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.
Highlights
Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is the etiological agent of porcine pleuropneumonia characterized as an exudative, fibrinous, hemorrhagic, and necrotizing pneumonia along with pleuritis [1]
Actinobacillus pleuropneumoniae ApxI induces expression of proinflammatory cytokine genes in porcine alveolar macrophages To evaluate the effect of A. pleuropneumoniae serotype 10-derived ApxI on proinflammatory cytokine gene expression, porcine alveolar macrophages (PAMs) were incubated with 0-2 cytotoxic unit (CU)/mL of ApxI for 2 h and subjected to real-time quantitative PCR (RT-qPCR) analysis
It was noted that 1 CU/mL ApxI preparation without polymyxin B (PMB) elicited mRNA expression of interleukin1 beta (IL-1b), IL-8, and tumor necrosis factor alpha (TNF-a) at a level ~8, 2, and 13-fold higher than that treated with ApxI in the presence of PMB, respectively (Figure 1A)
Summary
Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is the etiological agent of porcine pleuropneumonia characterized as an exudative, fibrinous, hemorrhagic, and necrotizing pneumonia along with pleuritis [1]. Multiple factors of A. pleuropneumoniae including lipopolysaccharide (LPS), A. pleuropneumoniae exotoxins (Apx), polysaccharide capsule and etc. Apx toxins are members of the “Repeats in Toxin” (RTX) family that are widespread in Pasteurellaceae which cause infectious diseases, most often in animals and in humans [8]. Mannheimia haemolytica RTX leukotoxin (Lkt) has been identified as a potent inducer on the gene expression of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-a), interleukin (IL)-1, and IL-8 in bovine alveolar macrophages [9,10]. A study on porcine alveolar macrophages indicated multiple components of A. pleuropneumoniae, e.g., killed bacteria, bacterial culture supernatant, crude surface extract, or lipopolysaccharide (LPS), are potent stimulants for IL-1, IL-8 and TNF-a expression [3]. Up to now, the role of Apx toxins in proinflammatory cytokine expression remains unidentified
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