Introduction, Epidemiology and New Insights into the Pathogenesis of Lupus

#### **Chapter 1**

## Introductory Chapter: Recent Advances in Systemic Lupus Erythematosus

*Sophia Lionaki and Ioannis Boletis*

#### **1. Introduction**

Systemic lupus erythematosus is the prototype of autoimmune disorder, which is typically manifesting in multiple organ systems and running a chronic course, affecting primarily females. It is associated with significant public health impact in affected individuals, with highly heterogenous presentation and progression. During the last two decades, substantial progress has been made in our knowledge on systemic lupus erythematosus incidence, pathogenesis, therapeutic interventions, and long-term outcomes. However, it remains a challenging area of research, especially considering the genetic, epigenetic, and environmental factors that have been found to play a crucial role in disease prognosis [1]. Estimates on the worldwide incidence and prevalence of lupus revealed that the highest incidence was found in North America, while the lowest rates have been reported in Northern Australia [2]. Interestingly, a registry from the island of Crete reported that the overall age-adjusted/sex-adjusted incidence is growing among males [3]. Factors such as age, gender, ethnicity, genetics, hormonal status and environmental factors appear to have a central function in the development of the disease [2].

#### **2. The new era of systemic lupus erythematosus**

Despite the significant improvements which have been achieved in the field of lupus, including the overall management and immunosuppressive agents used for therapy, mortality rates of affected patients remain three times higher than those in the general population [2]. In particular, patients with lupus nephritis, who end up in end-stage kidney disease, inquire a huge burden of morbidity, related not only to the dialysis procedure but also to the inflammatory background, the impact of cumulative immunosuppression, and the phenomenon of accelerated atherosclerosis which results in cardiovascular death [4]. Still the performance of each patient is variable. For instance, neurologic and psychiatric manifestations of systemic lupus erythematosus appear to have an increasing rate in recent reports although they are found in different frequencies across lupus cohorts, depending on the methodology used to define the related signs/symptoms and the screening practice [5]. Likewise, family planning becomes a crucial problem for women with systemic lupus erythematosus, considering the fact that females of reproductive age are the most frequently affected patients. Pregnancies in patients with active lupus and especially in those with renal involvement have been associated with significant morbidity and mortality for both the mother and the fetus [6]. Moreover, the interplay between

environmental factors and the genetic profile of each individual appear to be of great importance with respect to the onset and the progression of this disorder [7, 8]. Given these circumstances, we consider systemic lupus erythematosus a challenging field of research, which enquires continuing updating in order to illustrate all current knowledge regarding disease pathogenesis and provide guidelines for clinical practice employing all newer immunosuppressive agents.

### **Author details**

Sophia Lionaki\* and Ioannis Boletis Department of Nephrology and Transplantation, Faculty of Medicine, Laiko Hospital, National and Kapodistrian University of Athens, Athens, Greece

\*Address all correspondence to: sofia.lionaki@gmail.com

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Introductory Chapter: Recent Advances in Systemic Lupus Erythematosus DOI: http://dx.doi.org/10.5772/intechopen.88226*

#### **References**

[1] Parks CG, de Souza Espindola Santos A, Barbhaiya M, Costenbader KH. Understanding the role of environmental factors in the development of systemic lupus erythematosus. Best Practice & Research. Clinical Rheumatology. 2017;**31**(3):306-320

[2] Stojan G, Petri M. Epidemiology of systemic lupus erythematosus: An update. Current Opinion in Rheumatology. 2018;**30**(2):144-150

[3] Gergianaki I, Bortoluzzi A, Bertsias G. Update on the epidemiology, risk factors, and disease outcomes of systemic lupus erythematosus. Best Practice & Research. Clinical Rheumatology. 2018;**32**(2):188-205

[4] Mageau A, Timsit JF, Perrozziello A, Ruckly S, Dupuis C, Bouadma L, et al. The burden of chronic kidney disease in systemic lupus erythematosus: A nationwide epidemiologic study. Autoimmunity Reviews. Jul 2019;**18**(7):733-737. pii: S1568-9972(19)30114-4

[5] Tani C, Palagini L, Moraes-Fontes MF, Carli L, Mauri M, Bombardieri S, et al. Neuropsychiatric questionnaires in systemic lupus erythematosus. Clinical and Experimental Rheumatology. 2014;**32**(5 Suppl 85):S-59-S-64

[6] Nahal SK, Selmi C, Gershwin ME. Safety issues and recommendations for successful pregnancy outcome in systemic lupus erythematosus. Journal of Autoimmunity. 2018;**93**:16-23

[7] Javinani A, Ashraf-Ganjouei A, Aslani S, Jamshidi A, Mahmoudi M. Exploring the etiopathogenesis of systemic lupus erythematosus: A genetic perspective. Immunogenetics. 2019;**71**(4):283-297

[8] Patrias K. Citing Medicine: The NLM Style Guide for Authors, Editors, and Publishers [Internet]. Bethesda, MD: National Library of Medicine (US); 2007. [Updated: 2 October 2015; Cited: 13 October 2016]. Available from: http://www.nlm.nih.gov/citingmedicine

#### **Chapter 2**

## Epidemiology of Systemic Lupus Erythematosus

*Masakazu Washio, Chikako Kiyohara and Akiko Ohta*

#### **Abstract**

Epidemiology is the study of the frequency and distribution of diseases and factors related to the development of diseases. Systemic lupus erythematosus (SLE) is a rare, chronic inflammatory autoimmune disease that affects many tissues and organs, whose female-to-male incidence ratio is 6:10 for childbearing age. Its chronic intractable nature has a significant impact on medical care utilization, activities of daily living, and quality of life. However, the etiology of SLE has not yet been elucidated in detail, although genetic factors as well as environmental factors are thought to play a role in its development. In this chapter, we introduce the incidence and the prevalence of SLE as well as factors related to the development of SLE and discuss how to prevent the development of SLE.

**Keywords:** SLE, epidemiology, incidence, prevalence, risk factor

#### **1. Introduction**

Systemic lupus erythematosus (SLE) is a rare, serious, chronic inflammatory autoimmune disease that affects many tissues and organs [1, 2]. The Japanese Ministry of Health and Welfare designated SLE as an intractable disease because there is no established way to cure or prevent it [3, 4]. Under a nationwide registration system for patients with intractable diseases, 55,021 SLE patients were eligible for financial aid from the Japanese government in 2007 and the prevalence of SLE was estimated to be 44 per 100,000 persons in Japan [5]. Females are 8.2 times more likely to suffer from SLE than males in Japan [5]. Serdula and Rhoads [6] reported that the age-adjusted prevalence of SLE was greater in Japanese (18.2/100,000 persons) than White People (5.8/100,000 persons) in Hawaii, but they could find no reason for the high prevalence of SLE in Japanese ancestry. The etiology of SLE has not yet been elucidated in detail, although genetic factors as well as environmental factors are thought to play a role in its development [1]. The discrepancies of rates (i.e., higher rates in certain ethnic groups) are in part due to genetic factors as well as due to environmental factors such as smoking and dietary habits [7].

In this chapter, we would like to show the incidence and prevalence of systemic lupus erythematosus (SLE) and the findings from epidemiological studies on the risk/preventive factors for SLE.

#### **2. Diagnosis criterion of SLE (case definition)**

The established diagnosis criterion of SLE is needed to estimate the frequency and distribution of the patients with SLE. However, case definition is one of the

important factors, which may influence the results of epidemiological studies. Currently, the American College of Rheumatology (ACR) 1982 revised criteria for the classification of SLE [8], as modified in 1972 (ACR-97) [9], are widely used for the diagnosis of SLE. The diagnosis of SLE requires the presence of four or more of the following 11 criteria, which are (1) malar rash, (2) discoid rash, (3) photosensitivity, (4) oral ulcer (usually painless, observed by a physician), (5) arthritis (nonerosive arthritis 2 or more peripheral joints), (6) serositis (a. pleuritis or b. pericarditis), (7) renal disorder (a. persistent proteinuria either 0.5 g/day or > 3+ if quantification not performed or b. cellular cast), (8) neurologic disorder (a. seizures or b. psychosis in the absence of offending drugs or metabolic disorders), (9) hematologic disorder (a. hemolytic anemia with reticulocytosis or b. leukopenia <4000/mm3 or c. lymphopenia <1500 mm3 or d. thrombocytopenia <100,000 mm3 in the absence of offending drugs), (10) immunologic disorder (a. antibody to native DNA in abnormal titer or b. presence of antibody to Sm nuclear antibody or c. positive finding of antiphospholipid antibody), and (11) positive antinuclear antibody test result. Although the presence of four or more ACR-97 criteria is required for SLE classification, all other reasonable diagnoses of diseases other than SLE (e.g., neurologic disorder due to uremia, acidosis, or electrolyte imbalance) must be excluded [7]. Among the 11 ACR criteria, positive antinuclear antibody test result, hematologic disorder, immunologic disorder, and arthritis are the four most common criteria seen in SLE patients at the time of diagnosis [10–13] (**Table 1**).

When epidemiological studies are conducted based on the rheumatologist definition, biopsy-proven lupus nephritis patients may be considered to have SLE even though they satisfy fewer than four ACR-97 criteria. In these cases, the rates of SLE will be greater than the rates based on the ACR-97. Recently, the Systemic Lupus International Clinics (SLICC), which is an international group for the clinical research of SLE, presented a new criterion for the classification of SLE in 2012 (SLICC-12) [14]. They also validated the ACR-97 and the SLICC-12. The SLICC-12 resulted in fewer misclassification than the ACR-97 [14]. Compared with the ACR-97, the SLICC-12 had greater sensitivity but less specificity [14, 15]. The SLICC case definition of


#### **Table 1.**

*Distribution of clinical manifestation and laboratory findings at the diagnosis of SLE.*

*Epidemiology of Systemic Lupus Erythematosus DOI: http://dx.doi.org/10.5772/intechopen.84146*

SLE yielded higher incidence and prevalence estimates than the ACR-97 case definition [15]. Thus, the incidence and prevalence of SLE are influenced by the diagnosis criterion of SLE. Therefore, interpretation of incidence and prevalence of SLE also take into account differences in the methodology used to determine these rates.

#### **3. Incidence and prevalence of SLE**

In the United Kingdom, Rees et al. [16] found that the incidence and prevalence of SLE in White People (6.73/100,000 person-years and 134.5/100,000 persons) were smaller than those in other ethnic groups such as Black African (13.78/100,000 person-years and 179.8/100,000 persons), Black Caribbean (31.46/100,000 personyears and 517.5/100,000 persons), and Indian (9.9/100,000 person-years and 193.1/100,000 persons) (**Table 2**). In addition to the United Kingdom, American epidemiologists also reported that the incidence and prevalence of SLE in White


**Table 2.**

*Incidence and prevalence of SLE by ethnic group in the United Kingdom/the United States.*


*Epidemiology of Systemic Lupus Erythematosus DOI: http://dx.doi.org/10.5772/intechopen.84146*



#### **Table 3.**

*Incidence and prevalence of SLE in females and males in selected countries.*

People were smaller than those in other ethnic groups in the United States [6, 12, 17, 18] (**Table 2**). The disease burden of SLE is highest in Black People [17, 18], followed by Asian/Pacific islanders [17] and White People in the United States [17, 18], which may be related to genetic and environmental factors.

As shown in **Table 3**, the incidence and prevalence of SLE are greater in females than in males in all studies regardless of ethnic group or countries [11–13, 16–27]. Age-adjusted incidence of SLE in females was 8.8–14.5 times greater than in males in California, the United States (i.e., 12.3-fold female excess in all population, 8.8-fold female excess in White People, 14.5-fold female excess in Black People, and 12.0-fold female excess in Asian/Pacific islanders) [18], while the age-adjusted incidence of SLE in females was 7.8–14.8 times greater than that in males in East Asia (i.e., 7.8-fold female excess in Taiwan [23], 8.5-fold female excess in South Korea [24], and 14.8-fold female excess in Japan [27]).

SLE is more common in women than men across all age groups, and this female predominance is especially noteworthy in the 15- to 64-year age group, wherein the male-to-female ratios of age-group incidence show a 6- to 10-fold female excess [28], which suggests that female sex hormones may play an important role in the development of SLE [28]. The Nurses' Health Study [29] revealed that oral conceptive use increased the risk of SLE in the United States, whereas Bernier et al. [30] reported that it was not past use but current use of oral contraceptive pills that increased the risk of SLE in the United Kingdom. These studies [29, 30] also suggest that female sex hormones such as estrogen may play an important role in the development of SLE. In addition to sex hormones, both X-linked and autosomal immune genes are also regulated epigenetically and likely contribute to the sex difference in the incidence of SLE [31].

#### **4. Factors related to the development of SLE**

Although genetic factors are suggested to play an important role in the development of SLE, nongenetic factors are also suggested to play a role in the development of SLE [1, 7]. In addition to genetic susceptibility, hormonal and reproductive exposures (e.g., endogenous estrogens, estrogen replacement therapy), occupational and environmental exposures (e.g., silica, ultraviolet light), and infectious exposures (e.g., Epstein-Barr virus) are suggested to influence the risk of SLE [1, 7]. Complex interactions between genetic and environmental factors are thought to play a role in the development and progression of SLE [7].

#### **4.1 Sex hormones and reproductive issues in females**

The incidence of SLE is greater in females than in males in all studies regardless of ethnic group or countries [11–13, 16–27]. Although SLE occurs predominantly in females, the incidence of SLE is low before puberty and after menopause (i.e., outside the reproductive ages) [32]. Sex difference in susceptibility is largest during the reproductive ages [33], which suggests that high endogenous estrogen concentrations may increase the risk for the development of SLE. Estrogens enhance B cell activation (e.g., immunoglobulin production including anti-ds-DNA), while they suppress T cell activity (e.g., proliferative response to mitogens and antigens, interleukin 2 production) [32].

Costenbader et al. [29] reported that menarche at a younger age (10 years old or younger vs. 12 years old: RR 2.1, 95% CI = 1.4–3.2) increased the risk for the development of SLE in the NHS 1976–2002 and the NHSII 1989–2003. In addition, they also reported that age at menarche was inversely associated with a risk for the development of SLE (vs. 12 years old: RR = 2.1 for 10 years or younger, RR = 1.2 for 11 years old, RR = 1.0 for 12 years old, 1.1 for 13 years old, and RR = 1.1 for 14 years old, and RR = 1.0 for 14 years old or older, p for trend = 0.02) [29]. These findings suggest that the exposure to high concentrations of endogenous estrogen at early age may increase the risk for the development of SLE.

On the other hand, Bernier et al. [30] reported that current use of combined oral contraceptives increased the risk of SLE (RR 1.54, 95% CI = 1.15–2.07), but past use of combined oral contraceptives did not increase the risk (RR 1.06, 95% CI = 0.85–1.33). In addition, they also reported that the risk of SLE increased with the dose of ethinyl estradiol (vs. nonusers: RR 1.42 for 30 μg or less, RR 1.63 for 31–49 μg, and RR 2.92 for 50 μg), while Costenbader et al. [29] reported that use of oral conceptive (vs. never: RR 1.5, 95% CI = 1.1–2.1) and use of postmenopausal hormones (vs. never: RR 1.9, 95 % CI = 1.2–3.1) increased the risk for development of SLE in the Nurses' Health Study. These findings suggest that use of exogenous estrogens may increase the risk for the development of SLE.

Costenbader et al. [29] also reported that postmenopausal women primary after surgical menopause (vs. premenopausal: RR 2.3, 95% CI = 1.2–4.5) and early age of menopause (younger than 47 years old vs. 53 years old and older: RR 2.2, 95% CI = 0.9–5.4) showed an increased risk for the development of SLE. In their study, most of females who developed SLE after menopause were those with surgical menopause (i.e., bilateral oophorectomy) and were more likely to have taken postmenopausal hormones [29]. The increased risk of developing SLE among postmenopausal females in their study may be partly explained by the use of postmenopausal hormones (RR 1.9, 95% CI = 1.2–3.1) [29] and the surgery (vs. no surgery: surgery without blood transfusion: OR 1.54, 95% CI = 1.05–2.26; surgery with blood transfusion: OR 4.46, 95% CI = 1.99–10.00) [34].

Ulff-Møller et al. [35] reported that live birth showed a decreased risk of SLE among Danish females (RR 0.74, 95% CI = 0.64–0.86), while Washio et al. [34] reported that live birth (OR 0.23, 95% CI = 0.09–0.59) decreased the risk of SLE and found a positive association between the risk of SLE and the number of living children delivered among Japanese females (vs. 0; OR 0.27 for one to two children, and OR 0.14 for three or more children, p for trend <0.01). On the other hand, Cooper et al. [36] could not find any meaningful association between the risk of SLE and number of live births. However, they found that breast-feeding was associated with a decreased risk of SLE (OR 0.6, 95% CI = 0.4–0.9) among females in the United States [36]. These findings suggest that lactation may play an important role in reducing the risk of SLE among women with live-born children because serum estrogen levels are usually at or below the lower range for the early follicular phase of the normal menstrual cycle during the lactation [37].

#### **4.2 Tobacco smoking and alcohol drinking**

Several researchers suggested that smoking increased the risk of SLE [38–42]. Ghaussy et al. [39] reported a significantly increased risk of SLE in both current and former smokers compared with never smokers (current smokers: OR 6.69, 95% CI = 2.59–17.28, former smokers: OR 3.62, 95% CI = 1.22–10.70) in the United States. On the other hand, others reported no association with smoking history (i.e., current, former, or never-smoker) and the risk of SLE in the United States [43, 44]. A metaanalysis by Costenbader et al. [45] revealed an increased risk of SLE among current smokers compared with nonsmokers (summary OR 1.50, 95% CI = 1.09–2.08).

The Kyushu Sapporo SLE study (i.e., the KYSS Study) was a hospital-based casecontrol study to evaluate nongenetic and genetic risk factors for the development of SLE among Japanese females [42]. All SLE patients fulfilled the American College of Rheumatology 1982 revised criteria for SLE [8]. In the KYSS study, Kiyohara et al. [46] reported that (1) compared with nonsmokers, smokers showed an increased risk of SLE (vs. nonsmokers: OR 2.49 for former smokers, and OR 3.06 for current smokers, p for trend <0.01). In addition, the risk of SLE increased with number of cigarettes smoked/day during peak smoking period (vs. 0/day: OR 2.77 for 1–19/day, and OR 3.29 for 20+/day, p for trend<0.01) [46]. Since hydrazine, a drug containing aromatic amines, is a known inducer of SLE [47], aromatic amines in cigarette smoke may partly explain the association between smoking and the risk of SLE.

Some studies suggested that alcohol consumption may decrease the risk of SLE [38, 40, 41]. Hardy et al. [38] reported a dose-response negative association between alcohol drinking and SLE risk (vs. 0 unit of alcohol: OR 0.73 for 1–2 units, OR 0.41 for 3–5 units, OR 0.47 for 6–10 units, and OR 0.30 for more than 10 units, p for trend <0.01). On the other hand, other studies failed to show an inverse association between alcohol drinking and SLE risk [37, 40]. A meta-analysis by Wang et al. [48] demonstrated that moderate alcohol drinking might have a protective effect on the development of SLE (vs. none: summary OR 0.73, 95% CI = 0.547–0.954). In the KYSS study, Kiyohara et al. [46] found a U-shape relationship between alcohol consumption and SLE risk among Japanese females (vs. 0 ml/week: OR 0.52, 95% CI = 0.31–0.86 for 1–70 ml/week, OR 0.38, 95% CI = 0.19–0.76 for 71–210 ml/week, and OR 0.67, 0.31–1.46 for 211 ml/week or more). These findings suggest that light to moderate alcohol consumption may decrease the risk of SLE.

Although there are potential biases associated with retrospective assessment of exposures and selection of cases and controls in a case-control study [49], Kiyohara et al. [46] reported that ever-smokers with drinking alcohol (OR 3.44, 95% CI = 2.03–5.82), nonsmokers without drinking alcohol (OR 2.56, 95% CI = 1.57–4.17), and ever-smokers without drinking alcohol (OR 6.98, 95% CI = 2.87–17.0) showed a greater risk of SLE than nonsmokers with drinking alcohol in Japanese women.

#### **4.3 Occupational exposures and chemicals**

Crystalline silica exposure is known to increase the risk of SLE [50, 51]. Finckh et al. [52] reported that exposure to silica for more than 1 year increased the risk of SLE (OR 4.3, 95% CI = 1.7–11.2). They also reported that the risk of SLE was associated with the duration of exposure to silica (vs. less than 1 year: OR 4.0 for 1–5 years, and OR 4.9 for more than 5 years, p for trend = 0.01) [52]. Parks et al. [53] reported a positive relationship between a history of silica exposure and SLE risk (vs. none: OR 1.6 for low, and OR 3.1 for medium or high, p for trend = 0.003).

On the other hand, Cooper et al. [54] reported that occupational silica exposure increased the risk of SLE among never-smokers (vs. no-silica exposure: OR 2.6, 95% CI = 1.2–5.7) but not among ever-smokers (vs. no-silica exposure: OR 0.99, 95% CI = 0.46–2.1), which suggests that smoking may play a more important role in the development of SLE than silica exposure.

Cooper et al. [43] reported that any use of permanent dyes increased the risk of SLE (OR 1.5, 95% CI = 1.0–2.2) in the United States. On the other hand, Sanchez-Guerrero et al. [44] failed to find a positive association between use of permanent hair dye and SLE risk (ever-users vs. never-users: OR 0.96, 95% CI = 0.63–1.47) in the Unites States.

#### **4.4 Ultraviolet radiation exposure**

Washio et al. [42] reported that walking increased the risk of SLE in Kyushu, southern Japan with a temperate climate (30 min/day or more vs. less than 30 min/day: OR 2.07, 95% CI = 1.14–3.76) but failed to increase the risk of SLE in Hokkaido, northern Japan with a subarctic climate (30 min/day or more vs. less than 30 min/day: OR 1.13, 95% CI = 0.46–2.79). In this study, walking may be a surrogate of staying outdoors with exposure to strong sunlight [42]. On the other hand, Cooper et al. [54] reported that outdoor work in the 12 months preceding diagnosis (OR 2.0, 95% CI = 1.0–3.8) increased the risk of SLE. In their study, a larger variation in the association between outdoor work and SLE risk was seen when examined within categories of sun reaction to midday sun (vs. none; OR 0.75 for tan or darken without burning, OR 2.7 for sunburn, and OR 7.9 for sunburn with blistering or rash) [54]. However, it is controversial whether ultraviolet (UV) radiation exposure itself plays a role in the development of SLE although UV radiation exposure may exacerbate preexisting SLE [50].

#### **4.5 Family history**

Family history of SLE [40, 55] as well as family history of connective tissue diseases/autoimmune diseases [40, 41, 55] is reported to increase the risk of SLE. Alarcón-Segovia et al. [56] reported that there was familial aggregation of SLE and of RA in SLE patients. These findings suggest that predisposing genes of autoimmune diseases as well as environmental risk factors sharing in family members may play a role in the development of autoimmune diseases including SLE.

#### **4.6 Genetic susceptibility**

It is widely accepted that SLE development requires environmental factors acting on a genetically predisposed individual. Studies of twin concordance are commonly used in epidemiology to estimate the role of genetics and the influence of environmental factors on disease susceptibility. Disease concordance is much higher in monozygotic twins (24–57%) than in dizygotic twins (2–5%),

suggesting that a genetic factor may play a role in the development of SLE [57, 58]. The genetic basis of SLE is very complex; it has been estimated that over 100 genes may be involved in SLE susceptibility [59], but it is difficult to predict how many genes contribute to SLE susceptibility. Exposure to reactive oxygen species (ROS) via cigarette smoking is thought to contribute to the development of SLE. ROS is considered to promote the autoimmune response [60]. The cytochrome P450 (CYP)1A1 and glutathione S-transferase (GST) M1 enzymes are critical for the functionalization of genotoxic substances in cigarette smoke. The CYP1A1 enzyme contributes to the phase I metabolic activation and formation of ROS, whereas the GSTM1 enzyme plays a critical role for phase II detoxification of activated carcinogens or ROS [61, 62]. Extensive studies have been performed on the possible associations between polymorphisms of *CYP1A1* and *GSTM1* and cancer susceptibility [63–65]. Similarly, the N-acetyltransferase (NAT) enzyme is involved in the metabolism and detoxification of cytotoxic and carcinogenic compounds as well as ROS [66]. It has been suggested that N-acetylation of polycyclic aromatic hydrocarbons (PAHs) by the NAT2 enzyme may be associated with ROS production [67]. ROS increase immunogenicity of DNA, LDL, and IgG, generating ligands for which autoantibodies show higher avidity [60]. Tumor necrosis factor r superfamily member 1B (TNFRSF1B) is a receptor for TNF-α and is considered to mediate various biological effects including generation of ROS and the subsequent intracellular proinflammatory signaling events [68]. Furthermore, cigarette smoking has been suggested to influence TNFRSF1B production [69, 70]. Representative functional polymorphisms of the *CYP1A1*, *GSTM1*, *NAT2*, and *TNFRSF1B* genes are *CYP1A1* rs464903, *GSTM1* deletion, *NAT2* genotypes determined by *NAT2*\*4, \*5B, \*6A, or \*7B allele and *TNFRSF1B* rs1061622. Considering that exposure to ROS via cigarette smoking may be contributed to the development of SLE, it is important to study the association between SLE and the polymorphisms involved in metabolism of tobacco smoke and ROS production. We conducted candidate gene association studies (hypothesis-driven approach) of SLE in female Japanese subjects with special reference to the interaction between the polymorphisms involved in ROS production and cigarette smoking [71–74]. *CYP1A1* rs4646903 (OR of the CC genotype = 2.47, 95% CI = 1.28–4.78) [71] and *NAT2* genotypes (OR of the intermediate acetylator and slow acetylator genotypes combined = 2.34, 95% CI = 1.36–4.02) were significantly associated with SLE risk [72]. *TNFRSF1B* rs1061622 was marginally associated with an increased risk of SLE (OR of the G allele possession = 1.56, 95% CI = 0.99–2.47) [71]. There were significant additive interactions between smoking and any one of the following: *CYP1A1* rs4646903, *NAT2*, or *TNFRSF1B* rs1061622 [72–74]. Replication of findings is very important before any causal inference can be drawn. Testing replication in different populations is an important step. Future studies involving larger control and case populations, precisely and uniformly defined clinical classification of SLE and better exposure histories, will undoubtedly lead to a more thorough understanding of the role of the genetic polymorphisms involved in ROS production in SLE development.

#### **5. Applications of findings in the epidemiological studies**

Descriptive epidemiologic studies of SLE have been conducted not only in the Western countries (e.g., the United Kingdom, France, the United States, Canada) but also in Asian countries (e.g., China, South Korea, Japan). The prevalence of SLE provides useful information for the needs of health services for SLE patients. Information of the age- and sex-specific incidence and prevalence of SLE can be used to estimate the number of newly diagnosed SLE patients and the total number

#### *Epidemiology of Systemic Lupus Erythematosus DOI: http://dx.doi.org/10.5772/intechopen.84146*

of SLE patients in a community whose age and sex structure is known. On the other hand, the discrepancies of rates between different groups (e.g., different ethnic groups in the same country, different countries), which may be partly due to genetic factors as well as due to environmental factors [6], may give epidemiologists clues to plan epidemiological studies to determine a risk factor for SLE.

Observational studies such as case-control studies and cohort studies have been conducted to determine factors related to the development of SLE (i.e., risk factors, preventive factors) [49, 75]. After determining risk factors, preventive action will be started to control the level of exposure to a risk factor for SLE (i.e., reducing the risk of SLE) as well as to undergo a medical examination for the early detection of SLE for persons who are at special risk (e.g., silica [50–54]) (i.e., high risk strategy [76]). The size of relative risk/odds ratio indicates the strength of association between an exposure and a risk of SLE. For a public health perspective, however, the attributable risk of SLE is more important than the relative risk. The attributable risk is the difference in the risk of SLE between the exposed and the unexposed persons [49, 75]. The population attributable risk is the incidence of SLE in a population that is associated with an exposure to a risk factor, which is useful for determining the relative importance of exposures for the entire population [49, 75]. When the proportion of exposed persons is large, the population attribute risk is high even if the relative risk is small. More cases of SLE may develop in a large number of persons who are at a small risk than in the small number who are at high risk.

Smoking is an avoidable risk factor for SLE [38–42, 45] as well as for cancer [77] and cardiovascular diseases [78]. Therefore, antismoking education for both smokers and nonsmokers throughout lifetime (i.e., population strategy [76]) is important to reduce the incidence of SLE as well as the incidence of cancer and cardiovascular diseases in the general population.

#### **6. Summary**

The incidence and prevalence of SLE vary with sex, age, ethnicity, and the way how to detect SLE patients (e.g., case definition). SLE is more common in women than men across all age groups, and this female predominance is especially noteworthy during the reproductive ages [28], which suggests that female sex hormones may play an important role in the development of SLE.

A lower incidence and prevalence of SLE has been constantly observed in White People than in Black People [12, 17, 18] as well as Asian/Pacific Islanders [6, 17, 18] in the United States, while the incidence and prevalence of SLE is lower in White People than in Black African, Black Caribbean, and Indian [16]. The discrepancies of rates between ethnic groups are in part due to genetic factors as well as due to environmental factors such as smoking and dietary habits [7].

There are worldwide differences in the incidence and prevalence of SLE [79]. In addition to genetic factors and environmental factors, the way to detect SLE patients (e.g., case definition) is an important factor, which influences the incidence and prevalence of SLE. Ighe et al. [15] reported that the SLICC case definition of SLE yielded higher incidence and prevalence estimates than the ACR-97 case definition.

In this chapter, we introduce factors related to the development of SLE as well as incidence and prevalence of SLE. Among the reproductive issues, menarche at a younger age [29], use of contraceptive [29, 30], and use of postmenopausal hormones [29] increase the risk of SLE, while breast-feeding is associated with a decreased risk of SLE. Among environmental factors, tobacco smoking increases the risk of SLE [38–42, 46], while light to moderate alcohol drinking decreases

the risk of SLE [46]. On the other hand, the exposure to crystalline silica [50, 51], silica [52, 53], strong sunlight [42, 54], and ultraviolet radiation [50] increase the risk of SLE. Among genetic factors, *CYP1A1* rs4646903 and *NAT2* genotypes are associated with an increased risk of SLE, while *TNFRSF1B* rs1061622 is suggested to increase the risk of SLE [71–74]. In order to reduce the risk of SLE, we should reduce the exposure to avoidable risk factors such as smoking, contraceptives, crystalline silica, silica, strong sunlight, or ultraviolet radiation.

### **Author details**

Masakazu Washio1 , Chikako Kiyohara<sup>2</sup> \* and Akiko Ohta3

1 Department of Community Health and Clinical Epidemiology, St. Mary's College, Kurume City, Fukuoka, Japan

2 Department of Preventive Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka City, Fukuoka, Japan

3 Division of Public Health, Department of Social Medicine, Faculty of Medicine, Saitama Medical University, Moroyama-machi, Saitama, Japan

\*Address all correspondence to: chikako@phealth.med.kyushu-u.ac.jp

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Epidemiology of Systemic Lupus Erythematosus DOI: http://dx.doi.org/10.5772/intechopen.84146*

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**Chapter 3**

## A Spontaneous Mouse Model of Lupus: Physiology and Therapy

*Gabriela Tejon, Yessia Hidalgo, Maria Rosa Bono and Mario Rosemblatt*

#### **Abstract**

Spontaneous models of lupus were recognized four decades ago beginning in the early 1960s with the NZB/NZW F1 (NZB/W F1) mouse, an F1 hybrid between the New Zealand Black (NZB) and New Zealand White (NZW) mice. Although the parental strains display limited autoimmunity, the NZB/W F1 develops severe lupuslike features similar to that of human lupus patients. Here, we will address the genetic characteristics of the model and discuss its main characteristics such as the presence of serum antinuclear autoantibodies (ANA) including anti-dsDNA, mild vasculitis, and the development of immune complex-mediated glomerulonephritis. Similar to human lupus, the disease develops primarily in female mice after six months of age, with a lesser percentage and severity in male mice. The relation of this phenomenon will be examined in the context of estrogen levels. The participation of both innate and adaptive immunity will be addressed as well as the contribution of both T and B cells in the development of the clinical aspects of the disease. We will focus on the use of the model as a valuable tool for elucidating the pathogenic mechanisms of the disease, as well as its use as preclinical testing of therapeutic for human use.

**Keywords:** lupus, mouse model, histopathology, autoreactive cells and antibodies, genetics, sex

#### **1. Introduction**

Autoimmune diseases are generally defined by the existence of autoantibodies and the presence of autoreactive T and B lymphocytes. More than 80 different autoimmune disorders have been described, including systemic lupus erythematosus (SLE). Animal models of human diseases are an invaluable tool for defining pathogenic mechanisms, finding novel therapeutic targets, and testing new therapies. These models have the advantage of having a shorter lifetime, a characteristic that allows to study the full cycle of the disease and to test for the possible therapies in much shorter period. Although using animal models may have some disadvantages due to the obvious genetic and physiological differences with humans, they have been an invaluable tool to study human diseases, especially in autoimmunity. Although the exact etiology of SLE has not yet been identified, there is a consensus that numerous factors such as genetics, environment, and hormonal aspects are involved in the development of this disease. Several mouse models resemble specific elements of the human disease and have been employed to understand the cellular and genetic treats linked to SLE susceptibility. Most of them, share in common, the development of glomerulonephritis and


#### **Table 1.**

*Main mouse models used to study lupus.*

autoantibodies against autoantigens. In **Table 1**, we summarize the principal characteristics of the most extensively studied mouse strains of both spontaneous and induced murine lupus models. Additionally, there are genetically modified mouse models in which researchers inactivate, express, or overexpress a gene product or protein to recognize their single role in lupus and immunity in general such as transgenic-induced lupus and gene knockout-induced lupus [1–3]. In this chapter, we will refer in detail to the NZB/W F1 mice, which are the oldest classic spontaneous models of lupus used to study, on the one hand, the numerous susceptibility loci from which several candidate genes have emerged. Also, it has allowed to address important issues such as physiological aspects of the disease, antibody specificities, the role of antigen-presenting cells, the participation of B and T lymphocytes, and drug responses in many preclinical studies. This model was generated by the cross between the NZB and NZW strains. Both NZB and NZW display limited autoimmunity, as will be discussed here, while the NZB/W F1 hybrids develop severe lupus-like phenotypes resembling that of lupus patients. The purpose of this chapter is to summarize the contributions and significant advances in the understanding of lupus pathogenesis by the use of the NZB/W F1 murine model.

#### **2. Histopathology characteristics of NZB/W F1 mice**

In pre-autoimmune NZB/W F1 mice, *in vivo* expression of IFN-α precipitates the autoimmune process and kidney damage, leading to premature death from severe immune complex glomerulonephritis. This fact does not happen in nonautoimmune BALB/c mice. These findings support the notion that sustained IFN-α production in susceptible individuals may be sufficient to generate all the characteristics of SLE [4]. Interestingly, Liu et al. demonstrated that IFN-α accelerates murine systemic lupus erythematosus in NZB/W mice in a T cell-dependent manner [5].

The major cause of death in the NZB/W F1 female is chronic glomerulonephritis with heavy mesangial deposits before 5 months of age, tubular cast formation, proliferation of glomerular cells, prominent crescent formation, and a significant periglomerular and interstitial monocytic infiltrate. Extraglomerular renal deposits of IgG2a and C3 are present in the peritubular tissue and arterioles, and increase in frequency with age.

Diseased mice develop splenomegaly and progressive thymic cortical atrophy that begins very early in the disease and results in nearly complete loss of the thymic cortex as the disease progresses. In many mice, the loss of cortex is accompanied by medullary atrophy. Additionally, females have lymphoid hyperplasia with nodes rarely exceeding 2–3 times the average size [6].

#### **3. Serologic characteristics of NZB/W F1 mice**

Interestingly very early, it was reported that repeated administration of dsDNA or ssDNA to NZB/W F1 mice has a tolerogenic and long-lasting effect in this strain of mice that otherwise are susceptible to developing lupus [7]. Autoimmune-prone NZB mice mainly produce anti-DNA antibodies IgM and develop a mild SLE. NZB/W F1 females develop a fulminant SLE at 6–9 months associated with a decrease in IgM and an increase in anti-DNA IgG antibodies. These results helped to elucidate the role of the H-2 complex in the anti-DNA antibody production, leading to the conclusion that the production of IgG anti-DNA antibodies observed in NZB/W F1 hybrid mice is restricted to the H-2d/H-2z heterozygous mice [8].

NZB/W F1 mice present high levels of circulating autoantibodies. Antibodysecreting cells (ASCs) from these mice produce antinuclear antibody (ANA) and anti-dsDNA predominantly, the majority of them being the IgG2a and IgG3 classes [3, 5, 9]. NZB/W F1 mice also produce other extractable nuclear antigens (ENA) autoantibodies such as anti-small nuclear ribonucleoprotein (snRNP) and anti-heterogeneous nuclear ribonucleoproteins (hnRNP) [10]. All these autoantibodies form immune complexes that are deposited in different organs like liver, kidney, and skin. Moreover, Brick et al. have described the presence of anti-histone antibodies in the serum of autoimmune NZB/NZW F1 mice and in MRL/lpr mice [11]. On the other hand, dietary fat affects antibody levels to lipids and cardiolipin in autoimmune-prone NZB/W F1 mice. Antibodies to cardiolipin have been reported to play an important role in thrombus formation and an increase in the rate of abortions, both in human lupus patients and in murine lupus [12].

CD5+ B-1 cells have attracted much attention, because of their involvement in both autoimmunity and B cell-type chronic lymphocytic leukemia (B-CLL). It has been demonstrated that elimination of B-1 cells prevents autoimmune symptoms in autoimmune-prone mice [13]. CD5+ B cells seem to be the precursors of CD5- anti-DNA IgG antibody-producing B cells in autoimmune-prone NZB/W F1 mice [14]. However, whether B-1 cells in the peritoneum are generally involved in the pathogenesis of the autoimmune disease remains controversial.

#### **4. Cellular abnormalities**

Systemic lupus erythematosus (SLE) produces alterations in the organism that affect cells of the innate and adaptive immune systems. In this section, we will

summarize the modifications described in diseased NZB/W F1 mice in different immune cell populations.

#### **4.1 Dendritic cells**

Dendritic cells (DCs) are the cellular sentinels of the organism, important orchestrators of immune responses, and key components in fine-tuning the balance between tolerance and immunity.

Two major subsets of DCs are described: conventional DCs (cDCs) and plasmacytoid DCs (pDCs), although other subsets of DCs have been described from DCs generated from bone marrow cultures [15]. Tissue-derived pDCs are considered to be the major IFN-α source in SLE; however, diseased NZB/W F1 mice show an increase in the frequency and absolute numbers of both cDCs and pDCs in spleen and blood compared to healthy mice. Also, compared to healthy mice, diseased mice present alterations in both types of DCs since they display an abnormal phenotype characterized by an overexpression of the co-stimulatory molecules CD80, CD86, PD-L1, and PD-L2. Homing experiments demonstrate that DCs from lupusdiseased mice migrate preferentially to the spleen compared to DCs from control mice. This preferential recruitment and retention of DCs in the spleen are related to altered expression of different chemokine and chemokine receptors on both DCs and spleen stromal cells [16]. Recently, pDCs from spleen and bone marrow have been compared in several models of lupus-prone mice without clear results concerning the role of pDC in the development of lupus [17].

In NZB/W F1 mice, the spleen is the principal organ, where nucleosome-specific T cells are stimulated. Splenic antigen-presenting cells, including macrophages, contribute significantly to the production of autoantibodies and in the development of the disease [18]. On the other hand, anti-apoptotic molecules such as Bcl-2 inhibitors selectively kill pDCs, but not cDCs, reducing IFN-α production [19].

#### **4.2 Macrophages**

Macrophages are professional antigen-presenting cells and play an essential role in the activation of the adaptative immune response. Macrophages usually eliminate circulating apoptotic bodies and pathogens. Macrophages from diseased NZB/W F1 lupus mice have reduced phagocytic capacity. The impaired ability of resident peritoneal macrophages from lupus-prone mice to engulf apoptotic cells has been demonstrated by *in vivo* and *in vitro* cell clearance assays [20, 21]. Some studies have shown defective Fc-mediated phagocytosis by peritoneal macrophages [22] making more autoantigens available that favor an autoimmune response. In this regard, it was shown that spleen F4/80high macrophages could present autoantigen efficiently to T cells, thus giving help to autoantibody-producing B cells in lupus-prone mice [18].

F4/80high macrophages reside in healthy kidneys. In NZB/W F1, there is an increasing number of macrophages during nephritis. However, these macrophages do not show a pro-inflammatory (M1) or anti-inflammatory (M2) phenotype upon cytokine stimulation. Instead, they acquired a mixed functional phenotype that resembles gut F4/80high macrophages constitutively activated [23]. Macrophages from diseased NZB/W F1 mice differ in the expression of some inflammatory genes, chemokine receptors, and TLRs, which are consistent with their heterogeneity and variability in renal location, further supporting the idea that ineffective macrophage function may contribute to glomerulonephritis in NZB/W F1 mice.

Macrophages produce a broad array of cytokines that can affect the immune response. For example, macrophages from peritoneal cavity upon stimulation with DNA secrete high amounts of IL-6 and TNF-α [24], two cytokines that participate in B cell proliferation and function. Very early, it was reported that IL-6 secretion by peritoneal and not by spleen macrophages have an active role in the production of anti-DNA autoantibodies in NZB/W F1 mice [25].

#### **4.3 T cells**

In the NZB/W F1 lupus mice, spleen CD4<sup>+</sup> T cells exhibit an activated phenotype characterized by high expression of PD-1, CD25, CD69 and increased secretion of IFN-γ and IL-10 [16, 26]. The primary function of T cells in lupus is to help B cells in the production of autoantibodies [27], thus, avoiding the interaction between T and B cells may decrease the signs of the disease. Treatment with an anti-CD4 monoclonal antibody dramatically reduced glomerular immunoglobulin, complemented deposition, and diminished lymphocytic infiltration and vasculitis in the kidneys [28]. CD28 blockade decreased the production of anti-ds DNA autoantibody, prevented the development of lupus nephritis, and prolonged animal survival [29].

Regulatory CD4<sup>+</sup> T cells (Tregs) are essential players in the maintenance of peripheral immune tolerance. Usually, Tregs suppress the activity of specific T helper (Th) cells, but in NZB/W F1 mice, a homeostatic state of imbalance between regulatory and effector T cells is produced due to a decrease of IL-2, an essential cytokine for the maintenance of Tregs [30]. On the other hand, the levels of the adipocytokine leptin are elevated in diseased mice and correlate with the production of autoantibodies and renal disease. Although leptin can promote effector T cell responses to self-antigens, it also inhibits Treg activity [31]. On the other hand, Likuni et al. demonstrated that Tregs could directly suppress B cells in NZB/W F1 lupus mice through cell-to-cell contact-mediated mechanisms, thus directly regulating auto-antibody-producing B cells, including those B cells that increase in number during active disease [32].

Follicular helper T cells are CD4+ T cells population that supports the activation and differentiation of previously class-switched B cells to long-lived antibodysecreting plasma cells. Recent reports show that follicular helper T cells contribute to the pathogenesis of lupus through the ICOS/ICOSL pathway in NZB/W F1 mice [33]. Also, the activation through the Ox40/Ox40L pathway increases the number of follicular helper T cells and promotes cellular and humoral autoimmune responses in NZB/W F1 mice [34]. Interestingly, Cortini et al. showed that, reciprocally, B cells support the follicular helper T cells development in NZB/W F1 mice through the OX40L expression on B cells [35].

Although CD8+ T cells have not been directly implicated in SLE, sick NZB/W F1 mice show an impaired expansion of CD8+ T cells, as well as the acquisition of memory, secretion of cytokine, and suppression of autoimmunity [36].

#### **4.4 B cells**

Participation of B cells in lupus implicates several of its cellular functions. Besides the secretion of autoantibody against a panoply of antigens, B cells contribute in other ways to the pathogenesis of lupus, including antigen presentation to T cells, follicular helper T cell differentiation, and cytokine secretion. Although the phenotype of resting B cells isolated from NZB/W F1, and non-autoimmune mice do not show significant differences, B cells from lupus mice are hyper-responsive to T cell-derived stimuli *in vitro.* T cell-derived cytokines and signals delivered through CD40 crosslinking induce higher levels of proliferation, IgM secretion, and enhanced expression of costimulatory molecules in NZB/W F1 B cells [37].

B cell-activating factor (BAFF) and a proliferation-inducing ligand (APRIL) play key roles in peripheral B cell survival, maturation, and differentiation. In NZB/W F1 mice, chronic activation of the immune system induced an increase in the levels of circulating BAFF and APRIL. The continuous activation of B cells and thus overexpression of BAFF and APRIL may contribute to an increase in the generation of autoreactive B cells and a thus furthering the development of autoimmune disease [38].

B cells activation by T cells leads to the differentiation of B cells into long-lived plasma cells. However, continuous activation in autoimmune NZB/W F1 mice also generates short-lived plasmablasts. The number of splenic antibody-secreting cells (ASC) increases in NZB/W F1 mice aged 1–5 months and stabilizes after this period. Less than 60% of the splenic auto-ASCs are short-lived plasmablasts, whereas 40% are non-dividing, long-lived plasma cells with a half-life of 6 months. Although anti-proliferative immunosuppressive therapy depleted short-lived plasmablasts, long-lived plasma cells survived and continued to produce autoantibodies [39]. Additionally, Cheng et al. demonstrated that autoantibodies from long-lived "memory" plasma cells of NZB/W F1 mice drive complex immune nephritis [40].

#### **5. Genetic characteristics: susceptibility loci in NZB and NZW mice and in the NZB/W F1 hybrid**

Several chromosomal regions containing genes affecting lupus susceptibility or resistance have been identified pointing that murine lupus is genetically complex and mediated by a combination of genes.

In NZB/W F1 hybrids, genetic interactions between alleles present in NZB and NZW are the causes of the severe systemic autoimmunity found in these mice, due to the generation of a phenotype that is absent in both parental strains.

To search for contributing loci in this model of SLE, investigators backcrossed NZB/W F1 mice to NZW, then used brother-sister mattings to generate 27 substrains, termed New Zealand mixed (NZM) mice [41]. Further analysis of these 27 substrains led to the selection of NZM2410 as a lupus model. Susceptibility to lupus in NZM2410 is predominantly due to genes localized to the telomeric region of chromosome 1 (Sle1), the middle of chromosome 4 (Sle2), and the centromeric segment of chromosome 7 (Sle3) [42]. To study the contribution of each of these loci to pathogenesis, congenic strain construction was performed by transferring each of these intervals from NZM2410 onto the B6 background. Phenotypic analysis of congenic mice revealed that each locus contributes a unique component phenotype to the disease [43]. Although the B6.Sle congenic strains express phenotypes relevant to autoimmunity, none develop severe pathology, indicating that individual genes are not sufficient to cause lupus. The co-expression of these three major loci is necessary and sufficient for the development of a fully penetrant disease. These studies demonstrated that susceptibility to lupus involves both genetic interactions and additive effects of individual genes.

Additionally, to the Sle susceptibility loci, other loci present on chromosomes 1, 4, 7, and 17 have been associated with susceptibility in multiple lupus-prone strains including the NZB/W F1 model, an indication that genes in these regions may be necessary for immune regulation and function.

#### **5.1 Susceptibility loci for systemic lupus on chromosome 1: Sle1, Nba2, Lbw7, Sbw1, and Cgnz1**

The congenic strain, B6.Sle1, develops autoantibodies against nuclear autoantigens and displays spontaneous T cell activation without developing glomerulonephritis [44]. Fine mapping of the Sle1 locus determined that four loci within this congenic interval, termed Sle1a, Sle1b, Sle1c, and Sle1d, are implicated in the loss of tolerance to chromatin [45, 46].

Analyses of NZB congenic mice, (NZB X SM/J)F1 X NZB, revealed that the Nba2 lupus susceptibility locus is associated with hypergammaglobulinemia and the development of various autoantibodies, including anti-DNA, anti-chromatin, and anti-gp70 [47]. In these studies, mice congenic for the Nba2 locus did not develop significant renal disease on a B6 background but developed severe lupus nephritis when crossed with NZW mice [48], consistent with the need of multiple susceptibility genes for full expression of lupus.

The susceptibility loci, Sle1 and Nba2, overlap in the same region of chromosome 1, suggesting that some susceptibility genes may be shared among lupusprone strains. Within The Nba2 and Sle1 genetic segment there are genes encoding for the inhibitor type IIFcγR (FcγR IIB) [49], members of the SLAM/CD2 family of immunomodulatory receptors (Cd244, Cd229, Cs1, Cd48, Cd150, Ly108, and Cd84) [45] and members of the IFN-inducible (Ifi) family [48] all of which can regulate cell proliferation and survival. Analysis of congenic strains demonstrated that the presence of nuclear antigens and the severity of renal disease are linked with the FcγR and SLAM gene clusters with little involvement from the Ifi interval [50].

The inhibitory receptor for IgG, FcγRIIB, appears to be a fundamental regulator of B cell as well as myeloid cell activation [51]. Deficiencies in these routes result in heightened humoral and inflammatory responses, further contributing to lupus pathology [52].

The complement receptor 2 (CR2) gene, which encodes the complement receptor type 2 that acts as a B cell co-receptor is also in the Sle1c interval [53].

Theofilopoulos and colleagues identified Sbw1 and Lbw7 in chromosome 1 during their original linkage analysis of (NZB X NZW) F2 progeny [54]. Sbw1 defines a locus associated with splenomegaly, while Lbw7 defines a locus associated with anti-chromatin autoantibodies. Lbw7 of NZW origin is likely to be identical to Nba2 from NZB [54]. Additionally, Cgnz1 was detected in lupus-prone NZM2338 mice and significantly linked to chronic glomerulonephritis, severe proteinuria, and early mortality in female mice [55].

#### **5.2 Susceptibility loci for systemic lupus on chromosome 4: Sle2, Nba1, Sgp4, Lbw2, Sbw2, and Adnz1**

The congenic strain, B6.Sle2, displays lowered B cell activation thresholds coincident with the appearance of polyclonal IgM in the sera and expansion of the B1a cell compartment, in the absence of glomerulonephritis [43]. Interestingly, combining this locus with Sle1, resulted in glomerulonephritis and enhanced mortality compared with the single congenic strains alone [56].

Another susceptibility locus present on chromosome 4 is the Nba1 locus from NZB and the Lbw2 susceptibility locus from NZB/W F1. Both are associated with kidney disease, while another locus, sbw2, is associated with splenomegaly. The Sbw2 locus mapped to the same region as Lbw2, suggesting a single locus with pleiotropic effects [54]. The Nba1/Lbw2 interval contains the C1qa gene encoding the first component of complement C1q. It has been shown that an insertion polymorphism in the NZB sequence upstream of C1q gene may be related to a limited degree of C1q production, which may confer a risk for lupus nephritis by reducing IC clearance and promoting IC deposition in the glomeruli [57].

Overlapping with the Nba1 locus, there is a locus designated Sgp4, which was linked to the production of nephritogenic gp70 antigens. Production of autoantibodies to the retroviral envelope glycoprotein gp70, and the generation of

gp70-anti-gp70 immune complexes (gp70 IC) have been implicated in the development of nephritis in these lupus models [58, 59].

An additional study using NZM2328 mice found that the NZB-derived locus Adnz1 also contributed to the production of anti-DNA autoantibodies but not to lupus nephritis [55].

#### **5.3 Susceptibility loci for systemic lupus on chromosome 7: Sle3, Lbw5, Nba5, and Aia3**

Chromosome 7 contains several susceptibility genes regulating nephritis and autoantibodies. Among them are the Sle3 and Lbw5 loci, both derived from the NZW strain and the Nba5 locus from the NBW strain. A candidate gene present in this region is Cd22, which functions as a negative regulator of BCR signaling transduction.

Sle3 appears to be responsible for the hyperactive and pro-inflammatory antigen-presenting capacity of dendritic cells and macrophages [60].

The Nba5 susceptibility locus was associated with higher titers of anti-gp70 autoantibodies [61], while Aia3 with autoimmune hemolytic autoimmunity in a linkage analysis of NZB [62].

#### **5.4 Susceptibility loci for systemic lupus on chromosome 17: Lbw1 (MHC)**

The contribution of MHC haplotype to disease was first reported in the NZB/ NZW F1 model [63]. These genes are located in chromosome 17. Several studies demonstrated a strong association of H2d/z heterozygosity with the development of SLE, indicating a co-dominant contribution from each strain, H2d from NZB and H2z from NZW [64].

#### **6. Influence of sex**

Differences between female and male responses to foreign and self-antigens have been well-documented. It was suggested that genes and hormones are involved in the differences found in their innate and adaptive immune responses. Generally, females mount higher immune responses than males, which can contribute to the increased susceptibility to autoimmune diseases in females [65].

Similar to humans, within the NZB/W F1 mouse model lupus develops primarily in females with a lesser percentage and severity in male mice. In female mice, lupus signs appear after 6 months of age, with 50% mortality at 8.5 months and 90% mortality at 12.8 months. Male mice develop the disease after a year of age with 50% mortality at about 15 months of age [66]. Accordingly, early studies performed in NZB/W F1 mice showed that estrogen supplementation is associated with a worsening disease and shorter lifespan than untreated littermate. In contrast, supplementation of a female with the male sex hormone 5α-dihydrotestosterone reduce immune complex deposition and prolong survival despite the presence of high levels of IgG antibodies to DNA. Additionally, castrated or 17β-estradiol-treated NZB/W F1 male mice have an earlier onset of lupus and accelerated mortality, suggesting a suppressive effect of androgen [67, 68]. Data accumulated during the past few years provide evidence that female hormones, particularly estrogens, promote lupus pathogenesis. However, some opposite results are suggesting that sexual dichotomy is due to protective effects of androgens. The mortality induced by estrogens may be due to toxic effects rather than accelerated autoimmunity [69].

#### *A Spontaneous Mouse Model of Lupus: Physiology and Therapy DOI: http://dx.doi.org/10.5772/intechopen.85938*

Cells of the immune system, including B cells, express the cellular receptors for estrogens, estrogen receptor-α (ERα), and estrogen receptor-β [70]. Global disruption of the ERα gene in NZB/W F1 causes a significant reduction in the concentration of anti-histone/DNA and anti-double-stranded DNA IgG antibodies, which are associated with glomerulonephritis. This loss of tolerance was observed in female mice whereas, more modest effects are seen in males [71] suggesting that the ability of ERα signaling to enhance autoantibody production and lupus pathogenesis is more pronounced in females than in males. Additionally, specific deletion of ERα in B cells retards the production of autoantibodies and the development of nephritis in NZB/W F1 mice, demonstrating that ERα acts in a B cell-intrinsic manner to control B cell activation, autoantibody production, and lupus nephritis [72].

B cells with the CD5 marker, which spontaneously produce IgM, are found in higher numbers in NZB mice and have been implicated in lupus [73]. Treatment of lupus-prone female NZB/W F1 mice with tamoxifen (TAM), a synthetic antiestrogen with high affinity for the estrogen receptor, decreases the percentage of B cells and CD5+ B cells in the spleen. Also, TAM-treated mice had less severe proteinuria and increased survival rate compared to controls [74].

On the other hand, it has been described that NZB/W F1 males have higher levels of a population of Gr1highLy-6G + CD11b + myeloid cells that protect them against lupus development [75]. This population is testosterone-regulated and suppresses autoantibody production *in vivo*. Additionally, Gr1+ cells from NZB/W F1 males suppress the differentiation and effector function of CXCR5+ PD-1+ T follicular helper cells, germinal center formation, and plasma cell differentiation [76].

Since sex hormones can bind transcription factors, they might affect autoimmunity via their effects on gene transcription. Accordingly, it has been demonstrated that estrogen upregulates the expression of IFN-γ through the ERα [71].

Additionally, the expression of interferon regulatory factor 5 (IRF5), a lupus susceptibility factor that controls the expression of type I IFNs, is higher in NZB/W F1 females than in males. IRF5 expression also depends on ERα expression, because of splenic cells from ERα knockout female express lower levels of IRF5 [77]. This suggests a (positive) feedback loop between the IFNs and estrogens since activation of type I IFNs or IFN-γ signaling upregulates the expression of ERα [78].

Other studies have provided evidence that lupus-associated miRNAs are differentially expressed in splenocytes of NZB/W F1 male and female mice. Additionally, these miRNAs were upregulated by estrogen treatment [79]. miRNAs regulate the expression, mainly at the post-transcriptional level, of some genes that are important in the development of the innate and adaptive immune system and the maintenance of immune homeostasis. Dysregulation of miRNAs impacts the function of different types of immune cells causing a breakdown of immune tolerance and ultimately the development of autoimmune-related disorders such as SLE [80].

#### **7. Treatment of murine SLE**

Different treatments to improve lupus have been evaluated in the NZB/W F1 murine model. In this section, we will review some well-documented procedures.

Interleukin-6 (IL-6) is a multifunctional cytokine synthetized by macrophages, monocytes, and B and T cells. IL-6 is critical for B cell differentiation and maturation, immunoglobulin secretion, cytotoxic T cell differentiation, acute-phase protein production, bone marrow progenitor stimulation, renal mesangial cell

proliferation, and macrophage/monocyte functions. Lupus mice treated with anti-IL-6 mAb reduce B cell proliferation, the ds-DNA antibodies production, and kidney damage [81]. Additionally, treatment with antibodies against the IL-6 receptor (IL6R-mAb) inhibits the production of anti-DNA and anti-TNP IgGs antibodies, and consequently, this treatment increases the survival of the mice [82]. Tocilizumab, an anti-IL6R-mAb commercialized mainly for the treatment of rheumatoid arthritis [83], has been evaluated in SLE patients. This procedure decreases anti-dsDNA antibody levels and circulating plasma cells and improves arthritis and medical scores [84].

Interleukin-10 (IL-10) is a cytokine produced by subsets of activated T cells and macrophages. It mediates a variety of both immunostimulatory and immunosuppressive properties. IL-10 neutralization with anti-IL-10 delays the onset of the disease, increasing survival from 10 to 80% in mice at 9 months. Autoimmunity protection by IL-10 antagonism appeared to be due to an upregulation of endogenous tumor necrosis factor alpha (TNF-α) [85].

TNF-α is a pleiotropic cytokine with immunostimulatory and proinflammatory activities. TNF-α stimulates T and B cell proliferation, immunoglobulin synthesis, enhances natural killer (NK) cell activity, and boosts neutrophil activation. The NZB/W F1 mice have reduced levels of TNF-α, and their treatment with recombinant TNF-α increased their survival [86]. Infliximab, a TNF-α blocking antibody, was evaluated in short- and long-term therapy in SLE patients showing several adverse effects in long-term therapy [87]. Infliximab and Etanercept are another TNF-α blockers commercialized mainly to treat rheumatoid arthritis [88, 89].

Type I interferons (IFN) are primarily regarded as inhibitors of viral replication. However, type I IFN, mainly IFN-α, plays a major role in activation of both the innate and adaptive immune system [90]. IFN-α signature precedes the onset of lupus in NZB/W F1 mice and in humans. Treatment with a vaccine that induces the secretion of anti-IFN-α neutralizing antibodies causes a delay in proteinuria development, low deposits of immune complexes, and increases survival [91]. Two antibodies against IFN-α, Sifalimumab and Rontalizumab, evaluated in SLE patients correlate with improvements in disease activity [92, 93].

BAFF is a B cell-activating factor essential for the survival of B cells. BAFF is produced predominantly by myeloid cells and binds to three distinct receptors on the B cell surface; the transmembrane activator and calcium modulator ligand interactor (TACI), the B cell maturation antigen (BCMA), and the BAFF receptor. Treatment with soluble TACI-Ig fusion protein inhibits the development of proteinuria and prolongs animal survival [94]. Besides, a short course of TACI-Ig and CTLA4-Ig induces a profound depletion of splenic B cells, prolong life, and even reverse proteinuria in aged NZB/W F1 mice [95]. Atacicept is a recombinant fusion protein that blocks activation of B cells by binding to TACI ligands. In SLE patients, the Atacicept treatment favors the reductions in disease activity and severe flares [96].

CD20 is a transmembrane phosphoprotein specifically expressed on B cells. Depletion of B cells with a monoclonal antibody against CD20 favors the survival of aged NZB/W F1 mice [97]. Rituximab, an anti-CD20 monoclonal antibody frequently used in SLE patients improves lupus nephritis, arthritis, serositis, cutaneous vasculitis, mucositis, rashes, fatigue, and neurologic symptoms [98]. Although rituximab's mechanisms of action are not known, its effects are likely mediated by antibody-dependent cell-mediated cytotoxicity and the induction of apoptosis on B cells [99].

Mammalian target of rapamycin (mTOR) is a protein kinase that regulates different cellular processes such as cell proliferation, growth, motility, cell survival, *A Spontaneous Mouse Model of Lupus: Physiology and Therapy DOI: http://dx.doi.org/10.5772/intechopen.85938*

protein synthesis, and transcription. NZB/W F1 mice treated with rapamycin (a drug used in rejection prophylaxis in solid organ transplantation) from 12 to 37 weeks of age inhibit the production of autoantibodies, development of proteinuria, and prolong mouse survival [100]. Moreover, in mice with established nephritis, rapamycin suppressed the interstitial infiltration of T cells, B cells, and macrophages [101].

Antigen presentation process involves costimulatory molecules CD28, and CTLA4 expressed on T cells, representing activation or inhibitory signals to T cells. CD28 and CTLA4 bind with medium or high affinity, respectively to B7, i.e., expressed on antigen-presenting cells (APCs) [102]. Abatacept is a fusion CTLA4-Ig protein that interrupts the interaction of B7 with CD28. NZB/W F1 mice that express murine CTLA4-Ig exhibit an improvement in all of lupus symptoms increasing survival [103]. In humans, Abatacept is mainly used in rheumatoid arthritis [104], although there are some SLE studies, one of them showing improvement in skin lesions in SLE patient [105].

Based on studies done in mouse models, most clinical trials have focused on agents that control B and T lymphocytes activations and functions. **Figure 1** shows some therapeutic targets investigated in mouse models of SLE (as described in [82, 85, 91, 95, 97, 103, 106–110]), many of which where then follow up in clinical trials [88, 89, 92, 98, 104, 111–118].

#### **Figure 1.**

*Immune cells contribution to SLE and potential targets for lupus therapies, as tested in mouse models: Defects in phagocytosis of apoptotic cells leads to the presentation of autoantigens by APC to naive CD4 T cells. Activated T cells help the differentiation of B cell into plasma cells that secrete high levels of autoantibodies. These autoantibodies form immune complexes by binding to autoantigens, and engaging Fcγ receptors on different cell types. This supports inflammation and tissue destruction through the recruitment of inflammatory cells to tissues. APC: Antigen-presenting cell, IC: Immune complexes, mAb: monoclonal antibody. Texts on the right side of the figure show the different targets tested for lupus therapy. Drug names are shown in brackets*

#### **8. Conclusions**

The spontaneous mouse model of lupus NZB/W F1 has been important to elucidate the pathogenesis of SLE. In this model, the lupus-like phenotypes include lymphadenopathy, splenomegaly, elevated serum antinuclear autoantibodies including anti-dsDNA IgG, and immune complex-mediated glomerulonephritis that are remarkably similar to the pathology described in human lupus. Consequently, it has provided a powerful tool to our knowledge on human lupus disease and the development of novel therapies. Additionally, similar to humans, lupus develops primarily in female NZB/W F1 mice with lesser percentage and severity in male. The female predominance of the disease remains poorly understood; however, hormonal contributions to immune system activation and X chromosome gene-dose effect have been proposed to be the important contributor to sex bias [66]. On the other hand, unlike SLE patients, NZB/W F1 mice do not manifest skin disease or arthritis [3].

Furthermore, human and murine lupus is characterized by a deregulation in autoreactive T helper cells, B and DC cells activation, and cytokine production. Defective function of regulatory T cells, inefficient clearance of immune complex and biological waste, nucleic acid sensing and IFN production pathways are also involved in the loss of tolerance and tissue damage associated to lupus [119]. The use of mouse models has allowed the study of the mechanisms involved in the cellular immune abnormalities, providing a powerful tool to identify novel pathways and targets for disease therapies. Several components of the immune system, such as cytokines, B cells, T cells, and hormones have been identified as potential targets for novel drugs. The side effects, dosage regimens, and response to treatment are first tested on murine models of lupus prior they go to clinical trials. Murine models of disease represent genetically homogeneous populations and in contrast to humans that take chronic doses of immunosuppressant, they allow for examination in the absence of any therap. Despite favorable results in mouse studies, many therapies have failed to meet clinical end points. This is probably because of the complexity of the disease, which involves the contribution of environmental and genetic susceptibility factors [119]. However, some of the therapeutic approaches have been successful recommended for SLE treatment, like Belimumab, a humanized monoclonal antibody directed against B cell activating factor. Additionally, other available agents such as rituximab, tacrolimus, azathioprine, methotrexate, cyclophosphamide, and mycophenolate mofetil are widely used off-label in SLE [9, 120].

The use of murine models has identified several novel candidate genes, and some of them have been associated to SLE in humans. An important contribution of the genetic studies in NZB/W F1 was the identification, in chromosome 1, of Sle1 and Nba2 loci, which are responsible for the production of autoantibodies. Sle1 and Nba2 encode members of the FcγR, SLAM, and IFN-inducible receptor families.

As sustained above, all the mouse models, and specifically the NZB/W F1, have the benefit of having a shorter evolution of the disease, allowing to investigate the full progression of the disorder and its pathophysiology and to test for possible therapies in a much shorter time period. In spite of their limitations and the fact that one cannot readily extrapolate to the human disease, mouse models of lupus have significantly helped researchers to advance our knowledge on this syndrome, adding relevant data on the pathogenesis of lupus and providing investigators with a valuable preclinical model for the design of future therapies. In spite of the various differences found between the human and mouse immune systems, there are sufficient similarities in the manifestation of the disease to be optimistic regarding

the use of this mouse model to further advance in our understanding of the physiology of the human disease and the formulation of creative new therapies.

### **Acknowledgements**

This work was supported by the Government of Chile through the Programa de Apoyo a Centros Científicos y Tecnológicos de Excelencia con Financiamiento Basal AFB 170004, from the Postdoctoral Fondecyt Project 3160224 and CONICYT doctoral fellowship 21130598.

### **Conflict of interest**

The authors declare no competing or financial interests.

### **Author details**

Gabriela Tejon1 , Yessia Hidalgo1 , Maria Rosa Bono1 and Mario Rosemblatt1,2,3\*

1 Department of Biology, Faculty of Sciences, University of Chile, Santiago, Chile

2 Faculty of Life Sciences, Andres Bello University, Santiago, Chile

3 Sciences and Life Foundation, Santiago, Chile

\*Address all correspondence to: mrosemblatt@cienciavida.org

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### Section 2
