Insights into the knowledge of complex diseases: Environmental infectious/toxic agents as potential etiopathogenetic factors of systemic sclerosis.

Systemic sclerosis (SSc) is a connective tissue disease secondary to three cardinal pathological features: immune-system alterations, diffuse microangiopathy, and fibrosis involving the skin and internal organs. The etiology of SSc remains quite obscure; it may encompass multiple host genetic and environmental -infectious/chemical-factors. The present review focused on the potential role of environmental agents in the etiopathogenesis of SSc based on epidemiological, clinical, and laboratory investigations previously published in the world literature. Among infectious agents, some viruses that may persist and reactivate in infected individuals, namely human cytomegalovirus (HCMV), human herpesvirus-6 (HHV-6), and parvovirus B19 (B19V), and retroviruses have been proposed as potential causative agents of SSc. These viruses share a number of biological activities and consequent pathological alterations, such as endothelial dysfunction and/or fibroblast activation. Moreover, the acute worsening of pre-existing interstitial lung involvement observed in SSc patients with symptomatic SARS-CoV-2 infection might suggest a potential role of this virus in the overall disease outcome. A variety of chemical/occupational agents might be regarded as putative etiological factors of SSc. In this setting, the SSc complicating silica dust exposure represents one of the most promising models of study. Considering the complexity of SSc pathogenesis, none of suggested causative factors may explain the appearance of the whole SSc; it is likely that the disease is the result of a multifactorial and multistep pathogenetic process. A variable combination of potential etiological factors may modulate the appearance of different clinical phenotypes detectable in individual scleroderma patients. The in-deep investigations on the SSc etiopathogenesis may provide useful insights in the broad field of human diseases characterized by diffuse microangiopathy or altered fibrogenesis.

Innate Immune Modulation Induced by EBV Lytic Infection Promotes Endothelial Cell Inflammation and Vascular Injury in Scleroderma.

Microvascular injury is considered an initial event in the pathogenesis of scleroderma and endothelial cells are suspected of being the target of the autoimmune process seen in the disease. EBV has long been proposed as a trigger for autoimmune diseases, including scleroderma. Nevertheless, its contribution to the pathogenic process remains poorly understood. In this study, we report that EBV lytic antigens are detected in scleroderma dermal vessels, suggesting that endothelial cells might represent a target for EBV infection in scleroderma skin. We show that EBV DNA load is remarkably increased in peripheral blood, plasma and circulating monocytes from scleroderma patients compared to healthy EBV carriers, and that monocytes represent the prominent subsets of EBV-infected cells in scleroderma. Given that monocytes have the capacity to adhere to the endothelium, we then investigated whether monocyte-associated EBV could infect primary human endothelial cells. We demonstrated that endothelial cells are infectable by EBV, using human monocytes bound to recombinant EBV as a shuttle, even though cell-free virus failed to infect them. We show that EBV induces activation of TLR9 innate immune response and markers of vascular injury in infected endothelial cells and that up-regulation is associated with the expression of EBV lytic genes in infected cells. EBV innate immune modulation suggests a novel mechanism mediating inflammation, by which EBV triggers endothelial cell and vascular injury in scleroderma. In addition, our data point to up-regulation of EBV DNA loads as potential biomarker in developing vasculopathy in scleroderma. These findings provide the framework for the development of novel therapeutic interventions to shift the scleroderma treatment paradigm towards antiviral therapies.

Impact of Human Cytomegalovirus and Human Herpesvirus 6 Infection on the Expression of Factors Associated with Cell Fibrosis and Apoptosis: Clues for Implication in Systemic Sclerosis Development.

Systemic sclerosis (SSc) is a severe autoimmune disorder characterized by vasculopathy and multi-organ fibrosis; its etiology and pathogenesis are still largely unknown. Herpesvirus infections, particularly by human cytomegalovirus (HCMV) and human herpesvirus 6 (HHV-6), have been suggested among triggers of the disease based on virological and immunological observations. However, the direct impact of HCMV and/or HHV-6 infection on cell fibrosis and apoptosis at the cell microenvironment level has not yet been clarified. Thus, this study aimed to investigate the effects of HCMV and HHV-6 infection on the induction of pro-fibrosis or pro-apoptosis conditions in primary human dermal fibroblasts, one of the relevant SSc target cells. The analysis, performed by microarray in in vitro HCMV- or HHV-6-infected vs. uninfected cells, using specific panels for the detection of the main cellular factors associated with fibrosis or apoptosis, showed that both viruses significantly modified the expression of at least 30 pro-fibrotic and 20 pro-apoptotic factors. Notably, several recognized pro-fibrotic factors were highly induced, and most of them were reported to be involved in vivo in the multifactorial and multistep pathogenic process of SSc, thus suggesting a potential role of both HCMV and HHV-6.

Epstein-Barr virus, cytomegalovirus and BK polyomavirus burden in juvenile systemic lupus erythematosus: correlation with clinical and laboratory indices of disease activity.

OBJECTIVES: Clinical and laboratory investigations have revealed that Epstein-Barr virus (EBV) is involved in altered immunological response of systemic lupus erythematosus (SLE). Higher seroprevalence rates of anti-EBV antibodies and increased viral load are demonstrated in adult SLE patients. The prevalence of BK polyomavirus (BKV) reactivation is also suggested to be higher in SLE. Herein, we aimed to evaluate the immune response of children with SLE to EBV antigens in addition to EBV and BKV DNA. We also tried to evaluate whether these serological results differ from another connective tissue disease - juvenile systemic sclerosis (jSS) - and healthy individuals. METHODS: Serum levels of EBV early antigen diffuse (EA-D) IgG, EBV nuclear antigen-1 IgG, EBV viral capsid antigen (VCA), cytomegalovirus (CMV) IgG, EBV DNA, CMV DNA and urinary BKV DNA were evaluated in healthy controls and in patients with a diagnosis of juvenile SLE (jSLE) and jSS. RESULTS: A total of 70 jSLE patients, 14 jSS patients and 44 sex-matched healthy individuals were involved in the study. EBV VCA was positive in 84.2% of jSLE patients, 85.7% of jSS patients and 36.3% of healthy controls. EBV EA-D IgG positivity was significantly higher in jSLE patients compared to jSS patients and healthy controls (20% vs. 7.1% and 0%, p = 0.005). EBV VCA positivity was associated with malar rash and immunological disorder, but there was no statistical significance in other antibody positivity in terms of clinical and haemogram findings and autoantibody positivity. CMV DNA positivity was present in only 2.8% of jSLE patients. None of the jSS patients or the healthy controls had CMV DNA positivity. EBV DNA and BKV DNA were also negative in all three groups. CONCLUSION: The results of our study assume a relationship between SLE and EBV, but we could not demonstrate an association between CMV and BKV. The negative DNA results in contrast to serological positivity can be interpreted as an altered and impaired immune system and increased viral susceptibility. These results suggest that EBV contributes to disease continuity, even if it does not directly cause development.

Parvovirus B19 activates in vitro normal human dermal fibroblasts: a possible implication in skin fibrosis and systemic sclerosis.

OBJECTIVE: Fibrosis is the most characteristic pathological hallmark of SSc, a connective tissue disease characterized by vascular and immunological abnormalities, inflammation and enhanced extracellular matrix production, leading to progressive fibrosis of skin and internal organs. We previously demonstrated that parvovirus B19 (B19V) can infect normal human dermal fibroblasts (NHDFs) and that B19V persists in SSc fibroblasts. In this study, we investigated whether parvovirus B19V is able to activate in vitro NHDFs and to induce in these cells some phenotypic features similar to that observed in the SSc fibroblasts. METHODS: We preliminarily analysed the time course of B19V infection in cultured NHDFs, then we investigated the ability of B19V to induce cell migration, invasive phenotype and mRNA expression of some profibrotic and/or proinflammatory genes. RESULTS: We confirmed our previous findings that B19V infects NHDFs, but the infection is not productive. After incubation with B19V, NHDFs showed a significant increase of both migration and invasiveness, along with mRNA expression of different profibrotic genes (α-SMA, EDN-1, IL-6, TGF-ß1 receptors 1 and 2, Col1α2), some genes associated with inflammasome platform (AIM2, IFI16, IL-1ß, CASP-1) and genes for metalloprotease (MMP 2, 9 and 12). CONCLUSION: These data suggest that B19V can activate dermal fibroblasts and may have a role in the pathogenesis of fibrosis. B19V-induced fibroblast migration and invasiveness could be due to the B19V-associated MMP9 overexpression and activation. Moreover, the up-regulation of MMP12, typical of SSc, could link the B19V infection of fibroblasts to the anti-angiogenic process.

Systemic sclerosis and the COVID-19 pandemic: World Scleroderma Foundation preliminary advice for patient management.

Due to the frequent presence of interstitial lung disease and widespread use of immunosuppressive treatment, systemic sclerosis (SSc) patients may be considered at risk for a more severe disease course and higher mortality when they develop Severe Acute Respiratory Syndrome - Coronavirus - 2 (SARS-CoV-2) virus infection. Therefore, with World Scleroderma Foundation endorsement, experts from different specialties including rheumatology, virology and clinical immunology gathered virtually to answer to the main practical clinical questions regarding SARS-CoV-2 infection coming from both patients and physicians. This preliminary advice is aligned with other national and international recommendations, adapted for SSc patients.

COVID-19 infection during autoimmune disease: study of 2 cases in Republic of Guinea.

INTRODUCTION: Coronavirus is a virus that can target the respiratory, musculoskeletal systems with a cascade of inflammatory processes. The objective of this work is to establish the link between autoimmune diseases and a COVID-19 infection in Guinea. METHODS: Retrospective patient data were obtained from medical records. Informed consent was obtained under the direction of the national health security agency (ANSS). RESULTS: We report the case of two patients aged 52 and 64 years respectively, known to have rheumatoid arthritis (RA) and systemic scleroderma (SDS) admitted with clinical signs suggesting underlying infection with COVID-19. They were tested with RT-PCR, which was positive within hours. CONCLUSION: In view of the rapid clinical worsening of patients with COVID-19 infection and autoimmune diseases, increased surveillance should be undertaken with abstinence of any factors that might weaken the immunity of these patients.

Retrotransposons shuttling genetic and epigenetic information from the nuclear to the mitochondrial compartment: Do they play a pathogenetic role in scleroderma?

Endogenous retroelements are a class of ancient defective viral insertions contained in the genome of host cells, where they account for up to 40% of all DNA. Centuries of co-existence in host genome have led to the development of immunotolerance to endogenous retroelements, most of which are defective and unable to replicate or transcribe functional proteins. However, given their capacity to move across the nuclear and mitochondrial genome and recombine, they could mix phenotypes and give rise to infections that may trigger innate and adaptive immune responses by sensing receptors capable of recognising foreign nucleic acids and proteins. It has recently been suggested that they play a role in the pathogenesis of autoimmune diseases on the grounds of their partial reactivation or the epigenetic control of host gene transcription. A number of studies have confirmed their contribution to the development of rheumatoid arthritis, multiple sclerosis and systemic lupus erythematosus, but there is still a lack of data concerning systemic sclerosis (SSc). Their role in the pathogenesis of SSc can be hypothesised on the basis of mitochondrial and nuclear chromatinic damage, and hyper-activation of the immune pathway involved in antiviral defense. SSc is characterised by genetic and immunological evidence of a viral infection but, as no viral agent has yet been isolated from SSc patients, the hypothesis that partial reactivation of endogenous retroviruses may trigger the disease cannot be excluded and deserves further investigation.

The association between hepatitis B, hepatitis C and systemic sclerosis: a cross-sectional study.

PURPOSE OF REVIEW: To investigate the association between systemic sclerosis (SSc) to chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) carriage. We utilized the database of Clalit Health Services, the largest healthcare organization in Israel and performed a cross-sectional study. RECENT FINDINGS: The study included 2431 SSc patients and 12 710 age-and-sex matched controls, HBV was found in 38 SSc patients (1.56%) and 64 controls (0.5%). HCV was found in 30 SSc patients (1.23%) and 83 controls (0.65%). In multivariable logistic regression model, HBV was found to be associated with smoking, dialysis treatment and SSc [odds ratio (OR) 2.97, 95% confidence interval (CI) 1.92-4.53]. HCV was found to be associated with dialysis treatment and SSc (OR 1.73, 95% CI 1.1-2.66). A trend was found between both HBV and HCV toward low socioeconomic status. SSc patients with HBV had demonstrated higher rates of end-stage renal disease requiring dialysis treatment. SUMMARY: In our study, HBV and HCV were found to be associated with SSc. Common immune mechanisms or therapeutic modalities may serve as mediators of this association.

Effects of Parvovirus B19 In Vitro Infection on Monocytes from Patients with Systemic Sclerosis: Enhanced Inflammatory Pathways by Caspase-1 Activation and Cytokine Production.

Parvovirus B19 (B19V) has been proposed as a triggering agent for some autoimmune diseases including systemic sclerosis (SSc). In this study, we investigated whether B19V infection in vitro differently activates inflammatory pathways, including those dependent on caspase-1 activation, in monocytes from patients with SSc and healthy controls. We showed that B19V can infect both THP-1 cells and primary monocytes but is not able to replicate in these cells. B19V infection increases the production of tumor necrosis factor-α and induces NLRP3-mediated caspase-1 activation in both THP-1 cells differentiated with phorbol 12-myristate 13-acetate and in monocytes from patients with SSc but not from healthy controls. B19V infection was sufficient for THP-1 to produce mature IL-1ß. Monocytes from patients with SSc required an additional stimulus, here represented by lipopolysaccharides, to activate cytokine genes. Following B19V infection, however, lipopolysaccharide-activated monocytes from patients with SSc strongly increased the production of IL-1ß and tumor necrosis factor-α. Altogether, these data suggest that viral components might potentiate the response to endogenous and/or exogenous toll-like receptor 4 ligands in monocytes from patients with SSc. The B19V-mediated activation of inflammatory pathways in monocytes might contribute to the disease progression and/or development of specific clinical phenotypes.

Increased antibody levels to stage-specific Epstein-Barr virus antigens in systemic autoimmune diseases reveal a common pathology.

The immune responses to antigens from different stages of the Epstein-Barr virus (EBV) life cycle were investigated in systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Sjögren's syndrome (SS), and systemic sclerosis (SSc) to gain knowledge of EBV's involvement in the etiology of systemic autoimmune diseases (SADs) and for an overview of the humoral immune responses against EBV. Investigations were performed by the use of ELISA. IgM, IgA, and IgG antibody binding to 11 EBV antigens: EBNA1, EBNA2, BALF5, EAD, BALF2, EA/R, VCA p18, VCA p23, gB, gp350, and gp42 were examined in serum pools from SAD patients and healthy controls (HCs). Increased antibody levels against the 11 EBV antigens in the SAD pools were seen compared to the HC pool. Specifically, SLE was characterized by strongly increased IgA to EAD both compared to HCs and other SADs, and RA was characterized by increased IgM levels to several EBV antigens. The SADs may be partly distinguished by their differential immune responses to various antigens in the EBV life cycle. All together, these findings support an association between EBV infection and SADs.

Cytomegalovirus aggravates the autoimmune phenomenon in systemic autoimmune diseases.

BACKGROUND: Human Cytomegalovirus (CMV), because of its ability to extensively manipulate host immunity during active infection, has been suggested to be involved in autoimmunity. However, its influence on T-cells and cytokines in systemic autoimmune diseases like systemic lupus erythematosus (SLE) and systemic sclerosis (SSc) is indistinct. METHODS: We investigated the in-vitro response of T lymphocytes from SLE and SSc patients to CMV antigen. Functional activity of T lymphocytes was determined by estimating Th1 (IL-2 and IFN-γ) and Th2 (IL-4 and IL-10) cytokines. RESULTS: We observed that CMV antigen stimulation in-vitro resulted in significant increase in CD4:CD8 T-cell ratio in peripheral blood mononuclear cells (PBMCs) from SLE and SSc patients; response dominated by CD4+ than CD8+ memory T-cells. SSc T-cell response was differentiated by aberrant increase in CD4+CD25+ T-cells. CMV antigen caused elevation in IL-4 and IFN-γ production in both patient PBMCs, whereas IL-2 was also raised in SLE PBMCs. The development of large pool of memory T-cells and overproduction of IFN-γ may result in flare-up of autoimmunity in these patients. CONCLUSION: Our study provides an insight into the immunopathological potential of CMV-reactive immune cells to develop new potential strategies for targeted therapeutic intervention.

Pandemic non-adjuvanted influenza A H1N1 vaccine in a cohort of patients with systemic sclerosis.

Objective: To assess the possible effect of therapy, disease subtype and severity on H1N1 immunogenicity in patients with SSc. Methods: Ninety-two patients and 92 age- and gender-matched healthy controls received adjuvant-free influenza A/California/7/2009 (pH1N1) vaccine. Blood samples were collected immediately before and 3 weeks after vaccination to evaluate antibody responses to the H1N1 virus. Efficacy was assessed by seroprotection (SP) and seroconversion (SC) rates and the factor increase in geometric mean antibody titre. Participants received a 21-day symptom diary card and were instructed to report local and systemic adverse events. Results: SSc patients were predominantly females (91%) and 61% had limited SSc, 12% had severe skin involvement and 57.6% were on immunosuppressive (IS) therapy. SSc patients and controls presented comparable overall SP (P = 0.20) and SC (P = 0.61) rates. Further evaluation of the possible effect of disease and therapy revealed similar rates of SP and SC in patients with dcSSc vs lcSSc (SP P = 0.62 and SC P = 0.66), severe vs mild/moderate skin involvement (SP P = 1 and SC P = 0.45) and with vs without IS (SP P = 0.26 and SC P = 0.10). The frequency of mild local and minor systemic reactions was similar in patients with dcSSC vs lcSSc (P = 0.70 vs 0.32) and in those with and without severe skin involvement (P = 0.59 vs 0.28). Conclusion: The non-adjuvanted influenza H1N1 virus vaccine proved to be safe and effective, independent of SSc clinical subtype, disease severity or therapy. These latter factors do not seem to contribute to mild adverse events observed in SSc. Our data support the annual influenza vaccination recommendation for these patients. Trial registration: ClinicalTrials.gov (http://clinicaltrials.gov), NCT01151644.

From microbiome to infectome in autoimmunity.

PURPOSE OF REVIEW: The current review discusses the pros and cons of the microbiome studies conducted in search of the association between microbiota and autoimmunity. RECENT FINDINGS: We focus on the role of infectome and autoinfectome as a bridge to link the findings of microbiome studies with those emerging from investigations of the role of specific viruses and antiviral responses as triggers of autoimmunity (through various mechanisms such as molecular mimicry). The 'usual suspects', such as herpetoviruses and Escherichia coli, are thoroughly discussed in light of the data emerged by the microbiome studies, using as examples specific autoimmune rheumatic diseases and inflammatory bowel diseases. SUMMARY: We conclude that the studies of the oral cavity, gastrointestinal, and urinary tract microbiome are informative but can only be useful if further explored from the infectome perspective. This means that the plethora of bacteria associated with autoimmune diseases from microbiome studies can be and must be tested experimentally. If certain bacteria are associated directly or indirectly with autoimmune diseases, specific immunological mechanisms must be identified.

Human cytomegalovirus (HCMV) UL44 and UL57 specific antibody responses in anti-HCMV-positive patients with systemic sclerosis.

The role of human cytomegalovirus (HCMV) has been postulated as a trigger of systemic sclerosis (SSc). The aim of the study was to assess the prevalence of antibodies against HCMV UL44 and UL57 antigens not tested in the past. Sixty SSc patients, 40 multiple sclerosis and 17 normal controls (NCs), all anti-HCMV positive, were tested by immunoblotting. Reactivity to HCMV antigens, expressed as arbitrary units (AUs), was assessed for correlation with clinical and immunological parameters, including types of SSc-related autoantibodies. Anti-UL44 and anti-UL57 HCMV antibodies were present in 3/60 (5%) and 58/60 (96.7%) SSc patients, respectively (p < 0.001). Anti-UL57 antibodies were present in 35/40 (87.5%) MS patients and 16/17 (94.1%) NCs (SSc vs MS, MS vs NC, p = ns). Strong (50-75 AU) and very strong (75-100 AU) anti-UL57 immunoreactivity was found in 24 (41.4%) and 22 (37.9%) SSc patients, respectively (p = ns). Dilution experiments showed anti-UL57 antibody persistence in up to 1/5000. Overall, there was no difference in the frequency or the magnitude of anti-UL57 immunoreactivity between diffuse cutaneous systemic sclerosis and limited cutaneous systemic sclerosis patients (96.67 vs 96.67%; 65.45 ± 20.19 vs 64.31 ± 21.11 AU, p > 0.05) but strong anti-UL57 reactivity were more frequent in SSc compared to NCs (p = 0.007). Anti-UL57 reactivity was not inhibited by SSc-specific autoantigens. Anti-UL57 seropositivity did not correlate with demographic, clinical or immunological features of SSc. Anti-HCMV UL57 antibodies are universally present in anti-HCMV-positive patients with SSc, while those against UL44 are rarely seen. Because anti-UL57 lack disease specificity and are not involved in cross-reactive responses, their immunopathogenetic potential is to be questioned.

Epstein-Barr virus infection induces aberrant TLR activation pathway and fibroblast-myofibroblast conversion in scleroderma.

Scleroderma (SSc) is a complex and heterogeneous connective tissue disease mainly characterized by autoimmunity, vascular damage, and fibrosis that mostly involve the skin and lungs. Epstein-Barr virus (EBV) is a lymphotropic γ-herpesvirus that has co-evolved with human species, infecting >95% of the adult population worldwide, and has been a leading candidate in triggering several autoimmune diseases. Here we show that EBV establishes infection in the majority of fibroblasts and endothelial cells in the skin of SSc patients, characterized by the expression of the EBV noncoding small RNAs (EBERs) and the increased expression of immediate-early lytic and latency mRNAs and proteins. We report that EBV is able to persistently infect human SSc fibroblasts in vitro, inducing an aberrant innate immune response in infected cells. EBV-Toll-like receptor (TLR) aberrant activation induces the expression of selected IFN-regulatory factors (IRFs), IFN-stimulated genes (ISGs), transforming growth factor-ß1 (TGFß1), and several markers of fibroblast activation, such as smooth muscle actin and Endothelin-1, and all of these genes play a key role in determining the profibrotic phenotype in SSc fibroblasts. These findings imply that EBV infection occurring in mesenchymal, endothelial, and immune cells of SSc patients may underlie the main pathological features of SSc including autoimmunity, vasculopathy, and fibrosis, and provide a unified disease mechanism represented by EBV reactivation.

Role of viral infections in the etiopathogenesis of systemic sclerosis.

Systemic sclerosis or scleroderma (SSc) is a clinically heterogeneous disease of the connective tissue characterised by vascular, immune/inflammatory and fibrotic manifestations. Despite extensive investigations, the key pathogenic links between these disease hallmarks remain obscure, as well as the etiology underlying the beginning of this complex disorder. As for other diseases characterised by prominent autoimmune phenomena, the search for infectious agents responsible for immune tolerance breaks or molecular mimicry events has been a long-pursued issue. In this review, we summarise the current knowledge regarding the association of different viral infections with SSc, focusing mainly on those reports describing a mechanistic interplay between the viral agents and the pathogenesis of SSc. Moreover, we speculate on how viral infections may trigger additional pathogenic mechanisms recently proposed to contributing to SSc phenotype.