Interaction of Mycoplasma gallisepticum with Chicken Tracheal Epithelial Cells Contributes to Macrophage Chemotaxis and Activation

Abstract

Mycoplasma gallisepticum colonizes the chicken respiratory mucosa and mediates a severe inflammatory response hallmarked by subepithelial leukocyte infiltration. We recently reported that the interaction of M. gallisepticum with chicken tracheal epithelial cells (TECs) mediated the upregulation of chemokine and inflammatory cytokine genes in these cells (S. Majumder, F. Zappulla, and L. K. Silbart, PLoS One 9:e112796, http://dx.doi.org/10.1371/journal.pone.0112796). The current study extends these observations and sheds light on how this initial interaction may give rise to subsequent inflammatory events. Conditioned medium from TECs exposed to the virulent Rlow strain induced macrophage chemotaxis to a much higher degree than the nonvirulent Rhigh strain. Coculture of chicken macrophages (HD-11) with TECs exposed to live mycoplasma revealed the upregulation of several proinflammatory genes associated with macrophage activation, including interleukin-1β (IL-1β), IL-6, IL-8, CCL20, macrophage inflammatory protein 1β (MIP-1β), CXCL-13, and RANTES. The upregulation of these genes was similar to that observed upon direct contact of HD-11 cells with live M. gallisepticum. Coculture of macrophages with Rlow-exposed TECs also resulted in prolonged expression of chemokine genes, such as those encoding CXCL-13, MIP-1β, RANTES, and IL-8. Taken together, these studies support the notion that the initial interaction of M. gallisepticum with host respiratory epithelial cells contributes to macrophage chemotaxis and activation by virtue of robust upregulation of inflammatory cytokine and chemokine genes, thereby setting the stage for chronic tissue inflammation.

FIG 1

Flow diagram depicting the coculture study setup.

FIG 2

Chemotaxis assays. HD-11 cells were assayed for chemotaxis in response to a chemotactic gradient obtained using conditioned medium collected after exposure of tracheal epithelial cells to M. gallisepticum R_low or R_high. Chicken recombinant MIP-1β was used as a positive (+ve) control, and DMEM-F12 or conditioned medium obtained from unexposed tracheal epithelial cells served as a negative (−ve) control. Results shown are means ± SD of total numbers of cells migrating in response to a chemotactic gradient (y axis). Various chemoattractants are shown on the x axis. n = 6 for all experiments. Means with different letters are significantly different (P < 0.05).

FIG 3

Kinetic analysis of differentially regulated genes in HD-11 cells during coculture study. Kinetics of mRNA fold differences in HD-11 cells cocultured with TECs exposed to R_low or R_high at 1.5, 6, and 24 h are shown. Fold change was determined using RT-qPCR (ΔΔC_T method). Data normalization was done using the GAPDH housekeeping gene, and HD-11 cells cocultured with TECs in the absence of any mycoplasma exposure were used as a control. n = 6 for all experiments. Results shown are fold changes ± SD, with all control values set to 1. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (A) mRNA fold differences for genes in HD-11 cells cocultured with R_low-exposed TECs. (B) mRNA fold differences for genes in HD-11 cells cocultured with R_high-exposed TECs.

FIG 4

Kinetic analysis of differentially regulated genes in HD-11 cells during exposure to live R_low. Kinetics of mRNA fold differences in HD-11 cells exposed to various MOIs of R_low at 1.5, 6, and 24 h are shown. Fold change was determined using RT-qPCR (ΔΔC_T method). Data normalization was performed using the GAPDH housekeeping gene with unexposed HD-11 cells used as a control. n = 6 for all experiments. Results shown are fold changes ± SD, with all control values set to 1. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (A) IFN-γ mRNA; (B) IL-1β mRNA; (C) IL-6 mRNA; (D) CCL-20 mRNA; (E) MIP-1β mRNA; (F) CXCL-13 mRNA; (G) IL-8 mRNA; (H) RANTES mRNA.

FIG 5

Kinetic analysis of differentially regulated genes in HD-11 cells during exposure to live R_high. Kinetics of mRNA fold differences in HD-11 cells exposed to various MOIs of R_high at 1.5, 6, and 24 h are shown. Fold change was determined using RT-qPCR (ΔΔC_T method). Data normalization was done using the GAPDH housekeeping gene, with unexposed HD-11 cells used as a control. n = 6 for all experiments. Results shown are fold changes ± SD, with all control values set to 1. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (A) IFN-γ mRNA; (B) IL-1β mRNA; (C) IL-6 mRNA; (D) CCL-20 mRNA; (E) MIP-1β mRNA; (F) CXCL-13 mRNA; (G) IL-8 mRNA; (H) RANTES mRNA.