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Genomic analysis of between-cow variation in dermal fibroblast response to lipopolysaccharide.

ABSTRACT: The innate immune response plays a major role in defense against mastitis-causing pathogens. Identification of existing variation in innate immune signaling among cows and the underlying molecular causes for the variation may help in design of new mastitis control strategies. The dermal fibroblast has been used as a model cell type to explore between-cow variation in the ability of cells to produce IL-8 in response to lipopolysaccharide (LPS) treatment, and this response appears related to an animal's ability to respond to in vivo challenge with LPS or Escherichia coli mastitis. In this study, primary dermal fibroblast cultures of cows and microarray-based genomic analysis were used to investigate the cause(s) for the variable response to LPS. Fibroblast cultures from 2 cows, one with a low response phenotype (LR(array)) and another with a high response phenotype (HR(array)), were selected from our collection of fibroblast cultures established from 88 cows. The LR(array) fibroblast culture produced approximately 5-fold less IL-8 and IL-6 protein in response to 24-h LPS treatment than the HR(array) fibroblast culture. Genomic analysis of RNA obtained from 3 replicates of the 2 cultures before and after 8-h LPS treatment revealed a combined LPS-induced differential expression of 321 transcripts, indicating the robust response capability of the fibroblast cell. Under basal conditions, the microarray analysis revealed 2-fold less expression of toll-like receptor 4 (TLR4) in the LR(array) fibroblasts compared with the HR(array) fibroblasts, and this was associated with a marked reduction in expression of genes regulated by the TLR4-MyD88-dependent and TLR4-TRIF-dependent pathways (IL-8, IL-6, SAA3, CCL20, MX1, IRF1, and ISG20). The between-culture differential expression of TLR4 was confirmed and extended by quantitative PCR analysis (QPCR) that revealed a 33-fold lower expression of TLR4 in the LR(array) fibroblast culture. After LPS treatment, the difference in TLR4 expression increased to almost 50-fold and was associated with more than 8-fold lower expression of IL-8 and IL-6. No DNA sequence variations were identified in the proximal 1,300-bp promoter region of the TLR4 gene, and microarray analysis did not reveal a molecular explanation for the reduced TLR4 expression under either basal conditions or following exposure to LPS. The attenuated innate immune response of the LR(array) fibroblast culture to LPS may be caused by reduced TLR4 receptor expression. Also, the primary dermal fibroblast cells can be used to examine underlying causes for between-cow variations in key immune response pathways.

SUBMITTER: Kandasamy S

PROVIDER: S-EPMC4235160 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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Genomic analysis of between-cow variation in dermal fibroblast response to lipopolysaccharide.

Kandasamy S S Kerr D E DE

Journal of dairy science 20120701 7

The innate immune response plays a major role in defense against mastitis-causing pathogens. Identification of existing variation in innate immune signaling among cows and the underlying molecular causes for the variation may help in design of new mastitis control strategies. The dermal fibroblast has been used as a model cell type to explore between-cow variation in the ability of cells to produce IL-8 in response to lipopolysaccharide (LPS) treatment, and this response appears related to an an ...[more]

PMID: 22720940

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Project description:We evaluated the between-cow (b-cow) variation and repeatability in omasal and milk fatty acids (FA) related to methane (CH4) emission. The dataset was originated from 9 studies with rumen-cannulated dairy cows conducted using either a switch-back or a Latin square design. Production of CH4 per mole of VFA (Y_CH4VFA) was calculated based on VFA stoichiometry. Experiment, diet within experiment, period within experiment, and cow within experiment were considered as random factors. Empirical models were developed between the variables of interest by univariate and bivariate mixed model regression analysis. The variation associated with diet was higher than the b-cow variation with low repeatability (< 0.25) for milk odd- and branch-chain FA (OBCFA). Similarly, for de novo synthesized milk FA, diet variation was ~ 3-fold greater than the b-cow variation; repeatability for these FA was moderate to high (0.34-0.58). Also, for both cis-9 C18:1 and cis-9 cis-12 cis-15 C18:3 diet variation was more than double the b-cow variation, but repeatability was moderate. Among the de novo milk FA, C4:0 was positively related with stoichiometric Y_CH4VFA, while for OBCFA, anteiso C15:0 and C15:0 were negatively related with it. Notably, when analyzing the relationship between omasal FA and milk FA we observed positive intercept estimates for all the OBCFA, which may indicate endogenous post-ruminal synthesis of these FA, most likely in the mammary gland. For milk iso C13:0, iso C15:0, anteiso C15:0, and C15:0 were positively influenced by omasal proportion of their respective FA and by energy balance. In contrast, the concentration of milk C17:0, iso C18:0, C18:0, cis-11 C18:1, and cis-9 cis-12 cis-15 C18:3 were positively influenced by omasal proportion of their respective FA but negatively related to calculated energy balance. Our findings demonstrate that for most milk FA examined, a larger variation is attributed to diet than b-cow differences with low to moderate repeatability. While some milk FA were positively or negatively related with Y_CH4VFA, there was a pronounced effect of calculated energy balance on these estimates. Additionally, even though OBCFA have been indicated as markers of rumen function, our results suggest that endogenous synthesis of these FA may occur, which therefore, may limit the utilization of milk FA as a proxy for CH4 predictions for cows fed the same diet.

Dataset Information

Genomic analysis of between-cow variation in dermal fibroblast response to lipopolysaccharide.

Publications

Genomic analysis of between-cow variation in dermal fibroblast response to lipopolysaccharide.

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