Highlights from the breakout session: cellular and molecular mechanisms of disease (II)

Abstract

The steps leading to key pathogenetic aspect of ANCA-associated vasculitis, such as lung nodules in granulomatosis with polyangiitis (GPA), onset of glomerulonephritis (GN) in experimental myeloperoxidase (MPO)-ANCA models and the implications of carrying staphylococcal superantigens, such as toxic shock syndrome toxin-1 (tsst-1), in GPA remain to be elucidated. The session ‘Cellular and molecular mechanisms of disease (II)’ was devoted to close some of these knowledge gaps and underlined that research in ANCA-associated vasculitis leads to more granular understanding of these complex diseases. Here, we present an overview of this session at a glance.

The first presentation ‘Lung granulomas in murine GPA are induced by MPO-ANCA and begin as microabscesses that evolve into granulomas’ focused on granuloma formation in GPA. This study aimed to answer how granulomatosis can be induced in a mouse model of ANCA-associated vasculitis, resembling GPA as a disease phenotype. There are vast differences in terms of lung manifestations, with some features being exclusively present in proteinase 3 (PR3)-ANCA (mainly GPA) vasculitis, namely granuloma formation, while microscopic polyangiitis (MPA) often presents with interstitial lung disease and eosinophilic GPA with adult-onset asthma. Reasons for these differences are unclear.

In this study, wild-type mice received MPO-ANCA IgG on days 0 and 1 and intratracheal lipopolysaccharide on day 1. Seven time points were studied and animals were euthanized accordingly. Lung infiltrates with a specific focus on neutrophils, macrophages and T cells were assessed by light microscopy and immunohistochemistry. All animals developed necrotizing GN and granulomatous lung disease by day 7. On immunohistochemistry, it was demonstrated that the initial formation of microabscesses and presence of capillarities progressed to granulomas during longer term follow-up. In accordance with these changes, there was a shift from neutrophils to macrophages as the predominant inflammatory cells by day 3. In further experiments in Rag2 knock-out mice, which are lacking functional T cells, the development of necrotizing GN and granuloma formation was confirmed, thus the observed changes are happening independent of T cells.

In conclusion, this study showed that the combination of MPO-ANCA IgG and lipopolysaccharide provokes the onset of a lung model resembling GPA. This pathological changes are independent of T cells, which do not seem to be involved in vasculitic granuloma formation.

The second presentation ‘Neutrophil proteases and free fatty acids mediate giant cell and granuloma formation in GPA: implications for therapy’ also focused on the pathogenesis of granuloma formation in an experimental setting. Prior research has focused on stimulation of monocytes and peripheral blood mononuclear cells (PBMCs) with PR3 and MPO obtained from patients with AAV, and found that PR3 promoted monocyte-derived multinucleated giant cell formation, which was restricted to PR3 obtained from patients with GPA and was not observed if PR3 was derived from MPA patients. This process was dependent on the presence of inflammatory stimuli and presence of the protease-activated receptor-2, while granuloma-like structures in PBMCs were surrounded by T cells [1].

In the current study, the authors study the role of PR3, elastase and their receptors as well as free fatty acids and CD36, which acts a multi-functional scavenger receptor and as a modulator of lipid homeostasis and immune response. It was demonstrated that elastase is also capable to induce multinucleated giant cell formation in vitro when extracted from patients with GPA. In addition, palmitic acid binding CD36, overexpressed on monocytes of patients and present at higher levels in blood samples of patients with GPA, induced multinucleated giant cell formation, but thereby stimulating macrophage inhibition factor rather than the expression of interleukin-6. The latter is prominently involved in the formation of multinucleated giant cells when PR3 and elastase from individuals with GPA are extracted to stimulate monocytes.

In conclusion, the current study expands on prior published experience to create a model of in vitro granuloma formation. The authors found that PR3, elastase and CD36 promote multinucleated giant cell formation, thereby involving different pathways. The effect of PR3 on granuloma formation could be confirmed in vivo using a Zebrafish model.

The third presentation ‘Human MPO-ANCA induces crescentic glomerulonephritis in mice expressing human MPO and FcγRIIa’ aimed to answer the question whether injection of human MPO-ANCA would induce crescentic GN in humanized mice knocked in for MPO and PR3 and transgenic for human FcγRIIa. Therefore, a set of experiments was carried out to provide evidence that human MPO-ANCA extracted from effluents of patients undergoing plasma exchange and exhibiting high ANCA titres is indeed pathogenic.

Primed neutrophils of healthy individuals had a more pronounced reactive oxygen species (ROS) production when stimulated with patient IgG. For subsequent in vivo experiments, the MPO-ANCA IgG fraction inducing the highest ROS production was chosen. Female hMPO.hPR3.hFcγRIIa mice received two doses of the human MPO-ANCA fraction, alongside an intraperitoneal injection of lipopolysaccharide. The experiments ended after 6 days when mice were euthanized. All nine animals had developed crescentic GN at that time with a mean number of 15 ± 4% of glomeruli developing crescents, and presence of microscopic haematuria and proteinuria. Control mice did not develop GN.

In conclusion, this is yet another study providing evidence that ANCA are pathogenic and disease manifestations, in this case crescentic GN, can be induced by the injection of MPO-ANCA IgG into humanized mice. This model resembling the human disease can be used for further investigations into potential druggable targets and development of therapies, which should aim at mitigating the disease progression.

The fourth presentation ‘The sequential carbonyl derivates and hydrazones adduct formation on myeloperoxidase contribute to development of auto-antigenicity’ aimed to understand why hydralazine, a drug used to manage hypertension in some countries, can induce ANCA-associated vasculitis. Drug-induced vasculitis is most often either MPO-ANCA positive or presents with dual positivity, defined as presence of PR3- and MPO-ANCA [2]. The proposed mechanism by which hydralazine can induce ANCA-associated vasculitis is hinging on formation of hydrazones adduct on MPO.

In vitro experiments revealed that carbonyl derivates are required for hydralazine to bind a protein, and it was demonstrated that hydrazones adduct was formed on MPO thereby enabling such conditions. In plasma samples of patients with hydralazine-induced vasculitis, hydrazones adduct on MPO could be detected, while they were undetectable in primary ANCA-associated vasculitis or healthy subjects. The use of multiple commercial anti-MPO antibodies provided evidence that hydrazones adduct formation on MPO results in conformational changes. Autoantibodies (IgG and IgM) obtained from patients with hydralazine-induced vasculitis were reactive against hydralazine-modified MPO.

In conclusion, this is an important study shedding light on hydralazine-induced vasculitis, a drug that is still in use to manage hypertension. Drug-induced vasculitis often requires shorter periods of immunosuppression once the offending agent is discontinued. The presence of hydrazones adduct formation on MPO as measured by these tests might be used to limit the exposure to immunosuppression in individuals with hydralazine-induced vasculitis.

The fifth presentation ‘Isolation and profiling of auto-antigen-specific B cells in MPO-ANCA vasculitis’ speculated whether circulating antigen-specific B cells can be isolated to profile relevant B-cell receptors, which might in turn be used to predict future relapse in MPO-ANCA vasculitis. Therefore PBMCs obtained from four patients with active disease (defined as BVAS > 0) and three healthy individuals were used to isolate and expand cells in vitro.

Antigen-specific B cells (MPO-tetramer positive) had higher anti-MPO IgG in comparison to MPO-tetramer negative ones or control populations. These cells did not share significant sequence overlap with MPO-tetramer negative populations. Further experiments are required to provide more information to whether these clonotypes can be used to monitor disease-specific B cell activity and thereby guide treatment, either in the induction phase or to tailor maintenance therapy according to the presence of these cells.

The sixth presentation ‘Unraveling the impact of TSST-1 on T and B cell responses in granulomatosis with polyangiitis: insights into disease pathogenesis and progression’ expanded on prior research which found an association between the staphylococcal superantigen tsst-1 and disease relapses of GPA [3,4]. This study investigated the impact of tsst-1 on T and B cell responses in GPA.

The authors included 63 patients with GPA in remission, of whom 30 (47.6%) were Staphylococcus aureus carriers, and 24 age- and sex-matched healthy controls. The frequencies of circulating tsst-1-reactive Vß2+ Th cells in PBMCs were significantly higher in GPA compared with controls. In vitro stimulation with S. aureus virulence factors induced a comparable intracellular cytokine production of Th cells between carriers and non-carriers, while the frequency of interleukin-21-producing Th cells was greater in response to tsst-1 in S. aureus carriers. Production of both, IgG and PR3-ANCA, was induced by tsst-1 in vitro.

This study underlines that the presence of tsst-1 contributes to ANCA production exerted by interleukin-21 secreting Th cells, which may contribute to the severity of the disease and disease progression.

In summary, these studies provide key insights into the complexity of microscopic polyangiitis and GPA, thereby identifying potential targets of future drug targets. The focus on understudied disease manifestations such as granuloma formation, drug-induced vasculitis, disease relapse and the study of ANCA pathogenicity underlines the importance of this session.

Data availability

Abstracts from the 21st International Vasculitis Workshop are available via doi: 10.5281/zenodo.11068008.

Funding

No specific funding was received from any funding body in the public, commercial and not-for profit sectors for this article.

Disclosure statement: Andreas Kronbichler received consultancy or speaking fees from Amgen, AstraZeneca, Boehringer Ingelheim, CSL Vifor, Delta4, GlaxoSmithKline, Miltenyi Biotech, Novartis, Otsuka, Roche, and Walden Biosciences. Kazuo Suzuki has declared no conflicts of interest.

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