The regulatory and effector functions of B cells in ANCA-associated vasculitis

Systemische auto-immuunziekten waarbij necrotiserende ontstekingen optreden in kleine en middelgrote bloedvaten kunnen gepaard gaan met de aanwezigheid van specifieke auto-antilichamen, ook wel anti-neutrofiele cytoplasmatische antilichamen (ANCA) genoemd. B-cellen spelen waarschijnlijk een belangrijke rol in deze ANCA-geassocieerde vasculitiden (AAV) vanwege hun productie van de ANCA. Weinig is echter bekend over de mechanismen die leiden tot ANCA-productie in B-cellen. In dit proefschrift wordt aangetoond dat interleukine-21 (IL-21) en B cel activerende factor (BAFF) de in vitro productie van ANCA kunnen stimuleren. Bovendien bleek het aantal IL-21 producerende T helper cellen verhoogd in ANCA-positieve patiënten ten opzichte van ANCA-negatieve patiënten en gezonde personen. Deze bevindingen suggereren dat IL-21 bijdraagt aan de productie van ANCA in patiënten met AAV. Hoewel B-cellen bijdragen aan het ontstaan van AAV door hun productie van ANCA, suggereren recente studies dat B-cellen afweerreacties in andere auto-immuunziekten kunnen onderdrukken. Deze onderdrukkende B-cellen, ook wel regulerende B-cellen genoemd, kunnen andere afweercellen remmen middels hun productie van IL-10. In het tweede deel van dit proefschrift wordt aangetoond dat er weliswaar een verstoorde homeostase van B-cellen is in AAV, maar dat de productie van IL-10 in B-cellen niet verstoord is. Bovendien bleken B-cellen van AAV-patiënten geen defecten te vertonen in het vermogen om andere afweercellen, in dit geval monocyten, te remmen. Derhalve werden geen aanwijzingen gevonden dat functionele defecten van regulerende B-cellen ten grondslag liggen aan het ontstaan van AAV. Samenvattend leveren de bevindingen uit dit proefschrift nieuwe inzichten op in de diverse functies van B-cellen in AAV-patiënten.Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV) are systemic autoimmune diseases characterized by necrotizing inflammation of small- to medium-sized blood vessels. In patients with AAV, B cells are considered pathogenic as they produce autoantibodies (ANCA) that mediate neutrophil activation which leads to vascular injury. Although ANCA play a pathogenic role in AAV, it is unclear what drives ANCA production. The first aim of this thesis was to study the mechanisms and factors that stimulate autoantibody production in patients with AAV. We demonstrated that interleukin (IL)-21 in combination with B cell activating factor (BAFF) stimulates ANCA production in vitro. In agreement, we demonstrated an increased frequency of IL-21-producing T helper cells in ANCA-positive patients when compared with ANCA-negative patients and healthy controls, suggesting that IL-21 might contribute to ANCA production in patients. Besides antibody production, B cells are known to affect immune responses by providing co-stimulatory signals and secretion of cytokines and growth factors. Recently a novel B cell subset, termed regulatory B cells, was described and reported to exert a suppressive effect on other immune cells. Therefore, the second aim was to characterize the circulating B cell subsets and evaluate the suppressive function of B cells in AAV patients. We found disturbed homeostasis of circulating B cells in AAV patients but the capacity of B cells in these patients to produce the anti-inflammatory cytokine IL-10 and suppress monocyte activation was not impaired.Overall, the data presented in this thesis contribute to the knowledge on the humoral and cellular functions of B cells in AAV

Celiac disease – causes and therapy, and optimization of anther culture method for intensification of Latvian wheat varieties breeding

Kopējā cilvēku populācijā ar celiakiju slimo 1% pasaules iedzīvotāju. Celiakijas slimnieki uzturā nedrīkst lietot glutēnu saturošus produktus, t.s. kviešus. Aktuāli ir pētījumi par celiakijas rašanās cēloņiem un celiakijas slimniekiem piemērotu kviešu izveidi. Pasaulē tiek īstenotas kviešu uzlabošanas programmas, tāpēc īpaša uzmanība tiek pievērsta speltas kviešiem. Par lielāko speltas kviešu vērtību tiek uzskatīta to spēja kodēt ļoti specifiskas pazīmes, kas nosaka, ka speltas kvieši ir mazāk toksiski celiakijas slimniekiem. Šī darba mērķis ir pilnveidot in vitro metodes, kas paredzētas Latvijas klimatiskiem apstākļiem piemērotu kviešu selekcijas intensifikācijai, kā arī darbā tika pētīti celiakijas cēloņi un terapija, un specializētās bezglutēna pārtikas pieejamība Latvijā. Tika konstatēts, ka putekšņmaciņu kultūras metode veiksmīgi var tikt izmantota paātrinātai selekcijas materiāla iegūšanai no Latvijas kviešu šķirnēm. Atslēgvārdi: celiakija, speltas kvieši, dubultotie haploīdi.Celiac disease is common in 1% of the general population. Celiac disease patients cannot use gluten containing products, including wheat. Current researches are realized to study the causes of celiac disease and to develop a wheat variety suitable for celiacs consumption. Wheat improvement programmes are implemented, the interest about spelt wheat has renewed as a result of research. Spelt wheat is valuable because of its ability to encode specific characteristics, which determine spelt wheat reduced toxicity in celiac disease. The main goal of this study was to improve in vitro methods to intensify breeding of wheat, which is suitable for cultivating in Latvia. In this paper the causes and the therapy of celiac disease were studied, and the availability of specialized gluten-free food in Latvia was researched. It was established that the anther culture method can be successfully used to obtain doubled haploids from Latvian wheat varieties. Keywords: celiac disease, spelt wheat, doubled haploids

The regulatory and effector functions of B cells in ANCA-associated vasculitis

Systemische auto-immuunziekten waarbij necrotiserende ontstekingen optreden in kleine en middelgrote bloedvaten kunnen gepaard gaan met de aanwezigheid van specifieke auto-antilichamen, ook wel anti-neutrofiele cytoplasmatische antilichamen (ANCA) genoemd. B-cellen spelen waarschijnlijk een belangrijke rol in deze ANCA-geassocieerde vasculitiden (AAV) vanwege hun productie van de ANCA. Weinig is echter bekend over de mechanismen die leiden tot ANCA-productie in B-cellen. In dit proefschrift wordt aangetoond dat interleukine-21 (IL-21) en B cel activerende factor (BAFF) de in vitro productie van ANCA kunnen stimuleren. Bovendien bleek het aantal IL-21 producerende T helper cellen verhoogd in ANCA-positieve patiënten ten opzichte van ANCA-negatieve patiënten en gezonde personen. Deze bevindingen suggereren dat IL-21 bijdraagt aan de productie van ANCA in patiënten met AAV. Hoewel B-cellen bijdragen aan het ontstaan van AAV door hun productie van ANCA, suggereren recente studies dat B-cellen afweerreacties in andere auto-immuunziekten kunnen onderdrukken. Deze onderdrukkende B-cellen, ook wel regulerende B-cellen genoemd, kunnen andere afweercellen remmen middels hun productie van IL-10. In het tweede deel van dit proefschrift wordt aangetoond dat er weliswaar een verstoorde homeostase van B-cellen is in AAV, maar dat de productie van IL-10 in B-cellen niet verstoord is. Bovendien bleken B-cellen van AAV-patiënten geen defecten te vertonen in het vermogen om andere afweercellen, in dit geval monocyten, te remmen. Derhalve werden geen aanwijzingen gevonden dat functionele defecten van regulerende B-cellen ten grondslag liggen aan het ontstaan van AAV. Samenvattend leveren de bevindingen uit dit proefschrift nieuwe inzichten op in de diverse functies van B-cellen in AAV-patiënten. Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV) are systemic autoimmune diseases characterized by necrotizing inflammation of small- to medium-sized blood vessels. In patients with AAV, B cells are considered pathogenic as they produce autoantibodies (ANCA) that mediate neutrophil activation which leads to vascular injury. Although ANCA play a pathogenic role in AAV, it is unclear what drives ANCA production. The first aim of this thesis was to study the mechanisms and factors that stimulate autoantibody production in patients with AAV. We demonstrated that interleukin (IL)-21 in combination with B cell activating factor (BAFF) stimulates ANCA production in vitro. In agreement, we demonstrated an increased frequency of IL-21-producing T helper cells in ANCA-positive patients when compared with ANCA-negative patients and healthy controls, suggesting that IL-21 might contribute to ANCA production in patients. Besides antibody production, B cells are known to affect immune responses by providing co-stimulatory signals and secretion of cytokines and growth factors. Recently a novel B cell subset, termed regulatory B cells, was described and reported to exert a suppressive effect on other immune cells. Therefore, the second aim was to characterize the circulating B cell subsets and evaluate the suppressive function of B cells in AAV patients. We found disturbed homeostasis of circulating B cells in AAV patients but the capacity of B cells in these patients to produce the anti-inflammatory cytokine IL-10 and suppress monocyte activation was not impaired. Overall, the data presented in this thesis contribute to the knowledge on the humoral and cellular functions of B cells in AAV.

Immune regulatory mechanisms in ANCA-associated vasculitides

A group of primary vasculitides is associated with the presence of anti-neutrophil cytoplasmic autoantibodies (ANCA). In these diseases, the contribution of ANCA to disease pathogenesis has been studied extensively. Also, in patients with ANCA-associated vasculitides, the T lymphocyte compartment is dysregulated, as aberrant distribution and function of T cell subsets has been shown. In this review we will discuss the putative role of T lymphocytes in inflammation and granuloma formation, as well as the involvement of B cell compartments in the pathophysiology of ANCA-associated vasculitis

Altered B cell balance, but unaffected B cell capacity to limit monocyte activation in anti-neutrophil cytoplasmic antibody-associated vasculitis in remission

OBJECTIVE: Regulatory B cells (Bregs) constitute a subset of B cells with immunomodulatory properties. Numerical and functional alterations in the Breg compartment have been associated with autoimmunity. The aim of this study was to assess the frequency and function of Bregs in patients with ANCA-associated vasculitis (AAV). METHODS: B cell subsets were determined in the peripheral blood of 48 AAV patients (12 active, 36 in remission) and 41 healthy controls (HCs) by flow cytometry. Bregs were defined within the CD19(+) population as CD24(hi)CD38(hi) or CD24(hi)CD27(+) cells. The percentage of IL-10-positive B cells in circulation was analysed by flow cytometry. Sorted CD19(+) B cells were co-cultured with monocytes to evaluate their capacity to inhibit monocyte TNF-α production upon lipopolysaccharide stimulation. RESULTS: The frequency of circulating CD19(+)CD24(hi)CD38(hi) cells was not different in AAV patients in remission compared with HCs, but was decreased in patients with active disease [mean in HCs 5.5% (s.d. 1.6) vs active 3.8% (s.d. 2.8), P = 0.0104]. Furthermore, the percentage of CD19(+)CD24(hi)CD27(+) cells was significantly decreased in both remission and active patients when compared with HCs [HCs 15.0% (s.d. 9.3) vs remission 6.6% (s.d. 4.4) (P < 0.0001) vs active 6.4% (s.d. 6.2) (P = 0.0006)]. The frequency of IL-10-positive B cells was comparable between patients and HCs. B cells from AAV patients suppressed monocyte TNF-α production to a similar extent to cells from HCs. CONCLUSION: Based on immunophenotypic classification, Bregs are numerically diminished in AAV patients. However, B cell function in terms of IL-10 production and their capacity to suppress monocyte activation is not compromised in AAV patients in remission

Toll-like receptor 9 activation enhances B cell activating factor and interleukin-21 induced anti-proteinase 3 autoantibody production in vitro

Objectives. Granulomatosis with polyangiitis (GPA) is a relapsing small-vessel vasculitis characterized by circulating ANCA against PR3. The mechanisms that trigger PR3-ANCA production are unknown. The aim of this study was to determine whether endogenous factors [B cell activating factor (BAFF) and IL-21] and exogenous factors [oligodeoxynucleotides containing CpG motifs (CpG-ODN)] synergize in stimulating PR3-ANCA production in GPA patients. Methods. Peripheral blood mononuclear cells from GPA patients and healthy controls (HCs) were cultured in the presence of BAFF and IL-21, with or without CpG-ODN, for 12 days. PR3-ANCA production in culture supernatants was quantified by Phadia EliA. Phenotypic characterization and the influence of CpG-ODN treatment on IL-21 receptor (IL-21R), transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI) and BAFF receptor (BAFF-R) expression on B cells was analysed by flow cytometry. Results. Stimulation with BAFF and IL-21 significantly increased ANCA production in patient samples, which could be augmented further by addition of CpG-ODN. Stimulation with CpG-ODN increased the percentage of IL-21R(+) and TACI(+) B cells but did not affect BAFF-R expression. GPA patients had an increased percentage of circulating IL-21R(+) and a decreased percentage of TACI(+) circulating memory B cells when compared with HCs. Additionally, patients had decreased expression of BAFF-R on B cells, which was inversely correlated with BAFF concentrations in plasma. Conclusion. Our data demonstrate that endogenous and exogenous factors can synergize to promote PR3-ANCA production. Mechanistically, CpG-ODN up-regulated IL-21R and TACI expression on B cells, possibly sensitizing these cells for IL-21-and BAFF-mediated signals. Agents inhibiting Toll-like receptor 9, BAFF and IL-21 signalling pathways may serve as potential therapeutics for intervention in GPA patients