Location: Food and Feed Safety Research
Project Number: 3091-32000-034-57-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Aug 1, 2017
End Date: Jul 31, 2020
Aim 1. Quantify the effects of an S. enterica serovar Heidelberg and serovar Enteritidis infection on T regulatory (Treg) cell properties in chickens. Aim 2. Determine the effects of an S. enterica serovar Heidelberg and serovar Enteritidis infection on interactions between Treg and other immune cells. Aim 3. Determine the molecular pathways through which Salmonella infections regulate Treg development.
We will study two distinct serovars of Salmonella, namely S. enterica serovar Heidelberg and serovar Enteritidis and correlate the Treg quantity and Treg-associated cytokine mRNA contents (high IL-10 and TGF-ß, and no IL-2) from local (cecal tonsils) and systemic (spleen, bone marrow, blood and thymus) organs with bacteriologic and pathologic outcomes of Salmonella infection. Chickens will be infected with S. enterica (serovars Heidelberg and Enteritidis) as described in experimental design section. Thymus, blood, spleen, cecal tonsils, liver, bone marrow, and bursa will be collected from S. enterica (serovars Heidelberg and Enteritidis)-infected and mock-infected chickens at each time point (0-35d) and analyzed for Treg percentage in a flow cytometer, as described by the PI ). Tregs (responder cells) from cecal tonsils from S. enterica-infected and mock-infected chickens will be flow sorted. CD4+CD25- effector cells will be collected from naïve chickens not infected with S. enterica. The suppressive properties of Tregs will be analyzed using the classical "suppression of naïve T cell proliferation" assay at effector:responder cell ratios of 1:1, 0.5:1, 0.25:1, or 0:1, as described by the PI. Tregs will be analyzed for IL-10, TGF-ß, CTLA-4, and IL-2 mRNA by real-time RT-PCR. Chickens will be infected with S. enterica (serovars Heidelberg and Enteritidis). CD8+ cells will be collected using magnetic beads, as described by the PI , and macrophages will be collected, as described by the PI from naïve chickens not infected with S. enterica. The effect of Treg-immune cell interactions will be studied in a transwell model described by the PI earlier, wherein one of the transwells will have Tregs (collected from cecal tonsils of S. enterica- and mock-infected chickens) and the other well will have either CD8+ cells or macrophages, and will be cultured for three days. Macrophages will be analyzed for IL-1, IL-10, IFN', and iNOS mRNA amounts by real-time RT-PCR as described by the PI. Cell supernatant from macrophage culture will be analyzed for nitric oxide production. CD8+ cells will be analyzed for perforin, granzyme, and IFN' mRNA. Chickens will be infected with S. enterica (serovars Heidelberg and Enteritidis), as described in the experimental design section. 1 X 108 CD4+CD25+ cells will be flow sorted from cecal tonsils of infected and control birds, as described by the PI . The PI has earlier collected 1 X 109 to 1011 cells from one cecal tonsil and hence do not expect any problems in collecting enough CD4+CD25+ cells for analysis. For each animal and each treatment, there are three intra-array replicates. The cells will be analyzed for kinome analysis.