ABSTRACT
Streptococcus suis serotype 2 is an important bacterial pathogen of swine and is also an emerging zoonotic agent that may be harmful to human health. Although the virulence genes of S. suis have been extensively studied, the mechanisms by which they damage the central immune organs have rarely been studied. In the current work, we wanted to uncover more details about the impact and mechanisms of S. suis on specific populations of thymic and immune cells in infected mice. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) assays revealed that S. suis infection induced apoptosis in CD3+, CD14+, and epithelial cells from the thymus. S. suis infection resulted in a rapid depletion of mitochondrial permeability and release of cytochrome c (CytC) and apoptosis-inducing factor (AIF) through upregulation of Bax expression and downregulation of Bcl-xl and Bcl2 expression in thymocytes. Moreover, S. suis infection increased cleavage of caspase-3, caspase-8, and caspase-9. Thus, S. suis induced thymocyte apoptosis through a p53- and caspase-dependent pathway, which led to a decrease of CD3+ cells in the thymus, subsequently decreasing the numbers of CD4+ and CD8+ cells in the peripheral blood. Finally, expression dysregulation of proinflammatory cytokines in the serum, including interleukin 2 (IL-2), IL-6, IL-12 (p70), tumor necrosis factor (TNF), and IL-10, was observed in mice after S. suis type 2 infection. Taken together, these results suggest that S. suis infection can cause atrophy of the thymus and induce apoptosis of thymocytes in mice, thus likely suppressing host immunity.
INTRODUCTION
Streptococcus suis is an important bacterial pathogen responsible for significant economic losses to the global swine industry, and it is also considered to be an emerging zoonotic pathogen (1). In pigs and humans, S. suis infection causes clinical signs that include meningitis, septicemia, arthritis, endocarditis, and pneumonia. In certain human cases, S. suis infection can be fatal due to streptococcal toxic shock syndrome, which is characterized by acute high fever, vascular collapse, hypotension, systemic shock, and multiple organ failure (2). S. suis can be divided into 29 serotypes, the most common being serotype 2, which is also considered to be the most virulent.
Although the mechanisms of virulence of S. suis infection are not fully understood, many virulence factors and stages of infection have been identified (3). To cause disease, S. suis must first colonize the host and then escape the host’s immune system, allowing the infection to persist and disseminate. The polysaccharide capsule plays a central role in protecting S. suis against phagocytosis and also helps it survive within neutrophils, monocytes, macrophages, and dendritic cells (4). Serine proteases secreted by S. suis can degrade chemokines such as CCL5 and interleukin (IL)-8, which are involved in the recruitment of phagocytes, thus preventing chemoattraction of phagocytes to the infection site. In addition, a factor H-binding protein (5), an IgA1 protease (6), and several DNases (7) are also believed to help S. suis escape the host’s defense mechanisms. S. suis can also deregulate the host’s inflammatory response, which must be regulated with precision to prevent harm to the host. S. suis infection may modulate immune responses in the following ways: (i) induction of the high plasma levels of interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), and IL-10 seen during the systemic phase of S. suis infection (8–10); (ii) transient splenic depletion of CD4+ T cells resulting in a poor memory response (11); and (iii) impairment of IL-12 production and the major histocompatibility complex class II (MHC-II)-restricted antigen presentation capacity of dendritic cells (12).
The thymus is a primary immune organ that can generate mature T cells and is therefore essential for peripheral T cell renewal (13). A number of pathogens, including Francisella tularensis, Listeria monocytogenes, Salmonella enterica serovar Typhimurium, Mycobacterium avium, and some parasites and viruses, can target the thymus and alter thymocyte development and export (14). Although thymic atrophy is considered a common feature in diseases caused by these pathogens, the precise underlying mechanisms in distinct diseases are varied (15, 16).
In this study, we wanted to determine the impact and mechanisms of S. suis on the thymus and on specific populations of thymic and immune cells in mice. S. suis type 2 induced thymus atrophy, with an up to 80% reduction in size accompanied by apoptosis of CD3+, CD14+, and thymic epithelial cells. In the infected thymus, the results obtained showed that S. suis triggered caspase-dependent and apoptosis-inducing factor (AIF)-mediated apoptosis in thymic cells in a p53-dependent manner. In addition, the thymus injury led to a dysregulated innate immune response. The new knowledge generated by the current study will help us better understand the mechanism of S. suis pathogenesis.
RESULTS
S. suis infection induces bacteremia and gross thymic atrophy in a mouse model.A total of 35 mice were infected with 5 × 107 CFU of S. suis, and 35 mice were mock infected as controls; 5 infected mice and 5 control mice were sacrificed at each time point postinfection. The infected mice showed intense clinical signs starting at 12 h postinoculation (hpi), including severe lethargy, anorexia, emaciation, rough appearance of hair coat, and swollen eyes. The clinical symptoms were maintained for 5 days postinoculation (dpi), with a gradual return to a normal appearance and stability by 14 dpi, when the experiment was terminated. During the experiment, two infected mice showed extreme lethargy and were humanely euthanized at 4 and 6 dpi, respectively.
Bacteremia induced by S. suis strain 700794 was monitored by counting colonies after plating 5 μl blood on Todd-Hewitt agar (THA). Mice showed high bacteremia, which peaked at 1 dpi with a mean of 2.95 × 106 CFU/ml. The mean titer dropped to 7.86 × 105 CFU/ml at 2 dpi and remained detectable through 14 dpi (data not shown).
The main gross lesions of infected mice were observed in the thymus (Fig. 1). The thymus of infected mice began to appear smaller from 1 dpi, and it showed a 50 to 80% reduction in size relative to control thymus on 2 dpi. From 4 to 7 dpi, infected mice developed serious thymic atrophy, and the normal morphology was not observed. The thymus of one mouse redeveloped from 7 dpi, and the thymi of all mice recovered normal morphology by 14 dpi (Fig. 1).
Pathological observations of the thymi from S. suis-infected and control mice. For comparison, thymi from S. suis-infected and control mice are shown at each time point. Compared to the thymi of control mice, thymi from S. suis-infected mice appeared relatively smaller at 1 dpi and developed serious atrophy from 4 to 7 dpi. From 10 dpi onward, the thymi from infected mice showed recovery. The data used for statistal analysis represent the mean ± standard deviation (SD) of thymus/body weight (mg/g) from 5 or 4 mice at each time point. *, P < 0.05; ***, P < 0.001; compared with control (one-way analysis of variance [ANOVA] and the Tukey’s multiple-comparison test).
S. suis infection caused histopathological lesions and induced apoptosis of thymocytes in a mouse model.The main histopathological lesions observed in the thymus of infected mice were a marked decrease in thymic lobule size, decreased lymphocyte numbers, and disintegration and necrosis of the boundaries of the thymic cortex. In addition, at 12 hpi, there was no significant change in the thymus (Fig. 2B) compared with those of the controls (Fig. 2A). However, the medulla boundary was blurred, with the medulla shrinking or disappearing from 1 to 7 dpi (Fig. 2C to F). Furthermore, after 7 dpi, a normal thymic cortex and medulla reappeared in half (2/4) of the infected mice (Fig. 2G and H). The most important finding here was thymocyte apoptosis and the presence of apoptotic bodies in the atrophied thymi (Fig. 2J).
Histopathological lesions induced in the thymi of mice infected by S. suis. (A to H) Mice were infected with 5 × 107 CFU S. suis, and thymus samples were collected at various times postinfection for histopathological lesion observation. (A) Thymus from control mouse and (B) thymus at 12 hpi, showing no significant changes in number of lymphocytes. The thymus samples had unclear cortical and medulla boundaries, atrophy, and great reduction of lymphocytes at (C) 1 dpi, (D) 2 dpi, and (E) 4 dpi. (F) By 7 dpi, the thymus had only mild lymphocyte reduction, and there were no significant differences in lymphocyte number relative to those of controls at (G) 10 dpi or (H) 14 dpi. Bar, 200 μm (×100). (I and J) Thymus from uninfected control mouse (I) compared to thymus at 1 dpi (J), showing unclear cortical and medulla boundaries (red rectangle), apoptotic bodies (red arrows), and macrophages swallowing apoptotic cells (yellow rectangle). Bar, 50 μm (×400).
The apoptosis induced in the thymus by S. suis infection was further examined by transmission electron microscopy (Fig. 3). Large numbers of apoptotic cells in the thymus and apoptotic bodies undergoing phagocytosis were observed at 1 and 2 dpi. At an ultrastructural level, observed apoptotic bodies consisted of membrane-bound fragments with condensed nuclei and cytoplasm (Fig. 3C to E). These were characterized by highly electron-dense and crescentic nuclear fragments, with nuclear segmentation and peripheral margination of chromatin. However, the regular arrangement of thymocytes began to recover by 7 dpi, and the number of necrotic and apoptotic cells was reduced through 10 dpi. No apoptotic thymocytes were observed at 12 or 14 dpi.
Electron micrographs of thymus showing histopathological lesions induced in mice infected by S. suis. Thymus samples were collected at various times postinfection for electron micrograph observation. (A) Thymus from uninfected mouse and (B) thymus from infected mouse at 12 hpi. At later stages of infection, various lesions, including early apoptotic cells (red arrows), apoptotic bodies (yellow arrows), and necrotic cells (red triangles), were seen, including at (C and D) 1 dpi, (E) 2 dpi, (F) 4 dpi, (G) 7 dpi, and (H) 10 dpi. (I) By 14 dpi, such lesions were no longer evident.
Changes in populations of apoptotic cells in the thymus of infected mice were further investigated using flow cytometry. The early stage of apoptosis involves the translocation of phosphatidylserine to the outer plasma membrane, and annexin V can be used to detect phosphatidylserine on the cell surface. Thus, early apoptotic cells were annexin V-positive/propidium iodide-negative, and late apoptotic or necrotic cells were annexin V-positive/propidium iodide-positive (17). After S. suis infection, at 12 hpi, the proportion of apoptotic thymocytes was significantly higher than that in control mice, including that of the early apoptotic cells (17.4% versus 10.6%; P < 0.05), that of the late apoptotic or necrotic cells (13.9% versus 5.2%; P < 0.01), and that of the total apoptotic cells (31.3% versus 15.8%; P < 0.01). This high proportion of apoptotic thymocytes remained elevated until 7 dpi (Fig. 4).
S. suis induces apoptosis in thymic cells of infected mice. Percentages of cells in (A) early, (B) late, and (C) total apoptosis in the thymi of control and S. suis-infected mice. Each number represents the mean ± SD generated from all mice in each group at the indicated time points. *, P < 0.05; **, P < 0.01; compared with controls (Wilcoxon rank sum test).
S. suis induces caspase-dependent and caspase-independent apoptosis in the thymus of infected mice.Next, we wanted to identify the types of cells that were undergoing apoptosis in the thymus after S. suis infection. Total thymocytes are composed of many T cells at various stages of differentiation during development, and >85% of them express CD3. In addition, thymus epithelial cells and macrophages (CD14+ cells) play an important role in positive and negative selection of thymocytes via major histocompatibility complex (MHC) antigens on the cell surface. These cells constitute the most important cell populations in the thymus (18). Thus, the types of cells undergoing apoptosis were also identified. CD3+ cells, CD14+ cells, and thymic epithelial cells were labeled using spectral fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse CD3+ antibodies, rabbit anti-mouse CD14+ antibodies, or anti-cytokeratin 7 monoclonal antibody (MAb), respectively. Apoptosis was detected using a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) assay, and cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). As shown in Fig. 5, we found that most apoptotic signals colocalized with CD3+ and CD14+ staining (Fig. 5a and b), with only a few apoptotic cells found that were thymic epithelial cells (Fig. 5c).
Identification of apoptotic cells in the thymi of infected mice. Appropriate FITC-conjugated antibodies were used to label (a) CD3+, (b) CD14+, and (c) cytokeratin-7-positive cells. Apoptotic cells (red) were detected by TUNEL assay, and cell nuclei (blue) were stained with 4′,6-diamidino-2-phenylindole (DAPI).
Since activation of caspase-3 is considered to be a biochemical hallmark of apoptosis (19, 20), we confirmed caspase-3 expression in apoptotic thymus cells by confocal microscopy (Fig. 6a). To determine the pathway of apoptosis activation initiated by S. suis, the initiators caspase-8 (Fas/TNF-mediated) and caspase-9 (mitochondrion-mediated) were evaluated. Both were shown to be activated by S. suis infection (Fig. 6b), with significant upregulation observed at 2 dpi, indicating stronger signals of both the mitochondrial and death receptor pathways. Western blot analysis further showed that S. suis dramatically induced the release of cytochrome c (CytC) from the mitochondria to the cytosol (Fig. 6d), again suggesting involvement and enhancement of the mitochondrion-mediated pathway.
S. suis induces caspase- and p53-dependent apoptosis in the thymus. (a) Using confocal laser scanning microscopy, cells were stained for caspase-3 antibody (green). Nuclei were stained by DAPI (blue). Bar, 10 μm. (b) Levels of proteins related to apoptosis in the thymus of mice at 1, 2, and 4 dpi. Thymus tissues were lysed, and Western blots were performed and analyzed digitally, and the optical density ratio was calculated. (c) Using confocal laser scanning microscopy, cells were stained for AIF antibody (red). Nuclei were stained by DAPI (blue). Bar, 10 μm. (d) Levels of cytochrome c (CytC), AIF, Bcl-XL, Bcl2, and Bax proteins in the thymus of mice at 1, 2, and 4 dpi. Thymus tissues were lysed and Western blotting was performed.
Apoptosis-inducing factor (AIF) is considered to be the main molecular effector and mediator of caspase-independent apoptosis (21). In many models of apoptosis, AIF translocates to the nucleus, where it induces chromatin condensation and DNA degradation (22). Western blot analysis showed that S. suis moderately increased the release of AIF from the mitochondria to the cytosol from 1 to 4 dpi (Fig. 6d). To verify the nuclear translocation of AIF, we carried out immunofluorescence staining using an AIF MAb. Fig. 6c shows nuclear localization of AIF signal in apoptotic cells from an infected thymus. Since nuclear translocation of AIF from mitochondria mediates the p53 pathway (23), which can lead to cell growth arrest, senescence, or apoptosis, the expression of p53 was also studied. As shown in Fig. 6b, p53 was activated in the thymus of infected mice, and the amounts of p53 cleavage peaked at 2 dpi. Furthermore, the expression of p53-inducible genes (PIGs) was investigated. Western blot analysis revealed that S. suis infection suppressed the expression of prosurvival Bcl-2 family proteins (Bcl-xl and Bcl-2) while increasing the levels of the proapoptotic Bax (Fig. 6d). These results suggest the involvement of an AIF-mediated p53 pathway in S. suis-induced apoptosis.
S. suis infection increases levels of CD4+ CD8− and CD4− CD8+ cells in the thymus while reducing those of CD4+ CD8+ cells, and reduces levels of CD4+ CD8− and CD4− CD8+ cells in peripheral blood leukocytes of mice.Thymocytes develop from immature CD4− CD8− (double-negative) cells into mature CD4+ and CD8+ cells via a CD4+ CD8+ (double-positive) intermediate. The transitions in the populations of CD4+, CD8+, and CD4+ CD8+ T cells in the thymus following S. suis infection were enumerated using flow cytometry at the designated time points. The most striking finding in this assay was that CD4+ CD8+ cell counts decreased significantly relative to those of the uninfected control mice (P < 0.05) from 12 hpi to 7 dpi. CD4+ CD8+ thymocytes constituted more than 97% of all thymocytes in control mice, but ∼97.8% of them were absent after 2 days of S. suis infection. In contrast, the percentages of CD4+ CD8− (Fig. 7A) and CD4− CD8+ cells (Fig. 7B) were significantly increased (P < 0.05). The most dramatic changes in the proportions of CD8+ and CD4+cells in the thymus were observed at 4 dpi and 12 hpi, respectively. For all of the cell types, no significant differences were observed from 10 dpi to 14 dpi between the infected group and control group. Therefore, S. suis infection impacted all CD3+ subsets in the thymus.
Lymphocyte subset changes in the thymus and the peripheral blood. Flow cytometry was used to determine the percentage of (A) CD4+ CD8− thymocytes, (B) CD4− CD8+ thymocytes, (C) CD4+ CD8+ thymocytes, (D) CD4+ CD8− lymphocytes in peripheral blood leukocytes (PBLs), and (E) CD4− CD8+ lymphocytes in PBLs. Each number represents the mean ± SD generated from all mice at each time point. *, P < 0.05; **, P < 0.01; compared with control (Wilcoxon rank sum test).
To further evaluate the dynamic of changes in T cells of the peripheral blood, peripheral blood leukocytes (PBLs) were analyzed by flow cytometry after S. suis infection. Compared to uninfected controls, the percentage of CD4+ CD8− cells was reduced significantly in infected mice from 1 to 14 dpi (P < 0.05; Fig. 7D), with the exception of that at 10 dpi. S. suis infection also significantly reduced the percentage of CD4+ CD8− cells from 20.6% at 12 hpi to 14% at 1 dpi. In addition, the percentages of CD4− CD8+ cells were significantly lower from 12 hpi to 4 dpi (P < 0.05, Fig. 7E), with the exception of that at 1 dpi. The most dramatic change in the proportion of CD4− CD8+ PBLs was observed at 2 dpi, with ∼6% less than that in control mice.
S. suis infection induces an abnormal serum cytokine dynamic.We used enzyme-linked immunosorbent assay (ELISA) kits to detect the cytokines IL-2, IL-4, IL-6, IL-10, IL-12 (p70), IFN-γ, TNF-α, and TNF-β at each time postinfection. S. suis type 2 infection also altered the expression of cytokines that can modulate innate and adaptive immune responses. As shown in Fig. 8, the levels of serum IL-2, IL-4, IL-6, and TNF-β increased significantly, peaking at 12 hpi (234.50, 9.68, 8.23, and 121.45 pg/ml, respectively) and returning to control levels by 2 dpi, with the exception of that of IL-6. IFN-γ expression was increased significantly at 12 hpi (2,329.24 pg/ml) and reached its peak on 1 dpi (2,510.29 pg/ml). Serum IL-10 and TNF-α were increased slightly at 12 hpi, were significantly higher at 1 dpi, peaked at 2 dpi (136.35 and 852.41 pg/ml, respectively), and finally returned to control levels by 14 dpi (36.37 and 260.69 pg/ml, respectively). No significant differences were observed for IL-12 at any of the time points in the study.
Expression kinetics of inflammatory mediators in C57BL/6 mice infected intraperitoneally (i.p.) with S. suis. ELISA kits were used to detect levels (pg/ml) of IL-2, IL-4, IL-6, IL-10, IL-12 (p70), IFN-γ, TNF-α, and TNF-β at each time postinfection. The red dots represent cytokine levels of control mice, and the green squares represent cytokine levels of infected mice. Results are expressed as means ± SD, and significance was determined using one-way ANOVA and Tukey’s multiple-comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant.
The capabilities to induce thymic atrophy vary among S. suis isolates.To investigate whether all S. suis isolates can cause atrophy of the thymus, we chose different serotypes to infect mice, and the degree of thymic atrophy was qualified using the average ratio of thymus/body weight (mg/g) for each group. As shown in Fig. 1 and 9, the highly virulent isolates 700794 (serotype 2) and BM0806 (serotype 7) induced severe thymic atrophy from 1 dpi, with thymus/body weight ratios of 2.694 (P < 0.001) and 2.012 (P < 0.05), respectively. The ratios decreased to 1.156 and 0.957 at 4 dpi, respectively, which is significantly (P < 0.001) different from that of control mice at the same time point. The moderately virulent isolates HG1210 (serotype 9) and M1302 (serotype 7) induced mild thymic atrophy from 1 dpi to 4 dpi, with thymus/body weight ratios of 3.498 and 2.288 at 4 dpi, respectively, which is lower than that of control mice at the same time point. The nonvirulent isolate W7119 (serotype 9) did not cause any thymic atrophy. Two highly virulent isolates, 700794 (serotype 2) and BM0806 (serotype 7), were further tested in a pig model. The results showed that both isolates could induce thymic atrophy during infection of swine, with thymus/body weight (g/kg) ratios of 0.464 and 0.73 at 5 dpi, respectively, which is significantly different from that of control piglets (Fig. 10).
Pathological observations of the thymus from mice infected with different serotypes of S. suis and control mice. For comparison, thymi from mice infected with S. suis isolates with different serotypes and control mice are shown at each time point. The data used for statistical analysis represent the mean ± SD of the thymus/body weight ratio (mg/g) from 5, 4, or 3 mice at each time point (in the BM0806-infected group, one mouse died after 1 dpi, and two mice died after 2 dpi). *, P < 0.05; ***, P < 0.001; compared with control (one-way ANOVA and Tukey’s multiple-comparison test).
Pathological observations of the thymus from pigs infected with different serotypes of S. suis and control pigs. For comparison, thymi from S. suis isolates with different serotypes (700794 and BM0806) and control pigs are shown at 5 dpi. The data used for statistical analysis represent the mean ± SD of the thymus/body weight ratio (g/kg). ***, P < 0.001; compared with control (one-way ANOVA and Tukey’s multiple-comparison test).
DISCUSSION
The thymus is a primary lymphoid organ responsible for generation of mature T cells and is thus important for both the innate and specific immunity of the host. It is a target of infectious diseases, and thymic atrophy has been reported in a variety of acute infections by both viral and bacterial pathogens. Although the pathogenesis of S. suis has been extensively studied, the mechanisms of thymic atrophy and its role in disease have been largely neglected. In this study, severe atrophy was associated with depletion of immature CD4+ CD8+ thymocytes after S. suis infection of mice. Apoptosis was detected in thymocytes, mediated by both caspase- and AIF-dependent p53 pathways. As a result, CD4+ CD8− and CD4− CD8+ cells were significantly decreased in peripheral blood, and cytokine secretion was greatly altered in the serum. This induction of thymic atrophy appears to be a previously unidentified virulence mechanism of S. suis type 2, probably leading to host immune suppression.
It is well documented that thymic atrophy is mainly due to depletion of immature CD4+ CD8+ thymocytes (14). The mechanisms responsible for such depletion are not completely understood and may vary in distinct diseases. A number of bacterial, viral, and parasitic infections deplete CD4+ CD8+ cells through apoptotic pathways (24). Thymocytes infected with type A Francisella tularensis appear necrotic, with disrupted nuclear and plasma membranes that induce thymocyte death by a mechanism that is distinct from apoptosis (25). In our study, S. suis-induced thymic atrophy was accompanied by thymocyte apoptosis, including that of CD3+, CD14+, and epithelial cells. In addition, CD4+ CD8+ thymocytes were not significantly increased in the peripheral blood, suggesting that immature CD4+ CD8+ thymocytes were not simply transported out of the thymus. These results suggest that apoptosis of thymocytes is very likely the direct cause of this depletion and atrophy. There are several pathways that can induce apoptosis (26), and the caspase-dependent pathway has previously been associated with thymic atrophy during acute infections. Our results are consistent with previous studies that showed that caspase-3, caspase-8, and caspase-9 were activated after S. suis infection in vitro (27, 28). Mitochondria have been shown to play an essential role in cell apoptosis (29). The mitochondrial AIF protein is a protein that triggers chromatin condensation and DNA fragmentation, which thus allow nuclei to undergo apoptotic changes (21). Our Western blot assay results showed that S. suis infection increased the release of AIF from the mitochondria to the cytosol, and immunofluorescence staining demonstrated that AIF colocalized with the host cell’s nucleus. The p53 pathway is mainly mediated by nuclear translocation of AIF from the mitochondria (23), and protein p53 is able to reduce Bcl-2 and promote Bax expression (19). Both Bcl-2 and Bax belong to the Bcl-2 family proteins, which precisely regulate mitochondrion-mediated apoptosis (30). In our study, p53 was activated in the infected thymus, and the cleavage of p53 was also significantly increased. Accordingly, the expression of Bcl-xl and Bcl2 was downregulated, and the expression of Bax was upregulated. These results indicated that the AIF-mediated p53 pathway of apoptosis, which has so far never been associated with pathogen-induced thymic atrophy, was also induced after S. suis infection. It represents a new mechanism for S. suis to induce apoptosis. Altogether, we provide evidence to show that S. suis triggers caspase- and p53-dependent apoptosis in thymocytes with the involvement of mitochondrial dysfunction.
S. suis infection induces a high level of systemic proinflammatory cytokines and chemokines (10). An increased production of proinflammatory mediators has also been associated with pathogen-induced thymic atrophy. IFN-γ contributes to the induction of thymic atrophy and T-cell depletion during viral and bacterial infections, including those caused by severe influenza A (H1N1) pdm09 virus, S. Typhimurium, and M. avium (16, 31, 32). Moreover, IFN-γ has been shown to increase apoptosis of thymocytes in vitro, and neutralization of IFN-γ in mice significantly alleviated thymic atrophy (33). In our current study, IFN-γ expression was significantly elevated from 12 hpi to 2 dpi and remained higher than that in the control group until 4 dpi. In addition, TNF-α is a critical molecular mediator of microbial pathogen-induced thymic involution (25). Our results showed significantly increased expression of TNF-α during the peak of thymic lesions, suggesting that it might also be important for the thymic atrophy in our model.
In total, 80% of the thymocytes in the outer cortex express both CD4 and CD8 molecules. TUNEL detection combined with flow cytometry showed a significant reduction in these cells in infected mice and led to the conclusion that the TUNEL-positive cells colocalized with CD3+ cells were CD4+ CD8+ cells. Apoptosis decreased the number of CD4+ CD8+ cells in the thymus, and consequently the numbers of CD4+ and CD8+ cells, which are key players in the development of host immune responses, were diminished in the peripheral blood. The disordered lymphocyte dynamics may have triggered negative feedback of the cell mediated-immune responses. The relationship between altered lymphocyte dynamics and the abnormal cytokine expression during S. suis infection will be further investigated in future studies.
Our results demonstrated that highly virulent isolates with different serotypes caused severe thymus atrophy, and moderately virulent isolates induced mild thymic atrophy, while nonvirulent isolate could not cause thymic atrophy. These results suggested that the capability of S. suis to induce thymus atrophy is possibly related to the virulence, but not the serotype, of the isolate. The underlying mechanism is undetermined and warrants further investigation.
In conclusion, S. suis induced severe thymic atrophy through induction of thymocyte apoptosis in infected mice. This apoptosis was induced not only by caspase-dependent pathways, but also by the AIF-mediated p53 pathway, resulting in abnormal T-cell populations in the peripheral blood and dysregulation of cytokine expression. Induction of thymic atrophy may lead to host immune depression and appears to be a new virulence mechanism of S. suis.
MATERIALS AND METHODS
Animal ethics statements.This study was carried out in accordance with recommendations from the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of China. The protocols were reviewed and approved by the Committee on the Ethics of Animal Experiments of the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences. Mouse challenge experiments (approval numbers SY-2018-MI-012 and SY-2019-MI-058) and pig challenge experiments (approval number SY-2019-SW-032) with S. suis were conducted within the animal biosafety level 2 facilities in the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences (CAAS).
Bacterial strains and animal tests.The S. suis type 2 strain 700794 used in this study was purchased from the China General Microbiological Culture Collection Center (CGMCC). According to previous studies (11), the strain was grown on sheep blood agar plates, and isolated colonies were used as inoculum for Todd-Hewitt broth (THB; BD Biosciences, Franklin Lakes, NJ); the number of CFU/ml in the final suspension was determined by plating samples onto Todd-Hewitt agar (THA). Seventy 6-week-old female C57BL/6 mice were randomly divided into two groups (35 mice per group), and 5 mice were kept in separate cages in isolated rooms for collection at each time point. Mice were inoculated by intraperitoneal (i.p.) injection with 0.25 ml of either the bacterial suspension (5 × 107 CFU) or the vehicle solution (sterile THB) at 0 days postinoculation (0 dpi). Mice were monitored daily for clinical signs that included depression, rough appearance of hair coat, and swollen eyes. Five mice from each group were humanely euthanized at 12 h postinoculation (hpi) and at 1, 2, 4, 7, 10, and 14 dpi, and thymic lesions were evaluated as thymus/body weight (mg/g) ratios. During the experiment, mice exhibiting extreme lethargy were considered moribund and were humanely euthanized.
To investigate the ability of different S. suis isolates to induce thymus atrophy, we tested five S. suis isolates with different serotypes and different levels of virulence in mice. Among these isolates, BM0806 (serotype 7) and 700794 (serotype 2) are highly virulent, HG1210 (serotype 9) and M1302 (serotype 7) are moderately virulent, and W7119 (serotype 9) is a nonvirulent strain (data not shown). Five mice from each infected group (2 × 107 CFU/mouse; i.p. injection) and five control mice were humanely euthanized at 1, 2, and 4 dpi, and thymic lesions were evaluated as the thymus/body weight (mg/g) ratio. In addition, five 4-week-old S. suis free piglets were infected i.p. with 700794 (serotype 2) or BM0806 (serotype 7) at 2 × 109 CFU/piglet, respectively. Five piglets were used as a control and received THB. These experimental piglets were humanely euthanized at 5 dpi, and thymic lesions were evaluated as the thymus/body weight (g/kg) ratio.
Blood and thymocyte collection.In order to monitor the level of infection, blood and thymocytes were collected at 12 hpi and at 1, 2, 4, 7, 10, and 14 dpi. Blood (5 μl) was collected via venipuncture of the tail vein, serially diluted in phosphate-buffered saline (PBS), and plated on THA. Thymus samples were dissected from each mouse, and thymocyte suspensions were prepared by pressing the tissue through a sieve in ice-cold PBS (34). The collected blood and thymocytes were also processed for flow cytometry analysis.
Histopathology examinations.Samples of the thymus were fixed in 10% phosphate buffered formalin, embedded in paraffin, cut in 2- to 4-μm-thick slices, and stained by hematoxylin and eosin (H&E). Lesions were observed in a blind manner by an experienced professional pathologist.
Electron microscopy.Cubes (∼1 mm3) of tissue from the thymus were fixed with cold 2.5% glutaraldehyde in 0.1 M PBS (pH 7.4) at room temperature and further processed for electron microscopy as described previously (35). Ultrathin sections were examined on a Hitachi H-7650 transmission electron microscope.
Flow cytometry analysis.Anti-mouse CD3ε MAb (C363.29B; Southern Biotech, Birmingham, AL) was conjugated with spectral red (SPRD). Anti-mouse CD4 MAb (GK1.5; Southern Biotech) was conjugated with fluorescein isothiocyanate (FITC). Anti-mouse CD8 MAb (53-6.7, Southern Biotech) was conjugated with R-phycoerythrin (R-PE). For the analysis of changes in the peripheral blood lymphocyte subsets and thymocyte subpopulations, cells were stained with anti-CD3-SPRD, anti-CD4-FITC, and anti-CD8-pE. Then, flow cytometry was used to identify total T cells (CD3+) and the CD4+ CD8− and CD4− CD8+ subpopulations, as previously described (24, 36). Thymic cell apoptosis and necrosis were evaluated by flow cytometry analysis as described previously (37), using the Annexin V-FITC apoptosis detection kit Ι (BD Biosciences) according to the manufacturer’s instructions.
Confocal microscopy.Thymus samples collected at necropsy were sectioned (8-μm-thick slices) on a cryostat and used for double-immunofluorescence staining. To confirm the types of cells that underwent apoptosis, thymus sections were stained with several different antibodies, including spectral FITC-conjugated rabbit anti-mouse CD3 antibody (1:50; Southern Biotech), rabbit anti-mouse CD14 antibody (1:50; MyBioSource, San Diego, CA), rabbit anti-mouse caspase-3 antibody (1:300; Cell Signaling, Danvers, MA), and FITC-conjugated goat anti-rabbit antibody (1:500; Sigma, Saint Louis, MO), and thymus epithelial cells were stained with mouse antibody against cytokeratin 7 (1:50; abcam, Cambridge, UK) and FITC-conjugated goat anti-mouse antibody (1:500; Sigma). Apoptosis was detected using a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) assay according to the instructions provided in the in situ cell death detection kit (Roche, Germany). To confirm that apoptosis-inducing factor (AIF) translocated to the nucleus, thymus sections were stained with rabbit anti-mouse AIF monoclonal antibody (1:50; abcam) and Alexa Fluor 568-conjugated goat anti-rabbit antibody (1:500; Sigma). Finally, nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma). Sections were observed using a laser scanning confocal microscope.
Western blotting.Thymus samples were washed three times with PBS and incubated on ice with cell lysis buffer containing a protease inhibitor cocktail (catalog no. 04693132001; Roche, Bern, Switzerland) and 0.1 mM phenylmethylsulfonyl fluoride (PMSF) for 2 h. Cell lysates were centrifuged at 13,000 × g for 20 min at 4°C, and protein concentration was determined by the Bradford assay (Thermo Fisher Scientific, Waltham, MA). Equal amounts of protein were loaded in 12% (wt/vol) SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes (catalog no. ISEQ00010; Millipore, Billerica, MA). After blocking with 10% dry milk dissolved in PBS with Tween 20 (PBS-T) at 4°C overnight, the membranes were incubated for 2 h at room temperature with the following different antibodies: anti-caspase-3, anti-caspase-8, or anti-caspase-9 (1:500; Cell Signaling), anti-P53 (1:1,000; Sigma), anti-CytC, anti-AIF, or anti-Bcl-2 (1:1,000; Abcam), or anti-β-actin (1:1,000; Sigma). After washing, the membranes were incubated with the appropriate secondary antibodies for 1 h, and immunoreactive bands were visualized using an enhanced chemiluminescence (ECL) system (PerkinElmer Life Sciences, Fremont, CA).
Cytokine analysis.Sera collected at 12 hpi and at 1, 2, 4, 7, 10, and 14 dpi were used for the detection of levels of IL-2, IL-4, IL-6, IL-10, IL-12 (p70), interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and TNF-β using commercial ELISA kits (Cusabio, Inc., Hangzhou, China) according to the manufacturer’s instructions. The amount of cytokines (pg/ml) was calculated according to a standard curve generated from recombinant mouse cytokines supplied in the kits.
Statistical analysis.Numerical data are expressed as the mean ± standard deviation (SD), and were analyzed using Prism software (version 5.02 for Windows; GraphPad Software, Inc.). Differences between groups were assessed using Wilcoxon rank sum tests or one-way analysis of variance (ANOVA) and Tukey’s multiple-comparison test. A P value of less than 0.05 was considered statistically significant.
ACKNOWLEDGMENTS
This work was supported by a Local Social Science Project (no. 2018QD0031) of Heilongjiang Province and the Heilongjiang Pig Modern Agricultural Technology Collaborative Innovation System.
FOOTNOTES
- Received 19 December 2019.
- Accepted 4 January 2020.
- Accepted manuscript posted online 13 January 2020.
- Copyright © 2020 American Society for Microbiology.
