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\title{Amoxicillin induced mast cell degranulation mediated by Fc\selectlanguage{greek}ε\selectlanguage{english}RI specific
IgG: a new insight to beta-lactam allergy\selectlanguage{english}}



\author[1]{Jiakai Wu}%
\author[1]{Miriam Bennett}%
\author[2]{Silvia Bulfone-Paus}%
\author[1]{Sara Kirkham}%
\author[1]{Clare Murray}%
\author[1]{Angela Simpson}%
\affil[1]{The University of Manchester Division of Infection Immunity and Respiratory Medicine}%
\affil[2]{The University of Manchester School of Biological Sciences}%


\vspace{-1em}



  
  \date{June 18, 2024}


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\selectlanguage{greek}\textbf{Amoxicillin induced mast cell degranulation mediated by FcεRI
specific IgG: a new insight to beta-lactam allergy}\selectlanguage{english}

To the editor,

Immediate drug hypersensitivity reactions (IDHR) to beta-lactams (BL),
referred to here as beta-lactam allergy (BLA), is defined as the
development of symptoms such as urticaria and bronchospasm within one
hour of beta-lactam exposure (1). Classical Type 1 immunoglobulin E
(IgE)-mediated allergy describes a response which occurs within minutes
of allergen exposure in a previously sensitised person. It has therefore
been widely accepted that BLA is mediated by beta-lactam specific IgE
(BL-sIgE) (1). In other Type 1 IgE driven allergies, such as food
allergy, sIgE to major allergen components can be shown to be highly
specific (2, 3). However, for BLA, BL-sIgE has poor specificity,
suggesting alternative mechanisms might be involved (4).

Type 1 IgE mediated reactions are mainly mediated by basophils and mast
cells (MCs). Previous studies have demonstrated the use of the basophil
activation test (BAT) in predicting BLA, with European allergy guidance
stating that BAT can aid BLA diagnosis (5). However, BAT requires fresh
whole blood which is not ideal for in depth mechanistic exploration.

In an \emph{in vitro} BLA model using human progenitor derived MCs, we
have shown that MCs sensitised with serum from a patient with confirmed
BLA, demonstrated amoxicillin (AMX) induced degranulation
(CD63\textsuperscript{+} positivity) after 30 minutes of stimulation
with AMX (Figure 1B). In contrast, sensitisation with serum from a
non-BLA control demonstrated no response (Figure 1C).

The mechanism of degranulation from the BLA model is very different from
the classical IgE-mediated Type 1 degranulation such as that seen in
food and inhalant allergy (6). In our \emph{in vitro} model of classical
IgE-mediated Type 1 degranulation, sIgE binds to Fce\selectlanguage{english}RI with high
affinity which is not disturbed by pipetting and washing (6). In our BLA
model, BLA serum sensitized MCs have a much weaker degranulation and can
easily lose their response to beta-lactam stimulation under physical
disturbance (Figure 1A).

We hypothesised that Fce\selectlanguage{english}RI was involved in the degranulation process in
BLA, not through the high affinity binding of IgE, but instead through
IgG binding. We produced a recombinant fragment representing the
extracellular portion of the Fce\selectlanguage{english}RI receptor alpha chain, which is mainly
responsible for antibody binding. When added to the serum, this fragment
binds and neutralizes Fce\selectlanguage{english}RI-specific antibody---IgE and IgG, by
preventing them binding to the cells. BLA serum was treated with
increasing concentrations of the Fce\selectlanguage{english}RI fragments and then added to the
MCs for sensitization. After stimulating the sensitized MCs using AMX,
we observed a dosage-dependent decrease of MCs degranulation, indicating
direct involvement of the Fce\selectlanguage{english}RI in the degranulation process of BLA
(Figure 2A).

To investigate whether degranulation was mediated by IgE, we neutralized
serum IgE by adding omalizumab before MC sensitisation. This showed no
impact on the AMX induced degranulation (Figure 2A). In contrast,
omalizumab inhibited MC degranulation in a classical IgE model, using
pooled serum from cat allergic patients stimulated with cat allergen
(Figure 2B).

To determine whether Fc\selectlanguage{greek}ε\selectlanguage{english}RI-specific IgG is functionally responsible for
the AMX induced MCs degranulation, we isolated IgG from BLA serum using
IgG-depletion spin columns. MCs sensitized with the IgG-only fraction
showed no response to anti-IgE, but retained a strong response to AMX
(Figure 2B). In comparison, in the IgG-depleted serum fraction
(\textasciitilde{}60\% IgG removed), we observed a strong degranulation
to anti-IgE stimulation but a weakened response to AMX (Figure 2C).

A limitation of this preliminary work is it used serum from only one BLA
patient.

In summary, we developed a cell model for BLA using \emph{in
vitro}differentiated human MCs. This BLA model has demonstrated the
involvement of Fc\selectlanguage{greek}ε\selectlanguage{english}RI-specific IgG in mediating AMX-induced MCs
degranulation. The results are worthy of further investigation in a
larger patient cohort.

Word Count 599

\emph{Figure legend (} Word count 98 and 98 respectively, max 100)

\textbf{Figure 1. Demonstration of \emph{in vitro} BLA model using MCs
derived from human progenitor cells.} A) After washing the sensitized
MCs with phosphate buffered saline (PBS), different cells responsiveness
to AMX and anti-IgE. B) When adding the AMX in the presence of BL IDHR
serum, cell degranulation was observed. C) No degranulation was observed
from AMX stimulation when serum from a non-BL IDHR subject was used. D)
Treatment of AMX in the absence of serum showed a mild increase of CD63
compared to cells without stimulation, Percentage
CD63\textsuperscript{+} cells is shown in the box. Representative
figures from three independent experiments.

\selectlanguage{greek}\textbf{Figure 2. Investigating the role of FcεRI-specific IgG in
BLA\emph{in vitro} model}\selectlanguage{english} A) the BLA \emph{in vitro} model, B) classical
type I IgE cat allergy model. AMX or cat allergen induced degranulation
was quantified on MCs sensitized with single BLA or pooled cat allergic
subject serum pre-treated with recombinant Fc\selectlanguage{greek}ε\selectlanguage{english}RI alpha chain fragment or
omalizumab. C) BLA \emph{in vitro} model; MCs sensitized with either
IgG-depleted serum fraction or IgG-only fraction. Sensitised MCs were
stimulated with anti-IgE, AMX or media control. Histogram demonstrating
fluorescence intensity of CD63 expression on MCs surface. Mean \selectlanguage{ngerman}±SD.
*=p\textless{}0.05 from three independent experiments, NS=not
significant.

\textbf{References}

1. Castells M, Khan DA, Phillips EJ. Penicillin Allergy. New England
Journal of Medicine. 2019;381(24):2338-51.

2. Van Gasse AL, Mangodt EA, Faber M, Sabato V, Bridts CH, Ebo DG.
Molecular allergy diagnosis: status anno 2015. Clin Chim Acta.
2015;444:54-61.

3. Santos AF, Du Toit G, O'Rourke C, Becares N, Couto-Francisco N,
Radulovic S, et al. Biomarkers of severity and threshold of allergic
reactions during oral peanut challenges. Journal of Allergy and Clinical
Immunology. 2020;146(2):344-55.

4. Kosnik M, Zidarn M, Korosec P. Over-reliance on assays for specific
IgE in diagnostics of penicillin allergy? Allergy. 2013;68(12):1626-7.

5. Romano A, Atanaskovic-Markovic M, Barbaud A, Bircher AJ, Brockow K,
Caubet J-C, et al. Towards a more precise diagnosis of hypersensitivity
to beta-lactams --- an EAACI position paper. Allergy.
2020;75(6):1300-15.

6. Bahri R, Custovic A, Korosec P, Tsoumani M, Barron M, Wu J, et al.
Mast cell activation test in the diagnosis of allergic disease and
anaphylaxis. J Allergy Clin Immunol. 2018;142(2):485-96 e16.

\textbf{Authors: Jiakai Wu \textsuperscript{1}, Miriam
Bennett\textsuperscript{1}, Silvia Bulfone-Paus\textsuperscript{2}, Sara
Kirkham\textsuperscript{1}, Clare Murray\textsuperscript{1, 3}, Angela
Simpson\textsuperscript{1, 3}}

\textbf{Affiliations: \textsuperscript{1} Division of Infection,
Immunity and Respiratory Medicine, School of Biological Sciences,
University of Manchester. \textsuperscript{2} Division of
Musculoskeletal \& Dermatological Sciences, School of Biological
Sciences, University of Manchester.}
\textsuperscript{\textbf{3}}\textbf{NIHR Manchester Biomedical Research
Centre, Manchester University NHS Foundation Trust, Manchester Academic
Health Science Centre, Manchester, United Kingdom.}

\textbf{Funding} : This work was funded by North West Lung Centre
Charity. MB was funded by NIHR ACF-2019-06-011 and serum sample storage
supported by access to ManARTS biobank under application number MANarts
M2019 86

All Authors have no conflicts of interest to declare.\selectlanguage{english}
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