Dysregulation of complement factor H in juvenile-onset systemic lupus erythematosus patients
1Department of Immunogenetics, National Research Centre, Cairo, Egypt
2Department of Rheumatology and Rehabilitation, Faculty of Medicine, Cairo University, Cairo, Egypt
Keywords: Complement factor H, factor H autoantibodies, juvenile-onset systemic lupus erythematosus, nephritis.
Objectives: This study aims to evaluate the expression pattern of factor H in peripheral blood and the frequency of factor H autoantibodies in plasma of juvenile-onset systemic lupus erythematosus (jSLE) patients compared to healthy controls.
Patients and methods: Between March 2019 and October 2019, a total of 30 healthy individuals (3 males, 27 females; mean age: 26±7.4 years; range, 18 to 40 years) and 65 jSLE patients (age of onset ≤16 years) (2 males, 63 females; mean age: 23.4±7 years; range, 15 to 38 years) were included. Factor H expression pattern was examined in blood of all subjects using quantitative real-time polymerase chain reaction and the frequency of factor H autoantibodies was estimated in plasma using enzyme-linked immunosorbent assay.
Results: Factor H expression was significantly downregulated in jSLE patients compared to healthy controls (p<0.01). A significant underexpression of factor H was observed in jSLE patients with nephritis compared to those without nephritis (p<0.03), while there was no association of factor H expression levels with any of the other clinical and serological features, disease activity or disease damage index of patients. Only 5% of jSLE patients were positive for factor H autoantibodies without any correlations with the clinical data or disease activity of patients.
Conclusion: Our study results suggest that factor H expression can be dysregulated in jSLE patients.
Juvenile-onset systemic lupus erythematosus (jSLE) affects up to 20% of all systemic lupus erythematosus (SLE) patients before the age of 16 years. It is characterized by more severe clinical courses and more common complications than adult-onset SLE. Its pathophysiology is different from that of adult-onset SLE, as indicated by the variability in sex and age distribution and also the presence of monogenic etiology of disease in early childhood.[1-3] The processes involved in the pathogenesis of SLE include generation of autoantibodies, deposition of immune complex, defective clearance of apoptotic cells, and chronic activation of complement.
Complement plays a central role in both innate and adaptive defense mechanisms, as it destroys microbes, and controls the inflammatory and adaptive immune response. It is also involved in the disposal of dead cells and misfolded proteins.[5-7] The complement system contains soluble and membrane-bound proteins that act through three (classical, alternative and lectin) pathways. Deficiency of complement components or regulatory complement proteins due to genetic deficiencies or a gene copy number variation or the presence of autoantibodies is strongly linked to lupus pathogenesis. [9-11]
Chronic activation of complement causes self-tissue damage due to a self-amplifying and aggressive inflammation that should be controlled by some circulating and membrane-associated regulators.[12,13] Failure to control complement that involves continuous activation or aberrant inhibition results in pathological processes that ends in some autoimmune and inflammatory diseases.[7,14-18] Several studies have shown that there is a strong correlation between alternative complement pathway activation and disease activity, or flares in SLE patients.[19-21] Factor H and factor H-like protein 1 are the most important soluble inhibitors of the alternative pathway.
Factor H, a soluble 150-kDa glycoprotein, regulates activation of the alternative pathway by inactivating complement component 3b (C3b) mediated by factor I, inhibiting the formation of complement 3 (C3)-convertase (C3bBb) and enhancing its degradation.[23-25] It has been demonstrated that factor H contributes to clearance of the apoptotic or necrotic cells by promoting their phagocytosis and inhibits cytokine release of macrophages, such as tumor necrosis factor alpha and interleukin-8.
Mutations and genetic variations of factor H and genes related to factor H and autoantibodies against factor H could result in its deficiency and dysfunction, dysregulation of the alternative pathway,[13,27] exhaustion of C3 in plasma and its deposition in the glomeruli, and these defects are associated with several diseases such as atypical hemolytic uremic syndrome (aHUS) and SLE.[13,27-29] In this study, we aimed to evaluate the expression pattern of factor H in peripheral blood and the frequency of factor H autoantibodies in plasma of jSLE patients compared to healthy controls.
Patients and Methods
This case-control study was conducted at Kasr Al Ainy Hospital, Cairo University clinics of Rheumatology and Rehabilitation outpatient between March 2019 and October 2019. A total of 30 healthy individuals (3 males, 27 females; mean age: 26±7.4 years; range, 18 to 40 years) and 65 jSLE patients (age of onset ≤16 years) (2 males, 63 females; mean age: 23.4±7 years; range, 15 to 38 years) were included. All patients fulfilled the 2012 Systemic Lupus International Collaborating Clinics (SLICC) classification criteria for SLE. Demographic and cumulative clinical manifestations were recorded, and laboratory investigations included complete blood count (CBC), erythrocyte sedimentation rate (ESR), urine analysis, serum creatinine, liver function tests, serum complement C3 and C4, antinuclear antibody (ANA) and anti-double stranded deoxyribonucleic acid (anti-dsDNA), as well as 24-h urinary protein. An ANA titer of >1/80 measured by enzyme-linked immunosorbent assay (ELISA) was considered positive. Disease activity at the last visit was assessed through the Systemic Lupus Erythematosus Disease Activity Index2000 (SLEDAI-2K). Disease damage index was determined through SLICC Damage Index (SDI)/ American College of Rheumatology (ACR) Damage Index (DI). Lupus nephritis (LN) was defined as clinical and laboratory manifestations meeting the American College of Rheumatology (ACR) renal criteria (persistent proteinuria >0.5 g per day (24-h urinary protein) or greater than 3+ dipstick, cellular casts including red cell, hemoglobin, granular, tubular or mixed, and renal biopsy for histopathological examination demonstrating immune complex-mediated glomerulonephritis compatible with LN).
Healthy controls were selected from age- and sex-matched individuals. Patients having chronic autoimmune, allergic or neoplastic disease and those having any acute infection within the past 15 days prior to study entry were excluded from the study.
Ribonucleic acid (RNA) extraction and quantitative real time polymerase chain reaction (qRT-PCR)
Total RNA was extracted and isolated from fresh blood of all individuals of the study populations using the QIAamp RNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. For reverse transcription, RNA was reversetranscribed to complementary DNA (cDNA) using HiSenScript™ RH[-] cDNA Synthesis Kit (iNtRON Biotechnology, Korea) according to the manufacturer’s instructions. Reverse transcription was performed under the following conditions: 5 min at 25°C, 45 min at 45°C, followed by 10 min at 85°C and the resulting cDNA was kept at -80°C until use.
The quantitative real-time PCR was carried out to quantify the expression levels in triplicate of factor H using TaqMan® factor H Assay kits and TaqMan® Universal Master Mix (Applied Biosystems) using 7500 fast real-time PCR system according to the manufacturer’s instructions. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as endogenous control to normalize the expression levels of factor H. Relative quantification (Rq) was calculated using the 2-ΔΔCT threshold cycle method. The ΔCT was determined by subtracting the cycle threshold (CT) values for GAPDH from the CT values for the gene of interest. The qRT-PCR was performed under the following conditions: 2 min at 50°C, 10 min at 95°C, followed by 45 cycles at 95°C for 15 sec and at 60°C for 1 min.[34,35]
Plasma complement factor H autoantibodies of all study individuals were determined using the human anti-complement factor H antibody IgG ELISA kit (Bioneovan Co., Ltd., Beijing, China) according to the manufacturer’s instructions.
Study power analysis and sample size calculation were performed using the G*Power version 3.1 software (Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany). Accordingly, a priori power analysis indicated a sample size of 31 subjects in each group for a power of 0.80 and a significance level of 0.05. Statistical analysis was performed using the IBM SPSS version 19.0 software (IBM Corp., Armonk, NY, USA). Descriptive data were expressed in mean ± standard deviation (SD), median (interquartile range [IQR]) or number and frequency, where applicable. A p value of <0.05 was considered statistically significant.
Clinical, laboratory and baseline characteristics of jSLE patients
Of a total of 65 jSLE patients, the mean age at onset and at time of sampling 13±2.7 and 23.4±7 years, respectively. The median disease duration was 120 (IQR: 110) months. Cumulative clinical and laboratory characteristics of the patients at the time of sampling are summarized in Table 1. Medications taken by lupus patients at time of sampling were as follows: glucocorticoids in 55 (100%), hydroxychloroquine in 45 (69.2%), azathioprine in 22 (33.8%), mycophenolate mofetil in 14 (21.5%), and cyclophosphamide in 10 (15.4%) of patients.
Expression pattern of factor H in peripheral blood of jSLE patients
Our study results showed that factor H expression was significantly downregulated in jSLE patients compared to healthy controls. A 2.9-fold downregulation of factor H was found in patients compared to healthy individuals. Relative quantification (median) of factor H expression was 0.35 at p<0.01 in jSLE patients (Figure 1a).
Frequency of factor H autoantibodies in jSLE patients
Our findings revealed that only 5% of our jSLE patients were positive for factor H autoantibodies (Figure 1b) while no autoantibodies were detected in healthy controls. In addition, there were no significant correlations between the factor H autoantibodies and the clinical data or disease activity of patients.
Association of factor H with different clinical characteristics and serological data
Our study showed a significant underexpression of factor H in jSLE patients with nephritis compared to those without nephritis (Table 2). On the other hand, there was no association of factor H expression levels with any of the other clinical and serological features (Table 2). Interestingly, there was a significant negative correlation between factor H expression levels and ESR of patients (r=-0.775, p=0.041). However, no associations were found between factor H expression levels with SLEDAI-2K, SDI, and proteinuria of patients.
In our study, dysregulation of factor H was associated with lupus patients. We found that factor H expression was significantly downregulated in jSLE patients compared to healthy controls. In addition, a significant underexpression of factor H was observed in jSLE patients with nephritis compared to those without nephritis. Our findings also revealed that only 5% of our jSLE patients were positive for factor H autoantibodies. Downregulation of factor H observed in patients may be due to its overconsumption during regulation of complement activation, generation of autoantibodies against it or a mutation affecting its expression or function.
The significant downregulation of factor H expression in our study agrees with the significant decrease in mean serum factor H level in SLE with LN patients compared to both SLE without renal involvement and control groups, in a study by Wang et al. They also observed an inverse correlation between serum factor H levels and disease activity scores in LN patients and in patients being positive compared to those being negative for ANA and anti-ds-DNA in LN. On the other hand, serum factor H levels increased significantly in the remission phase.
In addition, dysfunctions of factor H through the defective regulation of complement alternative pathway and the aberrant disposal of apoptotic cells were observed in some active LN patients and correlated with their clinical manifestations. Furthermore, Bao et al. showed that absence of factor H in Murphy Roths Large (MRL)/lymphoproliferation (lpr) mice promoted the development of LN in a markedly accelerated manner.
Furthermore, autoantibodies have been demonstrated to affect the role of complement factors and predispose to inflammatory diseases. It has been documented that autoantibodies produced toward complement factor H is associated with aHUS. In a study by Foltyn Zadura et al., plasma analysis of SLE patients showed positive results in 6.7% for the presence of autoantibodies against factor H. They found that factor H autoantibodies could only be found in active lupus patients without significant association between their titers and disease activity. They also showed a significant high percentage of factor H autoantibodies in rheumatoid arthritis patients from two independent cohorts compared to controls. In contrast, Wang et al. could not observe any factor H autoantibodies in serum of patients with LN and healthy controls.
The main limitations of our study include the limited number of individuals in the groups. Also, the drugs used might have affected the expression pattern of factor H or its autoantibody positivity rate. However, studying the association between factor H and lupus pathogenesis is of great importance in exploring the etiology of lupus.
In conclusion, factor H expression was significantly downregulated in jSLE patients compared to healthy controls, particularly in patients with nephritis compared to those without nephritis, while there was no association of factor H expression levels with any of the other clinical and serological features, disease activity or disease damage index of patients. Only 5% of jSLE patients were positive for factor H autoantibodies without any correlations with the clinical data or disease activity of patients. Taken together, we conclude that factor H expression can be dysregulated in jSLE patients. However, further well-designed, large-scale studies are needed to confirm these findings.
Citation: Eissa E, Morcos B, Dorgham D, Kholoussi N. Dysregulation of complement factor H in juvenile-onset systemic lupus erythematosus patients. Arch Rheumatol 2022;37(3):417-423.
The study protocol was approved by the National Research Centre, Cairo, Egypt Ethics Committee (16/109). The study was conducted in accordance with the principles of the Declaration of Helsinki.
A written informed consent was obtained from each patient.
Data Sharing Statement:
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Conception and design, laboratory analysis: E.E.; Laboratory analysis, drafting manuscript: B.M.; Collecting, drafting manuscript: D.D.; Supervision, revising, the article approving the final version: N.K.
The authors declared no conflicts of interest with respect to the authorship and/or publication of this article.
This study was funded by the National Research Centre, Cairo, Egypt.
- Hedrich CM, Smith EMD, Beresford MW. Juvenileonset systemic lupus erythematosus (jSLE) - Pathophysiological concepts and treatment options. Best Pract Res Clin Rheumatol 2017;31:488-504.
- Smith EMD, Lythgoe H, Midgley A, Beresford MW, Hedrich CM. Juvenile-onset systemic lupus erythematosus: Update on clinical presentation, pathophysiology and treatment options. Clin Immunol 2019;209:108274.
- Eissa E, Morcos B, Abdelkawy RFM, Ahmed HH, Kholoussi NM. Association of microRNA-125a with the clinical features, disease activity and inflammatory cytokines of juvenile-onset lupus patients. Lupus 2021;30:1180-7.
- Davidson A, Aranow C. Pathogenesis and treatment of systemic lupus erythematosus nephritis. Curr Opin Rheumatol 2006;18:468-75.
- Walport MJ. Complement. First of two parts. N Engl J Med 2001;344:1058-66.
- Trouw LA, Blom AM, Gasque P. Role of complement and complement regulators in the removal of apoptotic cells. Mol Immunol 2008;45:1199-207.
- Galindo-Izquierdo M, Pablos Alvarez JL. Complement as a therapeutic target in systemic autoimmune diseases. Cells 2021;10:148.
- Ricklin D, Hajishengallis G, Yang K, Lambris JD. Complement: A key system for immune surveillance and homeostasis. Nat Immunol 2010;11:785-97.
- Lewis MJ, Botto M. Complement deficiencies in humans and animals: Links to autoimmunity. Autoimmunity 2006;39:367-78.
- Demirkaya E, Zhou Q, Smith CK, Ombrello MJ, Deuitch N, Tsai WL, et al. Brief report: Deficiency of complement 1r subcomponent in early-onset systemic lupus erythematosus: The role of disease-modifying alleles in a monogenic disease. Arthritis Rheumatol 2017;69:1832-9.
- Lintner KE, Wu YL, Yang Y, Spencer CH, Hauptmann G, Hebert LA, et al. Early components of the complement classical activation pathway in human systemic autoimmune diseases. Front Immunol 2016;7:36.
- Wang FM, Yu F, Tan Y, Song D, Zhao MH. Serum complement factor H is associated with clinical and pathological activities of patients with lupus nephritis. Rheumatology (Oxford) 2012;51:2269-77.
- Foltyn Zadura A, Zipfel PF, Bokarewa MI, Sturfelt G, Jönsen A, Nilsson SC, et al. Factor H autoantibodies and deletion of Complement Factor H-Related protein-1 in rheumatic diseases in comparison to atypical hemolytic uremic syndrome. Arthritis Res Ther 2012;14:R185.
- Sjöberg AP, Trouw LA, Blom AM. Complement activation and inhibition: A delicate balance. Trends Immunol 2009;30:83-90.
- Okroj M, Heinegård D, Holmdahl R, Blom AM. Rheumatoid arthritis and the complement system. Ann Med 2007;39:517-30.
- Leffler J, Bengtsson AA, Blom AM. The complement system in systemic lupus erythematosus: An update. Ann Rheum Dis 2014;73:1601-6.
- Cavazzana I, Manuela N, Irene C, Barbara A, Sara S, Orietta BM, et al. Complement activation in antiphospholipid syndrome: A clue for an inflammatory process? J Autoimmun 2007;28:160-4.
- Roumenina LT, Loirat C, Dragon-Durey MA, Halbwachs-Mecarelli L, Sautes-Fridman C, Fremeaux-Bacchi V. Alternative complement pathway assessment in patients with atypical HUS. J Immunol Methods 2011;365:8-26.
- Buyon JP, Tamerius J, Belmont HM, Abramson SB. Assessment of disease activity and impending flare in patients with systemic lupus erythematosus. Comparison of the use of complement split products and conventional measurements of complement. Arthritis Rheum 1992;35:1028-37.
- Sturfelt G, Nived O, Sjöholm AG. Kinetic analysis of immune complex solubilization: Complement function in relation to disease activity in SLE. Clin Exp Rheumatol 1992;10:241-7.
- Nagy G, Brózik M, Varga L, Füst G, Kirschfink M, Kiss E, et al. Usefulness of detection of complement activation products in evaluating SLE activity. Lupus 2000;9:19-25.
- Ferreira VP, Pangburn MK, Cortés C. Complement control protein factor H: The good, the bad, and the inadequate. Mol Immunol 2010;47:2187-97.
- Rodríguez de Córdoba S, Esparza-Gordillo J, Goicoechea de Jorge E, Lopez-Trascasa M, SánchezCorral P. The human complement factor H: Functional roles, genetic variations and disease associations. Mol Immunol 2004;41:355-67.
- Jokiranta TS, Hellwage J, Koistinen V, Zipfel PF, Meri S. Each of the three binding sites on complement factor H interacts with a distinct site on C3b. J Biol Chem 2000;275:27657-62.
- Schmidt CQ, Herbert AP, Kavanagh D, Gandy C, Fenton CJ, Blaum BS, et al. A new map of glycosaminoglycan and C3b binding sites on factor H. J Immunol 2008;181:2610-9.
- Mihlan M, Stippa S, Józsi M, Zipfel PF. Monomeric CRP contributes to complement control in fluid phase and on cellular surfaces and increases phagocytosis by recruiting factor H. Cell Death Differ 2009;16:1630-40.
- Zhao J, Wu H, Khosravi M, Cui H, Qian X, Kelly JA, et al. Association of genetic variants in complement factor H and factor H-related genes with systemic lupus erythematosus susceptibility. PLoS Genet 2011;7:e1002079.
- Cserhalmi M, Uzonyi B, Merle NS, Csuka D, Meusburger E, Lhotta K, et al. Functional characterization of the disease-associated N-terminal complement factor H mutation W198R. Front Immunol 2017;8:1800.
- Wang FM, Song D, Pang Y, Song Y, Yu F, Zhao MH. The dysfunctions of complement factor H in lupus nephritis. Lupus 2016;25:1328-40.
- Petri M, Orbai AM, Alarcón GS, Gordon C, Merrill JT, Fortin PR, et al. Derivation and validation of the Systemic Lupus International Collaborating Clinics classification criteria for systemic lupus erythematosus. Arthritis Rheum 2012;64:2677-86.
- Gladman DD, Ibañez D, Urowitz MB. Systemic lupus erythematosus disease activity index 2000. J Rheumatol 2002;29:288-91.
- Gladman DD, Goldsmith CH, Urowitz MB, Bacon P, Fortin P, Ginzler E, et al. The Systemic Lupus International Collaborating Clinics/American College of Rheumatology (SLICC/ACR) damage index for systemic lupus erythematosus international comparison. J Rheumatol 2000;27:373-6.
- Dooley MA, Aranow C, Ginzler EM. Review of ACR renal criteria in systemic lupus erythematosus. Lupus 2004;13:857-60.
- Abdel Raouf H, Kholoussi NM, Eissa E, El Nady HG, Fayed DB, Abdelkawy RFM. MicroRNAs as immune regulators of inflammation in children with epilepsy. Int J Mol Cell Med 2020;9:188-97.
- Elnady HG, Sherif LS, Kholoussi NM, Ali Azzam M, Foda AR, Helwa I, et al. Aberrant expression of immune-related microRNAs in pediatric patients with asthma. Int J Mol Cell Med 2020;9:246-55.
- Faul F, Erdfelder E, Lang AG, Buchner A. G*Power 3: A flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav Res Methods 2007;39:175-91.
- Bao L, Haas M, Quigg RJ. Complement factor H deficiency accelerates development of lupus nephritis. J Am Soc Nephrol 2011;22:285-95.