Cytokine signaling in splenic leukocytes from vaccinated and non-vaccinated chickens after intravenous infection with Salmonella enteritidis

Abstract

In order to design a new Salmonella enterica vaccine, one needs to understand how naive and immune chickens interact differently when exposed to S. enterica. In this study we therefore determined the immune response of vaccinated and non-vaccinated chickens after intravenous infection with Salmonella enterica serovar Enteritidis (S. Enteritidis). Using flow cytometry we showed that 4 days post infection (DPI), counts of CD4 and B-lymphocytes did not change, CD8 and γδ T-lymphocytes decreased and macrophages and heterophils increased in the spleen. When vaccinated and non-vaccinated chickens were compared, only macrophages and heterophils were found in significantly higher counts in the spleens of the non-vaccinated chickens. The non-vaccinated chickens also expressed higher anti-LPS antibodies than the vaccinated chickens. The expression of interleukin (IL)1β, IL6, IL8, IL18, LITAF, IFNγ and iNOS did not exhibit any clear pattern in the cells sorted from the spleens of vaccinated or non-vaccinated chickens. Only IL17 and IL22 showed a differential expression in the CD4 T-lymphocytes of the vaccinated and non-vaccinated chickens at 4 DPI, both being expressed at a higher level in the non-vaccinated chickens. Due to a similar IFNγ expression in the CD4 T-lymphocytes in both the vaccinated and non-vaccinated chickens, and a variable IL17 expression oscillating around IFNγ expression levels, the IL17∶IFNγ ratio in CD4 T-lymphocytes was found to be central for the outcome of the immune response. When IL17 was expressed at higher levels than IFNγ in the non-vaccinated chickens, the Th17 immune response with a higher macrophage and heterophil infiltration in the spleen dominated. However, when the expression of IL17 was lower than that of IFNγ as in the vaccinated chickens, the Th1 response with a higher resistance to S. Enteritidis infection dominated.

Figure 1. Intravenous infection of chickens with S. Enteritidis.

Panel A, S. Enteritidis counts in the spleens of vaccinated and non-vaccinated chickens, 4 and 14 days after intravenous challenge, respectively. Panel B, serological response to the infection. nv-ni, non-vaccinated and non-infected chickens; v-i-4, vaccinated and infected and 4 days post challenge; nv-i-4, non-vaccinated and infected and 4 days post challenge etc. Table below – t-test comparison of biological relevant groups, ni, non-infected chickens; vi4, vaccinated and infected and 4 days post challenge; ni4, non-vaccinated and infected and 4 days post challenge etc. ns – non-significant difference, * P<0.05, ** P<0.01, *** P<0.001.

Figure 2. Gating strategy for the characterization of cellular infiltrates in the spleen and for the sorting of leukocyte subpopulations.

The leukocytes were gated based on the CD45 expression (P1) and only CD45+ cells were included in the sorting and analyses. Sorted populations were followed: CD4 T-lymphocytes (P4+P3), CD8+ T-lymphocytes (P5), γδ T-lymphocytes (P6), monocytes/macrophages (P7), B-lymphocytes (P8) and heterophils (P9).

Figure 3. Relative representation of leukocyte subpopulations in the spleens of infected chickens 4 and 14 days post infection.

Light blue columns - vaccinated and infected chickens 14 DPI, blue columns vaccinated and infected chickens 4 DPI, green columns – non-infected chickens, red columns non-vaccinated and infected chickens 4 DPI, pink columns - non-vaccinated and infected chickens 14 DPI. Table below – t-test comparison of biologically relevant groups, ni, non-infected chickens; vi4, vaccinated and infected and 4 days post challenge; ni4, non-vaccinated and infected and 4 days post challenge etc. ns – non-significant difference, * P<0.05, ** P<0.01, *** P<0.001.

Figure 4. Expression of IL1, IL6, IL8 and IL18 in sorted splenic leukocytes after intravenous S. Enteritidis challenge.

Light blue columns - vaccinated and infected chickens 14 DPI, blue columns vaccinated and infected chickens 4 DPI, green columns – non-infected chickens, red columns non-vaccinated and infected chickens 4 DPI, pink columns - non-vaccinated and infected chickens 14 DPI. Table below – t-test comparison of biologically relevant groups, ni, non-infected chickens; vi4, vaccinated and infected and 4 days post challenge; ni4, non-vaccinated and infected and 4 days post challenge etc. ns – non-significant difference, * P<0.05, ** P<0.01, *** P<0.001.

Figure 5. Cytokine expression of LITAF and iNOS in sorted splenic leukocytes after intravenous S. Enteritidis challenge.

Light blue columns - vaccinated and infected chickens 14 DPI, blue columns vaccinated and infected chickens 4 DPI, green columns – non-infected chickens, red columns non-vaccinated and infected chickens 4 DPI, pink columns - non-vaccinated and infected chickens 14 DPI. Table below – t-test comparison of biologically relevant groups, ni, non-infected chickens; vi4, vaccinated and infected and 4 days post challenge; ni4, non-vaccinated and infected and 4 days post challenge etc. ns – non-significant difference, * P<0.05, ** P<0.01, *** P<0.001.

Figure 6. Expression of IFNγ, IL17 and IL22 in sorted splenic leukocytes after intravenous S. Enteritidis challenge.

Light blue columns - vaccinated and infected chickens 14 DPI, blue columns vaccinated and infected chickens 4 DPI, green columns – non-infected chickens, red columns non-vaccinated and infected chickens 4 DPI, pink columns - non-vaccinated and infected chickens 14 DPI. Table below – t-test comparison of biologically relevant groups, ni, non-infected chickens; vi4, vaccinated and infected and 4 days post challenge; ni4, non-vaccinated and infected & 4 days post challenge etc. ns – non-significant difference, * P<0.05, ** P<0.01, *** P<0.001.

Figure 7. Transcription of IFNγ and IL17 in T-lymphocytes of vaccinated or non-vaccinated chickens 4 days post intravenous challenge.

A, B, C - individual chickens vaccinated or non-vaccinated chickens. The most right panel shows polarization towards IFNγ or IL17 transcription ratio in CD4 T-lymphocytes of vaccinated and non-vaccinated chickens, respectively. Squares, transcription of IL17 and triangles, transcription of IFNγ.