Lipopolysaccharide-pretreated plasmacytoid dendritic cells ameliorate experimental chronic kidney disease
Authors: Dong Zheng, Qi Cao, Vincent W.S. Lee, Ya Wang, Guoping Zheng, YuanMin Wang, Thian Kui Tan, Changqi Wang, Stephen I. Alexander, David C.H. Harris, Yiping Wang
Journal: Kidney International (2012)

Plasmacytoid dendritic cells play important roles in inducing immune tolerance, preventing allograft rejection, and regulating immune responses in both autoimmune disease and graft-versus-host disease. In order to evaluate a possible protective effect of plasmacytoid dendritic cells against renal inflammation and injury, we purified these cells from mouse spleens and adoptively transferred lipopolysaccharide (LPS)-treated cells, modified ex vivo, into mice with adriamycin nephropathy. These LPS-treated cells localized to the kidney cortex and the lymph nodes draining the kidney, and protected the kidney from injury during adriamycin nephropathy. Glomerulosclerosis, tubular atrophy, interstitial expansion, proteinuria, and creatinine clearance were significantly reduced in mice with adriamycin nephropathy subsequently treated with LPS-activated plasmacytoid dendritic cells as compared to the kidney injury in mice given naive plasmacytoid dendritic cells. In addition, LPS-pretreated cells, but not naive plasmacytoid dendritic cells, convert CD4+CD25- T cells into Foxp3+ regulatory T cells and suppress the proinflammatory cytokine production of endogenous renal macrophages. This may explain their ability to protect against renal injury in adriamycin nephropathy. Plasmacytoid dendritic cells play important roles in inducing immune tolerance, preventing allograft rejection, and regulating immune responses in both autoimmune disease and graft-versus-host disease. In order to evaluate a possible protective effect of plasmacytoid dendritic cells against renal inflammation and injury, we purified these cells from mouse spleens and adoptively transferred lipopolysaccharide (LPS)-treated cells, modified ex vivo, into mice with adriamycin nephropathy. These LPS-treated cells localized to the kidney cortex and the lymph nodes draining the kidney, and protected the kidney from injury during adriamycin nephropathy. Glomerulosclerosis, tubular atrophy, interstitial expansion, proteinuria, and creatinine clearance were significantly reduced in mice with adriamycin nephropathy subsequently treated with LPS-activated plasmacytoid dendritic cells as compared to the kidney injury in mice given naive plasmacytoid dendritic cells. In addition, LPS-pretreated cells, but not naive plasmacytoid dendritic cells, convert CD4+CD25- T cells into Foxp3+ regulatory T cells and suppress the proinflammatory cytokine production of endogenous renal macrophages. This may explain their ability to protect against renal injury in adriamycin nephropathy. Plasmacytoid dendritic cells (pDCs) were identified in 1958 as a cell type similar to plasma cells but lacking B cell and plasma cell markers. These cells were found in the T-cell zones of human lymphoid tissue.1.Colonna M. Trinchieri G. Liu Y.J. Plasmacytoid dendritic cells in immunity.Nat Immunol. 2004; 5: 1219-1226Crossref PubMed Scopus (1348) Google Scholar It has since been recognized that these cells correspond to circulating immature CD11c- dendritic cells (DCs) that can be induced to develop DC morphology.2.Nakano H. Yanagita M. Gunn M.D. CD11c(+)B220(+)Gr-1(+) cells in mouse lymph nodes and spleen display characteristics of plasmacytoid dendritic cells.J Exp Med. 2001; 194: 1171-1178Crossref PubMed Scopus (605) Google Scholar The murine equivalent of human pDCs are CD11c+CD11b-B220+Gr-1+ cells that are present within lymph nodes and spleen, and differ from myeloid dendritic cells (mDCs) that are CD11b+B220-. Besides these differences in phenotypic markers, pDCs and mDCs also differ from each other in terms of function. mDCs are professional antigen-presenting cells involved in T-cell activation and initiation of adaptive immune response, whereas pDCs are considered to be poor antigen-presenting cells and T-cell stimulators based on their low surface expression of major histocompatibility complex and classical costimulatory molecules. However, upon maturation with CD154 and Toll-like receptors (TLRs), pDCs can become potent antigen-presenting cells and are capable of stimulating T-cell proliferation and differentiation.3.Salio M. Palmowski M.J. Atzberger A. et al.CpG-matured murine plasmacytoid dendritic cells are capable of in vivo priming of functional CD8 T cell responses to endogenous but not exogenous antigens.J Exp Med. 2004; 199: 567-579Crossref PubMed Scopus (159) Google Scholar DCs can be licensed to become regulatory by a variety of cytokines including interleukin-10 (IL-10)/transforming growth factor-β (TGF-β),4.Steinbrink K. Wölfl M. Jonuleit H. et al.Induction of tolerance by IL-10-treated dendritic cells.J Immunol. 1997; 159: 4772-4780PubMed Google Scholar vasoactive intestinal peptide,5.Gonzalez-Rey E. Chorny A. Fernandez-Martin A. et al.Vasoactive intestinal peptide generates human tolerogenic dendritic cells that induce CD4 and CD8 regulatory T cells.Blood. 2006; 107: 3632-3638Crossref PubMed Scopus (129) Google Scholar and endogenous glycan-binding protein (galectin 1).6.Ilarregui J.M. Croci D.O. Bianco G.A. et al.Tolerogenic signals delivered by dendritic cells to T cells through a galectin-1-driven immunoregulatory circuit involving interleukin 27 and interleukin 10.Nat Immunol. 2009; 10: 981-991Crossref PubMed Scopus (351) Google Scholar pDCs are able to induce either T helper (Th)1- or Th2-type immune responses depending on the type of stimulation and the dose of antigen.7.Boonstra A. Asselin-Paturel C. Gilliet M.C. et al.Flexibility of mouse classical and plasmacytoid-derived dendritic cells in directing T helper type 1 and 2 cell development: dependency on antigen dose and differential toll-like receptor ligation.J Exp Med. 2003; 197: 101-109Crossref PubMed Scopus (482) Google Scholar Upon viral infection, pDCs produce large amounts of type I interferon and have therefore been regarded as an important component of innate immunity.8.Zuniga E.I. McGavern D.B. Pruneda-Paz J.L. et al.Bone marrow plasmacytoid dendritic cells can differentiate into myeloid dendritic cells upon virus infection.Nat Immunol. 2004; 5: 1227-1234Crossref PubMed Scopus (173) Google Scholar On the other hand, IL-3- and CD154-treated pDCs preferentially prime Th2 cells, possibly through engagement of costimulatory molecules such as OX40 ligand.9.Ito T. Amakawa R. Inaba M. et al.Plasmacytoid dendritic cells regulate Th cell responses through OX40 ligand and type I IFNs.J Immunol. 2004; 172: 4253-4259Crossref PubMed Scopus (157) Google Scholar pDCs can also be modified into both inflammatory and regulatory phenotypes by synthetic oligodeoxynucleotide containing unmethylated cytosine–guanine (CpG), and have been shown to induce allogeneic T-cell hyporesponsiveness and prolong cardiac graft survival by increasing expression of IL-10, TGF-β, and IL-6.10.Abe M. Wang Z. de Creus A. et al.Plasmacytoid dendritic cell precursors induce allogeneic T-cell hyporesponsiveness and prolong heart graft survival.Am J Transplant. 2005; 5: 1808-1819Crossref PubMed Scopus (110) Google Scholar Zwiorek et al.11.Zwiorek K. Bourquin C. Battiany J. et al.Delivery by cationic gelatin nanoparticles strongly increases the immunostimulatory effects of CpG oligonucleotides.Pharm Res. 2008; 25: 551-562Crossref PubMed Scopus (110) Google Scholar showed that CpG-stimulated pDCs can exhibit proinflammatory effects via their expression of IL-1, tumor necrosis factor-α, and interferon-γ. In contrast, pDCs modulated by lipopolysaccharide (LPS) have been shown to be capable of suppressing experimental autoimmune encephalomyelitis12.Ellestad K.K. Tsutsui S. Noorbakhsh F. et al.Early life exposure to lipopolysaccharide suppresses experimental autoimmune encephalomyelitis by promoting tolerogenic dendritic cells and regulatory T cells.J Immunol. 2009; 183: 298-309Crossref PubMed Scopus (54) Google Scholar via enhanced secretion of anti-inflammatory cytokines IL-10, TGF-β, and IL-13. Moreover human pDCs stimulated with CpG oligodeoxynucleotide have been shown to play important regulatory roles in autoimmune diseases and graft-versus-host diseases.13.Mazariegos G.V. Zahorchak A.F. Reyes J. et al.Dendritic cell subset ratio in peripheral blood correlates with successful withdrawal of immunosuppression in liver transplant patients.Am J Transplant. 2003; 3: 689-696Crossref PubMed Scopus (137) Google Scholar, 14.Waller E.K. Rosenthal H. Jones T.W. et al.Larger numbers of CD4(bright) dendritic cells in donor bone marrow are associated with increased relapse after allogeneic bone marrow transplantation.Blood. 2001; 97: 2948-2956Crossref PubMed Scopus (120) Google Scholar, 15.Farkas L. Beiske K. Lund-Johansen F. et al.Plasmacytoid dendritic cells (natural interferon- alpha/beta-producing cells) accumulate in cutaneous lupus erythematosus lesions.Am J Pathol. 2001; 159: 237-243Abstract Full Text Full Text PDF PubMed Scopus (646) Google Scholar, 16.Hadeiba H. Sato T. Habtezion A. et al.CCR9 expression defines tolerogenic plasmacytoid dendritic cells able to suppress acute graft-versus-host disease.Nat Immunol. 2008; 9: 1253-1260Crossref PubMed Scopus (250) Google Scholar, 17.Bailey-Bucktrout S.L. Caulkins S.C. Goings G. et al.Cutting edge: central nervous system plasmacytoid dendritic cells regulate the severity of relapsing experimental autoimmune encephalomyelitis.J Immunol. 2008; 180: 6457-6461Crossref PubMed Scopus (114) Google Scholar, 18.Moseman E.A. Liang X. Dawson A.J. et al.Human plasmacytoid dendritic cells activated by CpG oligodeoxynucleotides induce the generation of CD4+CD25+ regulatory T cells.J Immunol. 2004; 173: 4433-4442Crossref PubMed Scopus (534) Google Scholar pDCs may also exert effects on regulatory T cells (Tregs); a possible mediator of this effect of pDCs on Tregs is indoleamine 2,3–dioxygenase (IDO).19.Mellor A.L. Baban B. Chandler P. et al.Cutting edge: induced indoleamine 2,3 dioxygenase expression in dendritic cell subsets suppresses T cell clonal expansion.J Immunol. 2003; 171: 1652-1655Crossref PubMed Scopus (399) Google Scholar,20.Munn D.H. Sharma M.D. Hou D. et al.Expression of indoleamine 2,3-dioxygenase by plasmacytoid dendritic cells in tumor-draining lymph nodes.J Clin Invest. 2004; 114: 280-290Crossref PubMed Scopus (752) Google Scholar Whereas there is an increasing understanding of pDCs, their role in chronic renal disease remains poorly defined. Adriamycin nephropathy (AN) is a chronic kidney disease model characterized by proteinuria, progressive glomerulosclerosis, and tubulointerstitial damage, and it mimics human focal segmental glomerulosclerosis. Our previous studies have shown that reconstitution of Tregs expressing high levels of Foxp3 (forkhead box P3) in severe combined immunodeficient (SCID) mice with AN reduced glomerulosclerosis and tubular injury in comparison with control SCID mice with AN.21.Mahajan D. Wang Y. Qin X. et al.CD4+CD25+ regulatory T cells protect against injury in an innate murine model of chronic kidney disease.J Am Soc Nephrol. 2006; 17: 2731-2741Crossref PubMed Scopus (115) Google Scholar Our studies have also shown that macrophages modulated by IL-10/TGF-β or IL-13/4 are able to reduce renal injury in murine AN.22.Cao Q. Wang Y. Zheng D. et al.IL-10/TGF-beta-modified macrophages induce regulatory T cells and protect against adriamycin nephrosis.J Am Soc Nephrol. 2010; 21: 933-942Crossref PubMed Scopus (200) Google Scholar,23.Wang Y. Wang Y.P. Zheng G. et al.Ex vivo programmed macrophages ameliorate experimental chronic inflammatory renal disease.Kidney Int. 2007; 72: 290-299Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar In this study, we sought to determine whether or not adoptive transfer of splenic pDCs that were modified ex vivo to a regulatory phenotype and infused into AN mice could protect mice against progressive renal injury. Our results showed that adoptive transfer of pDCs modulated with LPS can in fact protect mice against AN, and suggested two novel mechanisms of protection. An increase in Foxp3+ Tregs by pDCs was observed and the induction of Tregs was found to be IDO dependent. In addition, proinflammatory cytokine production by endogenous renal macrophages was decreased in mice treated with LPS-pretreated pDCs. To characterize the phenotype of pDCs, the expression of the B7-H family of molecules in naive pDCs and LPS-pretreated pDCs was assessed. LPS-pretreated pDCs expressed higher levels of B7-H1 but not B7-H2, B7-H3, or B7-H4 than did naive pDCs (Figure 1; B7-H1 expression relative to β-actin, naive pDCs vs. LPS-pretreated pDCs 1.5±0.3 vs. 13.2±2.7, P<0.01). In addition, to determine the function of pDCs, the cytokine expression of pDCs was assessed; there was stronger expression of IL-10, TGF-β, and IDO but not IL-12 in LPS-pretreated pDCs and CpG-pretreated pDCs compared with naive pDCs (IDO mRNA expression relative to β-actin naive pDC vs. LPS-pretreated pDC 1.8±0.4 vs. 7.2±1.6, P<0.01). Also, the protein level of IL-10 and TGF-β of supernatant and cellular IDO from cultured LPS-pretreated pDCs was much higher than those from cultured naive pDCs (P<0.01; Figure 1d). As TLR4 functions as a LPS-sensing receptor, we tested TLR4 expression and found that TLR4 was expressed on significantly more LPS-pretreated pDCs than naive pDCs (0.6±0.1% vs. 18.2±2.7%, P<0.01; Figure 2). At 4 weeks after adriamycin, we examined the effect of pDCs on renal structural injury. In AN, renal injury is characterized by glomerulosclerosis, tubular atrophy, interstitial volume expansion, and interstitial fibrosis. Glomerulosclerosis was significantly reduced in AN mice transfused with LPS-pretreated pDCs compared with AN mice transfused with naive pDCs and untransfused AN mice (Figure 3). Similarly, tubulointerstitial injury was significantly improved in AN mice transfused with LPS-pretreated pDCs compared with mice transfused with naive pDC and untransfused AN mice (Figure 3; normal vs. AN control vs. AN+naive pDC vs. AN+LPS-pretreated pDC: glomerulosclerosis 2.3±0.3 vs. 28.0±4.6 vs. 28.4±3.7 vs. 12.6±1.8%, P<0.01, tubular cell height 7.3±0.4 vs. 1.5±0.2 vs. 1.1±0.3 vs. 5.7±0.9μm, P<0.05, interstitial volume 1.8±0.6 vs. 26.8±1.1 vs. 28.3±2.5 vs. 12.6±1.0%, P<0.01, interstitial fibrosis 2.3±0.6 vs. 20.1±0.5 vs. 18.9±1.4 vs. 12.3±1.0%, P<0.01). These results indicate that LPS-pretreated pDCs protected against renal injury in AN. To explore the effect of pDCs on renal function, serum creatinine and urinary protein were measured in each group at 4 weeks after adriamycin. In AN mice, serum creatinine and urinary protein excretion were significantly increased compared with normal mice, and were significantly improved in AN mice transfused with LPS-pretreated pDCs compared with untransfused AN mice or those transfused with naive pDCs. Creatinine clearance was also improved after pDC transfusion (normal vs. AN vs. AN+naive pDC vs. AN+LPS-pretreated pDC: serum creatinine 5.1±0.5 vs. 16.5±2.3 vs. 18.7±3.8 vs. 11.5±1.4μmol/l, P<0.05; urinary protein 50.7±26.3 vs. 403.5±104.3 vs. 458.8±49.2 vs. 134.5±23.6μg/12h, P<0.01; creatinine clearance 34.0±4.1 vs. 14.6±1.5 vs. 15.1±2.6 vs. 24.5±3.0μl/min, P<0.05; urine protein/urine creatinine ratio 20.8±2.6 vs. 938.4±98.4 vs. 1068.5±123.4 vs. 551.1±76.2μg/mmol, P<0.01; Figure 3). In AN, focal segmental glomerulosclerosis is accompanied by interstitial inflammation. Therefore, renal leukocyte infiltration at week 4 after adriamycin was examined using anti-F4/80, -CD4, and -CD8 antibodies. The numbers of macrophages, CD4+ T cells, and CD8+ T cells were increased in all AN mice. However, the numbers of these cells were markedly reduced in AN mice transfused with LPS-pretreated pDCs compared with untransfused AN mice and AN mice transfused with naive pDCs (Figure 4). To determine whether a difference in access to organs of interest explained the effects of LPS-pretreated vs. naive pDCs, tracking studies were performed. At day 28, fluorescently labeled cells were seen in kidney, kidney-draining lymph nodes (KDLNs), and spleen of AN mice transfused with LPS-pretreated pDCs and naive pDCs. Both DiI-stained LPS-pretreated pDCs and naive pDCs were located mostly in kidney interstitium and in paracortex of KDLNs in similar numbers (Figure 5). Thus, the protective effect of LPS-pretreated pDCs vs. naive pDCs was not explained by a difference in access. To further explore the mechanism by which LPS-pretreated pDCs was protective in AN, the number of Foxp3+ CD4+ cells was examined and found to be significantly increased in KDLNs and kidney of AN mice transfused with LPS-pretreated pDCs compared with those treated with naive pDCs or untransfused AN mice (naive pDCs vs. LPS-pretreated pDCs in KDLNs 6.67±0.8% vs. 18.69±2.5%, P<0.01; Figure 6a and b). In vitro, LPS-pretreated pDCs significantly increased the percentage of Foxp3+ T cells when cocultured with CD4+CD25- T cells for 7 days, whereas naive pDCs had no effect. This effect was found to be contact dependent in Transwell experiments (Figure 6c and d). The blockade of IDO expression on modified DCs with 1-methyl-D-tryptophan (D-1MT) eliminated induction of Foxp3+ T cells by LPS-pretreated pDCs, whereas blockade with antibodies against IL-10, TGF-β, or B7-H1 had no effect (Figure 6e). This suggested that at least part of the protective effect of LPS-pretreated pDCs in AN could be due to induction of Tregs in KDLNs and kidney in an IDO-dependent manner. In addition to induction of Foxp3+ Tregs, we explored other potential protective mechanisms by examining the interaction of pDCs with macrophages both in vitro and in vivo. LPS-pretreated pDCs suppressed proinflammatory cytokine production of effector macrophages (M1) in vitro and in vivo as shown by suppression of mRNA expression of proinflammatory cytokines tumor necrosis factor-α, IL-12, and inducible nitric oxide synthase, whereas naive pDCs had no effect. However, there were no differences in the expression of markers of protective macrophage (M2) mannose receptor, arginase, IL-10, and TGF-β on M1 cocultured with naive pDCs versus LPS-pretreated pDCs (Figure 7a). Endogenous macrophages isolated from kidneys of each of the four groups were examined. Similar to the in vitro studies, the mRNA expression of tumor necrosis factor-α, IL-12, and inducible nitric oxide synthase of endogenous kidney macrophages was reduced significantly in AN mice receiving LPS-pretreated pDCs, but not naive pDCs, and there was no change in expression of M2 macrophages markers (Figure 7b). Together, these data suggested a further mechanism by which LPS-pretreated pDCs could prevent renal injury, that is, by deactivation of host macrophages. In this study, the effects of splenic pDCs were examined in murine AN. We demonstrated that splenic pDCs are able to reduce renal structural and functional injury and monocyte infiltration in AN. The mechanisms by which pDCs protected against renal injury appeared to involve their ability to convert CD4+CD25- T cells into CD4+CD25+Foxp3+ Tregs in KDLNs and kidney via increasing the expression of IDO and to suppress proinflammatory cytokine production of endogenous renal macrophages. DCs largely reside in renal tubulointerstitium, forming an intricate immune sentinel network.24.Kruger T. Benke D. Eitner F. et al.Identification and functional characterization of dendritic cells in the healthy murine kidney and in experimental glomerulonephritis.J Am Soc Nephrol. 2004; 15: 613-621Crossref PubMed Scopus (212) Google Scholar A quantitative evaluation of DC subsets has shown that both mDCs and pDCs participate in inflammatory renal injury,25.Woltman A.M. de Fijter J.W. Zuidwijk K. et al.Quantification of dendritic cell subsets in human renal tissue under normal and pathological conditions.Kidney Int. 2007; 71: 1001-1008Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar suggesting that both mDCs and pDCs play a role in tubulointerstitial injury under pathophysiological conditions. In addition, a significant number of pDCs have been found at sites of inflammation in lupus nephritis. Inhibition of interferon-α produced by pDCs with a specific TLR7 inhibitor IRS954 has been shown to ameliorate disease progression in lupus-prone mice.26.Tucci M. Calvani N. Richards H.B. et al.The interplay of chemokines and dendritic cells in the pathogenesis of lupus nephritis.Ann NY Acad Sci. 2005; 1051: 421-432Crossref PubMed Scopus (41) Google Scholar Moreover, Coates et al.27.Coates P.T. Duncan F.J. Colvin B.L. et al.In vivo-mobilized kidney dendritic cells are functionally immature, subvert alloreactive T-cell responses, and prolong organ allograft survival.Transplantation. 2004; 77: 1080-1089Crossref PubMed Scopus (61) Google Scholar demonstrated that Fms-like tyrosine kinase 3 ligand increased the number of CD11c+CD45RA+ pDCs in glomeruli and interstitium of kidneys and that these pDCs were functionally immature and exhibited suppressive features. In general, pDCs play crucial roles in the development and resolution of renal inflammation. DCs are able to induce dynamic alterations in their function and phenotype to induce or suppress immune responses.28.John R. Nelson P.J. Dendritic cells in the kidney.J Am Soc Nephrol. 2007; 18: 2628-2635Crossref PubMed Scopus (67) Google Scholar For example, depletion of renal DCs enhanced renal injury in nephrotoxic nephritis29.Scholz J. Lukacs-Kornek V. Engel D.R. et al.Renal dendritic cells stimulate IL-10 production and attenuate nephrotoxic nephritis.J Am Soc Nephrol. 2008; 19: 527-537Crossref PubMed Scopus (101) Google Scholar,30.Tadagavadi R.K. Reeves W.B. Renal dendritic cells ameliorate nephrotoxic acute kidney injury.J Am Soc Nephrol. 2010; 21: 53-63Crossref PubMed Scopus (120) Google Scholar and impaired recovery from ischemia/reperfusion injury,31.Kim M.G. Su B.C. Sook K.Y. et al.Depletion of kidney CD11c+ F4/80+ cells impairs the recovery process in ischaemia/reperfusion-induced acute kidney injury.Nephrol Dial Transplant. 2010; 25: 2908-2921Crossref PubMed Scopus (72) Google Scholar indicating a protective role of DCs. However, depletion of renal activated DCs rapidly resolved established renal immunopathology in a mouse model of glomerular injury, suggesting a facilitatory role for DCs in the progression of renal diseases.32.Heymann F. Meyer-Schwesinger C. Hamilton-Williams E.E. et al.Kidney dendritic cell activation is required for progression of renal disease in a mouse model of glomerular injury.J Clin Invest. 2009; 119: 1286-1297Crossref PubMed Scopus (177) Google Scholar In the study of nephrotoxic nephritis, all DCs were depleted at an early point of disease and therefore the resident DCs were also removed.30.Tadagavadi R.K. Reeves W.B. Renal dendritic cells ameliorate nephrotoxic acute kidney injury.J Am Soc Nephrol. 2010; 21: 53-63Crossref PubMed Scopus (120) Google Scholar It is possible that depletion of only these resident DCs was responsible for the worse renal injury. Thus, the effect of DCs in kidney may depend on the particular type of renal disease (for example, immune vs. nonimmune) or the functional state of renal DCs (for example, activated or resting; mature or immature). An alternative way to clarify the role of DCs in renal disease is the use of adoptive transfer. We adoptively transferred unstimulated (naive) and pDCs stimulated with LPS into AN mice, and showed protective effects with stimulated but not naive pDCs. pDCs modified by CpG have been shown to induce allogeneic T-cell hyporesponsiveness and prolong cardiac graft survival.10.Abe M. Wang Z. de Creus A. et al.Plasmacytoid dendritic cell precursors induce allogeneic T-cell hyporesponsiveness and prolong heart graft survival.Am J Transplant. 2005; 5: 1808-1819Crossref PubMed Scopus (110) Google Scholar Furthermore, pDCs modified by LPS have also been shown to be capable of suppressing experimental autoimmune encephalomyelitis.12.Ellestad K.K. Tsutsui S. Noorbakhsh F. et al.Early life exposure to lipopolysaccharide suppresses experimental autoimmune encephalomyelitis by promoting tolerogenic dendritic cells and regulatory T cells.J Immunol. 2009; 183: 298-309Crossref PubMed Scopus (54) Google Scholar In this study, we examined the suppressive capacity of both LPS-pretreated and naive pDCs. pDCs pretreated with LPS showed high expression of B7-H1, IDO, and the anti-inflammatory cytokines IL-10 and TGF-β. Both CpG- and LPS-pretreated pDCs expressed high levels of B7-H1. B7-H1 has been shown to downregulate T-cell activation.33.Gao W. Demirci G. Strom T.B. et al.Stimulating PD-1-negative signals concurrent with blocking CD154 co-stimulation induces long-term islet allograft survival.Transplantation. 2003; 76: 994-999Crossref PubMed Scopus (140) Google Scholar,34.Ozkaynak E. Wang L. Goodearl A. et al.Programmed death-1 targeting can promote allograft survival.J Immunol. 2002; 169: 6546-6553Crossref PubMed Scopus (201) Google Scholar Increasing B7-H1 expression in pDCs has been shown to induce allogeneic T-cell hyporesponsiveness and prolong graft survival.10.Abe M. Wang Z. de Creus A. et al.Plasmacytoid dendritic cell precursors induce allogeneic T-cell hyporesponsiveness and prolong heart graft survival.Am J Transplant. 2005; 5: 1808-1819Crossref PubMed Scopus (110) Google Scholar pDCs also express high levels of IDO after LPS or CpG stimulation. IDO is a component of the enzymatic pathway involved in the catabolism of tryptophan. Tryptophan depletion has been shown to suppress proliferation of activated T cells.35.Munn D.H. Mellor A.L. Indoleamine 2,3-dioxygenase and tumor-induced tolerance.J Clin Invest. 2007; 117: 1147-1154Crossref PubMed Scopus (856) Google Scholar,36.Munn D.H. Zhou M. Attwood J.T. et al.Prevention of allogeneic fetal rejection by tryptophan catabolism.Science. 1998; 281: 1191-1193Crossref PubMed Scopus (2164) Google Scholar The role of IDO in regulatory function of pDCs has been addressed previously.37.Fallarino F. Asselin-Paturel C. Vacca C. et al.Murine plasmacytoid dendritic cells initiate the immunosuppressive pathway of tryptophan catabolism in response to CD200 receptor engagement.J Immunol. 2004; 173: 3748-3754Crossref PubMed Scopus (187) Google Scholar CpG oligodeoxynucleotide activation of pDCs significantly increased their expression of IDO and resulted in the generation of inducible Tregs from CD4+CD25- T cells.38.Chen W. Liang X. Peterson A.J. et al.The indoleamine 2,3-dioxygenase pathway is essential for human plasmacytoid dendritic cell-induced adaptive T regulatory cell generation.J Immunol. 2008; 181: 5396-5404Crossref PubMed Scopus (363) Google Scholar In this study, we found that LPS-modulated pDCs also expressed high levels of IDO. It has been shown that pDC were able to suppress autoimmune encephalomyelitis following their exposure to LPS,12.Ellestad K.K. Tsutsui S. Noorbakhsh F. et al.Early life exposure to lipopolysaccharide suppresses experimental autoimmune encephalomyelitis by promoting tolerogenic dendritic cells and regulatory T cells.J Immunol. 2009; 183: 298-309Crossref PubMed Scopus (54) Google Scholar and to induce tolerance in experimental models of asthma, and in allogeneic hematopoietic stem cell, heart, and liver transplantation.10.Abe M. Wang Z. de Creus A. et al.Plasmacytoid dendritic cell precursors induce allogeneic T-cell hyporesponsiveness and prolong heart graft survival.Am J Transplant. 2005; 5: 1808-1819Crossref PubMed Scopus (110) Google Scholar, 39.de Heer H.J. Hammad H. Soullie T. et al.Essential role of lung plasmacytoid dendritic cells in preventing asthmatic reactions to harmless inhaled antigen.J Exp Med. 2004; 200: 89-98Crossref PubMed Scopus (665) Google Scholar, 40.Arpinati M. Chirumbolo G. Urbini B. et al.Role of plasmacytoid dendritic cells in immunity and tolerance after allogeneic hematopoietic stem cell transplantation.Transpl Immunol. 2003; 11: 345-356Crossref PubMed Scopus (60) Google Scholar, 41.Tokita D. Mazariegos G.V. Zahorchak A.F. et al.High PD-L1/CD86 ratio on plasmacytoid dendritic cells correlates with elevated T-regulatory cells in liver transplant tolerance.Transplantation. 2008; 85: 369-377Crossref PubMed Scopus (129) Google Scholar However, whether pDCs can be used to treat chronic kidney disease is unknown. This study, for first time, showed that LPS-pretreated pDCs can reduce glomerulosclerosis, tubulointerstitial injury, and interstitial fibrosis, and improve renal function in a model of chronic proteinuric renal disease. pDCs, both naive and pretreated with LPS, were able to reach the site of injury within kidneys, and to migrate to KDLNs. However, pDCs modified with LPS ex vivo into a regulatory phenotype, but not unstimulated pDCs, could ameliorate renal injury and reduce renal infiltration with inflammatory cells. Thus, it is their phenotype and not access to sites of inflammation that distinguishes LPS-pretreated pDCs from naive pDCs, and determines their protective effect. In this study, LPS-pretreated pDCs showed inhibitory functions, yet the mechanism underlying pDC modulation by LPS into a regulatory phenotype is unknown. We found that some pDCs after LPS treatment had increased expression of the LPS receptor TLR4, consistent with its involvement in the effect of LPS on pDCs. This result is in line with that of others investigators who showed that TLR4 was significantly increased after LPS in murine liver and thymic pDCs.42.Castellaneta A. Sumpter T.L. Chen L. et al.NOD2 ligation subverts IFN-alpha production by liver plasmacytoid dendritic cells and inhibits their T cell allostimulatory activity via B7-H1 up-regulation.J Immunol. 2009; 183: 6922-6932Crossref PubMed Scopus (70) Google Scholar,43.Okada T. Lian Z.X. Naiki M. et al.Murine thymic plasmacytoid dendritic cells.Eur J Immunol. 2003; 33: 1012-1019Crossref PubMed Scopus (66) Google Scholar However, in our study and similar to that of others, only 18% of pDCs expressed TLR4, suggesting that the effects of LPS might be mediated by other unknown pa