Complement Deficiencies Result in Surrogate Pathways of Complement Activation in Novel Polygenic Lupus-like Models of Kidney Injury This information is current as of October 2, 2021. Sladjana Skopelja-Gardner, Lucrezia Colonna, Payton Hermanson, Xizhang Sun, Lena Tanaka, Joyce Tai, Yenly Nguyen, Jessica M. Snyder, Charles E. Alpers, Kelly L. Hudkins, David J. Salant, YuFeng Peng and Keith B. Elkon J Immunol published online 1 April 2020 Downloaded from http://www.jimmunol.org/content/early/2020/03/31/jimmun ol.1901473 Supplementary http://www.jimmunol.org/content/suppl/2020/03/31/jimmunol.190147 http://www.jimmunol.org/ Material 3.DCSupplemental Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published April 1, 2020, doi:10.4049/jimmunol.1901473 The Journal of Immunology Complement Deficiencies Result in Surrogate Pathways of Complement Activation in Novel Polygenic Lupus-like Models of Kidney Injury Sladjana Skopelja-Gardner,* Lucrezia Colonna,* Payton Hermanson,* Xizhang Sun,* Lena Tanaka,* Joyce Tai,* Yenly Nguyen,* Jessica M. Snyder,† Charles E. Alpers,‡ Kelly L. Hudkins,‡ David J. Salant,x YuFeng Peng,*,1 and Keith B. Elkon*,{,1 Lupus nephritis (LN) is a major contributor to morbidity and mortality in lupus patients, but the mechanisms of kidney damage remain unclear. In this study, we introduce, to our knowledge, novel models of LN designed to resemble the polygenic nature of human lupus by embodying three key genetic alterations: the Sle1 interval leading to anti-chromatin autoantibodies; Mfge82/2, leading to defective clearance of apoptotic cells; and either C1q2/2 or C32/2, leading to low complement levels. We report that Downloaded from proliferative glomerulonephritis arose only in the presence of all three abnormalities (i.e., in Sle1.Mfge82/2C1q2/2 and Sle1.Mfge82/2C32/2 triple-mutant [TM] strains [C1q2/2TM and C32/2TM, respectively]), with structural kidney changes re- sembling those in LN patients. Unexpectedly, both TM strains had significant increases in autoantibody titers, Ag spread, and IgG deposition in the kidneys. Despite the early complement component deficiencies, we observed assembly of the pathogenic terminal complement membrane attack complex in both TM strains. In C1q2/2TM mice, colocalization of MASP-2 and C3 in both the glomeruli and tubules indicated that the lectin pathway likely contributed to complement activation and tissue injury in this http://www.jimmunol.org/ strain. Interestingly, enhanced thrombin activation in C32/2TM mice and reduction of kidney injury following attenuation of thrombin generation by argatroban in a serum-transfer nephrotoxic model identified thrombin as a surrogate pathway for complement activation in C3-deficient mice. These novel mouse models of human lupus inform the requirements for nephritis and provide targets for intervention. The Journal of Immunology, 2020, 204: 000–000. ystemic lupus erythematosus (SLE) is a complex polygenic SLE: 1) low serum complement levels, 2) accumulation of AC/AC autoimmune disease associated with more than 90 genetic debris in the germinal centers (GC) and kidney tissue, and 3) risk loci (1). Despite the large number of genes implicated, presence of autoantibodies (3–7). How these pathways interact S by guest on October 2, 2021 distinctive pathways of immune function, such as complement and lead to lupus nephritis (LN), the main contributor to morbidity activation, immune complex (IC) and apoptotic cell (AC) pro- and mortality in SLE patients (8, 9), remains unclear. cessing, B cell tolerance, and IFN, are affected (2). Involvement Both animal models and evidence in humans indicate that de- of these pathways is commonly reflected in three hallmarks of fective AC clearance is an important component of SLE devel- opment (4, 6, 10, 11). Cells from SLE patients show accelerated death ex vivo (12) and impaired phagocytosis of AC (13), both of *Division of Rheumatology, University of Washington, Seattle, WA 98109; which could be the contributing factors to increased accumulation †Department of Comparative Medicine, University of Washington, Seattle, WA of cellular debris found in the GC of SLE patients (14). Multiple 98109; ‡Department of Nephrology, University of Washington, Seattle, WA 98109; x { opsonins, including milk fat globule epidermal growth factor 8 Division of Nephrology, Boston University, Boston, MA 02215; and Department of Immunology, University of Washington, Seattle, WA 98109 (MFGE8), and cell surface receptors have been shown to bind AC 1Y.P. and K.B.E. are joint last authors. and enable their clearance (4, 6, 15). MFGE8 is a soluble glyco- ORCIDs: 0000-0001-6350-9582 (S.S.-G.); 0000-0002-1859-1772 (C.E.A.). protein that binds to phosphatidylserine on AC, facilitating the removal of AC through its interaction with the a b integrin on Received for publication December 12, 2019. Accepted for publication March 15, v 3 2020. phagocytes (10, 16). Aberrant splicing of Mfge8 has been asso- This work was supported by National Institutes of Health (NIH)/National Institute of ciated with dysregulated MFGE8 function in SLE patients, as have Environmental Health Sciences Grant P30ES007033, Foundation for the NIH Grant genetic polymorphisms in Mfge8 (17, 18). MFGE8-deficient mice 5T32AR007108-40, and by the Lupus Research Alliance. accumulate AC in GC and develop a lupus-like disease on the Address correspondence and reprint requests to Dr. YuFeng Peng and Dr. Keith B. mixed C57BL/6 3 129 (10) but not on the pure C57BL/6 (B6) Elkon, Division of Rheumatology, University of Washington, 750 Republican Street E531, Seattle, WA 98109. E-mail addresses: [email protected] (Y.P.) and background (19). Therefore, defective AC clearance alone is not [email protected] (K.B.E.) sufficient to induce a lupus-like pathology but can fuel disease The online version of this article contains supplemental material. progression in a susceptible environment. Abbreviations used in this article: AC, apoptotic cell; DM, double-mutant; fibrin(ogen), The role of complement in kidney disease is complex: early fibrin/fibrinogen; GC, germinal center; IC, immune complex; IF, immunofluorescence; components (C1q, C2, C4, and C3) function to clear AC and ICs, LN, lupus nephritis; LP, lectin pathway; MAC, membrane attack complex; MASP-2, mannan-binding lectin-associated serine protease 2; MFGE8, milk fat globule epidermal whereas late components form the membrane attack complex growth factor 8; NTN, nephrotoxic nephritis; NTS, nephrotoxic serum; PAS, periodic (MAC) that acts as an effector of kidney injury in SLE (20–22). acid–Schiff; SLE, systemic lupus erythematosus; TF, tissue factor; TM, triple-mutant; Specifically, more than 90% of C1q-deficient individuals develop UACR, urine albumin/creatinine ratio. SLE, including LN in a subset of those patients (3), and anti-C1q Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 Abs in SLE patients are associated with more severe LN (23). C3 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1901473 2 SURROGATE PATHWAYS OF COMPLEMENT ACTIVATION IN LUPUS MODELS deficiency can also lead to SLE, characterized by LN in ∼26% of alkaline phosphatase–conjugated goat anti-mouse IgG (Sigma-Aldrich). C3-deficient patients (24). In addition, low circulating C3 levels For confirmation of anti-dsDNA IgG, Crithidia luciliae slides (Bio-Rad are strongly associated with active and recurrent renal disease in Laboratories) were used. Serum samples were diluted in PBS/FBS (1:50), and IgG binding was detected by Alexa 555–conjugated anti-mIgG Ab. To SLE patients, in part because of consumption (25, 26). Whereas analyze the autoantibody repertoire, pooled serum samples from six dif- previous studies have suggested that C1q and C3 play a role in ferent strains used in the spontaneous disease model (12-mo-old females, AC clearance and B cell tolerance (3, 27–29), the mechanisms n = 15–20) were examined for reactivity to a defined autoantigen array at that lead to kidney injury in low complement states in the presence the University of Texas Microarray Center (University of Texas South- western). Normalized data were clustered according to the multidimen- of autoantibodies and defective clearance of dead or dying cells sional principal component analysis and visualized in heatmap form using remain unknown. Qlucore Omics Explorer data analysis software; statistically significant To determine the mechanisms of kidney injury in a disease expression of strain-specific autoantigens was determined (Qlucore, Lund, model that closely mimics human SLE pathogenesis, we generated Sweden). SLE-like polygenic murine models that have defective clearance Immunohistochemistry and pathology evaluation of AC (Mfge8-deficiency),