Staphylococcus aureus (S. aureus) is a gram-positive bacterium that causes mastitis in bovines and leads to financial losses to the dairy industry. Although antibody response plays a role in immune defense against S. aureus, cellular responses are of interest for vaccine development. A vaccine that stimulates both antibody and cellular responses could promote memory cell formation and provide effective protection against S. aureus. The superantigens and virulence factors secreted by live S. aureus (LSA) can interfere with immune responses and memory cell formation. Because irradiation reduces the metabolic activity and secretion of proteins, including S. aureus superantigens and hemolysins, we hypothesized the irradiated S. aureus (ISA) could drive immune cell responses.
Dendritic cells (DC) were co-cultured with lymphocytes to study the cellular responses to ISA and LSA. Dendritic cells present antigens and polarize lymphocytes into different helper T (Th) cell types that drive cellular immune responses. The DC loaded with either ISA or LSA induced increased mRNA transcription of Th17-related cytokines and cytotoxic effector memory cell formation during antigen recall experiments. Lymphocytes co-cultured with LSA-loaded DC exhibited a higher fold-change in interferon (IFN) γ mRNA compared to ISA-loaded DC, suggesting the secreted antigens and the metabolic activity of S. aureus play a role in Th1 polarization.
Th1 polarization can drive excessive inflammation and suppress beneficial Th17 responses. Bovine DC were stimulated with a mutant α-toxin deletion S. aureus strain to evaluate if α-toxin-mediated NOD2 receptor signaling activates Th1 polarization in response to S. aureus, which revealed that NOD2 mRNA transcription in DC was independent of α-toxin and that the deletion of α-toxin had no effect on the transcription of the cytokine IL-12 or the production of IFNγ by lymphocytes, events that drive Th1 polarization, in co-cultures. The deletion of accessory gene regulator (agr), which controls α-toxin production, reduced IFNγ production in lymphocytes co-cultured with the S. aureus-loaded DC, indicating that agr controlled the ability of S. aureus antigens to drive the Th1 polarization of lymphocytes.
Overall, this thesis demonstrates that ISA is a promising source of antigens that stimulate memory cells formation and Th17 polarization in bovine immune cells. The reduced Th1 cytokine response to S. aureus was not dependent on α-toxin, but other virulence factors controlled by agr should be screened to determine the source of Th1 stimulation. / Ph. D. / Dairy cows’ health and productivity is negatively impacted by mastitis, infection starting at the mucosal surfaces of the udder. <i>Staphylococcus aureus</i> is a bacterium that can cause mastitis and there is no efficacious vaccine available. I explored the use of weakened <i>S. aureus</i> as a source of vaccine components and the α-toxins role in stimulating the immune cells like dendritic cells (DC) and lymphocytes. <i>S. aureus</i> was weakened using gamma irradiation to conserve the structural components of the bacterium and render it unable to secrete α-toxin. The DC were collected from dairy cows and stimulated with irradiated <i>S. aureus</i> and live <i>S. aureus</i> before lymphocytes were added to the cultures. The DC signaling, lymphocytes’ pro-inflammatory interferon gamma and mucosal immunity related interleukin responses were measured from RNA production. Memory cell formation and production of interferon gamma were measured from whole cells. The role of α-toxin in lymphocyte stimulation was further studied using a strain of bacterium that does not produce the toxin. Irradiated <i>S. aureus</i> induced low production of inflammatory interferon gamma compared to the live <i>S. aureus</i>. The α-toxin played no role in this, even if other components produced under the same regulatory element likely did, as shown by reduced interferon production in response to bacteria without the regulatory element. Irradiation of the bacterium did not reduce mucosal immunity related cytokine production or formation of memory cells. The irradiated <i>S. aureus</i> is a source for vaccine components that stimulate immune cells like DC and immunity to <i>S. aureus</i> on mucosal surfaces of the udder.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/75153 |
| Date | 24 February 2017 |
| Creators | Lehtimaki, Mari |
| Contributors | Dairy Science, Kanevsky, Isis, Luo, Xin, Akers, Robert Michael, Cranford, Jamie L. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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