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Differential expression of Toll-like receptors and inflammatory cytokines in ovine interdigital dermatitis and footrot.

ABSTRACT: Footrot is a common inflammatory bacterial disease affecting the health and welfare of sheep worldwide. The pathogenesis of footrot is complex and multifactorial. The primary causal pathogen is the anaerobic bacterium Dichelobacter nodosus, with Fusobacterium necrophorum also shown to play a key role in disease. Since immune-mediated pathology is implicated, the aim of this research was to investigate the role of the host response in interdigital dermatitis (ID) and footrot. We compared the expression of Toll-like receptors (TLRs) and pro-inflammatory cytokines and the histological appearance of clinically normal in comparison to ID and footrot affected tissues. Severe ID and footrot were characterised by significantly increased transcript levels of pro-inflammatory cytokines TNFα and IL1β and the pattern recognition receptors TLR2 and TLR4 in the interdigital skin. This was reflected in the histopathological appearance, with ID and footrot presenting progressive chronic-active pododermatitis with a mixed lymphocytic and neutrophilic infiltration, gradually increasing from a mild form in clinically normal feet, to moderate in ID and to a focally severe form with frequent areas of purulence in footrot. Stimulation with F. necrophorum and/or D. nodosus extracts demonstrated that dermal fibroblasts, the resident cell type of the dermis, also contribute to the inflammatory response to footrot bacteria by increased expression of TNFα, IL1β and TLR2. Overall, ID and footrot lead to a local inflammatory response given that expression levels of TLRs and IL1β were dependent on the disease state of the foot not the animal.

SUBMITTER: Davenport R

PROVIDER: S-EPMC4157958 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

Project description:BackgroundPorcine reproductive and respiratory syndrome virus (PRRSV) is largely responsible for heavy economic losses in the swine industry worldwide because of its high mutation rate and subsequent emergence of virulent strains. However, the immunological and pathological responses of pigs to PRRSV strains with different virulence have not been completely elucidated.MethodsTwenty-four piglets were divided into 4 groups (n = 6 each) and inoculated with highly pathogenic PRRSV isolate BB0907 (HP), low pathogenic PRRSV NT0801 (LP), LP derivative strain NT0801-F70 (LP-der), and DMEM medium (control), respectively. The changes in TLR2, 3, 7, and 8 gene expression and TNF-α, IL-1β, IL-6, IFN-γ, and IL-10 secretion were evaluated using real-time PCR and ELISA at 6, 9, and 15 days post inoculation (d.p.i.). The cytokine levels were evaluated in the supernatants of porcine alveolar macrophages (PAMs) and peripheral blood mononuclear cells (PBMCs) following stimulation with LTA, poly(I:C), CL097, and PRRSV individually.ResultsHP caused more severe clinical signs and pathological lesions in swine than LP and LP-der had almost no virulence compared with LP. The serum levels of IL-1β, IL-6, TNF-α, and IFN-γ were increased in HP-infected piglets, which were greater than in those infected with LP or LP-der. The mRNA levels of TLR3, 7, and 8 were significantly up-regulated in PAMs in HP-infected pigs compared to those in groups LP and LP-der. Furthermore, TNF-α and IL-1β secretion in PAMs from group LP was statistically greater than those from the control group after stimulation with either poly(I:C) or CL097. Meanwhile, TNF-α, IL-1β, and IL-6 levels in CL097-stimulated PBMCs from HP-infected pigs were markedly higher than those from the LP- and LP-der-infected groups.ConclusionsWe found that HP was a stronger inducer of TLR 3, 7, and 8 expression and IL-1β, IL-6, TNF-α, and IFN-γ production compared to LP and LP-der. HP enhanced production of TNF-α, IL-1β, and IL-6 in PBMCs following CL097-stimulation more than LP and LP-der, whereas LP enhanced the secretion of TNF-α and IL-1β in poly(I:C)- and CL097-stimulated PAMs. Our data regarding cellular reactivity to different isolates should be useful in the development of more efficacious vaccines.

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Differential expression of Toll-like receptors and inflammatory cytokines in ovine interdigital dermatitis and footrot.

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