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. 2021 Oct 5;37(1):109789.
doi: 10.1016/j.celrep.2021.109789.

Attenuation of apoptotic cell detection triggers thymic regeneration after damage

Sinéad Kinsella  1 , Cindy A Evandy  2 , Kirsten Cooper  2 , Lorenzo Iovino  2 , Paul C deRoos  2 , Kayla S Hopwo  2 , David W Granadier  2 , Colton W Smith  2 , Shahin Rafii  3 , Jarrod A Dudakov  4
Affiliations

Attenuation of apoptotic cell detection triggers thymic regeneration after damage

Sinéad Kinsella et al. Cell Rep. .

Abstract

The thymus, which is the primary site of T cell development, is particularly sensitive to insult but also has a remarkable capacity for repair. However, the mechanisms orchestrating regeneration are poorly understood, and delayed repair is common after cytoreductive therapies. Here, we demonstrate a trigger of thymic regeneration, centered on detecting the loss of dying thymocytes that are abundant during steady-state T cell development. Specifically, apoptotic thymocytes suppressed production of the regenerative factors IL-23 and BMP4 via TAM receptor signaling and activation of the Rho-GTPase Rac1, the intracellular pattern recognition receptor NOD2, and micro-RNA-29c. However, after damage, when profound thymocyte depletion occurs, this TAM-Rac1-NOD2-miR29c pathway is attenuated, increasing production of IL-23 and BMP4. Notably, pharmacological inhibition of Rac1-GTPase enhanced thymic function after acute damage. These findings identify a complex trigger of tissue regeneration and offer a regenerative strategy for restoring immune competence in patients whose thymic function has been compromised.

Keywords: NOD2; Rac1 GTPase; T cell development; TAM receptors; apoptotic cell death; lymphopenia; thymus; tissue regeneration.

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Conflict of interest statement

Declaration of interests S.K. and J.A.D. have filed a patent application on this work.

Figures

Figure 1.
Figure 1.. NOD2 limits thymus regeneration by inhibiting the production of regenerative factors
(A and B) Thymuses were pooled from 6-week-old C57BL/6 mice, and transcriptome analysis was performed on fluorescence-activated cell sorting (FACS)-purified ECs (A) or DCs (B) isolated from either untreated mice (d0) or 4 days after TBI (550 cGy, d0, n = 3; d4, n = 2; each n pooled from 5 mice). (A) Gene ontology (GO) pathway analysis was performed on upregulated genes in ECs at day 4 after SL-TBI using DAVID, and the top-ten pathways by FDR are displayed. Red bars represent pathways involved with immune function; dashed line at p = 0.05. Top-ten genes in ECs are shown by frequency of representation among all GO pathways with an FDR <0.05. (B) Gene ontology (GO) pathway analysis was performed on upregulated genes in DCs at day 4 after SL-TBI using DAVID, and the top-ten pathways by FDR are displayed. Red bars represent pathways involved with immune function; dashed line at p = 0.05. Top-ten genes in DCs are shown by frequency of representation among all GO pathways with an FDR <0.05. (C–I) 6- to 8-week C57BL/6 WT or Nod2−/− mice were given a sublethal dose of TBI (550 cGy), and the thymus was harvested and analyzed at the indicated time points. (C) Total thymic amounts of BMP4, IL-23, and IL-22 were assessed by ELISA at day 7 after SL-TBI (n = 4/group from a representative experiment of at least two independent experiments). (D) Total thymic cellularity at days 7 and 14 after SL-TBI (d7, n = 11–15; d14, n = 6–10; combined from two independent experiments). (E) Concatenated flow plots showing CD4 and CD8 expression at days 7 and 14 after SL-TBI. (F) Total number of CD4+CD8+ DP, CD4+CD3+ SP4, or CD3+CD8+ SP8 thymocytes (d7, n = 11–15; d14, n = 6–10; combined from two independent experiments). (G) Number of CD45+MHCII+CD11c+CD103+ cDC1 and CD45−EpCAM−CD31+PDGFRa− ECs at day 7 after SL-TBI (n = 11–15/group across two independent experiments). (H) Number of CD45–EpCAM+MHCII+UEA1loLy51hi cTECs and CD45−EpCAM+MH-CII+UEA1hiLy51lo mTECs at day 7 after SL-TBI (n = 11–15/group across two independent experiments). (I) Total thymic cellularity and absolute number of thymocyte subsets at day 150 after SL-TBI (n = 3–5). Graphs represent mean ± SEM; each dot represents a biologically independent observation. See also Figures S1 and S2.
Figure 2.
Figure 2.. miR29c mediates the effects of NOD2 in limiting production of regenerative factors in ECs and DCs
(A) Thymuses were isolated from 6- to 8-week C57BL/6 WT or Nod2−/− mice at day 3 following SL-TBI, and the expression of 3p and 5p arms of miR29a, miR29b, or miR29c was analyzed by qPCR (3p, n = 4/group; 5p, n = 7/group across two independent experiments). (B) DPs, ECs, and DCs were FACS purified from WT thymuses at day 0 or 3 after SL-TBI and expression of miR29c-5p was analyzed by qPCR (DCs, n = 4; ECs/DPs, n = 6–7/population/time point across two independent experiments). (C) Thymic exECs were generated as previously described (Seandel et al., 2008; Wertheimer et al., 2018) and transfected with a miR29c mimic. 20 h after transfection, the expression of Bmp4 was analyzed by qPCR (n = 7 independent experiments). (D) exECs were transfected with a miR29c inhibitor, and the expression of Bmp4 was analyzed by qPCR 20 h after transfection (n = 4 independent experiments). (E) CD11c+ DCs were isolated from untreated C57BL/6 thymuses and transfected with a miR29c mimic. 20 h after transfection, Il23p19 was analyzed by qPCR (n = 5 across three independent experiments). (F) HEK293 cells were co-transfected with either Bmp4-3′ UTR, Il12p40-5′ UTR, or Il23p19-3′ UTR luciferase constructs and a miR29c-5p mimic. Binding activity was quantified by measuring luciferase activity after 20 h (n = 5–7 independent experiments). Graphs represent mean ± SEM; each dot represents a biologically independent observation. See also Figure S3.
Figure 3.
Figure 3.. TAM receptor detection of phosphatidylserine mediates thymocyte suppression of regenerative factors
(A and B) Thymocytes were isolated from untreated C57BL/6 mice and incubated for 4 h with Dexamethasone (100 nM) or zVAD-FMK (20 μM). After 4 h, apoptotic thymocytes (ACs) were washed and co-cultured with exECs (A) or freshly isolated CD11c+ DCs (B) for 24 h after which Bmp4 expression was analyzed by qPCR (n = 7 across 5 independent experiments) or IL-23 was analyzed by intracellular cytokine staining (n = 5 across two independent experiments). (C–G) 6- to 8-week-old C57BL/6 mice were given sublethal TBI (550 cGy), and the thymus was harvested at days 0, 1, 2, 3, and 7. (C) Annexin V staining on DP thymocytes (displayed are concatenated plots from 3 individual mice, representative of three independent experiments). (D) Staining for CD4 and CD8 on thymus cells (displayed are concatenated plots from 3 individual mice, representative of three independent experiments). (E) Total number of DP thymocytes (solid line; left axis) compared with proportion of Annexin V+ DP thymocytes (red broken line; right axis) (n = 8 across 3 independent experiments). (F) Absolute binding of Annexin V in the thymus (n = 8 across 3 independent experiments). (G) Correlation of absolute thymic binding of Annexin V with total number of cells in the thymus (n = 3/time point comprising one of three independent experiments). (H) DP thymocytes, DCs, or ECs were FACS purified from untreated C57BL/6 mice, and expression of Axl, Mer, and Tyro3 was analyzed by qPCR (DP, n = 7; DC, n = 5; EC, n = 7 across two independent experiments). (I and J) Thymocytes were isolated from untreated C57BL/6 mice and incubated for 4 h with dexamethasone (100 nM). After 4 h, apoptotic thymocytes (ACs) were washed and co-cultured with exECs (I) or freshly isolated CD11c+ DCs. (J) in the presence or absence of the TAM receptor inhibitor RXDX-106 (25 μM) for 20 h after which Bmp4 expression was analyzed by qPCR (n = 6/treatment across 2 independent experiments) or IL-23 was analyzed by intracellular cytokine staining (n = 4). Graphs represent mean ± SEM; each dot represents a biologically independent observation. See also Figure S4.
Figure 4.
Figure 4.. Rac1-GTPase is activated by apoptotic thymocytes and suppresses production of IL-23 and BMP4
(A and B) exECs (A) or freshly isolated CD11c+ DCs (B) were incubated for 20 h in the presence of inhibitors (all at 50 μM) for RhoA (Rhosin), ROCK (TC-S 7001), Rac1 (EHT-1864), or Cdc42 (ZCL272) after which Bmp4 (ECs) or Il12p40 was analyzed by qPCR (exECs, n = 5 independent experiments; DCs, n = 6 across two independent experiments). (C) Thymocytes were isolated from untreated C57BL/6 mice and incubated for4 h with Dexamethasone (100 nM) or zVAD-FMK (20 μM). After 4 h, apoptotic thymocytes (ACs) were washed and co-cultured with exECs for 24 h after which Rac1-GTPase activation was measured using a GTPase ELISA specific for Rac1 (n = 5 across two independent experiments). (D) Thymocytes were isolated from untreated C57BL/6 mice and incubated for 4 h with dexamethasone (100 nM). After 4 h, apoptotic thymocytes (ACs) were washed and co-cultured with exECs in the presence or absence of the TAM receptor inhibitor RXDX-106 (25 μM) for 20 h after which Rac1-GTPase activation was measured using a GTPase ELISA specific for Rac1 (n = 5 across two independent experiments). (E) Mice deficient for Rac1 specifically in DCs were generated by crossing Rac1fl/fl mice with CD11c-Cre. WT or Rac1ΔDC mice were given SL-TBI and thymus cellularity was assessed at day 7 (n = 5–7/group combined from two independent experiments). Graphs represent mean ± SEM; each dot represents a biologically independent observation.
Figure 5.
Figure 5.. Rac1 inhibition enhances thymus regeneration and peripheral CD4+ naive T cell recovery after acute damage
6- to 8-week C57BL/6 WT or Nod2−/− mice were treated with the Rac1 inhibitor EHT1864 (40 mg/kg i.p. injection) at days 3, 5, and 7 following a sublethal dose of TBI (550 cGy), and the thymus was analyzed on day 14. (A) Total thymic cellularity at day 14 after SL-TBI (WT, n = 20/treatment across 4 independent experiments; KO, n = 9–12 across three independent experiments). (B) Flow plots showing CD4 and CD8 expression at day 14 after SL-TBI (gated on CD45+ cells; plots were concatenated of all samples in each treatment group from one experiment). (C) Total number of DP, SP4, or SP8 thymocytes (n = 20/treatment across 4 independent experiments). (D) Expression of miR29c analyzed by qPCR on whole thymic tissue (n = 5/group). (E) Total thymic amounts of BMP4 and IL-23 assessed by ELISA at day 7 (n = 4–5/group, representative of two independent experiments). (F) Total number of cTECs and mTECs (n = 15/group across three independent experiments). (G) Flow plots showing CD4 and CD8 expression in the spleen at day 56 after SL-TBI (gated on CD45+CD3+ cells). (H) Total number of CD4+ and CD8+ T cells in the spleen at d56 (n = 5/group). (I) Plots of CD62L and CD44 on CD4+ and CD8+ T cells (gated on either CD45+CD3+CD4+CD8− or CD45+CD3+CD4−CD8+) cells; plots concatenated of all samples in a given experiment). (J) Ratio of number of naive (CD62Lhi CD44lo) CD4+ or CD8+ to CD4+ or CD8+ EM (CD62Lhi CD44hi) T cells (n = 5/group). Experiments in (C)–(J) were performed only in WT mice. Graphs represent mean ± SEM; each dot represents a biologically independent observation. See also Figure S5.
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