CLINICAL RESEARCH www.jasn.org

Autoantibodies against C-Reactive Influence Complement Activation and Clinical Course in Lupus Nephritis

†‡ | †‡ | †‡†† Qiu-yu Li,* § Hai-yun Li, Ge Fu,¶ Feng Yu,* ** Yi Wu, and Ming-hui Zhao*

*Renal Division, Department of Medicine, Peking University First Hospital, Beijing, People’s Republic of China; †Key Laboratory of Renal Disease, Ministry of Health of China, Beijing, People’s Republic of China; ‡Key Laboratory of CKD Prevention and Treatment, Ministry of Education of China, Beijing, People’s Republic of China; §Department of Pneumology, Peking University Third Hospital, Beijing, People’s Republic of China; |Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education, School of Basic Medical Sciences, Xi’an Jiaotong University, Xi’an, Shanxi, People’s Republic of China; ¶State Key Laboratory of Natural and Biomimetic Drugs, Department of Chemical Biology, School of Pharmaceutical Science, Peking University, Beijing, People’s Republic of China; **Department of Nephrology, Peking University International Hospital, Beijing, People’s Republic of China; and ††Peking-Tsinghua Center for Life Sciences, Beijing, People’s Republic of China

ABSTRACT Autoantibodies against the major acute-phase reactant C-reactive protein (CRP) are frequently found in patients with lupus nephritis. Further defining the autoimmune on CRP may not only improve patient stratifi- cation but also, hint at mechanisms of CRP action. Herein, we show that amino acids 35–47 constitute the major recognized by anti-CRP autoantibodies in patients with lupus nephritis. Notably, the presence of autoantibodies against amino acids 35–47 associated with more severe renal damage and predicted worse outcome. This epitope is exposed on CRP only after irreversible structure changes, yielding a conformationally altered form termed modified or monomeric CRP (mCRP). ELISA and surface plasmon resonance assays showed that amino acids 35–47 mediate the interaction of mCRP with complement factor H, an inhibitor of alternative pathway activation, and this interaction greatly enhanced the in vitro cofactor activity of complement factor H. In contrast, autoantibodies against amino acids 35–47 inhibited these actions of mCRP. Our results thus provide evidence for the in vivo generation of mCRP in a human disease and suggest that mCRP actively controls the pathogenesis of lupus nephritis by regulating complement activation. Therefore, amino acids 35–47 constitute a functional autoimmune epitope on CRP that can be targeted therapeutically and diagnostically.

J Am Soc Nephrol 28: 3044–3054, 2017. doi: https://doi.org/10.1681/ASN.2016070735

SLE is an autoimmune disease characterized by mas- Received July 9, 2016. Accepted April 12, 2017. sive production of autoantibodies, excessive activa- Q.-y.L. and H.-y.L. contributed equally to this work. tion of complement, and defective clearance of cell Published online ahead of print. Publication date available at debris and immune complexes, leading to extensive www.jasn.org. tissue damage in multiple organs.1 C-reactive protein fl Correspondence: Dr. Feng Yu, Renal Division, Department of (CRP) is a serum marker of in ammation and a pu- Medicine, Peking University First Hospital; Institute of Nephrology, tative soluble pattern recognition receptor that con- Peking University; Key Laboratory of Renal Disease, Ministry of Health tributes to efficient removal of dead cells or invading of China; and Key Laboratory of CKD Prevention and Treatment, Ministry of Education of China, Beijing 100034, People’s Republic of 2,3 by activating complement. It is, there- China and Department of Nephrology, Peking University International fore, interesting to note the blunted CRP response in Hospital, Beijing 102206, People’s Republic of China, or Dr. Yi Wu, Key 4 Laboratory of Environment and Genes Related to Diseases, Ministry of active SLE and the presence of anti-CRP autoanti- Education, School of Basic Medical Sciences, Xi’an Jiaotong University, – bodies in a subset of patients.5 8 These might point to 76# Yanta West Road, Xi’an, Shanxi 710061, People’s Republic of the potential of CRP and its autoantibodies to be China. Email: [email protected] or [email protected] therapeutic targets and/or diagnostic indices. Copyright © 2017 by the American Society of Nephrology

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However, anti-CRP autoantibodies add little to the cur- Table 1. Clinical characteristics of patients with LN 9 rent diagnosis, despite their prevalence in patients with Characteristic Value 6–8 SLE. This would instead argue that the generation of Clinical evaluation CRP autoantibodies may merely be an epiphenomenon re- No. of patients 80 sulting from systemic inflammation. Alternatively, the Sex, men/women 7/73 actual pathogenetic and diagnostic significance could be ob- Age, mean6SD, yr 30.0612.1 scured by the polyclonal nature of CRP autoantibodies, Follow-up time median (range), mo 48 (34–58) which comprise both disease-specificandpassiveclones. Fever (noninfectious), no. (%) 27 (33.8) Identification of the immune epitopes on CRP and their re- Malar rash, no. (%) 33 (41.3) spective disease associations is, therefore, mandatory to ad- Photosensitivity, no. (%) 16 (20.0) dressthisissue. Oral ulcer, no. (%) 72 (90.0) In addition, accumulating evidence suggests that CRP Alopecia, no. (%) 16 (20.0) Arthralgia, no. (%) 32 (40.0) autoantibodies do not react with the native conformation Serositis, no. (%) 9 (11.3) but instead, recognize a conformationally altered variant Neurologic disorder, no. (%) 4 (5.0) fi 6–8 (i.e., monomeric or modi ed C-reactive protein [mCRP]). Anemia, no. (%) 43 (53.8) Whereas CRP is secreted by hepatocytes as a cyclic pen- ARF, no. (%) 13 (16.3) tamer,2,3 it has been proposed that pentameric CRP is in- Nephrotic syndrome, no. (%) 50 (71.4) duced to undergo irreversible conformation changes at sites Laboratory assessment of tissue injury, giving rise to mCRP with greatly enhanced No. of patients 80 bioactivities.10,11 Although in vivo support for this proposal Leukocytopenia, no. (%) 17 (21.3) has been lacking, the discovery of autoantibodies against Anemia, no. (%) 49 (61.3) mCRP in SLE6–8 strongly argues for the generation and Thrombocytopenia, no. (%) 16 (20.0) actions of mCRP in the disease. However, modest and re- Hematuria, no. (%) 64 (80.0) Leukocyturia (noninfection), no. (%) 43 (53.8) versible conformation changes in CRP could also occur as Hemoglobin, mean6SD, g/L 104.8622.2 12 fi implicated by the structural features and exempli ed ex- Urine protein (mean6SD), g/24 h 3.8 (1.7–6.9) 13 perimentally. Those changes may expose cryptic epitopes Serum creatinine median (range), mmol/L 70.8 (54.6–137.8) that are shared by mCRP but nevertheless, are not the result C3 (mean6SD), g/L 0.4 (0.3–0.6) of its generation. This uncertainty could also be addressed by Antinuclear (+), no. (%) 77 (96.3) the identification of autoimmune epitopes that are unique to Antidouble-stranded DNA antibody (+), no. (%) 63 (78.8) mCRP. Anti-SSA antibody (+), no. (%) 44 (55.0) The majority of patients with SLE are diagnosed with renal Anti-SSB antibody (+), no. (%) 8 (10.0) involvement (i.e., lupus nephritis [LN]).1 Herein, we identify Anti-Smith antibody(Sm) (+), no. (%) 18 (22.5) amino acids (a.a.) 35–47, a sequence exposed only in mCRP, Renal histopathology indices as the predominant epitope recognized by CRP autoanti- No. of patients 80 Activity indices score 6.562.8 bodies in patients with LN. Patients with anti-a.a. 35–47 au- Chronicity indices score 1 (1–2) toantibodies showed more severe renal damages and worse SSA, Sjögren’s syndrome A ; SSB, Sjögren’s syndrome B antigen. outcomes. Complement factor H (CFH) is an inhibitor of complement alternative pathway activation.14,15 Genetic polymorphism of CFH affects the susceptibility of SLE,16 them were identified as mCRP autoantibody positive (Figure and its deficiency accelerates the development of LN in 1A and Table 2). Six strong positive samples were then used mice.17 We show that mCRP binds CFH and enhances its to conduct epitope mapping against a panel of synthesized cofactor activity via a.a. 35–47, whereas autoantibodies CRP (Figure 1B); a.a. 35–47 and 199–206 emerged against this epitope inhibit these actions. Because comple- as the predominant epitopes recognized by autoantibodies. ment overactivation is a key driver of renal damage,1 our Interestingly, only a.a. 35–47 efficiently inhibited autoanti- findings suggest that anti-a.a. 35–47 autoantibodies may ad- body binding to immobilized mCRP (Figure 1C), although versely affect LN through interfering with the function of both epitopes are prevalent in patients with LN (Figure 1, D mCRP as a complement regulator. and E). The lack of inhibitory effects of a.a. 199–206 might be due to their low affinity to autoantibodies. We thus con- cluded that a.a. 35–47 were the major autoimmune epitope RESULTS on CRP. By alanine scanning and competition assays, Leu37, Phe39, a.a. 35–47 Are the Major Autoimmune Epitope on Tyr40, andLeu43 were determined as the key residuescritical to CRP in LN the antigenicity of a.a. 35–47 epitope (Figure 1F). Mutating We first screened for autoantibodies against immobilized each of these residues reversed the inhibition of the corre- mCRP in sera of 80 patients with LN (Table 1), and 24 of sponding mutant on autoantibody binding to

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Figure 1. The a.a. 35–47 are the major autoimmune epitope on CRP in LN. (A) Urea-denatured mCRP was immobilized onto microtiter wells andtestedforautoantibodybindingofserafrom 24 healthy controls and 80 patients with LN. Twenty-four patient sera were assigned as mCRP autoantibody positive using a cutoff given by the mean OD value obtained with the control sera plus two times the SD. (B) Synthesized CRP peptides were immobilized and tested for autoantibody binding of six sera from patients with LN with strong signals of mCRP autoantibody (patients 1–6). Apparent binding to a.a. 35–47 and 199–206 was consistently observed. The binding to other peptides, however, were comparable with that of CRP signal peptide, which is cleaved before secretion and considered as the background control. The cutoff was set as the interquartile mean of the OD values obtained with all peptides plus two times the SD. (C) The serum of patient 1 was mixed with the indicated peptides and added to immobilized mCRP; a.a. 35–47 were the only peptide that significantly reduced the binding of mCRP au- toantibodies. Similar results were obtained with the serum of the other patient (not shown). Sera of 24 patients with LN with mCRP autoan- tibodies and 24 healthy controls were tested for autoantibody binding to immobilized a.a. (D) 35–47 or (E) 199–206. Binding was normalized to that of patient 1. Autoantibodies against a.a. 35–47 and 199–206 were detected in 11 and 17 patients, respectively. (F) a.a. 35–47 mutants with a single residue replaced with alanine were tested for their inhibitory effects on the binding of autoantibodies in the serum of patient 1 to immobilized mCRP. Leu37, Phe39, Tyr40, and Leu43 were found to be critical residues. Similar results were obtained with the serum of the other patient (not shown). (G) The crystal structure of pentameric CRP (1B09)12;a.a.35–47 are shown in blue, with key residues colored red. Most parts of a.a. 35–47, including the three critical residues, were buried inside the native structure. In B, C, and F, each data point or bar represented the mean of three technical replicates. Dashed lines represent cutoff values. immobilized mCRP. Of note, most of a.a. 35–47, including the generation of mCRP.18,19 The exposure of a.a. 199–206, the Leu37, Phe39, and Tyr40, were buried in the native subunit sequence lining the subunit contact interface, however, may structure of CRP (Figure 1G) and would be exposed only after occur both in mCRP18,20 and on reversible conformation

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Table 2. Comparisons of clinical data between patients with LN with gross mCRP autoantibodies and patients with LN without gross mCRP autoantibodies Patients with LN, n=80 Clinical Feature Anti-mCRP Positive Anti-mCRP Negative P Value No. of patients 24 56 Age mean6SD, yr 30.42613.68 31.86611.57 0.46 Sex, men/women 11/13 6/50 0.60 No. of leukocyturia (%) 14 (58.3) 29 (51.8) 0.77 No. of hematuria (%) 19 (79.1) 45 (80.3) 0.80 No. of ARF (%) 5 (20.8) 8 (14.3) 0.42 Urine protein median (interquartile range), g/24 h 2.72 (0.62–4.88) 3.96 (2.15–7.66) 0.03 Serum creatinine median (interquartile range), mmol/L 69 (52.28–153) 71.7 (54.6–125.25) 0.59 Creatinine clearance rate mean6SD, ml/min 76.30643.77 81.84645.54 0.67 C3 median (interquartile range), mg/ml 0.31 (0.23–0.48) 0.44 (0.37–0.64) ,0.01 No. of anti–ds-DNA positive (%) 21 (87.5) 42 (75) 0.34 Pathologic AI score median (interquartile range) 7 (4–9) 6 (5–8) 0.94 Pathologic CI score median (interquartile range) 1 (1–2.5) 1.5 (1–2) 0.54 SLEDAI median (interquartile range) 19 (17–22) 17 (12–21) 0.13 ds-DNS, anti-double stranded DNA antibody; AI, activity index; CI, chronicity index; SLEDAI, systemic lupus erythematosus disease activity index. changes in CRP.12,13 Taken together, identification of a.a. CFH (Figure 3, B and C). We further showed that a.a. 35–47 35–47 as the predominant epitope recognized by autoanti- could directly bind CFH (Figure 3D), whereas mCRP mu- bodies strongly supports the in vivo generation of mCRP tant without this motif (mCRP D35–47) showed negligible in LN. binding (Figure 3E). The above results were independently replicated by surface plasmon resonance (SPR) assays (Fig- Anti-a.a. 35–47 Autoantibodies Are Associated with ure 4, A–E). These together show that mCRP binds CFH via Prognosis of LN a.a. 35–47. Further analysis indicated that patients with LN with anti-a.a. One major function of CFH is to act as the cofactor for 35–47 autoantibodies had more serve renal damage than those factor I–mediated cleavage and inactivation of , thus in- without as evidenced by the higher activity index (P=0.04) and hibiting the formation of C5 convertase and the overactiva- chronicity index (P=0.004) scores (Table 3). The average fol- tion of complement.14,15 We thus next examined whether a.a. low-up time of our patients was 48 months (34–58 months), 35–47-mediated mCRP binding affected the cofactor activity during which eight patients reached the secondary end point of CFH. Consistent with the previous report,21 mCRP but not of doubling of serum creatinine, two patients reached ESRD, CRP significantly enhanced the cofactor activity of CFH, re- and no one died. Importantly, the presence of anti-a.a. 35–47 sulting in increased cleavage of C3b as detected by immuno- autoantibodies was associated with poor outcome by log rank blotting (Figure 5, A–C). Of note, such enhancement was also test (P=0.02) (Figure 2) and univariate Cox hazard analysis observed with the peptide of a.a. 35–47 (Figure 5D). These (hazard ratio, 5.17; 95% confidence interval, 1.44 to 18.62; suggest that mCRP might be able to limit excessive comple- P=0.01) (Table 4). Anti-a.a. 35–47 autoantibodies remained ment activation through a.a. 35–47-mediated interaction as an independent risk factor even after multivariate adjust- with CFH. Consistent with such speculation, we found that ment (hazard ratio, 6.77; 95% confidence interval, 1.14 to CFH was enriched, whereas the terminal complement com- 39.92; P=0.04). By contrast, anti-a.a. 199–206 autoantibodies plex C5b-9 was frequently depleted at sites stained positive were not associated with either clinical characteristics or prog- with mCRP in kidney tissues of patients with LN (Figure 5E). nosis of LN. The inverse association with prognosis thus Toexclude the possibility that the colocalization of mCRP and suggests a pathogenic role of anti-a.a. 35–47 autoantibodies CFH in Figure 5E was not simply the result of the secondary in the disease. anti-IgG identifying the human IgG in the immune deposits, we further performed the additional control experiments Anti-a.a. 35–47 Autoantibodies Inhibit the Interaction omitting primary or using irrelevant primary of mCRP with CFH mAb (i.e., 8D8 recognizing native CRP but not mCRP), We and others have shown that mCRP can bind CFH21,22 yielding a faint background or no staining (Supplemental (Figure 3A), which may play a beneficial role in LN.16,17 Figure 1). However, it is unclear which sequence motif in mCRP me- The above findings would predict an inhibition of anti-a.a. diates the binding. By competition with peptides derived 35–47 autoantibodies on mCRP-CFH interaction. We thus from CRP sequences, only a.a. 35–47 were found to abro- isolated anti-a.a. 35–47 autoantibodies from a patient with gate the interaction of fluid-phase mCRP with immobilized LN undergoing plasma exchanging. The purified anti-a.a.

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35–47 autoantibodies bound specifically to a.a. 35–47 but did

Value not bind to a control CRP peptide (not shown). As expected, 0.90 0.64 0.70 0.46 0.29 0.46 0.47 0.40 0.99 P . – c mCRP anti-a.a. 35 47 autoantibodies markedly inhibited mCRP fi binding to immobilized CFH in both ELISA (Figure 3F) and SPR assays (Figure 4F) and reversed the enhancement of mCRP on CFH’s cofactor activity (Figure 5F). By contrast, 4.56) 0.90 94.75) 0.40 3.25) 0.73 – – – – 5.31 11.88 0.66 0.22 human IgG and anti-a.a. 199 206 autoantibodies showed little 544) 206 Negative – 7 9) 6 6 6 – 1/6 – effect. Similar results were also obtained for the interaction of fl 0.38 5 (71.4) 2 (28.6) 7 (100) 6 (3 5 (71.4) surface-bound mCRP with uid-phase CFH (not shown). 19.43 27.14 85 (43 71 (35.4 1.5 (0.75

2.51 (0.90 These results further corroborate the functional importance of a.a. 35–47 and imply a pathogenic role of autoantibodies against this epitope in LN. =24 n 6.63) 8.25) DISCUSSION – – 139) 115.6) 5.32 14.48 0.19 206 Positive Anti-a.a. 199 – – 2) – 6 6 6 – 17 0/17 This study has identified a.a. 35–47 as the predominant 9 (52.9) 3 (17.6) 0.40 16(94.1) autoimmune epitope on CRP associated with renal injury and prognosis of LN. Importantly, this epitope is unique to mCRP conformation and constitutes the major binding site. Because the presentation of a.a. 35–47 epitope fl

Value Anti-a.a.199 re ects both the generation and the function of mCRP, it is 0.93 0.45 0.830.66 12 (70.6) 0.04 7.5 (6.75 0.004 1 (1 0.16 0.42 19.43 P plausible that mCRP plays a direct role in renal injury. CRP transported from the circulation and produced locally by tubular epithelial cells23 likely are major sources for mCRP fl 5.86) 0.25 3.41 (0.48 0.42) 0.96 generation in in amed kidneys. Accordingly, mCRP has c mCRP autoantibodies and patients with LN without epitope-speci – – fi 109) 0.11 66 (53 23) 7.69 0.08 31.76 42.79 0.04 90 (43 Patients with LN and Anti-mCRP Positive, 24 47 Negative – – 8) 1) been found in renal tissues of diabetic kidney disease 6 6 – – – 13 0/13 and LN,25 although sample preparation procedure (e.g., 9 (69.2) 8 (61.5) 3 (23.1) 6 (2 1 (0 63 (44 13 (100) 20 (17 25.38 93.97 fixation or antigen retrieval) might affect the detected con- 0.31 (0.24 formationofCRP.Ongeneration,mCRPcouldthenpro- mote silent clearance of cell debris via activating the classic pathway of complement by interacting with C1q,19,22 while preventing complement overactivation by recruiting 21,22 3.41) 3.94 (1.21 0.5) 2.45) CFH. – – – 214) 22) 16.97 36.18 47 Positive Anti-a.a. 35 – – 9) – However, in situations where clearance mechanisms are 6 6 – 11 1/10 overwhelmed, mCRP accumulated on cell debris26,27 or im- 5 (45.4) 3 (27.2) 2 (18.2) 8 (72.7) 18 (16 36.36 54.71 28 8.5 (6 2.5 (1.75 mune complexes in kidney tissues would instead be trans- 0.39 (0.19 ported to lymph nodes and recognized by B cells,29 leading to Anti-a.a. 35 the production of autoantibodies. Of the two autoimmune epitopes identified herein, the exposure of a.a. 35–47 is a better proxy for mCRP generation, whereas a.a. 199–206 could be exposed on both mCRP and CRP with reversible12,13 or mod- erate conformation changes.30 Therefore, the levels of anti-a.a. 35–47 autoantibodies are more specific to the underlying mechanisms of tissue damage, but a large portion of anti-a. SD, ml/min

6 a. 199–206 autoantibodies is presumably derived from CRP conformations other than mCRP, likely induced by systemic autoimmune activation. This might account for the lack of association of a.a. 199–206-specific autoantibodies with renal Clinical Feature prognosis. Because the interactions of mCRP with C1q and CFH are SD, yr ds-DNA positive (%) Comparisons of clinical data between patients with LN with epitope-speci – 6 both mediated by a.a. 35–47 on the basis of our previous work31 and this study, autoantibodies recognizing this epitope could then interfere with these interactions as shown herein. Table 3. ds-DNS, anti-double stranded DNA antibody; AI, activity index; CI, chronicity index; SLEDAI, systemic lupus erythematosus disease activity index. autoantibodies Age mean No. of patients Sex, men/women, % No. of leukocyturia (%) No. of hematuria (%) No. of ARF (%) Urine protein median (interquartile range), g/24 h 1.46 (0.61 Serum creatinine median (interquartile range), mmol/L 94 (64 Creatinine clearance rate mean C3 median (interquartile range), mg/ml No. of anti Pathologic AI score median (interquartile range) Pathologic CI score median (interquartile range) SLEDAI median (interquartile range) Such a mistaken targeting by autoantibodies may thus

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Figure 2. Anti-a.a. 35–47 autoantibodies predict worse prognosis of patients with LN. Kaplan–Meier plots of composite end point events in 80 patients with LN with or without (A) gross anti-mCRP, (B) anti-a.a. 35–47 autoantibodies, or (C) anti-a.a. 199–206 autoantibodies. represent a vicious feedback loop that further aggravates renal application to other complement-mediated diseases indepen- damage. In this regard, other than being a potential marker of dent of mCRP involvement. diagnosis, the epitope of a.a. 35–47 might also be exploited Genetic association between noncoding polymorphism of therapeutically to offer additional benefit when combined CRP and SLE susceptibility in humans has been inconclusive.9 with conventional . Administration of a synthesized Previous studies have also reported inconsistent results on the peptide of a.a. 35–47 will (1) in principle, neutralize the cor- effects of overexpressed33,34 or administrated35,36 human CRP responding autoantibodies to release their inhibition on the in SLE and diabetic kidney disease in animal models. Here, we interactions of mCRP with CFH; (2) suppress mCRP-induced report the discovery of anti-a.a. 35–47 autoantibodies and proinflammatory cell responses31,32;and(3) directly enhance their close association with renal prognosis, suggesting a crit- the cofactor activity of CFH to limit excessive complement ical role of mCRP in LN. However, the clinical prognosis find- activation. Given the central role of CFH in complement reg- ings of anti-a.a. 35–47 autoantibodies are preliminary due to ulation,14,15 the third action of a.a. 35–47 peptide allows its the small number of patients, and larger validation cohorts are

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Table 4. Univariate analysis of renal survival of patients with LN controversies and require a refined animal Clinical Feature HR 95% Confidence Interval P Value model, in which the actions of different Age 0.11 0.02 to 0.79 0.003 CRP conformations could be faithfully re- Sex 2.64 1.34 to 5.72 0.01 capitulated and differentiated. Leukocyturia 1.33 0.38 to 4.72 0.66 Hematuria 4.28 0.76 to 24.10 0.10 CONCISE METHODS ARF 7.18 2.03 to 25.33 0.002 Urine protein 1.25 1.09 to 1.43 0.001 Serum creatinine 1.00 1.00 to 1.01 0.003 Reagents Creatinine clearance rate 0.96 0.93 to 0.99 0.01 Human native pentameric CRP (purity .99%; C3 0.44 0.03 to 7.06 0.56 purified from ascites) was purchased from the Anti–ds-DNA positive 0.57 0.15 to 2.22 0.42 BindingSite (Birmingham, United Kingdom; Pathologic AI score 1.39 1.06 to 1.83 0.02 catalog no. BP300.X). Urea-denatured mCRP Pathologic CI score 1.6 1.07 to 2.40 0.02 and recombinant mCRP mutants were pre- SLEDAI 1.01 0.97 to 1.05 0.79 pared as described.31 were dialyzed to Antigross mCRP autoantibodies 1.71 0.48 to 6.07 0.41 remove NaN3 and passed through Detoxi-Gel Anti-a.a. 35–47 mCRP autoantibody 5.17 1.44 to 18.62 0.01 Columns (catalog no. 20344; Thermo Fisher Anti-a.a. 199–206 mCRP autoantibody 0.98 0.21 to 4.64 0.98 Scientific, Rockford, IL) to remove the endo- HR, hazard ratio; ds-DNS, anti-double stranded DNA antibody; AI, activity index; CI, chronicity index; SLEDAI, systemic lupus erythematosus disease activity index. toxin where necessary. CRP peptides (purity .98%) were synthesized by Scilight Biotech- necessary to substantiate our findings. Furthermore, the lo- nology (Beijing, China) and Science Peptide Biologic Technology calized generation of mCRP in inflamed tissues and the con- (Shanghai, China). CFH was obtained from Calbiochem (Darm- formation-dependent actions may underlie the current stadt, Germany).

Figure 3. a.a. 35–47 mediate the binding of mCRP to CFH in . (A) Urea-denatured mCRP, recombinant Cys-mutated mCRP, or native CRP at the indicated concentrations was added to CFH immobilized onto microtiter wells. Cys-mutated mCRP represents an mCRP conformation with enhanced activities.19 The binding was detected with mCRP-specific mAb 3H12 or native CRP-specific mAb 1D6.18,20 mCRP bound strongly to CFH, whereas native CRP did not. (B) Urea-denatured mCRP or (C) Cys-mutated mCRP was added to immobilized CFH together with the in- dicated CRP peptides (n=3). Prominent inhibition of mCRP binding was only observed with a.a. 35–47. (D) a.a. 35–47 with a C-terminal biotin tag were added to immobilized CFH (n=3). Peptide binding was determined with HRP-labeled streptavidin. This confirmed the direct binding of a.a. 35–47 to CFH. (E) Wild-type and mutant mCRP lacking a.a. 35–47 were expressed in Escherichia coli. These proteins were purified, and their bindingtoimmobilizedCFHwasexamined(n=3). Wild-type mCRP showed strong binding as expected, whereas the binding capacity was lost on deletion of a.a. 35–47. (F) Purified anti-a.a. 35–47 but not anti-a.a. 199–206 autoantibodies inhibited the binding of mCRP to CFH. *P,0.05.

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Figure 4. a.a. 35–47 mediate the binding of mCRP to CFH in SPR assays. (A) Urea-denatured mCRP, (B) Cys-mutated mCRP, (C) a.a. 35–47 peptide, (D) mCRP D35–47, or (E) native CRP was injected in fluid phase to CFH conjugated to CM5 chips. CM5 chips without CFH conjugation served as controls. Dose-dependent binding was observed for mCRP and a.a. 35–47 but was not detected with mCRP D35–47 and native CRP. (F) Binding of 6.25 (blue), 12.5 (pink), 25 (green), or 50 nM (purple) mCRP to CFH in the absence or presence of 26.5 nM anti-a.a. 35–47 autoantibodies or human IgG. mCRP binding was markedly reduced by the coinjection of anti-a.a. 35–47 autoantibodies. Human IgG showed little inhibition.

3H12mAbrecognizing mCRPand1D6 and 8D8mAbs recognizing with matched sex and age distribution. All of the samples were stored CRP were prepared as described.18,20 Autoantibodies were purified at 280°C in aliquots. Informed consents for blood sampling and according to the protocol used in our previous work.37 Briefly, gross renal biopsy were signed by all of the participants. The research was IgG proteins were first purified from the serum of a patient with LN in compliance of the Declaration of Helsinki and approved by the undergoing plasma exchange through a protein G column. Epitope- local ethical committee. specific autoantibodies were then isolated from gross IgG proteins by The renal biopsy specimens were examined by light, immunoflu- a.a. 35–47- or 199–206-conjugated resins. orescence, and electron microscopy. LN was reclassified according to the International Society of Nephrology/Renal Pathology Society 2003 Patient Selection classification system.40 Pathologic parameters, including activity in- Eighty patients were enrolled with renal biopsy–proven LN diagnosed dices and chronicity indices, were determined by renal pathologists between January of 2003 and July of 2013 at Peking University First using a semiquantitative scoring system.41,42 Hospital. These patients fulfilled the 1997 American College of Rheumatology revised criteria for SLE,38 and their clinical disease Characterization of CRP Autoimmune Epitopes activities were assessed using the SLEDAI.39 They were followed up Autoantibody binding assays were performed according to our pre- by outpatient clinic. Death was defined as the primary end point, and vious work.8 Briefly, urea-denatured mCRP (2 mg/ml) or synthesized ESRD or doubling of serum creatinine was defined as the secondary CRP peptides (10 mM) were immobilized onto microtiter wells end point. The demographic and clinical data of the patients are (Nunc; Roskild, Denmark) in 100 ml 0.05 M bicarbonate buffer summarized in Table 1. (pH 9.6) for 1 hour at 37°C. Wells with buffer alone served as anti- The plasma samples of patients were obtained on the same day of gen-free controls. Test sera were diluted at 1:100 in PBS containing renal biopsy before initiation of immunosuppressive treatment. 0.05% Tween-20 and 0.2% BSA and added in duplicate to both an- Plasma samples of 24 healthydonors were collected as normal controls tigen-coated wells and antigen-free wells at 37°C for 1 hour followed

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by washing. In competition assays, test sera were added to immobilized mCRP together with syn- thesized CRP peptides. Autoantibody binding was determined with alkaline phosphatase– conjugated goat anti-human IgG (1:5000; Fc specific; Sigma Aldrich, St. Louis, MO). Wells were then developed, and absorbance at 405 nm was measured. Twenty-four sera from healthy donors were examined following the same pro- tocol, and the resultant mean plus 2 SDs of the absorbance values were set as the autoantibody- positive cutoff.

CFH Binding Assays ELISAs were performed as described31 with mi- nor modification. Briefly, 4 mg/ml CFH was immobilized onto microtiter wells in 100 ml TBS-Ca (10 mM Tris, 140 mM NaCl, and

2 mM CaCl2, pH 7.4) for 1 hour at 37°C. Wells were washed with TBS-Ca containing 0.02% Tween 20 and blocked with 1% BSA/TBS-Ca for 1 hour. CRP, mCRP, or mCRP mutants at the indicated concentrations were added for 1 hour. In competition assays, mCRP was added together with the indicated CRP peptides. The binding was detected with CRP or mCRP mAbs and horseradish peroxidase (HRP)–conjugated goat anti-mouse IgG antibodies (1:5000; Gibco BRL). Wells were developed, and absorbance at 450 nm was measured. The interactions of CFH with CRP, mCRP, and CRP peptides were also examined by SPR using a Biacore T200 (Biacore AB, Uppsala, Swe- den). CFH was immobilized onto CM5 chips to reach an response unit of 1000. Test ligands were

by the calculation of the ratio. mCRP and a.a. 35–47 significantly promoted the cofactor activity of CFH, whereas CRP did not. (E) Im- munostaining of mCRP, CFH, and C5b-9 in kidney tissues of patients with LN. The signals of mCRP colocalize with that of CFH but show little colocalization with that of C5b-9. The Pearson coefficients evaluating the extent of colocalization are also shown in the right panel. (F) The cofactor activity of CFH was measured by iC3b generation using a sand- Figure 5. a.a. 35–47 promote the cofactor activity of CFH. (A) Urea-denatured mCRP, wich ELISA after coincubation of the indicated (B) Cys-mutated mCRP (0–45 mg/ml), (C) native CRP (100–300 mg/ml), or (D) a.a. 35–47 components for 1 hour (n=3): C3b, 7.5 mg/ml; peptide (1–3 mg/ml) was added to the indicated reaction mixtures for 1 hour (n=3). CFH, 5 mg/ml; , 0.25 mg/ml; C3b cleavage was determined by immunoblotting; a9, b,anda0 represent uncleaved mCRP, 20 mg/ml; and autoantibodies, 15 mg/ C3b a9-chain, C3b b-chain, and cleaved fragment of C3b a9 chain, respectively. The ml. The enhancement of mCRP on CFH’sco- intensity ratio between bands a9 and a0 was calculated to quantify the cofactor activity factor activity was reversed by anti-a.a. 35–47 of CFH. This index is more reliable than the absolute intensity of a0, because variations but not anti-a.a. 199–206 autoantibodies. in loading and/or developing across samples and experiments are largely eliminated *P,0.05; **P,0.01.

3052 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 3044–3054, 2017 www.jasn.org CLINICAL RESEARCH then injected at a flow rate of 30 ml/mininTBS-Cacontaining DISCLOSURES 0.005% P-20. Injection of buffer alone served as negative control. None.

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