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Supplementary Figure 3. Effects of estrogen on IgG-Fc sialylation and IgG-Fc galactosylation in individual patients. Analysis of (a, b) IgG-Fc sialylation and (c, d) IgG-Fc galactosylation in postmenopausal women with active rheumatoid arthritis (RA) randomized to receive hormone replacement therapy (HRT) (a, c) or no HRT (b, d) in a controlled trial. Each line represents one patient. (PDF 2784 kb)

Supplementary methods. (DOCX 131 kb)

Supplementary Figure 4. Correlation between IgG glycosylation and estrogen levels and disease activity. Correlation between (a) IgG-Fc sialylation and (b) IgG-Fc galactosylation (y-axis) and estrogen level (x-axis). Correlation between (c) IgG-Fc sialylation and (d) IgG-Fc galactosylation (y-axis) and Disease Activity Score (DAS) (x-axis). Spearman’s correlation coefficients (r) and p values are shown. (PDF 2188 kb)

Supplementary Figure 2. Estrogen treatment increases IgG-Fc sialylation and IgG-Fc galactosylation in postmenopausal patients with rheumatoid arthritis (RA). Forty-nine postmenopausal women with active RA were included in a randomized controlled trial receiving either hormone replacement therapy (HRT) or no HRT. Serum samples were taken at baseline and after 1 year and 2 years of treatment. Mass spectrometry-based analysis of (a) sialic acids, (b) galactose, and (c) fucose of the Fc portion of IgG2/3 (n = 17–25). Statistical analysis was performed with t tests at selected time points, as well as in an investigation over time in individuals who received HRT. * p

Supplementary Figure 1. Estrogen effects on ovalbumin-specific IgG subclasses. Ovariectomized mice received slow-release treatment pellets with placebo (Pla) or estrogen (E2; 0.83 μg/day). Ten days after ovariectomy, the animals were immunized with ovalbumin (OVA), and 14 days later, they were boostered. On day 38, IgG subclasses of ovalbumin (OVA)-specific IgG were measured. A scatterplot with means indicated by lines is shown (n = 9–11 mice per group). Statistical analysis was performed with analysis of variance followed by the Bonferroni multiple-comparisons test. (PDF 1088 kb)

Bone remodeling is a continuous process to retain the structural integrity and function of the skeleton. A tight coupling is maintained between osteoclast-mediated resorption of old or damaged bones and osteoblast-mediated formation of new bones for bone homeostasis. While osteoblasts differentiate from mesenchymal stem cells, osteoclasts are hematopoietic in origin and derived from myeloid precursor cells. Osteoclast differentiation is driven by two cytokines, cytokine receptor activator of NF-κB ligand (RANKL), and macrophage colony-stimulating factor. Imbalances in the activity of osteoblasts and osteoclasts result in the development of bone disorders. Bacterially caused porcine atrophic rhinitis is characterized by a loss of nasal ventral conche bones and a distortion of the snout. While Bordetella bronchiseptica strains cause mild and reversible symptoms, infection of pigs with toxigenic Pasteurella multocida strains causes a severe and irreversible decay. The responsible virulence factor Pasteurella multocida toxin (PMT) contains a deamidase activity in its catalytical domain that constitutively activates specific heterotrimeric G proteins to induce downstream signaling cascades. While osteoblasts are inhibited by the toxin, osteoclasts are activated, thus skewing bone remodeling toward excessive bone degradation. Still, the mechanism by which PMT interferes with bone homeostasis, and the reason for this unusual target tissue is not yet well understood. Here, we show that PMT has the potential to differentiate bone marrow-derived macrophages into functional osteoclasts. This toxin-mediated differentiation process is independent of RANKL, a cytokine believed to be indispensable for triggering osteoclastogenesis, as addition of osteoprotegerin to PMT-treated macrophages does not show any effect on PMT-induced osteoclast formation. Although RANKL is not a prerequisite, toxin-primed macrophages show enhanced responsiveness to low concentrations of RANKL, suggesting that the PMT-generated microenvironment offers conditions where low concentrations of RANKL lead to an increase in the number of osteoclasts resulting in increased resorption. PMT-mediated release of the osteoclastogenic cytokines such as IL-6 and TNF-α, but not IL-1, supports the differentiation process. Although the production of cytokines and the subsequent activation of signaling cascades are necessary for PMT-mediated differentiation into osteoclasts, they are not sufficient and PMT-induced activation of G protein signaling is essential for efficient osteoclastogenesis.

The glycosylation of IgG greatly influences its interactions with Fc gamma receptors (FcγR) and, thereby, determines whether immune complexes mediate pro- or anti-inflammatory effects on their target cells. Especially the presence of terminal sialic acid constitutes a strong anti-inflammatory signal. The bone destructive autoimmune disease rheumatoid arthritis is strongly associated with the presence of low sialylated autoantibodies against citrullinated proteins (ACPA). We recently reported that those autoantibodies directly promote the development of bone resorbing osteoclasts by binding to their precursors. Osteoclasts develop by cells fusion from macrophage like precursors and have been shown to express several FcγR. It is hence likely that binding of (auto)-immune complexes to these receptors influences osteoclastogenesis. The aim of this thesis was to examine, whether osteoclastogenesis is affected by immune complexes in general and whether IgG sialylation plays a role in this process. As an antigen independent immune complex mimetic we used heat aggregated complexes of pooled human IgG. The importance of IgG sialylation was tested by the enzymatic cleavage of terminal sialic acid prior to the aggregation. Here we show that desialylated IgG complexes foster human osteoclastogenesis by binding to both FcγRII and FcγRIII on preosteoclasts. In contrast, untreated (sialylated) IgG complexes had no significant effect, demonstrating, that IgG sialylation regulates the pro-osteoclastogenic activity of IgG complexes. By injecting sialylated and desialylated murine IgG complexes into the knee joints of C57BL/6 wild type mice, we confirmed our in vitro results and demonstrated that the enhancement of osteoclastogenesis by desialylated immune complexes is independent of inflammation. Interestingly, coinjection of the inflammatory cytokine TNFα equalized the capacity of sialylated compared to desialylated IgG complexes to promote osteoclastogenesis. We further show that the sialylation levels of both, random IgG and ACPA, correlate with bone density in patients with rheumatoid arthritis and that the previously demonstrated pro-osteoclastogenic effect of ACPA relies on low Fc sialylation. In addition, the administration of the sialic acid precursor N-acetylmannosamine to DBA/J1 mice subjected to collagen induced arthritis did not only increase sialylation levels of IgG1, but also lowered the susceptibility of these mice to collagen induced arthritis with a significant negative correlation between the IgG sialylation levels and the final arthritis scores. From these data we conclude that IgG sialylation is a key factor determining the pro-osteoclastogenic capacity of immune complexes under homeostatic conditions. As we did not see any difference between sialylated and non-sialylated IgG complexes in activation of the FcγR-associated kinase Syk, we propose an active regulatory effect of sialylated IgG mediated by a second receptor. In summary we describe a novel mechanism how immune complexes promote bone loss independent of inflammation. We further propose the enhancement of IgG sialylation as an effective treatment of bone loss in rheumatic diseases. Interaktionen zwischen IgG und Fc gamma Rezeptoren (FcγR) werden neben anderen Faktoren auch von der IgG-Glykosylierung beeinflusst. Diese ist damit auch mitbestimmend, ob die Bindung von Immunkomplexen an Zielzellen eine pro- oder anti-inflammatorische Reaktion auslöst. So beinhaltet das Vorhandensein einer terminalen Sialylierung einen starken anti-inflammatorischen Effekt. Die knochenzerstörende Autoimmunerkrankung rheumatoide Arthritis ist stark mit der Anwesenheit von Autoantikörpern (sogenannte ACPA) korreliert, die gegen citrullinierte Proteine gerichtet sind. Wir haben kürzlich berichtet, dass diese Autoantikörper die Entwicklung knochenresorbierender Osteoklasten fördern, indem sie an die Oberfläche derer Vorläufer binden. Osteoklasten entstehen durch Zellfusion aus Makrophagen-ähnlichen Vorläuferzellen. Es wurde gezeigt, dass sie verschiedene FcγR besitzen, wodurch eine Beeinflussung der Osteoklastendifferenzierung durch (auto)-Immunkomplexe wahrscheinlich erscheint. Das Ziel dieser Doktorarbeit war, zu untersuchen, ob Osteoklastogenese von Immunkomplexen beeinflusst wird und inwieweit die IgG-Sialylierung bei diesem Prozess von Bedeutung ist. Als Antigen-unabhängiges Immunkomplexmodell wurden hitzeaggregierte IgG-Komplexe genutzt. Die Bedeutung der IgG-Sialylierung wurde durch enzymatisches Abspalten der teminalen Sialinreste vor der Hitzeaggregation untersucht. In dieser Arbeit zeigen wir, dass desialylierte IgG-Komplexe die Ausdifferenzierung humaner Präosteoklasten über die Bindung sowohl an FcγRII als auch an FcγRIII fördern. Unbehandelte (sialinhaltige) IgG-Komplexe hatten dagegen keinen signifikanten Effekt. Dies zeigt deutlich, dass die pro-osteoklastogene Aktivität von IgG-Komplexen durch die IgG-Sialylierung beeinflusst wird. Diese Ergebnisse wurden durch die intraartikuläre Injektion von sialylierten und desialylierten IgG-Komplexen in Mausknie in vivo bestätigt, wobei allerdings interessanterweise der Zusatz des inflammatorischen Zytokins TNFα die zuvor beobachteten Unterschiede in der Erhöhung der Osteoklastenzahl nach Injektion sialylierter oder desialylierter IgG-Komplexe nivellierte. Weiterhin zeigen wir, dass der Sialylierungsgrad sowohl von unspezifischem Serum-IgG als auch von ACPA mit der Knochendichte in Patienten mit rheumatoider Arthritis korreliert und dass der kürzlich veröffentlichte pro-osteoklastogene Effekt von ACPA von einem niedrigen Fc-Sialylierungsgrad abhängig ist. Desweiteren führte die Fütterung von DBA/J1-Mäusen, die einer Kollagen induzierten Arthritis ausgesetzt wurden, nicht nur zu einem Anstieg der IgG1-Sialylierung, sondern auch zu einer geringeren Anfälligkeit der Mäuse Arthritis zu bekommen. Der Grad der IgG-Sialylierung korrelierte dabei signifikant negativ mit dem finalen Arthritisgrad der Mäuse. Aus diesen Daten schließen wir, dass die IgG-Sialylierung eine Schlüsselrolle innehat, die bestimmt, ob Immunkomplexe Osteoklastogenese fördern oder nicht. Da wir keinen Unterschied in der Aktivierung der FcγR-assoziierten Kinase Syk nach Stimulation mit sialylierten oder desialylierten IgG-Komplexen entdecken konnten, stellen wir die Hypothese auf, dass sialylierte IgG-Komplexe einen aktiven regulatorischen Effekt ausüben, z.B. durch Bindung an einen weiteren Rezeptor. Zusammenfassend beschreiben wir einen neuen Mechanismus, durch den Immunkomplexe Knochenerosion unabhängig von Entzündungsreaktionen befördern. Desweiteren schlagen wir eine Erhöhung des IgG-Sialylierungsgrades als Behandlungsmöglichkeit des Knochenverlustes bei rheumatoider Arthritis vor.