Tolerogenic dendritic cells protect against acute kidney injury

Ischemia reperfusion injury is a common precipitant of acute kidney injury that occurs following disrupted perfusion to the kidney. This includes blood loss and hemodynamic shock, as well as during retrieval for deceased donor kidney transplantation. Acute kidney injury is associated with adverse long-term clinical outcomes and requires effective interventions that can modify the disease process. Immunomodulatory cell therapies such as tolerogenic dendritic cells remain a promising tool, and here we tested the hypothesis that adoptively transferred tolerogenic dendritic cells can limit kidney injury. The phenotypic and genomic signatures of bone marrow–derived syngeneic or allogeneic, Vitamin-D3/IL-10–conditioned tolerogenic dendritic cells were assessed. These cells were characterized by high PD-L1:CD86, elevated IL-10, restricted IL-12p70 secretion and a suppressed transcriptomic inflammatory profile. When infused systemically, these cells successfully abrogated kidney injury without modifying infiltrating inflammatory cell populations. They also provided protection against ischemia reperfusion injury in mice pre-treated with liposomal clodronate, suggesting the process was regulated by live, rather than reprocessed cells. Co-culture experiments and spatial transcriptomic analysis confirmed reduced kidney tubular epithelial cell injury. Thus, our data provide strong evidence that peri-operatively administered tolerogenic dendritic cells have the ability to protect against acute kidney injury and warrants further exploration as a therapeutic option. This technology may provide a clinical advantage for bench-to-bedside translation to affect patient outcomes. Ischemia reperfusion injury is a common precipitant of acute kidney injury that occurs following disrupted perfusion to the kidney. This includes blood loss and hemodynamic shock, as well as during retrieval for deceased donor kidney transplantation. Acute kidney injury is associated with adverse long-term clinical outcomes and requires effective interventions that can modify the disease process. Immunomodulatory cell therapies such as tolerogenic dendritic cells remain a promising tool, and here we tested the hypothesis that adoptively transferred tolerogenic dendritic cells can limit kidney injury. The phenotypic and genomic signatures of bone marrow–derived syngeneic or allogeneic, Vitamin-D3/IL-10–conditioned tolerogenic dendritic cells were assessed. These cells were characterized by high PD-L1:CD86, elevated IL-10, restricted IL-12p70 secretion and a suppressed transcriptomic inflammatory profile. When infused systemically, these cells successfully abrogated kidney injury without modifying infiltrating inflammatory cell populations. They also provided protection against ischemia reperfusion injury in mice pre-treated with liposomal clodronate, suggesting the process was regulated by live, rather than reprocessed cells. Co-culture experiments and spatial transcriptomic analysis confirmed reduced kidney tubular epithelial cell injury. Thus, our data provide strong evidence that peri-operatively administered tolerogenic dendritic cells have the ability to protect against acute kidney injury and warrants further exploration as a therapeutic option. This technology may provide a clinical advantage for bench-to-bedside translation to affect patient outcomes. Translational StatementDespite the ability to predict and identify acute kidney injury (AKI), clinicians can only offer supportive care and dialysis when the problem arises. Herein, we demonstrate the potential use of tolerogenic dendritic cells (tolDCs), demonstrating that these cells can alter the early immunopathology and severity of AKI. There is an impetus to further explore the mechanistic role of tolDCs in AKI and repair, and evidence from phase I/II clinical trials for transplant tolerance suggests that tolDCs are safe. This provides tolDCs with an immediate clinical advantage for bench-to-bedside translation to impact patient outcomes. Despite the ability to predict and identify acute kidney injury (AKI), clinicians can only offer supportive care and dialysis when the problem arises. Herein, we demonstrate the potential use of tolerogenic dendritic cells (tolDCs), demonstrating that these cells can alter the early immunopathology and severity of AKI. There is an impetus to further explore the mechanistic role of tolDCs in AKI and repair, and evidence from phase I/II clinical trials for transplant tolerance suggests that tolDCs are safe. This provides tolDCs with an immediate clinical advantage for bench-to-bedside translation to impact patient outcomes. Acute kidney injury (AKI) is a global disorder,1Susantitaphong P. Cruz D.N. Cerda J. et al.World incidence of AKI: a meta-analysis.Clin J Am Soc Nephrol. 2013; 8: 1482-1493Crossref PubMed Scopus (979) Google Scholar and epidemiological evidence clearly establishes that AKI is neither benign nor self-limited, and survivors are confronted with an increased risk of chronic kidney disease,2Chawla L.S. Eggers P.W. Star R.A. Kimmel P.L. Acute kidney injury and chronic kidney disease as interconnected syndromes.N Engl J Med. 2014; 371: 58-66Crossref PubMed Scopus (1346) Google Scholar infection,3Griffin B.R. You Z. Holmen J. et al.Incident infection following acute kidney injury with recovery to baseline creatinine: a propensity score matched analysis.PloS One. 2019; 14e0217935Crossref Scopus (16) Google Scholar cardiovascular morbidity,4Chawla L.S. Amdur R.L. 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Academic Press, 2021: 1-42Crossref Scopus (2) Google Scholar Dendritic cells (DCs) are potent antigen-processing/presenting cells that are recruited and activated into mature DCs by proinflammatory cytokines and danger-associated molecular patterns released by injured/dying renal tubular epithelial cells (RTECs).15Dong X. Swaminathan S. Bachman L.A. et al.Resident dendritic cells are the predominant TNF-secreting cell in early renal ischemia-reperfusion injury.Kidney Int. 2007; 71: 619-628Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar Mature DCs generate effector T-cell responses in the tubulointerstitium by presenting nonfilterable proteins,16Macconi D. Chiabrando C. Schiarea S. et al.Proteasomal processing of albumin by renal dendritic cells generates antigenic peptides.J Am Soc Nephrol. 2009; 20: 123-130Crossref PubMed Scopus (89) Google Scholar but the evidence for a direct effect of DCs on RTECs that is independent of changes in the T-cell subset or function is lacking, as is a comprehensive understanding of parenchymal molecular pathways changed in response to DC fluxes that occur in AKI. DCs can also be pharmacologically or genetically manipulated in vitro to generate a tolerogenic, or stable, semimature (alternatively activated) phenotype.17Morelli A.E. Thomson A.W. Tolerogenic dendritic cells and the quest for transplant tolerance.Nature Rev Immunol. 2007; 7: 610-621Crossref PubMed Scopus (757) Google Scholar Tolerogenic DCs (tolDCs) display low-level costimulatory molecule expression and enhanced anti-inflammatory cytokine secretion; they are capable of subverting effector T-cell responses and inducing regulatory T cells. Renewed interest in cellular therapies has facilitated successful translation of preclinical studies to phase I/II clinical trials for reduced dependence on immunosuppressive drugs or tolerance in autoimmune disease and transplantation, with promising feasibility and safety data.18Li J. Thomson A.W. Rogers N.M. Myeloid and mesenchymal stem cell therapies for solid organ transplant tolerance.Transplantation. 2021; 105: e303-e321Crossref PubMed Scopus (7) Google Scholar, 19Zahorchak A.F. Kean L.S. Tokita D. et al.Infusion of stably immature monocyte-derived dendritic cells plus CTLA4Ig modulates alloimmune reactivity in rhesus macaques.Transplantation. 2007; 84: 196-206Crossref PubMed Scopus (47) Google Scholar, 20Sawitzki B. Harden P.N. 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Regulatory macrophages and tolerogenic dendritic cells in myeloid regulatory cell-based therapies.Int J Mol Sci. 2021; 22: 7970Crossref PubMed Scopus (19) Google Scholar The attractiveness of tolDC-based therapy stems from the premise of antigen-specific immunosuppression although realistically both autoimmunity and alloreactivity in transplantation are characterized by responsiveness to a broad range of antigens due to epitope spreading. The application of tolDCs to clinical diseases lacking clear identification of antigenic specificity may still be beneficial given their anti-inflammatory mechanism of action. tolDCs represent a potential therapy for AKI, which is crucial for a disease with no effective treatment other than expectant management, but their efficacy has not been tested. In this preclinical study, we investigated whether tolDCs could limit RTEC damage in AKI, interrogated their mechanism of action, and provided essential transcriptional information that will guide an understanding of both tolDC biology and AKI. C57BL/6 and BALB/c mice obtained from Australian Bio-Resources (Garvan, Sydney, Australia) were housed in our animal facility (Westmead Institute for Medical Research), with 12-hour light/dark cycle, standard chow, and water ad libitum, approved under ethics protocol (Western Sydney Local Health District). Studies were performed in accordance with the Australian code for the care and use of animals for scientific purposes developed by the National Health and Medical Research Council of Australia. Aseptic mice bone marrow passed through 70 μm cell filter and treated with red cell lysis buffer (eBioscience). Cells were resuspended in DC media (Supplementary Methods). tolDCs were generated with the addition of 20 nM 1α,25-dihydroxyvitamin D3 (VitD3; Sigma-Aldrich) and 10 ng/ml recombinant murine interleukin-10 (IL-10; Peprotech) to DC media, beginning on day 2 of culture. Medium cytokines ± vitD3/IL-10 were renewed every other day. Lipopolysaccharide (LPS; InvivoGen) was used at 100 pg/ml in select flasks on day 6, before cell collection on day 7 for the LPS-bone marrow–derived DC (BMDC) and LPS-tolDC groups. Magnetic beads were used to enrich cells before in vivo studies using the MACS Dead Cell Removal Kit and CD11c microbeads (Miltenyi Biotec). Single-cell suspensions were washed in flow-wash buffer (phosphate-buffered saline [PBS], 2% fetal calf serum) and incubated with Fc block (antimouse CD16/32; BD Biosciences) and then with fluorescent antibody cocktails (Supplementary Material). Samples were analyzed using the LSR Fortessa flow cytometer (BD Biosciences) with appropriate bead- and cell-based compensation controls, and data analysis was performed in FlowJo (v10.8.1; BD Bioscience). The detection of mouse IL-10, IL12-p70, and transforming growth factor beta 1 (TGF-β1; enzyme-linked immunosorbent assay; Thermo Fisher) in cell culture supernatant was performed by enzyme-linked immunosorbent assays, and indoleamine 2,3-dioxygenase 1 (IDO1) activity (#MAK356; Sigma-Aldrich) was measured by a fluorogenic assay detecting N-formylkynurenine as per the manufacturer's instructions. The allostimulatory capacity of γ-irradiated, LPS-stimulated C57BL/6 BMDCs (irLPS-BMDCs) or tolDCs was tested in a 3-day mixed lymphocyte reaction after coculture with BALB/c splenocytes labeled with CellTraceViolet (Thermo Fisher). Positive controls were splenocytes exposed to 10 ng/ml phorbol myristate acetate and 1 μg/ml ionomycin (Sigma-Aldrich). Bulk RNA-sequencing of live+ CD11c+-enriched BMDCs, LPS-BMDCs, tolDCs, and LPS-tolDCs (n = 3 C57BL/6 mice per group) was sequenced using the NovaSeq 6000 platform (Illumina) with 100 bp single-end read length. Detailed description is available in Supplementary Methods. Primary C57BL/6 RTECs were isolated as described previously.24Rogers N.M. Zhang Z.J. Wang J.-J. et al.CD47 regulates renal tubular epithelial cell self-renewal and proliferation following renal ischemia reperfusion.Kidney Int. 2016; 90: 334-347Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar Kidneys were digested using the multitissue dissociation kit and GentleMacs (Miltenyi), incubated with CD326 microbeads (Miltenyi) and passed through LS columns. The positive cell fraction was suspended in the defined RTEC K1 medium (Supplementary Methods) and cultured on collagen-coated dishes (BD Biosciences). Cell passages 2 and 3 were seeded onto 6-well culture plates, and Transwell polyester inserts (0.4 μm pore; Corning) were added to the RTEC wells with either (i) DC media alone or either (ii) tolDCs or (iii) LPS-tolDCs and allowed to equilibrate for 24 hours. LPS (100 ng/ml) was added to the RTEC chamber and RTECs collected at 0, 2, 4, 6, and 24 hours after stimulation. Ten- to twelve-week-old male C57BL/6 mice were anesthetized using isoflurane/oxygen titrated to effect, with body temperature maintained at 36 °C for bilateral ischemia-reperfusion injury (IRI). A midline laparotomy facilitated access to occlude the renal pedicles using microaneurysm clamps for 20 minutes before releasing and abdominal closure with 5/0 monofilament. For adoptive cell transfer experiments, mice received PBS alone (control), syngeneic tolDCs, LPS-tolDCs, or allogeneic-tolDCs (1 × 106, live/CD11c+ cells in 150 μl PBS) via a retro-orbital approach on the day prior (D-1) or day of (D0) surgery. In addition, C56BL/6 mice received 0.1 ml/10 g body weight of liposome containing either control PBS or clodronate (Liposoma) by i.p. injection, followed by adoptive cell therapy and bilateral renal IRI 4 days later. All mice were killed after 24-hour reperfusion, with collection of blood by cardiac puncture and kidney tissue either snap-frozen, embedded in optimal cutting temperature compound, or fixed in 10% neutral-buffered formalin. Serum creatinine and urea levels were measured by a centralized lab (Westmead ICPMR). Kidneys embedded in paraffin were sectioned at 4 μm and stained with hematoxylin and eosin by standard methods.24Rogers N.M. Zhang Z.J. Wang J.-J. et al.CD47 regulates renal tubular epithelial cell self-renewal and proliferation following renal ischemia reperfusion.Kidney Int. 2016; 90: 334-347Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar Sections were scored for degree of acute tubular damage based on tubular dilatation, cell necrosis, infarction, and cast formation (Supplementary Methods). Kidneys preserved at optimal cutting temperature (OCT) media were sectioned at 5-μm thickness and stained with the tetramethylrhodamine red terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling in situ cell death detection kit (Roche). The average number of terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling–positive cells in a ×20 magnification field over 5 different regions were measured. Frozen sections of liposomal-treated PBS and clodronate-treated kidneys were stained with rabbit antimouse CD68 (ab125121; Abcam), antirabbit horseradish peroxidase antibody (Dako), and Vector NovoRed substrate (Vector Labs). The average number of CD68+ cells at ×40 magnification over 15 different regions were measured. All images were scored by 2 independent observers. Single-cell suspensions from collagenase/DNase-digested kidneys were incubated with Fc block before staining with conjugated antibodies (Supplementary Methods). Absolute cell counts (using BD TruCount; BD Bioscience) and relative proportions of live CD45+ cells were assessed using the LSR Fortessa flow cytometer. Cell tracking was performed using tolDCs or LPS-tolDCs labeled with CellTrace Violet before adoptive transfer of 2 × 106 cells/mouse on the day of IRI surgery. The kidneys, lungs, and spleen were retrieved 24 hours later, processed into a single cell suspension, and stained with Live/Dead fixable near-IR stain (L34976; Thermo Fisher) and CD45 antibody for analysis. RNA was extracted from either tissue or cell lysate using the ISOLATE II RNA Mini Kit (Bioline), quantified using a Nanodrop (BioTek), and reverse-transcribed using the SensiFAST cDNA Synthesis Kit (Bioline). cDNA was amplified in triplicate using the CFX384 real-time polymerase chain reaction machine (Bio-Rad) with SensiFAST No-ROX (Bioline) and targeted TaqMan primers purchased from Thermo Fisher (Supplementary Methods). Data were analyzed using the ΔΔCT method with expression normalized to the house-keeping gene and PBS-treated animals as the referent control. Spatial transcriptomics was performed on 6 fresh-frozen mouse kidney samples (n = 2 per group) using Visium slides (10x Genomics). Detailed description of optimized methods25Raghubar A.M. Pham D.T. Tan X. et al.Spatially resolved transcriptomes of healthy mammalian kidneys illustrate the molecular complexity and interactions of functional nephron segments.Front Med (Lausanne). 2022; 9873923PubMed Google Scholar and bioinformatics analysis is given in the Supplementary Methods. Data are represented as mean ± SD unless otherwise stated. Data were analyzed with the t test (parametric variables), Mann-Whitney U test (nonparametric variables) for means between 2 groups, or analysis of variance between multiple groups using Prism (v9; GraphPad) unless otherwise stated. A P value of <0.05 was deemed significant. BMDCs and tolDCs generated by exposure to IL-10/vitD3 ± LPS (Figure 1a) revealed distinct surface marker profiles (Figure 1b and c; Supplementary Figure S1A and B). Major histocompatibility complex II+, CD40+, CD86+, and PD-L1+ expression was similar for naïve BMDCs (gray, tinted) and tolDCs (dotted line) regardless of mouse genotype. These markers were upregulated after LPS exposure in both BMDCs (LPS-BMDCs) and tolDCs (LPS-tolDCs; Figure 1c and d; Supplementary Figure S1A and B). The increase in major histocompatibility complex II and CD86 expression was limited in LPS-tolDCs compared with LPS-BMDCs, but PD-L1+ expression was significantly greater in LPS-tolDC groups compared with LPS-BMDC. The PD-L1:CD86 mean fluorescence intensity ratio, a marker of tolerogenicity, was >2.0 for C57BL/6-derived LPS-tolDCs and BALB/c-derived LPS-tolDCs (Figure 1e; Supplementary Figure S1A and B). Robust T-cell proliferation in response to irradiated, allogeneic LPS-BMDC was abrogated in the presence of either tolDCs or LPS-tolDCs, and this suppressive effect was lost when the tolDC to stimulator cell ratio was decreased or when only tolDC culture supernatants were added to the mixed lymphocyte reaction (Figure 1f; Supplementary Figure S1C–E). We previously identified and validated a human tolDC transcriptomic signature26Robertson H. Li J. Kim H.J. et al.Transcriptomic analysis identifies a tolerogenic dendritic cell signature.Front Immunol. 2021; 12733231Crossref Scopus (5) Google Scholar and performed bulk RNA sequencing on live+CD11c+-enriched BMDCs, LPS-BMDCs, tolDCs, and LPS-tolDCs to characterize potential genomic signature(s) of our tolerized cells. There were over 4000 differentially expressed genes between the 4 groups (Supplementary Table S1), but when filtered for the absolute log2-fold change ≥1.5, a common set of 69 upregulated and 121 downregulated genes were found in the LPS-tolDC when compared with other groups (Figure 2a; Supplementary Table S2). To assess genes conserved after tolerogenic induction, we identified differential gene expression in tolDCs by comparing differential gene expression lists from LPS-tolDCs versus LPS-BMDCs and tolDCs versus LPS-BMDCs. Conserved genes were determined from upregulated or downregulated differential gene expression common to both comparator lists, despite changes in expression intensity in response to a Toll-like receptor 4 agonist (Supplementary Table S3). A heatmap of these conserved tolerogenic genes (Figure 2b) demonstrated high expression of Trem2, Arg1, Tgm2, Pecam1, Tim3, and Notch1 in tolDCs, and these genes remained elevated after LPS stimulation, but to a lesser degree. Fabp5, Kynu, CD40, CD80, CD86, and Rel were all suppressed in both the tolerogenic groups relative to mature DCs. Gene set enrichment analysis and gene concept network of conserved tolDC genes (tolDC or LPS-tolDC) had suppressed inflammatory pathways compared with LPS-BMDC (Figure 2c and d), pertaining to nuclear factor κB signaling, T-cell–mediated immunity, and cytotoxicity. This effect was partly reversed, with upregulated immune activation and cytokine production pathways when LPS-tolDCs were compared with tolDCs instead (Figure 2e and f), consistent with LPS-tolDCs representing an "alternatively activated" subset.26Robertson H. Li J. Kim H.J. et al.Transcriptomic analysis identifies a tolerogenic dendritic cell signature.Front Immunol. 2021; 12733231Crossref Scopus (5) Google Scholar Gene set enrichment analysis also demonstrated downregulation of "innate immune response" in tolDCs by negative normalized enrichment scores for the LPS-activated (LPS-tolDC vs. LPS-BMDC) and naïve (tolDC vs. BMDC) groups. Downregulated genes included Yes1, CCL5/12/17/22/24, Kynu, TLR9/11, S100a8/a9, CD1d1, CD40, NLRP1b, IL-12b/IL-18/IL-23, Marco, and C1q(a/b/c) and indicate that both tolDCs and LPS-tolDCs demonstrate restricted innate immune activation compared with their nontolerogenic counterparts (Figure 2g and h). In addition to the elevated PDL1:CD86 ratio and the ability to suppress lymphocyte proliferation, tolDC and LPS-tolDC supernatants demonstrated increased IL-10 and suppressed IL-12p70 production (Figure 3a–d). RNA-sequencing analysis showed that TGF-β1 was elevated in LPS-tolDCs versus BMDCs and IDO1 was elevated in LPS-tolDCs versus tolDCs (P < 0.001). These findings were confirmed with increased TGF-β1 and IDO1 activity in LPS-tolDCs (Figure 3e and f; Supplementary Table S4). The presence of tolDCs or LPS-tolDCs limited tumor necrosis factor alpha, lipocalin-2, and kidney injury molecule-1 mRNA expression in RTECs after LPS exposure, albeit with differences in temporal expression profiles over 24 hours (Figure 3g). RTEC expression of IL-10, TGF-β, IDO1, and IDO2 was not significantly different between control and treatment groups over the 24-hour period (Supplementary Table S5). C56BL/6 mice undergoing renal IRI received either PBS (control), syngeneic (C57BL/6), or allogeneic (Allo, BALB/c) adoptive cell therapy perioperatively (Figure 4a). LPS-tolDC and Allo-tolDC therapy was able to limit severe AKI compared with control, with lower serum creatinine (Figure 4b), blood urea nitrogen (Supplementary Figure S1G), and weight loss (Supplementary Tables S6 and S7). Similarly, these groups demonstrated less histologic injury and cell death by terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling staining (Figure 4c–f; Supplementary Table S8). The timing of administration did not influence outcomes. We next sought to determine if adoptively transferred cells could be found in the injured kidney, which would support a location-dependent cytoprotective effect. The intrarenal CD45+ absolute cell count was similar between groups after IRI; however, a greater percentage of LPS-tolDCs were found in the kidney but not lung or spleen compared with unstimulated tolDCs (Figure 4g; Supplementary Table S9). LPS-tolDCs had greater expression of CC chemokine receptor 7 (Figure 4h) and CC chemokine receptor 4 (Supplementary Table S10) compared with unstimulated tolDCs. Inflammatory cell influx and perturbed immunologic homeostasis is a hallmark of renal IRI, and tolDCs impart a robust anti-inflammatory stimulus that mitigates inflammation through a by-stander effect.27Unger W.W. Laban S. Kleijwegt F.S. et al.Induction of Treg by monocyte-derived DC modulated by vitamin D3 or dexamethasone: differential role for PD-L1.Eur J Immunol. 2009; 39: 3147-3159Crossref PubMed Scopus (347) Google Scholar,28Nikolic T. Roep B.O. Regulatory multitasking of tolerogenic dendritic cells—lessons taken from vitamin d3-treated tolerogenic dendritic cells.Front Immunol. 2013; 4: 113Crossref PubMed Scopus (87) Google Scholar We investigated differences in immune cell populations after renal IRI with LPS-tolDCs (vs. PBS), and there was no significant difference in the absolute CD45+, CD11b+, or CD3+ cell counts between groups (Figure 5a; Supplementary Table S11). The relative proportion of Ly6G+ neutrophils and CD3−B220−NK1.1−Ly6G− myeloid cells were also unchanged (Figure 5b). The CD11b+F4/80+ myeloid population displayed 3 distinct subsets, with similar CD11bhiF4/80lo, higher CD11bhiF4/80int, and lower CD11bloF4/80hi cell counts in the LPS-tolDC group (Figure 5c; Supplementary Table S12). These 3 subsets also demonstrated distinct costimulatory molecule expression profiles (Figure 5d–f). CD11bhiF4/80lo and CD11bloF4/80hi subsets were Ly6Clo but displayed similar CD40/CD86/PD-L1 expression. A greater proportion of the CD11bhiF4/80int subset was seen in LPS-tolDCs and likely represents the recruited, activated monocyte-derived myeloid subset characterized by high Ly6C, CD40, CD80, CD86, and PD-L1 expression (Figure 5e; Supplementary Figure S2). The original dogma of tolDC treatment in transplantation assumed immunosuppression by direct action on T cells in vivo, but this was debunked after evidence that Allo-tolDCs are the antigenic source for recipient DCs,29Divito S.J. Wang Z. Shufesky W.J. et al.Endogenous dendritic cells mediate the effects of intravenously injected therapeutic immunosuppressive dendritic cells in transplantation.Blood. 2010; 116: 2694-2705Crossref PubMed Scopus (68) Google Scholar and the latter compartment must remain functional for adequate antigen presentation. Although the timeframe for our adoptive transfer experiments was considerably shorter, we used liposomal clodronate to determine if recipient DC processing of apoptotic cells was responsible for renoprotection. Treatment with clodronate alone reduced injury from renal IRI in the absence of tolDCs (Figure 6a; Supplementary Table S13), so the model was readjusted to provide a greater injury stimulus. Allo-tolDCs were renoprotective with lower serum creatinine (Figure 6b) and reduced histologic injury (Figure 6f–h) despite the depletive effects of clodronate (Figure 6c–e). Kidney mRNA expression of proinflammatory cytokines tumor necrosis factor alpha, IL-1β, IL-6, CC chemokine ligand 2, and kidney injury molecule-1, a biomarker of tubular cell injury, was all lower in LPS-tolDC- and Allo-tolDC-treated mice compared with controls (Figure 7a–f; Supplementary Table S14). Superoxide dismutase 1 and inducible nitric oxide synthase were higher in the PBS-treated groups, with transcript levels mitigated after cell therapy; however, superoxide dismutase 3 was not different between the groups (Figure 7g–i). A total of 11,685 10× Visiu