Specificity of marker autoantibodies in Systemic Lupus Erythematosus: autoantibodies detected in SLE patients by a novel DNA/histone H4 peptide complex

(1)

i

GENERAL INTRODUCTION

Systemic Lupus Erythematosus (SLE) is a complex autoimmune rheumatic disease often described as a prototype of systemic autoimmune diseases, because of its broad spectrum of clinical manifestations and the involvement of almost all organs and tissues. SLE is characterized by the production and persistence of self-antigens in the blood, disruption of immune tolerance leading to biosynthesis of antibodies against those self- antigens, typically nuclear antigens (antinuclear antibodies or ANA), complement proteins (anti-C1q), ribosomal proteins, cell surface molecules and others. The disease is chronic and, in its clinical phase, characterized by a variable course, where periods of flares alternate to periods of remission. During its preclinical phase, autoantibodies common to other systemic autoimmune diseases can be detected in the patients’ sera, but the pattern of autoantibodies become more specific with the progression to the clinical phase [1], [2].

In the adult world population the SLE prevalence rate is estimated in the range from 20 to 70 per 100,000 person-years [3]. Women are affected much more than men, the female to male ratio being estimated as 10:1, and the disease onset occurs in their childbearing age. In fact, in 65% of patients, SLE manifestations begin from 16 to 55 years of age [1], although the disease may affect people of all ages.

Although the SLE etiology is still unknown, it is widely accepted that genetic, epigenetic, immunologic and environmental factors concur to the establishment of self-antigen overproduction and defective clearance, their recognition by T and B lymphocytes, the breaking of immunological tolerance leading to the appearance of the aforementioned autoantibodies and of immune complexes (ICs). Such immune complexes subsequently deposit in the target organs, leading to complement activation, inflammation, organ damage, disease onset and progression. Clinical manifestation of SLE include mucocutaneous (i.e. rashes), musculoskeletal (i.e. arthritis), central nervous system and hematologic features, along with cardiovascular and renal involvement (lupus glomerulonephritis) from mild to severe grade, and other manifestations affecting all organs. The clinical picture is usually complicated by several disease and/or therapy- related comorbidities, such as cardiovascular diseases, stroke, malignancies, infections

(6)

2

that can arise in patients even at young age.

Current therapy of SLE relays on non-steroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, antimalarial agents such as chloroquine and hydroxychloroquine, and immunosuppressive drugs such as cyclophosphamide, methotrexate and mycophenolate mofetil among others [2].

The burden of SLE disease activity and concurrent comorbidities may contribute to dramatically reduce the quality of life of patients, including their ability to work and to attend daily duties, but also their life expectancy, which may be shorter than the general population if the disease is not properly and early diagnosed and treated.

The diagnosis of SLE is based on a combination of clinical and laboratory findings and the clinical and serological criteria developed by the American College of Rheumatology (ACR) and revised in 1997 [4]. More recently, the Systemic Lupus Collaborating Clinics revised and validated such criteria in order to improve their clinical relevance [5]. Among the serological ACR criteria, the detection of marker autoantibodies such as anti-Sm antibodies and antibodies against native (double- stranded) DNA (anti-dsDNA) is included because of their high specificity for the disease and, in the latter case, also for their relation with disease activity [6]. At present, several assays based on different techniques are available for the

detection of anti-dsDNA autoantibodies, from Crithidia Luciliae

Immunofluorescence (CLIF) test to tests employing radioactive reagents such as the Farr assay, and solid phase ELISA and multiplex assays, but none of them permits to conciliate at the same time optimal specificity and sensitivity, good predictive and prognostic value, ease of use and cost saving. Such an assay would prevent the exclusion of large groups of patients from diagnosis and therapies, allowing the diagnosis of SLE in its preclinical stage or when the disease is still limited to few organs. Moreover it would make the differential diagnosis and the follow-up easier for all groups of patients, and spare patients the discomfort of more aggressive or unnecessary therapies.

(7)

3

CHAPTER 1 SYSTEMIC LUPUS ERYTHEMATOSUS

EPIDEMIOLOGY OF SLE

1.1 Incidence and Prevalence

Estimates of incidence and prevalence of SLE can vary greatly worldwide, as reviewed by Pons-Estel et al [3] and Carter et al [7]. The reported variability is the result of many factors: the study design (e.g. multi-center or single-center, prospective or retrospective), the choice of the observed population (e.g. hospital-based or community-hospital-based), the patients’ selection criteria (e.g. fulfilling the ACR/SLICC criteria only may exclude mild or sub-clinical SLE), the presence or absence of public health services in the countries involved in the study, the availability of reliable diagnostic tests and/or of wide and accurate SLE patients databases, to name but a few.

Overall SLE incidence ranges from 0.3 cases per 100,000 person-year in Ukraine to 31.5 cases per 100,000 person-year among Afro-Caribbean population in the UK, whereas overall prevalence ranges from 3.2 cases per 100,000 individuals in India to 517.5 among Afro-Caribbeans in the UK [7].

Focusing our attention on adult SLE and on a time period spanning the last thirty years, the overall incidence rates reported by several studies that use mostly ACR classification criteria [3] can be grouped by geographical areas as shown in Tab.1: the incidence in Europe ranges from 0.3 to 5 cases per 100,000 persons-years, from 3 to 25 in North America, 6.3-8.7 in Central-South America, 0.9-3.1 in the Asia-Pacific region including Australia. Similarly, overall prevalence in Europe ranges from 9 in Russian Federation to 97 cases per 100,000 inhabitants in UK in 2012, from 17.2 (Canada, Nova Scotia), to 178 in a study covering the populations of Alaska, Phoenix and Okhlahoma. The estimated prevalence among Central, South America and Caribbean Islands ranges from 58.6 in Argentina to 84.1 in Barbados. Available studies from Asia and from Pacific Countries report a relatively low prevalence in South Corea (18-20 cases per 100,000), intermediate (37.56) in China [8] and a maximum of 92.8 among Australian Aborigines (Table 2).

(8)

4 GEOGRAPHICAL AREA/COUNTRY OVERALL INCIDENCE

(cases per 100,000 persons-years) NORTHERN EUROPE Iceland 3.3 UK 5.1-4.6 Denmark 1.0-2.3 Norway 2.6 Sweden (Lund) 2.8-3.9 Finland 1.7 Estonia 1.5-1.8 EASTERN EUROPE Russian Federation Ukraine 1.4 0.3 SOUTHERN EUROPE

Spain, 2.2 (Asturias)- 3.6 (Lugo) France, All regions 3.3

Italy 2.0 (Valtrompia)-2.6 (Ferrara) Greece, Northwest 2.1

Turkey, Trace 4.4 NORTH AMERICA

USA 5.1 (Rural Wisconsin)

5.6 (Southeastern) 7.6 (Southeastern Michigan) 3.7-4.9 (Olmsted County, Minnesota) 7.4 (Alaska, Phoenix, Okhlahoma) 23.7 (47 States + Wahington DC) 5.6 (Georgia) Canada 3.0 (Quebec) 2.9-25.5 (Nova Scotia) CENTRAL AMERICA AND

CARIBBEAN ISLANDS

Barbados 6.8

SOUTH AMERICA Argentina, Buenos Aires 6.3 Brazil, Natal 8.7

ASIA

Asian-Pacific Region 0.9-3.1 China, Hong Kong 3.1

Kazakhstan 1.6

(9)

5

GEOGRAPHICAL AREA/COUNTRY OVERALL PREVALENCE (cases per 100,000 inhabitants) NORTHERN EUROPE Iceland 35.9 UK 64.9-97.0 Denmark 28.3 Norway 44.9 Sweden (Lund) 55-65 Finland 28 Estonia 39-48 EASTERN EUROPE Russian Federation 9 Ukraine 14.9 SOUTHERN EUROPE

Spain, 17.5 (Lugo), 34.1 (Asturias) France, All regions 47

Italy 71 (Florence), 39.2 (Valtrompia), 57.9 (Ferrara)

Greece, Northwest 39.5 Turkey, Thrace 51.7 NORTH AMERICA

USA 78.5 (Rural Wisconsin) 103 (Arizona) 72.1 (Southeastern) Southeastern Michigan 62.6 (AR+CH, n=54) 52.3 (AA, n=1356) 120.1 (C, n=886)

178 (Alaska, Phoenix, Okhahoma) 74.4 (Georgia)

Canada 32.8 (Quebec)/17.2 (2000)- 92.0 (2012) (Nova Scotia)

Mexico 70

CENTRAL AMERICA AND CARIBBEAN ISLANDS

Barbados 84.1

Martinique Islands 64.2 SOUTH AMERICA

Argentina, Buenos Aires 58.6

Venezuela 70 ASIA Asian-Pacific Region 4.3-45.3 China, Mainland 37.6 Iran, Teheran 40 Iran, Sanandaj 50 Kazakhstan 20.6 South Korea 18-8-21.7 OCEANIA

Australia 45.3 (overall), 92.8 (Aborigines) Table 2. Worldwide overall prevalence in SLE(adapted from refs. [3] and [7]).

(10)

6

Autoimmune diseases are generally considered to be rare in Africa for the high burden of infections, particularly malaria, that may have an immunosuppressive effect [9], [10]. Moreover a “prevalent gradient” has been hypothesized for SLE by which the disease prevalence would increase from Africans to African-Americans through Afro-Caribbeans, following the slave trade route from Western African countries [11], [12]. This hypothesis would stress the role of environmental triggers in developing the disease. However, cases of SLE are increasingly reported throughout the African continent, suggesting that the real impact of SLE in African countries could have been underestimated because of the lack of epidemiologic studies, the presence of general under-diagnosis due to poverty, paucity or absence of diagnostic tools [13], [14]. The recent African Lupus Genetics Network (ALUGEN) registry has the purpose to address many of these issues [15].

1.2 Mortality and Survival

As for incidence and prevalence, estimates of mortality and survival in SLE can vary basically for the same reasons: for example, hospital-based studies, a stricter application of disease classification criteria or the prevalence of older age patients in the cohort considered would tend to overestimate mortality and survival, whereas rural studies would tend to underestimate them because of the reduced availability of specialist care in that areas. Community-based studies could provide more reliable results, but they are not possible in many countries.

Nonetheless, many evidences are well established. In 2012 Mak and colleagues published a meta-analysis of seventy-seven cross-sectional, retrospective and prospective studies covering the period from 1 January 1950 to 31 July 2010, for a total of 18,998 SLE patients. The overall 5-year survival rate improved from 74.8% in 1950 to 94.8%, in 2000, whereas the overall 10-year survival rate improved from 63.2% in 1950, to 91.4% in 2000 [16]. Such achievements are attributable to more effective and better managed therapies and immunosuppressive regimens, to a better control of comorbidities but also to increasingly early diagnosis.

However, the life expectancy of SLE patients still remains lower than that of the general population: the overall Standard Mortality Ratio (SMR) is estimated to be

(11)

7

2.6-3 times higher among SLE patients, due to the contribute of comorbidities correlated to disease or therapy, such as cardiovascular diseases (SMR = 2.3), infections (SMR = 5) and renal disease (SMR= 4.7). Other authors report a higher contribute of renal disease to mortality (SMR= 7.9) [18].

1.3 Sex and Age.

SLE can affect subjects of all ages, but the most affected age group in women is the 20-40 one, followed by the menopausal one, albeit there are considerably variations to this pattern depending on countries and ethnicity: the lowest mean age at disease onset (20-25 yrs) has been found in Asia, some Arabian countries and North Africa, whereas the highest mean age has been reported in Europe and Canada [17], [19]. Men with SLE are generally older, presenting the disease after the age of 60. On the other hand, peak incidence after 40 years of age is reported for Caucasians, both women and men, in some studies in the UK [20] and in USA [21].

1.4 Ethnicity and socio-economic factors

It is widely accepted that ethnicity has a strong influence on SLE epidemiology as well as on its clinical features. There are several evidences supporting the fact that incidence and prevalence among non-white populations are two-threefold higher than in Whites [17] and that patients of African, Asian and indigenous ancestry (Native Americans, First Nations from Canada, Aborigines from Australia) are more at risk for the disease, experience more severe manifestations (particularly the renal ones) and are at higher mortality risk than that of European ancestry [7],[19]. Most of the epidemiological and genetic studies were traditionally carried out in the USA and Europe, but considerable efforts have been made by researchers in China, South America and in the African continent, particularly in South Africa. A population-based study performed in Georgia in the period 2002-2004 found an impressive disproportion in the number of female patients presenting the onset of the disease at young age between Blacks and Whites (20 cases among Blacks vs 4 cases among Whites) [22]. This trend is globally respected to a large extent by the vast majority of the studies, and similar results are reported for Afro-Caribbeans in Barbados [23] but also in the UK, where the higher number of enrolled Black,

(12)

8

Black-Caribbean and Asian subjects probably justify the increased prevalence reported in comparison with the majority of European countries [20]. On the contrary, a prevalence similar to that observed in Western Countries along with a delayed peak age around 50 has been observed for Asians in rural areas of China [8], and a much lower mortality was also found in Asian and Hispanic patients enrolled in the USA Medicaid system even respect to Whites (52% and 41% lower, respectively) [24]. Gómez-Puerta et al. described for the first time this “Asian-Hispanic paradox” for SLE after it was reported for other autoimmune diseases, e.g. rheumatoid arthritis (RA) in Texas. They hypothesized three reasons for it: firstly, the fact that the enrollment in Medicaid is reserved to low-income people, thus canceling the differences due to the lower socio-economic status of Hispanics; secondly, the SLE definition applied by Medicaid is less strict than that applied by academic health centers, so the latter usually include patients with more active disease and worse outcomes; thirdly, the higher cohesion in Asian and Hispanic communities may play a role in decreasing the impact of the disease [24], e.g. by favoring the adherence to therapies.

Ethnic differences exist with respect to organ involvement in SLE: for example, lupus nephritis and immunologic disorders seem to affect Black, Hispanic and Asian populations more than Whites, cutaneous manifestations such as malar rash and photosensitivity are more present among Caucasians, and neuropsychiatric involvement in Indian patients [7]. Inter and intra-ethnic differences have been examined by the LUMINA (Lupus in Minorities: NAture vs. Nurture) and GLADEL (Grupo Latino Americano De Estudio del Lupus) studies regarding Hispanics from Texas and Puerto Rico and Hispanics from South America, respectively. These studies notably highlighted that Hispanics are not a homogeneous group by the genetic, clinical and socio-economic points of view, in contrast to the approach often found in many studies from the USA [25], and that the socio-economic status of patients can have a considerable impact on the disease course. In effect, in the LUMINA cohort Hispanics from Texas, mainly of Native American origin, experienced a disease severity and a damage accrual comparable to that observed in the African-American patients of the cohort, whereas Hispanics from Puerto Rico exhibited a less severe clinical picture [26]. In the GLADEL cohort, composed mainly by Caucasian, African-Latin American (ALA) and Mestizo (defined as people

(13)

9

with European-Amerindian background in Latin America) patients, ALAs and Mestizos are younger at onset and at the diagnosis of SLE and have higher probability to suffer for acute or chronic renal failure than Hispanics of Caucasian origin, but the frequency of the nephrotic syndrome has been found to be comparable between Mestizos and Whites [26]. Despite some methodological differences between the two studies, intra-ethnic comparisons between Texan Hispanics from the LUMINA cohort and their Mestizos counterpart from the GLADEL cohort have also been made, and less favorable clinical outcomes (organ damage, survival) have been noted in the first group [27]. These data could be partly attributed to the higher proportion of Amerindian genes in Texan Hispanics with respect to the prevalence of European genes found in Mestizos [26], in agreement with the higher burden of SLE on Native Americans reported in the literature, but socio-economic factors have been also called into play, such as the limited accessibility to health care services in Texas by many first-generation Hispanic patients who are not completely integrated in the resident community [26]. The LUMINA study has postulated that non-Caucasian genetic background has a major role in acute onset and in the first phases of the disease activity, but these factors are replaced by low socioeconomic status features such as poverty and low education as for higher disease activity and lower survival outcomes [26].

ETIOPATHOGENESIS

Systemic Lupus erythematosus (SLE) is a chronic, multiorgan disease caused by immune dysregulation at several levels that results in the loss of tolerance in B and T cells, impairment of the clearance of apoptotic material with subsequent exposure of abnormal amounts of self-antigens to the actors of the adaptive immune response, leading to the hyperproduction of autoantibodies typically against nuclear antigens, but also against cytoplasmic and cell surface self-antigens. Subsequently, immune complexes form and/or deposit at the level of the target organs and tissues. SLE has been defined as a prototypic immune complex- mediated disease, in which the typical mechanisms of a type III hypersensitivity reaction play their role in the complex and still not fully understood disease pathogenesis [28]. Glomerulonephritis, arthritis, vasculitis, central nervous system, musculo-skeletal and pulmonary disorders are important manifestations of the

(14)

10

disease, that can exacerbate or ameliorate during the periods of flares or remission, respectively, typical of the disease course. The etiology of SLE remain unknown, but it is clear that the coexistence of genetic predisposition along with hormonal, epigenetic and environmental factors contributes to the establishing of the abnormal immune response and of the clinical picture.

1.5 GENETIC FACTORS

1.5.1 Familial and twin studies

Familial aggregation studies have shown that the relative risk (RR) of having SLE increases in members of SLE patients’ families, and that it is directly correlated with the genetic proximity among them. A nationwide study carried out in Taiwan[29] found that first- degree relatives of SLE patients have a RR of SLE 17-fold higher than the general population and that heritability of SLE among them (defined as the proportion of the phenotypic differences due to genetic factors) can vary from 44% to 56%, depending upon the correlation model used. Old studies set this estimate at 60% [30], probably because of lower population numerosity and the absence of a “correction factor” for shared environment, but the intra-familial susceptibility to the disease remains evident. Moreover, it has been shown that subjects having a familiar history of SLE are at higher risk for other autoimmune diseases such as rheumatoid arthritis (RA), Sjogren syndrome (SS), scleroderma (SSc) and autoimmune tyroiditis (AT) [31].

Twin studies are an important tool to evaluate the relative contribution of genetics and environment to the onset and progression of diseases, and SLE makes no exception. In the abovementioned study by Kuo and colleagues, covering the population of Taiwan, the observed relative risk in disease susceptibility is generally higher in twins than in siblings of patients (316 vs 24, respectively), suggesting a strong impact of genetic factors in disease onset [29]. Most studies observed a concordance for SLE in monozygotic (MZ) twins of 25-30% but only 0- 4% in dizygotic (DZ) twins [32]. Such concordance is similar to that observed in other autoimmune diseases such as rheumatoid arthritis and multiple sclerosis [33], [34] suggesting common genetic patterns contributing to disease susceptibility. On the other hand, several case- report studies account for a notable discordance between

(15)

11

monozygotic twins (69%), even if lower than that in dizygotic ones (96%), which can reflect into variations in disease onset, clinical manifestations and severity that can be accounted for by epigenetic changes, providing new insights into the pathogenesis of SLE [35], [36]. In addition, the long time often required for that subclinical lupus switches to full-blown SLE could partly account for the reported discordance rates in MZ twins [34], highlighting that long- term follow-up is required for this kind of analysis.

Evaluation of concordance rates in MZ and DZ twins strongly supported the research on genetic alterations predisposing to SLE, leading to a continuously growing set of findings since the last 30 years.

1.5.2 Sex and gender

SLE is usually considered a disease typically affecting women, because most studies along with the clinical experience report a female-to-male prevalence ratio close to 10:1. Such a remarkable disproportion between sexes is maintained across populations, with some differences among Caucasians that tend to a 6-7:1 ratio, and African-Americans/Aboriginals, that tend to the 9-10:1 ratio [19]. Nonetheless, male patients may suffer for a more severe disease, particularly with respect to renal and haematologic involvement, and higher mortality than females [37]. Moreover, the prevalence of certain clinical manifestations may differ between males and females, but variations among populations have been observed on the individual manifestations [37], [38].

Several factors has been investigated to account for a female predisposition to SLE, reviewed in [32]. It has been observed an increased prevalence of SLE and similar autoimmune disorders such as Sjogren Syndrome among subjects with chromosomal abnormalities, such as men with the 47XXY karyotype (Klinefelter’s syndrome, KS) and women with the 47XXX karyotype (trisomy X or Triple X syndrome). KS male patients exhibit 14-fold higher prevalence of SLE with respect to normal men, which make it closer to that observed in women with normal 46XX karyotype. Moreover their disease manifestations are more similar to that found in women with respect to severity and renal involvement. Women affected by Triple X Syndrome are more at risk for SLE than normal women, whereas only rare case reports exist about the association between 47X0 women (Turner’s syndrome) and

(16)

12

SLE [39]. These data suggest a X chromosome-linked gene-dose effect in the susceptibility to SLE.

1.5.3 Detection of genetic loci contributing to SLE susceptibility

The association of SLE with allelic variants has been extensively investigated by means of “traditional” techniques of high resolution molecular typing such as polymerase chain reaction (PCR) and high-throughput Sequence-Based Typing (SBT) as well as, by the last decade, with Genome-Wide Association Studies (GWAS). GWAS is a hypothesis -free approach consisting in the comparison and analysis of

hundreds or thousands of Single Nucleotide Polymorphisms (SNPs) among patients and controls at the same time by means of microarray technologies[40]. Such approach has boosted the identification of new genetic variants in SLE patients and confirmed most of that previously discovered: up to 2016, more than 60 loci correlating with lupus susceptibility across populations have been identified [41]. Another important aspect confirmed and highlighted by genome scanning approaches is that genetic associations may vary across populations. On the other hand, GWAS and molecular studies cannot give information about the function of allelic variants [41]: this can be achieved by in vivo studies using mice models of spontaneous lupus.

1.5.4 Genetic abnormalities within the Human Leucocyte Antigen (HLA) complex.

The Human Leucocyte Antigen (HLA) complex was the first genetic region associated with SLE and it still remains the strongest predictor of SLE susceptibility [42]. The highly polymorphic HLA region is the human counterpart of the Major Histocompatibility Complex (MHC) previously discovered in mouse and it is located in the short arm of chromosome 6 (6p21.3), spanning a trait of 7.6 Mbp hosting more than 200 genes [42]. It is subdivided in three regions, the telomeric class I (HLA-I) and the two centromeric class II and III (HLA-II and HLA-III) regions. The class I genes, named HLAA, B, C, and the class II genes, named HLADR, DQ, -DP, encode for proteins crucially involved in the process of antigen processing and presentation to T lymphocytes and in transplant compatibility, whereas the class III region encodes for the complement proteins C2, C4A and C4B of the classical pathway, along with the Tumor Necrosis Factor- α (TNF-α) cytokine, lymphotoxins, heat shock proteins, and Complement Factor B (CFB) [43]. Allelic variants in the

(17)

13

HLA-II and HLA-III regions have been proven to provide the strongest association with the development of SLE and specifically with the production of pathogenic autoantibodies. Among the HLA- II class, the extended haplotypes DR2 and DR3, including DRB1, DQA1 and DQB1 genes are particularly linked with the risk of SLE in Caucasians, Asians and Latin Americans of European origin [19], [40]. For example, the -DRB1*15:01 (DR2) and -DRB1*03:01 (DR3) haplotypes seem to increase the risk for SLE in Caucasians two-threefold [40]. Within this population, the haplotype HLA-A1, -B8, -DR3 is more present among North-Europeans. Interestingly, a protective role of some HLA-DR6 alleles (-DRB1*13:02 and -DRB1*14:03) has been hypothesized in a recent study on a Japanese cohort of patients, i.e. a lower frequency of the HLA-DRB1*15:01/13:02 or HLA-DRB1*15:01/14:03 genotypes was found to be negatively associated with the disease (p> 0.5, OR<1 in both cases) [44]. Thus, the protective effect of the –DRB1*13:02 and –DRB1*14:03 alleles seems to exceed the predisposition conferred by the presence of the –DRB1*15:01 allele in the cohort studied. Moreover, both –DRB1*13:02 and –DRB1*14:03 alleles were previously negatively associated with other conditions, i.e. human papilloma virus (HPV) infection-derived cervical cancer and hepatitis B, respectively [44].

The functional meaning of HLA-DR and DQ variants seem to consist in structural modifications in the peptide-binding groove of the HLA proteins and in the up-regulation of HLA-II genes transcription, finally resulting in the production of SLE autoantibodies: in Caucasians from North America and Europe, HLA-DR3 is associated with certain ANA specificities, i.e. anti-Ro/SSA and anti-La/SSB autoantibodies [45],[46]. A strong association has been demonstrated between the SNP rs2187668 within DR3 and anti-dsDNA autoantibodies [43], whereas HLA-DR4 is more frequent in patients with anti-Sm autoantibodies [46].

With regards to the class III alleles, deficiencies of C2 and C4 genes (C2 or C4 null alleles) are rare but they are strong risk factor for the disease: 80% of siblings homozygous for C4 (C4A along with C4B) are concordant for SLE [32]. The C4 gene can harbor genetic variations other than null alleles, i.e. Copy Number Variations (CNV), defined as duplications or deletions of DNA segments spanning from 100 bp to 3Mbp found by comparison with a reference genome having the normal copy number N=2 [47]: in Caucasians, low gene copy number of C4 is a risk factor for lupus, whereas high copy number seems to exert a protective effect [42]. The functional effect of complement deficiency or low copy number consists in the

(18)

14

contribution to the impairment of the removal of immune complex and apoptotic debris from the circulation, which in SLE prolongs the time of exposition of nuclear autoantigens to T cells, B cells and antigen-presenting cells (APCs) leading to their activation.

1.5.5 Genetic abnormalities in non -HLA genes

The influence of abnormalities in the complement system genes is also shown by the effects of C1q null allele. C1q is a subunit of the C1 protein which activates after the binding with IgM or IgG immunoglobulins associated with polyvalent antigens, starting the proteolytic cascade of the classical pathway by the cleavage of C4 in the C4a and C4b fragments. Unlike the HLA region-encoded complement proteins, C1q is encoded by three genes (C1qA, C1qB, C1qC) on chromosome 1, corresponding to the A, B, C chains composing the C1q heterotrimer. Hereditary homozygous point mutations in such genes have been described in families of SLE patients of Indian origin, each leading to premature stop codons and complete deficiency of C1q [48]. Such deficiency was found to be associated with early onset and cutaneous manifestations of SLE, positivity to Anti-nuclear Antibodies (ANA), including Anti-Ro/SSA, and to anti-cardiolipin antibodies (aCL) but not to anti-Sm, anti-RNP and anti-dsDNA autoantibodies [48]. In other studies, C1q deficiency was associated to the presence of severe form of lupus with early onset and rash, glomerulonephritis and CNS involvement, with a concordance of 90% among siblings homozygous for this defect [32]. Other C1 complex component genes may have defects consisting in polymorphisms, point mutations or partial gene deletions[49]. GWA studies found a SNP in the gene coding for the α-chain of the Complement Receptor type 3 (CR3), also called ITGAM (Integrin-α M) or C11b, in patients of African and European ancestry, that translates in a nonsynonymous Arg77-to- His substitution in the corresponding protein [50]. Normal ITGAM, present on the plasma membrane of neutrophils, macrophages, natural killer cells, is a receptor that binds Integrin- β2 (ITGB2) to form a heterodimer that binds microbes and particles opsonized by the C3 fragment iC3b, leading to their phagocytosis or destruction, so this modification produces a dysfunctional CR3 which impairs these processes. This effect has been demonstrated on human monocytes and macrophages from healthy subjects bearing or not the SNP and in simian fibroblasts [51]. The presence of this SNP was furtherly confirmed in Hong-Kong Chinese and in

(19)

15

Thai populations, but with only low frequency in China [40]. Moreover, two other variants of the same kind have been detected (Phe 941-to-Val and Gly1145-to-Ser) that, although rare, also confirm the importance of impairments in the CR3-mediated phagocytosis in the pathogenesis of SLE [52].

Other important genetic variations are SNPs in the Fcγ- receptor genes [42], such as the Phe176-to-Val mutation which produces a low IgG1 and IgG3- binding affinity form of the Fcγ-RIIIa receptor on monocytes, natural killer (NK) lymphocytes and macrophages in subjects homozygous for the Phe/Phe genotype. Analogously, the His131-to-Arg mutation in the Fcγ-RIIa gene, more represented in African-Americans, leads to a less efficient clearance of IgG2-bound immune complexes in subjects carrying the Arg/Arg alleles. Both have been associated with lupus nephritis [42].

The list of genetic variations potentially involved in SLE susceptibility lengthens continuously and includes also other factors involved in several steps of innate and specific immune responses, as listed in Tab.3:

Gene/Location Gene product Function Type of variation

O.R. Population ancestry

HLA-DR2,-DR3/6p21.3

HLA proteins Antigen processing and presentation

Haplotypes 2.4 European, Asian

C2/6p21.3 Complement protein C2 Clearance of immune complexes Deletion 5.0 European C4/6p21.3 Complement protein C4 Clearance of immune complexes CNV 4.3 European C1Q/1p36.12 Complement protein C1q Clearance of immune complexes Deletion 10 European

FCGR2A/1q23.3 Receptor for the Fc fragment of IgG-IIa Clearance of immune complexes H131R 1.6 European, Eastern Asian, African American

FCGR2B/1q23.3 Receptor for the Fc fragment of IgG-IIb Clearance of immune complexes I232T 1.7 European, Eastern Asian, African American

FCGR3A/1q23.3 Receptor for the Fc fragment of IgG-IIIa Clearance of immune complexes F176V 1.4 European, Eastern Asian, African American

(20)

16

(low affinity) complexes France) [53]

PDCD1/2q37.3 Programmed Cell Death Protein-1 Membrane receptor of the Ig superfamily; Regulation of apoptosis in T T-cells and Tregs SNP (intron) 1.1 European, Mexican [54] PTPN22/1p13.2 Protein Tyrosine Phosphatase, Non receptor, type 22 TCR and BCR signaling; inhibition of T cell activation R620W 1.35 European, Hispanic IRF5/7q32.1 Interferon Regulatory Factor-5 Activation of IFN-1, TNF-α, IL-6, IL-12 transcription SNPs 1.61 European, Eastern Asian, Hispanic, Latin American

STAT4/2q32.2 Signal Transducer and Activation of Transcription-4 Transcription factor regulating IFN-γ signaling; apoptosis SNPs 1.50 European, Eastern Asian, Hispanic, Latin American, African- American

IRAK1/Xq28 IL-1 Receptor-Associated Kinase-1

Toll, IL-1 and NF-kB signaling in B cells

SNPs 1.31 European, Eastern Asian, African American

BANK-1/4q24 B-cell scaffold protein with Ankyrin repeats-1 BCR signaling, B cell activation R61H 1.38 European, Eastern Asian, African- American

TREX1/3p21.31 Three Prime Repair Exonuclease 1 3’-5’-DNA exonuclease; ssDNA and dsDNA degradation SNPs 25 Europea, Asian, African- American [55] MECP2/Xq28 Methyl-CpG-binding Protein-2 Regulation of gene expression through DNA methylation SNPs 1.31 European, Eastern Asian, African- American

Table 3 (continued). Genetic variations associated with SLE susceptibility (adapted from [42] and [40]).

1.5.6 Murine models and genetics of SLE

Mouse models of spontaneous lupus have helped to shed a light on how single genetic variations contribute to build the “genetic predisposition” and disease phenotypes. The classical model is the F1 hybrid between the New Zealand Black and the New Zealand White strains, i.e. NZB/W F1, that develop a severe lupus-like disease very similar but not identical to the human SLE, characterized by

(21)

17

lymphadenopathy, splenomegaly, ANA, anti-dsDNA but not autoantibodies against immune- complexes containing RNA. They do not exhibit cutaneous and hematological diseases, but immune complex-mediated glomerulonephritis develops at 5 months of age and leads to end-stage renal disease at 10-12 months, so they have been used prevalently to study lupus nephritis[56]. Like human lupus, the disease is highly prevalent in female animals and at least partly dependent on estrogen levels. The NZ Mixed strain NZM2410 and NZM2328 arise from a backcross between the NZB/W F1 and the NZW strains and are largely used for studying genetic influences on SLE because of their homozygosity [56]. Three susceptibility loci were found in NZM2410, named Sle1, Sle2 and Sle3. Congenic C57BL/6 strains (also termed B6) carrying Sle1-3 loci individually develop patterns of autoimmune activation that make it possible to identify the role played by each of these loci in the full disease phenotype: Sle1 is associated with loss of tolerance in T and B cells leading to ANA production; Sle2 is associated with B cell hyperreactivity, high levels of B-1 cells and IgM antibodies; Sle3 reduces activation- mediated apoptosis in CD4+ T cells [56], but full lupus occurs only after the three loci are transferred in the same C57BL/6 genotype and it occurs in a stepwise fashion. Moreover, Sle1-mediated breaking of immune tolerance in B and T cells has been found critical for the development of fatal disease, and it has been

associated with antibodies towards the H2A/H2B/DNA component of

nucleosomes[57]. Moreover, a Sle5 locus associated with anti-dsDNA autoantibodies has been described within the Sle3 trait [57]. The same is, in all probability, in humans, where the odds ratios for the majority of known SLE susceptibility genes (some of which are reported in tab.3) are quite low, except for HLA, complement and TREX1 genes, so the susceptibility to SLE is more likely polygenic rather than oligo-monogenic. In addition, a suppressive locus named Sles1, linked to the MHC-II locus H2z, has been found in NZM2410 mice, reflecting that disease results when other factors weights the balance in favour of susceptibility instead of resistance loci[56]. In this regard, many genetic modifications reside in non-coding regions of the genome, underscoring a regulatory role of epigenetic and environmental factors on establishing the SLE phenotype.

(22)

18

1.6 HORMONAL FACTORS

Several evidences call into cause the relevance of sex hormones as risk factors for the onset and the course of SLE: the noticeable disproportion in the female: male ratio, the fact that in females SLE incidence and severity are predominant in the reproductive age, the observation that in many patients the disease activity increases during pregnancy and decreases in menopause, the different pattern of the disease experienced by male patients. Many efforts have been done by researchers to elucidate this issue, but the role of sex hormones in SLE is still to be defined. Traditionally, estrogens are said to be a trigger or an aggravating factor for SLE, whereas androgens are said to be protective. For example, when treated with estrogen 17-β estradiol (estradiol), lupus-prone mice such as the abovementioned NZB/W F1 strain castrated before puberty showed consistently higher serum levels of anti-nucleic acid IgM and IgG (anti-DNA and anti- polyadenilic acid antibodies), enhanced immune complex deposition in the glomeruli with severe nephritis and poor survival compared to non-castrated and non-treated controls [58]. On the contrary, mortality and autoantibodies titres in mice castrated and treated with androgens (5-α-dihydrotestosterone) were significantly reduced [58]. With respect to mortality, a protective effect of androgens is observed especially in females, whereas the harmful effect of estrogens were markedly high in males [58], in agreement with the more severe disease often experienced by male human patients, especially those with Klinefelter’s syndrome, as explained above. Intracellular estrogen receptors ERα and ERβ are present in many cells of the immune system, including T helper and B lymphocytes, with pro-inflammatory and anti-inflammatory/immunosuppressive actions, whereas testosterone receptors has also been found intracellularly and on the membrane of T cells [59]. Estrogen and androgen receptors can explain why sex hormones have a role in inflammation and immunity. Some studies, reviewed by Mc Murray and May, have found that SLE female patients tend to have higher serum levels of estradiol and lower levels of androgens such as testosterone, dihydrotestosterone and precursors such as progesterone, dihydrohepiandrosterone (DHEA) and DHEA-sulphate (DHEAS) [60], whereas estradiol serum levels in male patients are only slightly increased and no differences was found in testosterone levels compared to healthy controls [60]. On the contrary, other studies report

(23)

19

significantly lower androgens and increased estradiol/testosterone ratio and luteinizing hormone (LH) in male patients, which was compatible with hypogonadism [61], [62]. Such imbalance could predispose to the development of the disease and contribute to explain the exacerbation of the disease during pregnancy in women, with a shift toward the Th2 lymphocyte response and inflammatory cytokines and the activation and expansion of autoreactive B cells, leading to IgG autoantibody production [59]. On the other hand, the increased levels of estradiol could be a consequence, instead of a cause, of inflammatory processes in SLE that increase the activity of aromatase, the enzyme converting testosterone to estradiol [59].

High levels of prolactin, whose production is stimulated by estradiol, have been also found in subgroups of SLE patients, but its action in SLE is controversial: a protective role has been suggested by the Carolina Lupus Study, a population-based, case-control study carried out in North and South Carolina between 1995 and 1999. This study found an inverse association between lactation, a period when women are exposed to high endogenous levels of this prolactin, and the risk of SLE [63], but other studies have found higher prolactin levels in Asians, Latin Americans and mixed populations, and a positive correlation with disease activity in Europeans, Asians and mixed populations [64], invoking genetic background in the frame. Early menopause have been found to be present in women who later developed SLE, even if one would expect a decreased risk of SLE with the considerable decrease of estrogens in menopause.

With respect to the role of exogenous hormones, many studies have investigated the effects of hormone replacement therapy (HRT) and the use of oral contraceptives on the risk for SLE, reviewed by Villarragua and colleagues in[65]: even if the association with SLE has not been demonstrated for oral contraceptive use [63], [65] such association have been found for HRT, contradicting previous studies which found only negligible association [63].

1.7 ENVIRONMENTAL FACTORS

Environmental factors along with epigenetic factors likely play a considerable role in the onset and course of lupus, as the aforementioned discordance among

(24)

20

identical twins suggests.

1.7.1 UV light

It has been suggested that exposure to UV light can trigger or exacerbate disease manifestations in patients by a dose-response effect, in which medium and high level of UV-B exposure would cause apoptosis of keratinocytes and other skin cells and the subsequent production and release of nuclear autoantigens [66]. Some case-control studies confirm such hypothesis showing a significant association between SLE and exposure to sunlight at midday in patients with skin reactions such as sunburn with blistering or rash [67], and in Sweden patients with skin type I or II and/or history of sunburn at young age [68] but others do not confirm such observations.

1.7.2 Silica

Among occupational factors, the largest number of evidences are related to exposure to silica dust due to work in mines, constructions, masonry etc. It is known that crystalline silica is responsible for chronic, inflammatory pulmonary diseases such as silicosis but it is also considered a risk factor for the development of autoimmune diseases such as ACPA-positive RA (ACPA stands for anti-citrullinated peptide/protein antibodies), SSc but also SLE [69]; NZM2410 mice administered intranasally with a suspension of 1 mg crystalline silica develop ANA, including anti-DNA and anti-histone autoantibodies, circulating ICs and glomerulonephritis sooner and at higher levels than control mice, with death occurring at 10 weeks of age instead of 16 weeks in controls [70]. In non-autoimmune rats, parenteral administration of silica as sodium silicate is able to induce ANA, including anti-dsDNA- and anti-Sm antibodies [71]. In humans, the level of the association of silica with SLE has been classified as “confident” by a panel of experts from the National Institute of Environmental Health Sciences (NIEHS) in the USA in 2010 [69], [72] after having examined three population-based case-control studies and three cohort studies enrolling 659 cases of occupational-derived exposure from Europe and North America [69]. The relative risk of developing SLE has been calculated as ranging from 1.6 in subjects with “any exposure” to 4.9 in highly exposed subjects; moreover, the relative risk was 7.5 in

(25)

21

silicosis patients and tenfold in subjects exposed to high levels of silica compared to non- exposed subjects [69].

The mechanisms of induction of SLE by silica are not clear, but the its ability of provoking inflammation and apoptosis in the cells of the respiratory tract [73] seems to have a role in T/B cell autoimmune activation. Studying the pathogenesis of silicosis has provided insights into the role of silica as a trigger of autoimmunity. Otsuki and colleagues found alterations in the expression of the Fas (First apoptosis signal) receptor, which triggers apoptosis after binding to its ligand FasL, as well as in the composition and activation profile of lymphocytes in PBMC extracted from peripheral blood of silicosis patients presenting ANAs without symptoms of SLE[74]. They hypothesized that chronic exposure to silica particles or silicates would disrupt the homeostatic balance between regulatory T lymphocytes (T regs), which normally suppress autoreactive lymphocytes, and activated CD4+T lymphocytes included autoreactive subsets [74].

1.7.3 Smoking

The association between SLE and smoking is not clear, because the available studies show conflicting results. Some studies have shown an association between SLE and previous or current smoking, others have found that the difference in the risk for SLE is low between current and never smokers [66], [72].

On the other hand, in 2010 the NIEHS expert panel inserted current cigarette smoke as a “likely” risk factor among the others for the development of SLE, on the basis of the published literature [69], [72]. Other studies have found that smoking can increase disease activity or worsen organ damage, particularly skin manifestations. Recently, Montes et al. performed a prevalence study in a cohort of more than one hundred SLE patients to evaluate a possible correlation between every kind of cigarette smoke exposure (current smokers, past smokers and second-hand smokers were considered), grouped as “ever exposed” , and organ damage measured by the SLICC/ACR-DI (Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index, also called SDI). The comparison was made with the “never exposed” group of patients. The authors found a significant association between the “ever exposed group” and cumulative organ damage: 82% of these patients had a SLICC/ACR-DI>0, whereas only 64% of non-exposed patients had this result. Moreover, the authors calculated a 22%

(26)

22

reduction in the risk of progression from score 0 to score>0 in the “never exposed” group[75]. As regards the mechanisms of action of smoking in SLE pathogenesis, there are not fully established hypothesis in the literature. Associations have been found between higher risk of SLE in smokers and polymorphisms of genes involved in reactive oxygen species (ROS) metabolism [66], [72]. Moreover, the abovementioned Carolina Lupus Study suggested a synergistic interaction between smoking habit and medium or high exposure to silica, hypothesizing that smoking could promote inflammation by hampering the clearance of inhaled silica particles, increasing soluble ICAM 1-concentration, complement activation and recruitment of inflammatory cells; in addition, T cell proliferation and B cell differentiation, including autoreactive subsets, could also be promoted by a mitogen polyphenol-rich glycoprotein present in tobacco leaves cured during the cigars manufacturing process [76].

1.7.4 Vitamin D

The role of Vitamin D deficiency as a risk factor for SLE onset and/or manifestations is not well established, because contradictory results are described in the literature. Low blood levels of 25-OH-hydroxyvitamin D3 are often found in patients because of darker skin, lifestyle, or because patients tend to avoid high levels of UV light in order to minimize cutaneous manifestations such as photosensitivity and malar rash and the risk of triggering disease flares. It is particularly observed in non-white patients, overlapping with the existing ethnic differences in prevalence and incidence of the disease [72]. A regulatory role of vitamin D in the innate and immune responses has been reported by several studies: vitamin D receptor (VDR) has been discovered on cells of the innate and specific immunity, such as machrophages, dendritic cells, B and T lymphocytes, especially CD8+ T lymphocytes [77]. A meta-analysis of clinical trials covering SLE and another autoimmune rheumatic diseases such as rheumatoid arthritis (RA) have found that supplementation of vitamin D reduces anti-dsDNA autoantibody levels, which are associated with high disease activity [78]. Vitamin D deficiency could be a risk factor for comorbidities in SLE such as cardiovascular disease, as shown by experiments on vitamin D- deprived MRL/lpr lupus-prone mice that developed defects in endothelial function and neo-angiogenesis. Moreover, an increase in the expression of genes of the IFN-1 signaling pathway, that is involved in abnormal

(27)

23

endothelial function, were also detected in the same study in mice and in humans as well [79]. On the other hand, a decrease of IFN gene expression (the “IFN-signature, as explained below) was not found in a previous clinical study in which three groups of patients were treated with a placebo and two increasing doses of cholecalcipherol respectively [80].

1.7.5 Infections

The relationship between pathogens and autoimmunity is thought to be somewhat bi-faceted: in subjects with an underlying immunological impairment the burden of infections throughout life can induce autoimmune diseases once the microbial/viral load, the intensity of the immune response and a certain combination of pathogens occur. On the other hand, the immune response against infections can also be protective against autoimmunity: in murine models, malaria due to the Plasmodium

berghei parasite confers protection from lupus [11], and the infection with the

helminth Schistosoma mansoni can retard or prevent type 1 diabetes in mouse models such as the NOD (Non Obese Diabetic) strain [81]. These and other evidences led to the formulation of the so-called “hygiene hypothesis” by Strachan in 1989, according to which the increased prevalence of autoimmune and allergic diseases in developed countries can be partially attributable to improved standards of antimicrobial therapies and prevention practices such as antibiotic drugs, vaccination policies, high hygienic standards that all together dramatically lowered the exposure of the populations to infectious diseases [82].

With regard to the role of pathogens in triggering autoimmune diseases, it is exemplified by acute rheumatic fever, which develops as a consequence of streptococcal A infections [82]. SLE association with bacterial, protozoan and viral diseases was suggested in early studies which observed virion-like tubular structures in endothelial cells in kidneys and in lymphocytes of SLE patients [83]; higher titers of antibodies against viral and parasitic antigens from Epstein-Barr virus (EBV), cytomegalovirus (CMV), toxoplasma were found in a cohort of over one-hundred patients and healthy controls [84]. Moreover, some author associated certain clinical manifestations with a particular microorganism in SLE: in patients with neuropsychiatric involvement, high titers of IgM against rubella virus have been found more often in the subgroup experiencing psychosis or depression than

(28)

24

in SLE patients suffering from other neuropsychiatric disorders [85]. The mechanisms by which pathogens could trigger an autoimmune disease are numerous, but one of the most supported is molecular mimicry: microbial or viral antigens (proteins, peptides, carbohydrates but also DNA) sharing structural similarity with self-antigens can induce the production of autoantibodies that cross-react with self-antigens[86].

Murine models help to elucidate the molecular and cellular factors involved in this mechanism, as reviewed by Christen et al in [87]. In these models, transgenic animals which express antigens known to be cellular and humoral targets in certain autoimmune diseases are infected with virus carrying the same (molecular identity) or similar (molecular mimicry) antigen to induce autoimmunity and the subsequent disease. Molecular identity models have been useful to analyze the elicited immune response on the basis of the entity of the cytotoxic T lymphocyte (CTL) response and the impact of central tolerance mechanisms. In the RIP-LCMV models of type 1 diabetes, the -GP strain expresses the glycoprotein (GP) of the lymphocytic choriomeningitis virus (LCMV) under the control of the promoter of the rat insulin gene (RIP), exclusively on its pancreatic islet β cells, whereas the -NP strain expresses the nucleoprotein (NP) of the same virus in the thymus. In both of them, the infection with the LCMV leads to a response against an autoantigen (the target) which is structurally identical to the viral antigen (the trigger). In both models an immune response characterized by high- avidity interaction between cytotoxic T cells and antigen presenting cells (APCs) via the respective T cell receptors (TCRs) and MHC/peptide antigen complex develops because of the “perfect fit” between the two, but in the RIP-LCMV-GP mice type 1 diabetes has a faster onset (10-14 days vs 1-6 months) and greater severity (higher organ damage and autoantibody titers) than that in in RIP-LCMV-NP mice [87]. Moreover, the help by CD4+Th cells is not necessary, because depletion of CD4+T cells does not change the severity of the disease. The fundamental reason for these differences is the frequency of circulating high-avidity cytotoxic T lymphocytes, which overcome a critical threshold (calculated as > 1%) in the LCMV-GP mice but not in the LCMV-NP mice. This frequency is clearly affected by central tolerance, massively intervening in the LCMV-NP mice expressing the target protein in the thymocytes, but not in the others[87]. Structural modifications introduced in the GP peptide have shown

(29)

25

that the affinity of the TCR-MHC/GP peptide complex directly correlates with the production of inflammatory cytokines (IFN-γ) and with cytotoxicity, both indicating organ damage. So, a reduced number of T cells assuring strong interaction with APCs leads to the development of milder diabetes in the LCMV-NP animals, due to low-avidity T cells left by central tolerance mechanisms which on the contrary effectively deleted the majority of high-avidity T cells. It is worth specifying that in this context the term “affinity” is properly referred to the strength of the chemical binding between the TCR and the MHC/peptide complex, whereas the term “avidity” refers to the global strength of such interaction which results from the additive effect of affinity and other factors, such as the early synthesis of important molecules of the TCR signalling cascade, increasing T cell responsiveness to activation.

The LCMV-NP model has been used to elucidate the role of molecular mimicry in

multiple infections, which probably reflects the real situation for the development

of autoimmune diseases in humans: when the LCMV-NP mice are infected with the LCMV virus bearing a subdominant epitope of the nucleoprotein, or with a different virus (Pichinde virus) bearing a nucleoprotein sharing an epitope very similar but not identical to that epitope, only slow onset diabetes occur (very slow in the Pichinde virus-infected mice), because the frequency of anti-LCMV-NP specific CTLs is only around the 1% threshold. However, when Pichinde virus infection is induced

after LCMV-NP infection, type 1 diabetes develops very rapidly, i.e. within only 1

week after the second infection, and with more severity. Such experiments demonstrate that molecular mimicry can accelerate an underlying autoimmune process by expanding the CTL repertoire, and this may occur by multiple rather than single infections, reflecting the expansion of specific T memory autoimmune cells.

Another piece to the puzzle is added by a murine model of autoimmune hepatitis type 2 (AIH-2), in which wild-type FVB animals expressing their own cytochrome P450 isoenzymes structurally mimicking the main antigen in AIH-2 (the human cytochrome P450 2D6 (CYP2D6) enzyme), are infected with an adenovirus expressing CYP2D6 as a trigger. These mice develop a persistent and aggressive autoimmune hepatitis in 2-4 weeks after the infection, with high titer of anti-CYP2D6 autoantibodies and specific T cells, along with profound liver damage. In

(30)

26

contrast with RIP-LCMV models, transgenic mice expressing CYP2D6 autoantigen develop only slow and mild hepatitis, with low autoantibody titer and reduced amount of anti- CYP2D6 CD8+ T cells. This model emphasizes the fact that molecular mimics of autoantigens may generate cross-reactive autoantibodies and autoreactive T cells that escape central and peripheral tolerance mechanisms more easily than autoreactive lymphocytes elicited by molecular identity. This is important because confirms an observation made on other models, i.e. that high-avidity, self-reacting T cells, which are more probable elicited by target-trigger molecular identity, are not always required for autoimmunity development, because they are more often eliminated or made functionally inert by anergy [87].

Molecular mimicry with Epstein-Barr virus peptides has been hypothesized in SLE for self-antigens recognized by some autoantibodies of the ANA (AntiNuclear Antibodies) class, which is the most represented in SLE, such as Anti-Sm (autoantibodies towards the Sm antigen of the pre-mRNA splicesoma), anti-Ro/SSA (autoantibodies towards the small Ro ribonucleoprotein complex, RoRNP) and anti-dsDNA antibodies [88]. For example, autoantibodies targeting the SmD ribonucleoprotein (anti-SmD) isolated from sera of SLE patients bind an epitope of the EBNA-1 protein of Epstein-Barr virus (the replication factor that maintains the viral genome in the latency phase of the infection). This epitope spans the EBNA-1 molecular trait between aminoacids 35-58, whose sequence is highly homologous to that of the peptide 95-119 of the SmD C-terminal region, and it acts as immunogen when administered to mouse models of SLE. Sera from immunized mice cross-react with non-self EBNA-1 and self SmD antigens, suggesting that infection with EBV virus could have a role in inducing SLE via molecular mimicry [89]. Other observations bring into play EBV in SLE pathogenesis: such as the resemblance of the latency and reactivation phases of EBV infection with the remission-and-flares course of SLE, the presence of similar symptoms in SLE and infectious mononucleosis, the detection of high titers of IgA antibodies against the viral EBNA-1 and EBV-VCA antigens in SLE patients with respect to controls [90]. On the other hand, this virus is equally present in the same proportion (95-99%) in the SLE and normal population, and no infectious agents have been identified as causes of SLE so far. It is thus evident that pathogens are only a part of the burden of environmental factors that together with individual susceptibility may lead to the

(31)

27

expression of the disease.

1.8 CELLULAR AND HUMORAL IMMUNE DYSREGULATIONS IN SLE

1.8.1 Cell death as a source of autoantigens

The key feature of lupus is the production of high titers of pathogenic IgG antibodies specific for nuclear antigens such as nucleic acids (single- stranded/ss-RNA, double- stranded/dsDNA), nuclear proteins or complexes among them (histones, nucleosomes, Smith antigens SmB and SmD), some of them arising years before disease onset. Normally, self nuclear antigens are present in the circulation as a result of cell turnover, infections or mechanical or physical stress, but cellular debris are removed in short time by phagocytic cells. If this clearance process is impaired, cellular self-components can remain available to autoreactive T and B cells in such amount and for enough time to be recognised and trigger inflammatory pathways.

In SLE, both cell death and clearance processes have been found to be altered in murine models and in patients: a mouse model of non-spontaneous lupus is created by injection of pristane (2,6,4,14-tethramethylpentadecane) intraperitoneally in BALB/c and C57BL/6 mice, causing a lupus-like inflammatory disease with severe nephritis, arthritis, high levels of type-1 IFN and ANAs [56] associated with high levels of dead cells in the bone marrow. Similarly, bone marrow samples from SLE patient with nephritis and anemia show an abnormal amount of dead cells and neutrophils with phagocytosed nuclear material, the so-called “LE cells” [91]. Moreover, sera from SLE patients but not from patients with other autoimmune diseases (RA, systemic vasculitis) or infectious diseases are capable of inducing apoptosis in monocytes and lymphocytes from healthy controls and in cell lines independently from disease activity [92].

Cell death can occur by apoptosis, necrosis or NET-osis, as reviewed in [93]. Apoptosis usually does not cause inflammation because during this process the cell membrane integrity remains, and apoptotic cells secrete chemokines to attract phagocytic cells (CX3CL1) and to keep out neutrophils (lactoferrine). Phagocytes bind apoptotic cells through their receptors specific for membrane molecules such as phopsphatidilserine, whose translocation to the outer layer of the apoptotic cell

Specificity of marker autoantibodies in Systemic Lupus Erythematosus: autoantibodies detected in SLE patients by a novel DNA/histone H4 peptide complex

CONTENTS

GENERAL INTRODUCTION

CHAPTER 1

SYSTEMIC LUPUS ERYTHEMATOSUS