Frequency of Th17 cells correlates with the presence of lung lesions in pigs chronically infected with Actinobacillus pleuropneumoniae

Abstract

Porcine contagious pleuropneumonia caused by Actinobacillus pleuropneumoniae (APP) remains one of the major causes of poor growth performance and respiratory disease in pig herds. While the role of antibodies against APP has been intensely studied, the porcine T cell response remains poorly characterized. To address this, pigs were intranasally infected with APP serotype 2 and euthanized during the acute phase [6-10 days post-infection (dpi)] or the chronic phase of APP infection (27-31 dpi). Lymphocytes isolated from blood, tonsils, lung tissue and tracheobronchial lymph nodes were analyzed by intracellular cytokine staining (ICS) for IL-17A, IL-10 and TNF-α production after in vitro stimulation with crude capsular extract (CCE) of the APP inoculation strain. This was combined with cell surface staining for the expression of CD4, CD8α and TCR-γδ. Clinical records, microbiological investigations and pathological findings confirmed the induction of a subclinical APP infection. ICS-assays revealed the presence of APP-CCE specific CD4⁺CD8α^dim IL-17A-producing T cells in blood and lung tissue in most infected animals during the acute and chronic phase of infection and a minor fraction of these cells co-produced TNF-α. APP-CCE specific IL-17A-producing γδ T cells could not be found and APP-CCE specific IL-10-producing CD4⁺ T cells were present in various organs but only in a few infected animals. The frequency of identified putative Th17 cells (CD4⁺CD8α^dimIL-17A⁺) in lung and blood correlated positively with lung lesion scores and APP-specific antibody titers during the chronic phase. These results suggest a potential role of Th17 cells in the immune pathogenesis of APP infection.

Figure 1

Microbiological investigation, lung pathology, clinical signs and antibody titers of APP-infected pigs. A The presence of APP was investigated at different host locations during necropsy (GM, Glandula mandibularis; TBLN, tracheobronchial lymph node; BALF, bronchoalveolar lavage fluid). Red boxes indicate APP detection by agar isolation, orange boxes indicate APP detection by PCR and orange boxes with red lines indicate positive results by both techniques. Green boxes indicate negative findings for APP. Results shown in the table refer to sampling on the day of euthanasia. The nasal swabs of animals #11, 9, 12, 16 and 17 were tested positive only on day 14 and/or 21 pi. This is indicated by red boxes with §. B Pathology of the lung was assessed by lung lesion score (LLS) for the lung tissue and by slaughterhouse pleurisy evaluation system (SPES) for the pleura. C Rectal temperatures were measured daily in both infected (colored lines) and control (black lines) animals. Body temperature of 40 °C or higher was considered as fever (red line). D Humoral response against APP serotype 2. Data are expressed as a ratio between optical density of the sample (ODs) and the mean of the optical density of the positive control (MODp). Colored lines in the left graph show ratios for infected animals, black lines in the right graph indicate ratios from sera of control animals.

Figure 2

APP-CCE specific IL-17A-producing CD4 ⁺ T cells in lung, blood, tracheobronchial lymph nodes and tonsils. Cells isolated from lung, blood (PBMC), tracheobronchial lymph nodes (TBLN) and tonsils were incubated overnight with APP crude capsular extract (APP-CCE), medium or PMA/Ionomycin. Living lymphocytes were gated (not shown; see Additional file 1) and further analyzed for the expression of IL-17A and CD4. A For the lung, data from representative animals from different groups are displayed: #5 and #8 for the acute phase, designated as “high responder” and “low responder” respectively; #12 and #16 for the chronic phase designated as “high responder” and “low responder” respectively; #6, designated as non-responder and control #23. Approximately 1 × 10⁶ (APP and medium) and 2 × 10⁵ (PMA/Ionomycin) cells are shown in the contour plots. Numbers displayed within the contour plots indicate the percentages of IL-17A⁺ CD4⁺ T cells within total CD4⁺ T cells. B Frequency of IL-17A⁺ CD4⁺ T cells within total CD4⁺ T cells in lung, blood (PBMC), tracheobronchial lymph node (TBLN) and tonsils of all infected animals (red dots) and control animals (blue dots) during acute and chronic phase. Numbers next to colored dots indicate numbers of individual animals. Median percent values are indicated by black bars. Medium-corrected percent values are presented (% of IL-17A⁺ CD4⁺ T cells within total CD4⁺ T cells for APP-CCE stimulation minus % of IL-17A⁺ CD4⁺ T cells within total CD4⁺ T cells for medium incubation).

Figure 3

Expression of CD8α by IL-17A ⁺ CD4 ⁺ T cells in the lung. Cells isolated from lung tissue were incubated overnight with APP crude capsular extract (APP-CCE), medium or PMA/Ionomycin and subsequently analyzed for CD4, CD8α and IL-17A expression by FCM. Living CD4⁺ T cells were gated (not shown; see Additional file 1) and subsequently investigated for expression of CD8α and IL-17A. Data from the same animals as in Figure 2 are shown. Approximately 1 × 10⁵ (APP and medium) and 5 × 10⁴ (PMA/Ionomycin) CD4⁺ T cells are shown in the contour plots.

Figure 4

Co-production of TNF-α and IL-17A by CD4 ⁺ T cells in the lung. Phenotyping and intracellular cytokine staining were performed on cells from lung tissue following overnight in vitro stimulation (APP-CCE, medium, PMA/Ionomycin). A Living CD4⁺ T cells were gated (not shown; see Additional file 1) and further analyzed for production of TNF-α and IL-17A. Data from the same animals as in Figure 2 are shown. Approximately 1 × 10⁵ (APP and medium) and 5 × 10⁴ (PMA/Ionomycin) cells are shown in the contour plots. B Frequency of IL-17A/TNFα co-producing CD4⁺ T cells in lung tissue of infected animals (red dots) and control animals (blue dots) during acute and chronic phase. Numbers next to colored dots indicate numbers of individual animals. Median percent values are indicated by black bars. Medium-corrected percent values are presented (% of IL-17A⁺ TNF-α⁺ cells within total CD4⁺ T cells for APP-CCE stimulation minus  % of IL-17A⁺ TNF-α⁺ cells within total CD4⁺ T cells for medium-incubation). Arrow heads are introduced in the main text.

Figure 5

Production of IL-17A by non-CD4 ⁺ cells and γδ T cells in the lung. Cells isolated from lung tissue were incubated overnight with APP-CCE, medium or PMA/Ionomycin and subsequently analyzed for CD4, TCR-γδ and IL-17A expression by FCM. Living lymphocytes excluding CD4⁺ T cells (not shown; see Additional file 1) were gated and further analyzed for expression of IL-17A and TCR-γδ. Data from the same animals as in Figure 2 are shown. Approximately 1 × 10⁶ (APP and medium) and 2 × 10⁵ (PMA/Ionomycin) cells are shown in the contour plots. Arrow heads are introduced in the main text.

Figure 6

APP-CCE specific IL-10-producing CD4 ⁺ T cells in lung, peripheral blood, tracheobronchial lymph nodes and tonsils. Cells isolated from lung, blood (PBMC), tracheobronchial lymph nodes (TBLN) and tonsils were incubated overnight with APP-CCE, medium or PMA/Ionomycin and subsequently analyzed by FCM. Living cells were gated (not shown; see Additional file 1) and further analyzed for expression of IL-10 and CD4. A For the lung, data from representative animals from different groups are displayed: #5 for the acute phase, designated as “responder”; #17 and #18 for the chronic phase, designated as “outlier” and “responder”, respectively; #6, designated as “non-responder” and control pig #23. Approximately 5 × 10⁵ (APP and medium) and 1.5 × 10⁵ (PMA/Ionomycin) cells are shown in the contour plots. Numbers displayed within the contour plots indicate the percentage of IL-10⁺ CD4⁺ T cells within total CD4⁺ T cells. B Frequency of IL-10⁺ CD4⁺ T cells in lung, PBMC, TBLN and tonsils of infected animals (red dots) and control animals (blue dots) during acute and chronic phase. Numbers next to colored dots indicate numbers of individual animals. Median percent values are indicated by black bars. Medium-corrected percent values are presented (% of IL-10⁺ CD4⁺ T cells within total CD4⁺ T cells for APP-CCE stimulation minus % of IL-10⁺ CD4⁺ T cells within total CD4⁺ T cells for medium incubation).

Figure 7

Correlation of the frequency of IL-17A ⁺ CD4 ⁺ T cells with lung lesion score and antibody titers during the chronic phase. Scatterplots show correlation of the frequency of IL-17A⁺ CD4⁺ T cells isolated from lung and blood with lung lesion score (top panel) and antibody titer (bottom panel) in chronically infected animals. Antibody titer is expressed as a ratio between optical density of the sample (ODs) and the mean of the optical density of the positive control (MODp). Spearman’s Rank Correlation coefficients (ρ) are displayed above each scatterplot.