Abstract
Mycoplasma gallisepticum-mediated respiratory inflammation in chickens is associated with accumulation of leukocytes in the tracheal submucosa. However the molecular mechanisms underpinning these changes have not been well described. We hypothesized that the initial inflammatory events are initiated upon ligation of mycoplasma lipid associated membrane proteins (LAMP) to TLRs expressed on chicken tracheal epithelial cells (TEC). To test this hypothesis, live bacteria or LAMPs isolated from a virulent (Rlow) or a non-virulent (Rhigh) strain were incubated with primary TECs or chicken tracheae ex vivo. Microarray analysis identified up-regulation of several inflammatory and chemokine genes in TECs as early as 1.5 hours post-exposure. Kinetic analysis using RT-qPCR identified the peak of expression for most genes to be at either 1.5 or 6 hours. Ex-vivo exposure also showed up-regulation of inflammatory genes in epithelial cells by 1.5 hours. Among the commonly up-regulated genes were IL-1β, IL-6, IL-8, IL-12p40, CCL-20, and NOS-2, all of which are important immune-modulators and/or chemo-attractants of leukocytes. While these inflammatory genes were up-regulated in all four treatment groups, Rlow exposed epithelial cells both in vitro and ex vivo showed the most dramatic up-regulation, inducing over 100 unique genes by 5-fold or more in TECs. Upon addition of a TLR-2 inhibitor, LAMP-mediated gene expression of IL-1β and CCL-20 was reduced by almost 5-fold while expression of IL-12p40, IL-6, IL-8 and NOS-2 mRNA was reduced by about 2–3 fold. Conversely, an NF-κB inhibitor abrogated the response entirely for all six genes. miRNA-146a, a negative regulator of TLR-2 signaling, was up-regulated in TECs in response to either Rlow or Rhigh exposure. Taken together we conclude that LAMPs isolated from both Rhigh and Rlow induced rapid, TLR-2 dependent but transient up-regulation of inflammatory genes in primary TECs through an NF-κB dependent pathway.
Highlights
Mycoplasma gallisepticum (M. gallisepticum) is an avian respiratory pathogen causing severe inflammation of the trachea, air sacs and lungs, especially in the presence of a co-infection [1,2,3]
Tracheal epithelial cell culture and immunocytostaining Primary tracheal epithelial cell cultures were established based on published methods [62,63]
Bacterial cell envelope components such as LPS, lipotechoic acid, peptidoglycan, flagella and lipoproteins are well characterized PAMPs that interact with host cell pattern recognition receptors such as TLRs, thereby contributing in part to the inflammation that ensues post-infection [20,43,65]
Summary
Mycoplasma gallisepticum (M. gallisepticum) is an avian respiratory pathogen causing severe inflammation of the trachea, air sacs and lungs, especially in the presence of a co-infection [1,2,3]. This pathogen is known to invade, survive and multiply inside a variety of non-phagocytic cells such as chicken RBCs, HeLa cells, and chicken fibroblasts, [4,5,6,7,8,9]. There is yet no clear evidence that M. gallisepticum invades tracheal epithelial cells in vivo [unpublished observations], as it predominantly colonizes the mucosal surface and only rarely is found inside phagocytic vacuoles [11]. The organism orchestrates immunopathological changes in the tracheal mucosa marked by infiltration of heterophils, macrophages and lymphocytes [2,12,13] soon after attachment and colonization of the respiratory surface
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