Cysteinyl cathepsin K (CatK) is expressed in osteoclasts to mediate bone resorption, but is also inducible under inflammatory conditions. Faslpr mice on a C57BL/6 background develop spontaneous systemic lupus erythematosus-like manifestations. Although normal mouse kidneys expressed negligible CatK, those from Faslpr mice showed elevated CatK expression in the glomeruli and tubulointerstitial space. Faslpr mice also showed elevated serum CatK levels. CatK deficiency in Faslpr mice reduced all tested kidney pathologies, including glomerulus and tubulointerstitial scores, glomerulus complement C3 and IgG deposition, chemokine expression and macrophage infiltration, and serum autoantibodies. CatK contributed to Faslpr mouse autoimmunity and pathology in part by its activity in TLR-7 proteolytic processing and consequent regulatory T (Treg) cell biology. Elevated TLR7 expression and proteolytic processing in Faslpr mouse kidneys and Tregs showed significantly reduced levels in CatK-deficient mice, leading to increased spleen and kidney Treg content. Purified CD4+CD25highFoxp3+ Tregs from CatK-deficient mice doubled their immunosuppressive activity against T effector cells, compared with those from CatK-sufficient mice. In Faslpr mice, repopulation of purified Tregs from CatK-sufficient mice reduced spleen sizes, autoantibody titers, and glomerulus C3 and IgG deposition, and increased splenic and kidney Treg contents. Tregs from CatK-deficient mice had significantly more potency than CatK-sufficient Tregs in reducing spleen sizes, serum autoantibody titers, and glomerulus C3 deposition, and in increasing splenic and kidney Treg content. This study established a possible role of CatK in TLR7 proteolytic activation, Treg immunosuppressive activity, and lupus autoimmunity and pathology.

Cathepsin K (CatK) is a lysosomal cysteine protease that is expressed primarily in osteoclasts (13). CatK deficiency leads to defects in bone resorption in humans and mice (4, 5). This protease, however, is also inducible under inflammatory conditions. For example, normal human aortas or primary cultured vascular cells show negligible expression of CatK, but its expression greatly increases in vascular cells in atherosclerotic human lesions and abdominal aortic aneurysms (69). Besides its prominent roles in bone resorption, CatK also participates in the pathogenesis of many other diseases that do not associate with its activity in osteoclasts, including atherosclerosis (8, 10), lung fibrosis (11), obesity and diabetes (12, 13), abdominal aortic aneurysm (14), learning and memory deficits (15), and ischemia (16).

CatK participates in the expression of most of these diseases through its activity on extracellular matrix protein degradation. Yet, recent data implicates this protease in the proteolytic activation of TLR-7 or TLR9 within the intracellular endolysosomes of macrophages (17), and dendritic cells (18, 19). Viral nucleic acids target activated TLR7 and TLR9, which share signaling pathways (20, 21). Ligand binding of TLR7 and TLR9 leads to the phosphorylation of downstream signaling adaptor molecule MyD88 and consequently the activation of NF-κB and IFN regulatory factor pathways that remain responsible for the production of inflammatory cytokines and type-1 IFNs (IFN-α and IFN-β), respectively (22, 23). Although no direct evidence currently exists, data suggest CatK activity in the proteolytic activation of TLR7, TLR8, and TLR9 plays a role in arthritis, experimental autoimmune encephalomyelitis (EAE), and psoriasis by inducing the production of IL-12, IL-6, and IL-23 by dendritic cells (18, 19). Inflammatory cytokines and type-1 IFNs interfere with regulatory T cell (Treg) function (24), Treg Foxp3 expression, and Treg immunosuppressive activity (25). These inflammatory molecules also increase the resistance of effector T cells (Teff) to Treg-mediated suppression (24), induce plasma cell and Teff differentiation (26), contribute to B-cell hyper-reactivity and autoantibody production (2628), and stimulate kidney mesangial cell proliferation (29), all of which contribute to systemic lupus erythematosus (SLE)–associated autoimmunity and pathology.

SLE, a prototypic systemic autoimmune disease, possesses B cell hyper-reactivity and production of autoantibodies against self-nuclear proteins and nucleic acids (30, 31). Although the role of TLR9 in SLE remains controversial (3234), TLR7 can promote the expression of SLE. Deficiency of TLR7 (32) or its signaling adaptor MyD88 (33) confers protection against autoimmunity in lupus-prone mice. TLR7-deficient mice display reduced autoantibodies against Smith Ag, and reduced lymphoid organ weight, CD4+ T cells, CD44+ and CD69+ B cells, and renal disease (32).

This study used both CatK-deficient and lupus-prone C57BL/6 Faslpr (B6.Faslpr) mice to test whether CatK plays a role in lupus autoimmunity and pathology. CatK-deficient B6.Faslpr mice displayed a less cleaved form of TLR7 and better functioning and suppressed glomerulonephritis and autoimmunity than CatK-sufficient B6.Faslpr mice.

Faslpr mice (C57BL/6, N11, The Jackson Laboratory, Bar Harbor, ME) were crossbred with Ctsk−/− (C57BL/6/129S) (5) or Ctss−/− mice to produce female FaslprCtsk−/− or FaslprCtss−/− mice and their littermate female FaslprCtsk+/+ control mice. To perform Treg adoptive transfer in FaslprCtsk+/+ mice, splenic CD4+CD25+Tregs from wild-type (WT) or Ctsk−/− mice were purified using magnetic beads according to the manufacturer’s instructions (Miltenyi Biotec, San Diego, CA). The resulting CD4+CD25+Tregs were further purified with a cell sorter (the BD FACSAria Cell Sorter; BD Biosciences, San Jose, CA). Treg purity was confirmed by both FACS analysis and anti-Foxp3 Ab-mediated immunostaining. Each 9-wk old female FaslprCtsk+/+ recipient mouse received tail-vein adoptive transfer of 5 × 106 donor Tregs. The biweekly collection of blood biweekly samples began 3 wk after the adoptive transfer for 12 wk. At the age of 24 wk, mice were sacrificed, splenocytes were analyzed for CD4, CD25, and Foxp3 by FACS, and kidneys were collected for immunofluorescent double staining to detect CD4+Foxp3+ Tregs. All mouse procedures conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health and were approved by the Harvard Medical School Standing Committee on Animals.

Mouse kidney tissues were fixed overnight in 4% paraformaldehyde and paraffin embedded. Serial sections (6 μm) were prepared for periodic acid Schiff or H&E, or processed for immunohistochemical studies by immunoperoxidase or alkaline phosphatase anti-alkaline phosphatase techniques (35). The severity of renal lesions in murine lupus nephritis was graded from 0 to 3 (from normal, mild, moderate, or severe) using the activity index described for human lupus nephritis (36). For each mouse, at least 15 glomeruli, tubular, or interstitial areas were graded and evaluated for glomerular cellularity, infiltrating leukocytes, mesangial matrix expansion, crescent formation, interstitial mononuclear cell infiltrates in the medulla and cortex, hyaline deposits, fibrinoid necrosis, and tubular atrophy. Glomerular or tubulointerstitial scores for each mouse were calculated as the mean of the summed individual scores for each image, with scores for necrosis and crescent formation weighted by a factor of two.

For immunohistochemical analysis, primary Abs included the following: rabbit anti–human CatK (1:90; Biovision, Milpitas, CA) (2), rat anti–mouse macrophage (Mac-2, 1:1000; BD Biosciences), hamster anti–mouse MCP-1 (1:50; BD Biosciences), and MHC class II (1:100; BD Biosciences). Positive Mac-2+ cells were counted in 10 consecutive visual fields at the same magnification and presented as a positive number per square millimeters. The MCP-1 and MHC class II were measured as the immunostaining signal-positive area. Paraffin serial sections were also used for immunofluorescent staining to detect CD4+Foxp3+Tregs (CD4, 1:250; Abnova, Walnut, CA, and Foxp3, 1:100; eBioscience, San Diego, CA) followed by Alex Fluor 568 or 488-labeled secondary Ab detection. Sections were analyzed with a confocal microscopy for subcellular localization of Alex Fluor 568 or 488 (Olymbus Fluoview FV1000; Olymbus).

Kidney frozen sections (5 μm) were prepared for immunofluorescent staining using anti-mouse IgG and C3 Abs (1:250 and 1:100; Invitrogen, Carlsbad, CA). Stained specimens were then observed under a fluorescent microscope. Fluorescence intensity was graded as 0–3 (from normal, mild, moderate, or bright).

Serum autoantibodies were assessed by ELISA as described (37). NUNC maxisorp ELISA plates were precoated with ssDNA (100 μg/ml), dsDNA (100 μg/ml), histone (20 μg/ml), and RNP/Sm (20 μg/ml) in PBS at 4°C overnight. Plates were blocked with 3% FCS for 1 h at 37°C, washed, and incubated with 1/300–1/1000 dilutions of mouse sera for 1 h at 37°C. Anti-ssDNA Ab (clone TNT-3, IgG2a; Abcam, Cambridge, MA), anti-dsDNA Ab (clone HYB331-01, IgG2a; Abcam), anti-histones (clone 2Q2205, IgG2a; Abcam), and anti-RNP/Sm Ab (clone NB600-546, IgG3 κ; NOVUS Biologicals, Littleton, CO) were used as standards. Plates were washed, and a 1/1000 dilution of alkaline phosphatase-linked corresponding goat anti-mouse IgG2a or IgG3 secondary Abs (Santa Cruz Biotechnology, Santa Cruz, CA) in PBS was added for 1 h at 37°C and developed with a phosphatase substrate for 30 min at 37°C.

Mouse serum CatK and culture medium IL-2 and IFN-γ levels were determined according to the manufacturer’s instructions. The ELISA kits used in this study include: mouse CatK (ALPCO, Salem, NH), mouse IL-2 DuoSet (DY402; R&D Systems, Minneapolis, MN), and mouse IFN-γ ELISA (555138; BD Biosciences).

The following Abs were used for FACS analysis of splenocyte preparation: FcR-blocking Ab anti-CD16/32 (eBioscience), anti-CD4–Alexa Fluor 488, anti-CD25–PE, anti-mFoxp3–Alexa Fluor 647, anti-mouse–PE, and all isotype controls (all from BD Biosciences). To determine the proportion of CD4+CD25+Foxp3+Tregs in splenocytes, 100 μl of splenocyte suspension (∼1 × 107 cell) was incubated at 4°C in a phosphate buffered saline containing 2% FCS with the Alexa Fluor 488-conjugated anti-CD4 and PE-conjugated anti-CD25 fluorescent Abs followed by Alexa Fluor 647-conjugated Foxp3 intracellular staining. Staining for intracellular Foxp3 was performed using the fixation/permeabilization solution Kit (BD Biosciences). Isotype controls were used for each Ab. Cells were acquired and analyzed with a FACSCalibur flow cytometer using CellQuest research software (version 3.3; BD Biosciences).

Total RNA was prepared from kidney or Tregs using the Qiagen mini kit (Qiagen, Valencia, CA). RNA concentration and quality were evaluated using the Agilent 2100 bioanalyzer (Nano LabChip; Agilent Technologies, Santa Clara, CA). After the cDNA synthesis, gene expression was quantified by real-time PCR (RT-PCR) on the ABI Prism 7900 sequence detection system (Taqman; Applied Biosystems, Foster City, CA). The low-density array detected two genes in one run in triplicate including endogenous controls (β-actin) and the mRNA levels of TLR7.

For immunoblot analysis, an equal amount of protein from each cell type or tissue preparation was separated on a SDS-PAGE, blotted, and detected with different Abs, including CatK (1:1000; Millipore, Bedford, MA), TLR7 (1:500; Abcam), and β-actin (1:3000; Santa Cruz Biotechnology).

JPM probe labeling was used to detect active cathepsins in kidney tissue extracts or Treg lysates. Tissues or cells were lysed in a lysis buffer (pH 5.5) containing 1% Triton X-100, 40 mM sodium acetate, and 1 mM EDTA. Cathepsin active site JPM probe labeling was performed as described previously (8).

TLR7 digestion with recombinant CatK was performed using purified TLR7 from FaslprCtsk+/+ mouse kidney tissue extracts. Tissue extracts were separated on an 8% SDS-PAGE. Gel slices around 125 kDa were collected and the protein eluted. Equal amounts of protein that contained the full-length mouse TLR7 were incubated with and without a recombinant CatK in a lysis buffer (pH 5.5) for 4 h followed by immunoblot analysis with TLR7 Ab (1:500; R&D Systems).

CD4+CD25 Teffs and CD4+CD25+ Tregs in this study were purified from splenocytes. Briefly, splenocytes were collected from 6- to 10-wk old mice or 24-wk old WT, Ctsk−/−, Faslpr, and FaslprCtsk−/− mice. Single-cell suspensions were incubated with biotinylated-Ab mixture containing Abs against CD8a, CD11b, CD45R, CD49b, and Ter-119 to deplete macrophages, granulocytes, B cells, and CD8+ T cells by negative selection. CD25+ cells were isolated from the CD4+ T cell population by staining with PE-conjugated anti-CD25 Ab followed by incubation with MACS anti-PE microbeads (Miltenyi Biotec). CD4+CD25+ T cells were then positively selected on a MACS mini-separation magnetic column, and the flow-through fraction containing CD4+CD25 T cells was collected. More than 90% of these cells were Tregs that were positive for CD4 and CD25, as confirmed by FACS. Negative selection of CD25+ cells yielded CD4+CD25 Teffs. CD4+CD25+ Tregs underwent a second round of purification using a cell sorter with the magnetic bead-purified Tregs as starting materials.

To test the function of Tregs from WT, Ctsk−/−, Faslpr, and FaslprCtsk−/− mice, both CD4+CD25+ Tregs and CD4+CD25 Teffs were purified from these mice. Teffs (3 × 104 cells) were used as responder T cells and cocultured with or without CD4+CD25+ Tregs (3 × 104 cells) with or without anti-CD3 mAb (2 μg/ml, clone OKT3; eBioscience) and anti-CD28 mAb (2.5 μg/ml, clone L293; BD Biosciences) (38, 39). Cocultures were maintained in complete RPMI 1640 medium for 2 d. Culture media were collected for ELISA to determine IFN-γ and IL-2. All experiments were performed in triplicate.

Both MLR and autoantigen recall assays were performed using our previously published protocols (40). Ag-presenting splenocytes were prepared from Faslpr, FaslprCtss−/−, and FaslprCtsk−/− mice and pretreated with 2 μg/ml of C-myosin at 37°C for 30 min. Different numbers of presenters (0–1 × 106) were cocultured with 5 × 105 splenocytes from bm12 mice as responders. After 2 d, culture media IL-2 levels were determined by ELISA. Splenocytes were also isolated from 24-wk old Faslpr, FaslprCtss−/−, and FaslprCtsk−/− mice and incubated (5 × 105 per well) with 50 μg/ml of histone in 200 ml of complete RPMI 1640 for 2 d at 37°C. Culture medium IL-2 levels were determined by ELISA.

All mouse data, including those from serum samples, were analyzed using non-parametric Mann–Whitney U test followed by Bonferroni corrections due to small sample sizes and often skewed data distributions. To simplify the data presentation in autoantibody titers, we did not compare data from each time point, but rather compared all time points together as a whole from each group of mice using repeated-measure ANOVA. All data are presented as mean ± SEM. A p value < 0.05 is considered statistically significant.

B6.Faslpr mice develop SLE-like manifestations after 13–14 wk of age (30). We used 24-wk old Faslpr and B6 WT control mice to test whether development of SLE-like manifestations in Faslpr mice affected CatK expression. In serum, Faslpr mice contained five times more CatK than the age-matched WT control mice, as determined by ELISA (Fig. 1A). Immunoblot analysis detected the 28-kDa active form of CatK in kidney tissue extracts from Faslpr mice but not in extracts from WT control mice (Fig. 1B). CatK immunostaining yielded the same results. Negligible amounts of CatK were recorded in the kidneys of WT mice, whereas prominent CatK expression was detected in both the glomeruli and tubulointerstitial space of Faslpr mice. In the glomeruli, CatK was expressed mainly in the capillary endothelial cells whereas in the tubulointerstitial space it was found mainly in the infiltrated inflammatory cells and peri-tubular capillary endothelial cells (Fig. 1C).

FIGURE 1.

Increased serum and kidney CatK levels in B6.Faslpr mice. (A) ELISA determined serum CatK levels. n = 12 per group. Immunoblot of kidney lysates (B) and immunostaining of kidney sections (C) using an anti-CatK Ab.

FIGURE 1.

Increased serum and kidney CatK levels in B6.Faslpr mice. (A) ELISA determined serum CatK levels. n = 12 per group. Immunoblot of kidney lysates (B) and immunostaining of kidney sections (C) using an anti-CatK Ab.

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Increased CatK expression in both the circulation and the kidneys from 24-wk old Faslpr mice suggested that this protease participates in the expression of disease in these mice. The comparison of CatK-deficient FaslprCtsk−/− mice to their littermate CatK-sufficient FaslprCtsk+/+ control mice helped test this hypothesis. At 24 wk of age, FaslprCtsk+/+ mice demonstrated increased spleen size (Fig. 2A), kidney glomerular, and tubulointerstitial scores (Fig. 2B), complement C3 (Fig. 2C) and IgG (Fig. 2D) deposition in the glomeruli, increased chemokine MCP-1 expression (Fig. 2E), and kidney Mac-2+ macrophage accumulation (Fig. 2F) compared with B6 littermates. Interestingly, CatK-deficient Faslpr mice (Fig. 2) did not show any relevant signs of glomerulonephritis. These observations suggested a direct role of CatK in the expression of autoimmunity and related pathology.

FIGURE 2.

CatK deficiency protects B6.Faslpr mice from glomerulonephritis. (A) Kidney/body weight ratio and spleen/body weight ratio. (B) Kidney Periodic Acid-Schiff staining to determine kidney mesangial cell proliferation and inflammatory cell infiltration in the glomerulus as indicated by glomerular and tubulointerstitial scores. (C) Kidney glomerular score for C3 deposition. (D) Kidney glomerular score for IgG deposition. (E) Kidney chemokine MCP-1-positive area. (F) Kidney Mac-2–positive macrophage numbers per square millimeter. Representative graphs of each panel are shown to the right. Number of mice per group is shown in each bar. Scale bar, 50 μm.

FIGURE 2.

CatK deficiency protects B6.Faslpr mice from glomerulonephritis. (A) Kidney/body weight ratio and spleen/body weight ratio. (B) Kidney Periodic Acid-Schiff staining to determine kidney mesangial cell proliferation and inflammatory cell infiltration in the glomerulus as indicated by glomerular and tubulointerstitial scores. (C) Kidney glomerular score for C3 deposition. (D) Kidney glomerular score for IgG deposition. (E) Kidney chemokine MCP-1-positive area. (F) Kidney Mac-2–positive macrophage numbers per square millimeter. Representative graphs of each panel are shown to the right. Number of mice per group is shown in each bar. Scale bar, 50 μm.

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We previously showed that cathepsin S (CatS), a closely related member of the same cysteine protease family as CatK, participates in Ag presentation. CatS deficiency leads to impaired Ab production in mice (40, 41). We considered that CatK may contribute to the development of lupus in Faslpr mice by enhancing autoantigen presentation. To test this possibility, we performed both MLR and autoantigen recall assays. As expected, in the MLR assay, Ag-presenting splenocytes from FaslprCtss−/− mice yielded a significantly reduced ability to induce the production of IL-2 from responding cells from the bm12 mice. In contrast, splenocytes from age-matched FaslprCtsk−/− and Faslpr mice showed identical activity on the same bm12 responder cells (Fig. 3A). As expected, histone induced IL-2 production from splenocytes from Faslpr mice, but such an induction significantly decreased in cells from FaslprCtss−/− mice. However, splenocytes from FaslprCtsk−/− mice responded to histone identically to those from Faslpr mice (Fig. 3B). These data indicate that CatK does not appear to be involved in Ag presentation in a manner similar to that of CatS. This conclusion concurs with prior studies, in which a CatK inhibitor did not affect Ag uptake and presentation in dendritic cells (18). Therefore, because FaslprCtsk−/− mice do not express lupus disease (Fig. 2), CatK must operate through a different mechanism.

FIGURE 3.

CatK is involved in TLR7 expression and activation but not in Ag presentation. (A) MLRs. ELISA determined media IL-2 from splenocytes from Faslpr, FaslprCtss−/−, and FaslprCtsk−/− mice as presenters, and splenocytes from bm12 mice as responders, for 2 d. Data are from six repeated experiments. (B) Autoantigen presentation assay. ELISA determined IL-2 concentration in the supernatants of splenocytes from Faslpr, FaslprCtss−/−, and FaslprCtsk−/− mice cultured with or without histone for 2 d. Data are from six independent experiments. (C) Kidney tissue extract JPM labeling to detect active cathepsins. (D) Kidney tissue TLR7 mRNA levels as determined by RT-PCR. (E) Kidney tissue extract immunoblot to detect both full-length and cleaved (activated) TLR7. (F) TLR7 immunoblot analysis detected CatK digestion of gel-purified mouse full-length TLR7. Undigested and whole kidney tissue lysate were used as experimental controls. The undigested lane was fused from the same blot. (G) JPM probe labeling to determine active cathepsins in Tregs. (H) RT-PCR to determine TLR7 mRNA levels in Tregs. (I) Treg extract immunoblot to detect both the full-length and cleaved (activated) TLR7. In (E) and (I), data are presented as cleaved TLR7 gel density relative to β-actin. Representative immunoblots for (E) and (I) are shown to the left. β-actin immunoblots were used to ensure equal protein loading in all immunoblots. Number of mice per group is shown in each bar.

FIGURE 3.

CatK is involved in TLR7 expression and activation but not in Ag presentation. (A) MLRs. ELISA determined media IL-2 from splenocytes from Faslpr, FaslprCtss−/−, and FaslprCtsk−/− mice as presenters, and splenocytes from bm12 mice as responders, for 2 d. Data are from six repeated experiments. (B) Autoantigen presentation assay. ELISA determined IL-2 concentration in the supernatants of splenocytes from Faslpr, FaslprCtss−/−, and FaslprCtsk−/− mice cultured with or without histone for 2 d. Data are from six independent experiments. (C) Kidney tissue extract JPM labeling to detect active cathepsins. (D) Kidney tissue TLR7 mRNA levels as determined by RT-PCR. (E) Kidney tissue extract immunoblot to detect both full-length and cleaved (activated) TLR7. (F) TLR7 immunoblot analysis detected CatK digestion of gel-purified mouse full-length TLR7. Undigested and whole kidney tissue lysate were used as experimental controls. The undigested lane was fused from the same blot. (G) JPM probe labeling to determine active cathepsins in Tregs. (H) RT-PCR to determine TLR7 mRNA levels in Tregs. (I) Treg extract immunoblot to detect both the full-length and cleaved (activated) TLR7. In (E) and (I), data are presented as cleaved TLR7 gel density relative to β-actin. Representative immunoblots for (E) and (I) are shown to the left. β-actin immunoblots were used to ensure equal protein loading in all immunoblots. Number of mice per group is shown in each bar.

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Cysteine protease active site labeling using the JPM probe revealed selective elevation of the CatK but not cathepsin B activity in the kidney extracts from FaslprCtsk+/+ mice. CatK activity disappeared in FaslprCtsk−/− mice (Fig. 3C). Along with increased CatK activity, we also found elevated TLR7 mRNA levels and accumulation of the cleaved (activated) 72-kDa TLR7 fragments (17, 42) in kidney extracts from FaslprCtsk+/+ mice. Both TLR7 expression (mRNA) and cleaved TLR7 accumulation were significantly reduced in kidney extracts from FaslprCtsk−/− mice (Fig. 3D, 3E). Incubation of gel-purified, full-length TLR7 from FaslprCtsk+/+ mouse kidney extracts with recombinant CatK tested a direct cleavage of TLR7 by CatK. After 2 h of incubation, CatK produced the same size (72 kDa) cleaved fragment from the full-length mouse TLR7 as the cleaved TLR7 fragment from the crude FaslprCtsk+/+ mouse kidney extracts (Fig. 3F). We also made similar observations in Tregs from WT, FaslprCtsk+/+, and FaslprCtsk−/− mice. Elevated CatK activity (Fig. 3G), TLR7 mRNA levels (Fig. 3H), and cleaved TLR7 accumulation (Fig. 3I) in Tregs from FaslprCtsk+/+ mice were all greatly reduced in cells from FaslprCtsk−/− mice. Therefore, CatK mediated TLR7 proteolytic processing in Tregs and possibly other kidney cells in addition to previously implicated in dendritic cells (19).

Prior studies showed reduced circulating CD4+CD25highFoxp3+ Tregs in SLE patients with reduced Treg immunosuppressive activity (43, 44). CD4+CD25highFoxp3+ Treg numbers were also reduced in spleens from 24-wk old FaslprCtsk+/+ mice, compared with those in age-matched WT mice. However, there were twice as many Tregs in FaslprCtsk−/− mice (Fig. 4A). There were no detectable Tregs in the kidneys of the WT and FaslprCtsk+/+ mice despite the fact that FaslprCtsk+/+ mice had increased numbers of total CD4+ T cells (Fig. 4B). CatK deficiency in Faslpr mice did not change the total number of CD4+ T cells in the kidney, but significantly increased the number of Tregs (Fig. 4B). CatK deficiency not only increased spleen and kidney tissue Treg numbers, but also enhanced Treg immunosuppressive activity. When Teffs from healthy WT mice were used, Tregs from both Ctsk−/− and FaslprCtsk−/− mice suppressed the production of IFN-γ and IL-2 by Teffs. Tregs from Ctsk−/− mice appeared more potent than those from FaslprCtsk−/− mice in suppressing Teffs (Fig. 4C, 4D). When Teffs from FaslprCtsk+/+ mice were used, we found that Tregs from WT mice were more potent than those from FaslprCtsk+/+ mice in suppressing Teff IFN-γ and IL-2 production (Fig. 4E, 4F). However, Tregs from both Ctsk−/− and FaslprCtsk−/− mice were equally potent to those from WT mice in suppressing IFN-γ and IL-2 productions by Teffs from Faslpr mice (Fig. 4E, 4F). Thus, deficiency of CatK potentiates the suppressive function of Tregs.

FIGURE 4.

CatK deficiency promotes Foxp3 expression and increases Treg suppressive function. (A) FACS analysis to determine splenic Foxp3+ cell percentage among CD4+ T cells from different groups of mice. (B) CD4 and Foxp3 coimmunostaining (immunofluorescent) to determine CD4+Foxp3+ Treg numbers in kidneys. Representative immunostaining graphs are shown to the right. Number of mice per group is shown in each bar. Scale bar, 50 μm. Media IFN-γ (C) and IL-2 (D) levels from WT CD4+CD25 Teffs after 2 d of incubation with or without anti-CD3 mAb and different Tregs from WT (control), Ctsk−/−, and FaslprCtsk−/− mice, as indicated. Media IFN-γ (E) and IL-2 (F) levels from CD4+CD25 Teffs from Faslpr mice after 2 d of incubation with or without anti-CD3 mAb and different Tregs from WT (control), Faslpr, Ctsk−/−, and FaslprCtsk−/− mice, as indicated. Data are mean ± SEM of six independent experiments. All mice were 24 wk of age.

FIGURE 4.

CatK deficiency promotes Foxp3 expression and increases Treg suppressive function. (A) FACS analysis to determine splenic Foxp3+ cell percentage among CD4+ T cells from different groups of mice. (B) CD4 and Foxp3 coimmunostaining (immunofluorescent) to determine CD4+Foxp3+ Treg numbers in kidneys. Representative immunostaining graphs are shown to the right. Number of mice per group is shown in each bar. Scale bar, 50 μm. Media IFN-γ (C) and IL-2 (D) levels from WT CD4+CD25 Teffs after 2 d of incubation with or without anti-CD3 mAb and different Tregs from WT (control), Ctsk−/−, and FaslprCtsk−/− mice, as indicated. Media IFN-γ (E) and IL-2 (F) levels from CD4+CD25 Teffs from Faslpr mice after 2 d of incubation with or without anti-CD3 mAb and different Tregs from WT (control), Faslpr, Ctsk−/−, and FaslprCtsk−/− mice, as indicated. Data are mean ± SEM of six independent experiments. All mice were 24 wk of age.

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In Fig. 2, we demonstrated that CatK deficiency significantly suppressed glomerulonephritis in Faslpr mice. CatK deficiency also reduced serum autoantibody titers against histone, dsDNA, ssDNA, and RNP/Sm in Faslpr mice (Fig. 5A–D). To assess whether increased immunosuppressive activity of Tregs from WT and Ctsk−/− mice affected autoimmunity in FaslprCtsk+/+ mice, we performed adoptive transfer experiments in which we transferred Tregs from WT and Ctsk−/− mice to 9-wk old FaslprCtsk+/+ mice. Three weeks after the transfer, we started monitoring serum autoantibody titers. In vitro prepared Tregs from both WT and Ctsk−/− mice significantly reduced all tested serum autoantibody titers, affirming an important role of Tregs in the expression of systemic autoimmunity. WT and Ctsk−/− mice-derived Tregs similarly suppressed serum titers of the anti-histone, dsDNA, and ssDNA autoantibodies (Fig. 5A–C). Compared with WT Tregs, however, those from Ctsk−/− mice displayed significantly higher potency in suppressing serum autoantibodies against RNP/Sm (Fig. 5D). Kidney glomerular depositions of C3 (Fig. 5E) and IgG (Fig. 5F) were also improved significantly in mice receiving Tregs from WT and Ctsk−/− mice. Compared with Tregs from WT mice, those from Ctsk−/− mice showed significantly more reduction in C3 deposition, although IgG deposition between WT Treg- and Ctsk−/− Treg-reconstituted mice showed comparable results (Fig. 5E, 5F). Transfer of Tregs from either WT or Ctsk−/− mice did not affect kidney sizes, but reduced the size of the spleens in FaslprCtsk+/+ recipient mice (Fig. 5G).

FIGURE 5.

Transfer of CatK-deficient Tregs into B6.Faslpr suppresses autoimmunity. Serum anti-histone autoantibody (A), anti-dsDNA autoantibody (B), anti-ssDNA autoantibody (C), anti-RNP/Sm autoantibody (D), glomerular C3 deposition score (E), glomerular IgG deposition score (F), kidney/body weight ratio and spleen/body weight ratio (G), FACS analysis of splenic Foxp3+ cell percentage in CD4+ T cells (H), and CD4 and Foxp3 coimmunostaining (immunofluorescent) to detect kidney Foxp3+CD4+ Treg numbers (I) in 24-wk old WT mice, FaslprCtsk+/+ mice, FaslprCtsk−/− mice, and FaslprCtsk+/+ mice receiving WT Tregs or Ctsk−/− Tregs. Representative graphs for (E) (scale bar, 150 μm), (F) (scale bar, 150 μm), and (I) (scale bar, 50 μm) are shown to the right. Number of mice per group is shown in each bar or parentheses.

FIGURE 5.

Transfer of CatK-deficient Tregs into B6.Faslpr suppresses autoimmunity. Serum anti-histone autoantibody (A), anti-dsDNA autoantibody (B), anti-ssDNA autoantibody (C), anti-RNP/Sm autoantibody (D), glomerular C3 deposition score (E), glomerular IgG deposition score (F), kidney/body weight ratio and spleen/body weight ratio (G), FACS analysis of splenic Foxp3+ cell percentage in CD4+ T cells (H), and CD4 and Foxp3 coimmunostaining (immunofluorescent) to detect kidney Foxp3+CD4+ Treg numbers (I) in 24-wk old WT mice, FaslprCtsk+/+ mice, FaslprCtsk−/− mice, and FaslprCtsk+/+ mice receiving WT Tregs or Ctsk−/− Tregs. Representative graphs for (E) (scale bar, 150 μm), (F) (scale bar, 150 μm), and (I) (scale bar, 50 μm) are shown to the right. Number of mice per group is shown in each bar or parentheses.

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Donor Tregs also changed target organ Treg numbers. Transfer of Tregs from Ctsk−/− mice yielded increased Treg numbers in the spleens and the kidneys of Faslpr mice (Fig. 5H, 5I). Transfer of Tregs from WT mice also increased Treg numbers in the spleens and kidneys of Faslpr mice but to a lesser extent (Fig. 5H, 5I).

This study has presented evidence that CatK contributes to mouse SLE manifestations by regulating TLR7 expression, proteolytically processing TLR7, reducing Treg numbers, and suppressing Treg immunosuppression activity. CatK deficiency led to reduced production of cleaved TLR7 and enhanced Treg immunosuppression. Tregs ameliorate SLE by their direct immunosuppression activity against Teffs (45) and may also contribute to SLE by other indirect mechanisms, for example, by affecting dendritic cell immunologic tolerance, which may in turn expand host Tregs (46, 47). CatK remains increased in lupus-prone Faslpr mice and if genetically deleted almost eliminates the production of autoantibodies and glomerulonephritis. Although previous data implicate CatK in TLR7 activation in dendritic cells (19), this study demonstrates that CatK produces cleaved TLR7 in vitro (Fig. 3F), and in Tregs (Fig. 3G–I) and kidneys (Fig. 3C–E).

Previous reports have presented evidence that CatK is involved in the expression of adjuvant-induced arthritis, EAE, and psoriasis (18, 19). This study expands on these observations to document the importance of CatK in the expression of systemic autoimmunity through a mechanism that involves direct expression and processing of TLR7 and suppression of Treg function, although it still remains unknown how CatK expression affected TLR7 transcription (Fig. 3D, 3H) or via other proteolytic enzymes (14, 48). Several lines of evidence demonstrated that deficiency of CatK (13, 14, 49) or other cathepsins (48, 50) altered the expression of other proteins. Such a role of CatK may not simply relate to its proteolytic activity, a longstanding question that has still remained unanswered. The process behind the regulation of CatK activation and expression during SLE in this study also remains unknown. An autoactivation mechanism (51, 52) and possibly other proteases may mediate CatK activation. It is also possible that proteolytic processing of TLR7 by CatK (Fig. 3E, 3F, 3I) may mediate downstream cell signaling and inflammatory cytokine expression, such as IL-6, IFN-γ, TNF-α, and IL-1β (53, 54). In turn, these cytokines serve as a feedback mechanism to stimulate CatK expression (55). These possibilities merit further investigation.

Unlike CatS, which has been shown to be involved in Ag presentation (40, 41), CatK does not participate in Ag presentation in both the MLR assay and response to histone (Fig. 3A, 3B). This finding concurs with a previous report, which came to the same conclusion using bone marrow-derived dendritic cells (18). Although prior studies showed that TLR9 ligand-mediated CpG-induced dendritic cell activation can be inhibited by a CatK inhibitor in vitro and proposed a role for CatK-mediated TLR9 activation in arthritis and EAE (18), there was no direct proof of this conclusion. Further, the role of TLR9 in SLE has been controversial and studies using TLR9-deficient Tlr9−/− mice have yielded mixed conclusions (3234). In addition, we have shown (Y. Zhou and G.-P. Shi, unpublished observations) that CD4+ T cells from Tlr9−/− mice differentiate to Tregs in the presence of IL-2 and TGF-β similarly to the differentiation seen when CD4+ T cells from WT mice were used. In contrast, CD4+ T cells from Tlr7−/−and Ctss−/− mice differentiated to Tregs in the presence of IL-2 and TGF-β 100% better than CD4+ T cells from WT or Tlr9−/− mice. TLR7 expression and activation determine Treg numbers and function. TLR7 stimulation reduces Treg de novo generation (54), or releases IL-6 and other cytokines to block Treg function (24). Therefore, it is possible that CatK enhances organ-specific and systemic autoimmunity by activating TLR7 and suppressing Treg immunosuppressive activity. Reduced production of the cleaved form of TLR7 in the kidneys (Fig. 3E) and Tregs (Fig. 3I) from FaslprCtsk−/− mice suggests an indirect role of CatK in generating the cleaved form of TLR7. Digestion of in vitro prepared full-length 125 kDa TLR7 with a recombinant CatK and production of the same size 72 kDa cleaved form of TLR7 as that from the FaslprCtsk+/+ mice (Fig. 3F) suggest a direct role of CatK in producing the cleaved form of TLR7 in lupus-prone FaslprCtsk+/+ mice, although we did not test whether the 72 kDa cleaved form of TLR7 from CatK in vitro digestion was an active form of TLR7. The role of CatK activity in TLR7 processing and in regulating Treg immunosuppression activity may be one of several possible mechanisms whereby CatK activity contributed to SLE-like manifestations in FaslprCtsk+/+ mice. This hypothesis is supported by the observations that systemic depletion of CatK nearly completely suppressed glomerulonephritis (Fig. 2), but the WT and Ctsk−/− Tregs in suppressing SLE-like manifestations in FaslprCtsk+/+ mice differed only in suppressing serum anti-RNP/Sm autoantibodies and glomerular C3 deposition (Fig. 5D, 5E). Other variables, including serum anti-histone, anti-dsDNA, and anti-ssDNA autoantibodies, and glomerular IgG deposition were comparable in mice receiving WT and Ctsk−/− Tregs (Fig. 5A–C, 5F). Therefore, CatK contributes to the expression of SLE-like phenotypes in Faslpr mice through additional unknown mechanisms.

SLE patient blood CD4+CD25high Treg numbers and suppressive capacity show reduced levels and inversely correlate with the clinical disease activity (44). In contrast, Teffs in SLE patients and animals also contain elevated Th17 cells (56) or have altered TCR expression, signaling, and inflammatory cytokine expression (57). This study showed a similar pattern of Treg immunosuppressive activity to that of humans. Tregs from FaslprCtsk−/− mice had lower immunosuppressive activity than those from Ctsk−/− mice, and the same cells from Faslpr mice also had lower immunosuppressive activity than those from WT mice. Further, the responses of Teffs to different Tregs differed between Teffs from WT and Faslp mice (Fig. 4C–F), although this study did not explore further the Teff types, TCR expression, and signaling profile.

The pathogenesis and progression of human SLE can differ from that of SLE-prone Faslp mice, but the current study opens up the possibility of using cellular treatment in patients with SLE, where self-Tregs can be treated in vitro with a CatK inhibitor to increase their suppressive ability. The study also offers the possibility of giving directly to the patients a CatK-selective inhibitor. The fact that lupus manifestations in the Faslpr mice are suppressed in both CatS (Y. Zhou and G.-P. Shi, unpublished observations) and CatK (Fig. 2) deficiencies raises interesting questions about the possible redundancy of these two cathepsins or the possible dependence on one another to promote autoimmunity. Regardless of the answers to these questions, consideration to the therapeutic targeting of cathepsins remains vital.

We thank Eugenia Shvartz for technical assistance and Chelsea Swallom for editorial assistance.

This work was supported by grants from the National Institutes of Health (HL81090, HL60942, HL123568 to G.-P.S.; and HL34636, and HL80472 to P.L.).

Abbreviations used in this article:

CatK

cathepsin K

CatS

cathepsin S

EAE

experimental autoimmune encephalomyelitis

RT-PCR

real-time PCR

SLE

systemic lupus erythematosus

Teff

effector T cell

Treg

regulatory T cell

WT

wild type.

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G.-P.S. has a patent application pending. The remaining authors have no financial conflicts of interest.