Natural Mycoplasma Infection Reduces Expression of Pro-Inflammatory Cytokines in Response to Ovine Footrot Pathogens

Abstract

Ovine footrot is a complex multifactorial infectious disease, causing lameness in sheep with major welfare and economic consequences. Dichelobacter nodosus is the main causative bacterium; however, footrot is a polymicrobial disease with Fusobacterium necrophorum, Mycoplasma fermentans and Porphyromonas asaccharolytica also associated. There is limited understanding of the host response involved. The proinflammatory mediators, interleukin (IL)-1β and C-X-C Motif Chemokine Ligand 8 (CXCL8), have been shown to play a role in the early response to D. nodosus in dermal fibroblasts and interdigital skin explant models. To further understand the response of ovine skin to bacterial stimulation, and to build an understanding of the role of the cytokines and chemokines identified, primary ovine interdigital fibroblasts and keratinocytes were isolated, cultured and stimulated. The expression of mRNA and protein release of CXCL8 and IL-1β were measured after stimulation with LPS, D. nodosus or F. necrophorum, which resulted in increased transcript levels of IL-1β and CXCL8 in the M. fermentans-free cells. However, only an increase in the CXCL8 protein release was observed. No IL-1β protein release was detected, despite increases in IL-1β mRNA, suggesting the signal for intracellular pre-IL-1β processing may be lacking when culturing primary cells in isolation. The keratinocytes and fibroblasts naturally infected with M. fermentans showed little response to the LPS, a range of D. nodosus preparations or heat-inactivated F. necrophorum. Primary single cell culture models complement ex vivo organ culture models to study different aspects of the host response to D. nodosus. The ovine keratinocytes and fibroblasts infected with M. fermentans had a reduced response to the experimental bacterial stimulation. However, in the case of footrot where Mycoplasma spp. are associated with diseased feet, this natural infection gives important insights into the impact of multiple pathogens on the host response.

Keywords: cell culture; footrot; sheep.

Figure 1

Characterisation of isolated ovine skin cells using immunofluorescence staining. Keratinocytes (left column; (B,D,F)) and fibroblasts (right column; (C,E,G)) were isolated from ovine interdigital skin and displayed typical cell morphologies in culture. (A) primary isolation showing mixed islets of cobblestone-like keratinocytes (blue arrow) and spindle-shaped fibroblasts (orange arrow). (B) purified keratinocyte culture. Cell types were stained for typical lineage markers, i.e., (D) cytokeratin (CK) 14 and (F) pan-cytokeratin in keratinocytes and (C) vimentin in fibroblasts, respectively. Proliferation was shown using an antibody directed against the typical proliferation marker, Ki67 (E) merged picture and the extracted FITC channel in (G)). The nuclear counterstain ensued with bisBenzimide Hoechst 33342; all staining was accompanied by a secondary antibody control (omission of first antibody). All scale bars represent 100 µm.

Figure 2

CXCL8 mRNA expression in interdigital ovine keratinocytes. Primary keratinocytes free of M. fermentans (n = 5, (A–C)) or naturally infected with M. fermentans (n = 4, (D–F)) were stimulated with 1 µg/mL E. coli LPS, heat-inactivated D. nodosus (HK), UV-inactivated D. nodosus (UV), formalin-fixed D. nodosus (FF) or heat-inactivated F. necrophorum (Fnec) (cell culture medium was used as diluent and control) for 2 h (A,D), 8 h (B,E) and 24 h (C,F). Horizontal line indicates the mean. Each replicated symbol represents a different cell line from an individual sheep. One-way-ANOVA, followed by Dunnett’s multiple comparisons test: * p ≤ 0.05, ** p ≤ 0.01.

Figure 3

CXCL8 mRNA expression in interdigital ovine fibroblasts. Primary fibroblasts free of M. fermentans (n = 6, (A–C)) or naturally infected with M. fermentans (n = 5, (D–F)) were stimulated with 1 µg/mL E. coli LPS, heat-inactivated D. nodosus (HK), UV-inactivated D. nodosus (UV), formalin-fixed D. nodosus (FF) or heat-inactivated F. necrophorum (Fnec) (cell culture medium was used as diluent and control) for 2 h (A,D), 8 h (B,E) and 24 h (C,F). Horizontal line indicates the mean. Each replicated symbol represents a different cell line from an individual sheep. One-way-ANOVA, followed by Dunnett’s multiple comparisons test: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 4

IL-1β mRNA expression in interdigital ovine fibroblasts. Primary fibroblasts free of M. fermentans (n = 6, (A–C)) or naturally infected with M. fermentans (n = 5, (D–F)) were stimulated with 1 µg/mL E. coli LPS, heat-inactivated D. nodosus (HK), UV-inactivated D. nodosus (UV), formalin-fixed D. nodosus (FF) or heat-inactivated F. necrophorum (Fnec) (cell culture medium was used as diluent and control) for 2 h (A,D), 8 h (B,E) and 24 h (C,F). Horizontal line indicates the mean. Each replicated symbol represents a different cell line from an individual sheep. One-way-ANOVA, followed by Dunnett’s multiple comparisons test: * p < 0.05, ** p < 0.01, *** p ≤ 0.001.

Figure 5

CXCL8 protein released into cell culture medium by interdigital ovine keratinocytes and fibroblasts. Primary keratinocytes free of M. fermentans (n = 4, (A)) or naturally infected with M. fermentans (n = 5, (B)) and primary fibroblasts free of M. fermentans (n = 6, (C)) or naturally infected with M. fermentans (n = 6, (D)) were stimulated with 1µg/mL E. coli LPS, heat-inactivated D. nodosus (HK), UV-inactivated D. nodosus (UV), formalin-fixed D. nodosus (FF) or heat-inactivated F. necrophorum (Fnec) for 24 h (cell culture medium was used as diluent and control). Horizontal line indicates the mean. Each replicated symbol represents a different cell line from an individual sheep. One-way-ANOVA, followed by Dunnett’s multiple comparisons test: * p < 0.05.