The lymphotoxin pathway regulates Aire-independent expression of ectopic genes and chemokines in thymic stromal cells - PubMed (original) (raw)

The lymphotoxin pathway regulates Aire-independent expression of ectopic genes and chemokines in thymic stromal cells

Natalie Seach et al. J Immunol. 2008.

Abstract

Medullary thymic epithelial cells (mTEC) play an important and unique role in central tolerance, expressing tissue-restricted Ags (TRA) which delete thymocytes autoreactive to peripheral organs. Since deficiencies in this cell type or activity can lead to devastating autoimmune diseases, it is important to understand the factors which regulate mTEC differentiation and function. Lymphotoxin (LT) ligands and the LTbetaR have been recently shown to be important regulators of mTEC biology; however, the precise role of this pathway in the thymus is not clear. In this study, we have investigated the impact of this signaling pathway in greater detail, focusing not only on mTEC but also on other thymic stromal cell subsets. LTbetaR expression was found in all TEC subsets, but the highest levels were detected in MTS-15(+) thymic fibroblasts. Rather than directing the expression of the autoimmune regulator Aire in mTEC, we found LTbetaR signals were important for TRA expression in a distinct population of mTEC characterized by low levels of MHC class II (mTEC(low)), as well as maintenance of MTS-15(+) fibroblasts. In addition, thymic stromal cell subsets from LT-deficient mice exhibit defects in chemokine production similar to that found in peripheral lymphoid organs of Lta(-/-) and Ltbr(-/-) mice. Thus, we propose a broader role for LTalpha1beta2-LTbetaR signaling in the maintenance of the thymic microenvironments, specifically by regulating TRA and chemokine expression in mTEC(low) for efficient induction of central tolerance.

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Figures

FIGURE 1

FIGURE 1

Expression of LT_β_R and its ligands on thymic cell populations. A, Thymic sections from C57BL/6 mice stained with anti-LT_β_R or isotype control Abs (green). B, Flow cytometric analysis of LT_β_R expression (solid line) compared with isotype (dashed line), on CD45− stroma gated on MHCIIhigh (hi), low (lo), or negative (−) cells, as quantified by the numerical ratio of LT_β_R to isotype control median expression levels for each gated population. C, PCR of LTα, LTβ, LIGHT, and LTβR expression in purified thymic cell populations (see Table II), relative to whole TSC expression, standardized to 1. Mean and SE were determined from two to three experiments for each population.

FIGURE 2

FIGURE 2

Reduced UEA-1 but not Aire expression in the thymic medulla of _Lta_−/− and _Ltb_−/− mice. Thymic sections from _Lta_−/−, _Ltb_−/−, and control mice were stained for medullary epithelial markers UEA-1 (A; green), Aire (B; red), and merge of UEA-1/Aire and pan epithelial marker keratin (C; blue), including higher magnification (second row). Images are representative of three to four experiments.

FIGURE 3

FIGURE 3

Flow cytometric analysis of TSC subsets in Lta_−/− and Ltb_−/− mice. A, UEA-1 expression on CD45−, EpCAM+ TEC with regions gating UEA-1high and UEA-1low subsets. B, UEA-1 expression on CD45−MHCII+ TEC with regions gating MHChighUEA-1high and MHClow UEA-1high TEC subsets. C, Expression of Ly-51 and UEA-1 positively identify cTEC and mTEC populations, respectively, on CD45−EpCAM+ TEC. D, UEA-1 and Aire expression on CD45−EpCAM+ TEC with regions gating UEA1highAire+ and UEA1highAire− populations. E, Regions show MTS-15+PDGFR_α+ and MTS15−PDGFR_α+ thymic fibroblast populations, gated on CD45− TSC. Dot plots are representative of 6–10 individual thymic digests. F, Enumeration of TSC populations, defined in Table II, in _Lta_−/−, _Ltb_−/− and control mice. Mean and SE were generated from two experiments each using five individual thymus digestions per group. *, p < 0.05.

FIGURE 4

FIGURE 4

TRA expression in _Lta_−/− and _Ltb_−/− TEC subsets. A, Dot plots gated on CD45−MHCII + TEC showing regions used for sorting of mTEChigh, mTEClow, and cTEC populations for control and _Lta_−/− mice. B, PCR analysis of: Aire, casein α (Csnα), casein γ (Csng), insulin 2 (Ins2), salivary protein 1 (Sp1), casein β (Csn β), casein κ (Csnk), glutamic acid decarboxylase 1 (GAD1), fatty acid-binding protein 9 (Fabp9), type 2 collagen (Col2), C-reactive protein (CRP), thyroglobulin (Tgn), and keratin 14 (K14) in control TEC subsets, relative to highest expression level, standardized to 1. C–F, PCR analysis of TRA expression in _Lta_−/− mTEChigh (C), _Lta_−/− mTEClow (D), _Ltb_−/− mTEChigh (E), and _Ltb_−/− mTEClow (F). Fold change in transcript levels are shown relative to age-matched controls, standardized to 1 (dashed line). Means and SE generated were from three to four experiments for each population.

FIGURE 5

FIGURE 5

Chemokine and cytokine expression in _Lta_−/− and _Ltb_−/− TSC populations. PCR analysis of chemokine and cytokine transcription in _Lta_−/− mTEChigh (A), _Lta_−/− cTEC (B), _Lta_−/− mTEClow (C), _Lta_−/− non-TEC (D), _Ltb_−/− mTEChigh (E), and _Ltb_−/− mTEClow (F). Transcript levels are shown relative to age-matched wt coda standardized to 1 (dashed line). Means and SE were generated from three to four different experiments for each populations.

FIGURE 6

FIGURE 6

Direct stimulation of LT_β_R restores mTEClow defects in _Lta_−/− mice. _Lta_−/− mice were injected with a LT_β_R agonist (LT_β_R-ag) and changes in the spleen and thymic stroma were assessed 8 h later. Whole wt spleen (A), _Lta_−/− mTEChigh (B), and _Lta_−/− mTEClow (C) subsets with fold changes for each are shown relative to _Lta_−/− mice injected with isotype control Ab, standardized to 1 (dashed line). Means and SE generated from two experiments. D, Flow cytometric analysis of UEA-1 binding in _Lta_−/− mTEClow showing the percentage of UEA1highmTEClow cells after a one-time LT_β_R injection (8 h later) or three-time daily LT_β_R-ag injections compared with injection with isotype control (isotype-Ig).

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