PREVALENCE OF AUTOANTIBODIES REVEALS A PREDOMINANT SMA AND ANCA-PR3/MPO PATTERN IN HIV INFECTION AND SMA IN HAV-INFECTED CHILDREN

Hepatitis A Virus (HAV) and Human Immunodeficiency Virus (HIV) have been associated with development of autoantibodies and autoimmune manife stations in children. Autoimmune Hepatitis (AIH) is particularly aggressive in children/adolescents with a more severe outcome. Thus, studying the mechanisms of virus-related autoimmune disorders in ch ldren is a relevant topic of research. We aimed to study the prevalence of autoantibodies in plasma of children infected with either HAV or HIV comparing to healthy children. The relationship bet ween the presence of autoantibodies and biochemical markers of hepatic damage was also investigated. De tection of autoantibodies (SMA) was associated with HAV infection with a prevalence of 35%. Similar lev els of hepatic enzymes were observed in sera of HAV-infected patients with reactivity against autoa ntigens as compared to those without autoantibodies . On the other hand, HIV infection showed broader aut o n ibodies reactivities than HAV-infected patients and was associated with SMA (18%), ANCA (20%), ANCA -PR3 (15%) and ANCA-MPO (13%). Moreover, either RF or ANA was detected in 8% of HI V-infected children. Prevalence of autoantibodies was not associated with either gender or age of inf ected children. A high prevalence of SMA was observed in HAVand HIV-infected patients. As HAV and SMA may persit in some patients and AIH can develop in susceptible children, it is recommen ded a follow up of virus infected patients. Since ANCA-PR3 and ANCA-MPO have been shown to be pathoge nic, proinflammatory and associated with symptomatic HIV infection, further studies are requ ired to determine the role of these autoantibodies in the pathogenesis associated with viral infection in children.

HAV has been suggested as a factor contributing to development AIH in both adults and individuals that are susceptible for autoimmune diseases (Muñoz Bertrán et al., 2002). AIH is particularly aggressive in children/adolescents with a more severe outcome (Pando et al., 1999;Floreani et al., 2013). In addition, AIH Type 1 is the major cause of chronic liver disease in Argentinian children (Cuarterolo et al., 1995). Moreover, the presence of autoantibodies has been correlated positively with the severity of hepatic lesions in AIH type-1 (Matsuo et al., 2000). On the other hand, a small number of studies have reported the presence of autoantibodies and a lower prevalence of autoimmune diseases in HIV-infected children as compared to adults (Argüello et al., 2012). Thus, studying the mechanisms of virus-related autoimmune disorders in children is a relevant topic of research.
Detection of autoantibodies is an important criterion for diagnosis and classification of extrahepatic manifestations associated with HAV infection as well as type 1 and 2 AIH . Besides, it is also relevant to decide optimal treatment options against HAV and HIV. Furthermore, studying the prevalence of autoantibodies in sera of patients with these viral infections is relevant to elucidate the mechanisms of viral infection-related autoimmune manifestations induction. Therefore, the main objective of this study was to study the prevalence of autoantibodies in sera of patients infected with either HAV or HIV as compared to healthy individuals. In addition, the relationship between the presence of autoantibodies and biochemical markers of hepatic damage was investigated.

Patients
Data of patients studied in this study are shown in Table 1 to 5. Control group was negative for: HAV, Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), HIV, Human T-Lymphotropic Virus 1 (HTLV-1), chagas disease and toxoplasmosis. Written informed consent was obtained from the parents of each patient and the study was approved by the Ethics Committee of Medical Research from the Hospital Central in accordance with the 1975 Declaration of Helsinki.
Either HAV or HIV children coinfected with HBV and HCV were excluded from this study. In addition, children with previous hepatic diseases (including AIH, cholestasis or infectious hepatitis) were excluded from this study.
TGA and RF were assayed by particle aglutination.

Statistics
Graph Prism 5.0 statistical software package was used for data analysis. Data was expressed as Mean ± SD for quantitative measures and both number and percentage for categorized data. Comparison between two independent means for parametric data was done using student test. Comparison between two independent groups for non-parametric data was done using Mann Whitney test. The fisher exact test used to study the association between 2 variables, or comparison between 2 independent groups as regards the categorized data. The probability of error at 0.05 was considered significant.

RESULTS
Presence of at least one autoantibody was detected in 19 (35%) out of 54 samples of HAV-infected children (p<0.0001, Fisher exact test) ( Table 1 and  3). In addition, concomitant detection of more than one antibody showed a prevalence of 3.7%. HAV infection was associated with SMA (35%, p<0.0001 fisher exact test) (Table 1 and 3). Either RF or APC reactivities was observed in one patient. Moreover, no reactivities were detected for ANA, AMA, TGA, LKM, DNAds and ANCA. Prevalence of autoantibodies (SMA), on the other hand, was not associated with either gender or age of HAV-infected children ( Table 6). Furthermore, levels of biochemical markers of liver damage (AST and ALT) showed no difference between samples of HAVinfected patients reactive to autoantibodies and those no-reactive ( Table 7).
It is interesting to note that HIV-infected children showed broader autoantibodies reactivities than HAVinfected patients. The presence of autoantibodies was observed in 18 out of 39 children (46%) and was not associated with either gender or age of HIV-infected children ( Table 2, 4 and 8). Concomitant detection of more than one antibody was observed in 3 HIVinfected children (7.7%). HIV infection was associated with SMA (18%, p = 0.0037) and ANCA (20%, p = 0.0016) ( Table 2 and 4). Notably, prevalence of both ANCA-PR3 (15%, p = 0.0086) and ANCA-MPO (13%, p = 0.0198) was associated with HIV-infection. Interestingly, a mixed pattern of ANCA-PR3 and ANCA-MPO was observed in 3 patients (8%) ( Table 4). Moreover, either RF or ANA was detected in 8% of HIV-infected children. Prevalence of SMA, on the other hand, was not associated with either gender (p = 0.3863) or age (p = 1) of infected children. In addition, prevalence of ANCA was neither associated with gender (p = 1) nor age (p = 0.6857) of patients. No reactivities were detected for TGA, APC, AMA, LKM and DNAds. Finally, similar SMA titers were observed in HAVand HIV-infected children (p = 0.4119, Mann Whitney test) ( Fig.  1).
Several mechanisms have been proposed to explain HAV-related induction of autoantibodies and autoimmune diseases including impaired function of regulatory T cells. HAV have been suggested to impair CD4+/CD25+ T regulatory lymphocytes in self-limited infection (Perrella et al., 2008). Interestingly, it has been shown that interaction of HAV with its Cellular Receptor Science Publications AJI 1 (HAVCR1) inhibited regulatory T cell functions and reduced production of Transforming Growth Factor-β (TGF-β) and IL-10 (Manangeeswaran et al., 2012). Moreover, individuals with a defect in suppressorinducer T lymphocytes (that control immune responses to the asialoglycoprotein receptor) have been suggested to develop AIH type 1 after HAV infection (Vento et al., 1991). In addition, antibodydependent citotoxicity has been involved in AIH (Vergani and Mieli-Vergani, 2008).
In this study, high prevalence of SMA (35%) was associated with HAV+ patients. This is in agreement with previous reports showing similar prevalence of SMA in HAV+ children (Fainboim et al., 2001;Elfaramawy et al., 2010). Although transient SMA reactivities have been observed in self-limited HAV infection, persistence of SMA has been associated with proctacted forms of paediatric infection (Fainboim et al., 2001;Elfaramawy et al., 2010). Interestingly, HLA DRB1*1301 haplotype has been associated with a higher susceptibility to develop prolonged HAV infection in children (Fainboim et al., 2001). Notably, HLA DRB1*13 haplotype has shown a strong association with paediatric type 1 AIH in high endemicity regions for HAV infection (Elfaramawy et al., 2010;Bittencourt et al., 2008;Fainboim et al., 1994;Bittencourt et al., 1999). In addition, prevalence of AIH type 1 with a pattern SMA/ANA reactivities is higher than AIH type 2 (LKM-1 reactivity) in children (Vergani and Mieli-Vergani, 2008). On the other hand, HAV vaccination has been associated with development of autoantibodies and rarely with autoimme hepatitis (Karali et al., 2011;Berry and Smith-Laing, 2007;Perumalswami et al., 2009). These experimental evidences suggest that AIH type 1 may develop after prolonged HAV infection in susceptible individuals (Huppertz et al., 1995;Skoog et al., 2002;Vento et al., 1991;Singh et al., 2007). Patients in this study were studied in the acute phase of infection and not at later times after infection. So, neither the prolonged nature of detected autoantibodies nor its association with viral presistance could be assessed. As both HAV and autoantibodies (SMA) may persit in some patients and AIH can develop in susceptible children, a follow up of virus infected patients is important.
Some studies have shown SMA associated with biochemical and histological features of disease activity in adult HAV infection and AIH (Dan and Yaniv, 1990;Couto et al., 2014). However, in the current study SMA was not related to the levels of biochemical markers of liver damage in infected children. Different experimental conditions and susceptibilities between children and adults may explain these discrepancies. In fact, different gene susceptibilities have been shown in children with AIH (associated with HLA DRB1*13 haplotype) as compared to adults (associated with HLA-DRB1*0405 haplotype) (Pando et al., 1999). Different autoantibody profiles have also been reported, with higher prevalence of SMA in paediatric AIH and of ANA in adult AIH (Pando et al., 1999). In addition, HLA-DRB1*12 haplotype has been associated with paediatric HAVrelated AIH type 1 (Elfaramawy et al., 2010).
On the other hand, HIV-infected individuals with various levels of immunological control have been associated with different autoimmune diseases (Iordache et al., 2014). However, few studies have reported the presence of autoimmune diseases in HIVinfected children. These studies have shown a lower prevalence of autoimmune diseases in children as compared to adults (Adebajo, 1997;Nguyen and Reveille, 2009;Schuval et al., 2001;Martínez-Rojano et al., 2001;Rodríguez-Mahou et al., 1994;Jarvis et al., 1993;Rodriguez et al., 1993;González et al., 1998;Stricker et al., 1998;Eley et al., 1999). Long term HIV infection leading to chronic activation and exhaustion of the host immune system and increased expression of autoantigens has been proposed to promote development of autoimmune diseases (Gaddi et al., 2000). Several autoantibodies have been associated with HIV infection in children including ANA, SMA, DNAds and RF. It has been suggested that polyclonal B cell activation, increased levels of total immunoglubulins and dysfunction of CD4+ T regulatory cells contribute to development of autoantibodies in HIV-infected children in the absence of clinical autoimmune disease (Argüello et al., 2012;Rodriguez et al., 1993;González et al., 1998;Stricker et al., 1998;Eley et al., 1999). Interestingly, low absolute levels of CD4+FoxP3+T cells and enhanced frequencies of a subset of dysfunctional regulatory T cells (CD4+FoxP3+CD25-T cells) have been associated with a higher prevalence of autoantibodies and hypergammaglobulinaemia in HIV-infected children with severe immunosuppression (Argüello et al., 2012).
In this study, the presence of autoantibodies was associated with HIV infection (46%). Prevalence of SMA (18%), ANA and RF (8%) in HIV-infected children is in agreement with previous reports Science Publications AJI (Argüello et al., 2012;Schuval et al., 2001;Rodríguez-Mahou et al., 1994;Jarvis et al., 1993). Interestingly, a previous study showed that SMA and RF were detected more frequently in HIV-infected children with low CD4+ T cell counts (Argüello et al., 2012). Detection of SMA in adult HIV-patients with low CD4+ T cells but not in patients with high CD4+ T cells has also been reported (Chretien et al., 2003). In the present study, CD4+ T cells levels were not assayed because of constrains in the samples available. Therefore, the relationship between autoantibodies reactivities and CD4+ T cells was not determined. Another limitation of this study was the lack of information on clinic manifestations.
So, the relationship between autoantibodies reactivities and clinic manifestations could not be evaluated.
Interestingly, ANCA (20%) was associated with HIV infection and ANCA-PR3/ANCA-MPO were observed in 6 (15%) and 5 (13%) patients, respectively. Moreover, a mixed pattern of ANCA-PR3 and ANCA-MPO were detected in 3 patients. ANCA have been associated with the pathogenesis of systemic vasculitis (Hu et al., 2009). Several experimental evidences support a pathogenic role for ANCA in the pathogenesis of ANCA-Associated Vasculitis (AAV). It has been shown that stimulation of neutrophils with pro-inflammatory cytokines (ex. TNFα) induced translocation of ANCA antigens (PR3 and MPO) to the neutrophil surface. Binding of ANCA IgG to its antigens and Fc receptors activate neutrophils and release free oxygen radicals and various proteases contributing to pathogenesis in vasculitic lesions (Chen and Kallenberg, 2010). In addition, a crucial role for the alternative pathway of complement activation in ANCA-mediated neutrophil activation and AAV has been reported (Schreiber et al., 2009). Moreover, it has been observed in mice models that administration of anti-MPO induced leukocytes recruitment to sites affected in human ANCA-associated vasculitis such as kidney and lung (Nolan et al., 2008). It is interesting to note that PR3-specific Th17 cells have also been involved in ANCA-mediated autoimmune diseases pathogenesis (Abdulahad et al., 2008).
On the other hand, ANCA have been associated with symptomatic HIV infection in adults (prevalence 18%-41.9%) (Habegger de Sorrentino et al., 1997;Kamat et al., 2010;Cornely et al., 1999;Klaassen et al., 1992;Savige et al., 1994;Nikolova et al., 2002). Interestingly, ANCA have been positively correlated with pulmonary infection and Mycobacterium tuberculosis in HIV-infected patients (Habegger de Sorrentino et al., 1997). Nevertheless, prevalence of ANCA is low in asymptomatic HIV infection and ANCAassociated vasculitides are rare in infected individuals (Iordache et al., 2014;Habegger de Sorrentino et al., 1997;Cornely et al., 1999). In addition, most of reported studies have shown low prevalence of ANCA in children infected with HIV (Argüello et al., 2012). A recent study showed ANCA reactivities in 3 out of 15 (20%) immunosupressed HIV-infected children (CD4+ T cells<15%) but no reactivity in children with no evidence of immunosuppression (CD4+ T cells >25%) (Argüello et al., 2012). Immunosupressed children showed low absolute levels of regulatory T cells and enhanced frequencies of a subset of dysfunctional regulatory T cells (CD4+FoxP3+CD25-T cells) in the absence of autoimmune disease. It is interesting to note that increased levels of the alternative complement pathway (complement activation fragments C3a and Bb), that is involved in ANCA pathogenesis, have been observed in HIV-infected children (Jarvis et al., 1993). Thus, ANCA (including ANCA-PR3, ANCA-MPO and a mixed pattern ANCA-PR3/MPO in infected children) could contribute to pathogenesis of HIV-related diseases including autoimmunity.

CONCLUSION
In summary, detection of autoantibodies was associated with both HAV and HIV infection in children.
HIV-infected patients showed broader reactivities for autoantibodies than HAV-infected children. HAV infection was associated with SMA while HIV infection was associated with SMA, ANCA-PR3 and ANCA-MPO. Similar levels of biochemical markers of liver damage were observed in HAV-infected patients with or without reactivity against autoantibodies. As HAV and SMA may persit in some patients and AIH can develop Science Publications AJI in susceptible children, it is recommended a follow up of virus infected patients. Children infected with HIV are an especially susceptible population. Since ANCA-PR3 and ANCA-MPO have been shown to be pathogenic, proinflammatory and associated with symptomatic HIV infection, further studies are required to determine the role of these autoantibodies in the pathogenesis associated with HIV infection in children.

ACKNOWLEDGEMENT
We would like to thanks Prof Virginia Rivero for her contribution to this study.