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Sjögren syndrome
Original research
Hyperactivation and altered selection of B cells in patients with paediatric Sjogren’s syndrome
  1. Alessandra Boni1,
  2. Rebecca Nicolai1,
  3. Ivan Caiello2,
  4. Francesca Marinaro2,
  5. Luciapia Farina2,
  6. Denise Pires Marafon1,
  7. Rita Carsetti3,
  8. Fabrizio De Benedetti1,
  9. Claudia Bracaglia1 and
  10. Emiliano Marasco1
  1. 1Division of Rheumatology, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy
  2. 2Laboratory of Immuno-Rheumatology, Bambino Gesù Children's Hospital, IRCCS, Roma, Italy
  3. 3B cell unit, Immunology Research Area, Bambino Gesù Children's Hospital, IRCCS, Roma, Italy
  1. Correspondence to Dr Claudia Bracaglia; claudia.bracaglia{at}opbg.net

Abstract

Objectives Paediatric Sjögren’s syndrome (pSS) is a rare chronic autoimmune disorder, characterised by inflammation of exocrine glands. B cell hyperactivation plays a central role in adult-onset Sjogren. This study was designed to analyse B cell and T cell phenotype, levels of BAFF, and selection of autoreactive B cells in patients with pSS.

Methods A total of 17 patients diagnosed with pSS and 13 healthy donors (controls) comparable for age were enrolled in the study. B cell and T cell subsets and frequency of autoreactive B cells in peripheral blood were analysed by flow cytometry. Levels of BAFF were analysed by ELISA.

Results The relative frequency of total B cells, transitional, naïve and switched memory B cells was similar between pSS patients and controls. In patients with pSS, we observed a reduction in the frequency of unswitched memory B cells, an increased frequency of atypical memory B cells and an expansion of PD1hi CXCR5− T peripheral helper cells. Levels of BAFF were higher in patients with pSS compared with controls and correlated with serum levels of total IgG and titres of anti-Ro antibodies. The frequency of autoreactive B cells in the transitional, unswitched memory and plasmablast compartment was significantly higher in pSS patients than in controls.

Conclusions Our results point to a hyperactivation of B cells in pSS. Current therapies do not seem to affect B cell abnormalities, suggesting that novel therapies targeting specifically B cell hyperactivation need to be implemented for paediatric patients.

  • B cells
  • Autoantibodies
  • Cytokines

Data availability statement

Data are available on reasonable request. All data relevant to the study are included in the article or uploaded as online supplemental information. The authors confirm that the data supporting the findings of this study are available within the article and/or its online supplemental materials. Data not shown in the paper will be made available on request.

http://creativecommons.org/licenses/by-nc/4.0/

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/.

https://doi.org/10.1136/rmdopen-2023-003800

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    WHAT IS ALREADY KNOWN ON THIS TOPIC

    • Clinical and laboratory evidence suggests that, similarly to adult-onset Sjögren syndrome (SS), B cells play a central role in the pathogenesis of paediatric-onset SS (pSS), although the molecular mechanisms of this are not clearly understood yet.

    WHAT THIS STUDY ADDS

    • We provide evidence of a hyperactive B cell phenotype in patients with pSS with an increased frequency of atypical memory B cells and an expansion of PD1hiCXCR5− T peripheral helper cells. We also show that B cell tolerance checkpoints are impaired both at the central and peripheral level.

    HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

    • Our data show that current therapies are not reverting B cell abnormalities in pSS. Thus, patients with pSS may benefit from new drugs targeting specifically the activation or selection of B cells.

    Introduction

    Paediatric Sjögren’s syndrome (pSS) is a rare chronic autoimmune disorder, characterised by inflammation of exocrine glands, predominantly salivary and lacrimal glands, with varying degrees of systemic involvement.1 Patients with pSS present with a different clinical picture from adult patients with SS, with classic ‘sicca symptoms’ being less frequent and with a higher proportion of glandular swelling (i.e., recurrent parotitis) and extraglandular manifestations.1 2 The lack of validated paediatric diagnostic criteria for pSS and of specific biomarkers translates to a diagnostic delay and an underdiagnosis of pSS.3 4

    The pathogenesis of pSS is complex and incompletely understood: environmental factors and predisposing genetic factors participate in triggering an autoreactive immune response.5 Patients with SS show biological signs of B cell hyperactivation, including polyclonal hypergammaglobulinaemia, presence of autoantibodies (antinuclear antibodies (ANA) and rheumatoid factor (RF)), increased levels of survival and differentiation factors for B cells, such as BAFF and interleukin-21, disturbances in B cell subsets6 and increased antibody responses to vaccination.7 Moreover, B cells from treatment naïve SS patients show an increased ability to differentiate into plasmablasts and class switch to IgG.7 Adult patients with SS are also at increased risk of B cell lymphoma (mainly mucosa-associated lymphoid tissue lymphomas).5 B cells infiltrate the salivary glands of patients with SS and can be found organised in ectopic germinal centres (GCs).5 The precise generation of autoreactive B cells in SS has not been determined yet. Mouse models of autoimmunity and analysis of B cell distribution in patients with SS showed that autoreactive B cells can be generated by activated B cells in GC or in the extrafollicular (EF) areas and also positively selected from autoreactive B cell precursors.8 9 High levels of B cell activating factor (BAFF) alter the selection of B cell precursors reducing the threshold for B cell activation and support autoreactive B cells reducing B cell apoptosis.10 Transgenic mice overexpressing BAFF develop features of systemic autoimmunity similar to systemic lupus erythematosus (SLE) and SS.11

    Further research is needed to better understand the pathological process underlying pSS, in order to develop early robust biomarkers for diagnosis and disease monitoring and to develop effective targeted therapies. We set out to investigate the distribution of B cells subsets and of T follcular helper icular helper (TFH) cells, the levels of B cell-cytokine and tolerance checkpoints in patients with pSS.

    Methods

    Patients and study design

    A total of 17 patients diagnosed with SS before the age of 18 and fulfilling the criteria proposed by Bartůnková et al3 were enrolled in the study. Patients were enrolled at the division of Rheumatology of Bambino Gesù Children’s Hospital. For each patient, clinical data and laboratory parameters at disease onset and during follow-up visits were recorded. Systemic activity was evaluated by European League Against Rheumatism (EULAR) Sjogren’s Syndrome Disease Activity Index (ESSDAI) according to EULAR recommendations.12 Demographic and clinical features and serological parameters are shown in table 1 and online supplemental table 1.

    View this table:
    Table 1

    Demographic, clinical and laboratory characteristics of patients with pSS at disease onset and controls (Ctrl)

    Biological samples were retrieved retrospectively from our biobank for the 17 patients with pSS. For seven patients, we identified peripheral blood mononuclear cell (PBMC) samples taken at diagnosis and before the initiation of any immunosuppressive treatment; these samples were used to evaluate B cell phenotype at disease onset. For six of these patients, we identified a sample of PBMC obtained a median of 13.1 (IQR 12.0–16.2) months after disease onset and after initiation of treatment (one patient was lost at follow-up and samples after therapy initiation were not available). For 10 patients, we identified in our biobank plasma samples collected before the initiation of any treatment; samples were used to assess levels of BAFF at disease onset. For 12 patients, we identified PBMC samples taken after the initiation of therapy and these were used to perform TFH phenotyping and B cell autoreactivity assessment (patients receiving rituximab (RTX) were excluded from this analysis). Control subjects (Ctrl) (n=13) comparable for age were identified among children and adolescents referred to our unit for evaluation because of musculoskeletal pain, who were confirmed not to have rheumatological diseases or immunodeficiencies.

    Immunophenotyping

    PBMCs were isolated by Ficoll density gradient centrifugation and frozen. PBMCs were thawed, counted and assessed for viability and stained with a cocktail of antibodies. The list of antibodies for staining is shown in online supplemental table 2. Samples were run on a LSR Fortessa-X-20 (BD). Data were analysed with FlowJo V.8.3 (TreeStar). Gating strategy is shown in online supplemental figure 1.

    Flow cytometry ANA staining was performed as previously described.13 14 HeLa cells were cultured in Dulbecco's Modified Eagle Medium (DMEM), 10% Fetal Calf Serum, 1% glutamine, 1% antibiotics/antimycotics. Before preparing nuclear extracts, cell lines were screened for the presence of mycoplasma, and only mycoplasma-free cell lines were used for nuclei extract preparation. Nuclei were isolated from cultured HeLa cells using nuclei EZ prep (Sigma-Aldrich). The isolated nuclei were lysed by mechanical force and the lysed product was cleared by centrifugation at 14 000 rpm for 10 min. The supernatant was labelled with Sulfo-NHS-LC-biotin (Thermoscientific) for 30 min.

    Cytokine and autoantibodies measurement

    Human BAFF ELISA kits were purchased from R&D Systems, Minneapolis, Minnesota, USA. All cytokines were quantified according to the manufacturers’ instructions. Anti-Ro autoantibodies were assayed with a fluorescence enzyme immunoassay (EliA, Phadia AB, Sweden).

    Statistical analyses

    All statistical analyses were performed using software R version R-4.0.3 (R Core Team, 2020). Data were checked for normality. For not normally distributed data, non-parametric statistical tests were employed. Spearman’s correlation coefficients (r) were calculated to describe correlations between variables. We set significance levels at p<0.05.

    Results

    B cell subsets and TFH cells in patients with paediatric-onset SS

    Analysing B cell phenotype of pSS patients at disease onset and before initiation of any treatment, we observed a reduction in the frequency of unswitched memory B cells and an increased frequency of atypical memory B cells compared with controls (figure 1A). We further analysed the phenotype of the atypical memory B cells: they lacked the expression of CXCR5 and expressed CD11c (figure 1C). The relative frequency of total CD19+ B cells, transitional, naïve and switched memory B cells and plasmablasts was similar between pSS patients and controls (figure 1A). We did not observe any correlation between B cell subsets and disease activity measured by ESSADI (data not shown).

    Figure 1
    Figure 1

    B cell and T cell phenotype in patients with pSS at disease onset and controls. (A) Percentage of CD19+, transitional, naïve, unswitched memory (Unsw) and switched memory (Sw), atypical memory B cells and plasmablasts (PBs) in patients with pSS (n=7) and controls (Ctrl) (n=13). (B) Percentage of PD1hi CXCR5+ TFH cells and of PD1hi CXCR5− TPH cell, and ratio of PD1hi CXCR5+ and PD1hi CXCR5− T helper cells in patients with pSS (n=7) and controls (Ctrl) (n=13). (C) Expression of CXCR5 and CD11c by atypical memory, naïve, Sw and unswitched memory B cells. Each dot represents the frequency of an individual subject. *p<0.05, **p<0.01. pSS, paediatric Sjögren’s syndrome.

    We then evaluated if these alterations in B cell subsets were modified by treatment. For six patients, samples of PBMC were available in our biobank after treatment initiation. As shown in figure 2, no statistically significant changes were observed before and after treatment, except for a reduced frequency of total CD19+ B cells after treatment. This difference was driven by the two patients treated with RTX.

    Figure 2
    Figure 2

    Percentage of CD19+ total B cells, transitional, naïve, unswitched memory (Unsw), switched memory (Sw), atypical memory B cells and plasmablasts (PBs) in patients with pSS at disease onset (pre) and at follow-up after the initiation of therapy (post) (n=6). *p<0.05. pSS, paediatric Sjögren’s syndrome.

    We, then, assessed the frequency of circulating TFH cells in patients with pSS. Two main TFH cell subsets were defined as shown in online supplemental figure 1: PD1hi CXCR5+ conventional TFH and PD1hi CXCR5− T peripheral helper (TPH) cells.15 We observed a significant expansion of PD1hi CXCR5− TPH cells in patients with pSS compared with controls; the difference was even more pronounced when we analysed the relative frequency of PD1hi CXCR5− T cells to PD1hi CXCR5+ (figure 1B). Thus, in patients with pSS, we observed a reduction in unswitched memory B cells and an expansion of atypical memory B cells and of TPH cells. B cell alterations were not affected by treatment.

    Levels of BAFF in patients with paediatric-onset SS

    At disease onset, before the initiation of treatment, plasma levels of BAFF were higher in patients with pSS compared with controls (figure 3A). We analysed the correlation of BAFF levels with clinical and laboratory features of pSS patients: we observed a significant positive correlation between levels of BAFF and plasma levels of total IgG (R2=0.67, p=0.0068) and titres of anti-Ro antibodies (R2=0.44, p=0.037) (figure 3B); we did not observe any correlation with disease activity measured with ESSDAI (data not shown). We, then, investigated if levels of BAFF correlated with B cell subsets: levels of BAFF strictly correlated with the frequency of transitional B cells (R2=0.81, p=0.0056) (figure 3C).

    Figure 3
    Figure 3

    Levels of BAFF in patients with pSS at disease onset and controls. (A) Plasma levels of BAFF in patients with pSS (n=10) and controls (n=13). (B) Correlation of levels of BAFF with levels of total serum IgG and titres of anti-Ro autoantibodies (n=10). (C) Correlation of levels of BAFF with frequency of tTransitional B cells (expressed as % of CD19+ B cells). Each dot represents the levels of BAFF of an individual subject. *p<0.05. BAFF, B cell activating factor; pSS, paediatric Sjögren’s syndrome.

    Defective B cell tolerance checkpoints in paediatric-onset SS

    In patients with adult-onset SS, both central9 and tissue-restricted defective B cell tolerance checkpoints8 have been described. In order to analyse B cell tolerance checkpoints in our cohort of patients with pSS, we analysed the frequencies of ANA+ B cells within the transitional, naïve, unswitched memory, switched memory and plasmablast compartment (figure 4A). In controls, the frequency of ANA positivity was greatest in the transitional compartment and declined as B cells matured into naive and memory cells (figure 4A,B). In patients with pSS, the frequency of ANA+ B cells showed the same trend; however, in the transitional and unswitched memory compartment, the frequency of ANA+ B cells was significantly higher in pSS patients than controls (figure 4B). Interestingly, the frequency of ANA+ plasmablasts was higher in patients than in controls (figure 4B). In summary, this analysis revealed for the first time that negative selection of ANA+ B cells in the transitional, unswitched memory and plasmablast compartments is less stringent in patients with pSS compared with controls.

    Figure 4
    Figure 4

    Antinuclear reactivity in peripheral B cell subsets from pSS patients and controls. (A) Contour plots of ANA+ cells for a representative pSS patient and a control subject. B cells recognising nuclear antigens (ANA+B cells) were identified within each B cell subset. (B) Violin plots depicting the frequency of ANA+ B cells within each B cell subset in pSS patients (n=12) and healthy controls (n=13). Each dot represents the frequency in an individual subject. **p<0.01, ***p<0.001. ANA, antinuclear antibody; PBs, plasmablasts; PSS, paediatric Sjögren’s syndrome; Sw_M, switched memory B cells; Unsw_M, unswitched memory B cells.

    Discussion

    We showed that patients with pSS have an altered B cell phenotype at disease onset, with a reduction in unswitched memory B cells and an expansion of atypical memory B cells. Patients with pSS showed higher levels of BAFF, and these correlated with serum levels of total IgG, with titres of anti-Ro autoantibodies, and, markedly, with frequency of transitional B cells. Both central and peripheral tolerance checkpoint of B cells appeared to be defective in patients with pSS: we observed an expansion of ANA+ B cells in the transitional, unswitched memory and plasmablast compartments, whereas the frequency of ANA+ naïve and switched memory B cells was similar between pSS patients and controls. Finally, we also observed an expansion of TPH cells, a subset of T cells that provides B cell help outside the B cell follicle and in inflamed tissues. To the best of our knowledge, this is the first description of a hyperactive B cell phenotype in patients with pSS.

    B cells hyperactivation plays a central role in the pathogenesis of Sjogren; however, less is known about the role of B cells in the pathogenesis of Sjogren in children. Clinical features are different between adulthood-onset Sjogren and pSS,1 suggesting potentially different pathogenetic mechanisms between the two groups. While adults typically experience the classical ‘sicca syndrome’, these symptoms are noticeably less common in the paediatric population.1 2 Children are more likely to experience glandular inflammation, exemplified by recurrent episodes of parotitis; they also have a greater incidence of systemic manifestations.1 2 As a consequence of this, in a study by Basiaga et al, around two-thirds of young patients identified with SS failed to fulfil the 2016 American College of Rheumatology (ACR)/EULAR classification criteria for Sjogren.4 This discrepancy may arise from true biological differences in the disease’s pathophysiology between children and adults or could be attributed to the earlier stage at which SS is detected in children, where inflammatory symptoms predominate before the emergence of the ‘sicca syndrome’ typically observed at a more advanced stage of the disease.16

    We showed that in pSS, similarly to adult patients,17 a reduction in unswitched memory B cells is present. We also observed an expansion of atypical memory B cells in pSS patients. Unswitched memory B cells develop in the spleen in infants and mature in the GC throughout life.18 They provide a first line of defences against pathogens and secrete antibodies that recognise T-independent antigens like polysaccharides.18 They are also a source of RF, one of the autoantibodies present in Sjogren, that can also be the precursors of the B cell lymphoma clones observed in Sjogren.8 Two main reasons could explain this reduction in unswitched memory B cells: this subpopulation migrates into target tissues, infiltrating salivary glands.8 Another explanation is that B cell development and homoeostasis are altered in pSS, leading to a reduced generation of unswitched memory B cells, for example, through reduced splenic functions.

    In our cohort of patients with pSS, we observed an expansion of atypical memory B cells. Memory B cells with an atypical phenotype were found expanded in patients with chronic infections, immunodeficiency and autoimmunity.19 In the context of SARS-CoV-2 infection, atypical memory B cells expand and display features of EF responses.20 EF responses play a crucial role in the activation of autoreactive B cells, for example, in mouse models of autoimmunity, RF+ B cells are activated in EF areas.21 We showed that atypical memory B cells lacked the expression of CXCR5, the receptor that, binding to CXCL13, drives B cells and TFH cells to the B cell area and the GC.22 Thus, atypical memory B cells do not have the potential to migrate to the B cell follicle; they probably home to the EF areas and/or to inflamed tissues. BAFF has been implicated in the activation of B cells in the EF areas.11 We observed increased levels of BAFF that correlated with total IgG and anti-Ro autoantibodies, pointing to a role of this cytokine in the activation of autoreactive B cells in the EF areas. Interestingly, in a longitudinal analysis, B cell abnormalities were not affected by immunosuppressive therapy; specifically, the reduction in unswitched memory B cells and the expansion in atypical memory B cells are still present after treatment. This mirrors the evidence that autoantibodies (ANAs, anti-Ro and anti-La antibodies) persist over time and after treatment in patients with pSS (manuscript in preparation). In summary, it appears that while existing immunosuppressive therapies are beneficial for managing inflammation, they do not substantially impact the B cell abnormalities associated with pSS and their production of autoantibodies.

    TFH cells, a specific subset of T cell, provide help to B cells for their activation, selection and differentiation. Several types of TFH cells have been described: classical TFH cells migrate to the B cell areas, TPH cells provide help to B cells in the EF areas and in the inflamed tissues.15 In our cohort of pSS patients, we observed an expansion of TPH cells that may provide help to autoreactive B cells in EF areas and in inflamed salivary glands, further supporting the importance of both the EF responses and the tissues in the autoreactive responses in pSS.8

    The impaired removal and the positive selection of autoreactive B cells is central in the pathogenesis of SS. Alterations in both central and peripheral tolerance mechanisms have been described in autoimmune diseases, including SS.9 Autoreactive B cells were found to be expanded in salivary glands in SS, where they are present within a polyclonal repertoire; in peripheral blood no clones recognising Ro and La antigens were observed.8 Different autoantigens showed different degrees of antigen-dependent affinity maturation and class-switching in tissues.8 In our cohort of pSS patients, B cells showed defects in both central and peripheral checkpoints: transitional B cells showed higher frequency of autoreactive B cells than controls, indicating loose central tolerance checkpoints. BAFF may play a role in survival and selection of transitional B cells: we indeed observed that levels of BAFF correlated positively with the frequency of transitional B cells (figure 3C), but not with other B cell subsets (data not shown). Unswitched memory B cells showed a higher frequency of autoreactivity: this may reflect an altered selection process either in the generation of unswitched memory B cells from the transitional and naïve compartment, or from activated B cells in the periphery, for example, in the EF areas. Moreover, as in SLE, we observed a defective tolerance checkpoint in the differentiation of activated B cells to plasmablasts.13 Increased levels of BAFF may play a role in the activation and differentiation of autoreactive B cells (but also non-autoreactive B cells) into plasmablasts as suggested by the correlation in pSS patients of BAFF levels with total IgG and anti-Ro antibodies titres. We did not observe an increased frequency of autoreactive switched memory B cells, indicating a proper negative selection process in the GC. One of the limitations of our study is that we analysed peripheral B cells, and not B cells in target tissues; thus, analysing tolerance checkpoints in the periphery may not reflect defective tolerance mechanisms occurring in tissues. One explanation for this is that B cells reactive to autoantigens specific for Sjogren (Ro, La, RF) were detected in salivary glands but not in the periphery.8 In tissues, B cells reactive to Ro and La showed signs of antigen-dependent affinity maturation, thus pointing to an altered selection process in GC.8 We did not observe impaired selection processes in peripheral switched memory B cells, the output of GC. However, this cannot rule out that in tissues, autoreactive clones expand and are positively selected in local GC in pSS.

    We did not observe an association of B cell subsets and BAFF levels with disease activity and clinical feature of pSS (data not shown): this can be due to the relatively small number of patients enrolled in this study, a factor that is largely due to the rarity of pSS. The B cell abnormalities described here were observed in patients at disease onset before the initiation of immunosuppressive treatment, thus, we are confident that our results on B cell phenotype and BAFF levels are due to the disease itself with no effect from therapy. As a matter of fact, current therapeutic strategies did not affect the decrease in unswitched memory B cells or the increase of atypical memory B cells. This fact underscores the need for novel treatments that specifically target the excessive activation of B cells in pSS. For instance, regulating BAFF levels with the use of belimumab, moderating B cell activation through BTK inhibitors, or disrupting the interaction between B cells and T cells by using drugs that inhibit costimulation—such as those that block CD40:CD40L or ICOS:ICOSL interactions or by using CTLA4-fusion proteins such as abatacept—might prove effective. While some of these drugs are undergoing trials in adult SS, the distinct clinical profile in children, characterised by more pronounced inflammatory symptoms and less damage, suggests that these medications might yield even greater efficacy if administered early in the course of the disease in the paediatric group.

    In summary, our data point to a hyperactivation of B cells in pSS. A reduction in unswitched memory B cells and an expansion of atypical memory B cells and TPH cells suggest that an aberrant EF response plays a role in the pathogenesis of pSS. We observed increased levels of BAFF that is involved in both B cell selection and activation. Both central and peripheral tolerance checkpoints are altered in patients with pSS, including the differentiation of autoreactive B cell clones into plasmablasts, allowing secretion of autoreactive antibodies. Current therapies do not seem to affect B cell abnormalities, suggesting that novel therapies targeting specifically B cell hyperactivation in pSS need to be implemented.

    Data availability statement

    Data are available on reasonable request. All data relevant to the study are included in the article or uploaded as online supplemental information. The authors confirm that the data supporting the findings of this study are available within the article and/or its online supplemental materials. Data not shown in the paper will be made available on request.

    Ethics statements

    Patient consent for publication

    Ethics approval

    This study involves human participants and the study was approved by the Bambino Gesù Children Hospital ethics committee (protocol number 1666_OPBG_2018). All patients and their families provided written informed consent. Participants gave informed consent to participate in the study before taking part.

    Acknowledgments

    We thank Mrs Aurora Pucacco for her help in patient enrollment and sample collection.

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    Supplementary materials

    Footnotes

    • AB and RN contributed equally.

    • Contributors AB collected the data, performed the analyses and wrote the manuscript. RN collected the data, performed the analyses and wrote the manuscript. IC collected the data, performed the analyses and wrote the manuscript. FM collected the data, performed the analyses and wrote the manuscript. LF collected the data, performed the analyses and wrote the manuscript. DPM performed the analyses, interpreted the results and wrote the manuscript. RC supervised the work, interpreted the results and wrote the manuscript. FDB supervised the work, interpreted the results and wrote the manuscript. CB performed the analyses, interpreted the results and wrote the manuscript. EM collected the data, performed the analyses and wrote the manuscript. EM, CB, AB, RN and FDB conceived and designed the analyses, supervised the work and wrote the manuscript. EM, CB and RN are responsible for the overall content as guarantor.

    • Funding This work was supported by the Italian Ministry of Health with 'Current Research funds'.

    • Competing interests None declared.

    • Provenance and peer review Not commissioned; externally peer reviewed.

    • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.