Association of NOX2 subunits genetic variants with autoimmune diseases

Author’s Accepted Manuscript Association of NOX2 subunits genetic variants with autoimmune diseases Jianghong Zhong, Lina M. Olsson, Vilma Urbonaviciute, Min Yang, Liselotte Bäckdahl, Rikard Holmdahl www.elsevier.com

PII: DOI: Reference:

S0891-5849(18)30108-4 https://doi.org/10.1016/j.freeradbiomed.2018.03.005 FRB13652

To appear in: Free Radical Biology and Medicine Received date: 15 December 2017 Revised date: 25 February 2018 Accepted date: 4 March 2018 Cite this article as: Jianghong Zhong, Lina M. Olsson, Vilma Urbonaviciute, Min Yang, Liselotte Bäckdahl and Rikard Holmdahl, Association of NOX2 subunits genetic variants with autoimmune diseases, Free Radical Biology and Medicine, https://doi.org/10.1016/j.freeradbiomed.2018.03.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Review Article Association of NOX2 subunits genetic variants with autoimmune diseasesყ Jianghong Zhonga, Lina M. Olssona, Vilma Urbonaviciutea, Min Yanga, Liselotte Bäckdahla, Rikard Holmdahla,* a

Medical Inflammation Research, Department of Medical Biochemistry and

Biophysics, Karolinska Institutet, Stockholm 17177, Sweden

Keywords: NOX2; Ncf1; polymorphism; ROS; Autoimmune diseases

Abbreviations: NOX2, NADPH oxidase isoform 2; Ncf1, neutrophil cytosolic factor 1; ROS, reactive oxygen species; SNP, single nucleotide polymorphism; CNV, copy number variation; RA, rheumatoid arthritis; PsA, psoriatic arthritis; MS, multiple sclerosis; SLE, systemic lupus erythematosus.

ყThis

article is part of a special issue entitled: Oxidative stress and altered redox

signalling in autoimmune and connective tissue diseases, edited by Jeremy Pearson and Justin Mason. *Corresponding author. E-mail address: [email protected]

(R. Holmdahl).

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ABSTACT A single nucleotide polymorphism in Ncf1 has been found with a major effect on chronic inflammatory autoimmune diseases in the rat with the surprising observation that a lower reactive oxygen response led to more severe diseases. This finding was subsequently reproduced in the mouse and the effect operates in many different murine diseases through different pathogenic pathways; like models for rheumatoid arthritis, encephalomyelitis, lupus, gout, psoriasis and psoriatic arthritis. The human gene is located in an unstable region with many variable sequence repetitions, which means it has not been included in any genome wide associated screens so far. However, identification of copy number variations and single nucleotide polymorphisms has now clearly shown that major autoimmune diseases are strongly associated with the Ncf1 locus. In systemic lupus erythematosus the associated Ncf1 polymorphism (leading to an amino acid substitution at position 90) is the strongest locus and is associated with a lower reactive oxidative burst response. In addition, more precise mapping analysis of polymorphism of other NOX2 genes reveals that these are also associated with autoimmunity. The identified genetic association shows the importance of redox control and that ROS regulate chronic inflammation instead of promoting it. The genetic identification of Ncf1 polymorphisms now opens for relevant studies of the regulatory mechanisms involved, effects that will have severe consequences in many different pathogenic pathways and understanding of the origin of autoimmune diseases.

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1.

Introduction The phagocytic NADPH oxidase isoform 2 (NOX2) complex is an enzyme

response for one-electron reduction of molecular oxygen to superoxide [1]. Amount of reactive oxygen species (ROS) formation is derived from superoxide anion production, which is the so-called oxidative burst. The scope of this review is to describe the structural background and recent findings of polymorphisms in the genes encoding the NOX2 components and in particular of the essential subunit Ncf1 (Ncf1, neutrophil cytosolic factor 1 also named p47phox). We assume that the observed large impact of Ncf1 polymorphisms will bring another angle in the understanding of the pathogenesis of autoimmune diseases. 2.

Structural and genetic insights into the NOX2 complex The initial event of the membrane complex NOX2 activation is the

phosphorylation of its components in the cytosol during activation of the cell. Upon stimulation, the cytosolic components, Ncf1 (p47phox), Ncf2 (neutrophil cytosolic factor 2, p67phox), Ncf4 (p40phox), and the small GTPase Rac1 (in monocytes) [2] or Rac2 (in neutrophils) [3, 4] are tethered, translocate to the membrane and associate with the transmembrane cytochrome b558 to assemble an active oxidase [5, 6]. The heterodimeric flavocytochrome b558 is composed of both a 22–kDa protein Cyba (also named p22phox) and a glycosylated 91–kDa protein Cybb (gp91phox or Nox2) [5]. Cybb is the electron transfer chain of the active NOX2 complex, containing binding sites for FAD, NADPH and two hemes [5]. The whole membrane complex with all involved proteins are denoted the NOX2 complex as shown in Fig. 1.

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Fig. 1. An illustration of the induced activation of the NOX2 complex in terms of structural interactions. Placements of the oxidase protein subunits are done with help of earlier reported studies [5].

2.1 The role of Ncf1 in the NOX2 complex Ncf1 is a 390 amino acids protein with a molecular mass of 47 kDa [7–9]. Ncf1 comprises an N-terminal phox homology (PX) domain (amino acids 4–121), two src homology 3 (SH3) domains (amino acids 159–214, SH3A; amino acids 229–284, SH3B), and the C-terminal auto-inhibitory region (AIR) (amino acids 292–340) and proline rich domain (PRR) (amino acids 363–368) [8]. In the resting state, the Ncf1 PRR domain connects with the C-terminal Ncf2 SH3 domain as a complex in the cytosol [5]. Ncf1 can also be unbound in Ncf2 deficient cells [10]. Upon activation, the Ncf1 is phosphorylated on selective sites located between Ser303 and Ser379 by different type of protein kinases. The

phosphorylation

phosphatidylinositol

of

Ncf1

3,4–biphosphate

leads

to

binding

(PI(3,4)P2),

to

the

phosphatidic

membrane acid,

and

phosphatidylserine with its PX domain [11, 12], which plays a role in assembly of the NOX2 complex. A naturally occurring single-nucleotide polymorphism (SNP) of Ncf1

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gene in rats results in replacement of the threonine at position 153 (T153M) with a methionine in the linker region between the PX and SH3A domains, which leads to a reduced ROS response [13, 14]. Taura and colleagues confirmed that the replacement of a threonine at position 153 in the linker region prevents oxidase activation, but do not affect Ncf1 translocation properties upon stimulation of phorbol myristate acetate (PMA) [15]. Glycine mutation of residues 151–158 in the linker region releases the PX domain from an autoinhibitory interaction, enhances PI(3,4)P2 binding and leads to nearly 10–fold less NADPH oxidase activity than wild-type Ncf1 in the presence of the lipid activators [16]. The phosphorylation of Ncf1 causes the internal binding of the Ncf1 SH3 domain to switch from binding the AIR domain of Ncf1 to the PRR of Cyba [11]. Interaction of the Ncf1 SH3 domain with Cyba induces the translocation of Ncf1 and Ncf2 to the neutrophil membrane [17], and 10%-20% of the Ncf1 proteins migrate to the plasma membrane upon activation [18]. Rac and/or Ncf2 can also bind directly to b558 to activate superoxide generation independently of Ncf1 in vitro [19]. The importance of Ncf1 translocation was consequently validated for ROS production by the cell system in Ncf1 mutant mice. The most commonly used mouse strain with a point mutation of the Ncf1 gene at the -2 position of exon 8, expresses a mutant Ncf1 protein [20, 21]. The spontaneous mutant Ncf1 protein lacks 8 residues 228–235 in the Ncf1 SH3B domain, which is completely defective in activating the NOX2 complex to produce ROS. When the deleted region was narrowed down to Val232 and Thr233, the short Ncf1 protein fails to translocate to the membrane, defects in binding to Cyba, and does not activate the NOX2 complex to produce extracellular ROS in response to PMA [22]. 2.2 The role of Ncf4 in the NOX2 complex

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Ncf4 is a 40 kDa, 339 amino acids protein, composed of PX, SH3 and the Nterminal Phox and Bem1p (PB1) domains [23]. The PX domain of the cytosolic PHOX protein has at least two interaction surfaces, one for F-actin and a second for phospholipids. If areas of high F-actin polymerization are present at the phagosomal membrane in the cell, Ncf4 and Ncf1 will become targeted to these areas via a lipidindependent direct interaction between F-actin and the PX domain [24]. Ncf1 is recruited at early stages of phagosome formation and Ncf4 at later stages, as a consequence of availability of different types of phosphoinositides [25]. The PX domain of Ncf4 specifically interacts with phosphatidylinositol 3-phosphate (PI3P), and binds with a stronger affinity to PI(3,4)P2 as compared with Ncf1 [26–28]. PI3P accumulates in the phagosomal membrane and is essential for phagosome maturation [29]. PI3P also accumulates in early endosomes, where Ncf4 translocates with Ncf2 [30]. When the endosomes become fused to phagosomes, these subunits may form an active NOX2 complex. Animal studies showed that binding to PI3P is selectively prevented by a point mutation of R58A in the Ncf4 PX domain, leading to inhibition of phagosomal PI3P accumulation, resulting in significant reductions in intracellular but not extracellular ROS production of murine neutrophils in response to PMA and select stimuli [26, 31–33]. On the other hand, both the SH3 domain of Ncf4 and C-terminal SH3 domain of Ncf2 (amino acids 458-526) can directly interact with the same PRR domain of Ncf1 (amino acids 358-390) [34]. Ncf4 was thus shown to down-regulate oxidase function by potentially competing for this domain interaction between Ncf1 and Ncf2. Although the affinity of the Ncf4 SH3 domain for the Ncf1 PRR is significantly lower than that of the Ncf2 SH3 domain, Ncf4 can bind to Ncf1 in solution in absence of phosphorylation [34]. In the dormant state, the Ncf1 SH3 domains are normally

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masked through an intra-molecular interaction between its N-terminal SH3 and sequences in its C-terminus. The intra-molecular interaction does not prevent potential binding between Ncf1 and Ncf2 or Ncf4, because the binding sites are different [11]. Thirdly, the SH3 domains of Ncf4 and Ncf1 competitively bind to PRR of Cyba. Ncf4 by itself was reported as an alternative organizer to activate the oxidase together with Ncf2 and Rac in absence of Ncf1, and the activation by Ncf4 was about 70% of the activation by Ncf1 [35]. Although Ncf4 is not required for NADPH activity in cell-free assays and whole cell models [36], the role of Ncf4 in the activation of NOX2 complex remains ambiguous and controversial. The PB1 domain of Ncf4 connects with that of Ncf2. The Ncf4 protein is constitutively associated Ncf2 in the cytosol of resting phagocytes, and this interaction is maintained even in activated cells [37]. However, mRNA and protein for Ncf4 were expressed by the pro-myelocyte stage together with Cyba and Rac2, whereas mRNA and proteins for Cybb, Ncf1 and Ncf2 were expressed after the myelocyte stage [38]. Before this finding, Ncf4 was indeed shown alone in a stable presence in the B cells of a CGD patient lacking Ncf2 [39]. 2.3 Mutations of Ncf1 versus Ncf4 gene modulating ROS production Naturally occurring polymorphisms in the NCF1 and NCF2 genes are associated with ROS production in autoimmune diseases [40–42]. Although the mechanisms linking the clinical significance of such deficits in the NOX2 complex to autoimmunity are so far incompletely understood, animal studies may deliver new insights to understand human diseases. The natural mutation at position T153M in the hinge region of Ncf1 protein, enhancing the binding of its PX domain to PI(3,4)P2 but reducing ROS production,

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leads to a severe destructive arthritis in rats [13, 43]. The spontaneous mutant mice with deletion of residues 228–235 in the Ncf1 protein, absence of NOX2-derived ROS, display a significantly more severe both collagen-induced-arthritis (CIA) [43, 44] and mannan induced psoriasis/psoriatic arthritis (MIP) [45], compared with the wild type mice; macrophage-restricted expressing of functional Ncf1 recovers arthritis resistance by oxidative burst [44, 45]. Additionally, the deficiency of ROS production is equivalent for T153M Ncf1 knock-in mice in comparison to the spontaneous mutant mice [46, 47]. It suggests that residues 228–235 of the Ncf1 SH3B domain may indirectly regulate lipid binding of the PX domain in vivo. It is known that both Ncf1 and Ncf4 directly bind to lipid products of phosphoinositide-3-OH kinase on subcellular membranes through their N-terminal PX domains [48], and that the R58A mutation directly regulates the PI3P binding and ROS production with stimulus-selective response plasticity [31, 49, 50]. The R58A mutant mice, of which the neutrophils display a lower production of intracellular ROS in response to PMA, enhances susceptibility to CIA but not MIP, compared with the wild type mice [32]. Thus, binding specificities of the Ncf1 and Ncf4 PX domains suggests different roles for Ncf1 and Ncf4 in how they modify innate versus adaptive immune responses through extracellular and/or intracellular ROS. 3.

The positional cloning of Ncf1 The changed paradigm on the importance of ROS as an important regulatory

factor of autoimmune diseases was originally derived from a hypothesis-free genetic analysis of rats susceptible to arthritis. In the early 1990s, an initial question was raised that was to identify the genes that cause the development of chronic inflammation in rats [51]. 3.1 Positional identification of a polymorphism in Ncf1

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Positioning genetic polymorphism for complex traits in autoimmunity is essentially dependent on comparing genetically segregated genetic polymorphism in individuals expressing a stable and reproducible phenotype. Pristane-induced arthritis (PIA) was identified as being appropriate for the analysis [52, 53]. Pristane is a defined component of mineral oil, which after a single subcutaneous injection into DA rats induces a disease mimicking rheumatoid arthritis (RA) [54]. To identify genes involved in the control of PIA, crosses were made between susceptible DA rats and resistant E3 rats and analysed the progeny with microsatellite markers covering the entire rat genome [55]. Regions on the chromosomes are statistically determined to control specific quantifiable traits (so-called quantitative trait locus, QTL) such as arthritis severity, arthritis onset, and rheumatoid factor production. Through a straightforward recombinant congenic approach, the underlying gene was identified to be Ncf1 from a QTL with the strongest impact on inflammation [13]. The allelic differences between DA and E3 rats were then pinpointed to a naturally occurring amino acid replacement, i.e. T153M, which causes an impaired ROS production [14]. A mutation in the murine Ncf1 gene and its consequences confirmed the findings in rats [21]. However, it remained to be investigated how the downstream mechanisms were operating, by which the Ncf1 polymorphism affected chronic inflammation and arthritis. 3.2 Regulation of autoimmune arthritis and encephalomyelitis The finding that impaired ROS production is associated with more severe disease was, however, in contradiction to the paradigm that the release of ROS is proinflammatory and that anti-oxidative agents were expected to have therapeutic potential. It raised the question how to explain the dramatic effect of autoimmune responses in ROS compromised rats and mice. Obviously, Ncf1 mediated oxidative

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regulation is associated with many different types of pathways. PIA in rat is one of the most reproducible models of RA with high incidence and little variation in severity. In rats affected with PIA, ROS derived from antigenpresenting cells (APCs) was shown to modify surface thiol groups on T cells, the lower capability to produce ROS the higher numbers of surface thiol groups on T cells. ROS derived from the NOX2 complex on APCs, most likely H2O2, can act as an immunological transmitter, onto T cells and affect T cell signalling [43, 56]. CIA in rat is a model of RA that is characterized by both the T cell dependent inflammatory response and the collagen antibody dependent B cell response. After immunization of autologous rat type II collagen (CII), Ncf1 mutation enhances the severity and chronicity of arthritis in rats as well as observed from the PIA model [13]. Using the murine CIA model, it was further confirmed that Ncf1 mutated mice also had more severe arthritis [21]. In a recent study, Ncf1 knock-in mice with inducible Ncf1 expression provide a unique opportunity to determine the critical time window for NOX2-derived ROS regulating on-going diseases. When Ncf1 is activated before immunization with CII, these mice develop only mild clinical symptoms with reduced CII specific IL-17A-producing T cells. When Ncf1 is activated after the priming phase, Ncf1-dependent protection of autoimmune arthritis is still observed together with reduced number of splenic monocytes but not correlated with CII specific T cell response. These results thus suggests clearly that NOX2derived ROS regulates both priming and effector phases of CIA in mice but most likely via different mechanisms [47]. Since macrophages have the highest burst capacity among APCs [44], transgenic mice were introduced with functional Ncf1 expression restricted to macrophages by inserting the human CD68 promoter. Functional Ncf1 in macrophage restored the resistance of arthritis to the level of wild

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type mice in a T-cell dependent CIA model and T cell activation was down regulated in transgenic mice [44]. Thus, the downstream T cell response seemed to operate along several different pathways. T-cell responsiveness was suppressed by ROS in auto-reactive T cells but variable n hetero-reactive T cell responses. In a mouse model where the major CII epitope was mutated to mimic the rat CII (MMC mouse) used for immunization, which led to protection against CIA [57, 58], T-cell tolerance was broken in Ncf1-mutated mice [59, 60]. These results suggest that Ncf1 controlled auto-reactive T-cell activation and thereby T cell tolerance and autoimmunity. Considering the role of ROS in B-cell regulation, B10Q.ACB mouse is a newly introduced model in which the VH VDJ region of a germline encoded and pathogenic antibody binding to the C1 triple helical epitope of CII is inserted, resulting in a spontaneously high frequency of CII-specific self-reactive B cells [61]. B10Q.ACB mice are protected from CIA. Introducing a mutation in the Ncf1 gene, leading to ROS deficiency, breaks this strong arthritis tolerance. In mechanisms, the development of CIA in Ncf1-mutated B10Q.ACB mice is associated with an enhanced germinal centre formation, increased T-cell responses, and intra-molecular epitope-spreading [62]. Glucose-6-phosphate isomerase peptide (G6PI) induced arthritis (GIA) in mouse is also a both T and B cells dependent model of RA. From differences in disease phenotype, Ncf1 mutation enhances disease severity and prolongs disease progression in murine GIA model, which can be reverted by Ncf1 expression macrophages as well [60]. In the study of molecular dynamics, G6PI contains redox-sensitive amino acids that may form the internal disulfide bonds. IFN-gamma-inducible lysosomal thiol reductase (GILT) is known important in processing of disulfide bond containing antigens [64, 65]. The expression of GILT in macrophages was indeed enhanced due

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to Ncf1 mutation, and the antigen presentation of G6PI was further demonstrated more efficient in Ncf1 mutated mice than that of the wild type controls [66]. Therefore, NOX2-dependent antigen processing could modify the antigen-specific Tcell activity and then regulate arthritis development by macrophages. Collagen antibody induced arthritis (CAIA) in mice is an arthritis model caused by innate effector cells, independent of both T and B cells [67]. Interestingly, Ncf1 mutated mice develop more severe arthritis merely after antibody transfer, suggesting that lack of ROS could enhance inflammation independent of antigen-specific T and B cells [68]. In contrast, both lipopolysaccharide (LPS) and lipomannan enhanced CAIA more potently in the presence of functional phagocyte ROS production than in its absence. The ROS-dependent enhancement of CAIA is regulated by TLR2, but not TLR4 stimulation, and driven by granulocytes, whereas macrophages do not contribute to the phenotype [68]. MIP in mice is an interleukin 17-pathway dependent model for psoriasis (Ps) and psoriatic arthritis (PsA) [45]. The disease severity of MIP was pronouncedly exacerbated in Ncf1 mutated mouse strains. Restoration of ROS production in macrophages ameliorates both skin and joint manifestations. Interestingly, diseases susceptibility was associated with the major histocompatibility complex (MHC) region but not conventional T cells. Instead, mannan-mediated stimulation of macrophages led to TNF-a secretion and stimulation of local unconventional T cells secreting IL-17A and further drove neutrophil infiltration in the skin, leading to disease symptoms [45]. Experimental autoimmune encephalomyelitis (EAE) is a commonly used model for neuroinflammatory diseases like multiple sclerosis (MS) [69]. The Ncf1 mutation enhanced the disease severity of EAE in MHC-H2q haplotype mice induced by

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recombinant rat myelin oligodendrocyte glycoprotein (MOG)1–125 protein, but suppressed the disease induced by mouse MOG79-96 peptide [21]. These conflicts in observations indicate that the functional Ncf1 has a unique role in immune regulation of native epitope or uptake and processing of MOG recombinant proteins. One underlying mechanism could be that administration of MOG peptides bypasses such steps before binding to the MHC molecular on the specific APC, and a hypothesis addressed in C57BL/6 mice [70]. In brief, NOX2-derived ROS limits the activation of cathepsin L and S, and wild type macrophages can efficiently present MOG35-55 to effector T cells. NOX2-deficient macrophages cannot oxidatively inactivate capthepsin L and S, which prevents efficient MHC class II presentation to effector T cells. Furthermore, NOX2 does not uniformly affect processing of a given antigen, but differentially affects relative epitope generation from a single antigen [70]. Taken together, the Ncf1/NOX2 control of chronic inflammation in experimental autoimmune arthritis and encephalomyelitis is complex. In these disease models, NOX2 regulation was interacting with different mechanisms, involving both adaptive and innate immune responses. All of these immune pathways pointed towards ROS regulated chronic inflammation. It is likely that different quality of the ROS response, i.e. in different cells, different sources and affecting different compartments could affect the final downstream mechanisms and thereby the outcome of a particular inflammatory disease. 4.

The polymorphisms of NOX2 genes in human autoimmune diseases The genes of the NOX2 complex are highly conserved among species including

humans and rodents [71, 72]. The importance of the different subunits of the NOX2 complex can be seen in patients with chronic granulomatous disease (CGD), a rare but severe immunodeficiency disorder caused by mutations in the subunits of the NOX2

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complex. Most (65%) of the CGD patients have mutations in the Cybb subunit encoded by the gene CYBB, which is located on the X chromosome. X-linked CGD patients usually have a severe form of CGD, reflecting the importance of Cybb for the function of NOX2. Mutations in CYBB more often lead to a greater reduction in ROS production compared to mutations in the other NOX2 complex genes [73]. Mutations in NCF2, encoding the Ncf2 subunit, and CYBA, encoding Cyba, are found in 5% of CGD patients respectively, whereas only one patient with mutations in NCF4 has been reported [74]. Compared to X-linked CGD, these patients often have a milder disease and higher residual ROS production [74]. The same is seen for patients with mutations in NCF1, however NCF1 stands out, as mutations in NCF1 accounts for 2530% of all CGD patients. What is even more striking is that almost all CGD-Ncf1 patients have the same mutation, a 2 base-pair (bp) deletion in exon 2, resulting in a shift of the reading frame and a truncated, non-functional protein [75]. Considering the wide range of mutations in the other NOX2 genes, why is this specific deletion so common in CGD-Ncf1 patients? Unlike in rodents, the human genomic region surrounding the NCF1 gene is very complex. In addition to the functional NCF1 gene, there are two non-functional NCF1-pseudogenes, located 360 kbs telemeric (NCF1C) and 1.5 Mbs centromeric (NCF1B) of the functional NCF1 gene. Both of the pseudogenes have the same 2 bp deletion seen in CGD-Ncf1 patients, and encode the same non-functional Ncf1 protein. In fact the CGD-Ncf1 patients seem to have lost their two functional NCF1 genes and instead have six pseudogenes on the two chromosomes [76]. The locus where the NCF1 genes are located in the 7q11.23 chromosomal region is characterized by high genome plasticity and have several large duplications, called low-copy repeat (LCR) regions, as well as inversions and smaller deletions and

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duplications [77, 78]. The NCF1 genes are each located on a separate LCR and in between the LCRs harbouring NCF1B and NCF1, is a single-copy region containing 26 genes. Due to the extremely high sequence similarity between the LCRs (99.6%), un-equal crossover events between them can occur during meiosis, leading to both deletions and duplications of varying sizes [78]. Patients with the neurodevelopment disorder Williams-Beuren syndrome (WBS), have large deletions of the single-copy region located in between the LCRs, due to non-allelic homologous recombination (NAHR) between the LCR regions on each side of the single-copy region. The NAHR that occurs in WBS patients leads to large-scale deletions. But when it comes to CGD patients, the crossover events between NCF1 and the pseudo genes are smaller [78] and the end result is not deletions but rather the creation of fusion-genes, consisting of parts of NCF1 and parts of one of the pseudo genes [75, 79]. It is not yet known through what type of crossover event these fusion-genes are created or exactly where the break points are located but the result is seen in CGD-Ncf1 patients. The sequence covering exon 2 in the functional NCF1 gene has been substituted with the reciprocal sequence of one of the pseudogenes, creating a non-functional pseudo-like version of NCF1. The patients have two copies of this pseudo-like version of NCF1 instead of normal NCF1 genes, and thus six genes that have the 2 bp deletion in exon 2, on the two chromosomes. Interestingly, the opposite outcome of this crossover event has also been detected, suggesting that it could be a reciprocal crossover event [78, 79]. This NCF1-pseudo fusion gene (NCF1/psd) has the functional NCF1 sequence in exon 2 instead of the 2 bp deletion, turning one the pseudogene into a functional NCF1 gene. Others and we have shown that the NCF1/psd gene is common in Caucasians [80], with 11% of healthy individuals of Swedish origin having one copy [42, 81]. A recent study by Zhao and colleagues found a similar frequency in European (16.5%) and

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African (13.5%) Americans, but in Asian populations the frequency was lower (<2%) [41]. The difference in frequency of the NCF1/psd gene between populations is interesting and could suggest a benefit of having the NCF1/psd gene under certain environmental conditions. The question if the NCF1/psd gene is fully functional has not been conclusively determined yet, but in a small study we did not detect a difference in ROS production in individuals who had one copy of the NCF1/psd gene in addition to the standard two NCF1 genes. The same was seen in a study of immortalized B-cells from individuals with the NCF1/psd gene [80]. Interestingly, we have also seen that having a copy of the NCF1/psd gene can restore reduced ROS production caused by a polymorphism in NCF1, suggesting that the NCF1/psd gene can function as a back-up to ensure full NOX2 capacity [42]. As mentioned earlier, in the genomes of both rats and mouse, there is only copy of the NCF1 gene and no NCF1 pseudogenes. The same is seen in all primates [82], however in the Neanderthal genome both pseudogenes appear to be present [83]. The duplications therefore seem to predate modern humans and have been suggested to occur 1.4 (NCF1B) million and 0.7(NCF1C) million years ago [84]. However, more detailed analyses of genomes of early humans are needed to conclusively determine when the duplications of the pseudogenes occurred. Because of the complexity of the NCF1 gene and the chromosomal region surrounding it, whole genome association analyses have been unable to evaluate association of NCF1 with autoimmune diseases. The first discoveries linking NOX2 genes with autoimmunity in humans have instead been found in NCF2 and NCF4. We have previously published an association of an intronic SNP in NCF4 with Rheumatoid factor negative male RA patients, but the causative variant remains elusive [40]. Interestingly, mutations affecting NCF4 are important for arthritis

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susceptibility in animal models [32]. A number of genome-wide association studies (GWAS) have identified an association of the intronic SNP rs4821544 in NCF4 with Crohn’s Disease (CD) [85, 86]. Subsequent replication studies have been inconsistent [87, 88], but might reflect that the association is specific for the subset of patients with ileal CD [86, 89]. Somasundaram and colleagues showed that the associated Callele of rs4821544 lead to reduced ROS production by GM-CSF primed and fMLP stimulated neutrophils [90]. Interestingly, CGD patients often develop intestinal inflammation, similar to that seen in patients with inflammatory bowel disease (IBD) and a study by Muise and colleagues, showed that a rare missense variant in NCF2 is associated with very early onset IBD by reducing the binding of Ncf2 to Rac2 leading to reduced ROS production [89]. However, the most established link between the genes of NOX2 and autoimmune disease is with systemic lupus erythematous (SLE). First reported as a SLE association in the GWAS [91], Jacob and colleagues managed to pinpoint the causal variant to a missense SNP in NCF2, strongly associated with both childhood and adult onset of SLE [92]. The SNP encodes an amino acid shift at a binding site to the Vav1 protein, important for propagating the NOX2 activation signal from the Fcγ receptor. The loss in binding efficiency resulted in a reduction of Fcγ receptor stimulated ROS production in transfected K562 cells. An additional missense SNP in NCF2, which results in a conformational change of the NOX2 complex, is associated with SLE in Hispanic American, but not European American SLE patients [93]. Despite of the complexity of the NCF1 gene, two studies published this year have independently identified a strong association of a SNP in NCF1 with SLE. Following our findings in animal models, we became interested in a missense SNP located in exon 4 (rs201802880, here denoted NCF1-339) because of its position as an

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important binding site of Ncf1 to the plasma membrane and because we found that it was relatively common in Swedish Caucasians [81]. NCF1-339 encodes an amino acid shift from Arginine to Histidine at position 90 in the Ncf1 protein, which corresponds to the PX domain. It is highly conserved and mutational analysis shows a reduced functionality of NOX2 if mutated [94]. To be able to genotype the NCF1-339 SNP exclusively in the functional NCF1 gene, and excluding the pseudogenes, we designed a nested PCR genotyping strategy based on pyrosequencing [81]. The method not only allowed us to genotype the normal NCF1 gene, but also the NCF1/psd gene, in those individuals that have one or two copies. Genotyping 973 Swedish SLE patients, we found a striking increase in frequency of the T-allele of the NCF1-339 SNP, 11% compared to 4% in controls. The odds ratio (OR) was 3.0 (95% CI 2.4 to 3.9), suggesting a very strong effect on SLE susceptibility [42]. We could also see that the SLE patient group had a higher frequency compared to controls, of CGD-Ncf1 carriers, meaning that they have a copy of the pseudo-like fusion gene instead of a functional NCF1 gene. When we calculated the total genetic effect, taking the effects of the NCF1/psd gene into account, 17% of the SLE patients compared to only 6% of the controls had a T-allele genotype (OR 3.7). We saw that the NCF1-339 T-allele lead to a reduced extracellular ROS production in neutrophils and an increased expression of type 1 IFN regulated genes. The NCF1-339 T-allele also reduced the age of disease diagnosis in SLE patients. Independently of our discovery, Zhao and colleagues found an association of NCF1-339 in a large study of Asian SLE patients (OR 3.5), which is at the top of the SLE susceptibility loci list (Fig. 2) [41, 95, 96]. They replicated the association in European American (OR 2.6) and African American (OR 2.0) SLE patients, and also found associations of NCF1339 with Sjögrens syndrome (OR 2.4) and with RA in Korean patients (OR 1.7). The

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finding of an association with Korean RA patients is interesting, because we did not detect association of NCF1-339 with Swedish RA patients [81]. We did however detect a protective effect against RA of having one copy of the NCF1/psd gene. Zhao and colleagues reported the same protective effect in SLE patients, but did not investigate RA patients.

Fig. 2. SLE susceptibility loci with common variants, which have been identified through GWAS, meta-analysis, fine-mapping, or replication studies yielding p < 5 × 10−8 in at least one ancestry. Loci labelled in red color contain genes coding for the NOX2 complex; loci labelled in purple color contain genes coding for antibody Fc receptors; loci labelled in green color contain genes associated with type I interferon 19

(IFN-I) signalling; loci labelled in yellow color contain genes associated with TLR signalling; loci labelled in blue color contain genes associated with NF-kB signalling, and loci labelled in white color contain genes associated with immune cells signalling and migration. 5.

Influence of ROS on lupus SLE is a complex systemic autoimmune disease that is characterized by

breakdown of tolerance to nuclear antigens, immune complex deposition in tissues, and multiorgan involvement [97]. Organ damage occurs as a consequence of the chronic autoimmune response directed against ubiquitous, mostly nuclear, selfantigens, or secondarily because of ischemia. Although candidate gene and genomewide association studies have successfully identified more than 100 robust SLE risk loci, the association intervals need to be further elucidated within immune pathways [96]. It is clear however, that the major and most frequent genetic association is mediated by polymorphism of Ncf1 and Ncf2, and a low ROS response is associated with the development of lupus. 5.1 NETs and type I Interferon signatures in lupus Common hypotheses on SLE pathogenesis suggest that genetic predisposition together with environmental factors, such as smoking, UV exposure and infections cause dysregulation of innate and adaptive immunity, leading to the clinical disease [98]. The sources of autoantigens in SLE are not known. Several lines of evidence suggest that neutrophil extracellular traps (NETs), web-like structures composed of chromatin backbones coated with neutrophil granule proteins, may be a source of autoantigens in patients with SLE and mouse models of lupus. An imbalance between NET formation [99] and clearance [100] could play a prominent role in the perpetuation of autoimmunity and the exacerbation of lupus disease.

20

Firstly, the formation of chromosomal derived NETs was shown to be dependent on ROS produced by the NOX2 complex [101, 102]. The oxidative burst induces a selective release of granular proteins into the cytoplasm. For the spontaneous NET activation and release, or NETosis, of low-density granulocytes from individuals with lupus, mitochondrial ROS is required for maximal NETs stimulation together with ribonucleoprotein immune complexes [103]. The functional NETs production is also demonstrated in neutrophils from CGD patients in an alternative NOX2-independent pathway [104], suggesting NETosis could occur through different signalling mechanisms in humans and animal disease models with different disease outcomes. On the other side, two independent expressing deoxyribonucleases (DNases), DNase I and DNase I-like 3, degraded NETs in vivo [105] and inhibited

extracellular ROS

production of the NOX2 complex in response to LPS and PMA [106]. Thus, both formation and disassembly of NETs are related with ROS production and the NOX2 complex, and both disease-promoting and protective effects of NETs have been reported [107]. Alternatively, another prominent feature, linked with NETs formation in patients with SLE [108], is an exaggerated and continuous activation of the type I interferon (IFN) system, which manifests as increased serum levels of IFNα and/or an increased expression of type I IFN-induced genes, a so-called type I IFN signature. The type I IFNs act as an immune adjuvant and stimulate T cells, B cells, and monocytes, which all play an important role in the loss of tolerance and persistent autoimmune reaction in SLE [109]. 5.2 NOX2 in murine models of lupus Spontaneous lupus disease models in mouse strains that are NOX2 deficient supports the regulatory role of ROS. NOX2-deficient mice on lupus-prone MRL/Faslpr background have markedly exacerbated lupus, reflected by increased

21

glomerulonephritis,

splenomegaly,

elevated

and

altered

lupus-characteristic

autoantibody profiles with a shift towards RNA-containing autoantigens. An increased number of antibody-forming cells and an expanded myeloid compartment were also observed in the spleen of NOX2-deficient MRL/Faslpr mice. Since, the NET formation, a potential source of self-antigen in SLE, relies on activity of NOX2 in neutrophils, this study suggests that the disease exacerbation in NOX2-deficient MRL/Faslpr mice occurs without classical NET formation [110, 111]. Insertion of the previously characterized Ncf1 mutation [20–22] into Balb/c background induced the spontaneous formation of lupus-associated anti-dsDNA, anti-Sm/ribonucleoprotein (SM/RNP) and anti-histone autoantibodies as well as glomerular deposition of IgG and complement C3. This study suggests that lack of phagocyte-derived oxidative burst is associated with spontaneous SLE-related autoimmunity. Of interest, prominent type I IFN response signature, which is observed in patients with SLE and correlates with the disease severity, was identified in Ncf1-mutated mice lacking a functional NOX2 complex. Expression analysis of germ-free Ncf1-mutated mice demonstrated that upregulated IFN signaling pathway is of endogenous origin [112, 113]. However, the mechanisms behind the spontaneous activation of type I IFN response remain to be explained. In the induced disease model of lupus, Kienhöfer and colleagues demonstrate a regulatory role of ROS in the Balb/c Ncf1 mutant mice by intraperitoneal injection of pristane [114]. Mice injected with pristane produce autoantibodies, similar to human lupus patients, develop immune complex–mediated glomerulonephritis. Pristaneinduced lupus is known to be associated with the type I IFN response [115]. The Ncf1 mutated mice developed aggravated pristane-induced lupus, reflected by increased levels of anti-dsDNA, anti-histone, anti-Sm/RNP and other antibodies, together with

22

an enhanced glomerulonephritis. The Ncf1 mutated mice with deficient NOX2derived ROS production displayed reduced ability to form pristane-induced NET formation in vivo. Conversely, treatment with a specific chemical NOX2 activator RE-02 induced NET formation and ameliorated pristane-induced lupus. Of interest, blood neutrophils from Ncf1-mutated mice and patients with SLE exhibited enhanced spontaneous NET formation, in contrast to the pristane induced NET formation. Moreover, Ncf2-null mice on the NZM 2328 background, a polygenic model in which mice spontaneously develop lupus, displays accelerated full-blown lupus [116]. This was characterized by more rapid development of hyperactive B cell and T cell immune compartments and significantly accelerated kidney disease. Of note, Ncf2deficient NZM mice exhibit increased expression of type I IFN-responsive genes, suggesting that activation of type I IFN axis is one potential mechanism by which NOX2 deficiency contribute to promotion of lupus disease. In contrast to a previous report by Campbell and colleagues studying Cybb deficient mice [111], the Ncf2-null lupus prone mice had an increased formation of NETs even in the absence of NOX2 activity [116]. These discrepancies might be due to different NET formation-inducing stimuli/conditions used in the different studies. For instance, PMA-induced NETosis was analysed in vitro in the studies of Campbell et al [111]. Jacob and colleagues used stimulation with lupus serum from NZM mice [116]. Once more, the relationship between ROS generation and the NETosis within lupus development appears complex [117] and, remains incompletely understood. Taken together, these findings in patients with SLE and in lupus mouse models suggest that NOX2-derived ROS may act as essential regulatory molecules involved in a variety of cellular processes of importance for immune regulation and limiting inflammation. These studies also contribute to understanding of lupus pathogenesis

23

and may lead to development of future therapeutic approaches for the subgroup of patients with NOX2 complex genes variants. 6.

Concluding remarks Is lack of ROS a key for understanding complex autoimmune diseases? The

recent studies of SLE clearly show that polymorphisms in genes determining NOX2 mediated ROS response represents the major genetic control and the lack of regulatory effects of ROS a key for the pathogenesis. Clearly, data from animal models indicate a role of STAT1 pathway and the involvement of NET formation and disassembly but the exact mechanism remains to be understood. The question arises also whether Ncf1 and NOX2 play a similar role in other autoimmune diseases and also in a broader sense to regulate chronic inflammation? The effect was first identified in the PIA model but the mechanisms in the RA models or in human diseases seem to be different from what is observed in SLE. Likewise, different pathways operate in models for the other chronic inflammatory diseases such as psoriasis, psoriatic arthritis and gout. In a wider perspective it could have relevance for both degenerative and malignant diseases in which ROS could have a regulatory role [118, 119]. It could of course be due to effects of interacting genes as well as different environmental induced factors. However, it might also be due to different qualities in ROS production and signalling within different cell types and cellular compartments. An interesting observation along the line is that the mouse with a mutation in the Ncf4 gene, which leads to a normal extracellular but abnormal intracellular production of ROS in response to select stimuli, showed a pronounced enhancement of autoimmune arthritis [32]. A model for psoriasis/psoriatic arthritis, which is independent of adaptive immunity, was not significantly affected by the Ncf4 mutation [32]. Thus, even a small quantity or location defect of outcomes derived

24

from the NOX2 complex could have dramatic differences for disease control. Disclosure RH is a cofounder of Pronoxis, a small company devoted to develop therapy based on small molecule stimulators of ROS response. Otherwise the authors have no conflicts of interest to disclose.

Acknowledgements This work was supported by grants from the Swedish Foundation for Strategic Research (SSF), the Knut and Alice Wallenberg Foundation, and the Swedish research Council.

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Highlights The Ncf1 gene polymorphism is associated with a lower oxidative burst response. The lower reactive oxygen response leads to more severe autoimmune diseases. Polymorphism in NOX2 genes is strong associated with systemic lupus erythematosus.

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NF-kB Signaling

Immune Cell Signaling

IFN-I Signaling

TLR Signaling

NOX2 Pathways

Fc γR2a CD11b Signaling

Ncf1 Ncf2 Tnfaip3 Itgam Irf5 Blk Tnfsf13b Fcgr2a Ifih1 Irak1 Ptpn22 Bank1 Ikzf1 Ets1 Mir146a Irf7 Tlr7 Ube2l3 Pxk Il10 Csk 1

2

3

Odds Ratio