Project description:The domestic pig (Sus scrofa) provides a large animal model for human innate immune responses and inflammation that is also economically important in its own right. Results: We demonstrate that macrophages can be harvested from 3 different compartments of the pig (lungs, blood and bone-marrow), cryopreserved and subsequently recovered and differentiated in CSF-1. We have performed surface marker analysis and gene expression profiling on macrophages from these compartments, comparing 25 animals from 5 different breeds and their response to lipopolysaccharide. The results provide a clear distinction between alveolar macrophages (AM) and monocyte-derived (MDM) and bone-marrow-derived macrophages (BMDM). In particular, the lung macrophages express the growth factor, Flt1 and its ligand, VEGFA at high levels, suggesting a distinct pathway of growth regulation. We confirm that pig macrophages more closely resemble human, than mouse, in their set of LPS-inducible genes. Relatively few genes showed breed-specific differential expression, notably CXCR2 and CD302 in alveolar macrophages. In contrast, there was substantial inter-individual variation between pigs within breeds, mostly affecting genes annotated as being involved in immune responses. Conclusions: Future research will address whether such variation is heritable, and might form the basis for selective breeding for disease resistance or functional genomics. The domestic pig (Sus scrofa) provides a large animal model for human innate immune responses and inflammation that is also economically important in its own right. We demonstrate that macrophages can be harvested from 3 different compartments of the pig (lungs, blood and bone-marrow), cryopreserved and subsequently recovered and differentiated in CSF-1. We have performed surface marker analysis and gene expression profiling on macrophages from these compartments, comparing 25 animals from 5 different breeds and their response to lipopolysaccharide. The results provide a clear distinction between alveolar macrophages (AM) and monocyte-derived (MDM) and bone-marrow-derived macrophages (BMDM). In particular, the lung macrophages express the growth factor, Flt1 and its ligand, VEGFA at high levels, suggesting a distinct pathway of growth regulation. We confirm that pig macrophages more closely resemble human, than mouse, in their set of LPS-inducible genes. Relatively few genes showed breed-specific differential expression, notably CXCR2 and CD302 in alveolar macrophages. In contrast, there was substantial inter-individual variation between pigs within breeds, mostly affecting genes annotated as being involved in immune responses. Future research will address whether such variation is heritable, and might form the basis for selective breeding for disease resistance or functional genomics.

Project description:The domestic pig (Sus scrofa) provides a large animal model for human innate immune responses and inflammation that is also economically important in its own right. Results: We demonstrate that macrophages can be harvested from 3 different compartments of the pig (lungs, blood and bone-marrow), cryopreserved and subsequently recovered and differentiated in CSF-1. We have performed surface marker analysis and gene expression profiling on macrophages from these compartments, comparing 25 animals from 5 different breeds and their response to lipopolysaccharide. The results provide a clear distinction between alveolar macrophages (AM) and monocyte-derived (MDM) and bone-marrow-derived macrophages (BMDM). In particular, the lung macrophages express the growth factor, Flt1 and its ligand, VEGFA at high levels, suggesting a distinct pathway of growth regulation. We confirm that pig macrophages more closely resemble human, than mouse, in their set of LPS-inducible genes. Relatively few genes showed breed-specific differential expression, notably CXCR2 and CD302 in alveolar macrophages. In contrast, there was substantial inter-individual variation between pigs within breeds, mostly affecting genes annotated as being involved in immune responses. Conclusions: Future research will address whether such variation is heritable, and might form the basis for selective breeding for disease resistance or functional genomics. The domestic pig (Sus scrofa) provides a large animal model for human innate immune responses and inflammation that is also economically important in its own right. We demonstrate that macrophages can be harvested from 3 different compartments of the pig (lungs, blood and bone-marrow), cryopreserved and subsequently recovered and differentiated in CSF-1. We have performed surface marker analysis and gene expression profiling on macrophages from these compartments, comparing 25 animals from 5 different breeds and their response to lipopolysaccharide. The results provide a clear distinction between alveolar macrophages (AM) and monocyte-derived (MDM) and bone-marrow-derived macrophages (BMDM). In particular, the lung macrophages express the growth factor, Flt1 and its ligand, VEGFA at high levels, suggesting a distinct pathway of growth regulation. We confirm that pig macrophages more closely resemble human, than mouse, in their set of LPS-inducible genes. Relatively few genes showed breed-specific differential expression, notably CXCR2 and CD302 in alveolar macrophages. In contrast, there was substantial inter-individual variation between pigs within breeds, mostly affecting genes annotated as being involved in immune responses. Future research will address whether such variation is heritable, and might form the basis for selective breeding for disease resistance or functional genomics. 140 Affymetrix Snowball microarray were analysed from 5 different breed (DR, LR, LW, PIE and HAM). 5 pig per breed were used and cells were harvested from Lungs, blood and bone-marrow (AM, MDM and BMDM). Cells were left untreated (0h) or stimulated with LPS Salmonella enterica serotype minnesota Re 595 - 100ng/ml (7h)

Project description:Innate immune response to a lipopolysaccharide (LPS) challenge varies among sheep breeds. How different breeds respond to bacterial infections impacts management practices of sheep producers. Hence, clinical response, acute-phase response, and gene expression of pro- and anti-inflammatory markers in peripheral white blood cells (WBCs) were examined after an LPS challenge in Dorset and Suffolk ewes. Ewes received either PBS or 2.5 µg/kg LPS (i.v.) 4 to 5 d after onset of synchronized estrus. Blood was collected via jugular venipuncture intermittently for 24 h to determine WBC counts. Rectal temperatures and observations of behavioral/physical appearances were recorded hourly. After LPS, WBCs decreased the first hour (P = 0.0001) and rectal temperatures (P < 0.0001) increased through 4 h; both returned toward normal 6 h after challenge. Suffolk ewes exhibited greater changes in temperature (P = 0.03) and behavioral/physical responses (P < 0.0001) than Dorset ewes and had an enhanced acute-phase response demonstrated by increased concentrations of plasma haptoglobin (P = 0.04), as well as cortisol concentrations (P = 0.03). Real-time PCR was completed on buffy coat homogenates for expression of pro-inflammatory [CXCL8, IL-6, interferon gamma (IFNG), complement component 3 (C3), toll-like receptor 4 (TLR4), prostaglandin synthase 2 (PTGS2)] and anti-inflammatory [IL-10, superoxide dismutase 2 (SOD2), forkhead box P3 (FOXP3), peroxisome proliferator-activated receptor gamma (PPARG), mannose receptor C type 1 (MRC1), transforming growth factor ? (TGF?)] genes. After LPS treatment, gene expressions increased for CXCL8 (P = 0.0003), TLR4 (P = 0.004), SOD2 (P < 0.0001), and C3 (P = 0.003), while PPARG (P = 0.006) and MRC1 (P = 0.003) decreased. Overall, Dorset ewes had greater expression of TLR4 (P = 0.003), IL-10 (P = 0.045), PPARG (P = 0.002), FOXP3 (P = 0.001), and SOD2 (P = 0.0002), whereas Suffolk ewes had greater expression of IL-6 (P = 0.0007), IFNG (P = 0.02), PTGS2 (P = 0.0002), and C3 (P = 0.008). Suffolk ewes also displayed greater expression of IL-6 (P = 0.002) and C3 (P = 0.0004) in response to LPS. In conclusion, differences in gene expression may explain the enhanced inflammatory response in Suffolk ewes and may predispose Suffolk ewes to be more responsive to bacterial infection than Dorset ewes.

Dataset Information

The impact of breed and tissue compartment on the response of pig macrophages to lipopolysaccharide.

Publications

The impact of breed and tissue compartment on the response of pig macrophages to lipopolysaccharide.

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