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, 21 (12), 3414-3426

Extracellular ATP Activates the NLRP3 Inflammasome and Is an Early Danger Signal of Skin Allograft Rejection

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Extracellular ATP Activates the NLRP3 Inflammasome and Is an Early Danger Signal of Skin Allograft Rejection

Joaquín Amores-Iniesta et al. Cell Rep.

Abstract

Immune cells are equipped with a number of receptors that recognize sterile injury and pathogens. We find that host immune cells release ATP as an inflammatory signal in response to allogeneic transplantation. ATP then acts via a feedback mechanism on the P2X7 channel to activate the NLRP3 inflammasome and subsequently process and release interleukin (IL)-18. This process is a necessary stage in the deleterious Th1 response against allotransplantation via interferon-γ production. Lack of IL-18 resulted in a decrease in graft-infiltrating CD8 cells but an increase in regulatory T cells. In human liver transplant patients undergoing progressive immunosuppressive drug withdrawal, we found that patients experiencing acute rejection had higher levels of the P2X7 receptor in circulating inflammatory monocytes compared to tolerant patients. These data suggest that the pharmacological inhibition of the P2X7 receptor or the NLRP3 inflammasome will aid in inducing transplant tolerance without complete immunoparalysis.

Keywords: ATP release; IL-18; NLRP3 inflammasome; P2X7 receptor; allotransplantation; caspase-1; danger signal; macrophages; purinergic signaling; skin transplant.

Figures

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Figure 1
Figure 1
Allogeneic Transplants Are Associated with Extracellular ATP (A) Representative images of extracellular ATP 3 days after syngeneic or allogeneic skin transplantation, using C57BL/6 as recipient and C57BL/6 (syngeneic) or BALB/c (allogeneic) skin as donor. (B) Quantification of graft-associated bioluminescence as the average radiance (p/s/cm2/sr) at day 3 after transplantation (n = 8). (C) Extracellular ATP quantification at 3, 7, and 14 days after syngeneic or allogeneic transplantation (n = 7−10). (D) Representative images of H&E-stained grafts. Arrows denote tissue necrosis and the detachment of epidermal layer. Scale bar, 50 μm. (E) CD3+ cells 3 or 7 days after transplantation (n = 12−18 fields of view from 3–4 transplants). (F) Relative gene expression for the indicated genes in grafts 3 or 7 days after transplantation (n = 4–5). (G) IFN-γ ELISA produced by axillar lymph node lymphocytes collected 7 days after syngeneic or allogeneic transplantation and subjected to mixed lymphocytic reaction with allogeneic splenocytes (n = 3–6). Data are represented as mean ± SEM (B, C, and E–G). Student’s t test was used for (B) and ANOVA with Bonferroni post-test was used for (E)–(G). See also Figure S1.
Figure 2
Figure 2
Allografts Induce ATP Release via P2X7 Receptor in Antigen-Presenting Cells (A) Extracellular ATP associated with allografts 3 days after transplantation, using C57BL/6 wild-type or P2rx7−/− as recipient and BALB/c skin as donor. Left: representative mouse per group is shown. Right: average radiance (p/s/cm2/sr) of n = 5−8 transplants is shown. (B) Extracellular ATP associated with allografts 3 days after transplantation in wild-type mice treated with vehicle or A438079 (100 μmol/kg). Left: representative mouse per group is shown. Right: average radiance (p/s/cm2/sr) of n = 5−6 transplants is shown. (C and D) ATP release from C57BL/6 bone marrow-derived macrophages (BMDMs), (C) incubated for 2 hr with C57BL/6 or BALB/c skin (n = 4) or (D) incubated for 30 min in the absence or presence of ovalbumin (OVA, 10 mg/mL) and A438079 (25 μM) (n = 3). (E) Yo-Pro uptake kinetic in BMDMs treated or not for 2 hr with OVA (10 mg/mL) or LPS (1 μg/mL) and then stimulated as indicated by an arrow with ATP (3 mM) in the presence or absence of A438079 (25 μM) (n = 6). (F) Extracellular ATP associated to allografts 3 days after transplantation in P2rx7−/− mice treated with 106 wild-type (P2rx7+/+) or P2rx7−/− BMDMs applied under the skin graft during the surgical procedure. Data are represented as mean ± SEM. For (A), (B), and (F), left shows representative mouse per group, and right shows p/s/cm2/sr of n = 5−6 transplants. Student’s t test was used for (A), (B), and (F); ANOVA with Bonferroni post-test was used for (C) and (D) and with Tukey post-test for the slopes represented in (E). See also Figure S2.
Figure 3
Figure 3
P2X7 Receptor Blockage Reduces Allograft Rejection (A) Representative images of H&E-stained allogeneic skin grafts using C57BL/6 wild-type or P2rx7−/− as recipient and BALB/c skin as donor. Scale bar, 50 μm. (B) Epidermal thickness at 3 days after transplantation. A438079 was intraperitoneally (i.p.) injected (100 μmol/kg/day) (n = 19−71 measurements from 3–4 transplants). (C) CD3+ or F4/80+cells 7 days after transplantation (n = 6−26 fields of view from 3–4 transplants). (D) IFN-γ in serum 7 days after transplantation (n = 3–5). (E) IFN-γ ELISA produced by axillar lymph node lymphocytes collected 7 days after transplantation and subjected to mixed lymphocytic reaction (MLR) with allogeneic splenocytes (n = 3–7). (F and G) Measurement of transplanted skin by (F) ultrasound or (G) skin transplant survival (n = 6−11 transplants). (H) Activity of plasma liver enzymes in human serum and mean fluorescence intensity for P2X7 receptor surface expression in inflammatory monocytes (CD14+CD16high) from liver-transplanted patients (tolerant or non-tolerant) during immunosuppression and after immunosuppression withdrawal (rejection) (n = 4 patients/group). (I) Correlation between the fold change of plasma ATP concentration and liver enzymes during immunosuppression and after immunosuppression withdrawal in the 4 non-tolerant patients. For (B), (F), and (H), data are represented as mean ± SEM. One-way ANOVA with Bonferroni post-test was used for (B)–(E), two-way ANOVA with Bonferroni post-test was used for (F) and (H) (differences between allogeneic and isogeneic; §differences between allogeneic and allogeneic + A438079; ns, no significant difference between isogeneic and allogeneic + A438079), and Gehan-Breslow-Wilcoxon test was used for (G). See also Figures S3 and S4 and Table S1.
Figure 4
Figure 4
The NLRP3 Inflammasome Is Not Involved in ATP Release in Allografts but Is Activated in Allografts (A) Extracellular ATP associated with allografts 3 days after transplantation in C57BL/6 wild-type (Nlrp3+/+) or Nlrp3−/− mice as recipients and BALB/c skin as donor. Left: luciferase signal representing extracellular ATP in one representative mouse per group is shown. Right: average radiance (p/s/cm2/sr) of n = 7−8 transplants is shown. (B) Extracellular ATP associated with allografts 3 days after transplantation in C57BL/6 wild-type (Casp1/11+/+) or Casp1/11−/− mice as recipients and BALB/c skin as donor. Left: luciferase signal representing extracellular ATP in one representative mouse per group is shown. Right: average radiance (p/s/cm2/sr) of n = 3−4 transplants is shown. (C) Relative gene expression for the indicated genes in grafts 3 days after transplantation, using C57BL/6 as recipient and C57BL/6 (syngeneic) or BALB/c (allogeneic) skin as donor. A438079 was i.p. injected (100 μmol/kg/day) (n = 5−6). Gene expression of healthy skin is presented with a dashed line. (D) IL-18 ELISA in serum 3 days after transplantation (n = 3−5). Data are represented as mean ± SEM. ns, no significant difference (p > 0.05); Student’s t test was used for (A) and (B); ANOVA with Bonferroni post-test was used for (C) and (D).
Figure 5
Figure 5
Inflammasome Is Upregulated during Graft Rejection (A) Casp1 gene expression in grafts (n = 3−6). (B and C) Relative gene expression for (B) Nlrp3, (C) Il1b, and Il18 in grafts (n = 3–6). Data are represented as mean ± SEM. ANOVA with Bonferroni post-test was used for (A)–(C), using C57BL/6 wild-type, Nlrp3−/−, or Casp1/11−/− as recipient and C57BL/6 (syngeneic) or BALB/c (allogeneic) skin as donor. WT, wild-type.
Figure 6
Figure 6
NLRP3 Inflammasome Deficiency Reduces Allogeneic Immune Response (A) Representative images of H&E-stained skin grafts, using C57BL/6 wild-type, Nlrp3−/−, or Casp1/11−/− as recipient and BALB/c skin as donor. Scale bar, 50 μm. (B) Epidermal thickness at 3 days after transplantation (n = 20−70 measurements from 3–4 transplants). (C) IL-18 ELISA in serum at 3 days post-transplantation (n = 4−6). (D) CD3+ or F4/80+cells 7 days after transplantation (n = 6−20 fields of view from 3–4 transplants). (E and F) IFN-γ (E) relative gene expression or (F) serum concentration 7 days after transplantation (n = 3−4, E; n = 3–8, F). (G) IFN-γ ELISA produced by axillar lymph node lymphocytes collected 7 days after transplantation and subjected to mixed lymphocytic reaction (MLR) with allogeneic splenocytes (n = 4–7). For (F) and (G), each circle represents an individual mouse. For (B)–(G), data are represented as mean ± SEM. ANOVA with Bonferroni post-test was used for (B)–(G). See also Figure S5.
Figure 7
Figure 7
IL-18 Mediates Inflammasome Alloimmune Response (A) IL-18-binding protein (IL-18BP) (left) and the ratio of IL-18/IL-18BP (right) in serum 7 days after transplantation (n = 4–5) using C57BL/6 as recipient and C57BL/6 (syngeneic) or BALB/c (allogeneic) skin as donor. (B) Relative gene expression for Il12b in grafts 3 or 7 days after transplantation using C57BL/6 wild-type or Il18−/− as recipient and BALB/c skin as donor (n = 3–4). (C) IFN-γ in serum 7 days after transplantation (n = 3–4). (D) IFN-γ ELISA produced by axillar lymph node lymphocytes collected 7 days after transplantation and subjected to mixed lymphocytic reaction (MLR) with allogeneic splenocytes (n = 4). (E) Representative images of H&E-stained skin grafts, using C57BL/6 wild-type or Il18−/− as recipient and BALB/c skin as donor. Scale bar, 50 μm. (F) F4/80+, CD3+, CD8+, or FOXP3+ cells 7 days after transplantation (n = 24–51 fields of view from 3 transplants). For (C) and (D), each circle represents an individual mouse. For (A)–(D) and (F), data are represented as mean ± SEM. Student’s t test was used for (A)–(D) and (F). See also Figure S6.

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