Chagasic Thymic Atrophy Does Not Affect Negative Selection but Results in the Export of Activated CD4+CD8+ T Cells in Severe Forms of Human Disease

Extrathymic CD4+CD8+ double-positive (DP) T cells are increased in some pathophysiological conditions, including infectious diseases. In the murine model of Chagas disease, it has been shown that the protozoan parasite Trypanosoma cruzi is able to target the thymus and induce alterations of the thymic microenvironment and the lymphoid compartment. In the acute phase, this results in a severe atrophy of the organ and early release of DP cells into the periphery. To date, the effect of the changes promoted by the parasite infection on thymic central tolerance has remained elusive. Herein we show that the intrathymic key elements that are necessary to promote the negative selection of thymocytes undergoing maturation during the thymopoiesis remains functional during the acute chagasic thymic atrophy. Intrathymic expression of the autoimmune regulator factor (Aire) and tissue-restricted antigen (TRA) genes is normal. In addition, the expression of the proapoptotic Bim protein in thymocytes was not changed, revealing that the parasite infection-induced thymus atrophy has no effect on these marker genes necessary to promote clonal deletion of T cells. In a chicken egg ovalbumin (OVA)-specific T-cell receptor (TCR) transgenic system, the administration of OVA peptide into infected mice with thymic atrophy promoted OVA-specific thymocyte apoptosis, further indicating normal negative selection process during the infection. Yet, although the intrathymic checkpoints necessary for thymic negative selection are present in the acute phase of Chagas disease, we found that the DP cells released into the periphery acquire an activated phenotype similar to what is described for activated effector or memory single-positive T cells. Most interestingly, we also demonstrate that increased percentages of peripheral blood subset of DP cells exhibiting an activated HLA-DR+ phenotype are associated with severe cardiac forms of human chronic Chagas disease. These cells may contribute to the immunopathological events seen in the Chagas disease

Ultrastructure of medullary thymic epithelial cells of autoimmune regulator (Aire)-deficient mice

The significance of the autoimmune regulator (Aire) transcription regulator in establishing central tolerance has recently been elucidated in great detail. Still, the role of Aire in medullary thymic epithelial cell (mTEC) physiology is not fully understood. To shed more light on this issue, we studied the ultrastructure of mTECs in Aire-deficient thymus. We show that all types of mTECs show ultrastructural signs of activation and increased intracellular traffic, which suggests that in the absence of Aire their physiology is impaired. Type 6 ‘large’ mTECs are fully developed in Aire-deficient mice and more frequent than in the normal thymus. The frequency of type 5 ‘undifferentiated’ mTECs is also increased. Collectively, our results suggest that the role of Aire in the physiology of mTECs could be more profound and not restricted only to the presentation of self-tissue-restricted antigens and/or apoptosis of end-stage fully mature cell types.Živana Milićević, Novica M. Milićević, Martti Laan, Pärt Peterson, Kai Kisand, Hamish S. Scott and Jürgen Westerman

Lipopolysaccharide induces tumor necrosis factor receptor-1 independent relocation of lymphocytes from the red pulp of the mouse spleen

It is well known that bacterial lipopolysaccharide (LPS) induces migration of several cellular populations within the spleen. However, there are no data about the impact of LPS on B and T lymphocytes present in the red pulp. Therefore, we used an experimental model in which we tested the effects of intravenously injected LPS on the molecular, cellular and structural changes of the spleen, with special reference to the red pulp lymphocytes. We discovered that LPS induced a massive relocation of B and T lymphocytes from the splenic red pulp, which was independent of the tumor necrosis factor receptor-1 signaling axis. Early after LPS treatment, quantitative real-time PCR analysis revealed the elevated levels of mRNA encoding numerous chemokines and proinflammatory cytokines (XCL1, CXCL9, CXCL10, CCL3, CCL4, CCL5, CCL17, CCL20, CCL22, TNFα and LTα) which affect the navigation and activities of B and T lymphocytes in the lymphoid tissues. An extreme increase in mRNA levels for CCL20 was detected in the white pulp of the LPS-treated mice. The CCL20-expressing cells were localized in the PALS. Some smaller CCL20-expressing cells were evenly dispersed in the B cell zone. Thus, our study provides new knowledge of how microbial products could be involved in shaping the structure of lymphatic organs

LTβR dependent growth suppressive activity also affects fully developed splenic tissue.

<p>(A) WT recipients without prior splenectomy were either sham operated or received WT or LTβR^-/- splenic implants. Eight weeks later weight (left side) and cell number (right side) of the endogenous spleen was determined. Only WT splenic regenerates significantly reduced weight and cell number of the endogenous WT spleen. Indicated are means and standard deviation (n = 4–11, * = p < 0.05, ** = p < 0.01). (B) LTβR^-/- recipients without prior splenectomy were either sham operated or received WT or LTβR^-/- splenic implants. Eight weeks later weight (left side) and cell number (right side) of the endogenous spleen was determined. Only WT splenic regenerates significantly reduced weight and cell number of the endogenous LTβR^-/- deficient spleen. Indicated are means and standard deviation (n = 5–8, ** = p < 0.01). These experiments were 2 times independently performed.</p

LTβR expression by non-splenic tissues suppresses growth of regenerating splenic tissue.

<p>(A) Macroscopic appearance of splenic regenerates 8 weeks after implantation of wild-type splenic tissue (WT) into wild-type recipients (WT), LTβR deficient splenic tissue (LTβR^-/-) into WT recipients, and WT splenic tissue into LTβR deficient recipients (LTβR^-/-). (B) Weight (left side) and cell number (right side) of splenic regenerates. Indicated are means and standard deviation (n = 4–9, ** = p < 0.01). (C) Microscopic appearance of splenic regenerates. Cryostat sections were stained by immunohistochemistry for T cells (brown; TCRβ⁺) and B cells (blue; B220⁺). Red pulp (RP), T-cell zone (T), and B-cell zone (B) are well developed except in the LTβR^-/- into WT combination where T and B cells are intermixed and only the red pulp (RP) is clearly recognizable (bar: 100 μm). This experiment was 3 times independently performed.</p

Architecture and cellular structure of WT spleen and WT into LTβR^-/- splenic regenerates are comparable.

<p>(A) The compartment sizes of normal WT spleens were analyzed and compared to that of splenic regenerates (WT→LTβR^-/-) 8 weeks after implantation. Indicated is the size of individual splenic zones as percent of total section area (n = 6–7 animals per group; RP: red pulp, MZ: marginal zone, WP: white pulp, T: T-cell zone, B: B-cell zone, T/B: mixed T/B-cell zone). (B) Frequency of leukocyte subsets in normal WT spleens and splenic regenerates (WT→LTβR^-/-) as determined by flow cytometry (n = 5–8 animals per group; B: B cells, IgD^- B: memory B cells, T: T cells; CD8: CD8⁺ T cells, CD4: CD4⁺ T cells, CD25: CD25⁺CD4⁺ -regulatory- T cells). (C) Expression of cytokines and chemokines in normal WT spleens and splenic regenerates (WT→ LTβR^-/-). Shown is the relative expression level normalized to the housekeeping gene (MLN 51) and relative to WT (fold expression change). The bars represent the mean values ± SD. Mann-Whitney <i>U</i> test was used to indicate a significant difference between WT spleens and splenic regenerates (*p < 0.05; ***p < 0.001).</p

MFAP4 does not suppress the regeneration of splenic implants.

<p>(A) WT and MFAP4 deficient recipients were splenectomized and then received a WT splenic implant. Eight weeks later the weight of the splenic regenerate was determined. No difference was seen between WT and MFAP4 deficient recipients. Indicated are means and standard deviation (n = 6). (B) WT and MFAP4 deficient recipients without prior splenectomy received a WT splenic implant. Eight weeks later the weight of the splenic regenerate was determined. No difference was seen between WT and MFAP4 deficient recipients. In addition, the suppressive activity of the endogenous spleen was similar in both groups (compare weights to the weights under A). Indicated are means and standard deviation (n = 6). These experiments were 2 times independently performed.</p