Abstract
The polymorphic membrane protein D (PmpD) is a highly conserved outer membrane protein which plays an important role in pathogenesis during Chlamydia psittaci infection. In this study, we evaluated the ability of the N-terminus of PmpD (PmpD-N) to modulate the functions of chicken macrophages and the signaling pathway(s) involved in PmpD-N-induced Toll-like receptors (TLRs), as well as interleukin (IL)-6 and IL-10 cytokine secretions. Thus, HD11 macrophages were treated with exogenous and intracellular PmpD-N of C. psittaci. The chlamydial growth was evaluated by enumeration of chlamydial loads in the infected macrophages. The phagocytic function of macrophages following PmpD-N treatment was detected by fluorescein-labeled Escherichia coli (E. coli). The concentration of nitric oxide (NO) secreted by HD11 macrophages was measured by the amount of NO2- in the culture supernatant using the Griess method. The cytokine secretions were assessed using multiplex cytokine ELISA kits. Expression levels of TLRs, myeloid differentiation factor 88 (MyD88), and nuclear factor kappa B (NF-κB) were analyzed by a Western blotting assay, as well as a luciferase assay, while NF-κB p65 nuclear translocation was assessed by confocal microscopy. The nuclear translocation of the transcription factor NF-κB was confirmed by evaluating its ability to combine with the corresponding promoter using the electrophoretic mobility shift assay (EMSA). After treatment with exogenous or endogenous PmpD-N, chlamydial loads and phagocytic functions were reduced significantly compared with those of the plasmid vector group, while NO secretions were reduced significantly compared with those of the lipopolysaccharide (LPS) treatment. Stimulation of HD11 cells with PmpD-N provoked the secretion of the Th2 cytokines, IL-6, and IL-10 and upregulated the expression of TLR2, TLR4, MyD88, and NF-κB. Furthermore, inhibition of TLR2, MyD88, and NF-κB in HD11 cells significantly decreased IL-6 and IL-10 cytokine levels, while NO production and phagocytosis increased significantly, strongly suggesting their involvement in PmpD-N-induced Th2 cytokine secretion and macrophage dysfunction. Our data indicate that C. psittaci PmpD-N inhibited macrophage functions by activating the Th2 immune response and the TLR2/MyD88/NF-κB signaling pathway.
Highlights
Chlamydia psittaci is an obligate intracellular Gram-negative bacterium that can cause chlamydiosis, which is characterized by fever, chills, headache, dyspnea, and cough in psittacine birds, domestic poultry, as well as wild fowl [1]
Based on RT-PCR, the pmpD-N gene was confirmed to express in HD11 cells (Figure 1C), and Polymorphic membrane protein D (PmpD)-N expression was identified by a Western blotting assay to react with goat anti-rabbit HRP-conjugated antibody (Figure 1D)
A more recent report indicates that birds inoculated with the high-virulence C. psittaci strain had suppressed immune response, leading to secondary infection of avian influenza virus H9N2 and high mortality in poultry [23]
Summary
Chlamydia psittaci is an obligate intracellular Gram-negative bacterium that can cause chlamydiosis, which is characterized by fever, chills, headache, dyspnea, and cough in psittacine birds, domestic poultry, as well as wild fowl [1]. Genome sequencing has revealed that the pmp gene encodes the largest membrane protein family—polymorphic membrane proteins (Pmps)—in Chlamydia species [5,6]. The Pmps family, in all chlamydial genomes, suggests an important function in chlamydial biology [8]. The observed diversity in the number of allele genes and protein sequence sizes (90–190 kDa) and the expression levels within and across Chlamydia species suggest that Pmps could be responsible for the observed differences in pathogenesis across Chlamydia species [9]
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