FIGURE 2 | C3G autoantibodies of group 1 and group 2 influence C3 convertase assembly and dissociation by Factor H. (A) Two group 1 autoantibodies from patients #A and #J with high C3 convertase binding IgGs enhance C3-convertase formation. This enhancing effect is even stronger than the C3 convertase stabilizer and complement activator properdin. In contrast, group 2 autoantibodies from patients #a and# r do not enhance C3 convertase formation. Their effect is comparable to IgGs derived from NHS or from a DEAP-HUS patient with autoantibodies to Factor H or to buffer. (B) The same group1 antibodies when bound to the 3 convertase stabilize the enzyme and enhances resistance to factor H mediated dissociation. C3-convertase assembly in vitro and stabilization by autoantibodies are measured over 45 min by detection of convertase Bb using ELISA. (C) The same experiments as outlined in panel (A) are performed with all 33 patients derived autoantibody fractions and bound IgGs are identified after 45 min. All 12 group 1 antibodies enhance C3-convertase formation and again all group 2 antibodies have no or rather low effects (\*\*\*p < 0.001). (D) All group 1 autoantibodies stabilize the C3 convertase from dissociation by factor H and all group 2 antibodies lack this activity (\*\*\*p < 0.001).

was added to the LPS coated wells and the reaction was incubated further at 37◦C for 1 h. Following washing surface deposited C3b or C5b-9 were quantified by ELISA at 450 nm using C3b mAb (1:1,000, Fitzgerald) and anti C5b-9neo (1:1,000, Comptech) and secondary anti-mouse (1:1,000, Dako) or anti-goat antiserum (1:1,000, Dako).

#### Hemolytic Assay

Purified patient derived IgGs (10, 30, or 40 ug) were added to NHS (10%) and the mixtures incubated for 20 min at 37◦C with agitation (550 rpm). Rabbit erythrocytes (2∗ 108 /ml) were washed, suspended in GVB∗∗ buffer (Comptech), added to IgGs-NHS and the mixture was then incubated for additional 30 min at 37◦C with agitation (450 rpm). Cells were centrifuged and erythrocyte lysis was evaluated by measuring the absorbance in supernatant at 414 nm.

### Statistical Analysis

Significant differences between two groups were analyzed using the unpaired two tailed Student's t-test of GraphPad Prism 5 for Windows. Values of ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001 were considered as statistically significant.

### RESULTS

### Identification of C3G Patients With Autoantibodies

To identify and characterize autoimmune C3G patients in the European/Jena cohort in more detail, serum samples of 250 nephritis patients were analyzed for autoantibodies which bind to the in vitro assembled alternative pathway C3-convertase. Thirty-three C3G patients were identified having C3bBb binding antibodies. Twelve sera showed strong binding (arbitrary units: 80 ± 4.8) and 21 patients showed weak binding (arbitrary units: 34 ± 2.6) as compared to IgGs from 44 NHS control samples (arbitrary units: 8.5 ± 1.2, p < 0.001) (**Figure 1**). Based on the different binding intensities two groups were separated, patient with high binding-, termed group 1 and patients with low binding antibodies, as group 2. The demographic data of the patients in these subgroups were rather homogenous for age and gender (**Table 1**).

All 33 sera were further analyzed using a standard assay for C3 Nephritic factor. Fifteen sera (42%) were positive for C3Nef which were identified in both group 1 and group 2 patients. Thus, in group 1 sera from seven individuals were negative and five sera were positive for C3Nef. Among group 2, 11 sera were negative and 10 were positive for C3Nef (**Figure 1** and **Table 1**).

In DEAP-HUS the presence of Factor H antibodies is associated with homozygous CFHR1-CFHR3 deficiency. Copy number variations in the CFHR gene cluster were determined for patients with C3-convertase antibodies. In particular no homozygous CFHR3/CFHR1 deficiency was detected; seven patients showed heterozygous CFHR3-CFHR1 deficiency (**Table 1**). Thus, autoimmune antibodies in C3G patients develop independently of CFHR3/CFHR1 homozygous deficiency.

### Estimating Binding Affinity of Autoantibodies

All here tested autoantibodies bound to the assembled C3 convertase, but with different intensity. These differences can be explained either by higher autoantibody titers in group 1 sera, or by a higher binding affinity to the C3-convertase. To find out if the autoantibodies bind stronger or weaker, binding of representative IgG preparations of group 1 or group 2 with a C3Nef negative and C3Nef positive variant of the C3-convertase was followed in real time and the apparent affinity was evaluated. The C3-convertase was assembled on the surface of a Ni(II)- NTA coated biosensor and purified IgGs from group 1 patients (#A C3Nef pos and #J C3Nef neg) of group 2 patients (#a C3Nef pos and #r C3Nef neg) were added as analyte. Association and dissociation was followed for 320 s. IgG fractions isolated from the patients harbored autoantibodies that bound to the assembled convertase. They showed a strong association and a slow dissociation upon removal of the analyte. In contrast, IgGs isolated from the NHS pool, from DEAP-HUS patient #1552, and also BSA did not bind to the C3 convertase. To compare the binding strength and to define an apparent KD, serial dilutions ranging from 25 to 75 nM were tested. Assuming that 10% of total IgG isolation represent C3-convertase binding autoantibodies the apparent KD values were calculated and identified for group 1 as 911 and 853 nM and for group 2 as 416 and 559 nM (**Table 2**). Control IgGs prepared from the NHS pool or from DEAP-HUS patient #1552 showed no, or background binding (KD > 2.5µM). The results demonstrate that autoantibodies from representative patients from both groups bind with rather similar affinity and binding under the assumption of 10% C3 convertase antibodies is in the nanomolar range. The differences in binding could be explained by the recognition of different epitopes.

### Group 1 but Not Group 2 Autoantibodies Enhance C3-Convertase Formation and Block Dissociation by Factor H

To analyze whether all C3-convertase binding autoantibodies have a stabilizing and activating effect we analyzed if the autoantibodies influence C3-convertase formation. First, purified IgGs from representative sera of group 1 (#A and #J) and group 2 (#a and #r) patients were tested in a functional kinetic assay. Again, the C3-convertases were assembled in presence of purified IgGs and convertase formation was followed by measuring attached Bb. IgGs from the two group 1 patients (#A and #J) enhanced C3-convertase assembly within the first 15 min and over time more C3-convertases were formed. The effect was more pronounced as that of properdin, the known C3-convertase stabilizer. In contrast, IgGs from the two group 2 patients (#a and #r) did not affect convertase formation in a significant manner. Their effect was lower than that of properdin and was almost comparable to NHS or buffer control (**Figure 2A**). Thus, group 1 IgGs allow faster C3-convertase formation and the formation of more enzyme (**Figure 2A**).

Next we asked if the IgG autoantibodies influence convertase dissociation by the inhibitor Factor H. To this end, C3-convertases were assembled in presence of the autoantibodies and Factor H. In this setting both group 1 autoantibodies (#A and #J) inhibited convertase dissociation by Factor H and the stabilizing effect on the assembled C3-convertase was even stronger than that of properdin (**Figure 2B**). In contrast, both group 2 antibodies (#a and #r) did not or weakly block C3-convertase dissociation by factor H (**Figure 2B**).

To evaluate if these differences are characteristic for the two groups, all IgGs were evaluated. All twelve group 1 autoantibodies efficiently stabilized the C3-convertase as shown by Bb binding (mean absorbance group 1: 1.1, p < 0.001, compared to NHS pool) (**Figure 2C**) even in the presence of Factor H (**Figure 2D**). All group 2 autoantibodies, had a rather low or no stabilizing effect on the C3-convertase (mean absorbance group 2: 0.5, n.s. difference to NHS pool) (**Figure 2C**) and no major influence on Factor H stabilization (**Figure 2D**). The effect by group 2 autoantibodies was comparable to IgGs derived from the control NHS pool or buffer. In summary, group 1 but not group 2 autoantibodies increased C3-convertase formation and stabilized the enzyme from decay by Factor H. A significant difference exists between group 1 and group 2 antibodies. IgGs from group 1, but not the group 2 group stabilized the C3-convertase. In each group, no significant difference was detectable between the C3Nef positive or the C3Nef negative samples. The effect of all group 1 autoantibodies was stronger than that of properdin, the known C3-convertase stabilizing protein and complement enhancer. This may explain the pathologic role of the antibodies.

#### Effect of Autoantibodies on C3-Convertase Activity

As group 1 autoantibodies enhanced C3-convertase formation, stabilized the enzyme and blocked the dissociation, we asked

FIGURE 3 | Group 1 but not group 2 antibodies enhance C3a generation. Fluid phase C3-convertase is assembled in presence of autoantibodies followed by addition of substrate C3. Following incubation for 45 min, C3a generation is monitored by ELISA. (A) Group 1 autoantibodies increase C3a generation and the effect is dose dependent. In contrast, group 2 antibodies do not enhance C3a formation. (B) When all autoantibodies were evaluated group 1 but not group 2 autoantibodies enhance C3a generation (\*\*\*p < 0.001). C3a generation by NHS is shown as a stippled line.

if the IgG autoantibodies influence C3-convertase activity. Therefore, C3-convertases were assembled in fluid phase in presence of the autoimmune IgGs, and after 30 min incubation generated C3a was quantitated by ELISA. The selected group 1 antibodies (i.e., #A and #J), but not the two group 2 antibodies (#a and #r) enhanced C3a generation, and the effect was dose dependent (**Figure 3A**). IgGs from C3Nef negative patient #r, had some activating activity at higher concentrations, but IgGs prepared from the C3Nef pos patient #a lacked any activity (**Figure 3A**).

Similar results were obtained when IgGs from all patients were analyzed. All high binding C3-convertase group 1-IgGs enhanced C3a formation (mean absorbance G1: 156, mean absorbance G2: 84; p < 0.0001 Group 1 compared to group 2). The group 2 IgGs had minor or no effects on C3a release (p = n.s. compared to NHS pool) (**Figure 3B**). Again, no difference between C3Nef positive and C3Nef negative samples was detectable. Thus, the high binding group 1 antibodies enhanced C3-convertase action also in the fluid phase which supports their role in pathology even further. Identified C3Nef in group 1 but not C3Nef in group 2 did activate the C3-convertase. These results are in agreement with C3 concentrations in patients' samples. G1 samples showed very low C3 amounts (103 ± 17µg/ml) compared to G2 samples (331 ± 80µg/ml; p < 0.0001).

#### Both Group 1 and Group 2 Autoantibodies Bind to the C5-Convertase

Group 1 and group 2 antibodies both bind to neoepitopes of the C3-convertase. As the AP C5-convertase, C3bBbC3b, with one additional C3b attached has a related structure or composition, we asked if the autoantibodies also bind to this enzyme. Therefore, autoantibody binding to AP C5-convertases assembled on the surface of a microtiter plate was evaluated by ELISA. Autoantibodies derived from all patients of the cohort bound to the assembled C5-convertase. Also for C5 convertase interaction, the high C3-convertase binding group 1-autoantibodies bound also more to the alternative pathway C5-convertase than group 2 antibodies (mean absorbance group 1: 1.2; mean absorbance group 2: 0.8; p < 0.01 group 1 compared to group 2) (**Figure 4**). In this set up both group 1 and group 2 antibodies bound with significantly higher intensity to the C5-convertase as NHS derived IgGs (p < 0.01).

### Most Autoantibodies Activate the C5-Convertase

Based on binding to the C5-convertase we asked if the autoantibodies affect C5-convertase activity. First, IgGs from the four selected patients were added to NHS and after 45 min incubation newly generated C5a was determined in the supernatant by ELISA. All four antibody IgG preparations (#A, #J, #a, and #r) increased C5a generation (p < 0.05 to 0.001 as compared to NHS-IgGs) (**Figure 5A**). When IgGs from all patients were tested all, except one sample (# F) from group 1 and two samples from group 2 (#b and #s) increased C5a generation. The IgGs from both groups generated rather similar C5a levels, however, the range was higher for group 1 autoantibodies (mean absorbance group 1: 58, mean absorbance group 2: 48, G1 to G2 p = n.s.; G1 to buffer p < 0.05; G2 to buffer p < 0.005) (**Figure 5B**). Again, this effect was independent and did not correlate with the presence or absence of C3Nef.

#### IgG Antibodies Induce C3b Deposition

Next the effect of the autoantibodies on fluid phase C3-convertase mediated C3b opsonization was evaluated. Purified patients IgGs were added to NHS and after 30 min surface deposited C3b was quantitated by ELISA. Both group 1 antibodies (#A and #J) reduced C3b surface deposition and this inhibitory effect was already detected with 10µg/ml IgG (**Figure 6A**). In contrast,

# 328, #2367 (both group 2), enhance C5a generation in a significant manner (\*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001). Purified IgGs from each patient was added to complement active NHS at 37◦C and the mixture was incubated for 45 min. C5a generation was monitored by ELISA and compared to C5a generation in NHS (stippled line).

both group 2 antibodies (#a and #r), as well as NHS-derived IgGs did not affect C3b deposition (**Figure 6A**).

The inhibitory effects were specific for group 1. All group 1 antibodies, but no group 2 antibody blocked C3b deposition (mean absorbance G1: 0.2, mean absorbance G2: 2.2; p < 0.001 group 1 vs. group 2) (**Figure 6B**). These results reveal a functional difference between group 1- and group 2 autoantibodies in C3b deposition.

#### Autoantibodies Influence TCC Deposition

The effect of the autoantibodies on TCC formation was evaluated by following C5b-9 deposition and using a hemolytic assay. First, the representative antibodies from group 1 (#A and #J) and group 2 (#a and #r) were evaluated. The IgGs were added to NHS and the deposition of C5b-9 was measured by ELISA. IgGs derived from the group patients (#A and #J) decreased C5b-9 deposition in a dose dependent manner, whereas group 2 autoantibodies (#a and #r) enhanced C5b-9 deposition when compared to NHS derived IgGs (**Figure 7A**). sC5b-9 concentration in the samples ranged from 3.4 ±1.3µg/ml in group 1 to 7.4 ± 2.3µg/ml in group 2.

The differences between group 1 and group 2 antibodies was confirmed for all autoantibodies using a hemolysis assay. The IgGs derived from the patients were added to NHS and then rabbit erythrocytes which represent activators of human complement were challenged with the supplemented serum and after 20 min incubation lysis of erythrocytes was followed. All group 1, but no group 2 antibody blocked hemolysis (mean

were tested in the same set up. Again, all group 1, but not group 2 antibodies block C3b surface deposition (\*\*\*p < 0.001). Purified IgG in (A,B) were added to NHS, and the supplemented serum was added to a LPS-coated microtiter plate. After 1 h C3b deposition was assayed by ELISA. Hemolysis by NHS is marked by a stippled line.

absorbance group 1: 0.3, mean absorbance group 2: 1.0, p < 0.001 group 1 compared to group 2; p = not significant group 2 to NHS pool) (**Figure 7B**). This clear difference between group I and group II antibodies was again independent of the presence or absence of C3Nef. Thus, a clear functional difference between group 1 and group 2 antibodies in autoimmune C3G affects complement mediated TCC deposition and hemolysis.

### Eculizumab Inhibits C5a Generation in All Probes

Given that all C3G autoantibodies activate the C5-convertase we asked whether the C3G autoantibodies influence the action of Eculizumab, the therapeutic C5 binding complement inhibitor. Fluid phase C3-convertases were assembled in presence of autoantibodies or in presence of autoantibodies and Eculizumab. After addition of C3 to the mixture and 45 min incubation, C3a as well as C5a, generated in fluid phase, was quantitated by ELISA. As expected, Eculizumab did not influence C3a release (**Figure 8A**). However, Eculizumab blocked C5a release in presence of both group 1 as well as group 2 autoantibodies (**Figure 8B**). Thus, C5 binding Eculizumab blocks the action of a C5-convertase which has the C3G autoantibody attached. Eculizumab and autoantibodies bind to different proteins. Eculizumab binds to the substrate

C5 and the autoantibodies bind to different neoepitopes of the C5-convertase.

erythrocytes was assayed by measuring the absorbance. Lysis of erythrocytes in NHS is marked as stipled line.

### DISCUSSION

Here we identified and characterized autoimmune forms of the kidney disease C3G. A cohort of 33 C3G patients displayed autoantibodies which bound to both the C3- and the C5 convertase of complement. All autoantibody isolates activated the C5-convertase (C5Nef), but only half of them activated in addition the C3-convertase (C3Nef). IgG fractions with C3Nef plus C5Nef (group 1 antibodies) had stronger complement activating functions in in vitro assays compared to fractions with C3Nef alone (group 2 antibodies). Group 1 antibodies induced a fast consumption of the complement components C3 and C5 in hemolytic assays. Eculizumab, the therapeutical C5 monoclonal antibody inhibits C5 cleavage and C5a generation triggered by each group 1 or group 2 antibody fraction, but as expected has no effect on enhanced C3 cleavage.

About 45% of all autoantibodies in our cohort were identified as C3Nef using the classical hemolytic assay. This assay identifies autoantibodies that bind to a neoepitope formed in the C3 convertase. However, additional autoimmune antibodies than C3Nef add to C3G, which are not identified by the classical C3Nef assay. These antibodies bind to the assembled C3-convertase or components thereof. In addition, several C3Nef antibodies bound to the C3-convertase but did not lead to the activation of the enzyme in vitro. In contrast, initially identified C3Nef antibodies bound/activated the C5-convertase.

Based on the reactivity to the assembled C3-convertase, two groups of autoantibodies were identified. Group 1 and group 2 antibodies, which both had high affinities in binding to the

C5a generation of C5-convertases with either group 1 or group 2 antibodies attached (\*\*\*p < 0.001). Eculizumab was added to NHS together with group 1 or group 2

assembled C3-convertase, but exclusively group 1 antibodies also activated the C3-convertase. As the binding affinities of IgGs to the C3-convertase were very similar in both groups, the different functional effects between the groups are explained by either a higher titer of the autoantibodies in each isolated IgG fraction of group 1 patients or by a higher C3-convertase stabilizing effect by group 1 antibodies compared to group 2. Different antibody titers between the two groups were excluded, as dilutions of group 1 antibody preparations did neither influence nor diminish the stabilizing effect or the resistance to convertase decay by Factor H. Thus, the functional differences between group 1 and group 2 antibodies are likely independent of the autoantibody titers and rely on the recognition of different epitopes which are recognized in the C3- and C5-convertases. All autoantibody fractions from the patients bound to the C5 convertase and as most of them enhanced the activity, they are likely similar to previously defined C5Nef (46). This is surprising as group 1 and group 2 autoantibodies contained C3Nef (according the C3Nef assay) which stabilize the C3 convertase but not the C5-convertase. Therefore, we conclude that two different types of antibody carriers were identified in this cohort of C3G patients: C3Nef+/C5Nef+ (stabilizing the C3-convertase and also the C5-convertase) and C3Nef– /C5Nef+ (stabilizing the C5 convertase) patients. A similar classification was found in a cohort study described by Donadelli et al. (47), who in addition identified patients with exclusively C3Nef antibodies. Thus, C3G patients are heterogeneous and include patients with C3Nef or C5Nef or combinations thereof. This heterogeneity results in presence of nephritic factors with different functional activities and consequences in vivo for the patient. In the Jena cohort, nearly all patients were C5Nef positive and show that a large and substantial fraction of autoimmune C3G patients are not identified by only a standard C3Nef assay.

antibodies (each 30 µg). Following incubation C5a generation was measured by ELISA.

Genetic copy number analyses of the CFHR gene cluster revealed for all 33 autoimmune C3G patients no homozygous deletion in the CFHR gene cluster. This lack of correlation with CFHR copy number variations is in clear contrast to DEAP-HUS patients, i.e., patients who present with the autoimmune form of hemolytic uremic syndrome. DEAP-HUS patients who developed autoantibodies to Factor H present frequently with a homozygous deletion of the CFHR1- CFHR3 segment or alternatively but rarely by a compound heterozygous CFHR1-CFHR3, together with CFHR3-CFHR4 deletions (48).

There is no clear therapy regiment for C3G today. As serum derived autoantibodies, as well as purified IgGs in our cohort bound to the in vitro assembled C5-convertase and enhanced fluid phase C5a generation, the question arose whether inhibition of C5 by Eculizumab is a therapeutic option. Off-label use of Eculizumab in C3G revealed promising results in some, but not all patients (49–51). In vitro assays in our cohort confirmed inhibition of C5 by Eculizumab and reduced hemolysis. However, the inhibitory effect was exclusively present in group 2, with C5Nef only. In group G1 carriers with C3Nef plus C5Nef, complement activation is likely too strong to be restricted by Eculizumab alone. Whether these patients benefit or profit from a stratified immunosuppressive therapy needs to be determined in the future in clinical practice.

### ETHICS STATEMENT

The study was approved by the ethics committee of the University Hospital of Jena, Germany.

## AUTHOR CONTRIBUTIONS

FZ, MK, SA, SL, IL, and AH performed experiments and analyzed the data. BN and KE helped in C3Nef assays. LW, SH, and GS provided patient's samples. All authors read the manuscript and discussed the data. PZ and CS designed the study, analyzed data, and wrote the manuscript.

#### FUNDING

The research leading to these results has received funding from the European Community's Seventh Framework Programme under grant agreement (2012-305608) European Consortium for High-Throughput Research in Rare Kidney Diseases (EURenOmics). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The study was supported by German

#### REFERENCES

Research Foundation Deutsche Forschungsgemeinschaft, SFB 1192 Immune mediated glomerular diseases, project B06 and SK46/2.

#### ACKNOWLEDGMENTS

We thank Marina Noris and Beppe Remuzzi for their contribution of serum samples from patients.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Zhao, Afonso, Lindner, Hartmann, Löschmann, Nilsson, Ekdahl, Weber, Habbig, Schalk, Kirschfink, Zipfel and Skerka. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

,

# Clinical and Immunological Profile of Anti-factor H Antibody Associated Atypical Hemolytic Uremic Syndrome: A Nationwide Database

#### Edited by:

Bradley Patton Dixon, Children's Hospital Colorado, United States

#### Reviewed by:

Christoph Licht, Hospital for Sick Children, Canada Mihály Józsi, Eötvös Loránd University, Hungary Pilar Sánchez-Corral, University Hospital La Paz Research Institute (IdiPAZ), Spain

#### \*Correspondence:

Arvind Bagga<EMAIL_ADDRESS>
†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Autoimmune and Autoinflammatory Disorders, a section of the journal Frontiers in Immunology

> Received: 23 February 2019 Accepted: 20 May 2019 Published: 07 June 2019

#### Citation:

Puraswani M, Khandelwal P, Saini H, Saini S, Gurjar BS, Sinha A, Shende RP, Maiti TK, Singh AK, Kanga U, Ali U, Agarwal I, Anand K, Prasad N, Rajendran P, Sinha R, Vasudevan A, Saxena A, Agarwal S, Hari P, Sahu A, Rath S and Bagga A (2019) Clinical and Immunological Profile of Anti-factor H Antibody Associated Atypical Hemolytic Uremic Syndrome: A Nationwide Database. Front. Immunol. 10:1282. doi: 10.3389/fimmu.2019.01282 Mamta Puraswani 1†, Priyanka Khandelwal 1†, Himanshi Saini 1 , Savita Saini 1 , Bahadur Singh Gurjar 2 , Aditi Sinha1 , Rajashri Pramod Shende3 , Tushar Kanti Maiti 4 , Abhishek Kumar Singh4 , Uma Kanga5 , Uma Ali 6 , Indira Agarwal 7 , Kanav Anand8 , Narayan Prasad9 , Padmaraj Rajendran10, Rajiv Sinha11, Anil Vasudevan12, Anita Saxena13 Sanjay Agarwal 14, Pankaj Hari 1 , Arvind Sahu3 , Satyajit Rath3,15 and Arvind Bagga1 \*

1 Division of Nephrology, Department of Pediatrics, All India Institute of Medical Sciences, New Delhi, India, 2 Immuno Biology Laboratory II, National Institute of Immunology, New Delhi, India, 3 National Centre for Cell Science, Pune, India, 4 Regional Center for Biotechnology, Faridabad, India, 5 Department of Transplant Immunology and Immunogenetics, All India Institute of Medical Sciences, New Delhi, India, 6 Department of Pediatrics, BJ Wadia Hospital for Children, Mumbai, India, 7 Department of Pediatrics, Christian Medical College, Vellore, India, 8 Division of Pediatric Nephrology, Sir Ganga Ram Hospital, New Delhi, India, 9 Department of Nephrology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India, 10 Department of Pediatric Nephrology, Institute of Child Health and Hospital for Children, Madras Medical College, Chennai, India, 11 Department of Pediatrics, Institute of Child Health, Kolkata, India, 12 Department of Pediatric Nephrology, St. Johns Medical College and Hospital, Bengaluru, India, 13 Department of Cardiology, All India Institute of Medical Sciences, New Delhi, India, 14 Department of Nephrology, All India Institute of Medical Sciences, New Delhi, India, 15 Translational Health Science and Technology Institute, Faridabad, India

Background: Atypical hemolytic uremic syndrome (aHUS), an important cause of acute kidney injury (AKI), is characterized by dysregulation of the alternative complement pathway. Autoantibodies to factor H (FH), a chief regulator of this pathway, account for a distinct subgroup. While high anti-FH titers predict relapse, they do not correlate well with disease activity and their functional characterization is required.

Methods: Of 781 patients <18-year-old of aHUS in the nationwide database from 2007 to 2018, 436 (55.8%) had anti-FH antibodies. Clinical features and outcome of patients managed in the last 6-year (n = 317) were compared to before (n = 119). In plasma samples of 44 patients, levels of serial circulating FH immune complexes (CIC), free FH, soluble terminal complement complex (sC5b-9), sheep red blood cell (SRBC) lysis and epitope specificity (n = 8) were examined. Functional renal reserve, ambulatory hypertension, left ventricular hypertrophy (LVH), and proteinuria were evaluated in a subset.

Results: Patients presented with markedly elevated anti-FH titers (10,633.2 ± 998.5 AU/ml). Management varied by center, comprising plasma exchange (PEX; 77.5%) and immunosuppression (73.9%). Patients managed in the last 6-year showed better renal survival at mean 28.5 ± 27.3 months (log rank P = 0.022). Mean anti-FH titers stayed 700–1,164 AU/ml during prolonged follow-up, correlating with CIC. Patients with relapse had lower free-FH during remission [Generalized estimating equations (GEE), P = 0.001];

**92**

anti-FH levels ≥1,330 AU/ml and free FH ≤440 mg/l predicted relapse (hazards ratio, HR 6.3; P = 0.018). Epitope specificity was similar during onset, remission and relapse. Antibody titer ≥8,000 AU/ml (HR 2.23; P = 0.024), time to PEX ≥14 days (HR 2.09; P = 0.071) and PEX for <14 days (HR 2.60; P = 0.017) predicted adverse renal outcomes. Combined PEX and immunosuppression improved long-term outcomes (HR 0.37; P = 0.026); maintenance therapy reduced risk of relapses (HR 0.11; P < 0.001). At 4.4±2.5 year, median renal reserve was 15.9%; severe ambulatory, masked and pre-hypertension were found in 38, 30, and 18%, respectively. Proteinuria and LVH occurred in 58 and 28% patients, respectively.

Conclusion: Prompt recognition and therapy with PEX and immunosuppression, is associated with satisfactory outcomes. Free-FH predicts early relapses in patients with high anti-FH titers. A significant proportion of impaired functional reserve, ambulatory hypertension, proteinuria and LVH highlight the need for vigilant long-term follow-up.

Keywords: atypical hemolytic uremic syndrome, factor H, plasma exchange, renal reserve, thrombotic microangiopathy

#### INTRODUCTION

Hemolytic uremic syndrome (HUS) is an important cause of acute kidney injury (AKI) in children (1, 2). While the majority of patients follow gastrointestinal infection with Shiga toxin associated organisms, abnormalities in the complement and coagulation pathways are associated with atypical hemolytic uremic syndrome (aHUS) (1, 3). Although 5–25% patients in European cohorts show antibodies to factor H (FH) (4– 6), this subset of illness is common in India accounting for ∼50% cases (7). Recent data from the global aHUS registry, from centers in Europe, North America and Australia, confirm the presence of anti-FH antibodies in 24% children and 19% adults (8).

The pathogenesis of anti-FH associated aHUS and reasons for its high frequency in south Asia are unclear. While more than 80% patients show a homozygous deletion in the gene encoding FH related protein 1 (CFHR1), the deletion is present in 5– 10% healthy people across the world. High levels of antibodies at disease onset or relapse are believed to induce functional deficiency of FH; their decline in response to plasmapheresis is associated with disease remission (7, 9, 10). The antibodies bind chiefly to the C-terminus of FH, inhibiting its cell surface regulatory functions (11, 12). A dose-response relationship is not established as many patients show high antibody levels even during remission, emphasizing the need to evaluate other markers of complement activation. Studies relating antibody titers to functional assays of FH inhibition, such as level of sheep red blood cell (SRBC) lysis, free FH, soluble terminal complement complex (sC5b-9) and epitope specificity of antibodies are limited (9, 13, 14).

We report the clinical features and outcomes of a large nationwide database of patients with anti-FH associated HUS. We also examined the functional implications of anti-FH antibodies and biomarkers that might enable prediction of a relapse.

#### METHODS

Since March 2007, 781 patients younger than 18-year-old with aHUS have been enrolled in a prospective multicenter nationwide database at the All India Institute of Medical Sciences (AIIMS). Of these, 436 patients with anti-FH antibody associated aHUS, diagnosed in presence of microangiopathic hemolytic anemia (hemoglobin <10 g/dl, schistocytes ≥2%, lactate dehydrogenase >450 U/l), thrombocytopenia (platelets <150,000/µl), AKI and anti-FH antibody titers >150 AU/ml (15) were included. Clinical features of patients enrolled until February 2013 have been reported earlier (7). Patients with septicemia, disseminated intravascular coagulation, and thrombotic microangiopathy secondary to medications, lupus, HIV infection, and following bone marrow transplantation were excluded. Institute ethics committee approval was obtained and informed written consent was taken prior to enrolment.

#### Investigations

Anti-FH antibodies were screened in plasma samples by enzyme linked immunosorbent assay (ELISA) (7). Antibody titer, determined at serial dilutions, was expressed as arbitrary units (AU)/ml at 1:50 dilution; values >150 AU/ml were considered abnormal (7). Investigations included urinalysis, blood levels of complement C3, antinuclear antibody, and antineutrophil cytoplasmic antibody. Leptospirosis, dengue, malaria, and rickettsia might rarely mimic clinical and laboratory features of HUS; we therefore screened for these infections. Levels of anti-FH antibodies, creatinine and urinalysis were estimated every 3–6 months. Studies for functional effects of anti-FH antibodies, including circulating FH immune complexes (CIC), free FH, sC5b-9, and SRBC lysis, were performed in 44 consecutive patients managed at AIIMS. Epitope specificity of FH-antibodies was determined during onset, remission, and relapse, in a subset of eight patients.

#### Circulating FH Immune Complexes (CIC)

Briefly, 96-well plates (Nunc-Immuno Micro Well, Sigma-Aldrich, MO), coated with sheep anti-FH polyclonal antibody (AbD Serotec, Hercules, CA) diluted 1:10,000 in 0.5 M carbonate buffer (pH 9.6), were incubated overnight at 4◦C (13). After washing, plates were blocked with 1% non-fat milk (Sigma-Aldrich) for 2-h at 37◦C. Serial dilutions of plasma samples and controls were incubated for 1-hr, followed by incubation with goat anti-human IgG conjugated with horse radish peroxidase (HRP; Sigma-Aldrich) and titers reported at 1:100 dilution. For color development, H2O2 substrate and 3,3′ ,5,5 ′ tetramethylbenzidine were added; optical density was read at 450 nm. Titers of immune complexes, expressed as AU/ml, were calculated based on reference plasma (courtesy Marie Agnès Dragon-Durey). Screening of 100 healthy donors was used to establish the positive threshold of 110 AU/ml, corresponding to mean + 2 SD; intra- and inter-assay coefficient of variation was <10%.

#### Free Factor H

Plasma samples diluted to 1:100 were incubated with Protein Gcoated beads (10:1; Sigma-Aldrich) for 1-h at room temperature, followed by centrifugation. The supernatant was used for CIC ELISA to confirm removal of all IgG-bound FH. ELISA plates, coated with sheep anti-FH polyclonal antibody (AbD Serotec) and blocked with 1% bovine serum albumin (BSA), were incubated for 1-h at room temperature with the supernatant diluted to 1:5000. The plate was washed and monoclonal anti-FH antibody (AbD Serotec) added, followed by washing and addition of anti-mouse IgG labeled with HRP with color development as described above. Purified FH (Calbiochem, Meudon, France) was used as the calibrator.

#### Sheep Red Blood Cell (SRBC) Hemolysis

Patient plasma was serially diluted to 1:1, 1:2, and 1:4 with buffer (20 mM Hepes, 7 mM MgCl2, 10 mM EGTA, 144 mM NaCl, 1% BSA; pH 7.4). Diluted plasma (10 µl) was incubated with 10 µl sheep erythrocytes (108 cells/ml) at 37◦C for 45 min [modified from (11)]. Following addition of 280 µl normal saline, the samples were centrifuged and absorbance read at 414 nm. Complete hemolysis with water was considered as 100%. Hemolysis was calculated as follows:

$$\text{SRBC lysis} = \frac{\text{(OD of patient sample - OD of blank)} \times 100}{\text{OD of well labeled 100\%}} \quad \text{(1)}$$

Mean SRBC lysis in 20 healthy controls was 16.9±2.1%; the positive threshold was 21%.

#### Soluble Terminal Complement Complex (sC5b-9)

sC5b-9 was quantitated by ELISA using the MicroVue kit (Quidel Corp, San Diego, CA). Diluted plasma specimens were added to 96-well plate pre-coated with monoclonal antibody against C9 ring and incubated at room temperature for 1-hr. After addition of biotinylated antibody specific to sC5b-9, HRP-conjugated streptavidin and tetramethylbenzidine, optical density was read at 450 nm. sC5b-9 levels were calculated from a standard curve, limits of detection being 3.7–170 ng/ml.

#### Production of FH Fragments

Short consensus repeats (SCR) 1–4, 5–8, 9–12, 13–16, and 17– 20 were PCR amplified with specific primers from the FH cDNA (OriGene, Rockville, MD) and cloned in pET28 and pET29 expression vectors (16). The clones were verified by DNA sequencing (ABI 3730 DNA analyzer; Applied Biosystems), products were transformed in E. coli BL21 and expressed in inclusion bodies generated after induction with 1 mM isopropyl-1-thio-b-D-galactopyranoside and purified using Ni-NTA resin. The eluted fragments were purified by gel filtration using Superdex75 columns (GE Healthcare) and their concentration determined by Bradford assay (Bio-Rad).

#### Epitope Specificity

Plasma samples were incubated with FH fragments for 1-h and added to ELISA plates pre-coated with purified FH (Calbiochem) and blocked with 1% BSA. Bound antibodies were detected by HRP conjugated goat anti-human IgG with color development, as above.

#### Therapy and Outcomes

The management of patients across centers was at the discretion of treating physicians, depending on their experience and available facilities. Renal replacement therapy was provided, when required. Specific management varied across centers and included plasma exchanges (PEX) and/or immunosuppressive therapies (9). Hematological remission was defined as platelet count >100,000/mm3 , schistocytes <2% and LDH less than upper limit of normal on two consecutive days. Disease relapse was considered when there was a new episode of illness after the patient had achieved remission for ≥2 weeks. Outcomes at 3-months and last follow-up were assessed, in terms of estimated glomerular filtration rate (eGFR) (17), hypertension (18), dipstick proteinuria and relapses. Adverse outcome was defined as eGFR <30 mL/min/1.73 m2 on follow-up or patient death.

#### Renal and Cardiovascular Outcomes

Long term outcomes were evaluated in 50 consecutive patients with eGFR more than 60 ml/min/1.73 m2 and two or more years' follow-up. Blood pressure was recorded and classified using standard guidelines (18). First morning urine protein-tocreatinine ratio >0.15 mg/mg was considered abnormal (19). On echocardiography, left ventricular mass index (LVMI) was defined as ratio of left ventricular mass to height2.7; LVH was LVMI >95th centile for age and sex (20). Dyslipidemia was defined as fasting total cholesterol >170 mg/dl, LDL cholesterol >110 mg/dl, non-LDL cholesterol >120 mg/dl, triglycerides >75 mg/dl or HDL cholesterol <40 mg/dl (21).

#### Ambulatory Blood Pressure Monitoring (ABPM)

Ambulatory blood pressure was recorded by oscillometry (90207, Spacelabs Medical, Redmond, WA). Data was reported as standard deviation scores (SDS) based on height and sex specific normative data (22). Patients were classified as having ambulatory hypertension if they showed clinic hypertension (blood pressure ≥95th percentile), mean ambulatory systolic or diastolic blood pressure exceeding 95th percentile for sex and height, and systolic or diastolic load >25% (severe if load >50%) (22). Masked hypertension was defined as ambulatory hypertension with normal clinic blood pressure (22).

#### Functional Renal Reserve

Functional renal reserve was evaluated following administration of oral trimethoprim (10 mg/kg/day) for 5 days (23), discontinuation of ACE-inhibitors, and a vegetarian diet for 48-h (24). Patients voided completely at 7:00 am on the day of test; residual urine, estimated by ultrasonography, was required to be below 10 ml. They were instructed to drink 5 ml/kg water every 30 min throughout the study period. An accurately timed urine collection of 2-h duration was obtained and residual urine rechecked; serum creatinine was estimated at its midpoint. A protein meal (1 g/kg; RiteBite Max bar containing 20 g casein and whey protein) was ingested. Following complete voiding, 40 min later, similar 2-h urine collection and measurement of serum creatinine were done. Renal clearance of creatinine (CrCl) and functional renal reserve were calculated:

$$\text{CrCl} = \frac{\text{UCr} \times \text{V}}{\text{sCr} \times \text{t}} \times \frac{1.73}{\text{BSA}} \tag{2}$$

UCr urine creatinine, V urine volume over 2-h, t duration of collection (minutes), BSA body surface area, m2

$$\text{Functional real reservoir} = \frac{(\text{CrCl after protein load - Baseline CrCl})}{\text{Baseline CrCl}} \times 100 \text{ (3)}$$

#### Statistical Analysis

Six-year cohorts of March 2007 to December 2012 and January 2013 to August 2018 were compared in terms of clinical features and outcomes. Data is presented as proportions, and median (interquartile range, IQR) or mean ± SD, based on distribution and analyzed using Stata version 14.0 (Stata Corp, College Station, TX). Anti-FH antibodies were expressed as mean ± SEM. Tests for significance included t-test, Wilcoxon signed rank and rank sum tests, and chisquare test; correlation was measured by Spearman coefficient. Anti-FH and free FH levels were log transformed to satisfy normality. Repeated measures analyses by generalized estimating equations (GEE) approach was used to compare serial anti-FH and free FH concentrations in sustained remission or subsequent relapse; receiver operator characteristic (ROC) curves were used for threshold of relapse. Determinants of adverse outcome and relapse were estimated as odds and hazards ratios, by univariate and multivariable analyses. Functional renal reserve was normalized by Box-Cox transformation. Linear regression analyses were used to evaluate predictors of renal reserve, LVMI and proteinuria; two tailed P < 0.050 was considered significant.

#### RESULTS

From March 2007 to August 2018, 436 (55.8%) of 781 patients from 30 centers in the nationwide database were diagnosed as having anti-FH associated aHUS (**Supplementary Figure 1**). Proportion of patients with anti-FH antibodies younger than 4-year, between 4–11 and 11–18 year at presentation were 20.8, 73.8, and 52.0%, respectively; five patients presented in infancy (**Supplementary Figure 2**). Patients between 4 and 11 year had higher antibody titers (11,127 ± 1,170 AU/ml vs. 8,870 ± 1,890 AU/ml; P = 0.025). There was seasonal variation, with peak between December and April (**Supplementary Figure 3**). Prodromal illness included fever (54.6%), upper respiratory tract infection (10.3%), and diarrhea (6.7%). Eight of 282 patients (2.8%) showed antinuclear antibodies, 3 of 219 (1.4%) had antineutrophil cytoplasmic antibodies. Three of 197 (1.5%) showed antibodies to leptospira; vivax or falciparum malaria was present in fifteen.

Five adult patients (aged 22–48 years) also showed anti-FH antibodies (854–60,032 AU/ml) and presented with similar clinical features, chiefly following a febrile illness. One patient each with systemic lupus erythematosus and following bone marrow transplant also showed elevated levels (852 and 4,264 AU/ml, respectively). These seven-patients have not

TABLE 1 | Clinical and biochemical features in patients with anti-FH associated hemolytic uremic syndrome in two 6-year cohorts.

Normal range: hemoglobin 11.0–15.8 g/dl, platelet count 190–590 × 103 /mm3 , reticulocyte count 3.6–11%, blood creatinine: 0.17–1.01 mg/dl, lactate dehydrogenase 195–314 IU/L, C3 90–130 mg/dl.

aDays between disease onset and evaluation.

Data shown as N (%), mean ± standard deviation or \*mean ± standard error of mean; AU arbitrary units; FH factor H.

been included to maintain homogeneity within a pediatric aHUS population.

**Table 1** shows that patients presented earlier during the illness in the last 6-year compared to before 2013; stage 2 hypertension and seizures were also fewer in the latter cohort. Sixteen (3.6%) patients did not have thrombocytopenia, including six with mildly deranged renal function. Neurological features (31.3%) comprised seizures and/or hypertensive encephalopathy (12.4%). Imaging showed posterior reversible encephalopathy (n = 15), infarcts (n = 7) and intracranial hemorrhage (n = 2). Other features included cardiogenic shock (n = 16), pulmonary edema and hemorrhage (n = 8), and pancreatitis, mesenteric ischemia and peripheral gangrene (n = 3, each).

#### Anti-FH Titers and Their Functional Characterization

Anti-FH titers at onset negatively correlated with serum C3 (P < 0.001), platelets (P = 0.013), and hemoglobin level (P = 0.057), and positively with LDH (P = 0.010; **Supplementary Figure 4**).

P < 0.001 for all comparisons from onset.

Patients requiring dialysis had higher mean antibody titers than those not dialyzed (11,287 ± 1,173 vs. 8,198 ± 2,017 AU/ml; P = 0.015). **Figure 1** shows that anti-FH titers remained detectable during remission, with mean titers varying between 700 and 1,164 AU/ml on follow-up. Mean anti-FH titers during remission in patients with or without subsequent relapse are shown in **Figure 2A**. Using GEE approach, we found that patients with relapses had significantly higher antibody titers 1 month preceding a relapse compared to those in sustained remission (ß = 0.18; P = 0.023). ROC curves showed that titers ≥1,330 AU/ml at 6-months predicted the occurrence of a relapse (sensitivity 75%, specificity 81.4%; area under curve 0.86). However, 27 (15.8%) patients with sustained remission had antibody levels above this cut-off.

**Table 2** shows serial levels of CIC, free FH, C3, sC5b-9 and SRBC lysis from onset to 12-months in 44 patients. While median CIC declined from 20,000 AU/ml at onset to 806 AU/ml at remission (P < 0.001), it remained detectable and correlated with antibody titers at 6- and 12-months (r = 0.44 and 0.40; P = 0.007 and 0.067, respectively). SRBC lysis reduced from 72.8% at onset to 16.9% during remission, and levels of free FH increased from 64 to 553 mg/l. SRBC lysis and free FH correlated with CIC (r = 0.68 and r = −0.63, P < 0.001) and anti-FH titers (r = 0.60 and r = −0.55, P < 0.001) at all times points. While blood levels of sC5b-9 declined significantly during remission compared to onset (**Table 2**), they were high compared to controls (P < 0.001).

Since anti-FH titers ≥1,330 AU/ml were a predictor of relapse, we examined levels of free FH, sC5b-9 and CIC in patients with sustained remission but having anti-FH titers persistently above (n = 11) or below (n = 33) this cut-off. Median sC5b-9 and CIC were 709 ng/ml and 2,196 AU/ml in the former vs. 329.9 ng/ml and 594 AU/ml (P = 0.067 and P = 0.060, respectively); levels of free FH were also similar (P = 0.66).

We also examined serial free FH levels during remission in 44 patients, including 10 who later relapsed (**Figure 2B**). Free FH levels were significantly lower at 6-months in patients who relapsed compared to those in sustained remission (ß = 0.29; P = 0.001). ROC curves showed that free FH ≤440 mg/l at 6-months predicted occurrence of relapse (sensitivity 70%, specificity 100%; area under curve 0.91). Among patients with TABLE 3 | Therapy and outcomes in patients with anti-factor H associated hemolytic uremic syndrome.

Data shown as median (interquartile range) or N (%); CKD, chronic kidney disease; HTN, hypertension; PEX, plasma exchange.

anti-FH levels ≥1,330 AU/ml, free FH ≤440 mg/l at 6-months discriminated between patients with a relapse and those with sustained remission, with sensitivity of 75%, positive predictive value of 79% and negative predictive value of 91%; area under curve = 0.91; hazards 6.3, 95% CI 1.7–23.8 (P = 0.018).

Epitope specificity (n = 8 patients) showed similar pattern of antibody binding during onset, remission and relapse. Antibodies

and 59.8% renal survival at 1-, 5-years and at last follow up (interrupted line). Corresponding renal survival in patients managed during 2013-18 (continuous line) was 78.7, 77.1, and 77.1% (log rank P = 0.022).

demonstrated strongest binding to SCR 17–20 (n = 8), moderate binding to SCR 9–12 and SCR 13–16 (n = 7, each), and also to SCR 1–4 (n = 3), and SCR 5–8 (n = 4; **Supplementary Table 1**).

#### Therapy

PEX was initiated earlier (median 11 vs. 17 days from onset of illness) and dialysis requirement was briefer (median 13 vs. 26 days) in the last 6-year (**Table 3**). PEX were done for at least 14 days in 72.7%; 42 patients received plasma infusions only. Based on center preference, initial immunosuppression comprised prednisolone with IV cyclophosphamide (n = 171) or rituximab (n = 43). Maintenance immunosuppression included prednisone alone (16), and with mycophenolate mofetil (133) or azathioprine (40).

#### Outcome

Information on outcome at 3-months or longer was available for 356 (81.7%) patients. During the last 6-year, patients achieved earlier hematological remission and showed better outcomes at 3-months (**Table 3**) and last follow-up (**Figure 3**). In the subset of 196 patients, managed with PEX and immunosuppression, adverse outcomes occurred in 12.2% at 3-months (**Supplementary Table 2**); survival free of adverse outcomes was 86, 86, and 78% at 1-year, 5-year and last-follow up at mean 28.5 ± 27.3 months, respectively. Sixty-one (17.1%) patients relapsed at mean 11.0 ± 12.9 months from onset; relapse free survival was 86.4, 73.5, and 68.3% at 1-year, 5-year and last follow up, respectively. Relapses were early, with 46% occurring within the first 6-months and 95% within 2-years.

### Determinants of Outcome

Mean antibody titer was 22,801 ± 6,712 AU/ml in 19 patients who died in the first month of illness compared to 9,851 ± 1,058 AU/ml in those who survived (P = 0.002). On multivariable analysis, antibody titer at onset ≥8,000 AU/ml (OR 6.1, 95% CI 0.88–43.0; P = 0.066) predicted mortality within 30 days of onset, while daily PEX for 7 days (OR 0.14, 95% CI 0.02– 0.95; P = 0.044) and concomitant immunosuppression (OR 0.07, 95% CI 0.01–0.71; P = 0.024) were protective (data not shown). Independent predictors of adverse outcome were antibody titer ≥8,000 AU/ml (HR 2.23, 95% CI 1.11–4.48; P = 0.024), longer time (≥14 days) between onset of illness and initiation of PEX (HR 2.09, 95% CI 0.93–4.69; P = 0.071), and short duration PEX <14 days (HR 2.60, 95% CI 1.19– 5.67; P = 0.017). Therapy with PEX and induction therapy (HR 0.37; 95% CI 0.16–0.89; P = 0.026) and maintenance immunosuppression (HR 0.02; 95% CI 0.001–0.413; P = 0.011) protected against adverse outcomes (**Supplementary Table 3**). Maintenance immunosuppression was associated with relapse free survival (HR 0.11; 95% CI 0.05–0.27; P < 0.001).

#### Renal, Cardiovascular Outcomes

Fifty consecutive patients with eGFR 100.2 ± 21.1 (range 67– 156) ml/min/1.73 m2 were screened for renal and cardiovascular outcomes at mean 4.4 ± 2.5 (range 2–15) year from onset. Seventeen patients had CKD stage 2 and proteinuria was present in 29. Despite 37 patients receiving antihypertensive agents, 24 had clinic hypertension. ABPM showed normal blood pressure (14%), severe ambulatory hypertension (38%), masked hypertension (30%), pre-hypertension (18%), and abnormal nocturnal dipping (76%). Mean systolic and diastolic blood pressures were at 77th and 87th percentile with mean load 43 ± 26 and 40 ± 26%, respectively. LVH was present in 28% and 5 patients had dyslipidemia.

Median renal reserve was 15.9 (6.3, 28.5)% in 41 patients (**Supplementary Figure 5**). Proportion of patients with renal reserve below 20, 10, and 5% were 49, 27, and 15%, respectively. Renal reserve was 10.3 (−5.3, 31.5)% in patients with stage 2 CKD compared to 27.9 (16.9, 40.4)% with eGFR >90 ml/min/1.73 m2 (P = 0.010). Factors independently and inversely associated with renal reserve were mean systolic ambulatory pressure (ß = −0.49, 95% CI −0.85,−0.12; P = 0.010), number of relapses (ß = −0.65, 95% CI −1.23, −0.07; P = 0.030), urine protein-to-creatinine ratio >1 (ß = −0.9, 95% CI −0.1, −1.71; P = 0.031) and increased LVMI (ß = −0.04, 95% CI −0.08, −0.001; P = 0.044). Proteinuria was associated with ambulatory systolic (ß = 0.19, 95% CI 0.07, 0.32; P = 0.003) and diastolic hypertension (ß = 0.16, 95% CI 0.03, 0.3; P = 0.021).

### DISCUSSION

We report the clinical features and outcomes of a large, prospective multicenter cohort of 436 children with anti-FH associated aHUS managed across the country over a period of 12-year. Although the management was heterogeneous and based on center practices and physician preference, patients managed in the recent years show overall better renal survival, with decline in proportion of adverse outcomes from 36.2 to 21.1%. Prompt PEX, performed for at least 2-weeks, combined with immunosuppressive medications resulted in renal survival of 86% at 5-year. Serial examination showed that anti-FH titers and circulating FH immune complexes persisted well above the normal range during remission. Among patients with high anti-FH level exceeding 1,330 AU/ml, level of free FH below 440 mg/l predicted a 6.3-fold risk of later relapse. On long-term follow-up, patients show significant sequelae in terms of severe and masked ambulatory hypertension, left ventricular hypertrophy and proteinuria (summarized in **Table 4**).

While 24% children and 19% adults in the global aHUS Registry had anti-FH antibodies (8), these autoantibodies were present in 55.8% patient in the current cohort, confirming the increased frequency of this condition in India (7). In conformity with previous reports, predominantly children between 4 and 11 years were affected (4, 8, 25). The reason for high prevalence of anti-FH antibody associated HUS in Indian children is not clear. The population frequency of homozygous CFHR1 deletion is similar in India (9.5%) compared to elsewhere (2– 10%) (7, 9). The high prevalence of the illness in school going children, predilection for the cold weather, and associated prodromal symptoms indicate a possible infectious trigger. While a gastrointestinal prodrome is reported by others (1, 25), the chief preceding illness in the present patients was low-grade fever (55%) or a respiratory tract infection. A previous study from this center, using multiplex polymerase chain reaction on stool specimens, showed multiple gastrointestinal pathogens in 35 patients predominantly in patients with anti-FH antibodies (26).

Anti-FH titers were related to disease severity as evidenced by relationship with platelet count, hemoglobin, blood levels of LDH, dialysis requirement, mortality, and renal outcome. As shown previously, we found that CIC and SRBC lysis were markedly elevated during active disease compared to remission (12, 13, 27–29); the decline in CIC during remission was more than that of free antibodies, perhaps due to change in avidity of antibodies for free FH (13). While our finding of persistently high antibodies during remission has been reported (4, 25), the present report emphasizes that levels of CIC continued to be high during follow-up. Other authors suggest that CIC correlate better with disease activity, than do antibody titers (13).

The present analysis confirms our previous findings on the association of high antibody titers (≥1,330 AU/ml at 6-months) with subsequent relapse (30). However, 15.8% patients with quiescent disease had persistently high titers, suggesting that elevated levels are not always pathogenic. While free FH has been measured in few patients (31, 32), we determined serial levels in a larger cohort. Among patients with high anti-FH titer, reduced free FH concentration (≤440 mg/l) predicted a 6.3-fold higher risk of subsequent relapse with a negative predictive value of 91%. These findings suggest that formation of CIC reduce availability of free FH, impairing cell surface protection. This was shown previously in vitro as dose-dependent reduction in SRBC lysis on addition of FH to sera of anti-FH positive patients (11). Our findings suggest that estimation of free FH is therefore promising for indicating the risk of relapses.

Previous studies on levels of sC5b-9, which assesses activity of the soluble terminal complement pathway, show variable levels during active disease and remission (29, 33–36). While all the present patients had elevated sC5b-9 at onset that declined during remission, the levels were higher than normal, as shown previously in some but not all studies (29, 36). Serial estimation of blood levels of sC5b-9 did not therefore predict relapses, limiting its utility as a biomarker of disease activity. In contrast, free FH levels were normal in patients with sustained remission but having high anti-FH antibody titers, suggesting its potential role as a biomarker.

While free FH is presently a research tool and not available widely, close monitoring of anti-FH antibody titers is required with more careful assessment if elevated >1,300 AU/ml during the first 12–24 months (37). Relapses usually follow minor infectious illnesses during which close clinical and biochemical monitoring is required.

Using FH fragments that were generated in E. coli and purified by gel filtration, Gurjar et al. used inhibition ELISA to determine epitope specificity in 21 patients of this cohort with anti-FH associated HUS (16). Antibodies showed strong binding to SCR 17–20; binding with lower affinity was present to SCR 5–8 (16). We extended this work to examine whether there was altered epitope specificity to FH at onset of the illness, remission and during relapse in eight more patients. Similar to the previous work, we found binding to SCR 17–20 in all patients at onset; binding to SCR 9–12 and SCR 13–16 was also present in most patients. There were no significant changes in TABLE 4 | Summary of key findings in patients with anti-factor H (FH) associated hemolytic uremic syndrome (HUS).

AUC, area under the curve; eGFR, estimated glomerular filtration rate; FH, factor H; LDH, lactate dehydrogenase; PEX, plasma exchange; SCR, short consensus repeats; sC5b-9, soluble terminal complement complex.

epitope binding between onset, remission and relapse, as shown previously (14). The small number of patients studied limits conclusions regarding epitope specificity. **Table 5** summarizes findings from various reports on epitope specificity of anti-FH antibodies, emphasizing predominant binding to the C-terminal, and also other domains on FH (5, 11, 13, 14, 16, 25, 38–40).

Education and dissemination of management protocols through scientific meetings and efforts for consensus guidelines (37) has resulted in prompt recognition, early referral, and protocol based management, improving patient outcomes. While PEX and immunosuppression are considered primary therapies for patients with anti-FH associated HUS, the duration of therapy is empirical (41). The present report suggests that prompt and adequate duration of PEX was associated with decreased mortality and improved renal outcomes. Since most relapses occurred in the initial 2 years, immunosuppression is recommended for this period. We did not find increased rates of infections with use of immunosuppressive agents, as was a concern in a previous study (25). Our findings also suggest that strategies like PEX with corticosteroids alone, or immunosuppression with/without plasma infusions had limited benefit on long-term outcome.

An audit on safety of PEX from centers in Europe and North America showed procedure related complications and hypersensitivity to plasma in one-third patients, limiting the safety of this procedure in children (42). A similar audit of 2024 PEX sessions in 109 patients in New Delhi showed chiefly self-limiting adverse events (9.1%), including chills, vomiting, abdominal pain, and urticaria; hypotension (1.6%), hypocalcemia, tachycardia, seizures (0.2%, each), and hemorrhage (0.1%) were rare and catheter-related adverse events comprised only bloodstream infection (1.45/1,000 catheterdays). Hematological remission was achieved in 93.4% of patients within a fortnight of initiating PEX, with 80% and 90% patients discontinuing dialysis by 1 and 3 months, respectively (43). PEX was therefore overall safe and effective with satisfactory short-term outcomes.

Inhibition of the complement pathway with eculizumab is the standard of care for aHUS in developed countries (15). Most patients with anti-FH associated illness are treated similarly (25), although international pediatric and KDIGO guidelines suggest the use of PEX and immunosuppressive therapy for this disorder (15, 44). However, eculizumab does not impact generation of antibodies and additional immunosuppression might still be required. On the other hand, present findings show that despite PEX and immunosuppressive therapy almost one-quarter of all patients with anti-FH associated HUS had an adverse outcome. Some

TABLE 5 | Epitope specificity of anti-factor H (FH) antibodies to short consensus repeats (SCR) of FH.

#Short consensus repeats (SCR) 8–15; \*SCR 16–18.

of these patients might have benefited from prompt use of eculizumab, especially if hematological remission was delayed beyond 7–10 days. There is need for a prospective study examining the efficacy and safety of eculizumab in this specific condition.

The rates of persistent proteinuria (15–30%) and ambulatory hypertension (10–46%) following Shiga toxin HUS are similar to the present cohort (45–48). Masked hypertension and abnormal dipping of blood pressure are proposed to be associated with adverse cardiovascular outcomes and microalbuminuria (49, 50). The findings of abnormal dipping in three-quarter of all patients, and masked hypertension, LVM in one-third are therefore important. We also found reduced renal functional reserve in one-third patients, similar to 24–65% in patients with Shiga toxin HUS (51– 54). Conforming to previous reports, functional reserve was inversely associated with proteinuria and ambulatory hypertension (53, 55–57). Since we assessed functional reserve in patients with eGFR >60 ml/min/1.73 m2 , its overall magnitude is perhaps higher. The present findings emphasize that patients recovering from anti-FH associated HUS require long-term assessment for cardiovascular and renal outcomes.

#### DATA AVAILABILITY

The datasets generated for this study are available on request to the corresponding author.

#### ETHICS STATEMENT

Institute ethics committee approval was obtained from All India Institute of Medical Sciences, New Delhi and informed written consent was taken prior to enrolment.

#### AUTHOR CONTRIBUTIONS

MP: performed experiments, manuscript preparation. PK: patient care, data collection and analysis, manuscript preparation. HS, BG, RS, TM, AKS, and SS: laboratory work for the study. AnS, SA, AdS, and PH: patient care, critical review of manuscript. UA, IA, KA, NP, PR, RS, and AV: patient care. ArS, SR, UK, and AB: supervision of experiments. AB: study design, patient care, manuscript preparation and also is the guarantor for this paper. All authors approved the manuscript before it was submitted.

#### CONTRIBUTING CENTERS

Indira Agarwal, Christian Medical College, Vellore; Vinay Aggarwal, BL Kapoor Hospital, New Delhi; Uma Ali, Bai Jerbai Wadia Hospital for Children, Mumbai; Kanav Anand, Sir Ganga Ram Hospital, New Delhi; Arvind Bagga, All India Institute of Medical Sciences, New Delhi; Chinmay Behera, Kalinga Institute of Medical Sciences, Bhubaneswar; Gurdeep Singh Dhooria, Dayanand Medical College & Hospital, Ludhiana; Sanjeev Gulati, Fortis Escorts Heart Institute, New Delhi; NK Hase, King Edward Memorial Hospital, Mumbai; Madhuri Kanitkar, Armed Forces Medical College, Pune; Sriram Krishnamurthy, Jawaharlal Institute of Post-graduate Medical Education & Research, Puducherry; Manish Kumar, Chacha Nehru Bal Chikitsalaya, Delhi; Amarjeet Mehta, Sawai Man Singh Hospital, Jaipur; Mahantesh Patil, KLES Dr. Prabhakar Kore Hospital & Medical Research Center, Karnataka; Saroj. K. Patnaik, Army Hospital Research and Referral, New Delhi; Ravindra Prabhu, Kasturba Medical College, Manipal; Narayan Prasad, Sanjay Gandhi Post-Graduate Institute of Medical Sciences, Lucknow; Padmaraj Rajendran, Institute of Child Health, Chennai; Abhijeet Saha, Lady Hardinge Medical College, New Delhi; VK Sairam, Kanchi Kamacoti Child Trust Hospital, Chennai; T. Saravanan, PSG Coimbatore; Sidharth Kumar Sethi, Medanta The Medicity, Gurugram; Fagun Shah, Child's Kidney Care Center, Surat; Rajiv Sinha, Institute of Child Health, Kolkata; Deepti Suri, Post-Graduate Institute of Medical Education and Research, Chandigarh; Nilam Thaker, Ahmedabad; AS Vasudev, Indraprastha Apollo Hospital, New Delhi; Anil Vasudevan, St. John's Medical College and Hospital, Bangalore; M Vijayakumar (late), Mehta Children Hospital, Chennai; Susan Uthup, Trivandrum Medical College, Thiruvananthapuram.

#### ACKNOWLEDGMENTS

Support for collaborative research studies and consensus development by: Indo-French Center for Promotion of

#### REFERENCES

Advanced Research (CEFIPRA) [IFC/A/4703-1/2015/1562; IFC/Network 1/2185]; Department of Biotechnology, Government of India [BT/PR14651/MED/30/566/2010]; Indian Council of Medical Research [Center for Advanced Research in Kidney Diseases; 5/7/1090/2013-RHN]; Department of Science and Technology, Government of India [EMR12016/002781]; and All India Institute of Medical Sciences, New Delhi [A-386].

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2019.01282/full#supplementary-material

phase of the autoimmune form of atypical hemolytic uremic syndrome. J Immunol. (2012) 189:3528–37. doi: 10.4049/jimmunol.1200679

diarrhea negative (atypical) hemolytic uremic syndrome. Pediatr Nephrol. (2014) 29:1967–78. doi: 10.1007/s00467-014-2817-4

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Puraswani, Khandelwal, Saini, Saini, Gurjar, Sinha, Shende, Maiti, Singh, Kanga, Ali, Agarwal, Anand, Prasad, Rajendran, Sinha, Vasudevan, Saxena, Agarwal, Hari, Sahu, Rath and Bagga. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Anti-double Stranded DNA Antibodies: Origin, Pathogenicity, and Targeted Therapies

#### Xiaoyu Wang and Yumin Xia\*

Department of Dermatology, The Second Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an, China

Systemic lupus erythematosus (SLE) is characterized by high-titer serological autoantibodies, including antibodies that bind to double-stranded DNA (dsDNA). The origin, specificity, and pathogenicity of anti-dsDNA antibodies have been studied from a wider perspective. These autoantibodies have been suggested to contribute to multiple end-organ injuries, especially to lupus nephritis, in patients with SLE. Moreover, serum levels of anti-DNA antibodies fluctuate with disease activity in patients with SLE. By directly binding to self-antigens or indirectly forming immune complexes, anti-dsDNA antibodies can accumulate in the glomerular and tubular basement membrane. These autoantibodies can also trigger the complement cascade, penetrate into living cells, modulate gene expression, and even induce profibrotic phenotypes of renal cells. In addition, the expression of suppressor of cytokine signaling 1 is reduced by anti-DNA antibodies simultaneously with upregulation of profibrotic genes. Anti-dsDNA antibodies may even participate in the pathogenesis of SLE by catalyzing hydrolysis of certain DNA molecules or peptides in cells. Recently, anti-dsDNA antibodies have been explored in greater depth as a therapeutic target in the management of SLE. A substantial amount of data indicates that blockade of pathogenic anti-dsDNA antibodies can prevent or even reverse organ damage in murine models of SLE. This review focuses on the recent research advances regarding the origin, specificity, classification, and pathogenicity of anti-dsDNA antibodies and highlights the emerging therapies associated with them.

Keywords: anti-dsDNA antibody, catalysis, lupus nephritis, peptide, suppressor of cytokine signaling 1 (SOCS1), systemic lupus erythematosus (SLE)

#### INTRODUCTION

Systemic lupus erythematosus (SLE) is a severe disease that involves dysregulation of the immune system, excessive production of pathogenic autoantibodies (and their upregulation in serum), and multiple immune-system-mediated injuries. Anti-double-stranded (ds)DNA antibodies have been some of the classic diagnostic and nosological criteria of SLE since 1982. In 2012, the high titer of anti-dsDNA antibodies in serum accompanied by biopsy-proven lupus nephritis (LN) was accepted as an independent classification criterion for SLE by the Systemic Lupus International Collaborating Clinics (1). However, the wide spectrum of molecular antibody specificity and complex contexts of antibody generation, as well as the diverse antigen structures to which these antibodies bind, make anti-dsDNA antibodies difficult to accept without further distinction as a classification criterion for SLE (1). Pathogenic anti-dsDNA autoantibodies react with DNA but are

#### Edited by:

Marie-Agnes Dragon-Durey, Université Paris Descartes, France

#### Reviewed by:

Umesh S. Deshmukh, Oklahoma Medical Research Foundation, United States Batteux Frederic, Université Paris Descartes, France

\*Correspondence:

Yumin Xia<EMAIL_ADDRESS><EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Autoimmune and Autoinflammatory Disorders, a section of the journal Frontiers in Immunology

> Received: 02 November 2018 Accepted: 03 July 2019 Published: 17 July 2019

#### Citation:

Wang X and Xia Y (2019) Anti-double Stranded DNA Antibodies: Origin, Pathogenicity, and Targeted Therapies. Front. Immunol. 10:1667. doi: 10.3389/fimmu.2019.01667

**104**

not strictly specific to it. Multiple self-antigens can be recognized by anti-dsDNA antibodies, subsequently triggering apoptosis, inflammatory responses, and tissue fibrosis. Recently, synthesized peptides that mimic a molecular DNA structure were reported to specifically recognize and interact with the anti-dsDNA antibodies, thus pointing to a novel therapeutic opportunity via inhibition of the antigen recognition of anti-dsDNA antibodies (2, 3). This review sheds light on the involvement of anti-dsDNA antibodies in the progression of SLE and provides new insights into the therapies for SLE.

#### THE ORIGIN OF ANTI-DSDNA ANTIBODIES

Normally, nuclear antigens, such as dsDNA, are not accessible to the immune system because they are restricted to the nucleus and mitochondria and are quickly degraded by DNases in the cytoplasm and endosomes. However, nuclear materials can be released from apoptotic cells after exposure to ultraviolet light, infection, and drugs. Cells undergo death via different processes. During NETosis, cells extrude DNA and neutrophil extracellular traps (NETs), in which DNA is covered by anti-bacterial substances (4, 5). In apoptosis, DNA cleavage leads to the formation of apoptotic bodies, which contain DNA-bound microparticles (6). In necrosis, high-molecular-weight DNA is liberated after cell lysis.

The released DNA can be recognized by anti-DNA antibodies and compose immune complexes. Extracellular nucleic acids containing immune complexes can be captured by immature dendritic cells (iDCs) via FcγRs; these cells subsequently get activated and migrate from peripheral tissues to lymphatic organs, where iDCs undergo maturation (7). Activated DCs induce the over-representation of costimulatory molecules, such as CD80, CD86, and PD-L1 (8, 9). Furthermore, these DCs trigger redistribution of the MHC II molecule and production of proinflammatory cytokines, such as interferon α (IFN-α), tumor necrosis factor (TNF)-α, and interleukin 6 (IL-6), which are interrelated with the activation of B cells and T cells (10, 11). The immune complexes act as an antigen to stimulate B cells by activating the recognition receptors, such as Toll-like receptors (TLRs). TLR7 and TLR9 are the key receptors for the recognition of self-DNA or immune complexes and trigger the production of IFN-1 and inflammatory responses (12, 13). There are also some intracellular sensors in B cells and in other immune cells that can identify foreign DNA or self-DNA, including TLR9 and cyclic guanosine monophosphate–adenosine monophosphate, contributing to the overproduction of IFN-1 (14–16). Multiple signaling pathways are involved in the IFN-1 production process, which are activated after the interaction between sensors and DNA or another nucleic acid, such as pathways dependent on TLR9 and on stimulator of interferon genes (16, 17). As a key factor in the regulation of the innate immune response, IFN-1 can upregulate B cell-activating factor, promote B-cell differentiation, and suppress regulatory T cell (Treg) function (18, 19). With the help of IFN-1, autoreactive B cells undergo amplification, somatic mutation of immunoglobulin variable region genes, and class switch recombination, resulting in highaffinity immunoglobulin G (IgG) (20). In addition to suppressing Treg cells, IFN-1 can promote Th17 differentiation and increase the number of activated T cells (21). The insufficiency of Treg function contributes to the loss of immune tolerance in SLE via the TLR pathway (22). TLR9 can recognize dsDNA with a CpG motif and is dynamically upregulated in B cells (23). After a knockdown of TLR9, B cells produce fewer anti-dsDNA antibodies, and SLE syndrome is ameliorated in mice (24).

Moreover, autophagy is associated with the immune regulation and is essential for the homeostasis in immune cells. LC3-associated phagocytosis is an autophagy pathway that participates in the endocytosis of DNA or immune complexes by immune cells, especially plasmacytoid DCs. LC3 induces production of IFN-α via the TLR9 pathway in plasmacytoid DCs, whereas beclin-1, another member of the autophagy pathway, inhibits the production of IFN-α (25, 26). Beclin-1 knockdown macrophages can remarkably reduce antibody production and deposition of renal immune complexes, suggesting that autophagy may be an additional mechanism for the production of these antibodies (27).

Anti-dsDNA antibodies recognize diverse DNA structures, including single-stranded (ss)DNA, Z-DNA, bent or elongated dsDNA, DNA:RNA hybrids, locked-nucleic acids, and peptide: nucleic acid hybrids (28). Anti-dsDNA antibodies can bind to a complex of native dsDNA or modified DNA that contains a thymine dimer (generated during DNA photodamage). The chemically modified dsDNA may acquire a higher affinity for autoantibodies and form more stable complexes (29). Otherwise, antibodies can react with many non-DNA antigens in target cells and tissues: annexin II, α-actinin, laminin, collagen III, collagen IV, entactin, complement receptor type 1 (C1q), Nmethyl-d-aspartate receptor (NMDAR), ribosomal P proteins, heparan sulfate, and others (30–39) (**Table 1**). Although there are no obvious similarities between DNA and potential crossreactive antigens, some peptides can bind to both murine and human anti-dsDNA antibodies by mimicking a molecular DNA structure (2, 3).

Anti-dsDNA antibodies have different subclasses, including IgA, IgE, IgG, and IgM. Nonetheless, not all of them contribute equally to tissue injuries in SLE. It is IgG and IgA but not IgM that correlate with disease activity in humans. Most pathogenic antibodies are affiliated with the IgG class in SLE patients (47). IgM antibodies seem to be protective by enhancing the elimination of apoptotic material and via immunomodulatory effects, thereby alleviating renal dysfunction (48). The class switch and somatic mutations of the IgG variable chain can increase the risk of pathological changes. Activation-induced deaminase (AID) in B cells is a key enzyme required for such processes, as confirmed in transgenic or non-transgenic murine models, where a loss of tolerance is accompanied by high production of class-switched IgG (49–51). When lacking AID, lupus-prone mice produce more anti-dsDNA IgM, but not the high-affinity IgG, and show substantial attenuation

of glomerulonephritis and longer survival than do the wildtype mice (52). In SLE, DNA emerges as an inducer of an antigen-driven selection of B cells and is essential for a somatic mutation. During this process, the insertion of positively charged amino acids (for example, asparagine, arginine, and lysine residues) into complementarity-determining regions (CDRs) is critical for the binding of high-affinity dsDNA to the negatively charged phosphodiester backbone (53, 54). Among almost all IgG antibodies, anti-DNA IgG may have the potential for recognizing somatically produced idiotypes (53). Besides, the IgA and IgE subclasses of anti-DNA antibodies are implicated, independently or in combination with IgG, in lupus nephritis (LN) (47, 55, 56).

### THE DETECTION OF ANTI-DSDNA ANTIBODIES

Tests for anti-DNA antibodies can be positive at least 2 years before clinical diagnosis of SLE, and a surge in serum levels can present as a predictor for a severe SLE flare-up in the following 6 months (57). Subsequently, patients may undergo a course of relapsing–remitting with differences in the possibility of a flare between individuals. Anti-dsDNA antibodies emerge as a heterogeneous population in SLE, whereas they can be detected in some non-SLE patients with rheumatic symptoms, indicating that the employment of anti-dsDNA antibodies as a criterion for diagnosis and classification without subdivision may result in misdiagnosis and misclassifying of SLE (58, 59).

There are a variety of tests for anti-dsDNA antibody detection, such as the Farr radioimmunoassay (FARR-RIA), Crithidia luciliae indirect immunofluorescence test (CLIFT), and enzymelinked immunosorbent assays (ELISA) (**Table 2**). The former two assays have been well-demonstrated for diagnosis and prognosis, whereas ELISAs are more valuable for detecting high-avidity anti-dsDNA antibodies in clinical laboratories and most closely associated with SLE activity (60). However, the materials used in assays limit their utilization, and researchers have been working on developing new suitable assays with high sensitivity and specificity. Panza et al. explored a novel assay that uses complexed histone peptides (PK201/CAT plasmid) with a fragment from Crithidia luciliae, which has more simple procedures (61). We utilized trypoanosoma equiperdum (TE) that containing uniformed dsDNA but no histone in the cell nucleus or kinetonucleus, which is much easier for purification with a simpler structure (62). Poulsen et al. developed a flowinduced dispersion analysis, offering a more sequence-specific antibody characterization via utilizing short and well-defined dsDNA sequences (63). It also exhibits the advantages of shorter analysis time and less sample volume consumption. Although the association between high levels of anti-DNA antibodies and disease activity has been widely appreciated, no signal-detecting approach can reliably evaluate disease activity in SLE. The current assays are not sufficient for detecting all of the antibodies in sera, especially the low-level antibodies, or the immune complex. Thus, at least two assays should be used for better evaluation and higher accuracy.

### THE PATHOGENICITY OF ANTI-DSDNA ANTIBODIES

#### Anti-dsDNA IgG Correlates Closely With LN

Among the affected organs, renal involvement indicates major internal damage in SLE patients. Anti-dsDNA antibodies are present in serum in nearly 80% of patients with LN. AntidsDNA antibodies directly or indirectly interact with renal antigens, thus producing immune complexes (32) Nevertheless, renal damage is not initiated solely by the complexes of

TABLE 3 | Effects on renal cell types.

a chromatin fragment and IgG depositing in the mesangial matrix or glomerular basement membranes. In some studies, enrichment of anti-dsDNA antibodies was not present in all extracted samples from the patients with proliferative LN and accounted for <10% of all antibodies, where overexpression of antibodies to C1q, Sm, SSA, and SSB was also responsible for the process (64). Similarly, researchers also discovered that a mouse strain (NZM.C57Lc4) with genetic modification has a severe renal disorder and the serological testing for antidsDNA antibodies yielded negative results (65). Moreover, antidsDNA IgG does not exert nephritogenic effects without the exposure of chromatin fragments in glomerular membranes and matrices (66). These results confirm that the initiation of LN is promoted by multiple autoantibodies, not by their single type. Reasonable factors that can explain this discrepancy including the subtypes of antibodies with differences in reactive specificity and affinity that have been described previously, differences in mouse strains utilized for the creation of animal models and other factors need to be illustrated in the future. Despite the doubts about their nephritogenic role in launching LN, the pivotal role of anti-dsDNA IgG and immune complexes in the acceleration of the LN process has been underscored by a wealth of evidence, where their effects on renal resident cells are well-documented (**Table 3**).

Most immunoglobulins are unable to penetrate into living cells (67). However, anti-dsDNA antibodies can penetrate into cells and engage in an interplay with targets. Glomerular cells, hepatocytes, monocytes, fibroblasts, and neuronal cells are vulnerable to penetration by anti-DNA antibodies, mirroring the findings in the liver, spleen, and skin after treatment with penetrating antibodies in vivo (68, 69). The cellular penetration of antibodies is assisted by F(ab)′ 2 fragments with the mediation of the antigen-antibody binding region, which is also temperature-dependent and energy-consuming (70). Although the Fc fragment of an anti-DNA antibody contributes to its binding activity with monogamous bivalency, in which both Fab combining sites come into contact with DNA, the binding is not inhibited by the blockage of Fc receptor in mesangial cells (71, 72). In addition, the existence of manifold charged amino acids in CDRs is a specific feature of penetrating antibodies, which is explained by the energy-independent electrostatic interactions of arginine residues in the CDR2 and CDR3 with the negatively charged sulfated polysaccharides on the cell surface (68, 73), suggesting that cell penetration is an intrinsic property of anti-dsDNA antibodies. The DNA– histone complexes binding to purified cell-penetrating antibodies extracellularly can significantly enhance their subsequent cell entry depending on the environment (70). Besides, a wide variety of cognate nuclear antigens can function as endocytic receptors and promote subsequent internalization of antibodies into the cell, e.g., ribosomal P proteins (37), C1q (36), annexin II (30), α-actinin (38), and heparan sulfate (35, 46), further confirmed that the antigen-antibody mediation is crucial for the penetration mechanism. Although it is still unclear how anti-dsDNA antibodies actually cross the cell membrane, these data provide novel insights into antibodybinding endocytic receptors and elucidate their pathogenic role in SLE.

The penetrating anti-DNA antibodies can affect the proliferation and apoptosis of resident cells in LN, but the exact effect remains controversial. The anti-DNA IgG purified from the serum of LN patients has been shown to downregulate miR-10a and to trigger the proliferation of mesangial cells by targeting HOXA1, KLF4, and CREB1. Concomitantly, the expression of IL-6 is also enhanced, which is a crucial cytokine for promotion of the processes of cell proliferation and inflammation (74). Similar effects on cell proliferation are also seen in proximal renal tubular epithelial cells (PTECs) after exposure to the human anti-DNA antibodies; this treatment is associated with upregulated secretion of TNF-α, IL-1β, and IL-6 (75). These findings indicate that such antibodies can promote the proliferation of renal cells mainly by modulating the relevant inflammatory cytokines. These findings are also supported by the characteristic biopsy lesions of LN with deposition of immune complexes, hypercellularity of mesangial cells and epithelial cells, and infiltration by inflammatory cells. However, monoclonal and polyclonal anti-dsDNA antibodies show the ability to induce apoptosis, either by reacting with antigens on the cellular membrane or by entering the cell (76). Apoptosis can also be induced by these autoantibodies via upregulation of extracellular regulated kinase (ERK) activation and Bcl-2 production in murine mesangial cells. Endoplasmic-reticulum stress is upregulated in human mesangial cells after stimulation by anti-dsDNA antibodies, with activation of nuclear factor-kappa B (NF-κB) and increased expression of relevant inflammatory cytokines (72). This phenomenon may account for the apoptosis of mesangial cells, because enhanced endoplasmic-reticulum stress can cause cell death by changing the expression of multiple factors, including CHOP and Bcl-2, as discussed by Iurlaro (77). This discrepancy may be a result of the differences in the methods or circumstances of in vitro studies, but the diffuse proliferation can be observed in renal biopsy specimens from LN patients. Upregulation of apoptosis can manifest itself as DNA cleavage in inflammatory lesions as well as increased lysis of renal proximal tubular cells in the presence of complement (78). The apoptosis of podocytes leads to the destruction of glomerular basement membranes, and this event is accompanied by the aggravated progression of inflammation and fibrogenesis in renal tissues with the proliferation of mesangial cells and PTECs.

It is well-known that accumulation of inflammatory cytokines is sufficient for accelerating the recruitment of immune cells and the induction of inflammatory and fibrogenic processes promoting kidney damage. A wealth of proinflammatory factors, including monocyte chemotactic protein 1 (MCP-1), TNFα, IL-1β, IL-6, IL-8, hyaluronan, lipocalin-2, and inducible nitric oxide synthase, are overexpressed in both human and murine mesangial cells upon stimulation with anti-dsDNA antibodies (72, 79, 80). Consistently with this evidence, the amounts of these cytokines have been shown to increase in human PTECs under the influence of anti-dsDNA antibodymediated activation of the mitogen-activated protein kinase (MAPK) pathway; these factors further trigger the infiltration of monocytes and macrophages (81). These cytokines not only augment the infiltration by inflammatory cells but also increase the synthesis of fibronectin, eventually reproducing the features of tubulointerstitial fibrosis (41). Anti-dsDNA antibodies also sustain endoplasmic-reticulum stress and activated NFκB accompanied by overexpression of cytokines in mesangial cells, resulting in a chronic inflammatory response and renal tissue damage (72). The high-titer anti-dsDNA antibodies are always accompanied by overexpression of TLR9 in podocytes, leading to increased production of TNF-α, IL-6, and IFN-γ (82). Besides, a multitude of signaling pathways are activated or involved in the autoantibody-mediated proinflammatory response. Excessive or persistent activation of tumor necrosis factor-like weak inducer of apoptosis (TWEAK)/fibroblast growth factor-inducible 14 (Fn14) signaling cascade positively correlates with the progression of LN. Anti-DNA IgG enhances the expression of Fn14 in keratinocytes through recognizing and binding to self-antigen, such as collagen III, indicating a relation between anti-DNA antibodies and the TWEAK/Fn14 signals (33). The mRNA expression levels of TWEAK and Fn14 increase in the glomeruli and tubulointerstitium in both LN patients and in a mouse model and act as an inducer of constitutive nuclear factor (NF-κB) activation, which mediates the expression of MCP-1, interferon γ-induced protein 10 (IP-10), macrophage inflammatory protein 1a, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 (83, 84). Additionally, MAPK, epithelial-to-mesenchymal transition (EMT), c-Jun N-terminal kinase, ERK, p38, and serine/threonine kinase Akt are also involved in TWEAK/Fn14 activation and downstream inflammatory and fibrotic responses (85, 86). Although the precise mechanisms of interaction of anti-DNA IgG and TWEAK/Fn14 have not been directly confirmed, anti-TWEAK antibodies, as well as Fn14 deficiency, are known to ameliorate renal damage induced by anti-dsDNA IgG in a murine model. Inhibition of TWEAK/Fn14 decreases renal IgG deposition without influencing serum anti-DNA IgG levels by relieving inflammation and protecting the filtration barrier, further confirming the crucial participation of the TWEAK/Fn14 signaling pathway in anti-DNA IgG-mediated LN (87–89).

A persistent chronic inflammatory response is always followed by fibrogenesis when there is no effective intervention, thereby leading to abnormal structure and dysfunction of kidneys with characteristic oversecretion of profibrogenic chemokines. Fibronectin is critical for fibrotic progression in glomerulonephritis and is distributed widely in the mesangial matrix, glomerular, and tubular basement membranes, and Bowman's capsule. The formation of glomerular fibronectin is enhanced both in patients and mice with active LN (90, 91). We have demonstrated that anti-dsDNA IgG contributes to renal fibrosis by downregulating suppressor of cytokine signaling 1 (SOCS1) and activating Janus kinase/signal transducer and activator of transcription 1 signals, which modulate the expression of profibrotic genes, transforming growth factor beta 1 (TGF-β1), platelet-derived growth factor B, and connective tissue growth factor (92, 93). In addition to SOCS1, protein kinase C signaling is also simulated by anti-dsDNA antibodies with increased secretion of TGF-β1 (94). As a profibrotic growth factor, TGF-β1 is responsible for the cellular myofibroblastlike phenotype switch, in which the assembly of the actin cytoskeleton induces the synthesis of plasminogen activator inhibitor 1 through the TGF-β1/MAPK pathway in mesangial cells, with an accumulation of the extracellular matrix (95– 97). Moreover, pathogenic anti-dsDNA antibodies cross-react with alpha-actinin 4; these data account for the promotion of myofibroblast-like phenotypic changes via upregulation of fibrogenic factors in mesangial cells, including F-actin and TGFβ1, as confirmed both in vitro and in vivo (38, 98). Anti-DNA antibodies can also enhance the phenotypic changes or EMT in cultured PTECs. The activation of EMT with increased mesenchymal markers contributes to the interstitial infiltration by leukocytes and to tubule-interstitial fibrosis (75). Therefore, we can conclude that anti-dsDNA antibodies play a crucial part in the inflammatory and fibrogenic mechanisms of LN (**Figure 1**); however, the complicated network of inflammatory cytokines and intricate signaling pathways should be investigated further.

### Anti-dsDNA IgG Participates in Other Injuries

Skin is frequently affected by SLE and can be the only affected organ. The serum positivity for an anti-dsDNA antibody is observed in only a subgroup of patients with cutaneous lupus erythematosus. The binding specificity of anti-DNA IgG to skin sections and keratinocytes has been observed in vitro, which can be promoted by ultraviolet light irradiation, resulting in keratinocyte apoptosis via antibody-dependent and cellmediated cytotoxicity (33, 99). The deposition of an immune complex at the dermoepidermal junction has been confirmed in both murine models and patients with serological positivity for anti-DNA IgG (33). Actually, the mild inflammatory response in combination with the presence of autoantibodies is necessary for further cross-reactions with collagen III, collagen IV, and SOCS-1 and−8 for induction of antibody accumulation in skin tissue (42, 100). In addition to LN, the role of TWEAK/Fn14 signals in cutaneous lupus erythematosus has been discovered in murine lupus models (87, 101–103). Ultraviolet B irradiation promotes the anti-DNA IgG binding to keratinocytes as well as the expression of Fn14, which engages in an interplay with TWEAK and subsequently enriches the expression of proinflammatory factors and induces apoptosis (33, 103). These induced factors, including IL-6, IL-8, MCP-1, and RANTES (regulated upon activation, normal T cell expressed and secreted), further amplify the inflammatory responses by attracting immune cells (101).

Central nervous system (CNS) diseases are associated with a poor prognosis among patients with SLE and have been increasingly reported during the past decade. Neuropsychiatric complications occur in 30–40% of patients with SLE and may constitute the initial presentation or a flare (89). Antiphospholipid, anti-N-methyl-D-aspartate receptor (NMDAR), anti-ribosomal-P, and anti-dsDNA antibodies account for the progression of CNS disease (43). Anti-dsDNA antibodies can cross-react with NMDAR on neurons. An extracellular epitope has been identified near amino acid residue 369 of GlyN1 in NMDAR and decreases the density of the surface receptor, causing further neuronal disorders, such as abnormalities of cognition and memory (43, 44). Enriched serum anti-dsDNA and anti-NMDAR antibodies are associated with the emergence of neuropsychiatric lupus symptoms (45). Nevertheless, an increased titer of an anti-DNA antibody is not always consistent with neurological dysfunctions (104). The inconsistency may be due to the differences in cognitive assessments applied in diverse studies and the samples tested. The anti-NMDAR/dsDNA antibodies can generate and gain access to the brain tissue after the destruction of the blood-brain barrier, leading to neuronal cell death and abnormalities in mice (45). Injection of mouse brains with an anti-DNA antibody (R4A) causes apoptosis of hippocampal cells (105). Besides, we observed enhanced expression of proinflammatory factors as well as abnormal cognition and behavior in wild-type mice, while the cognition and integrity of the blood-brain barrier are attenuated in Fn14 deficient mice, indicating the amplification of TWEAK/Fn14 signals in the pathogenesis of CNS diseases (106).

### The Constant Region Contributes to the Antigenic Specificity and Pathogenicity of Anti-DNA Antibodies

Classically, the variable region is the only part of an antibody that is credited with the antigenic specificity, whereas the functions of antibodies are determined by the constant region. These two domains are believed to be structurally and functionally independent. However, the specificity and affinity of antibodies are believed to be determined by both regions but not solely by the variable one (107). The variety of isotypes of anti-DNA antibodies is generated through the isotype switch recombination in the sequence from IgM to IgG, whose variable regions are identical to those of the initial antibody. Particular subclasses of anti-DNA antibodies are more closely associated with a pathogenic potential (47). In lupus patients, serum anti-DNA IgG1 is always elevated before the occurrence of renal relapse, while IgG2a, IgG2b, and IgG3 are more frequently eluted from kidneys with active nephritis (78, 108). In addition, different IgG subclasses with similar specificity for DNA show a remarkable difference in the binding properties for basement membranes, the formation of immune-complex deposits, and the severity of the induced proteinuria (109).

To validate the association between subclasses and pathogenicity, we found that different isotypes of anti-dsDNA antibodies differ from each other in the recognition specificity of nuclear and renal antigens when we generated a panel of murine anti-DNA antibodies (IgM, IgG1, IgG2a, IgG2b, and IgG3), which contain identical variable-joining-diversity (VDJ) regions, suggesting that the effect of the constant region on antibody binding is directly associated with autoimmunity (34). In fact, the factors responsible for the pathogenicity of anti-DNA antibodies are diverse, including their binding avidity for DNA, cross-reactivity for self-antigens, characteristics of the idiotype, and localization of specific amino acid residues in CDRs (110). Additionally, the diverse subclasses of autoantibodies that determine the complement fixation and Fc binding are crucial in LN. The isotypes that form the immune complex can fix complement—in a process involving IgG1 and IgG3 in humans and IgG2a and IgG2b in mice—upregulated in the renal glomerulus (111, 112). IgG2a and IgG2b manifest the most robust complement activity and binding affinity for triggering Fc receptors III and IV and are the most potent autoantibodies during the mediation of hemolytic anemia in vivo (108). Although IgG2a is the predominant subclass among IgG-reactive IgGs and is present as the most pathological subclass in murine lupus models (113), IgG3 derived from MRL/lpr mice is crucial for the spontaneous glomerular disruption and dysfunction (109). Besides, the constant region of antibodies affects their binding ability toward DNA and histones not only by varying the environments of their paratopes but also by altering their secondary structures (34, 114). Nevertheless, the prognostic value of these subclasses of anti-dsDNA antibodies remains to be illustrated.

### The Catalytic Properties of Anti-dsDNA IgG

The first IgG antibody with DNA-related catalytic activity in SLE was found in 1992 (115). Antibodies binding to nucleoprotein complexes, to DNA, and enzymes that account for nucleic acid metabolism may acquire the feature of the primary antigen-catalytic activity. The amino acid residues of catalytic anti-dsDNA antibodies and DNA enzymes may have similarities because anti-dsDNA antibodies in patients with SLE have been shown to hydrolyze plasmid DNA (116–118). The catalytic activity of anti-DNA antibodies is associated with the binding of magnesium and calcium ions, and their cytotoxicity results from the hydrolysis of nuclear matrix proteins (**Figure 2**) (116). Moreover, the variety of light chains of anti-DNA IgG, the tolerance of temperature, optimum pH, and hydrolysis rate are thought to be responsible for distinct activities of anti-DNA antibodies and their capacity for DNA hydrolysis (119).

In the sequence analysis of anti-DNA antibodies from humans and mice, there is a high frequency of somatic mutations in the VH and VL sequences of anti-dsDNA IgG with high affinity (53). Such somatic mutations result in a higher proportion of certain amino acids, especially arginine, asparagine, lysine, and tyrosine in the CDRs; this phenomenon promotes the formation of electrostatic interactions and hydrogen bonds between these amino acids and DNA (53). In our study, anti-dsDNA antibodies of various isotypes (PL9-11 clone) that share identical variable regions but different constant regions could cleave not only dsDNA but also peptide antigens depending on the isotype, e.g., the ALWPPNLHAWVP peptide, indicating that the catalytic cleavage of DNA can be regarded as a natural property of these antibodies (120). The specific affinity of anti-DNA antibodies to antigens probably determines the possibility of catalysis, but not the catalytic efficiency of the reaction. Although antibodies derived from various sources contain similar variable-chain sequences, these antibodies have different catalytic activities. In addition, in the context of this specific autoantibody system, a specific mutation results in the reduction of DNA binding as well as weakened catalytic activity (121). The DNA binding and hydrolytic activities of anti-DNA antibodies are well-conserved

in both VH and VL. The catalytic characteristics of anti-DNA antibodies are isotype-dependent, and constant regions have an influence on the autoantibody-antigen interaction presumably by modifying the secondary structure of antibodies (34, 114, 120).

### THE BLOCKADE OF PATHOGENIC ANTI-DSDNA IGG

#### An iDC Vaccine Suppresses the Production of Anti-dsDNA IgG

This therapeutic strategy against SLE is focused on eliminating or controlling active B cells that offer limited benefits but hamper the advancement of treatment for SLE (122, 123). Recently, the significance of iDCs in the induction of clonal anergy and immune tolerance was highlighted. iDCs participate in the anergy of T cells by inducing the apoptosis and promoting the differentiation of Treg cells because the expression of costimulatory factors and MHC molecules on the surface of iDCs decrease considerably (124). As a key cytokine for inhibiting excessive immune responses, IL-10 can reduce the production of relevant cytokines (such as TNF-α and IL-1β,) as well as increase production of inhibitory factors (such as programmed death ligand 1) in DCs, thereby inhibiting Treg responses and the function of effector T cells (125–127). Among these factors, programmed death ligand 1 in combination with TGFβ is particularly important for promoting the conversion of CD4+ T cells to Foxp3+ CD4+ Treg cells, whereas blockade of programmed death ligand 1 can reduce anti-dsDNA IgG production and immune-complex formation in lupus-prone mice by suppressing the synthesis of IL-10 in CD4+ Treg and B cells, thus further validating the therapeutic potential of iDCs (128, 129). After the induction of clonal anergy, T cells fail to bind to cognate antigens that are presented by DCs.

The iDCs exert weaker activating effects on T cells by expressing lower amounts of costimulatory cytokines, including IL-6 and IL-12 in patients with SLE; this observation is consistent with iDCs' effects in lupus-like mice where iDCs lower the responses of Th1/Th2 cells and thus inhibit the secretion of IL-2, IL-4, IL-12, and interferon γ and the formation of anti-dsDNA IgG (130–132). Besides, iDCs from hemopoietic stem cells can be loaded with nuclear antigens, thereby serving as a live cell vaccine (131, 132). Thus, iDCs are emerging as promising immunomodulators in SLE. Nonetheless, there are many challenges regarding the appropriate production protocols and administration route and timing of an iDC vaccine. Moreover, the exact mechanism by which iDCs interact with Treg cells, B cells, and other cells, as well as the possible side effects are largely unclear. Therefore, the translational application of the iDC vaccine in clinical settings requires multidisciplinary efforts.

#### DNA-Mimicking Peptides Block the Binding of Anti-dsDNA IgG

Based on the interactions between self-antigens and anti-dsDNA antibodies, novel therapeutic peptides that bind to autoantibodies have attracted increasing attention. These peptides can be synthesized by combining desired amino acid residues or by chemical modification of certain sequences, to ensure higher activity and specificity. To date, therapeutic peptides have proven to be effective against experimental autoimmune diseases, for example, rheumatoid arthritis and multiple sclerosis (133). Moreover, some therapeutic peptides have been demonstrated to prevent anti-dsDNA antibodies from binding and reacting with target antigens and tissues in the studies on SLE.

Peptide DWEYS (D/E W D/E Y S/G), a part of NMDAR expressed on neurons, has been shown to bind to anti-dsDNA antibodies, thereby disrupting the neurotoxicity of anti-DNA antibodies through the NMDAR-activated pathway, in which circulating antibodies penetrate the hippocampal pyramidal cells followed by impairment of spatial cognition (134). In addition, peptide DWEYS can prevent the proliferation of both B cells and T cells and ameliorate the production of anti-DNA IgG and proteinuria in lupus mice by selectively suppressing the autoreactive B lymphocytes via cross-reaction with the immunoglobulin receptors on B cells (135). Although the DWEYS peptide is certainly beneficial for therapeutic applications owing to the small molecular weight and nonimmunogenicity, it is not highly useful due to its short half-life.

FISLE-412, an analog of a reduced protease inhibitor for the human immunodeficiency virus, has the potential to block DNA recognition of anti-dsDNA antibodies (136). FISLE-412 can reduce glomerular deposition of IgG, protect kidneys from damage, and suppress the onset of SLE in murine models. Moreover, the FISLE-12-bound anti-dsDNA antibodies exert no neurotoxic effects on C57BL/6 mice; this finding shows a greater neuroprotective effect than that of the DWEYS peptide (136). As compared with DWEYS, FISLE-412 has higher affinity and a more stable structure and is more suitable for oral administration (136). The study on the structure-activity relationship of several synthesized analogs of FISLE-412 has revealed that the hydrophobic portion is the key region for this therapeutic function (137). In addition, the quinaldic region contributes to the binding activity of FISLE-412 (137).

ASPVTARVLWKASHV is a 15-mer peptide selected by purified polyclonal anti-dsDNA IgG in lupus patients and can inhibit antigen-IgG binding (138). ALW (ALWPPNLHAWVP) is a 12-mer peptide mimic originating from a panel of murine anti-DNA IgG isotypes; it can bind to all four IgG isotypes and prevent IgG binding to DNA or glomerular antigens (3). ALW is physiologically stable and resistant to oxidation, cyclization, and degradation because it lacks a sequence of certain amino acid residues (3). Although ALW and its mimics are receiving increasing attention as therapeutic molecules with less toxicity for lupus patients, a combination of different peptides should be taken into consideration because of the variability and crossreactivity of anti-DNA antibodies. In addition, peptides have low toxicity when administered orally or subcutaneously and would not be expected to be immunogenic.

pCons (FIEWNKLRFRQGLEW) is a 15-mer peptide derived from murine anti-dsDNA antibodies; it exerts therapeutic effects by preventing antibody-antigen binding (139). pCons significantly enhances the survival rate and alleviates glomerulonephritis in NZB/W lupus mice; this beneficial action may be mediated by the regulation of T-cell autoreactivity (140, 141). The protective effects have been validated in lupus-prone mice treated with a B lymphocyte gene vaccine that codes for IgG1 Fc-pCons, where early and repeated administration of the vaccine delayed proteinuria and enhanced survival remarkably as well as the expansion of Foxp3+ CD4+ Treg cells (142).

hCDR1 (GYYWSWIRQPPGKGEEWIG) is a 19-mer peptide that is based on CDR1 sequences from human anti-DNA antibodies. hCDR1 has suppressive effects on T cells by reducing apoptosis of T cells with less secretion of interferon-γ and upregulation of IL-10 in lupus-prone mice (143). The effects of hCDR1 on B cells are also associated with downregulated levels of anti-DNA antibodies in lupus-prone mice (144). In an early clinical study, weekly subcutaneous administration of different doses of hCDR1 (Edratide)-−0.5, 1.0, and 2.5 mg was found to be effective in ameliorating the manifestations and in downregulating the production of proinflammatory and proapoptotic cytokines in SLE patients; the affected proapoptotic proteins include IL-1β, TNF-α, IFN-γ, IL-10, caspase 3, and caspase 8 (145). The efficacy of hCDR1 in the treatment of SLE was questioned because of the limited sample size (9 patients) and disease severity from mild to moderate among the patients. In a later clinical phase II trial, the injection of hCDR1 once a week at a dose of 0.5 mg in SLE patients with a larger sample size (340 patients) was found to be safe and effective, with a decreased SLE disease activity index (SLEDAI-2K). However, there was no remarkable difference between the hCDR1 group and placebo group. In addition, the shortest course of efficacy proven by this trial was nearly 1 year, and the primary endpoint was not reached; this factor may be responsible for the insignificance of the therapeutic effects (146). Thus, a longer-term and appropriate endpoint should be scheduled in later studies on hCDR1.

### MAIN CONCLUSIONS AND FURTHER INVESTIGATIONS

Anti-dsDNA antibodies, the hallmark of SLE, constitute a potent parameter for classifying and diagnosing SLE. Their contribution to damage to the kidneys, skin, and brain in SLE has been welldocumented. The production of anti-dsDNA antibodies results from combining multiple factors, including abnormalities of dendritic cells, B cells, or T cells and deficiency of a DNase that leads to failure of cleaning released nuclear materials; however, it still need further studies. The effects of penetrating autoantibodies on triggering a complicated inflammatory and fibrotic process underlie the role of anti-DNA antibodies in damaging target cells and organs. The pathogenicity of antibodies highlights the promising therapeutic potential of DNA-mimicking peptides that react with these autoantibodies in SLE, which can ameliorate the manifestations of SLE in murine models. However, the clinical trials progress slowly in which the timing, dosages, route of administration, alteration of bioactivity, and possible adverse effects of such peptides should be taken into account for developing more efficient therapies for SLE.

### AUTHOR CONTRIBUTIONS

YX conceived this paper. XW wrote this manuscript. XW and YX read and approved the final manuscript.

### FUNDING

This study was supported by the National Natural Science Foundation of China (Projects No.81472876 and No.81874241) and the Fundamental Research Funds for the Central Universities (No.2015qngz01).

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Wang and Xia. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Immunological Pathogenesis of Membranous Nephropathy: Focus on PLA2R1 and Its Role

Wenbin Liu1,2, Chang Gao1,3, Haoran Dai 4 , Yang Zheng1 , Zhaocheng Dong1,3, Yu Gao1,2 , Fei Liu3 , Zihan Zhang3 , Zhiyuan Liu5 , Weijing Liu6 , Baoli Liu1 \* † , Qingquan Liu1 \* † and Jialan Shi 7 \* †

1 Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, Beijing, China, 2 Capital Medical University, Beijing, China, 3 Beijing University of Chinese Medicine, Beijing, China, 4 Shunyi Branch, Beijing Hospital of Traditional Chinese Medicine, Beijing, China, 5 Taishan Medical University, Shandong, China, 6 Key Laboratory of Chinese Internal Medicine of Ministry of Education, Beijing, China, 7 Departments of Medicine, VA Boston Healthcare System, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, United States

#### Edited by:

Marie-Agnes Dragon-Durey, Université Paris Descartes, France

#### Reviewed by:

Renato Alberto Sinico, University of Milano-Bicocca, Italy Barbara Seitz-Polski, Centre Hospitalier Universitaire de Nice, France

#### \*Correspondence:

Baoli Liu<EMAIL_ADDRESS>Qingquan Liu<EMAIL_ADDRESS>Jialan Shi<EMAIL_ADDRESS>
†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Autoimmune and Autoinflammatory Disorders, a section of the journal Frontiers in Immunology

> Received: 06 February 2019 Accepted: 17 July 2019 Published: 06 August 2019

#### Citation:

Liu W, Gao C, Dai H, Zheng Y, Dong Z, Gao Y, Liu F, Zhang Z, Liu Z, Liu W, Liu B, Liu Q and Shi J (2019) Immunological Pathogenesis of Membranous Nephropathy: Focus on PLA2R1 and Its Role. Front. Immunol. 10:1809. doi: 10.3389/fimmu.2019.01809 Membranous nephropathy (MN) is the major cause of nephrotic syndrome with special pathological features, caused by the formation of immune complexes in the space between podocytes and the glomerular basement membrane. In idiopathic membranous nephropathy (IMN) the immune complexes are formed by circulating antibodies binding mainly to one of two naturally-expressed podocyte antigens: the M-type receptor for secretory phospholipase A2 (PLA2R1) and the Thrombospondin type-1 domain-containing 7A (THSD7A). Formation of antibodies against PLA2R1 is much more common, accounting for 70–80% of IMN. However, the mechanism of anti-podocyte antibody production in IMN is still unclear. In this review, we emphasize that the exposure of PLA2R1 is critical for triggering the pathogenesis of PLA2R1-associated MN, and propose the potential association between inflammation, pollution and PLA2R1. Our review aims to clarify the current research of these precipitating factors in a way that may suggest future directions for discovering the pathogenesis of MN, leading to additional therapeutic targets and strategies for the prevention and early treatment of MN.

#### Keywords: membranous nephropathy, immunological pathogenesis, PLA2R1, PM2.5, lung, kidney

## INTRODUCTION

Membranous nephropathy (MN) is an immune-mediated glomerular disease that affects 12 new cases out of every million adults every year globally (1, 2). MN is characterized by a pathological change in the glomerular basement membrane (GBM) caused by the accumulation of immune complexes which appear as granular deposits of immunoglobulin (Ig)G when imaged with immunofluorescence and as electron-dense deposits under electron microscopy. These immune deposits in the space between podocytes and GBM contain the complement membrane attack complex (C5b-9). MN can be either idiopathic (idiopathic membranous nephropathy or IMN) or caused partially by clinical disease such as hepatitis B, systemic lupus erythematosus, cancer, or drug side-effect (secondary membranous nephropathy or SMN) (1). However, it is not clear whether these clinical diseases are direct causes of MN or merely triggers, such as the potential association between cancer and MN (3–5). In addition to the typical GBM changes, SMN exhibits mesangial deposits, "full-house" immunostaining (IgG, IgA, IgM, C3, and C1q, mesangial deposits, and Liu et al. Immunological Pathogenesis of MN

reticular aggregates) in membranous lupus nephritis, and amyloid or fibrillary glomerulonephritis which are seen as fibrils under electron microscopy (**Table 1**) (6). The deposition of immune complexes in SMN may be caused by exogenous antigens "pre-planted" between podocytes and the GBM which bind to circulating antibodies, such as in hepatitis B virusassociated MN (7). The formation of glomeruli subepithelial immune complex deposits in IMN is now believed to be mediated by specific intrinsic podocyte antigens and their corresponding autoantibodies in humans, such as neutral endopeptidase (NEP), M-type receptor for secretory phospholipase A2 (PLA2R1), and Thrombospondin type-1 domain-containing 7A (THSD7A) (8– 10). PLA2R1 (70–80% of IMN) and THSD7A (3–5% of IMN), the two major podocyte antigens identified in adult IMN, can be detected through both direct immunofluorescence staining of renal tissue and detection of their autoantibodies in serum for diagnosis and prognosis (1).

In recent years in China, the morbidity of MN has been gradually increasing possibly due to long-term exposure to air pollution, mainly fine particulate matter of <2.5µm (PM2.5). Each 10 mg/m3 increase in PM2.5 concentration over 70 mg/m3 is associated with 14% higher odds for MN (11). However, the association between the air pollution and the pathogenesis of MN remains unclear. It is worth noting that PLA2R1 and THSD7A are naturally expressed in other parts of the human body, such as the lungs (3). In this review, we have focused on the possible mechanisms of anti-podocyte antibody production, particularly in PLA2R1-associated MN, and proposed several hypotheses, which may be beneficial to further exploration of the pathogenesis of IMN.

#### THE DEFINITION AND PATHOLOGY OF MEMBRANOUS NEPHROPATHY

Membranous nephropathy, originally called membranous glomerulonephritis (MGN), was first described in 1946 by Bell as a type of glomerular disease, which manifests pathologically as a thickening of the GBM and is clinically characterized by marked proteinuria and edema (12). In the later half of the 1950s, understanding of the pathology of MN developed rapidly, driven by several outstanding pioneers. In 1956, Mellors and Orgeta discovered immunoglobulins containing in the glomeruli deposits using immunofluorescence. In 1957, Jones demonstrated the presence of silver-positive rods projecting from the GBM using periodic acid silver methenamine stain. And in 1959, Movat defined the causal relationship between thickening of the GBM and protein deposition between the GBM and the podocyte using electron microscopy (12). In this way, these pioneers identified the basic features of glomerular lesions in MN, consisting of changes in GBM structure caused by subepithelial electron-dense deposits (**Figure 1A**). Moreover, in 1968, Ehrenreich used repeated renal biopsy to describe the four stages of glomerular lesions in MN: Stage I consists of only a few small subepithelial deposits such that the GBM may appear normal or be slightly thickened under light microscopy; in stage II spikes protruding from the GBM can be observed using appropriate staining; in stage III the deposits are incorporated within the GBM; in stage IV the GBM appears to be irregularly thickened by reabsorbed deposits, and in complete clinical remission the deposits may disappear and leave some areas lucent or the GBM may return to normal (13) (**Figure 1A**). These pathological discoveries in the middle of the last century not only defined MN as a unique type of renal pathology, but also guided the development of clinical practices that are still in use today (6).

#### ENLIGHTENMENT BROUGHT BY HEYMANN NEPHRITIS

#### Megalin, the Rat Podocyte Antigen

In 1959, Heymann et al. established an animal model that develops pathological manifestations similar to human MN by immunizing rats with tissue components of the proximal tubular brush border (Heymann nephritis or HN) (**Figure 1B**) (14). This animal model relies on the active immune response of the rat and therefore is often referred to as active Heymann nephritis (AHN). In passive Heymann nephritis (PHN), an analogous lesion can be induced more rapidly by administering heterologous antibody to tubular brush border (anti-Fx1A antibody) (15). At the time, it was thought that the deposition of subepithelial immune complex was caused by circulating immune complexes because Fx1A and its antibody are simultaneously present in glomerular deposits and circulation in HN (16, 17), and the development of immune complexes in circulation was found to be coincident with the glomerular deposits (18). However, in 1978, Couser et al. and Hoedemaeker et al. simultaneously induced HN pathological changes in isolated rat kidneys using anti-Fx1A antibodies suggesting that IMN is not caused by the deposition of circulating immune complexes, but in instead by in situ immune complex formed by the autoantibodies binding to antigens located on podocytes (19, 20). In the same year, Makker et al. further confirmed that the podocyte antigen in HN is a mannose-containing glycoprotein (21). And then in 1982, Kerjaschki et al. identified megalin as an antigen on the rat podocyte membrane and tubular brush border (22) (**Table 2**). Consistent with previous studies, the pathogenicity of megalin is related to glycosylation (23), and anti-megalin autoantibodies fail to react with megalin in Western blots run under reducing conditions, indicating that autoantibodies recognize the spatial conformation of their epitopes (24). As the rat-specific podocyte target antigen in HN, megalin contains multiple epitopes (25–27), and demonstrates intramolecular epitope spreading during the progression of HN proteinuria (28). Intermolecular epitope spreading has also been observed with the development of autoantibodies to plasminogen, a known ligand of megalin (24). Although megalin is not the pathogenic antigen in human MN, this series of animal studies reveals the possible pathogenesis of human MN, suggesting that autoantibodies bind to podocyte intrinsic antigens to form in situ immune complexes in the glomerular subepithelial. Thus, the search for human podocyte-specific target antigens has become the focus of further studies (29).

TABLE 1 | Pathogenic factors, histopathological features, and clinical outcomes of secondary and idiopathic membranous nephropathy.

MN, Membranous nephropathy; HIV, Human Immunodeficiency Virus; HLA-DQA1, The gene encoding HLA complex class II HLA-DQ alpha chain 1(SNP rs2187668); PLA2R1, The gene encoding M-type phospholipase A2 receptor (SNP rs4664308); SNP, Single-nucleotide polymorphisms; IgG, immunoglobulin G; IgA, immunoglobulin A; IgM, immunoglobulin M; C1q, C3, and C4 are all complement components; ESRD, End-stage renal disease.

#### Human Podocyte Antigens

The results from animal model studies have since been validated in humans (**Figure 1C**). In 2002, Ronco et al. found that the pathogenic antigen in three families of neonatal MN was neutral endopeptidase (NEP) located on the foot process membrane of the podocytes and the brush border of the renal tubules, while the pathogenic antibody derived from NEP deficiency in the mother. This was the first confirmation that the immune complex antigen of human MN could be an intrinsic component of the podocyte membrane (8, 30). More importantly, in 2009 Beck et al. discovered PLA2R1 and its circulating autoantibodies, the first podocyte-targeted antigen-antibody system found in adult IMN (9) (**Table 2**). The presence of circulating anti-PLA2R1 antibodies can be detected in 70–80% of IMN (31–33). As with megalin, PLA2R1 has multiple antigenic epitopes (34–36), and the epitopes spread as the disease progresses (36). The second target podocyte antigen, THSD7A, was discovered in 2014 by Tomas et al. (10) (**Table 2**). These findings not only demonstrate that human MN immune complexes form by the same mechanism as seen in HN, but also gives us reason to believe that there are still undiscovered pathogenic podocyte antigen-autoantibody pairs in IMN (37). Other proteins postulated to serve as autoantigens in adult MN include superoxide dismutase (SOD2), aldose reductase (AR), and alfa-enolase. While these proteins typically localize inside the cell, AR, and SOD2 are also expressed on the plasma membrane of podocytes in patients with MN (24, 38, 39). However, the clinical significance and pathogenic role of these cytoplasmic antigens, and the differences between them and the podocyte membrane antigens (PLA2R1 or THSD7A) in pathogenic autoimmune responses has yet to be clearly demonstrated.

### Complement Activation and Podocyte Injury

Another important area of research regarding MN is the mechanism of kidney injury following the formation of immune complexes (**Figure 1B**). In 1980, Salant et al. found that C3 deficient rats treated with cobra venom factor did not produce proteinuria within 5 days after PHN was induced with sheep antirat Fx1A antibody injection (40). Similar results were obtained when HN was established in C6 and C8-deficient rats (41, 42). These findings suggest that complement activation to produce C5b-9 is a key factor in the development of proteinuria in HN. Subsequent studies on experimental MN have further clarified that the pathogenic role of C5b-9 is mainly on podocytes because of its non-dissolving activity. The mechanisms involved include: (1) Inducing podocytes to produce oxygen free radicals; (2) Stimulating podocytes to produce various proteases to cause GBM damage; (3) Influencing a microfilament skeleton structure in podocytes, by separating and redistributing the proteins nephrin and podocin, which are major components of the membrane; (4) Upregulating cyclooxygenase 2 (COX-2) in podocytes, causing damage to the endoplasmic reticulum; (5) Increasing the extracellular matrix by promoting the production of TGF-β by podocytes, leading to GBM thickening and glomerular sclerosis; (6) Promoting podocyte apoptosis and shedding from GBM (43). In addition to the above findings from experimental MN, C5b-9 is also found in the glomeruli and urine of patients with MN, with levels of urine C5b-9 correlating with disease severity and patient prognosis (44, 45), indicating that complement activation is involved in the pathogenesis of human MN. The complement system mainly consists of three early activation pathways (classical pathway, alternative pathway,

understanding of MN. Three major podocyte antigens found in human MN (left), and other major work on MN since 2000 (right).