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The Contribution of Innate Immunity to the Pathogenesis of ANCA-associated Vasculitis
Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) constitute a group of vasculitides characterized by neutrophil-rich necrotizing inflammation of small vessels and the presence of ANCA in the circulation. Dying neutrophils surrounding the walls of small vessels are a histological hallmark of AAV. Traditionally it has been assumed that these neutrophils die by necrosis, but neutrophil extracellular traps (NETs) have recently been visualized at the sites of vasculitic lesions. NETs were first described to be involved in capture and elimination of pathogens but dysregulated production and/or clearance of NETs are thought to contribute to vessel inflammation in AAV; directly by damaging endothelial cells and indirectly by acting as a link between the innate and adaptive immune system through the generation of pathogenic PR3-ANCA and MPO-ANCA that can activate neutrophils. ANCA can, however, be found in all individuals and are therefore suggested to belong to the repertoire of natural antibodies produced by innate-like B cells, implying that not all ANCA are pathogenic. 

In paper I, we found neutrophils in patients to be more prone to undergo NETosis/necrosis spontaneously compared with neutrophils in healthy controls (HC), as well as that active patients possessed elevated levels of NETs in the circulation. Our results also suggest that ANCA during remission could contribute to the clearance of NETs as we observed an inverse relation between ANCA and NETs. In paper II, we observed neutrophils in patients to be more easily activated upon ANCA stimulation as they produced more ROS than neutrophils in HC. In paper III, we showed for the first time that cells of adaptive immunity (B and T cells) in addition to cells of innate immunity can release ET-like structures, in this case consisting of mitochondrial (mt) DNA. mtDNA can act as a damage-associated pattern molecule (DAMP) and promote inflammation, and increased levels of mtDNA has been observed in AAV. Our finding broadens our perspective of the possible roles of T and B cells in immunological responses, and should be further investigated in AAV. In paper IV, we observed reduced frequencies of MZ-like B cells, considered to be innate-like B cells that produce natural antibodies, and of the proposed regulatory B (Breg) cell populations CD24highCD27+ and CD25+CD27+ B cells in patients, particularly in those with active disease. We also observed the phenotypes of these different Breg cell populations to be different from the corresponding cells in HC.

We hypothesize that the increased activation potential by neutrophils in AAV to produce ROS and undergo NETosis/necrosis contribute to the excessive inflammation as well as an increased antigen load of PR3 and MPO, and that this in combination with dysregulation of innate-like B cells and Breg cells could lead to break of tolerance to these antigens and production of pathogenic autoantibodies. ANCA can in turn activate neutrophils to release NETs, suggesting a vicious circle in disease development.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2016. , 91 p.
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1545
National Category
Immunology in the medical area Urology and Nephrology
URN: urn:nbn:se:liu:diva-132327DOI: 10.3384/diss.diva-132327ISBN: 9789176856598 (print)OAI: diva2:1043807
Public defence
2016-12-01, Berzeliussalen, Campus US, Linköping, 13:00 (English)
Available from: 2016-11-01 Created: 2016-11-01 Last updated: 2017-01-11Bibliographically approved
List of papers
1. Increased levels of neutrophil extracellular trap remnants in the circulation of patients with small vessel vasculitis, but an inverse correlation to anti-neutrophil cytoplasmic antibodies during remission
Open this publication in new window or tab >>Increased levels of neutrophil extracellular trap remnants in the circulation of patients with small vessel vasculitis, but an inverse correlation to anti-neutrophil cytoplasmic antibodies during remission
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2015 (English)In: Rheumatology, ISSN 1462-0324, E-ISSN 1462-0332, Vol. 54, no 11, 2085-2094 p.Article in journal (Refereed) Published
Abstract [en]

Objectives. Neutrophil extracellular traps (NETs) have been visualized at the site of ANCA-associated vasculitis (AAV) lesions. Increased levels of NET remnants in the circulation have been reported in some AAV patients with active disease. The aim of the present study was to analyse NET remnants in a larger cohort of AAV patients with varying degrees of disease activity and to elucidate possible factors responsible for remnant variation. Methods. Levels of NET remnants in the circulation of healthy controls (HCs; n =31) and AAV patients (n =93) were determined with ELISA. NET remnants were then correlated with ANCA levels, spontaneous and induced cell death (NETosis/necrosis) in vitro, neutrophil count and corticosteroid therapy. Results. Patients with active disease showed higher levels of circulating NET remnants compared with patients in remission (P=0.026) and HCs (P=0.006). From patients sampled during both remission and active disease, we found increased levels during active disease (P=0.0010). In remission, ANCA-negative patients had higher levels of NET remnants than ANCA-positive patients and a negative correlation was observed between NET remnants and PR3-ANCA (rs = 0.287, P=0.048). NET remnants correlated with neutrophil count in HCs (rs =0.503, P=0.014) but not in patients during remission. Neutrophils from patients showed enhanced spontaneous cell death (P=0.043). Conclusion. We found increased levels of circulating NET remnants in patients with active AAV. Furthermore, AAV patients exhibited an increased propensity for spontaneous cell death. NET remnant levels seem to be positively related to disease activity and neutrophil count, but inversely related to ANCA at least during remission.

Place, publisher, year, edition, pages
small vessel vasculitis; ANCA-associated vasculitis; neutrophil extracellular trap (NET); NET remnants; ANCA
National Category
Clinical Medicine
urn:nbn:se:liu:diva-123334 (URN)10.1093/rheumatology/kev217 (DOI)000364760700023 ()26170375 (PubMedID)
Available from: 2015-12-14 Created: 2015-12-11 Last updated: 2016-11-01
2. Neutrophils from vasculitis patients exhibit an increased propensity for activation by anti-neutrophil cytoplasmic antibodies
Open this publication in new window or tab >>Neutrophils from vasculitis patients exhibit an increased propensity for activation by anti-neutrophil cytoplasmic antibodies
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2014 (English)In: Clinical and Experimental Immunology, ISSN 0009-9104, E-ISSN 1365-2249, Vol. 176, no 3, 363-372 p.Article in journal (Refereed) Published
Abstract [en]

Anti-neutrophil cytoplasmic antibodies (ANCA) are thought to be pathogenic in ANCA-associated vasculitis (AAV) by stimulating polymorphonuclear leucocytes (PMNs) to degranulate and produce reactive oxygen species (ROS). The aim of this study was to investigate if PMNs from AAV patients are stimulated more readily by ANCA compared with PMNs from healthy controls (HCs). Differences in ANCA characteristics that can account for different stimulation potential were also studied. PMNs from five AAV patients and five HCs were stimulated with 10 different immunoglobulins (Ig)Gs, purified from PR3-ANCA-positive patients, and ROS production, degranulation and neutrophil extracellular trap (NET) formation was measured. ANCA levels, affinity and clinical data of the AAV donors were recorded. The results show that PMNs from AAV patients produce more intracellular ROS (P=0 center dot 019), but degranulate to a similar extent as PMNs from HCs. ROS production correlated with NET formation. Factors that may influence the ability of ANCA to activate PMNs include affinity and specificity for N-terminal epitopes. In conclusion, our results indicate that PMNs from AAV patients in remission behave quite similarly to HC PMNs, with the exception of a greater intracellular ROS production. This could contribute to more extensive NET formation and thus an increased exposure of the ANCA autoantigens to the immune system.

Place, publisher, year, edition, pages
Wiley-Blackwell, 2014
ANCA-associated vasculitis; autoimmunity; degranulation; reactive oxygen species
National Category
Clinical Medicine
urn:nbn:se:liu:diva-109138 (URN)10.1111/cei.12301 (DOI)000337516700008 ()24666336 (PubMedID)
Available from: 2014-08-13 Created: 2014-08-11 Last updated: 2016-11-01Bibliographically approved

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