Lara A. Aqrawi

The Expression of BAFF Is Controlled by IRF Transcription Factors

Patients with systemic lupus erythematosus (SLE) and primary Sjögren’s syndrome (pSS) are typically characterized by the presence of autoantibodies and an IFN-signature. The strength of the IFN-signature positively correlates with disease severity, suggesting that type I IFNs are active players in these diseases. BAFF is a cytokine critical for development and proper selection of B cells, and the targeting of BAFF has emerged as a successful treatment strategy of SLE. Previous reports have suggested that BAFF expression is directly induced by type I IFNs, but the precise mechanism for this remains unknown. In this article, we demonstrate that BAFF is a bona fide ISG and that IFN regulatory factors (IRFs) control the expression of BAFF. We identify IRF1 and IRF2 as positive regulators of BAFF transcription and IRF4 and IRF8 as potent repressors; in addition, we have mapped the precise binding site for these factors in the BAFF promoter. IFN-β injections induced BAFF expression mainly in neutrophils and monocytes, and BAFF expression in neutrophils from pSS patients strongly correlated with the strength of the IFN-signature. In summary, we show that BAFF expression is directly induced by type I IFNs via IRF1 and IRF2, whereas IRF4 and IRF8 are negative regulators of BAFF expression. These data suggest that type I IFN blockade in SLE and pSS patients will lead to downregulation of BAFF and a consequential reduction of autoreactive B cell clones and autoantibodies.

Ductal epithelial expression of Ro52 correlates with inflammation in salivary glands of patients with primary Sjögren's syndrome

Ro52 is an E3 ubiquitin ligase with a prominent regulatory role in inflammation. The protein is a common target of circulating autoantibodies in rheumatic autoimmune diseases, particularly Sjögrens syndrome (SS). In this study we aimed to investigate the expression of the SS target autoantigen Ro52 in salivary glands of patients with primary Sjögrens syndrome (pSS). Ro52 expression was assessed by immunohistochemical staining of paraffin‐embedded and frozen salivary gland biopsies from 28 pSS patients and 19 non‐pSS controls from Swedish and Norwegian registries, using anti‐human Ro52 monoclonal antibodies. The degree and pattern of staining and inflammation was then evaluated. Furthermore, secreted Ro52 protein was measured in saliva and serum samples from the same individuals through a catch‐enzyme‐linked immunosorbent assay (ELISA). Ro52 was highly expressed in all the focal infiltrates in pSS patients. Interestingly, a significantly higher degree of Ro52 expression in ductal epithelium was observed in the patients compared to the non‐pSS controls (P < 0·03). Moreover, the degree of ductal epithelial expression of Ro52 correlated with the level of inflammation (Spearmans r = 0·48, P < 0·0120). However, no secreted Ro52 protein could be detected in serum and saliva samples of these subjects. Ro52 expression in ductal epithelium coincides with degree of inflammation and is up‐regulated in pSS patients. High expression of Ro52 might result in the breakage of tolerance and generation of Ro52 autoantibodies in genetically susceptible individuals. We conclude that the up‐regulation of Ro52 in ductal epithelium might be a triggering factor for disease progression in SS.

Low number of memory B cells in the salivary glands of patients with primary Sjögren's syndrome

We have previously shown that patients with primary Sjögrens Syndrome (pSS) show a significant reduction of autoantigen specific CD27+ memory B cells and an abnormally elevated level of autoantibody producing plasma cells in peripheral blood (PB) compared to controls. Because both memory B cells and plasma cells have been detected in salivary glands (SG) of pSS patients, we aimed to study the B cell pattern in SG biopsies. Double immunohistochemical staining of CD20 and CD27 was carried out on paraffin-embedded SG tissue from 10 pSS patients to distinguish CD20+/CD27+ memory B cells, and identify the CD20+ glandular B cell zones (BCZ). Given that plasma blasts and plasma cells are CD27++ and CD20− , additional CD138 single staining of serial sections allowed the distinction of CD27++/CD138− plasma blasts located within the BCZ from CD27++/CD138+ plasma cells that were found mostly on the periphery of the BCZ and also observed interstitially. Both BCZ and the memory B cell populations were then quantified. Contrary to what has been reported earlier through immunoflourescent staining of memory B cells in SG tissue, we have shown that there is a low number of memory B cells located within the glandular BCZ. Plasma blasts and plasma cells, however, were more abundant in the SG. Together our findings suggest that these low numbers of memory B cells in both PB and SG of pSS patients may be the result of activation of these cells into plasma cells at the site of inflammation.

Adipose tissue is prominent in salivary glands of Sjögren's syndrome patients and appears to influence the microenvironment in these organs.

Abstract A minor salivary gland (SG) biopsy with focal lymphocytic sialadenitis and a focus score of ≥1 is today’s widely accepted pathological finding confirming the SG component of Sjögren’s syndrome (SS). Adipocytes can occupy a large percentage of the SG area although little is known about their significance in SS lesions. This study aimed to characterise adipose tissue infiltration in labial SG biopsies from 27 SS patients and 28 non-SS sicca controls. Biopsies were evaluated by one oral pathologist and assessed for focus score, acinar atrophy, fatty replacement and non-specific chronic inflammation. Moreover, to explore the SG microenvironment, immunohistochemical staining of paraffin-embedded SG tissue was performed using interleukin-6 (IL-6). The fatty replacement was evident in all SS patients possessing autoantibodies (Ro/SSA and/or La/SSB) as well as a positive SG biopsy (focus score ≥1). Additionally, 62% of SS patients having autoantibodies but a negative biopsy showed fatty infiltration (FI) while non-SS controls demonstrated fatty replacement in only 32% of the cases. Overall, the SS group (mean age 53.0 years) had a significantly higher incidence (p value 0.005) of FI than the non-SS controls (mean age 54.8 years). Interestingly, adipocytes were located in IL-6 rich areas, and IL-6 positive adipocytes were detected. As fat deposition seems to be more recurrent in SGs affected by SS, we propose the assessment of adipose tissue replacement as a helpful tool for diagnostic evaluation in SS. Detection of IL-6 positive adipocytes suggests their involvement in immune reactions. Still, functional studies are needed to investigate the SG microenvironment further.

Identification of potential saliva and tear biomarkers in primary Sjögren’s syndrome, utilising the extraction of extracellular vesicles and proteomics analysis

BackgroundThere is a long-lasting need for non-invasive, more accurate diagnostic techniques when evaluating primary Sjögren’s syndrome (pSS) patients. Incorporation of additional diagnostics involving screening for disease-specific biomarkers in biological fluid is a promising concept that requires further investigation. In the current study we aimed to explore novel disease biomarkers in saliva and tears from pSS patients.MethodsLiquid chromatography-mass spectrometry (LC-MS) was performed on stimulated whole saliva and tears from 27 pSS patients and 32 healthy controls, and salivary and tear proteomic biomarker profiles were generated. LC-MS was also combined with size exclusion chromatography to isolate extracellular vesicles (EVs) from both fluids. Nanoparticle tracking analysis was conducted on joint fractions from the saliva and tears to determine size distribution and concentration of EVs. Further EV characterisation was performed by immunoaffinity capture of CD9-positive EVs using magnetic beads, detected by flow cytometry. The LC-MS data were analysed for quantitative differences between patient and control groups using Scaffold, and the proteins were further analysed using the Database for Annotation, Visualization and Integrated Discovery (DAVID), for gene ontology overrepresentation, and the Search Tool for the Retrieval of Interacting Genes/Proteins for protein-protein interaction network analysis.ResultsUpregulation of proteins involved in innate immunity (LCN2), cell signalling (CALM) and wound repair (GRN and CALML5) were detected in saliva in pSS. Saliva EVs also displayed biomarkers critical for activation of the innate immune system (SIRPA and LSP1) and adipocyte differentiation (APMAP). Tear analysis indicated overexpression of proteins involved in TNF-α signalling (CPNE1) and B cell survival (PRDX3). Moreover, neutrophil gelatinase-associated lipocalin was upregulated in saliva and tears in pSS. Consistently, DAVID analysis demonstrated pathways of the adaptive immune response in saliva, of cellular component assembly for saliva EVs, and of metabolism and protein folding in tears in pSS patients.ConclusionsLC-MS of saliva and tears from pSS patients, solely and in combination with size-exclusion chromatography allowed screening for possible novel biomarkers encompassing both salivary and lacrimal disease target organs. This approach could provide additional diagnostic accuracy in pSS, and could possibly also be applied for staging and monitoring the disease.

Autoantigen-Specific Memory B Cells in Primary Sjögren’s Syndrome

Sjögren’s syndrome (SS) is a systemic rheumatic autoimmune disease affecting the exocrine glandular function and is characterized by the presence of autoantibodies against the ribonucleoprotein particles, SS‐A/Ro and SS‐B/La, and mononuclear cell infiltration of exocrine tissues. Our aim is to characterize memory B cell pattern and function in relation to the progression of the disease, by analysing samples from a well‐defined cohort of patients with primary SS. We have measured the number of Ro/La‐specific plasma cells in peripheral blood mononuclear cells (PBMC) from 23 patients and 20 healthy controls by direct enzyme‐linked immunospot (ELISPOT) assay. Furthermore, we quantified the Ro‐ and La‐specific memory B cells in these individuals by a 6‐day in vitro polyclonal stimulation of PBMC followed by an antigen‐specific ELISPOT assay for the detection of memory B cells. In addition to this, ELISA profiling of autoantibodies was carried out using patients’ plasma and supernatant, collected post‐mitogen stimulation of PBMC. The average Ro60‐, Ro52‐ and La48‐specific plasma cells in PB was 9, 17 and 13 cells in 105 PBMC, respectively. After in vitro stimulation, these numbers increased to 43, 50 and 26 for Ro60, Ro52 and La48, correspondingly. However, the fraction of memory B cells activated into antibody‐secreting cells was lower than the overall IgG B cell population. We conclude that these lower Ro/La‐specific memory B cell levels may indicate that a greater portion of the Ro‐ and La‐specific B cells are in an activated stage. This is in tune with previous reports.

Ro52- and Ro60-specific B cell pattern in the salivary glands of patients with primary Sjögren's syndrome

Primary Sjögrens syndrome (pSS) is characterized by the presence of autoantibodies against the ribonucleoprotein (RNP) particles Ro/SSA and La/SSB, and mononuclear cell infiltration of exocrine tissues, especially salivary and lachrymal glands. Low numbers of autoantigen‐specific memory B cells and elevated levels of plasma cells have been detected previously in the peripheral blood (PB) of pSS patients compared to controls. As both Ro52 and Ro60‐specific cells have been detected in the salivary glands (SG) of pSS patients, we aimed to characterize the SSA‐specific B cell pattern in SG biopsies. A series of double immunohistochemical stainings were performed on paraffin‐embedded tissue from 10 well‐characterized pSS patients for each Ro52 and Ro60 along with CD19, CD5, CD20 or CD27, respectively. Ro52 and Ro60‐specific cells detected in SG tissue were found to be CD19+ B cells located outside the CD19+/CD20+ B cell zones (BCZ) and also interstitially. These SSA‐specific cells were also quantified. No SSA‐specific cells were CD5+, indicating that they do not belong to the B‐1 B cell subset. Furthermore, no SSA‐specific cells were observed within the CD20+ BCZ. Hence, no SSA‐specific memory B cells were detected in these individuals. Contrary to this, SSA‐specific cells were found to be CD19+/CD27++, demonstrating that they are differentiating short or long‐lived plasma cells. Taken together, our findings suggest that these lower levels of SSA‐specific memory B cells in PB and absence of SSA‐specific memory B cells in SG of pSS patients could result from activation of these cells into plasma cells at the site of inflammation.

Induction of Local Secretory IgA and Multifunctional CD4+ T-helper Cells Following Intranasal Immunization with a H5N1 Whole Inactivated Influenza Virus Vaccine in BALB/c Mice

Avian influenza subunit vaccines have been shown to be poorly immunogenic, leading to the re‐evaluation of the immunogenic and dose‐sparing potential of whole virus vaccines. In this study, we investigated the immune responses after one or two doses of intramuscular or intranasal whole inactivated influenza H5N1 virus vaccine in BALB/c mice. Serum samples and nasal washings were collected weekly post‐vaccination and analysed using enzyme‐linked immunosorbent assay (ELISA). Sera were also analysed by the haemagglutination inhibition (HI) assay. Antibody‐secreting cells were measured in lymphocytes from spleen and bone marrow via enzyme‐linked immunospot (ELISPOT). Splenocytes were stimulated in vitro, and T‐helper profiles were measured through multiplex bead assay in the supernatants, or intracellularly by multiparametric flow cytometry. Both vaccine routes induced high HI titres following the second immunization (intramuscular = 370, intranasal = 230). Moreover, the intramuscular group showed significantly higher levels of serum IgG (P < 0.01), IgG1 (P < 0.01) and IgG2a (P < 0.01) following the second vaccine dose, while the intranasal group exhibited significantly higher levels of serum IgA (P < 0.05) and local IgA (P < 0.01) in the nasal washings. Also, IgA antibody‐secreting cells were found in significantly higher numbers in the intranasal group in both the spleen (P < 0.01) and the bone marrow (P < 0.01). Moreover, Th1 (TNF‐α, IL‐2, IFN‐γ) and Th2 (IL‐4, IL‐5, IL‐10) cytokines were expressed by both groups, yet only the intranasal group expressed the Th17 marker IL‐17. As the intranasal vaccines induce local IgA and are easily administered, we suggest the intranasally administered whole virus vaccine as a promising candidate for a pandemic H5N1 vaccine.

Non-proliferating plasma cells detected in the salivary glands and bone marrow of autoimmune NOD.B10.H2b mice, a model for primary Sjögren’s syndrome

Abstract Autoantibody secreting plasma cells (PCs) are essential contributors in the development of autoimmune conditions such as primary Sjögren’s syndrome (pSS). Particularly, the long-lived PC subset residing in the bone marrow has shown to continuously produce autoantibodies, whilst remaining unaffected by immunosuppressive treatment. We have previously shown accumulation of potentially long-lived PCs in chronically inflamed salivary glands of pSS patients. In this study, we aimed to characterise the PC compartment in the salivary glands (the target organ for pSS) and bone marrow before the onset of the murine pSS like disease versus advanced diseases progression. Bromodeoxyuridine (BrdU) was incorporated to distinguish the long-lived PCs. Double immunohistochemical staining and immunofluorescence were then conducted on submandibular gland and bone marrow sections from 8- and 40-week-old mice to identify BrdU and CD138. BrdU+ cells were detected in the submandibular glands of 8-week-old mice, and observed within all focal infiltrates by 40 weeks of age. Most CD138+ PCs were however BrdU− and located predominantly on the periphery of these infiltrates. This observation was verified through immunofluorescence. A comparable staining pattern was observed in the bone marrow of 8- and 40-week-old NOD.B10.H2b mice, where some of the CD138+ cells also expressed BrdU. Interestingly, megakaryocytes in the bone marrow of NOD.B10.H2b mice were detected in close proximity to CD138+ cells, illustrating a possible presence of PC survival niches. Our results demonstrate the presence and accumulation of potentially long-lived PCs in NOD.B10.H2b mice as the disease advances.

The expression of B & T cell activation markers in children's tonsils following live attenuated influenza vaccine

Live attenuated influenza vaccines (LAIV) can prevent influenza illness and death in children. The absence of known correlates of protection induced by LAIV requires human studies of underlying mechanisms of vaccine-induced immunity, to further elucidate the immunological processes occurring. In this study, children scheduled for elective tonsillectomy were enrolled in a clinical trial to evaluate the immune response to LAIV, in order to compare T and B cell gene expression profiles. Twenty-three children (aged 3–17 years) were divided into 4 groups; unvaccinated controls, or vaccinated intranasally with LAIV at days 3–4, 6–7, and 12–15 before tonsillectomy. Total RNA extraction was performed on tonsillar tissue and high RNA quality was assured. The samples were then analyzed using a validated RT2 Profiler PCR Array containing 84 gene-specific primers involved in B and T cell activation, proliferation, differentiation, regulation and polarization. The gene expression after LAIV vaccination was subsequently compared to the controls. We observed that at d 3–4 post vaccination, 6 genes were down-regulated, namely APC, CD3G, FASLG, IL7, CD8A and TLR1. Meanwhile at 6–7 days post vaccination, 9 genes were significantly up-regulated, including RIPK2, TGFB1, MICB, SOCS1, IL2RA, MS4A1, PTPRC, IL2 and IL8. By days 12–15 the genes RIPK2, IL4, IL12B and TLR2 were overexpressed. RIPK2 was upregulated at all 3 time points. Our data suggests an overall proliferation, differentiation and regulation of B and T cells in the tonsils following LAIV, where the majority of genes were up-regulated at days 6–7 and normalized by days 12–15. These findings may provide a first step into defining future biomarkers or correlates of protection after LAIV immunization.