Alterations in Thymocyte Populations Under Conditions of Endotoxin Tolerance

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Alterations in Thymocyte Populations Under Conditions of Endotoxin Tolerance

Endotoxin tolerance (ET) is a protective mechanism in which prior exposure to low doses of lipopolysaccharide (LPS) reduces the severity of subsequent inflammatory responses to high-dose LPS challenges. While peripheral T cells are known to play a role in ET induction, the impact of ET on thymocyte development remains poorly understood. This study systematically investigates dynamic changes in thymocyte populations, T-cell receptor (TCR) diversity, and associated molecular mechanisms during LPS-induced ET in mice.

Thymic Atrophy and Recovery During ET

A murine ET model was established by intraperitoneal injection of LPS (5 mg/kg). Thymus weight and total cellularity decreased significantly at 72 hours post-LPS injection (34.0 ± 4.9 mg vs. 16.0 ± 3.8 mg; P < 0.01; Figure 1C) but partially recovered by day 8 (24.0 ± 3.8 mg; P < 0.01 vs. control). Similarly, total thymocyte counts dropped from 37.2 ± 8.2 × 10⁶ cells in controls to 6.0 ± 1.5 × 10⁶ cells at 72 hours (P < 0.01), recovering to 22.3 ± 7.9 × 10⁶ cells by day 8 (P < 0.01 vs. control; Figure 1D). Histological analysis revealed disrupted cortical-medullary boundaries at 72 hours, with lymphocyte depletion and scattered Hassall’s corpuscles, followed by structural restoration by day 8 (Figure 1E).

Dramatic Shifts in Thymocyte Subsets

Flow cytometry revealed profound changes in thymocyte composition. Double-positive (CD4+CD8+ DP) cells, which constituted 72.1% ± 4.1% of thymocytes in controls, plummeted to 10.6% ± 3.5% at 72 hours (P < 0.01) but rebounded to 84.8% ± 2.2% by day 8 (P < 0.05 vs. control; Figure 2B). Conversely, single-positive (SP) populations exhibited inverse trends: CD4+ SP cells increased from 15.8% ± 4.4% (control) to 44.7% ± 3.1% at 72 hours (P < 0.01), then dropped to 6.4% ± 0.5% by day 8 (P < 0.01 vs. 72-hour). CD8+ SP cells followed a similar pattern, rising from 7.0% ± 1.9% to 34.0% ± 3.9% (P < 0.01) at 72 hours before declining to 5.1% ± 0.6% (P < 0.01; Figure 2B). Absolute cell counts mirrored these changes: DP cells decreased from 30.0 ± 7.1 × 10⁶ to 0.6 ± 0.2 × 10⁶ at 72 hours (P < 0.01), recovering to 18.9 ± 0.5 × 10⁶ by day 8. SP subsets showed sustained depletion, with CD4+ SP counts falling from 5.8 ± 1.6 × 10⁶ (control) to 2.8 ± 0.1 × 10⁶ at 72 hours (P < 0.05), then to 1.4 ± 0.1 × 10⁶ by day 8 (P < 0.01 vs. 72-hour; Figure 2C).

TCR β-Chain CDR3 Diversity Remodeling

Capillary electrophoresis of TCR β-chain complementarity-determining region 3 (CDR3) revealed dynamic repertoire changes. Control thymocytes displayed Gaussian CDR3 length distributions spanning 4 amino acids (Supplementary Table 1). At 72 hours, 45% of TRBV families exhibited oligoclonal peaks with reduced polymorphism (2–3 amino acid spans). By day 8, 80% of families showed restored polyclonality with extended CDR3 lengths (5–6 amino acids; Figure 3A). Heatmap analysis confirmed reduced CDR3 diversity at 72 hours and partial recovery by day 8 (Figure 3B).

Altered Apoptosis, Proliferation, and Activation

Early ET (72 hours) reduced apoptosis in SP cells: CD4+ SP apoptosis decreased from 3.0% ± 0.4% (control) to 1.7% ± 0.1% (P < 0.01), while CD8+ SP apoptosis dropped from 4.0% ± 0.8% to 1.1% ± 0.6% (P < 0.01; Figure 4B). Conversely, day 8 saw increased CD8+ SP apoptosis (8.9% ± 0.7%; P < 0.01 vs. control) and reduced DP apoptosis (0.5% ± 0.2% vs. 1.8% ± 0.2% control; P < 0.01). Proliferation (Ki-67+ cells) was suppressed across all subsets at 72 hours: CD4+ SP proliferation fell from 7.0% ± 1.9% to 2.3% ± 1.7% (P < 0.05), CD8+ SP from 6.7% ± 0.1% to 2.6% ± 1.4% (P < 0.01), and DP cells from 4.6% ± 1.0% to 2.3% ± 1.2% (P = 0.054; Figure 4E).

Activation markers showed subset-specific changes. DP cells at 72 hours exhibited elevated CD44 (13.0% ± 3.6% vs. 2.0% ± 0.9% control; P < 0.01) and CD69 (19.2% ± 5.5% vs. 6.3% ± 1.6%; P < 0.05), which normalized by day 8 (Figure 4F–H). CD4+ SP cells displayed increased CD62L (81.5% ± 1.4% vs. 52.3% ± 0.3% control; P < 0.01) at 72 hours, indicating enhanced maturation.

Cytokine Production and ERK Signaling

Interleukin-4 (IL-4) secretion increased in DP cells (1.97% ± 0.06% vs. 0.33% ± 0.15% control; P < 0.01) and SP subsets at 72 hours (Figure 5C). By day 8, IL-4 production surged in CD4+ SP (19.4% ± 3.0%; P < 0.01 vs. control) and CD8+ SP cells (16.9% ± 0.5%; P < 0.01). Interferon-γ (IFN-γ) showed delayed recovery: CD4+ SP IFN-γ+ cells decreased from 1.7% ± 0.3% (control) to 0.1% ± 0.01% at 72 hours (P < 0.01), rebounding to 1.1% ± 0.4% by day 8 (P < 0.05 vs. 72-hour; Figure 5D).

ERK pathway activation differed temporally. Phosphorylated ERK (p-ERK) increased in DP (12.8% ± 3.2% vs. 0.6% ± 0.1% control; P < 0.01) and SP cells at 72 hours (Figure 5G). While CD8+ SP and DP p-ERK normalized by day 8, CD4+ SP cells maintained elevated p-ERK (3.5% ± 0.4%; P < 0.01 vs. control), suggesting persistent signaling in this subset.

Mechanistic Implications

The data demonstrate three-phase thymic remodeling during ET:

  1. Acute phase (72 hours): LPS triggers thymic atrophy with DP depletion, SP accumulation, and oligoclonal TCR repertoires. Reduced apoptosis in SP cells and ERK hyperactivation suggest disrupted negative selection.
  2. Recovery phase (day 8): DP compartment regeneration coincides with polyclonal TCR diversification. Increased IL-4 production and persistent CD4+ SP ERK signaling may facilitate tolerance-associated Th2 polarization.
  3. Long-term adaptation: Sustained CD62L expression in SP cells implies enhanced migratory capacity, potentially exporting tolerance-committed T cells to periphery.

These findings bridge thymic development with ET establishment, proposing that LPS reprograms thymocyte selection to generate tolerance-prone T cell repertoires. The dynamic TCR diversity changes highlight the thymus’s role in reshaping immune responsiveness during repeated microbial challenges.

doi.org/10.1097/CM9.0000000000001598

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