Effects of nuclear factor-κB on regulation of cytokine expression and apoptosis in bovine monocytes exposed to Mycobacterium avium subsp paratuberculosis

Douglas J. Weiss Department of Veterinary and Biomedical Science, College of Veterinary Medicine, University of Minnesota, Saint Paul, MN 55108

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Cleverson D. Souza Department of Veterinary and Biomedical Science, College of Veterinary Medicine, University of Minnesota, Saint Paul, MN 55108

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Oral A. Evanson Department of Veterinary and Biomedical Science, College of Veterinary Medicine, University of Minnesota, Saint Paul, MN 55108

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Abstract

Objective—To evaluate the role of the nuclear factor-κB (NF-κB) in the response of bovine monocytes to exposure to Mycobacterium avium subsp paratuberculosis (MAP).

Sample Population—Monocytes from healthy adult Holstein cows that were known to be negative for MAP infection.

Procedures—Monocytes were incubated with MAP organisms with or without a specific inhibitor of the NF-κB pathway (pyrrolidine dithiocarbamate), and activation of the NF-κB pathway was detected by use of an electrophorectic mobility shift assay. The capacities of monocytes to express tumor necrosis factor (TNF)-α, interleukin (IL)-10, and IL-12; to acidify phagosomes; to phagocytize and kill MAP organisms; and to undergo apoptosis were evaluated.

Results—Addition of MAP organisms to monocytes activated the NF-κB pathway as indicated by increased NF-κB–DNA binding. Addition of pyrrolidine dithiocarbamate prevented nuclear translocation of NF-κB, decreased expression of TNF-α and IL-10, and increased IL-12 expression. Treatment of MAP-exposed monocytes with pyrrolidine dithiocarbamate increased the rate of apoptosis but failed to alter phagosome acidification, organism uptake, or organism killing by those cells.

Conclusions and Clinical Relevance—Results indicated that NF-κB rapidly translocated to the nucleus after exposure of bovine monocytes to MAP organisms. These data suggest that NF-κB is involved in initiation of inflammatory cytokine transcription and inhibition of apoptosis but that it is not directly involved in phagosome acidification or organism killing.

Abstract

Objective—To evaluate the role of the nuclear factor-κB (NF-κB) in the response of bovine monocytes to exposure to Mycobacterium avium subsp paratuberculosis (MAP).

Sample Population—Monocytes from healthy adult Holstein cows that were known to be negative for MAP infection.

Procedures—Monocytes were incubated with MAP organisms with or without a specific inhibitor of the NF-κB pathway (pyrrolidine dithiocarbamate), and activation of the NF-κB pathway was detected by use of an electrophorectic mobility shift assay. The capacities of monocytes to express tumor necrosis factor (TNF)-α, interleukin (IL)-10, and IL-12; to acidify phagosomes; to phagocytize and kill MAP organisms; and to undergo apoptosis were evaluated.

Results—Addition of MAP organisms to monocytes activated the NF-κB pathway as indicated by increased NF-κB–DNA binding. Addition of pyrrolidine dithiocarbamate prevented nuclear translocation of NF-κB, decreased expression of TNF-α and IL-10, and increased IL-12 expression. Treatment of MAP-exposed monocytes with pyrrolidine dithiocarbamate increased the rate of apoptosis but failed to alter phagosome acidification, organism uptake, or organism killing by those cells.

Conclusions and Clinical Relevance—Results indicated that NF-κB rapidly translocated to the nucleus after exposure of bovine monocytes to MAP organisms. These data suggest that NF-κB is involved in initiation of inflammatory cytokine transcription and inhibition of apoptosis but that it is not directly involved in phagosome acidification or organism killing.

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