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. 2017 Jan 26:8:78.
doi: 10.3389/fmicb.2017.00078. eCollection 2017.

Chlamydial Lipoproteins Stimulate Toll-Like Receptors 1/2 Mediated Inflammatory Responses through MyD88-Dependent Pathway

Yong Wang  1 , Qiong Liu  2 , Ding Chen  3 , Jie Guan  1 , Linghui Ma  1 , Guangming Zhong  3 , Hengping Shu  1 , Xiang Wu  1
Affiliations

Chlamydial Lipoproteins Stimulate Toll-Like Receptors 1/2 Mediated Inflammatory Responses through MyD88-Dependent Pathway

Yong Wang et al. Front Microbiol. .

Abstract

Chlamydiae are very important pathogens which could cause several types of diseases in human, but little is known about its pathogenic mechanism. In order to elucidate host inflammatory response and the signal pathway induced by Chlamydial lipoproteins, the predicted lipoproteins of Chlamydia trachomatis were tested for their ability to induce the release of proinflammatory cytokines by mouse macrophages or human TLR (Toll-Like Receptor) expressing cell lines. The results showed that recombinant proteins of C. trachomatis D381, D541, D067, and D775 displayed a strong ability to induce the release of IL-8 in TLR expressing cell line. The signal pathways involved TLR1/2 and TLR2/CD14 but not TLR4. Moreover, except D067, the proinflammatory cytokine induction by D381, D541, and D775 required the thioacylation site (cysteine) for lipid modification and the induction was through MyD88-mediated pathway. Our data supported that lipoproteins played a vital role in pathogenesis of C. trachomatis-induced inflammatory responses via TLR pathway. It was the first study to characterize other chlamydial lipoproteins after identifying the role of MIP (D541) on pathogenesis of Chlamydial diseases.

Keywords: Chlamydial trachoma; Toll-Like Receptor; cytokine; immune response; lipoproteins.

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Figures

Figure 1
Figure 1
Cytokine profile of mouse macrophage after Chlamydia trachomatis lipoprotein stimulation. (A) Cytokine productions by mouse macrophages in response to lipoproteins of Chlamydia trachomatis. Mouse macrophages (105 cells/ml per well) were stimulated by recombinant C. trachomatis lipoproteins (D381, D541, D067, D775), lipobox mutated proteins (D381C24A, D541C20A, D067C22A, D775C13A, D775C20A, and D775C13AC20A), Hsp60, E. coli LPS, pam3, and GST. The stimulators were incubated with or without polymycin B (50 μg/ml), and supernatants were collected and the cytokine levels were analyzed by sandwich ELISA. (B) Cytokine productions by TLR4−/− mouse macrophages in response to lipoproteins of Chlamydia trachomatis. Macrophages (105 cells/ml per well) were stimulated by Hsp60, D541, D541 mutant (D541C20A), D381, D381 mutant (D381C24A), D067, D067 mutant (D067C22A), D775, D775 mutants (D775C13A, D775C20A, and D775C13AC20A), E. coli LPS, Pam3, and GST. After 24 h culture, supernatants were collected to analyze mMIP-2 levels by ELISA. D381, D541, D067, and D775 could elicit TLR4−/− mouse macrophages to release proinflammatory cytokine, mouse Macrophage Inflammatory Protein-2 (mMIP-2), which was not through TLR4 signal pathway. Except D067, the other 3 proteins required lipobox cysteine for inflammatory cytokine stimulation.
Figure 2
Figure 2
Toll-Like receptors required for the recognition of chlamydial inflammatory lipoproteins. (A) IL-8 productions of stimulated TLR2 and TLR2/CD14 expressing cells. (B) IL-8 productions of stimulated TLR2/6 and TLR1/2 expressing cells. Cytokine productions of HEK-293 cell lines transfected with human TLR1/2, TLR2, TLR2/6, or TLR2/CD14 genes (105 cells/ml per well) were tested, respectively, after stimulation of Chlamydia trachomatis lipoproteins, D541, D541 mutant, D381, D381 mutant, D067, D067 mutant, D775, D775 mutants, Hsp60, E. coli LPS, pam3, and GST. The stimulators were diluted in D10 medium with 20 μg/ml gentamycin. After 24 h of culture, supernatants were collected and human IL-8 were analyzed by ELISA.
Figure 3
Figure 3
Production of proinflammatory cytokines induced by D775 required lipid modification while D541 did not. TLR4−/− mouse macrophage were incubated with lipoproteins treated with lipase, lipoproteins treated with proteinase, lipase alone, or proteinase alone. Lipoprotein+lipase groups: D775 and D541 were incubated with1000 U/ml pig pancreas lipase at 37°C for 16 h, followed by heating at 100°C before beginning cytokine stimulation assay. Lipoprotein+ proteinase groups: D775 or D541 with 100 μg/ml proteinase K at 37°C for 2 h, followed by addition of 200 mM PMSF. Lipoprotein groups: D775 and D541 were incubated in enzyme free buffer as controls. Cell-free supernatants were harvested after 4 h incubation for cytokine mMIP-2 measurement by ELISA. It confirmed that lipid modification of D775 was essential for its stimulation of proinflammatory cytokine production, but D541 stimulation was not greatly affected by lipase.
Figure 4
Figure 4
Cytokine productions by myD88−/−, Tirap/Mal−/−, and Trif−/−mouse macrophages in response to Chlamydia trachomatis lipoproteins. The mouse macrophages (105 cells/ml per well) were stimulated by recombinant C. trachomatis lipoproteins D541 and D775 as well as E. coli LPS and Pam3, and then mMIP-2 level was determined by ELISA. The “+” symbol represents the addition of polymycin B (50 μg/ml) while the “−” symbol in the figure indicates that no polymycin B is supplemented. After 24 h culture, supernatants were collected and the mMIP-2 were analyzed. Results were obtained from 6 different dilution concentrations of stimulators and cell cultures performed in triplicates. The results suggested the proinflammatory cytokine production induced by D775 and D541 relied on MyD88 while D541 stimulation depended on both MyD88 and Tirap/Mal.
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