Visualization of autoantibodies and neutrophils in vivo identifies novel checkpoints in autoantibody-induced tissue injury

Abstract

In several autoimmune diseases, e.g., pemphigoid disease (PD), autoantibodies are the direct cause of pathology. Albeit key requirements for antibody-mediated diseases were identified, their interactions and exact temporal and spatial interactions remained elusive. the skin is easily accessible for imaging. Thus, we selected epidermolysis bullosa acquisita (EBA), a PD with autoantibodies to type VII collagen (COL7), to visualize interactions of autoantibodies, target tissue and effector cells (neutrophils). Following injection into mice, anti-COL7 IgG bound to the dermal-epidermal junction (DEJ) within minutes. We unexpectedly observed an inhomogeneous distribution of autoantibodies along the DeJ. Thus, we hypothesized that specific external triggers may affect autoantibody distribution. Indeed, mechanical irritation led to an increased autoantibody binding along the DEJ. Subsequently, anti-COL7 IgG was injected into mice expressing green fluorescent protein under the LysM promoter (LysM-eGFP) mice. This allows to visualize myeloid cells in vivo in these animals. Using multiphoton imaging, we observed a limited extravasation of LysM-eGFP + cells into skin was observed within 24 hours. Intriguingly, LysM-eGFP + cells did not immediately co-localize with autoantibodies, which was only noted at later time points. Of note, interactions of LysM-eGFP + with the autoantibodies at the DeJ were short-lived. Collectively, our results define the following checkpoints for autoantibody-induced tissue injury: (i) autoantibody egress to target tissue influenced by mechanical trigger factors, (ii) neutrophil recruitment into the vicinity of autoantibody deposits and (iii) short-term neutrophil localization to these deposits, as well as (iv) delayed recruitment of neutrophils with subsequent autoantibodyinduced inflammation.

Prevalence and presumptive triggers of localized bullous pemphigoid

“…More specifically, in experimental bullous pemphigoid-like inflammatory epidermolysis bullosa acquisita, caused by autoantibodies targeting type VII collagen (COL7), 10 mechanical irritation experienced a more rapid autoantibody deposition in the skin and a more severe clinical disease manifestation in contrast to sites which were not mechanically irritated. 11 Two previous studies addressed the topic of LBP: Chang et al…”

Epidermal Damage Induces Th1 Polarization and Defines the Site of Inflammation in Murine Epidermolysis Bullosa Acquisita

“…In addition, we identified the disruption of the epidermal layer as an additional trigger for EBA skin wound development after autoantibody had bound (Figure 2 and Supplementary Figure S2). Whether other kinds of mechanical manipulations, such as removing only the horny layers, (Pinkus, 1951) or even mechanical irritations without epidermal disruption result in the characteristic trauma-induced lesions in inflammatory and classical EBA (Hundt et al, 2020) will be an aim of further studies.…”

Therapeutic effects of Fc gamma RIV inhibition are mediated by selectively blocking immune complex-induced neutrophil activation in epidermolysis bullosa acquisita

“…Visualization experiments were performed on day 1, 3 and 8 of autoantibody-induced experimental EBA. An in-depth method description is detailed in our recent publication ( 12 ). Mice were anesthetized using 10 µg/kg fentanyl, 16.8 mg/kg midazolam and 1.7 mg/kg medetomidine hydrochloride.…”