Thymic function is maintained during Salmonella-induced atrophy and recovery

Abstract

Thymic atrophy is a frequent consequence of infection with bacteria, viruses, and parasites and is considered a common virulence trait between pathogens. Multiple reasons have been proposed to explain this atrophy, including premature egress of immature thymocytes, increased apoptosis, or thymic shutdown to prevent tolerance to the pathogen from developing. The severe loss in thymic cell number can reflect an equally dramatic reduction in thymic output, potentially reducing peripheral T cell numbers. In this study, we examine the relationship between systemic Salmonella infection and thymic function. During infection, naive T cell numbers in peripheral lymphoid organs increase. Nevertheless, this occurs despite a pronounced thymic atrophy caused by viable bacteria, with a peak 50-fold reduction in thymocyte numbers. Thymic atrophy is not dependent upon homeostatic feedback from peripheral T cells or on regulation of endogenous glucocorticoids, as demonstrated by infection of genetically altered mice. Once bacterial numbers fall, thymocyte numbers recover, and this is associated with increases in the proportion and proliferation of early thymic progenitors. During atrophy, thymic T cell maturation is maintained, and single-joint TCR rearrangement excision circle analysis reveals there is only a modest fall in recent CD4(+) thymic emigrants in secondary lymphoid tissues. Thus, thymic atrophy does not necessarily result in a matching dysfunctional T cell output, and thymic homeostasis can constantly adjust to systemic infection to ensure that naive T cell output is maintained.

FIGURE 1. Infection with STm induces thymic atrophy.

Mice were infected with 5×10⁵ STm i.p. and responses evaluated at discrete times after infection. A, Bacterial burdens in the spleen and thymus. B, Spleen mass and total cellularity were determined (left) and numbers of total (top right) and naive (CD62L^hi; bottom right) CD4⁺ and CD8⁺ T cells were enumerated. C, Thymic mass and total cellularity before and at defined times after infection. D, Apoptosis in the CD45⁺ thymocyte population determined by incorporation of the dead cell stain, Sytox Blue, by flow cytometry. Graphs in A-C are representative of 3 independent timecourses, and show mean ± SD (n ≥ 4 mice per group). Graphs in D are representative of two independent timecourses, and show mean ± SD (n=4). **p ≤ 0.001; *p ≤ 0.05 or NS compared to non-infected (n.i.) controls. n.d. = not detectable.

FIGURE 2. Thymic atrophy requires systemic dissemination of live bacteria.

A, Mice were immunized for 7 d with 5×10⁵ heat killed (H.K. STm) or live STm, and thymus cellularity and mass assessed. B, Mice were infected i.p. for 24 h with either PBS, 5×10⁵ attenuated STm strain SL3261 (as used in all other experiments) or 5×10⁵ virulent SL1344 STm. Induction of thymic atrophy was assessed by measuring thymic mass, cellularity and levels of apoptosis in thymocytes (as described in Fig. 1D). C, Mice were infected s.c. in the foot pad with 5×10⁵ STm and contralateral foot with PBS and popliteal LN and thymic mass recorded as well as was thymic cellularity. D, Thymic mass and cellularity in mice infected with STm i.p. or i.v. for 7 d. Graphs show mean ± SD of 4 mice per group and are representative of data from at least two independent experiments; *p≤ 0.05 or NS compared to PBS or non-infected (n.i.) controls.

FIGURE 3. Effects of STm infection on distinct thymocyte populations.

Mice were infected with 5×10⁵ STm i.p. and responses evaluated at discrete times after infection. A, Total numbers of thymocytes in the four major thymocyte populations (based on the expression of CD4, CD8 and TCRβ) were quantified by flow cytometry (CD8SP were gated on TCRβ^hi cells to exclude DN cells maturing to the DP stage). B, Thymocyte numbers in the four DN subsets (DN1-4) were defined using CD44 and CD25 and quantified by flow cytometry. C, Apoptosis in the four major thymocyte subsets after infection was quantified by Sytox Blue uptake and flow cytometry. D, The effect of STm infection on CD4SP and CD8SP maturation was quantified by flow cytometry using markers to distinguish total numbers of immature (Qa2^loCD69^hiHSA^hiCD62L^lo) and mature (Qa2^hiCD69^loHSA^loCD62L^hi) cells per thymus. E, The proportion of immature and mature cells in the total CD4SP pool per thymus was assessed. Graphs show mean ± SD of at least 4 mice per group (* p ≤ 0.05 or NS compared to non-infected (n.i.) controls) and are representative of data from three independent timecourses in (A-C) and two in (D + E).

FIGURE 4. Thymic architecture is maintained during STm induced thymic atrophy.

Frozen sections of thymuses from non-infected and mice infected i.p. with 5×10⁵ STm for discrete times were stained to identify the distinct areas within the thymus. A, H&E stains define cortical (light areas) and medullary regions (dark areas). B, Sections were stained by immunofluorescence using anti-β5T (blue) to identify cortical epithelial cells and anti-ERTR5 to identify medullary (red) epithelial cells and analysed by confocal microscopy. C, Sections were stained by immunofluorescence to identify mature, single positive thymocytes were labelled using anti-CD4 (red) and anti-CD8 (green) antibodies, with double positive thymocytes stained in yellow. In A-C, images shown are representative of at least 4 individual mice per time-point. Scale bars shown in A-C represent 100μm.

FIGURE 5. Thymic output is maintained during the immune response to STm.

Mice were infected with 5×10⁵ STm i.p. and responses evaluated at discrete times after infection. CD4 T cells were purified by cell sorting from secondary lymphoid tissues at the times described, and the number of RTEs containing sjTREC sequences was quantified by real time PCR. Graphs show the total number of sjTREC⁺ CD4 T cells per tissue (top), and the proportion of sjTREC⁺ cells per 10⁶ CD4 T cells (bottom). Graphs shown are mean ± SD of 4 mice per group (* p ≤ 0.05 or NS compared to non-infected (n.i.) controls), and are representative of at least two independent time-course experiments.

FIGURE 6. Infection does not ablate numbers of ETPs.

A, ETPs were quantified by flow cytometry using the gating strategy shown, and total numbers of ETPs in the thymus (left), and the proportion (right) of ETPs relative to the DN1 subset during infection are shown. B, The percentage of ETPs in cycle at 14 d post infection as assessed by BrdU incorporation and flow cytometry. C, ETPs were purified by cell sorting from STm experienced (10 d post infection) or control (PBS immunised) mice and co-cultured in vitro with OP9-DL1 stromal cells either alone or mixed together at 1:1 ratio. After culturing for 14 days, expansion of CD45+ cells was enumerated by flow cytometry. D, CCR9KO and CCL25KO mice were infected i.p. with 5×10⁵ STm and thymic mass and cellularity assessed at 14 and 35 d post infection. Graphs shown in A-B and D are mean ± SD (n=4 mice per group), and are representative of at least two independent time-course experiments. Data shown in C is combined from two independent experiments. *p ≤ 0.05 or NS compared to PBS or non-infected (n.i.) controls.