Mechanisms underlying Actinobacillus pleuropneumoniae exotoxin ApxI induced expression of IL-1β, IL-8 and TNF-α in porcine alveolar macrophages

Abstract

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) causes fibrino-hemorrhagic necrotizing pleuropneumonia in pigs. Production of proinflammatory mediators in the lungs is an important feature of A. pleuropneumoniae infection. However, bacterial components other than lipopolysaccharide involved in this process remain unidentified. The goals of this study were to determine the role of A. pleuropneumoniae exotoxin ApxI in cytokine induction and to delineate the underlying mechanisms. Using real-time quantitative PCR analysis, we found native ApxI stimulated porcine alveolar macrophages (PAMs) to transcribe mRNAs of IL-1β, IL-8 and TNF-α in a concentration- and time-dependent manner. Heat-inactivation or pre-incubation of ApxI with a neutralizing antiserum attenuated ApxI bioactivity to induce cytokine gene expression. The secretion of IL-1β, IL-8 and TNF-α protein from PAMs stimulated with ApxI was also confirmed by quantitative ELISA. In delineating the underlying signaling pathways contributing to cytokine expression, we observed mitogen-activated protein kinases (MAPKs) p38 and cJun NH2-terminal kinase (JNK) were activated upon ApxI stimulation. Administration of an inhibitor specific to p38 or JNK resulted in varying degrees of attenuation on ApxI-induced cytokine expression, suggesting the differential regulatory roles of p38 and JNK in IL-1β, IL-8 and TNF-α production. Further, pre-incubation of PAMs with a CD18-blocking antibody prior to ApxI stimulation significantly reduced the activation of p38 and JNK, and subsequent expression of IL-1β, IL-8 or TNF-α gene, indicating a pivotal role of β2 integrins in the ApxI-mediated effect. Collectively, this study demonstrated ApxI induces gene expression of IL-1β, IL-8 and TNF-α in PAMs that involves β2 integrins and downstream MAPKs.

Figure 1

A. pleuropneumoniae exotoxin ApxI induces IL-1β, IL-8 and TNF-α mRNA expression of porcine alveolar macrophages (PAMs) in a concentration- and time-dependent manner. (A) PAMs were stimulated with 0-2 CU/mL ApxI in the presence (w/) or absence (w/o) of polymyxin B (PMB) for 2 h. Total RNA of PAMs was extracted for cDNA synthesis and subjected to RT-qPCR. (B) PAMs were treated with 1 CU/mL ApxI in the presence of PMB for 0-12 h and subjected to RT-qPCR. The expression levels of cytokines were further normalized to corresponding survival rate of PAMs at each time point. Data are representative of three independent experiments of at least triplicate determinations. The error bars are SEM.

Figure 2

Bioactivity of ApxI is required for induction of proinflammatory cytokine gene expression. PAMs were treated with 0.5 CU/mL of ApxI, heat-inactivated ApxI (Δ), or ApxI pre-incubated with an antiserum raised against recombinant subunit ApxI protein (rA). Medium without ApxI toxin served as a negative control (C). Two hours after stimulation, the total RNA of PAMs was extracted for RT-qPCR analysis. Data are representative of three independent experiments of at least triplicate determinations.

Figure 3

Protein levels of proinflammatory cytokines secreted from PAMs treated with ApxI. PAMs were stimulated with 1 CU/mL of ApxI, heat-inactivated ApxI (Δ), or control medium (C) for 4 h. Subsequently, culture supernatants were collected and the protein levels of IL-1β (A), IL-8 (B), TNF-α (C) and IL-6 (D) determined by ELISA. The results are the average of three independent experiments of triplicate determinations. **p < 0.01; ***p < 0.001; n.s.: no significant difference from each other.

Figure 4

p38 and JNK mediate ApxI-induced proinflammatory cytokine production. (A) PAMs were stimulated with 0.5 CU/mL of ApxI, heat-inactivated ApxI (Δ), or control medium (C) for 1 h. Subsequently, cells were lysed and subjected to Western blot analysis using an antibody specific to phospho-p38 (p-p38) or phospho-JNK (p-JNK). (B-D) PAMs were pre-incubated with a p38 inhibitor SB203580 (SB), a JNK inhibitor SP600125 (SP), or vehicle DMSO (V) for 1 h, followed by stimulation with 0.5 CU/mL ApxI for 2 h. PAMs were subjected to RT-qPCR analysis for mRNA levels of IL-1β (B), IL-8 (C), and TNF-α (D). (E) PAMs were pre-incubated with SB, SP, or DMSO for 1 h, followed by stimulation with 0.5 CU/mL of ApxI for 8 h. Subsequently, the culture supernatants were collected and the protein levels of IL-1β, IL-8, and TNF-α were determined by ELISA. Data are representative of three independent experiments of at least triplicate determinations. ***p < 0.001.

Figure 5

CD18 mediates p38 and JNK activation and proinflammatory cytokine mRNA expression. (A and B) PAMs were pre-incubated with CD18 blocking antibody (Anti-CD18) for 1 h prior to stimulation with 0.5 CU/mL ApxI or control medium (C) for an additional 1 h. Thereafter, cells were lysed and subjected to Western blot analysis with an antibody to phospho-p38 (p-p38) or phospho-JNK (p-JNK). The average intensity of p-p38 or p-JNK isoforms (p46 and p55) was quantified and normalized to the intensity of β-actin (A and B, lower panel). (C) PAMs were pre-incubated with anti-CD18 antibody and stimulated with 0.5 CU/mL ApxI for 2 h, followed by RT-qPCR analysis of cytokine mRNA expression. Data are representative of three independent experiments of at least triplicate determinations.