MO263: Multi-Antibody Composition in Lupus Nephritis: Isotype Prevalence and Clinical Oucome
Authors: Angeletti Andrea , Bruschi Maurizio , Alvise Ramirez Giuseppe , Manfredi Angelo , Meroni Pierluigi , Marco Ghiggeri Gian 
Published: May 2022

Abstract

BACKGROUND AND AIMS
Lupus nephritis (LN) is the most serious organ manifestation of systemic lupus erythematosus (SLE), occurring in 40%–60% of subjects with SLE with worse outcomes. LN is mediated by antibody deposition in glomeruli, but the mechanisms leading to the immune deposits and renal lesions are not clarified. Previous findings suggested a role for circulating anti–double stranded DNA (dsDNA). However, anti-DNA deposition in LN accounts for not more than 10%–20% of eluted IgG. Therefore, renal targets of autoimmunity in LN are largely unknown.

METHOD
We studied 1052 patients with SLE, 459 with and 573 without LN, enrolled at different times from diagnosis (0–1 month, 2–12 m, 13–24 m, 25–48 m, 48–96 m and &gt; 96m). These cohorts are part of a nation-wide study (The Zeus study, NCT02403115) to characterize the antibody multi-components in SLE and LN. A total of 92 LN patients recruited at T0, had a follow-up of 36 months.
Laser capture microdissection were performed from fresh frozen renal samples of 20 patients with LN (classes III–V), and eluted Igs were analyzed by Western blot using podocyte extracts as fixed antigens. Recognized proteins were characterized by matrix-assisted laser desorption ionization (MALDI)–mass spectrometry (MS) and liquid chromatography (LC)-MS.
Levels of anti-dsDNA, anti-ENO1, anti-AnnexinA1, anti-SOD, anti-C1q, anti-H2A histone were determined using non-commercial ELISA.

RESULTS
Glomerular IgGs recognized 11 proteins (Fig. 1A), which resulted positive also in serum of same subjects (Fig. 1B). Isotype analysis of glomerular eluates showed prevalence of IgG2 for anti-dsDNA, anti-Histone H2, anti-C1q, anti–α-enolase and anti-Annexin AI (Fig. 1C). Fig. 1D shows representative images of patients with LN class V: colocalization with IgG2 is positive for dsDNA, Histone H2, C1qα-enolase and annexin. Prevalence of IgG2 for the same proteins were confirmed also in serum samples of the same subjects (Fig. 1E).
The presence in the serum of the IgG2 panel was highly discriminatory for SLE/LN versus healthy subjects (Fig. 2A).
Heat map in Fig. 2B reports the association between the levels of circulating IgG2 antibodies and the clinical parameters for both LN and SLE together. While in Fig. 2C, the heat maps indicate the correlation between two parameters (an antibody and an organ pathology). Volcano plots indicate that anti-ENO1 and anti-Histone H2 IgG2 had the most significant changes in LN patients.
In the prospective analysis, considering the 91 LN with 36 months of follow-up, decrements of each antibody at T12 related to proteinuria are summarized in Fig. 2E: major decrements were for anti-ENO1 and anti-Annexin A1 (−55% and − 70%, respectively) and 0% for anti-dsDNA.
Serum levels of anti-SOD IgG2 at T0 resulted significantly higher in LN compared to SLE (Fig. 2F). The kinetics of reduction of anti-SOD IgG2 was in accordance with reduction of proteinuria (Fig. 2G). Of interest, anti-dsDNA IgG2 serum levels persistently maintained at high values during the same period (Fig. 2H).

CONCLUSION
Our study demonstrated higher sensitivity of anti-dsDNA IgG2 than classical IgGs antibodies for marginal SLE diagnosis. Positivity for the whole panel of circulating IgG2 antibodies is a specific signature of SLE/LN and distinguishes them from healthy people. Moreover, anti-ENO1 and anti-Histone H2 IgG2 serum levels represent the best discriminatory element for distinguishing between LN and SLE. From prospective analysis, we report that circulating anti-ENO1 IgG2 may represent a valuable markers of remittent LN. Similarly, circulating anti-SOD2 IgG2 are elevated in new onset LN. It might be hypothesized that increase is related to the activation of SOD2, as response to the oxidative stress induced by the active disease.