key: cord-1040039-v3qhdtd4
authors: Verstappen, Gwenny M.; Pringle, Sarah; Bootsma, Hendrika; Kroese, Frans G. M.
title: Epithelial–immune cell interplay in primary Sjögren syndrome salivary gland pathogenesis
date: 2021-04-28
journal: Nat Rev Rheumatol
DOI: 10.1038/s41584-021-00605-2
sha: a48a8e1003e6264a1a35287ab99e4260575a9458
doc_id: 1040039
cord_uid: v3qhdtd4

In primary Sjögren syndrome (pSS), the function of the salivary glands is often considerably reduced. Multiple innate immune pathways are likely dysregulated in the salivary gland epithelium in pSS, including the nuclear factor-κB pathway, the inflammasome and interferon signalling. The ductal cells of the salivary gland in pSS are characteristically surrounded by a CD4(+) T cell-rich and B cell-rich infiltrate, implying a degree of communication between epithelial cells and immune cells. B cell infiltrates within the ducts can initiate the development of lymphoepithelial lesions, including basal ductal cell hyperplasia. Vice versa, the epithelium provides chronic activation signals to the glandular B cell fraction. This continuous stimulation might ultimately drive the development of mucosa-associated lymphoid tissue lymphoma. This Review discusses changes in the cells of the salivary gland epithelium in pSS (including acinar, ductal and progenitor cells), and the proposed interplay of these cells with environmental stimuli and the immune system. Current therapeutic options are insufficient to address both lymphocytic infiltration and salivary gland dysfunction. Successful rescue of salivary gland function in pSS will probably demand a multimodal therapeutic approach and an appreciation of the complicity of the salivary gland epithelium in the development of pSS.

Nearly 100 years ago, Henrik Sjögren described a dis ease whereby patients had a reduced functionality of the salivary glands (xerostomia) and lacrimal glands (xerophthalmia), with accompanying disturbances of the cornea and the conjunctiva (keratoconjunctivitis sicca) 1 . Sjögren syndrome, named after his work, has since been extensively characterized and is now considered a sys temic autoimmune disease. Primary Sjögren syndrome (pSS) typically refers to the occurrence of Sjögren syn drome as the first clinically presenting autoimmune con dition in a patient. In terms of clinical presentations, pSS is associated with dryness of the mouth, eyes and vagina, chronic pain and fatigue, and numerous possible extrag landular organ manifestations (including neuropathies, pulmonary manifestations and nephritis) 2 .

pSS is mirrored in pathological and biological analy sis by the presence of lymphocytic infiltration in the salivary glands (focal lymphocytic sialadenitis) and autoantibodies in the blood. The salivary gland biopsy and its analysis have an important role in the diagno sis and classification of pSS 3 . Introduced in 1974, the focus score (that is, the number of lymphocytic foci per 4 mm 2 tissue) has been relied upon heavily for the histological assessment of salivary gland involvement in pSS 4 . Infiltrating cells, congregated in foci around the striated ducts, consist mostly of CD4 + T cells and B cells, although other immune cells, including (but not lim ited to) myeloid dendritic cells, plasmacytoid dendritic cells (pDCs) and follicular dendritic cells, might also be present 5, 6 . These infiltrates can develop into ectopic lymphoid structures, even comprising ectopic germinal centres 7 . Particular attention has been paid to the role of (glandular) B cells in pSS pathogenesis. These cells are hyperactive in pSS and responsible for the formation of autoantibodies, hypergammaglobulinaemia, lymphoepi thelial lesions (LELs) and pSSrelated mucosaassociated lymphoid tissue (MALT) lymphoma [8] [9] [10] [11] .

Regarding autoantibodies, antiSSA/Ro (and to a lesser extent antiSSB/La) antibodies are present in the majority of patients with pSS 3 . Serum levels of antiSSA/Ro and antiSSB/La antibodies correlate with the number of corresponding plasma cells in the minor salivary glands of patients with pSS, suggest ing that salivary glands form an important niche for autoantibodysecreting cells 12 . The SSA/Ro antigen com prises two ribonucleoproteins, Ro52 and Ro60, which are both present in the cytoplasm of all mammalian cells. Although a direct contribution of autoantibodies to exo crine gland dysfunction in humans has not been proven, they have been linked to glandular immune activation 13 .

Epithelial-immune cell interplay in primary Sjögren syndrome salivary gland pathogenesis

The fact that autoantibodies, encompassing antiSSA/Ro antibodies, antiSSB/La antibodies and rheumatoid factor, can be present years before pSS diagnosis sug gests that an additional trigger is needed in the devel opment towards clinical disease 14 . The journey towards impaired function of the salivary glands in pSS, resulting in reduced saliva production and a notable decrease in patient quality of life, is most probably multifactorial and still somewhat enigmatic.

Far from being only a site where infiltrating immune cells assemble, the ductal epithelium has a central role in disease pathogenesis. In this Review, we discuss how interactions between the epithelium and the immune system in pSS contribute to both disease initiation and throughout different stages of disease development in patients with pSS. We focus on new insights into the development of abnormalities in epithelial cells, as well as innate immune signalling and T cell and B cell acti vation within the salivary glands. All studies cited are based on human samples, unless specifically otherwise stated.

Salivary gland epithelium abnormalities A complete discussion of the deterioration of salivary gland function in pSS necessitates comprehension of the foundation of the salivary gland: the epithelium. The salivary gland epithelium comprises several cell types, namely acinar, myoepithelial and ductal cells (Fig. 1a) . Acinar cells produce and secrete either watery or mucousrich saliva (referred to as serous or mucous acinar cells, respectively). This secretion is facilitated by the contraction of myoepithelial cells that envelope the acinar cell clusters. The secreted saliva is channelled and simultaneously modified through small interca lated ducts into striated ducts (both consisting of basal and luminal cell types), and finally through the larger excretory ducts into the mouth. This architecture can be applied to both the minor and major (parotid, subman dibular and sublingual) salivary glands. Although all salivary glands can be affected in pSS, the minor glands of the lower lip (the labial salivary glands (LSGs)) are most often used in diagnosis and research owing to their relative ease of access. Notably, minor salivary glands, including the LSGs, account for less than 10% of unstim ulated whole saliva production 15 . This caveat aside, the literature suggests that the volume of LSG lymphocytic infiltration correlates only weakly with the reduction in salivary gland function in pSS 16, 17 , implying that addi tional mechanisms or epitheliumspecific deficits are probably important in decreased salivary gland function in pSS.

Acinar cells are the salivaproducing workhorses of the salivary glands, controlling both the volume and pro tein content of saliva. All the acinar cells in the parotid gland, the major gland responsible for saliva production after stimulation, are serous cells. The action of chew ing or exposure of taste receptors to acidic substances stimulates these cells to secrete the digestive enzyme αamylase. pSS is associated with a decrease in the amount and activity of αamylase, strongly implying that serous acinar cells have a less than optimal func tion in pSS [18] [19] [20] . Despite a notable reduction in saliva production in pSS, the number and gross morphology of acinar cells in both the LSGs and the parotid sali vary gland often appears normal, implying that, even if defective, acinar cells remain viable in pSS. In support of this idea, various data show that the levels of FAS-FAS ligand (FASL)induced apoptosis in acinar cells is low in patients with pSS, although the levels are higher than those of control populations (such as healthy individ uals) [21] [22] [23] . However, data from studies in mice suggest that TNF is capable of inducing apoptosis of major sal ivary gland acinar cells; notably, acinar cells from the NOD mouse, a model of pSS, are more likely to undergo TNFinduced apoptosis than cells from agematched BALB/c (control) mice 24 . Conversely, treatment of human LSG acinar cells with TNF and IFNγ seems to induce the expression of the antiapoptotic genes ATF6 and ERAD 25 . To complicate the situation further, levels of sex hormones might also affect acinar cell apoptosis 26 . Taken together, no firm conclusions can be drawn from the available data regarding the degree of acinar cell apoptosis that occurs in the salivary glands in pSS.

The secretion of saliva by acinar cells begins with the engagement of the muscarinic 3 receptors on acinar cells by muscarinic neurotransmitters such as acetyl choline. Downstream of this event, an increase in cytosolic levels of Ca 2+ in these cells occurs, originating from both out side the cells and from intracellular internal endoplas mic reticulum stores 27 . The presence of autoantibodies directed against extracellular loops of the muscarinic 3 receptor implies that these very first stages of secretion might already be disturbed in pSS, or at least targeted by the immune system 28 (Fig. 1b) . Muscarinic receptor binding and the subsequent increase in cytosolic Ca 2+ culminates in inositol 1,4,5trisphosphate receptor (IP3R) signalling, via the secretory machinery com ponents phosphatidylinositol 4,5bisphosphate (PIP2) and synaptotagmin 1. Notably, the levels of IP3R and the expression of synaptotagmin 1 are decreased in salivary gland acinar cells of patients with pSS compared with

• Dysregulation of the functional machinery of acini, activation and apoptosis of ductal cells and defects in progenitor cell homeostasis all contribute to salivary gland dysfunction in primary Sjögren syndrome (pSS). • Unknown trigger(s) in salivary gland epithelial cells might activate innate immune responses and result in adaptive immune responses towards self-antigens, making epithelial cells both mediators and targets of the response. • Dysregulation of innate immune signalling pathways in salivary gland epithelial cells and consequent pro-inflammatory cytokine production by the epithelium probably contributes to salivary gland dysfunction. • Direct or indirect interference of autoantibodies with innate immune responses could perpetuate type I interferon activity in salivary gland epithelial cells and immune cells. • Increased activation of nuclear factor-κB and expression of pro-survival factors, together with enhanced proliferation, might predispose certain intraepithelial B cells to neoplastic changes, promoting mucosa-associated lymphoid lymphoma development. • The restoration of salivary gland function in patients with pSS might require treatment tailored to glandular pathology and multimodal therapeutic approaches, for example, a combination of immunotherapy and cell therapy.

www.nature.com/nrrheum in healthy individuals 29, 30 . Furthermore, both PIP2 and synaptotagmin 1 seem to localize to the acinar cell baso lateral membrane in pSS rather than to the apical mem brane, suggesting at least dysfunction of the secretory machinery 30 . Hence, various critical components of the secretory machinery in acinar are dysregulated in pSS.

Another mechanism proposed to interfere with the calcium signalling phase of saliva production in pSS involves the microRNA miR1423p. This microRNA is present in secreted exosomes derived from infiltrating T cells in the glands and is transferred to acinar cells. miR1423p targets important components of intracel lular Ca 2+ signalling, ultimately resulting in decreased saliva production 31 . In healthy scenarios, successful Ca 2+ signalling results in the activation of aquaporin (AQP) water channels, and water is transported out of the apical membrane of acinar cells. Notably, the expression of the various AQPs is increased (AQP5) or decreased (AQP1, AQP3 and AQP5) in patients with pSS compared with healthy individuals, and the ability of these channels to respond to muscarinic stimuli are decreased compared with individuals without pSS 32, 33 .

The function of saliva depends not only on water secretion but also on its protein content. Mucins, produced by the salivary gland mucous acinar cells, provide essential lubrication to facilitate swallowing, and are secreted via the apical pole of acinar cells 34 . Mucousproducing acinar cells are found in the LSGs, as well as in the submandibular and sublingual major salivary glands 34 . In pSS, the salivary mucin compo nents MUC5B and MUC7 are found outside the basal pole of LSG mucous acinar cells 35, 36 . Interestingly, MUC5B and MUC7 are capable of Tolllike receptor 4 (TLR4)mediated autocrine stimulation of CXCL8, TNF, IFNα, IFNβ, IL6 and IL1β production by LSG acinar cells 36 . Consequently, basally mislocalized mucins might contribute to glandular inflammation. However, the direc tion of causality between mucin disruption and acinar cell inflammation remains to be clarified, as does the relevance of mucin disruption in the larger, nonmucinproducing parotid salivary gland. The basal lamina, which is nor mally strictly localized to the basal pole of acinar cells to provide anchorage and polarization, is also disorganized in the salivary glands in pSS 37 . This change might contrib ute to the secretion of mucins at the wrong cellular pole, although further evidence is needed to support this idea. Intriguing further data suggest that the cytoplasm and endoplasmic reticulum are overloaded with a different mucin, MUC1, in LSG acinar cells in pSS, which might cause cellular stress and potentially increase the likelihood of apoptosis 35, 36, 38 . In a separate study, incubation of LSG acinar cells with TNF or IFNγ triggered both increased nuclear factorκB (NFκB) activity and overexpression and aberrant localization of MUC1 (reF. 38 ), implying that the aberrant distribution of mucins might be a conse quence of the inflammatory environment in the salivary glands in pSS, potentially culminating in cellular stress and cell death.

pSS is associated with the presence of autoantibod ies, most often with antibodies directed against SSA/Ro proteins. However, autoantibodies directed against the acinar cellassociated proteins parotid secretory protein, salivary protein 1 and carbonic anhydrase 6 have also been reported in some patients with pSS, in addition to those antibodies against the muscarinic 3 receptor mentioned above [39] [40] [41] . These findings open up debate as to whether acinarspecific autoantibodies have a poten tial role in the induction of cell death in acinar cells, or whether these antibodies are a downstream consequence of that process.

Acinar cells might thus contribute to the expression of proinflammatory cytokines in pSS and are grossly dys regulated in terms of the functional machinery necessary for saliva production (Fig. 1b) . The extent of apoptosis of the cells, and its relation to the development of salivary gland disease, requires further clarification. Despite the observed abnormalities in acinar cells of patients with pSS, these abnormalities do not cumulatively result in appreciable numbers of immune cells surrounding the acini, as the infiltrating cells accumulate mostly around the striated ducts. However, it remains possible that cytokines produced by periductal infiltrates affect acinar cells. Furthermore, the potentially pathogenic role of the small number of interstitial immune cells and immune cells located within the acini (for example, CD8 + T cells) 37 needs further investigation.

Complementing the acinar cells responsible for making saliva are the ductal cells. Aside from the channelling of saliva into the mouth, ductal cells are also respon sible for the extraction of sodium from, and the addi tion of potassium to, the saliva. This process, mediated via calciumactivated sodium and potassium channels, culminates in attainment of the hypotonic sodium and potassium concentrations necessary for the slightly acidic antimicrobial functions of saliva, and for the prevention of mineral loss from dental hard tissues 42 . In patients with pSS, the concentrations of sodium and chloride in the saliva are higher than in healthy individu als, potentially owing to a reduction in ion resorption by ductal cells and/or because the volume of water secreted from acinar cells is reduced 19, 20, 43, 44 . The investigation of the role of ductal cells in pSS has centred historically around the striated ducts in histologybased studies or in vitro culture systems of salivary gland epithelial cells (SGECs). Although cultured SGECs might represent ductal cells in general (that is, both striated and interca lated ductal cells), whether they represent striated duct cells specifically has never been proven 45 . For the pur pose of this Review, however, we will treat SGEC cultures as a model for striated duct cells, referred to as SGECs (to mean LSGderived SGECs) throughout unless mentioned otherwise.

SGECs from patients with Sjögren syndrome respond aberrantly to extrinsic stimuli. For example, SGECs from patients with pSS are more sensitive to FAS/FASLmediated apoptosis in the presence of TNF and IFNγ than SGECs from healthy individuals 46, 47 . These cells are also more susceptible to other mecha nisms of programmed cell death, such as IFNγinduced or poly (I:C)induced anoikis (that is, apoptosis due to loss of attachment to the basal membrane) 48 . However, data from the literature suggest that striated duct cells 22, 49 and SGECs 50 are both resistant and susceptible to apoptosis.

In addition to disturbed apoptosis, SGECs express various TLRs (TLR1, TLR2, TLR3, TLR4 and TLR7) and other pattern recognition receptors (PRRs), implying that these cells have an ability to process pathogenassociated molecular patterns (PAMPs) and dangerassociated molecular patterns (DAMPs).

Furthermore, TLR3 activation can stimulate the expr ession of pSSassociated autoantigens SSA/Ro60, SSA/Ro52 and SSB/La by SGECs 51 . SGECs can also express receptors for cytokines, including receptors for type I interferons and TNF [52] [53] [54] . The chemokine recep tor CXCR3 might function as a scavenging receptor in the healthy salivary gland, preventing a buildup of surplus proinflammatory chemokines such as CXCL9, CXCL10 and CXCL11. In pSS, the function of this recep tor is thought to be impaired in the ductal cells; indeed, in SGECs from patients with Sjögren syndrome, intra cellular levels of CXCL10 are reduced compared with SGECs from healthy individuals, which is presumed to be a consequence of impaired CXCR3 functionality permitting the accumulation of these chemokines 55 . In addition to responding aberrantly to extrinsic stimuli, striated duct cells 56, 57 and SGECs 52,58 from patients with Sjögren syndrome also express many cytokines and chemokines (for example, IL1, IL6, IL7, IL18, TNF, B cellactivating factor (BAFF), CXCL10, CXCL12 and CXCL13) at higher levels than those observed in healthy individuals. Thus, striated ducts and their presumed in vitro counterpart SGECs are capable of regulating the immune response (as discussed in more detail in the section on epithelial cell-lymphocyte crosstalk).

Considering the involvement of striated duct cells in, for example, LELs, the NFκB pathway and inflammas ome activation (as discussed in detail in later sections), we would argue that the role of striated duct cells in the development of pSS pathology cannot be understated. The further roles of smaller, intercalated ducts remain until this point less well understood, and will require further investigation.

Mouse studies have shown that a small number of immune cells, including but potentially not limited to tissueresident memory T (T RM ) cells, macrophages, group 1 innate lymphoid cells (ILC1s), natural killer cells and dendritic cells, patrol the salivary glands, scanning for hallmarks of infection and/or epithelial cell damage 59,60 ( Fig. 1c ). T RM cells and macrophages, in particular, have been identified in close association with the mouse acinar epithelium 60 . In patients with pSS, this homeostatic surveillance system seems to be disturbed, and the sali vary gland immune landscape is unbalanced owing to an infiltration of mainly CD4 + T and B cells as well as, in lower abundance, pDCs and other immune cells 9 . Besides their immune function, these infiltrating cells can affect salivary gland homeostasis in multiple ways, including crosstalk with the salivary gland epithelium (which will be discussed in more detail in the section on epithelial cell-lymphocyte crosstalk).

In normal salivary gland homeostasis, the afore mentioned immune cell sentinels are complemented by salivary gland progenitor cells (SGPCs). SGPCs prolif erate and differentiate into fresh acinar and ductal cells, to replenish damaged cells and cells reaching the end of their lifespan [61] [62] [63] [64] . In the major salivary glands, the ductal compartment (both the striated and intercalated ducts) has been proposed to house SGPCs, although progenitor cells might also be present in the acinar compartment 61,65-67 . These progenitor cells can be stud ied in vitro through organoid cultures 61, 65, 68 . The number of SGPCs is much lower in patients with pSS than in healthy salivary glands, and the few that remain have a lower differentiation capacity 68 . One explanation for this finding is that progenitor cells residing in the basal layer of the parotid gland striated ducts become senescent prematurely in pSS, as a result of the mitotic effects of proinflammatory cytokines 68 . The telomeres of SGPCs from patients with pSS are shorter, suggesting that some SGPCs have indeed undergone replicative senescence 68 . In one study, the degree of senescence of cells located in the basal layer of the striated ducts (where progenitor cells probably reside), as inferred from the expression of the senescence marker p16, correlated with the produc tion of saliva by the parotid salivary gland, the extent of CD45 + cell infiltration and the ultrasound score 69 . The expression of p16 by all acinar and ductal cells combined also correlated with saliva production and CD45 + cell infiltration, but to a lesser extent 69 . Senescent cells in general have a senescenceassociated secretory pheno type (SASP), consisting of the expression of a panel of proinflammatory cytokines (including IL1α, IL1β, IL6, IL7, IL13, IL15, TGFβ, GMCSF and TNF), chemokines (including IL8, GROα, GROβ, CCL2, CCL3, CCL5, CCL16, CCL26 and CCL20), growth fac tors (including HGF and FGF), matrix metalloproteases and proteases [70] [71] [72] . Most interestingly and potentially detrimentally, this SASP enables the active spreading of senescence directly to neighbouring cells 72 . Therefore, senescent cells might not only reduce the ability of the salivary gland to function, by disabling a potential pro genitor cell population, but also augment inflamma tion and disease progression. Coupled with this effect is the strange persistence of functionally defective aci nar cells, whose clearance would otherwise presumably trigger replacement with fresh counterparts by SGPCs, a curious dynamic that requires more investigation.

Of further interest, disruption of salivary gland homeostasis also occurs in patients undergoing immune checkpoint inhibitor (ICI) therapy for the treatment of cancer. Approximately 5% of ICItreated patients will experience some form of salivary gland dysfunction [73] [74] [75] [76] . Among these patents, 60% will progress to technically fulfil the ACR-EULAR 2016 criteria for pSS 3 , owing to the presence of sicca symptoms, immune foci in the sali vary glands and/or autoantibody positivity [73] [74] [75] [76] . The potential mechanism of ICIinduced salivary gland dysfunction is highlighted in Box 1.

Microorganisms are implicated as a potential trigger in the development of pSS. Activation of TLRs that reco gnize components of bacteria (such as TLR1, TLR2 and TLR4) or viruses (such as TLR3, TLR7 and TLR9) on salivary gland ductal cells, for example, might repre sent a first step in the initiation of inflammation in the salivary gland in the early stages of disease. However, evidence that conclusively demonstrates the contribu tion of specific bacteria to salivary gland dysfunction in pSS is lacking [77] [78] [79] [80] [81] . Indeed, the most likely hypothesis is that alterations in the bacteriome in the oral cavity Nature reviews | Rheumatology 0123456789();: represent a consequence, rather than a cause, of salivary gland dysfunction in pSS 82 . Nevertheless, the triggering of specific TLRs and other (cytosolic) PPRs on SGECs by viral material might result in type I interferon pro duction, and conceivably contribute to the wellknown type I interferon signature associated with pSS. Although intrinsic and other extrinsic triggers might also be responsible for interferon production by epithelial cells (as discussed in the later section on interferon pathways and as reviewed elsewhere 83 ), this common feature of pSS suggests the presence of an initial immune response against viral infection.

To date, infection with three different viruses has been associated with the development of pSS. First, some data suggest that past infection with hepatitis C virus (HCV) is associated with the development of pSS, with estimates suggesting that approximately 14% of patients with pSS test positive for antibodies against HCV in cer tain pSS populations [84] [85] [86] . However, it should be noted that HCV infection (latent or active) is an exclusion cri terion in the 2016 ACREULAR classification criteria for pSS and debate exists as to whether this patient sub group truly represents pSS or a Sjögren syndromelike condition, especially as HCV infection rates vary widely geographically 86 . Second, chronic infection with human T cell lymphotropic virus type 1 (HTLV1) has also been associated with lowgrade salivary gland damage in pSS and increased serum concentrations of TNF and IFNγ, although this virus is not necessarily associated with pSS development 87, 88 . Patients with sicca syndrome and HTLV1 antibody serum positivity, for example, do not tend to have antiSSA autoantibodies 87, 88 . Third, various evidence points to an association between Epstein-Barr virus (EBV) and pSS, although it should be noted that this virus is very common and present in up to 95% of the general population 89 . The EBV remains latent in resting memory B cells and requires B cell stimulation and plasma cell differentiation for lytic replication [89] [90] [91] . This ability to remain dormant in memory B cells means that the virus poses a persistent threat. EBV DNA has been detected at increased levels in the salivary glands of patients with pSS compared with healthy individu als, with Ro52reactive perifollicular plasma cells being frequently infected 92, 93 . Furthermore, patients who were also positive for antiSSA and/or antiSSB antibodies had higher titres of IgG antibodies against the EBV early antigen than those patients without antiSSA and antiSSB antibodies 91 .

EBV is capable of directly infecting epithelial cells; in these cells, the EBV glycoprotein BMRF2 mediates transport of the virus to the basolateral membrane and seems to facilitate spreading of progeny virions 94 . In one study, levels of the EBVderived microRNA ebvmiRBART133p were increased in the salivary glands of patients with pSS compared with healthy individuals 95 . Interestingly, ebvmiRBART133p was suggested to downregulate the expression of stromal interaction molecule 1 (STIM1), a component of the acinar cell secretory machinery, providing a plausi ble link between EBV and saliva production 95 . This microRNA can transfer between cells, such as between B cells (the preferred target cells of EBV) and SGECs, via microvesicles 95 . Notably, the EBV protein Epstein-Barr nuclear antigen 2 (EBNA2) has a marked degree of protein sequence similarity to Ro60 autoantigens and likewise the protein sequences of Epstein-Barr virus (EBV)encoded RNA 1 (EBER1) and EBER2 are strikingly similar to La autoantigens 96, 97 . Furthermore, antibodies to Ro60 can crossreact with EBNA1 (reF. 98 ), suggesting that molecular mimicry might trig ger immune system activation to selfantigens in pSS. However, no correlation has been found between EBV reactivation periods and the development of pSS symptoms 96 ; furthermore, the molecular mimicry hypothesis does not explain why such a common infec tion as EBV results in pSS development in only a small proportion of these individuals 89 .

How viral infections might contribute to pSS remains unclear. In addition to the often touted classical hypoth eses, including molecular mimicry or enhanced apop tosis of epithelial cells, evidence would also suggest that viruses are capable of inducing epigenetic changes in epi thelial cells 99 , an exciting new interaction that remains to be investigated in pSS. Virusinduced epigenetic changes might, for example, repress the expression of proapoptotic genes. Impaired viral clearance as a mech anism for pSS development has also been inferred from the presence of a less efficient splice variant of the clas sical type I interferon downstream mediator OAS1 in patients with pSS 100, 101 .

With respect to SARSCoV2 viral infections, it is worth considering that a potential pool of patients with sicca complaints might be emerging. The involve ment of the salivary gland as a reservoir of this virus has been proposed, following the identification of the ACE2 receptor (an entry receptor for SARSCoV2) on SGECs 102, 103 . Close monitoring of this patient pool over the coming years might provide crucial evidence regard ing the role of viral infections in the triggering of sicca symptoms and possibly also in the triggering of pSS.

Innate immune function of the epithelium As discussed in the previous sections, the salivary gland epithelium fulfils innate immune functions that are mainly mediated by the expression of PRRs (for exam ple, TLRs) 53,54 and the secretion of cytokines. In this section, we discuss the different innate immunological pathways that contribute to dysfunction of the salivary

Both the major and minor salivary glands contain a T cell-rich inflammatory infiltrate following immune checkpoint inhibitor (ICI) use, but the glands do not resemble classical salivary glands of patients with pSS on the infiltration level 74, 203 . Some evidence suggests that ICI therapy induces a considerable shift in the organization of the parenchyma (specifically, a loss of typical saliva-producing acinar cells) 203 . The mechanism underpinning this effect is as yet unclear, but might encompass acinar cell death followed by aberrant salivary gland progenitor cell compensation, or increased plasticity of salivary gland epithelial cell types. This plasticity could be induced, for example, by the presence of the type II interferon signature associated with cytotoxic T cell activation and checkpoint inhibitor therapy (as opposed to the type I interferon signature most commonly associated with classical pSS), and might explain the notably different morphology observed 204 . Although presenting potentially with a different phenotype to pSS, deciphering how this epithelial skewing occurs will probably shed light on the mechanism underpinning salivary gland dynamics in general.

www.nature.com/nrrheum gland epithelium in pSS. Although the initial events that cause innate immune activation in the salivary gland epithelium remain to be identified, several possibilities have been raised. These include the involvement of exog enous antigens (for example, PAMPs from ineffectively cleared viruses) or aberrant expression of endogenous factors (for example, retroelements) that stimulate innate immune responses. Another possibility is that these responses are triggered by DAMPs because of ineffi cient removal of epithelial cell debris. Interestingly, a patient's genetic background might predispose them to impaired clearance of cell debris. The genotype and copy gene number of the complement component C4 has a high sex bias and has been linked to pSS susceptibility 104 . The lower gene copy number and expression of C4A reported for women that increase the risk of SLE or pSS might reduce the capacity of the immune system to clear cell debris, thereby prolonging exposure of intracellular proteins to the immune system 104 . However, data on the relationship between the C4 genotype and salivary gland pathology are not yet available.

The NFκB family is a group of transcription factors capable of activating an array of inflammatory down stream targets, including proinflammatory cytokines 105 . The canonical NFκB pathway is activated by binding of PAMPs to the TLRs and by proinflammatory cytokines themselves binding to their respective receptors 105 . Engagement of TLR and cytokine receptors triggers activity of the IκB kinase complex, culminating in phosphorylation of IκBα. In the inactive state, IκBα is in complex with canonical NFκB members RelA and p50, whereas phosphorylation of IκBα results in deg radation of this inhibitor, enabling the release of the RelA-p50 heterodimer and its subsequent translocation to the nucleus, where the dimer activates target gene transcription 105 . Studies have reported NFκB activity in the epithelial cells of the salivary glands of patients with pSS, reflected by the expression of NFκB downstream target genes encoding cytokines such as IL1α, IL6 and TNF 57 . NFκB is also important for the regulation of cell-cell interactions and is capable of disrupting tight junction integrity 106 . Mutations that result in overactivity of the NFκB pathway have been reported in patients with pSS: for example, mutations in genes encoding IκBα or the inhibitor TNFαinduced protein 3 (TNFAIP3; also known as A20) [107] [108] [109] . Interestingly, in mice, consti tutive activation of the NFκB pathway via knockout of TNFAIP3 in cytokeratin 14-expressing epithelial cells (including those cells of the striated duct) is sufficient to result in reduced saliva production and infiltration of a T cellrich infiltrate into the salivary glands 110 . This find ing emphasizes the role of epithelial cells and the NFκB pathway in pSS development, and the apparent ability of activated epithelial cells to recruit immune cells.

The inflammasome is an intracellular complex of oli gomers that detect and respond to DAMPs and PAMPs to incite inflammation, functioning as an important innate immune system receptor and sensor. Inflammasomes require priming via classical innate immune activation pathways, such as the microbeactivated TLR pathway 111 . Subsequent to this signal, a second activation signal of inflammasome components via stimuli such as lyso somal disruption, reactive oxygen species production and release of oxidized mitochondrial DNA or micro bial nucleic acids is required 111 . Inflammasomes can be broadly divided into four types: the absent in melanoma 2 (AIM2) inflammasome, the NOD, LRR and pyrin domaincontaining protein 1 (NLRP1) inflammasome, the NLRP3 inflammasome and the NLRP4 inflammas ome. Inflammasome activation ultimately facilitates pro cessing of preformed proinflammatory cytokines such as proIL1β and proIL18 into their final active forms. Stimulation of the AIM2 inflammasome occurs follow ing binding by cytosolic free DNA (cfDNA) of viral, bac terial or mammalian origin, but not by other triggers 112 . Some data suggest that the AIM2 inflammasome is highly active in SGECs from patients with pSS, which could be because of defective functioning of DNase 1 resulting in the accumulation of cfDNA 113 . Patients with pSS and MALT lymphomas, and those patients with pSS judged to be at a high risk of developing MALT lympho mas, have high serum levels of cf DNA and extranuclear DNA accumulations in salivary gland tissue, mostly localized between the striated ducts and lymphocytic infiltrates 114 . This extracellular DNA, if transported into striated duct cells, might be an important stimulus for the AIM2 inflammasome.

Oligomerization of NRLP1, NRLP3 or NRLP4 results from upregulation of NRLP transcription following sig nals induced by cfDNA, as is described for AIM2, as well as signals induced by a panoply of DAMPs, PAMPs and other triggers, such as reactive oxygen species, oxidized mitochondrial DNA and lysosomal disruption. Various data suggest that the salivary glands of patients with pSS express higher levels of NLRP3 than healthy individuals, and P2X7, a component of the NRLP3 machinery, is also upregulated in the salivary gland tissue of patients with pSS 114, 115 . However, which specific cell types express the upregulated NRLP3 is unclear, as indeed is the expres sion of the NRLP1 and NRLP4 inflammasomes in pSS 115 . However, although perhaps less wellunderstood than the role of the TLR and NFκB systems, a central role for the inflammasome in pSS is emerging. Mirroring the NFκB pathway, the activity of the inflammasome in the salivary gland epithelium reinforces the role of the epithelium, and its crosstalk with the immune system, in pSS. The upregulated inflammasome might contribute to the sustained glandular inflammatory process in pSS, and represents a potential alternative therapeutic target.

Important signalling pathways that contribute to the dis ruption of the salivary gland epithelium in pSS involve interferons. The interferon family consists of type I (IFNα and IFNβ), type II (IFNγ) and type III (IFNλ) interfer ons, which have distinct as well as common functions. Interferons stimulate a wide variety of genes important in innate and adaptive immune responses 116 . In particular, type I interferons create an antiviral state by promoting immune responses to viruses 117 . The important role of interferons in the pathogenesis of pSS is reflected by the elevated transcript levels and altered epigenetic regula tion of interferonstimulated genes (ISGs) in both the blood and salivary gland tissue of patients [118] [119] [120] . These ISGs are upregulated in both SGECs and striated ducts, in addition to in immune cells 121, 122 , together account ing for the interferon signature of the glands. Indeed, both type I and type II interferoninduced proteins (IFNinduced protein with tetratricopeptide repeats 3 (IFIT3) and guanylatebinding protein 2 (GBP2), respec tively) are expressed in ductal epithelial cells, whereas infiltrating immune cells mainly expressed GBP2 (reF. 122 ). In the following subsections, we discuss both the path ways that result in interferon production (for example, PRR signalling and the overexpression of endogenous retroelements), and the pathways that result from stim ulation by interferon (for example, proinflammatory cytokine production) in the salivary glands.

pDCs are a potent source of IFNα within the inflamed salivary gland 6 . Activated pDCs are able to migrate to the salivary glands in response to chemokines such as CXCL12, CXCR3 ligands and CCR5 ligands 123, 124 . Within the glands, type I interferon production by pDCs can be sustained in the presence of TLR7 ligands, TLR9 ligands and immune complexes of autoantibodies and autoantigens that contain TLR7 ligands in the form of autoantigenassociated RNA 125 . Although a strong correlation exists between the pres ence of Ro/La autoantibodies and the expression of ISGs in the blood and tissue of patients with pSS, the sequence of appearance of these features and their contribution to dysfunction of the salivary gland epithelium need further investigation.

In addition to pDCs, epithelial cells themselves can produce type I interferon after stimulation via PRRs, as demonstrated, for example, in studies of mice and of a human submandibular gland cell line 36, 126 . Furthermore, SGECs respond to TLR signalling by pro ducing IFNβ 127, 128 . Interestingly, interferon production can be augmented by endogenous viruslike genomic repeat elements ('retroelements') 129 . The retroelement long interspersed nuclear element 1 (LINE1) is over expressed in the minor salivary gland tissue of patients with pSS, probably owing to hypomethylation in the pro moter region of the gene encoding LINE1 (reFs 129, 130 ). Importantly, LINE1 and IFNβ are expressed in the ductal epithelium 129, 130 . Mechanistically, LINE1 retroelements bind to cytosolic nucleic acid sensors, such as RIGllike receptors, and consequently trigger type I interferon production 131 . Vice versa, signalling downstream of extrinsic interferons can modulate the transcription of retroelements. For example, transcriptional modifi cations of Alu retroelements, induced by interferon sig nalling, enables this retroelement to bind to intracellular Ro60 (reFs 129, 132 ). Ro60 probably has an inhibitory role in type I interferon receptor signalling, as data have shown that deletion of Ro60 in an EBVtransformed B cell line increases interferoninduced proinflammatory cytokine production and the expression of retroelements 132 .

Similar to Ro60, Ro52 (later identified as TRIM21) has a multifaceted role in the innate immune response. TRIM21 primarily functions simultaneously as a cytosolic Fc receptor and as an E3 ubiquitinprotein ligase 133, 134 . An important antiviral role of TRIM21 is binding the Fc part of intracellular antibodies, direct ing endocytosed antibodybound viral particles to the proteasome for degradation and activation of innate immune signalling 134 . Conversely, TRIM21 can also function as a negative regulator of TLR signalling by mediating ubiquitination (protein inactivation) of interferonregulating factors (IRFs), such as IRF3, IRF5 and IRF7, thereby inhibiting interferon production [135] [136] [137] . An inhibitory role for TRIM21 is supported by studies of TRIM21deficient C57BL/6 mice, which develop features of systemic autoimmunity upon local tissue injury 138 . Furthermore, the amount of antidsDNA anti body production and plasmablast formation is higher in TRIM21deficient MRL/lpr mice than in wild type MRL/lpr mice, indicating enhanced B cell activation in the absence of TRIM21 (reF. 139 ). AntiRo52/TRIM21 antibodies from patients with pSS can specifically reco gnize the RING domain of the TRIM21 protein. This RING domain is essential for the E3 ubiquitin ligase activity of TRIM21 and antiRo52 antibodies could interfere with this activity in vitro 140 . Although direct interference of antiRo52/TRIM21 antibodies (via their antigenbinding fragment (Fab) domain) with intracel lular TRIM21 has not been proven in vivo, and is dif ficult to reconcile, aberrant function of TRIM21 upon uptake of autoantibodycontaining immune complexes cannot be ruled out. For example, a study of mice with lupuslike disease provided evidence of possible leak age of IgG antibodies from phagolysosomes containing endocytosed immune complexes into the cytosol, which stimulated the TRIM21mediated immune responses 141 . Furthermore, studies of patients with pSS have shown that TRIM21 can be expressed on the cell surface of antigenpresenting cells 142 , enabling transport of aggre gated immunoglobulins and immune complexes into the cell. In conclusion, aberrant expression of retroelements and (indirect) interference of autoantibodies with the regulation of innate immune responses by TRIM21 (and possibly also Ro60) might result in a feedforward loop of type I interferon activity in ductal epithelial cells, pDCs and B cells (Fig. 1c) . A main consequence for the glan dular epithelium is that type I interferons enhance the production of pathogenic chemokines and cytokines (for example, CXCL10, IL7 and BAFF) by ductal epithelial cells, resulting in the recruitment and activation of T cells and B cells and thereby amplifying the inflammatory response 58, 143, 144 .

In addition to type I interferons, type II interferons have also been implicated in pSS associated salivary gland pathology. IFNγ is mainly produced by natural killer cells and T cells as part of the innate and adaptive immune responses, respec tively. In the salivary glands of patients with pSS, IFNγ is abundantly produced by immune cells and the salivary gland tissue often has a mixed type I and type II interferon signature 57, 122 . IFNγ is not only produced by bona fide T H 1 cells in the salivary gland but also by T follicular helper (T FH )like cells that express both www.nature.com/nrrheum 0123456789();: programmed cell death protein 1 (PD1) and induci ble T cell costimulator (ICOS) and by CCR9 + T helper cells 145, 146 . IFNγ exerts proinflammatory effects on SGECs (including both labial and parotid gland derived SGECs) by stimulating the production of cytokines and chemokines 58, 143 . IFNγ also induces the expression of MHC class II and costimulatory mole cules on SGECs 47, 147, 148 . Collectively, these effects might result in immune cell activation 46 . IFNγ can also induce FASmediated apoptosis and anoikis in SGECs 47,48 , although an opposite effect was observed in acinar cells and in an immortalized salivary gland ductal cell line, where IFNγ induced apoptotic resistance 25, 46 . IFNγ has further been implicated in reducing the integrity of the glandular epithelium (that is, the tight junction barrier function), as has been observed in minor salivary gland tissue from patients with pSS 106 . Together, these results suggest that IFNγ negatively affects SGECs in various ways, dependent on the glandular cell type, and might contribute to salivary gland dysfunction in patients with SS.

Type III interferon has been added to the interferon family more recently 149 . Downstream effector genes of type III interferons are similar to those downstream of type I interferons and the functions of both interferon pathways largely overlap. However, an important difference is restricted expression of the type III interferon receptor by epithelial cells of mucosal sur faces and pDCs (reviewed elsewhere 150 ). In the ductal epithelium of patients with pSS, IFNλ2 is upregulated compared with individuals with nonSjögren syndrome sicca symptoms 151 . Similar to type I and type II inter ferons, IFNλ can promote the production of cytokines (for example, IL7, BAFF and CXCL10) by the salivary gland epithelium 58, 151 . Whether type III interferons nota bly contribute to salivary gland pathology, either inde pendent or in synergy with other interferons, remains to be elucidated.

In healthy salivary glands, a small number of lympho cytes (in the form of T RM cells) are present for immune surveillance 60 . By comparison, in pSS, CD8 + T cells are located in close association with acini or ducts with a disrupted basal lamina and CD4 + T cells are located in association with the ducts 37 . B cells can also infiltrate the ductal epithelium, a phenomenon that is specific to pSS 10 . Such histological findings suggest that crosstalk between the salivary gland epithelium and lymphocytes has a critical role in salivary gland pathology. In this section, we focus on crosstalk between epithelial cells of the salivary glands and lymphocytes (specifically, CD4 + T cells and intraepithelial B cells) in pSS.

The presence of MHC class II in combination with CD80 and CD86 molecules on the surface of SGECs upon immune activation (for example, following stimulation with IFNγ) infers the ability of the epithe lial cells to process antigens for presentation to CD4 + T cells 52, 152 . In the presence of T cell receptor stimulation, constitutive expression of CD86 by SGEC lines could promote CD4 + T cell proliferation by engaging with CD28 (reF. 153 ). SGECs from patients with pSS are also capable of expressing other costimulatory mole cules such as CD40 and ICOS ligand (ICOSL) 49, 152, 154 . In SGECs from patients with pSS, CD40 is spontane ously expressed (at higher levels than in SGECs from patients with sicca symptoms but not diagnosed with pSS) and this expression can be enhanced in the pres ence of IFNγ or IL1β 148 . Ligation of CD40 on SGECs activates the noncanonical NFκB pathway, resulting in proinflammatory cytokine production and either FASdependent apoptosis or apoptotic resistance 49, 155 , probably depending on the epithelial cell type and microenvironmental cues. However, although SGECs seem to express all molecules required for antigen pres entation, no formal proof is yet available showing that human SGECs actively present antigen to T cells via cognate interaction in vivo.

In addition to the provision of costimulatory sig nals to T cells, SGECs can produce several cytokines and chemokines, as shown for both labial and parotid glandderived SGECs. These can include chemokines that attract T cells (for example, CCL19 and CXCL10), as well as cytokines that promote local T cell proliferation and/or differentiation 56, 58, 143, 154 . For example, SGECs can produce IL6, a pleiotropic cytokine that can promote the differentiation of T FH cells (as well as B cells) 156 . IL6 production by SGECs, together with ICOSL expres sion on these SGECs, can support the differentiation of T FH cells in vitro 154 . This finding is notable as the expan sion of both glandular and circulating T FH cells in pSS is associated with more severe disease, that is, increased ESSDAI scores, IgG antibody production and ectopic lymphoid structure formation 146, 157, 158 .

Another potentially relevant cytokine for salivary gland epithelium-T cell crosstalk is IL7. This cytokine is produced by nonhaematopoietic cells and has an important role in T cell homeostasis 159 . Although a histological study in minor salivary glands found that IL7 was mostly expressed by cells with a fibroblast mor phology in the interstitium, and not by acinar or ductal cells 160 , more recent findings suggest that IL7 is also expressed by SGECs 58, 121 . Cultured SGECs from patients with pSS produce IL7 after stimulation with poly(I:C), IFNα or IFNγ 58 . Data from the B6.NOD-Aec mouse model of pSS suggest that IL7 also has an indirect role in salivary gland pathology, by enhancing T H 1 responses and IFNγ-dependent CXCL10 expression in the salivary gland 161 . Thus, activation of T H 1 cells by IL7 can pro pagate IFNγ production, creating a proinflammatory loop. Although the epithelial source of IL7 production (for example, ductal or acinar cells) has not been stud ied, various data suggest that the IL7-IL7Rα axis con tributes to T celldriven autoimmune pathology in pSS. For example, the amount of IL7Rpositive T cells in the salivary glands of patients with pSS correlates with IL7 expression and the severity of sialadenitis 162 . In addition, CCR9 + T helper cells typically express high levels of the IL7 receptor and stimulation of these cells with IL7 induces IFNγ, IL17 and IL21 production, thereby also supporting B cell activation 145 .

Available evidence indicates that the salivary gland epi thelium is able to promote B cell hyperactivity in pSS and forms a niche for autoantibodyproducing plasma cells. Chronic B cell hyperactivity in the salivary glands is demonstrated, amongst others findings, by the fre quent occurrence of ectopic lymphoid structures in the glandular tissue, which might even contain germinal centres 7 , and by the high risk of patients with pSS devel oping B cell nonHodgkin lymphomas in these glands 163 . Several mechanisms underlying B cell hyperactivity in pSS have been described and reviewed elsewhere 8,9 . In this section, we focus on the crosstalk between epi thelial cells and B cells and highlight novel insights into the development of LELs and MALT lymphoma in pSS.

Epithelial cells can contribute to B cell activity through the production of cytokines, including IL6 and BAFF 121, 154, 164 . Both IL6 and BAFF are involved in B cell activation and homeostasis, and BAFF is critically important for B cell survival 156, 165, 166 . Previous reports have shown that type I interferon is an important promoter of BAFF production by SGECs 6,144,167 . The expression of interferoninducible genes was indeed increased in EpCAMpositive epithelial cells in biopsy samples from patients with pSS compared with biopsy samples from individuals with nonSjögren syndrome sicca symptoms 121 . In one study that employed cocultures of B cells and SGECs, poly(I:C) stimulated the production of soluble factors by SGECs from patients with pSS, which enhanced the survival of the B cells 121 . Surprisingly, blockade of a proliferationinducing ligand (APRIL) or BAFF alone had no effect on B cell survival 121 , suggest ing that other factors might be responsible for enhanced B cell survival under this experimental condition.

The epithelium not only affects B cells, but B cells can also, vice versa, have effects on epithelial cells. For example, B cells can induce epigenetic modifications in SGECs 168 . B cellinduced transcriptional changes in epi thelial cells might contribute to the formation of LELs, as discussed in the next section.

The intimate relationship between the salivary gland epithelium and B cells in pSS is most clearly demonstrated by the presence of LELs (Fig. 2) . These LELs develop exclusively in the striated ducts and are a characteristic histological fea ture of pSS 11 . LELs are formed in close association with the periductal infiltrate 10, 11 . Although these lesions are found in both the minor and major salivary glands, they are more pronounced in the major (parotid) glands 10 . LELs consist of hyperplastic duct cells in the epithe lial cell lining and infiltrating lymphocytes within the contour of the basement membrane 11 . This hyperplasia might result in complete occlusion of the ducts, poten tially contributing to hyposalivation. Evidence suggests that LELs nearly always harbour B cells in between the hyperplastic basal cells and that a small proportion of the striated ducts without hyperplasia already contain some B cells 10 . In addition, some intraepithelial T cells are also found in LELs; however, unlike B cells, which are completely absent from the salivary gland ducts of patients with nonSjögren syndrome sicca symptoms, the presence of intraepithelial T cells is not specific to pSS 10 . Together these findings suggest that LEL forma tion starts with the infiltration of B cells into the ductal epithelium. The severity of the LEL (stage of duct occlu sion) increases with the absolute and relative number of intraepithelial B cells 10 . How B cells are attracted into the epithelium is not fully understood, but the expression of CXCR3 probably has an important role, as intraepithe lial B cells express CXCR3 and the salivary gland epi thelium produces CXCL10 and other CXCR3 ligands upon activation 143 . These intraepithelial B cells, which are probably already activated before they migrate to the epithelium, can expand locally, as demonstrated by the high proportion of Ki67staining cells and the expansion of clonal B cells within the striated ducts 169, 170 . The latter finding suggests that additional activation and proliferation signals might be derived from epi thelial cells, and possibly also from other intraepithelial immune cells, such as T cells and dendritic cells.

Interestingly, the majority, if not all, of the intraepi thelial B cells in the minor (labial) and major (parotid) glands express the inhibitory Fcreceptor like 4 (FcRL4) protein (Fig. 3) , which is abundantly expressed by MALT lymphoma B cells of patients with pSS 169 , and is also expressed on activated B cells 171 . Gene expression profil ing of FcRL4 + B cells from parotid gland tissue of patients with pSS has further revealed that these cells express transcripts of chronic activation markers, such as Tbet also known as PRDM1) 169 . Available evidence indicates that binding of soluble IgA to FcRL4 results in a switch from B cell receptor (BCR)mediated activation to TLRmediated activation 179 . This functional switch of the B cells probably results in enhanced NFκB pathway acti vation and cytokine production. Secreted cytokines of intraepithelial B cells, such as IL6, might affect epithelial homeostasis and lead to the proliferation of epithe lial cells and ultimately to LEL formation. Direct proof that B cells are involved in LEL formation comes from trials of rituximab (a B celldepleting agent) in patients with pSS. Treatment of patients with pSS with rituxi mab not only led to a strong reduction of B cells within the salivary gland, including B cells located within the epithelium, but concomitantly also led to a reduction in the severity of the LELs and the partial restoration of the epithelium after 12 weeks of treatment 180 . Notably, sali vary gland B cell depletion was more variable at week 24 in this study 181 , suggesting that in some patients, B cells have started to repopulate the glands at this time point. Although rituximab treatment had clear effects on salivary gland histopathology, in particular at early time points, these effects unfortunately did not translate into rituximab having a proven clinical efficacy in pSS [182] [183] [184] .

The effects of B cell depletion therapy on the epithe lium using rituximab, together with the cytokine profiles of intraepithelial B cells and SGECs, suggest that some crosstalk occurs between B cells and striated duct epithe lial cells, leading to sustained activation and proliferation of both cell types. Upregulation of CXCR3 and CD11c, and possibly also other integrins, on the B cell surface might have a role in keeping the cells within the epithe lial layer. However, in addition to being present in ductal areas, B cell clones are also present, to some extent, in the periductal areas, suggesting that some cellular exchange occurs between these areas 170 .

The lack of mRNA and protein expression of plasma cell markers by glandular FcRL4 + B cells suggest that these cells rarely differentiate into plasmablasts and instead the cells are maintained in a state of chronic activation and continue to proliferate at a high rate 169, 172 . These cells might undergo extrafollicular somatic hypermuta tion; indeed, activation induced deaminase (AID), the enzyme responsible for initiating diversity in immuno globulin genes during somatic hypermutation (and class switching), is expressed by FcRL4 + B cells in the tonsils (and possibly also in salivary glands) 185 . Notably, AID expression, which can be induced by Tbet 186 , might not only result in hypermutation of immunoglobulin variable region genes, but also in offtarget mutations (that is, nonimmunoglobulin genes) 187 . Thus, a path ogenic combination of increased NFκB activation, prosurvival factors, proliferation and possibly also AID expression in FcRL4 + intraepithelial B cells could make these cells prone to neoplastic changes and promote pro gression towards MALT lymphoma, as discussed in the next section.

NonHodgkin lymphomas arise in 5-10% of patients with pSS, and the majority of these lymphomas are MALT lymphomas that develop preferentially within the parotid glands 163 . A hallmark of salivary gland MALT lymphomas is their association with LELs, highlighting the dependency of these lymphomas on epithelial cells. MALT lymphomas are considered to arise as a conse quence of chronic B cell stimulation and, in pSS, these neoplastic cells often express stereotypic rheumatoid factors that have a high affinity for the Fc region of IgG antibodies [188] [189] [190] . In patients with pSS, nonneoplastic rheumatoid factorexpressing B cells are enriched within the circulating CD21 −/low B cell population 191 , a pheno type that is associated with impaired BCR stimulation (similar to FcRL4 + B cells) and frequent polyreactivity or selfreactivity 192, 193 . The phenotype of CD21 −/low B cells partially overlaps with DN2 B cells 194 . Notably, IL21 and TLR7 ligands together promote the expansion and dif ferentiation of DN2 B cells 178 and are both readily availa ble in the inflamed salivary gland in patients with pSS 146 . While the frequency of rheumatoid factorexpressing B cells in the inflamed salivary glands of patients with pSS without MALT lymphoma seems to be low 195, 196 , these cells might expand after dual engagement of the BCR and TLRs with immune complexes that contain RNAassociated autoantigens 197 . Although the role of the BCR in lymphomatous escape remains enigmatic, rheumatoid factor expressed on the surface of B cells might simply trap immune complexes of autoantibod ies and RNAassociated autoantigens, which stimulate TLRs, with or without further BCRmediated activation of the cells. In the inflamed salivary gland, these immune complexes might provide chronic B cell stimulation and a strong selection advantage of incidental stereotypic rheumatoid factorexpressing B cells. Simultaneous engagement of FcRL4 on these B cells might inhibit differentiation towards plasma cells and in turn enable ongoing proliferation.

Rheumatoid factorexpressing B cells frequently express IGHV169, a immunoglobulin heavychain variable region variant that can be detected by the mon oclonal antibody G6 (reF. 198 ). Compared with healthy individuals, patients with pSS and cryoglobulinaemic vasculitis have an increased frequency of G6positive memory B cells on the periphery, which exclusively express kappa light chains 191 . Interestingly, all MALT lymphomas that have rheumatoid factor activity are IgM clones with kappa light chains 188, 190 , suggesting that G6 + B cells might form a pool of potential precursor cells to MALT lymphomas. Furthermore, researchers have shown that G6 + B cells incidentally contain somatic mutations in genes involved in B cell proliferation, such as TNFAIP3 (reF. 191 ). A specific germline polymorphism of TNFAIP3 is associated with MALT lymphoma devel opment in pSS 199 , underlining the importance of intact NFκB regulation in preventing B cell dysregulation. Variations in B cellregulating genes, such as TNFAIP3, might result in the escape of autoreactive cells from tolerance checkpoints 191 . Although the presence of G6 + B cells that contain lymphomaassociated mutations in the peripheral blood of patients with pSS has not been linked directly to MALT lymphoma development, these cells might migrate to the salivary glands, where they could be further activated.

Taken together, an attractive hypothesis is that the highly proliferative, intraepithelial FcRL4 + B cells are the glandular counterparts of DN2 B cells, incidentally displaying rheumatoid factor reactivity and forming a pool of lymphoma precursor cells in the salivary glands of patients with pSS. The notion that FcRL4 + B cells are more frequently observed in parotid glands com pared with labial glands might explain the preferential development of MALT lymphoma in parotid glands 169 . Although the critical steps towards neoplastic dysreg ulation of glandular B cells remain unknown, somatic mutations within and outside of the immunoglobulin variable region genes are probably involved. We postu late that therapeutic targeting of intraepithelial B cells might prevent the development of MALT lymphoma in patients with pSS.

Over the past decade, the attenuation of sicca symptoms and, more recently, the attenuation of systemic disease activity have evolved as main treatment targets in pSS. As the epithelium is the functional backbone of the sali vary gland, its restoration must be a central determinant of future therapies aimed at targeting hyposalivation in pSS. We hypothesize that therapies that target, for exam ple, only the immune system might reduce glandular inflammation but not necessarily rescue salivary gland function, as demonstrated in existing clinical trials 200 .

Although the targeting of the immune system is a valid approach in pSS, considering the typical (auto)immune manifestations and systemic nature of the disease, more attention should be paid to the correction of epithelial defects. Multifaceted approaches that consist of both an antiinflammatory and a proepithelial component -for example, immunotherapy combined with cell therapymight provide the crucial regenerative stimuli required to correct these defects.

With regard to cell therapy, fresh, patientmatched induced pluripotent stem cells might be used to derive untainted salivary gland acinar and/or ductal cells, which could subsequently be transplanted back into the patient. The relatively new drug class senolytics (a class of drugs that selectively induce death of senescent cells) might also prove therapeutically useful in the depletion of senescent epithelial progenitor cells, limiting further damage that would otherwise be inflicted by these cells 201, 202 . The tim ing of therapy application is probably an important con sideration, given the general early loss of salivary gland function in pSS. More advanced stages of glandular dis ease are characterized by the dominance of B cells and the presence of LELs, and in these patients, glandular B cells are an important treatment target. As previously high lighted, B cell depletion therapy with rituximab reduced the severity of LELs 180 , but whether other B celldirected therapies also target these pathogenic structures remains to be examined. Indepth histological evaluation of glan dular tissue in clinical trials is, in this context, a valuable outcome measure of clinical trials.

Dissection and identification of specific defects at the individual patient level might be the key to successful treatment, given the variety of inflammatory pathways involved in the pathogenesis of pSS. Therefore, treat ment decisions guided by the clinical picture of the sal ivary gland hold promise. To define the clinical picture of the salivary gland in individual patients, imaging and indepth histopathological examinations of sal ivary gland biopsy samples are needed, going beyond measuring the focus score. In addition, tissue transcrip tomics, in particular at the singlecell level, might be useful to identify dysregulated pathways. Lastly, the sci entific community is advancing with the establishment of in vitro models of salivary gland disease (for exam ple, cocultures of organoids or explants with immune cells) 58, 68, 121 to screen for new drug targets and to examine the effect of immunomodulatory treatments on salivary gland function.

Overall, a multitude of pathways, systems and processes are probably dysregulated in the salivary gland in pSS, including defects that affect the epithelium, innate immune signalling and adaptive immune activation. In an ideal world, each defect would be corrected, and the salivary gland restored to its naive state. Although the initial triggers that activate the salivary gland epithelium remain undetermined, the nature of the local immune response, including dominant type I interferon activity, hints at the involvement of a viral infection. Subsequent dysregulation of the immune response, possibly owing to interference of type I interferon signalling by autoan tibodies and chronic autoantigen exposure by apoptotic epithelial cells, results in a proinflammatory feedback loop. Various immune cells (such as lymphocytes, den dritic cells and macrophages) might interact with the epithelium and contribute to the inflammatory response. In particular, crosstalk between the ductal epithelium and B cells, and consequently the formation of LELs, negatively affects salivary gland morphology and might result in MALT lymphoma development. Finally, as a scientific community, we need to think more abstractly, and consider combining immunotherapy with cell thera pies to provide an unfettered source of SGECs, although preventing continual resurgence of the same epithelial problems will remain a challenge.

Published online xx xx xxxx

Zur Kenntnis der Keratoconjunctivitis sicca (Keratitis filiformis bei Hypofunktion der Tränendrüsen)

Sjögren syndrome

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Autoantibodies from Sjögren's syndrome trigger apoptosis in salivary gland cell line

The role of intrinsic epithelial activation in the pathogenesis of Sjögren's syndrome

Toll-like receptor in salivary glands from patients with Sjögren's syndrome: functional analysis by human salivary gland cell line

Expression of functional Toll-like receptors by salivary gland epithelial cells: increased mRNA expression in cells derived from patients with primary Sjögren's syndrome

Epithelial CXCR3-B regulates chemokines bioavailability in normal, but not in Sjogren's syndrome, salivary glands

Lymphoid' chemokine messenger RNA expression by epithelial cells in the chronic inflammatory lesion of the salivary glands of Sjögren's syndrome patients: possible participation in lymphoid structure formation

Cytokine mRNA expression in salivary gland biopsies of Sjögren's syndrome

Interleukin-7/interferon axis drives T-cell and salivary gland epithelial cell interactions in Sjögren's syndrome

Transforming growth factor-β signaling guides the differentiation of innate lymphoid cells in salivary glands

Salivary gland macrophages and tissue-resident CD8 + T cells cooperate for homeostatic organ surveillance

Human salivary gland stem cells functionally restore radiation damaged salivary glands

Generation of a single-cell RNAseq atlas of murine salivary gland development

Rescue of salivary gland function after stem cell transplantation in irradiated glands

SOX2 regulates acinar cell development in the salivary gland

Long-term in vitro expansion of salivary gland stem cells driven by Wnt signals

Salivary gland homeostasis is maintained through acinar cell self-duplication

The contribution of specific cell subpopulations to submandibular salivary gland branching morphogenesis

Salivary gland stem cells age prematurely in primary Sjögren's syndrome

Progenitor cell niche senescence reflects pathology of the parotid salivary gland in primary Sjögren's syndrome

Senescent human hepatocytes express a unique secretory phenotype and promote macrophage migration

Hallmarks of cellular senescence

Cellular senescence: defining a path forward

Inflammatory arthritis and sicca syndrome induced by nivolumab and ipilimumab

Sicca syndrome associated with immune checkpoint inhibitor therapy

Autoimmune phenomena and disease in cancer patients treated with immune checkpoint inhibitors

Immune-related adverse events with immune checkpoint blockade: a comprehensive review

Saliva microbiome in primary Sjögren's syndrome reveals distinct set of disease-associated microbes

Oral microbial flora of patients with Sicca syndrome

Molecular analysis of oral microflora in patients with primary Sjögren's syndrome by using high-throughput sequencing

Next-generation sequencing of whole saliva from patients with primary Sjögren's syndrome and non-Sjögren's sicca reveals comparable salivary microbiota

Dysbiotic salivary microbiota in dry mouth and primary Sjögren's syndrome patients

Dysbiosis of the buccal mucosa microbiome in primary Sjögren's syndrome patients

Innate immunity and interferons in the pathogenesis of Sjögren's syndrome

Hepatitis C virus infection in 'primary' Sjogren's syndrome: prevalence and clinical significance in a series of 90 patients

How hepatitis C virus modifies the immunological profile of Sjögren syndrome: analysis of 783 patients

Sjögren syndrome associated with hepatitis C virus: a multicenter analysis of 137 cases

Initial human T-cell leukemia virus type 1 infection of the salivary gland epithelial cells requires a biofilm-like structure

Association of sicca syndrome with proviral load and proinflammatory cytokines in HTLV-1 infection

How compelling are the data for Epstein-Barr virus being a trigger for systemic lupus and other autoimmune diseases?

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

Discrepancy of serological and molecular patterns of circulating Epstein-Barr virus reactivation in primary Sjögren's syndrome

Detection of Epstein-Barr virus DNA by in situ hybridization and polymerase chain reaction in salivary gland biopsy specimens from patients with sjögren's syndrome

Implication of Epstein-Barr virus infection in disease-specific autoreactive B cell activation in ectopic lymphoid structures of Sjögren's syndrome

EBV BMRF-2 facilitates cell-to-cell spread of virus within polarized oral epithelial cells

Targeting the Ca 2+ sensor STIM1 by exosomal transfer of Ebv-miR-BART13-3p is associated with Sjögren's syndrome

The role of Epstein-Barr virus infection in primary Sjögren's syndrome

Viruses and autoimmunity: a review on the potential interaction and molecular mechanisms

Early events in lupus humoral autoimmunity suggest initiation through molecular mimicry

Infection-induced epigenetic changes and their impact on the pathogenesis of diseases

Identification of a Sjögren's syndrome susceptibility locus at OAS1 that influences isoform switching, protein expression, and responsiveness to type I interferons

Variants at multiple loci implicated in both innate and adaptive immune responses are associated with Sjogren's syndrome

Epithelial cells lining salivary gland ducts are early target cells of severe acute respiratory syndrome coronavirus infection in the upper respiratory tracts of rhesus macaques

Salivary glands: potential reservoirs for COVID-19 asymptomatic infection

Complement genes contribute sex-biased vulnerability in diverse disorders

New regulators of NF-κB in inflammation

Disruption of tight junction structure in salivary glands from Sjögren's syndrome patients is linked to proinflammatory cytokine exposure

Association of genes in the NF-κB pathway with antibody-positive primary Sjögren's syndrome

IkBα promoter polymorphisms in patients with primary Sjögren's syndrome

A failure of TNFAIP3 negative regulation maintains sustained NF-κB activation in Sjögren's syndrome

Dysregulation of NF-kB in glandular epithelial cells results in Sjögren's-like features

The NLRP3 inflammasome: molecular activation and regulation to therapeutics

AIM2 in regulatory T cells restrains autoimmune diseases

Cell-autonomous epithelial activation of AIM2 (absent in melanoma-2) inflammasome by cytoplasmic DNA accumulations in primary Sjögren's syndrome

Systemic activation of NLRP3 inflammasome in patients with severe primary Sjögren's syndrome fueled by inflammagenic DNA accumulations

The P2X7 receptor-inflammasome complex has a role in modulating the inflammatory response in primary Sjögren's syndrome

Mechanisms of type-I-and type-II-interferon-mediated signalling

Interferons and their actions

Genome-wide DNA methylation analysis in multiple tissues in primary Sjögren's syndrome reveals regulatory effects at interferon-induced genes

Type I and II interferon signatures in Sjogren's syndrome pathogenesis: contributions in distinct clinical phenotypes and Sjogren's related lymphomagenesis

Prevalence of interferon type I signature in CD14 monocytes of patients with Sjogren's syndrome and association with disease activity and BAFF gene expression

Salivary gland epithelial cells from patients with Sjögren's syndrome induce B-lymphocyte survival and activation

Precise probes of type II interferon activity define the origin of interferon signatures in target tissues in rheumatic diseases

CD8 + T cells orchestrate pDC-XCR1 + dendritic cell spatial and functional cooperativity to optimize priming

The inducible CXCR3 ligands control plasmacytoid dendritic cell responsiveness to the constitutive chemokine stromal cell-derived factor 1 (SDF-1)/CXCL12

Induction of interferon-α by immune complexes or liposomes containing systemic lupus erythematosus autoantigen-and Sjögren's syndrome auto antigen-associated RNA

Activation of innate immune responses through Toll-like receptor 3 causes a rapid loss of salivary gland function

Viruses induce high expression of BAFF by salivary gland epithelial cells through TLR-and type-I IFN-dependent and -independent pathways

Toll-like receptor 3 stimulation promotes Ro52/TRIM21 synthesis and nuclear redistribution in salivary gland epithelial cells, partially via type I interferon pathway

Expression of long interspersed nuclear element 1 retroelements and induction of type I interferon in patients with systemic autoimmune disease

Defective regulation of L1 endogenous retroelements in primary Sjogren's syndrome and systemic lupus erythematosus: role of methylating enzymes

LINE1 contributes to autoimmunity through both RIG-I-and MDA5-mediated RNA sensing pathways

The Ro60 autoantigen binds endogenous retroelements and regulates inflammatory gene expression

Cutting Edge: autoantigen Ro52 is an interferon inducible E3 ligase that ubiquitinates IRF-8 and enhances cytokine expression in macrophages

Intracellular antibody-bound pathogens stimulate immune signaling via the Fc receptor TRIM21

Self protection from anti-viral responses -Ro52 promotes degradation of the transcription factor IRF7 downstream of the viral Toll-like receptors

The E3 ubiquitin ligase Ro52 negatively regulates IFN-β production post-pathogen recognition by polyubiquitin-mediated degradation of IRF3

TRIpartite Motif 21 (TRIM21) differentially regulates the stability of interferon regulatory factor 5 (IRF5) Isoforms

Loss of the lupus autoantigen Ro52/ Trim21 induces tissue inflammation and systemic autoimmunity by disregulating the IL-23-Th17 pathway

TRIM21 dysfunction enhances aberrant B-cell differentiation in autoimmune pathogenesis

Anti-Ro52 autoantibodies from patients with Sjögren's syndrome inhibit the Ro52 E3 ligase activity by blocking the E3/E2 interface

Defects in lysosomal maturation facilitate the activation of innate sensors in systemic lupus erythematosus

Autoantigen TRIM21/Ro52 is expressed on the surface of antigen-presenting cells and its enhanced expression in Sjögren's syndrome is associated with B cell hyperactivity and type i interferon activity

Involvement of the interferon-γ-induced T cell-attracting chemokines, interferon-γ-inducible 10-kd protein (CXCL10) and monokine induced by interferon-γ (CXCL9), in the salivary gland lesions of patients with Sjögren's syndrome

B cell-activating factor of the tumor necrosis factor family (BAFF) is expressed under stimulation by interferon in salivary gland epithelial cells in primary Sjogren's syndrome

Increased CCL25 and T helper cells expressing CCR9 in the salivary glands of patients with primary Sjögren's syndrome: potential new axis in lymphoid neogenesis

Unique expansion of IL-21 + Tfh and Tph cells under control of ICOS identifies Sjögren's syndrome with ectopic germinal centres and MALT lymphoma

Expression of B7 costimulatory molecules by salivary gland epithelial cells in patients with Sjögren's syndrome

CD40 on salivary gland epithelial cells: High constitutive expression by cultured cells from Sjögren's syndrome patients indicating their intrinsic activation

IFN-λs mediate antiviral protection through a distinct class II cytokine receptor complex

Type-III interferons in Sjögren's syndrome

Expression of type III interferons (IFNλs) and their receptor in Sjögren's syndrome

Cytometry by time-of-flight immunophenotyping identifies a blood Sjögren's signature correlating with disease activity and glandular inflammation

Functional expression of a costimulatory B7.2 (CD86) protein on human salivary gland epithelial cells that interacts with the CD28 receptor, but has reduced binding to CTLA4

Differentiation of follicular helper T cells by salivary gland epithelial cells in primary Sjögren's syndrome

p38 mitogen-activated protein kinase and nuclear factor-kB facilitate CD40-mediated salivary epithelial cell death

IL-6: a cytokine at the crossroads of autoimmunity

Ratio of blood T follicular regulatory cells to T follicular helper cells marks ectopic lymphoid structure formation while activated follicular helper T cells indicate disease activity in primary Sjögren's syndrome

Is the T follicular regulatory/ T follicular helper cell ratio in blood a biomarker for ectopic lymphoid structure formation in Sjögren's syndrome? Arthritis Rheumatol

IL-7 signaling and CD127 receptor regulation in the control of T cell homeostasis

Increased expression of interleukin-7 in labial salivary glands of patients with primary Sjögren's syndrome correlates with increased inflammation

Interleukin-7 enhances the Th1 response to promote the development of Sjögren's syndrome-like autoimmune exocrinopathy in mice

Increased interleukin (IL)-7Rα expression in salivary glands of patients with primary Sjögren's syndrome is restricted to T cells and correlates with IL-7 expression, lymphocyte numbers and activity

Sjogren Syndromeassociated lymphomas: an update on pathogenesis and management

Aberrant expression of BAFF by B lymphocytes infiltrating the salivary glands of patients with primary Sjögren's syndrome

BAFF mediates survival of peripheral immature B lymphocytes

Cracking the BAFF code

JAK-1 inhibition suppresses interferon-induced BAFF production in human salivary gland: potential therapeutic strategy for primary Sjögren's syndrome

Epigenetic dysregulation in salivary glands from patients with primary Sjögren's syndrome may be ascribed to infiltrating B cells

FcRL4 + B-cells in salivary glands of primary Sjögren's syndrome patients

Repertoire analysis of B-cells located in striated ducts of salivary glands of patients With Sjögren's syndrome

Characterization of human FCRL4-positive B cells

Gene expression profiling of epithelium-associated FcRL4 + B cells in primary Sjögren's syndrome reveals a pathogenic signature

B cell IFN-γ receptor signaling promotes autoimmune germinal centers via cell-intrinsic induction of BCL-6

IFN-γ receptor and STAT1 signaling in B cells are central to spontaneous germinal center formation and autoimmunity

IL-27 directly enhances germinal center B cell activity and potentiates lupus in sanroque mice

Induction of IgG2a class switching in B cells by IL-27

Distinct effector B cells induced by unregulated toll-like receptor 7 contribute to pathogenic responses in systemic lupus erythematosus

IL-21 drives expansion and plasma cell differentiation of autoreactive CD11c hi T-bet + B cells in SLE

FcRL4 acts as an adaptive to innate molecular switch dampening BCR signaling and enhancing TLR signaling

Towards personalised treatment in primary Sjögren's syndrome: baseline parotid histopathology predicts responsiveness to rituximab treatment

Blood and salivary-gland BAFF-driven B-cell hyperactivity is associated to rituximab inefficacy in primary Sjögren's syndrome

Effectiveness of rituximab treatment in primary Sjögren's syndrome: a randomized, double-blind, placebo-controlled trial

Treatment of primary Sjogren syndrome with rituximab: a randomized trial

Randomized controlled trial of rituximab and cost-effectiveness analysis in treating fatigue and oral dryness in primary Sjögren's syndrome

Discriminating gene expression profiles of memory B cell subpopulations

Viral infection expression is required to control chronic intrinsic T-bet -cutting edge: B cell

A broad atlas of somatic hypermutation allows prediction of activation-induced deaminase targets

Salivary gland MALT lymphomas of Sjögren's syndrome patients in majority express rheumatoid factors affinity-selected for IgG

The spectrum of MALT lymphoma at different sites: biological and therapeutic relevance

Among B cell non-Hodgkin's lymphomas, MALT lymphomas express a unique antibody repertoire with frequent rheumatoid factor reactivity

Lymphoma driver mutations in the pathogenic evolution of an iconic human autoantibody

Expansion of autoreactive unresponsive CD21 -/low B cells in sjögren's syndrome-associated lymphoproliferation

Accumulation of antigen-driven lymphoproliferations in complement receptor 2/CD21 −/low B cells from patients with Sjögren's syndrome

Extrafollicular responses in humans and SLE

Stereotypic rheumatoid factors that are frequently expressed in mucosa-associated lymphoid tissue-type lymphomas are rare in the labial salivary glands of patients with Sjögren's syndrome

Acquisition of N-glycosylation sites in immunoglobulin heavy chain genes during local expansion in parotid salivary glands of primary Sjögren patients

RNA-associated autoantigens activate B cells by combined B cell antigen receptor/Toll-like receptor 7 engagement

Molecular characterization of the V H 1-specific variable region determinants recognized by anti-idiotypic monoclonal antibodies G6 and G8

Germline and somatic genetic variations of TNFAIP3 in lymphoma complicating primary Sjögren's syndrome

World workshop on oral medicine VII: immunobiologics for salivary gland disease in Sjögren's syndrome: a systematic review

Small-molecule inhibitors and the salivary gland epithelium in Sjögren's syndrome

Senolytic CAR T cells reverse senescence-associated pathologies

Lack of conventional acinar cells in parotid salivary gland of patient taking an anti-PD-L1 immune checkpoint inhibitor

Checkpoint inhibition-induced sicca: a type II interferonopathy?

The work of the authors is supported by a Dutch Arthritis Society (ReumaNL) Long Term Project Grant (LLP-29). The authors would like to thank M. S. van Ginkel and B. van der Vegt for providing the histology images.

G.M.V. and S.P. researched data for the article and wrote the article. All authors provided substantial contribution to the discussion of content. H.B. and F.G.M.K. reviewed and/or edited the manuscript before submission.

The authors declare no competing interests.

Nature Reviews Rheumatology thanks A. Tzioufas, G. Nocturne, B. Fisher and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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