BASIC RESEARCH www.jasn.org Thymic Deletion and Regulatory T Cells Prevent Antimyeloperoxidase GN † ‡ Diana S.Y. Tan,* Poh Y. Gan,* Kim M. O’Sullivan,* Maree V. Hammett, Shaun A. Summers,* † ‡ Joshua D. Ooi,* Brita A. Lundgren,§ Richard L. Boyd, Hamish S. Scott,§ A. Richard Kitching,* † ‡ Ann P. Chidgey, and Stephen R. Holdsworth* *Centre for Inflammatory Diseases, Monash University Department of Medicine, Clayton, Victoria, Australia; †Immune Regeneration Laboratory, Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, Australia; ‡Department of Nephrology, Monash Medical Centre, Clayton, Victoria, Australia; and §Department of Molecular Pathology, Centre for Cancer Biology, SA Pathology, Adelaide, South Australia, Australia ABSTRACT Loss of tolerance to neutrophil myeloperoxidase (MPO) underlies the development of ANCA-associated vasculitis and GN, but the mechanisms underlying this loss of tolerance are poorly understood. Here, we assessed the role of the thymus in deletion of autoreactive anti-MPO T cells and the importance of pe- ripheral regulatory T cells in maintaining tolerance to MPO and protecting from GN. Thymic expression of MPO mRNA predominantly localized to medullary thymic epithelial cells. To assess the role of MPO in forming the T cell repertoire and the role of the autoimmune regulator Aire in thymic MPO expression, we 2/2 2/2 compared the effects of immunizing Mpo mice, Aire mice, and control littermates with MPO. 2/2 2/2 Immunized Mpo and Aire mice developed significantly more proinflammatory cytokine-producing anti-MPO T cells and higher ANCA titers than control mice. When we triggered GN with a subnephrito- 2/2 genic dose of anti-glomerular basement membrane antibody, Aire mice had more severe renal disease +/+ than Aire mice, consistent with a role for Aire-dependent central deletion in establishing tolerance to MPO. Furthermore, depleting peripheral regulatory T cells in wild-type mice also led to more anti-MPO T cells, higher ANCA titers, and more severe GN after immunization with MPO. Taken together, these results suggest that Aire-dependent central deletion and regulatory T cell–mediated peripheral tolerance both play major roles in establishing and maintaining tolerance to MPO, thereby protecting against the development of anti-MPO GN. J Am Soc Nephrol 24: 573–585, 2013. doi: 10.1681/ASN.2012090898 Systemic autoimmunity to myeloperoxidase system to discriminate between self and non-self (MPO) is directly involved in causing the glomeru- antigens. Central tolerance involves thymic deletion lar and vascular inflammation of ANCA-associated of thymocytes with high-affinity interactions be- pauci-immune necrotizing autoimmune anti-MPO tween the T cell receptor and self-peptide MHC GN (AIMPOGN).1–3 ANCA induces neutrophil ac- complexes, preventing many potentially autoreac- tivation and endothelial cell adhesion, with the re- tive T cells from entering the periphery.9 The role of lease of neutrophil extracellular traps containing 4, MPO and proteases triggering endothelial injury. Received September 10, 2012. Accepted December 21, 2012. 5 Experimental studies demonstrate that autoim- Published online ahead of print. Publication date available at mune anti-MPO CD4+ T cells respond to glomer- www.jasn.org. ular MPO deposited by degranulating neutrophils, Correspondence: Prof. Stephen R. Holdsworth, Centre for In- directing injurious delayed type hypersensitivity flammatory Diseases, Monash University Department of Medi- (DTH)–mediated injury.6–8 cine, Monash Medical Centre, 246 Clayton Road, Clayton, VIC Immunologic tolerance is maintained by central 3168, Australia. Email: [email protected] and peripheral mechanisms, allowing the immune Copyright © 2013 by the American Society of Nephrology J Am Soc Nephrol 24: 573–585, 2013 ISSN : 1046-6673/2404-573 573 BASIC RESEARCH www.jasn.org central tolerance in the maintenance of tolerance to the po- low-expressing mTECs (mTEC-lo) (Figure 1B). Expression tential kidney autoantigen, MPO, is largely unknown. The in mTEC-hi is consistent with the known critical involvement autoimmune regulator (Aire) transcription factor is impor- of these cells in the development of the T cell repertoire.31 tant for the induction and regulation of tolerance.10–12 Aire Given that Aire is expressed only on the CD452 nonhemato- is primarily found in lymphoid organs, particularly in the poietic population, and predominantly by the mature mTECs thymus where it is predominantly found in the nuclei of (mTECs-hi) (Figure 1, C and D), we determined whether 2 2 mature, highly MHC II–expressing13–15 medullary thymic MPO expression would be altered in Aire / mice. We found epithelial cells (mTECs).16,17 Aire promotes the promiscu- that MPO expression was almost absent from the mTEC-hi 2 2 ous expression of tissue-restricted antigens (TRAs) in and mTEC-lo cells in Aire / mice (Figure 1B), suggesting a mTECs.13,16–18 However, the mechanisms by which Aire strong association with Aire-dependent peripheral antigen ex- controls the presentation of TRA expression in mTECs and pression. its effect on tolerance and autoimmunity remain to be fully defined. MPO Protein Expression Confined to Neutrophils and Despite central tolerance, some autoreactive cells escape Macrophages the selection process, entering the periphery where they may The presence of MPO protein was determined by immunohis- cause autoimmunity if activated.19,20 Naturally arising CD4 tology staining. We examined the thymi of 5-week-old Mpo+/+, 2 2 2 2 +CD25+Foxp3+ regulatory T cells (Tregs), mainly produced Mpo / , and Aire / mice (Figure 2, A–C, respectively). MPO 2 2 by the thymus by high-affinity interactions with thymic ep- protein is present in Mpo+/+ and Aire / mice, but as expected, 2 2 ithelial cells,21 are a distinct T cell population that plays a absent from Mpo / thymi (Figure 2, A–C). The MPO distri- pivotal role in the maintenance of self-tolerance. Several bution was along the corticomedullary junction. Colocaliza- studies demonstrate the importance of Tregs in the preven- tion studies demonstrated MPO localization to neutrophils tion of organ-specific autoimmunity by potently sup- (Figure 2, D–F) and macrophages (Figure 2, G–I) in the thymic pressing autoreactive T cells in a contact-dependent and compartment of all five thymus samples. Confocal immuno- cytokine-independent manner.22–26 Depletion of Tregs leads fluorescence microscopy revealed that all neutrophils and some to the spontaneous development of some autoimmune dis- macrophages are MPO positive. It was also evident that most of eases.27–29 the MPO positive cells are neutrophils. To assess the role of central and peripheral tolerance in regulating the development of autoimmunity to MPO, we Thymic Aire-Dependent MPO Expression Deletes used a validated model of MPO-induced autoimmunity.6,7,30 Potentially Autoreactive Anti-MPO T Cells and Protects Establishment of anti-MPO autoimmunity directs the devel- against AIMPOGN opment of focal necrotizing GN similar to that seen in human We assessed MPO-specific cell-mediated immune responses ANCA-associated GN. Our studies demonstrate the impor- with regard to the Th1 and Th17 subset differentiations in tance of both central and peripheral mechanisms in maintain- lymph node cells (LNs) draining the sites of MPO immuni- ing tolerance to MPO. Aire promotes thymic MPO expression zation and serum levels of MPO-ANCAtiters in groups of wild- 2 2 2 2 2 2 and enhances central deletion of autoreactive anti-MPO T type (WT), Mpo / ,andAire / mice. In both Mpo / and 2 2 cells, whereas peripheral Tregs suppress potentially autoreac- Aire / mice, the frequencies of anti-MPO autoreactive CD4+ tive MPO-specific CD4+ T cells. Both mechanisms limit anti- T cells by enzyme-linked immunosorbent spot (ELISPOT) MPO GN. assay and the titers of circulating MPO-ANCA were increased compared with Mpo+/+ (Figure3,A,B,F–H) and Aire+/+ lit- termate WT controls (Figure 3, C and I). The dermal DTH 2 2 RESULTS response to MPO was also increased in Aire / mice (Figure 3D). A control protein, ovalbumin (OVA) in Freund’scom- MPO mRNA Is Predominantly Expressed by MHC II– plete adjuvant (FCA), did not induce anti-MPO autoimmu- Expressing Medullary Thymic Epithelial Cells in an Aire- nity in Mpo+/+ mice. Circulating anti-MPO lgG, lgG1, and Dependent Manner lgG2c levels (Figure 3, E–G) were detected in normal Mpo+/+ After enzymatic digestion of thymic tissue and flow cytometric mice after MPO immunization (not in OVA-immunized mice), 2 2 2 2 sorting of thymic stromal cell (TSC) subsets, transcripts for but were significantly higher in Mpo / and Aire / mice (Fig- 2 2 2 2 MPO were detected in the Mpo+/+ mice, but not in Mpo / ure 3H). There was no difference between Mpo+/+ and Mpo / mice, which served as a negative control. Within the mouse mice in the frequency of Foxp3+ expression of the CD4+ T cells thymus, MPO mRNA is highly expressed in the CD452 TSC isolated from LNs draining the sites of injection (Figure 3, I and subpopulation, but was only minimally detected in the CD45+ J).TheabsenceofthymicMPOandAireaffectedtheinducible thymic hematopoietic subpopulation (Figure 1A). Of the repertoire of autoreactive anti-MPO CD4+ T cells and the levels CD452 population, the major cell subpopulation expressing of autoantibody in a manner consistent with a role for thymic MPO mRNA was the mTECs. MPO was expressed in both the expression of MPO in deleting potentially autoreactive anti-MPO MHC II high-expressing mTECs (mTECs-hi) and MHC II CD4+ T cells. 574 Journal of the American Society of Nephrology J Am Soc Nephrol 24: 573–585, 2013 www.jasn.org BASIC RESEARCH Figure 1. Expression of MPO and Aire mRNA by thymic cell populations, including hematopoietic TECs (CD45+), nonhematopoietic TECs (CD452), medullary TECs (mTECs), cortical TECs (cTECs), and non-TECs (nTECs) with either high (hi) or low (lo) coexpression of MHC II, and dendritic cells (DCs). (A) MPO is predominantly expressed on nonhematopoietic populations in the Mpo+/+ thymus. (B) 2 2 MPO is predominantly seen in MHC II coexpressing mTECs in the WT mice compared with the Aire / mice.