), critically ill patients can vary in sensitivity to VILI, suggesting gene-environment interactions could contribute to individual susceptibility. This study sought to uncover candidate genes associated with VILI using a genome-wide approach followed by functional analysis of the leading candidate in mice. Alveolar-capillary permeability after high tidal volume (HTV) ventilation was measured in 23 mouse strains, and haplotype association mapping was performed. A locus was identified on chromosome 15 that contained ArfGAP with SH3 domain, ankyrin repeat and PH domain 1 (Asap1), adenylate cyclase 8 (Adcy8), WNT1-inducible signaling pathway protein 1 (Wisp1), and N-myc downstream regulated 1 (Ndrg1). Information from published studies guided initial assessment to Wisp1. After HTV, lung WISP1 protein increased in sensitive A/J mice, but was unchanged in resistant CBA/J mice. Anti-WISP1 antibody decreased HTV-induced alveolar-capillary permeability in sensitive A/J mice, and recombinant WISP1 protein increased HTV-induced alveolar-capillary permeability in resistant CBA/J mice. HTV-induced WISP1 coimmunoprecipitated with glycosylated Toll-like receptor (TLR) 4 in A/J lung homogenates. After HTV, WISP1 increased in strain-matched control lungs, but was unchanged in TLR4 gene-targeted lungs. In peritoneal macrophages from strain-matched mice, WISP1 augmented LPS-induced TNF release that was inhibited in macrophages from TLR4 or CD14 antigen gene-targeted mice, and was attenuated in macrophages from myeloid differentiation primary response gene 88 gene-targeted or TLR adaptor molecule 1 mutant mice. These findings support a role for WISP1 as an endogenous signal that acts through TLR4 signaling to increase alveolar-capillary permeability in VILI.

Figure 1.

Ratio of Evan’s blue albumin (EBA) permeability in high tidal volume (HTV) ventilated mice compared with spontaneous breathing control (CON) mice in 23 mouse strains. EBA permeability index was calculated by dividing the pulmonary tissue EBA at 620 nm absorbance per gram of lung tissue by the plasma EBA at 620 nm absorbance. EBA permeability in each strain was normalized as permeability ratio of HTV mice to CON mice. Values are means (±SEM); n = 216 mice, four to six mice per mouse strain in each CON and HTV group.

Figure 2.

Haplotype association mapping of ventilator-induced lung injury (VILI) using single-nucleotide polymorphisms (SNPs) in mice. (A) Haplotype association map for VILI in mice. The scatter (Manhattan) plot of corresponding −log (P) association probability (ordinate) for each SNP at the indicated chromosomal location (abscissa). The horizontal dashed line indicates the significance level of −log (P). (B) Candidate genes on mouse chromosome 15 associated with VILI. Genes with one or more significant SNPs (−log (P) > 6.0) included ArfGAP with SH3 domain, ankyrin repeat and PH domain 1 (Asap1), adenylate cyclase 8 (Adcy8), WNT1-inducible signaling pathway protein 1 (Wisp1), and N-myc downstream regulated 1 (Ndrg1).

Figure 3.

Lung injury increased in sensitive A/J mice after HTV ventilation as compared with resistant CBA/J mice. (A) EBA permeability increased in sensitive A/J mice compared with CON, whereas HTV did not increase EBA permeability in resistant CBA/J mice after 4 hours of mechanical ventilation. Values are means (±SEM) (four to six mice/group). (B) HTV increased bronchoalveolar lavage (BAL) protein concentration in sensitive A/J mice, whereas it failed to increase BAL protein concentration in resistant CBA/J mice after exposing them to 4 hours of mechanical ventilation compared with CON. Values are means (±SEM) (four to six mice/group). (C) HTV increased BAL total cell number counts in sensitive A/J mice compared with CON after 4 hours, whereas HTV did not increase BAL total cell number counts in resistant CBA/J mice. (D) HTV increased BAL neutrophils in sensitive A/J mice compared with CON after 4 hours, whereas HTV did not increase BAL neutrophil counts in resistant CBA/J mice. (E) HTV increased BAL macrophages in sensitive A/J mice after 4 hours of HTV compared with CON. Values are means (±SEM) (four to six mice/group). Significant difference from control (**P < 0.01, ***P < 0.001), as determined by two-way ANOVA followed by Bonferroni’s multiple comparisons.

Figure 4.

Mechanical ventilation increased WISP1 protein in sensitive A/J mice. (A) HTV ventilation increased WISP1 protein level in lung tissues determined by immunoblot in sensitive A/J mice, whereas there was no change in WISP1 levels in resistant CBA/J mice exposed to 4 hours of HTV. β-actin was the loading control. Data are representative of three tests. (B) Immunohistochemistry of WISP1 protein in CON mice from the sensitive A/J strain. The localization of WISP1 protein was similarly detected in airway and alveolar epithelium (arrow) and alveolar macrophages (arrowhead) in both sensitive A/J and resistant CBA/J strains after CON and HTV. Data shown are representative of two tests. (C) Immunofluorescence staining for WISP1 protein (Alexa-488 fluorescently labeled antibody, green color) in alveolar macrophages increased more in sensitive A/J mice than in resistant CBA/J mice after HTV. (D) The mean fluorescence intensity for WISP1 immunostaining was determined by quantifying fluorescent intensity of 10–20 cells in 4–5 fields that were randomly selected with Nikon image software NIS-Element AR 3.2. Values are means (±SEM). Significant difference from control (***P < 0.001), as determined by two-way ANOVA followed by Bonferroni’s multiple comparisons. (E) Immunoblot of WISP1 protein in BAL fluid was elevated after 4 hours of HTV in the sensitive A/J strain compared with the resistant CBA/J strain.

Figure 5.

Anti-WISP1 monoclonal antibody (mAb) or recombinant mouse WISP1 protein (rmWISP1) reversed phenotype to VILI in sensitive A/J mice or resistant CBA/J mice, respectively. (A) A/J mice were intratracheally administered anti-WISP1 mAb or serum IgG (0.5 μg/g in 50 μl PBS) using a MicroSpray syringe before HTV mechanical ventilation. Lung injury was evaluated by measuring EBA permeability after 4 hours of HTV or CON. Anti-WISP1 mAb decreased EBA permeability after 4 hours of HTV-induced lung injury in sensitive A/J mice, whereas serum IgG did not decrease lung injury. Values are means (±SEM) (four to six mice/group). Significant difference from control (*P < 0.05, ***P < 0.001), as determined by one-way ANOVA followed by Tukey’s post test. (B) CBA/J mice were intratracheally instilled with rmWISP1 (0.5 μg/g in 50 μl PBS) or 50 μl PBS as a control using a MicroSpray syringe before HTV mechanical ventilation or CON. Values are means (±SEM) (n = four to six mice/group). Significant difference from control (*P < 0.05), as determined by two-way ANOVA followed by Bonferroni’s multiple comparisons.

Figure 6.

WISP1 interacted with Toll-like receptor (TLR) 4 in VILI. A/J mouse lung tissues obtained from 4-hour CON or HTV mechanical ventilation were homogenized and total lysates were immunoprecipitated (IP) with anti-WISP1 antibody or anti–TLR4–MD2 antibody before immunoblot (IB) was performed. (A) Glycosylated form of TLR4 (120 kD) was detected with anti-TLR4 antibody (IB: TLR4) either in the whole lung tissue lysates (Total Lysates) or after immunoprecipitation with anti-WISP1 antibody (IP: WISP1). (B) WISP1 was detected with anti-WISP1 antibody (IB: WISP1) either in the whole lung tissue lysates (Total Lysates) or after immunoprecipitation with anti–TLR4–MD2 antibody (IP: TLR4-MD2). Antibody species-matched serum (Rat IgG) was used as a negative control for coprecipitation experiments. Results are representative of tests performed on three occasions.

Figure 7.

WISP1 contribution to VILI is dependent on TLR4 signaling pathway. (A) HTV increased EBA permeability in TLR4^+/+ mice compared with CON, but did not increase EBA permeability in TLR4^−/− mice after 4 hours of mechanical ventilation. Values are means (±SEM) (n = 3 mice/group). Significant difference from control (*P < 0.05), as determined by two-way ANOVA followed by Bonferroni’s multiple comparisons. (B) WISP1 protein expression was determined in strain-matched control (TLR4^+/+) and TLR4 gene–targeted (TLR4^−/−) mice after 4 hours of HTV ventilation. HTV increased WISP1 protein expression in TLR4^+/+ mice, but not in TLR4^−/− mice compared with CON. β-actin was the loading control. Data are representative of three tests. (C) rmWISP1 enhanced LPS-induced TNF release in peritoneal macrophages via CD14–TLR4 signaling. Peritoneal macrophages were seeded in 96-well plates at a density of 6 × 10⁴ cells/well for 24 hours before treating with indicated concentrations of rmWISP1, LPS, and LPS + rmWISP1 for 22 hours, and TNF release in the culture medium was measured by ELISA. rmWISP1, by itself, caused minimal TNF release. rmWISP1, however, enhanced LPS-induced TNF release in peritoneal macrophages from C57BL/6J strain-matched control (WT-B6), but did not amplify LPS-induced TNF release in macrophages obtained from TLR4 gene–targeted (TLR4^−/−) mice or CD14 gene–targeted (CD14^−/−) mice. In addition, rmWISP1-enhanced LPS-induced TNF release was inhibited more in macrophages isolated from myeloid differentiation primary response gene 88 gene–targeted (MyD88^−/−) mice than in macrophages from TLR adaptor molecule 1 (TICAM1; a.k.a., TRIF) mutant (TRIF^−/−) mice. Values are means (±SEM) from three independent experiments in duplicate wells.