UVM Theses and Dissertations
Format:
Print
Author:
Kandasamy, Sukumar
Dept./Program:
Animal, Nutrition and Food Sciences Program
Year:
2011
Degree:
Ph. D.
Abstract:
Mastitis, the inflammation of mammary gland., affects approximately 20% of dairy cows at any given time in the United States of America. Enhancing mastitis resistance in dairy cows is considered as a promising method to reduce incidence of the disease. This can potentially be achieved by breeding cows for enhanced resistance to mammary infections. Among the different immune response mechanisms, the innate immune response plays a predominant role in resisting mammary infections in dairy cows. Studying both the natural variation in innate immune response among cows as well as the effects of this variation on mastitis may help to develop new strategies to control mastitis incidence. The extent of phenotypic variation in innate immunity that exists in a population of cows can be estimated using an in vitro cell-culture-based model system. In this study, the suitability of using dermal fibroblast cells to study between-cow variation in innate immune response was assessed.
Stimulation of the dermal fibroblasts with either E. coli lipopolysaccharide (LPS), or a synthetic mimic of Gram-positive organisms, Pam2CSK4, or a bovine cytokine, IL-1[Greek Beta], resulted in a marked increase in production of markers of the innate immune response such as IL-6 and IL-8. Genome-wide expression analysis of LPS-stimulated dermal fibroblasts revealed differential expression of approximately 300 genes. In addition, gene expression changes confirmed the activation of both MyD88-dependent-and TRIF-dependent-TLR4 pathways in LPS-stimulated fibroblasts. These results suggested that the dermal fibroblast cells can be used to study the innate immune response of cows under laboratory conditions. Subsequently, dermal fibroblast cultures were established from 88 lactating cows to explore between-cow variation in innate immune response. Substantial between-cow variation was observed in the fibroblast production of IL-8 or IL-6 proteins to LPS, allowing the cows to be ranked as high (IL-8 responses of cows that fall into top two quartiles) or low (lL-8 responses of cows that falls into bottom two quartiles) responders based on.the IL-8 response. The LPS-induced IL-8 response in the high responder fibroblasts (n=8) were approximately six-fold greater (P<0.01) than in the low (n=8) responder cells.
A similar pattern of variation was also observed in both IL-8 and IL6 responses to IL-1[Greek Beta] in most of the fibroblast cultures. Subsequently, potential underlying causes for the variable innate immune response were examined by analyzing the gene expression changes in one very low responding and one very high responding fibroblast culture using microarray and quantitative PCR analysis techniques. Results showed that decreased basal TLR4 expression was associated with the profound deficiency in the innate immune response of the low responding fibroblast culture. Subsequently, ability of cows ranked as high-or low-responders to defend against experimental E. coli infection of the mammary gland was assessed. In that study, eight high and eight low responder cows were identified from a pool of 80 animals, and challenged in late lactation with E. coli (strain P4; 200 cfu) in one quarter of the udder. Clearance of bacteria from the gland was similar between the high and low groups. However, the high responder cows had greater mammary inflammation, and delayed resolution of the inflammation as indicated by higher milk SCC, milk BSA content, and lower milk production. Results suggested that the innate response of the low responder cows is sufficient to resolve Gram-negative mastitis with less deterioration of milk quality. Together, the results of these studies in a newly defined model system to study the bovine innate immune system indicate that the model reveals the large degree of between-animal variation and that the in vitro responses in the model are reflected to some extent in vivo.
Stimulation of the dermal fibroblasts with either E. coli lipopolysaccharide (LPS), or a synthetic mimic of Gram-positive organisms, Pam2CSK4, or a bovine cytokine, IL-1[Greek Beta], resulted in a marked increase in production of markers of the innate immune response such as IL-6 and IL-8. Genome-wide expression analysis of LPS-stimulated dermal fibroblasts revealed differential expression of approximately 300 genes. In addition, gene expression changes confirmed the activation of both MyD88-dependent-and TRIF-dependent-TLR4 pathways in LPS-stimulated fibroblasts. These results suggested that the dermal fibroblast cells can be used to study the innate immune response of cows under laboratory conditions. Subsequently, dermal fibroblast cultures were established from 88 lactating cows to explore between-cow variation in innate immune response. Substantial between-cow variation was observed in the fibroblast production of IL-8 or IL-6 proteins to LPS, allowing the cows to be ranked as high (IL-8 responses of cows that fall into top two quartiles) or low (lL-8 responses of cows that falls into bottom two quartiles) responders based on.the IL-8 response. The LPS-induced IL-8 response in the high responder fibroblasts (n=8) were approximately six-fold greater (P<0.01) than in the low (n=8) responder cells.
A similar pattern of variation was also observed in both IL-8 and IL6 responses to IL-1[Greek Beta] in most of the fibroblast cultures. Subsequently, potential underlying causes for the variable innate immune response were examined by analyzing the gene expression changes in one very low responding and one very high responding fibroblast culture using microarray and quantitative PCR analysis techniques. Results showed that decreased basal TLR4 expression was associated with the profound deficiency in the innate immune response of the low responding fibroblast culture. Subsequently, ability of cows ranked as high-or low-responders to defend against experimental E. coli infection of the mammary gland was assessed. In that study, eight high and eight low responder cows were identified from a pool of 80 animals, and challenged in late lactation with E. coli (strain P4; 200 cfu) in one quarter of the udder. Clearance of bacteria from the gland was similar between the high and low groups. However, the high responder cows had greater mammary inflammation, and delayed resolution of the inflammation as indicated by higher milk SCC, milk BSA content, and lower milk production. Results suggested that the innate response of the low responder cows is sufficient to resolve Gram-negative mastitis with less deterioration of milk quality. Together, the results of these studies in a newly defined model system to study the bovine innate immune system indicate that the model reveals the large degree of between-animal variation and that the in vitro responses in the model are reflected to some extent in vivo.