RESUMO
Skin infection studies are often limited by financial and ethical constraints, and alternatives, such as monolayer cell culture, do not reflect many cellular processes limiting their application. For a more functional replacement, 3D skin culture models offer many advantages such as the maintenance of the tissue structure and the cell types present in the host environment. A 3D skin culture model can be set up using tissues acquired from surgical procedures or post slaughter, making it a cost effective and attractive alternative to animal experimentation. The majority of 3D culture models have been established for aerobic pathogens, but currently there are no models for anaerobic skin infections. Footrot is an anaerobic bacterial infection which affects the ovine interdigital skin causing a substantial animal welfare and financial impact worldwide. Dichelobacter nodosus is a Gram-negative anaerobic bacterium and the causative agent of footrot. The mechanism of infection and host immune response to D. nodosus is poorly understood. Here we present a novel 3D skin ex vivo model to study anaerobic bacterial infections using ovine skin explants infected with D. nodosus. Our results demonstrate that D. nodosus can invade the skin explant, and that altered expression of key inflammatory markers could be quantified in the culture media. The viability of explants was assessed by tissue integrity (histopathological features) and cell death (DNA fragmentation) over 76 h showing the model was stable for 28 h. D. nodosus was quantified in all infected skin explants by qPCR and the bacterium was visualized invading the epidermis by Fluorescent in situ Hybridization. Measurement of pro-inflammatory cytokines/chemokines in the culture media revealed that the explants released IL1ß in response to bacteria. In contrast, levels of CXCL8 production were no different to mock-infected explants. The 3D skin model realistically simulates the interdigital skin and has demonstrated that D. nodosus invades the skin and triggered an early cellular inflammatory response to this bacterium. This novel model is the first of its kind for investigating an anaerobic bacterial infection.
Assuntos
Dichelobacter nodosus/crescimento & desenvolvimento , Pododermatite Necrótica dos Ovinos/microbiologia , Infecções por Bactérias Gram-Negativas/veterinária , Cultura Primária de Células/métodos , Doenças dos Ovinos/microbiologia , Dermatopatias Bacterianas/veterinária , Animais , Biópsia , Infecções por Bactérias Gram-Negativas/microbiologia , Interleucina-1beta/análise , Interleucina-8/análise , Queratinócitos/metabolismo , Modelos Biológicos , Ovinos , Dermatopatias Bacterianas/microbiologia , Fatores de Tempo , Técnicas de Cultura de TecidosRESUMO
Chlamydophila abortus is a Gram-negative obligate intracellular bacterium that causes infectious abortion in sheep (ovine enzootic abortion, OEA) and humans. Infected placentas recovered from sheep that experience OEA have thickened membranes, contain dense inflammatory cellular infiltrates and show evidence of intravascular thrombosis. Despite widespread inflammation, chlamydial multiplication is restricted to the chorionic trophoblast cells. To investigate the potential role of trophoblast in the initiation and propagation of placental inflammation during OEA, the AH-1 ovine trophoblast cell line was experimentally infected with C. abortus and analysed for the release of pro-inflammatory mediators. C. abortus was found to induce the release of both tumour necrosis factor-alpha (TNFalpha) and CXCL8 (interleukin-8) from AH-1 cells in a dose- and time-dependent manner. Ultra-violet (UV)-killed organisms did not elicit this profile, indicating that intracellular multiplication of C. abortus was required for release of these pro-inflammatory mediators. Exposure of AH-1 cells to recombinant ovine TNFalpha alone resulted in the release of CXCL8, suggestive of a self-propagating inflammatory cytokine and chemokine cascade. These data indicate a primary role for trophoblast in the initiation and propagation of placental inflammation during chlamydial abortion.
Assuntos
Aborto Animal/imunologia , Infecções por Chlamydophila/veterinária , Chlamydophila/imunologia , Interleucina-8/metabolismo , Complicações Infecciosas na Gravidez/veterinária , Trofoblastos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Chlamydophila/crescimento & desenvolvimento , Infecções por Chlamydophila/imunologia , Infecções por Chlamydophila/patologia , Infecções por Chlamydophila/fisiopatologia , Relação Dose-Resposta Imunológica , Feminino , Homeostase/imunologia , Inflamação/imunologia , Interleucina-8/genética , Interleucina-8/imunologia , Gravidez , Complicações Infecciosas na Gravidez/imunologia , Complicações Infecciosas na Gravidez/patologia , Complicações Infecciosas na Gravidez/fisiopatologia , Ovinos , Trombose/imunologia , Trofoblastos/imunologia , Trofoblastos/microbiologia , Trofoblastos/patologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Upregulation of intelectin (ITLN) transcript and protein has previously been shown in intestinal nematode infections of resistant mice strains with immunolocalisation of protein to goblet cells and paneth cells. In man, intelectin expression has been shown in respiratory tract epithelium, with upregulation occurring in bronchoalveolar lavage fluid of asthmatic individuals. This study describes the expression of intelectin in the respiratory tract of sheep and the immunolocalisation to goblet cells using a novel affinity-purified chicken anti-intelectin peptide antibody. Furthermore we show that when sheep tracheal explants were cultured for 48 h+/- recombinant sheep IL-4, sheep ITLN transcripts were upregulated compared with controls. Putative roles for intelectin have included an antibacterial role and an alteration of the character of mucus. Our data suggest ITLNs may play an important role in the mucosal response in allergy and parasitic infections.
Assuntos
Células Caliciformes/efeitos dos fármacos , Células Caliciformes/metabolismo , Interleucina-4/farmacologia , Lectinas/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/metabolismo , Linhagem Celular Tumoral , Regulação da Expressão Gênica/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Ovinos , Células Th2/efeitos dos fármacos , Células Th2/fisiologia , Traqueia/citologiaRESUMO
Chlamydia/Chlamydophila are a family of intracellular gram-negative bacteria that infect their hosts primarily via mucosal epithelia. Chronic disease associated with bacterial persistence, inflammation and tissue damage are common sequelae of infection with these organisms. Human epithelial cell lines respond to infection by releasing pro-inflammatory cytokines and chemokines such as interleukin (IL)-6 and IL-8, and upregulating the expression of mRNA encoding Ikappa-Balpha, the endogenous inhibitor of NF-kappaB. However, Ikappa-Balpha is not upregulated in response to bacterial lipopolysaccharide (LPS). The failure of epithelial cells to respond to LPS is associated with the absence of surface expression of CD14. Identification of the components of Chlamydia/Chlamydophila that can induce pro-inflammatory mediators coupled with the mechanisms by which epithelial cells detect infection and respond accordingly will advance the development of preventative strategies.
Assuntos
Infecções por Chlamydia/imunologia , Chlamydia/imunologia , Infecções por Chlamydophila/imunologia , Chlamydophila/imunologia , Chlamydia/genética , Infecções por Chlamydia/genética , Regulação Bacteriana da Expressão Gênica , Células HeLa , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Interleucina-8/genética , Interleucina-8/imunologia , Inibidor de NF-kappaB alfaRESUMO
The BeWo trophoblast cell line does not constitutively express the tryptophan degrading enzyme indolamine 2,3-dioxygenase (IDO), nor can IDO expression be induced by gamma interferon. This correlates with the inability of BeWo cells to control the growth of Chlamydophila abortus, in contrast to effects observed in HeLa cells treated with gamma interferon.