Changes in cell migration-related molecules expressed by thymic microenvironment during experimental Plasmodium berghei infection: consequences on thymocyte development

Abstract

We previously showed alterations in the thymus during experimental infection with Plasmodium berghei. Such alterations comprised histological changes, with loss of cortical-medullary limits, and the intrathymic presence of parasites. As the combination of chemokines, adhesion molecules and extracellular matrix (ECM) is critical to appropriate thymocyte development, we analysed the thymic expression of ECM ligands and receptors, as well as chemokines and their respective receptors during the experimental P. berghei infection. Increased expression of ECM components was observed in thymi from infected mice. In contrast, down-regulated surface expression of fibronectin and laminin receptors was observed in thymocytes from these animals. Moreover, in thymi from infected mice there was increased CXCL12 and CXCR4, and a decreased expression of CCL25 and CCR9. An altered thymocyte migration towards ECM elements and chemokines was seen when the thymi from infected mice were analysed. Evaluation of ex vivo migration patterns of CD4/CD8-defined thymocyte subpopulations revealed that double-negative (DN), and CD4(+) and CD8(+) single-positive (SP) cells from P. berghei-infected mice have higher migratory responses compared with controls. Interestingly, increased numbers of DN and SP subpopulations were found in the spleens of infected mice. Overall, we show that the thymic atrophy observed in P. berghei-infected mice is accompanied by thymic microenvironmental changes that comprise altered expression of thymocyte migration-related molecules of the ECM and chemokine protein families, which in turn can alter the thymocyte migration pattern. These thymic disturbances may have consequences for the control of the immune response against this protozoan.

Figure 1

Extracellular matrix density in the thymus. (a) Immunofluorescence staining shows enhancement of fibronectin-relative contents in the thymus from Plasmodium berghei-infected mice compared with the control. Enhanced density was also observed for laminin staining (original magnification, × 100). (b) and (c) The graphics correspond to quantitative analysis of selected microscopic fields of thymi from control and infected mice in terms of fibronectin and laminin, respectively. Results are expressed in pixels per square millimetre and represent the mean ± SE for at least five animals. **P < 0·01, and ***P < 0·001.

Figure 2

Altered expression of fibronectin receptors (VLA4 and VLA5) and laminin receptor (VLA6) in total thymocytes and in CD4/CD8-defined subpopulations (DN: CD4⁻ CD8⁻, DP: CD4⁺ CD8⁺, CD4: CD4⁺ CD8⁻, CD8: CD4⁻ CD8⁺) from control and Plasmodium berghei-infected mice. (a), (c) and (e) Decreased percentage of thymocytes from infected mice that express VLA4, VLA5 and VLA-6, respectively. (b), (d), and (f) Decreased expression of fibronectin receptors VLA4 and VLA5, and laminin receptor VLA6 in thymocytes from infected mice. Results are expressed as mean ± SE for at least five animals. *P < 0·05, **P < 0·01.

Figure 3

Altered expression of chemokines CCL12 and CCL25, and their receptors (CXCR4 and CCR9, respectively) in total thymocytes and in CD4/CD8-defined subpopulations (DN: CD4⁻ CD8⁻, DP: CD4⁺ CD8⁺, CD4: CD4⁺ CD8⁻, CD8: CD4⁻ CD8⁺) from control and Plasmodium berghei-infected mice. (a) and (d) Relative gene expression of CXCL12 and CCL25, respectively, in control and P. berghei-infected mice. (b) and (e) Increased percentage of thymocytes from control and P. berghei-infected mice expressing the chemokine receptors, CXCR4 and CCR9. (c) and (f) Increased CXCR4 and CCR9 fluorescence intensity in thymocytes from control and infected mice. Results are expressed as mean ± SE for at least five animals. *P < 0·05, and **P < 0·001.

Figure 4

Altered thymocyte migration in Plasmodium berghei-infected mice. The graphic shows the numbers of total cells migrating towards fibronectin (FN) and laminin (LN), or CXCL12 and CCL25, as well as the chemokines combined with one given extracellular matrix (ECM) element. Results are expressed as mean ± SE derived from five experiments, each one obtained by a pool of at least five control and 10 infected mice. *P < 0·05 and **P < 0·01.

Figure 5

Absolute cell number of different T-cell subsets in spleens from control and Plasmocium berghei-infected mice. Increased numbers of CD4⁺ and CD8⁺ single-positive (SP) subpopulation, and CD4⁻ CD8⁻ cells (double-negative; DN) can be observed in infected animals. Results are expressed as mean ± SE for at least five animals. *P < 0·05, and ***P < 0·001.