Absence of insulin receptor substrate 2 (IRS2) in myeloid cells results in enhanced hypoxia-induced remodeling of the pulmonary vasculature and heart with modest effects on allergic lung inflammation

Figures

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  Figure 1. LysM-Cre-mediated deletion of Irs2 in macrophages. (A) Lung macrophages and M-CSF dependent, bone-marrow derived macrophages (BMM) were prepared from untreated Cre+/−, Cre−/−, or Cre+/+ as described in Materials and Methods. DNA was isolated in 50 mM NaOH. Primers located 1.1 kb upstream of the 5′ loxP site and 0.2 kb downstream of the 3′ loxP site in the targeted Irs2 gene were used in standard PCR using 100 ng input DNA. The amplified product was run on a 1.2% agarose gel and stained with ethidium bromide. The gel was imaged using the Amersham GE gel imaging system. (B) Total spleen cells and BMM were prepared from Cre+/− and Cre −/− littermates as indicated. They were deprived of serum and growth factors for 2 h before treatment with or without 20 ng/mL IL-4 for 30 min. IRS2 was immunoprecipitated with rabbit anti-IRS2. These IPs or whole cell lysates were prepared for Western blotting. Blots were probed with anti-phosphotyrosine (PY) or anti-phospho-STAT6 as indicated. The blots were stripped and re-probed with anti-IRS2 or anti-STAT6 as appropriate.

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  Figure 2. Effect of LysM-Cre-mediated deletion of Irs2 on OVA/Alum-induced lung inflammation and remodeling. Cre−/− or Cre+/− littermates were exposed to Alum or OVA/Alum as described in Materials and Methods. (A) Bronchoalveolar lavage (BAL) was performed to obtain cells and cell-free fluid. The cellular composition was evaluated by differential counting. The percentage of macrophages and eosinophils are shown. The levels of cytokines in the BAL fluid were evaluated as described in Materials and Methods and the abundance of IL-4 and IL-5 is shown. (B) Lung sections were stained with H&E or PAS and scored for inflammation or mucus production by an investigator blinded to the groups. (C) Representative photomicrographs for H&E and PAS are shown. The data show the mean ± the standard error of the mean (SEM) and were obtained from two independent experiments (n = 8–12 mice/group). Data statistics were calculated using one-way ANOVA with Tukey’s multiple comparison test.

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  Figure 3. Effect of LysM-Cre-mediated deletion of Irs2 on OVA/Alum-induced gene expression in the lung. Cre−/− or Cre+/− littermates were exposed to Alum or OVA/Alum as described in Fig. 2. Total lung RNA was extracted from the left lobe of the lung. The indicated specific primer pairs were used to amplify target genes as shown in Fig. S2. The expression of the target gene was calculated using the −ΔCt method, with the expression level relative to HPRT1 as the reference gene. The data show the mean ± SEM and were obtained from two independent experiments (n = 7–10 mice/group). Data statistics were calculated using one-way ANOVA with Tukey’s multiple comparison test.

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  Figure 4. Effect of LysM-Cre mediated deletion of Irs2 on HDM-induced lung inflammation and remodeling. Cre+/− or Cre−/− mice were exposed to 50 µg of HDM by intransal instillation 4 times as described in Materials and Methods. (A) Bronchoalveolar lavage (BAL) was performed to obtain cells and cell-free fluid. The cellular composition was evaluated by differential counting. The percentage of macrophages and eosinophils are shown. The levels of cytokines in the BAL fluid were evaluated as described in Materials and Methods and the abundance of IL-4 and IL-5 is shown. (B) Lung sections were stained with H&E or PAS and scored for inflammation or mucus production by an investigator blinded to the groups. (C) Representative photomicrographs for H&E and PAS are shown. The data show the mean ± the SEM and were obtained from two independent experiments (n = 9–12 mice/group). Data statistics were calculated using one-way ANOVA with Tukey’s multiple comparison test.

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  Figure 5. Effect of LysM-Cre-mediated deletion of Irs2 on HDM-induced gene expression in the lung. Cre−/− or Cre+/− littermates were exposed to HDM as described in Fig. 4. Total lung RNA was extracted from the left lobe of the lung. The indicated specific primer pairs were used to amplify target genes as shown in Fig. S2. The expression of the target gene was calculated using the −ΔCt method, with the expression level relative to HPRT1 as the reference gene. The data show the mean ± SEM and were obtained from two independent experiments (n = 8–10 mice/group). Data statistics were calculated using one-way ANOVA with Tukey’s multiple comparison test.

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  Figure 6. Effect of LysM-Cre-mediated deletion of Irs2 on HDM-induced vascular remodeling in the lung. Cre−/− or Cre+/− littermates were exposed to HDM as described in Fig. 4. (A) Lung sections were stained with anti-smooth muscle cell actin (SMA) and scored for staining of small pulmonary vessels by an investigator blinded to the groups as described in Material and Methods. (B) Representative digital images of small pulmonary blood vessels taken with a 40× or 100× oil objective are shown. The data show the mean ± the standard error of the mean (SEM) and were obtained from 2 independent experiments (n = 4–6 mice/group). Data statistics were calculated using one-way ANOVA with Tukey’s multiple comparison test.

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  Figure 7. LysM-Cre-mediated conditional deletion of Irs2 contributes to cardiopulmonary changes. (A) Mice with global deletion of IRS2 (Irs2−/−) or littermates were exposed to Alum or OVA/Alum as described in Fig. 2. Right ventricular systolic pressure (RVSP) was measured by in vivo by a pressure-volume catheter. Right ventricular hypertrophy was evaluated in isolated hearts and expressed as Fulton index (weight of RV/LV+septum). n = 3–6 **P < 0.01B. Mice with conditional deletion of Irs2 driven by LysM-Cre (Cre+/−), or Cre−/− littermates were exposed to room air or hypoxic conditions (10% O2) for 3 weeks as indicated. RVSP was measured by in vivo by a pressure-volume catheter. Right ventricular hypertrophy was evaluated in isolated hearts and expressed as Fulton index (weight of RV/LV+septum). Data are expressed as mean ± SEM n = 6–10. Data statistics were calculated using one-way ANOVA with Bonferoni’s multiple comparison test, ****P < 0.0001 *P < 0.01. The inset shows representative H&E stains of cross sections of hearts isolated from the indicated mice exposed to hypoxic conditions.