The Alarmin Concept Applied to Human Renal Transplantation: Evidence for a Differential Implication of HMGB1 and IL-33

<div><p>The endogenous molecules high mobility group box 1 (HMGB1) and interleukin-33 (IL-33) have been identified as alarmins, capable of mediating danger signals during tissue damage. Here, we address their possible role as innate-immune mediators in ischemia-reperfusion injury (IRI) following human kidney transplantation. We analysed serum and urinary HMGB1 and IL-33 levels, all determined by enzyme-linked immunosorbent assay, in a cohort of 26 deceased renal transplant recipients. Urinary HMGB1 and IL-33 levels were significantly increased as soon as 30 min after reperfusion, as compared to those before treatment. Moreover, both serum and urinary IL-33 (but not HMGB1) increase was positively correlated with cold ischemia time, from 30 min to 3 days post-transplantation. <i>In vitro</i>, human umbilical vein endothelial cells subjected to hypoxia conditions released both HMGB-1 and IL-33, while only the latter was further increased upon subsequent re-oxygenation. Finally, we postulate that leukocytes from renal recipient patients are targeted by both HMGB1 and IL-33, as suggested by increased transcription of their respective receptors (TLR2/4 and ST2L) shortly after transplantation. Consistent with this view, we found that iNKT cells, an innate-like T cell subset involved in IRI and targeted by IL-33 but not by HMGB1 was activated 1 hour post-transplantation. Altogether, these results are in keeping with a potential role of IL-33 as an innate-immune mediator during kidney IRI in humans.</p></div

Hypoxia/re-oxygenation-induced release <i>in vitro</i> of HMGB1 and IL-33.

<p>Confluent (≈95%) monolayer HUVEC were exposed to sixteen hours hypothermia/hypoxia in UW solution (H16h) followed by 1 hour (R1h), or 3 hours (R3h) of re-oxygenation in a new culture medium (Medium 200) at 37°C in 20% O₂. Confluent (≈95%) monolayer HUVEC were used as controls (Ctl). Early release of HMGB1 and IL-33 by HUVEC in response to in vitro hypoxia/re-oxygenation (A–B). HMGB1 (A) and IL-33 (B) in cell culture supernatants were quantified by ELISA. Increase of IL-33 but not HMGB1 mRNAs in HUVEC in response to <i>in vitro</i> hypoxia/re-oxygenation (C–D). Total RNA was extracted from monolayer HUVEC at the indicated time points and expression of HMGB1 (C) and IL-33 (D) mRNAs was quantified by RT-qPCR. Data are expressed as means ± SEM or of fold change relative to D0 and are representative of three separate experiments. *p<0.05, **p<0.01, ***p<0.001 <i>vs</i> Ctl by Mann-Whitney test.</p

Correlation of serum and urinary alarmin levels with cold ischemia time.

<p>POD: Post Operative Day. The correlation coefficient (<i>r</i>) is calculated by the non-parametric Spearman’s rank correlation test. A p-value<0.05 was considered significant.</p

Baseline demographic and clinical characteristics of recipients and donors.

(1)<p>BMI: Body Mass Index.</p>(2)<p>ECD: Expanded Criteria Donors.</p>(3)<p>DGF: Delayed Graft Function defined as a need for dialysis within the first week after transplantation.</p

Increased levels of HMGB1, IL-33 and sST2 in serum and urine shortly after renal IRI.

<p>HMGB1, IL-33 and sST2 levels were quantified by ELISA in serum and urine of kidney graft recipients (n = 26) before transplantation (D0) as control time, and 30 minutes (H0.5), 3 hours (H3), day 1 (POD1) and day 3 (POD3) after transplantation. Serum, urine and urinary molecule/creatinine ratio levels for HMGB1 (A–C), IL-33 (D–F) and sST2 (G–I). Note that serum samples from only 6 out of 26 transplanted patients contained measurable amounts of IL-33. Data are expressed as means ± SEM. *p<0.05, **p<0.01, ***p<0.001 by Wilcoxon or Mann-Whitney test, as appropriate. ns, no significant.</p

Early activation of iNKT cells after renal IRI: a potential role for IL-33.

<p>(A, B) PBMCs from kidney graft recipients were recovered before transplantation (D0), and 3 hours (H3) and day 3 (POD3) after transplantation. They were membrane-labelled with anti-CD3-FITC, anti-iNKT-PE 6B11 clonotype, and anti-CD69-PerCP/Cy5.5. CD69 analysis was performed by flow cytometry gating on CD3(+)6B11(+) cells, defined as iNKT cells: (A) Flow cytometry plot showing expression profiles of surface marker CD69 on iNKT cells <i>ex vivo</i> from one representative patient at D0 (filled histogram), H3 (bold line) and POD3 (dotted line). Numbers indicate MFI of CD69 expression on iNKT lymphocytes. (B) Mean Fluorescence Intensity (MFI) of CD69 expression on iNKT lymphocytes from the patient cohort (n = 16 at D0, H3, and POD3). (C, D) PBMCs from healthy adult donors (n = 6) were cultured with (black columns) or without (white columns) HMGB1(C) or IL-33 (D) for 3, 6 or 24 hours of culture. MFI of CD69 expression on iNKT lymphocytes was analysed by Flow cytometry as described in (A, B). Data are expressed as means ± SEM. **p<0.01, ***p<0.001 by Wilcoxon test.</p

Spearman’s correlation rank between variables.

<p>NS, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.</p>a<p>NS in Jα18-/-in individual analysis.</p>b<p>−0.53 (p<0.05) in WT, NS in Jα18-/-mice.</p>c<p>−0.52 (p<0.01) in WT, NS in Jα18-/-mic.</p>d<p>−0.74 (p<0.0001) in WT.</p>e<p>−0.41 (p<0.05) in WT.</p

Phenotypic characterization of the iNKT cell infiltrate in the liver of WT mice on day 15 after infection with <i>L.donovani.</i>

<p>Hepatic cell infiltration of C57BL/6 WT was analyzed by flow cytometry using anti-CD3-V500, anti-NK1.1-PerCP-Cy-5.5, anti-TCRβ-V450, anti TCR γδ-FITC, and αGalCer/CD1d tetramer-APC, anti-CD4-PE-Cy7, anti-CD8-APC-Cy7. (A) Gating strategy allowing to estimate the low percentage of NK1.1- and NK1.1+ cells among TCRβ+/αGalCer/CD1d tetramer+ cells. (B) Gating strategy and evaluation of the percentage of γδ-T cells and non iNKT cells among CD3+/NK1.1+. (C) Percentage of CD4+ and CD4-/CD8- cells among iNKT cells (TCRβ+/αGalCer/CD1d tetramer+). This panel is representative of three independent experiments.</p

Jα18^-/- mice display a qualitatively and quantitatively impaired hepatic granulomatous response after infection with <i>L.donovani</i>.

<p>Quantification of granuloma formation in the liver of <i>L. donovani</i> infected WT and Jα18^-/-mice by microscopic examination of tissue sections stained with HES, at D15, D30 and D60 post-infection. Representative granuloma foci from WT mice at D15 (A) and D60 (B), and from Jα18^-/- mice at D15 (C) and D60 (D) (×100 magnification). (E) Total number of granulomas in 100 microscopic fields (×400) at various time points. (F) Relative percentage of large granulomas (>25 cells) detected in each group of mice. (G) Relative percentage of mature granulomas detected in each group of mice.</p

Kinetics of selected chemokine and related chemokine receptors expression in liver extracts from WT and Jα18^-/- mice.

<p>mRNA expression of selected chemokines and their receptors was quantified by qPCR in liver extracts at various time points after infection. Quantification of mRNA for the T and NK cell-chemoattractant chemokines CXCL9, CXCL10 and their receptor CXCR3 (A), quantification of the PMN-chemoattractant chemokines MIP-2 and CXCL5 and their receptor CXCR2 (B) and of CCL2 and its receptor CCR2 (C). Data are the mean±SEM of 8 to 10 mice per group from two independent experiments and normalized on the expression of three housekeeping genes.</p