Protein kinase C epsilon is important in modulating organic-dust-induced airway inflammation

Abstract

Organic dust samples from swine confinement facilities elicit pro-inflammatory cytokine/chemokine release from bronchial epithelial cells and monocytes, dependent, in part, upon dust-induced activation of the protein kinase C (PKC) isoform, PKCε. PKCε is also rapidly activated in murine tracheal epithelial cells following in vivo organic dust challenges, yet the functional role of PKCε in modulating dust-induced airway inflammatory outcomes is not defined. Utilizing an established intranasal inhalation animal model, experiments investigated the biologic and physiologic responses following organic dust extract (ODE) treatments in wild-type (WT) and PKCε knock-out (KO) mice. We found that neutrophil influx increased more than twofold in PKCε KO mice following both a one-time challenge and 3 weeks of daily challenges with ODE as compared with WT mice. Lung pathology revealed increased bronchiolar and alveolar inflammation, lymphoid aggregates, and T cell influx in ODE-treated PKCε KO mice. Airway hyperresponsiveness to methacholine increased in PKCε KO + ODE to a greater magnitude than WT + ODE animals. There were no significant differences in cytokine/chemokine release elicited by ODE treatment between groups. However, ODE-induced nitric oxide (NO) production differed in that ODE exposure increased nitrate levels in WT mice but not in PKCε KO mice. Moreover, ODE failed to upregulate NO from ex vivo stimulated PKCε KO lung macrophages. Collectively, these studies demonstrate that PKCε-deficient mice were hypersensitive to organic dust exposure and suggest that PKCε is important in the normative lung inflammatory response to ODE. Dampening of ODE-induced NO may contribute to these enhanced inflammatory findings.

Figure 1. Absence of PKCε activity confirmed in lung slices from PKCε deficient mice

Precision-cut lung slices from wild-type (WT, Prkce^+/+) and knock-out (KO, Prkce^−/−) mice were ex vivo treated with phorbol 12-myristate 13-acetate (PMA, 200 ng/ml, positive control), organic dust extract (5% ODE), or media alone in submerged in vitro culture. PKCε activity was assayed from 1-24 hr. Results represent mean ± SEM (N=5-6 per condition) with statistical significance denoted by asterisks (**p<0.01, ***p<0.001) versus media alone, and hatch marks (###p<0.001) denotes difference between matched WT and KO conditions.

Figure 2. PKCε deficient mice demonstrate enhanced influx of neutrophils, but not cytokine/chemokine release, following a one-time challenge with ODE

Wild-type (WT, Prkce^+/+) and PKCε knock-out (KO, Prkce^−/−) mice were intranasally treated with saline (PBS) or ODE (12.5%) once and BALF was collected at 5 and 24 h following exposure. Results represent the mean ± SEM (N=5-8 mice/group) of the total cells and cell differential (A) and cell-free levels of lung lavage fluid supernatant cytokines/chemokines (B). Statistical significance is denoted by asterisks (*p<0.05, **p<0.01, **p<0.001) versus respective saline-treated groups, and hatch mark denotes statistical significance (#p<0.05, ##p<0.01) between WT and KO treated mice.

Figure 3. PKCε is important in mediating repetitive ODE-induced lung inflammation

Wild-type (WT, Prkce^+/+) and PKCε knock-out (KO, Prkce^−/−) mice were intranasally treated with saline or ODE (12.5%) daily (repetitive exposure) for 3 weeks whereupon BALF and lung tissue were collected 24 h following final exposure. A, Results represent the mean ± SEM (N=6 mice/group) of the total cells and cell differential recovered from the BALF of mice. B, Semi-quantitative inflammatory score (mean ± SEM, N=6 mice/group) of the degree and distribution of cellular aggregates, alveolar compartment, and bronchiolar compartment lung inflammation is shown. C, A representative 4- to 5-μm-thick section, H&E stained, of one of six mice per treatment group is shown at 20× magnification and 40× magnification where noted. All lung specimens were inflated to 15 cm H₂O pressure during fixation to avoid atelectasis artifact. Representative lung sections from saline control treated for comparison are also shown. Scale bar line represents 30 μm. Statistical significance is denoted by asterisks (*p<0.05, **p<0.001, ***p<0.001) between WT and KO-treated groups and hatch marks denote statistical significance between saline and ODE treated groups (##p<0.01, ###p<0.001).

Figure 4. Repetitive organic dust exposure induces the influx of phagocytes, T and B cells in PKCε deficient mice

Wild-type (WT, Prkce^+/+) and PKCε knock-out (KO, Prkce^−/−) mice were intranasally treated with ODE (12.5%) daily for 3 weeks. Lung sections (4- to 5-μm thick) were stained with anti-Mac-3 antibody for phagocytes, anti-CD3 antibody for T cells, and anti-murine CD45R/B220 antibody for B cells. Representative lung sections shown depict phagocyte influx into alveolar compartment and predominately T and B cell influx within cellular aggregates in both WT and KO animals repetitively treated with ODE at 40X magnification with scale bar line representing 30 μm. Isotype control antibody staining (negative) control is also shown. B, Quantification of positive staining of mouse lung tissue representing mean ± SEM of 20 fields of representative mouse per group. Statistical significance is denoted by asterisks (*p<0.05) between WT and KO-treated groups.

Figure 5. PKCε deficient mice demonstrate increased airway hyper-responsiveness (AHR)

Wild-type (WT, Prkce^+/+) and PKCε knock-out (KO, Prkce^−/−) mice were intranasally treated (tx) with ODE or saline (PBS) and total lung resistance (R_L) was directly measured using a mechanically ventilated mouse system 3 h following exposure. Data are expressed as means with standard error bars (N=3-4 mice/group). Letters denote statistical significance difference between groups. a: AHR is increased in KO + ODE (closed square, dotted line) as compared to WT + ODE (closed circle, solid line) at MCh 48 mg/ml (p<0.05) and 96 mg/ml (p<0.0001). b: AHR is increased in KO + ODE as compared to KO + PBS (open square, dotted line) at MCh 24 mg/ml (p<0.01) and 96 mg/ml (p<0.001) mg/ml. c: AHR is increased in WT + ODE as compared to WT + PBS (open circle, solid line) at MCh 48 (p<0.001) and 96 (p<0.001) mg/ml. d: AHR is increased in KO + PBS as compared to WT + PBS at methacholine (MCh) 48 (p<0.01) and 96 (p<0.01) mg/ml.

Figure 6. Nitric oxide pathway is dysregulated in PKCε deficient mice following ODE challenge

Wild-type (WT, Prkce^+/+) and PKCε knock-out (KO, Prkce^−/−) mice were treated (tx) with ODE (12.5%) or saline (PBS) once, and bronchoalveolar lavage fluid (BALF) and lung tissue were collected 24 h following exposure. Primary lung macrophages from WT and KO mice were ex vivo stimulated with 1% ODE or saline for 24 hr. A, Results represent the mean ± SEM (N=5 mice/group) of cell-free levels of lung lavage fluid supernatant total nitrate. B, Results represent the mean ± SEM (N=7/group) of total nitrate level in lung macrophage cell-free supernatants. The mean (± SEM) fold change in inducible nitric oxide synthase, NOS2 (B) and exogenous nitric oxide synthase, NOS3 (C) following ODE treatment as compared to PBS treatment from lung tissue are shown (N=5 mice/group, independent, matched experiments). Statistical significance is denoted by asterisks (*p<0.05, **p<0.001, ***p<0.001) between respective saline vs. ODE-treated groups, and hatch mark denotes statistical significance (#p<0.05) between WT and KO ODE-treated groups.