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

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

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

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

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

Up-regulation of alarmin-receptor mRNAs in leucocytes after renal IRI.

<p>Patient peripheral blood was recovered before transplantation (D0) as control time, and 30 minutes (H0.5), 3 hours (H3), day 1 (POD1) and day 3 (POD3) after transplantation. Total RNA was extracted from leucocytes at the indicated time points and expression of TLR2 (n = 24) (A), TLR4 (n = 25) (B), and ST2L (n = 14) (C) mRNAs was quantified by RT-qPCR. Results are expressed as means ± SEM of fold change relative to D0. *p<0.05, **p<0.01, ***p<0.001 <i>vs</i> D0 by Wilcoxon test. ND: not done.</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

Analysis of NK differentiation potential and NK progenitor cell expansion mediated by HOXB4.

<p>(A) Percentages of NK cells (CD56⁺CD3⁻CD19⁻) among nucleated cells collected at the end of 5 weeks of co-culture. Cells derived from the 2 weeks primary co-cultures with MS-5/SP-HOXB4 or MS-5/EGFP were then plated on unmodified MS-5 cells in conditions known to promote NK-cell differentiation and maintained during three weeks. At the end of the culture period, cells were analyzed by FACS for the expression of CD56 and CD3/CD19 markers. Un-co-cultured hEB cells were directly cultured under NK-cell differentiation conditions for 3 weeks (n = 5, *<i>p</i><0.05, **<i>p</i><0.01). (B) Fold increase of total NK cells. NK cells were derived from total cells isolated from the primary 2-week co-cultures of hEB-derived cells with either MS-5/SP-HOXB4 or MS-5/EGFP control and then cultured under NK-cell differentiation condition for three weeks. NK cells were then numbered. Bars represent fold amplifications relative to day-0 control (un-co-cultured hEBs) (designated as 100%) (n = 5, *<i>p</i><0,05).</p

Functional activity of NK cells derived from co-culture with MS-5/SP-HOXB4.

<p>(A) The functional activity of cells was assessed in the presence of IL-12 and against K562 target cells; the NK-cell activity was evaluated by the mobilisation of the CD107a antigen on the cell surface and the intra-cytoplasmic expression of IFNγ. Cells were gated on CD56⁺ cells (not shown). (B) Cytotoxic activity of NK effector cells against target K562 cells. a) Target K562 cells were labelled by CFSE. b) Percentage of 7AAD⁺ death cells among the CFSE⁺ target cells at various Effector-Target (E/T) ratios (one experiment out of two). c) Percentage of cytotoxicity (<i>i.e</i> percentage of 7AAD⁺ death cells among the CFSE⁺ target cells) according to the ratio E/T (dotted line and bold line represent two independent experiments).</p

Expression of inhibitory and activating receptors and of the cytotoxic arsenal of NK cells derived from co-culture with MS-5/SP-HOXB4.

<p>NK cells were obtained from NK progenitors derived from hEB cells co-cultured with MS-5/SP-HOXB4. Cells were FACS analyzed for surface expression of CD56, CD16, CD94, the mix of CD158a, h, CD158b1/b2, j, CD158e1/e2 and CD158i (referred as CD158) and of CD159, CD335, CD336 and CD337. For CD159, CD94, CD335, CD336 and CD337 expression, cells were gated on CD56⁺ cells (not shown). NK cells obtained using the HOXB4 co-culture model were also analyzed for the intra-cytoplasmic expression of Perforin, Granzyme-A and Granzyme-B. Cells were gated on CD56⁺ cells (not shown). Data are from one experiment out of two.</p