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\begin{document}

\title{Distinct and mutually exclusive Ca ++ flux- and adenyl cyclase-inducing
gene expression profiles of G-Protein-Coupled Receptors on human
antigen-specific B cells}



\author[1]{Iris Chang}%
\author[1]{Abhinav Kaushik}%
\author[1]{Pattraporn Satitsuksanoa}%
\author[1]{Minglin Yang}%
\author[1]{Laura Buergi}%
\author[1]{Stephan Schneider}%
\author[1]{Cezmi Akdis}%
\author[2]{Kari Nadeau}%
\author[1]{Willem van de Veen}%
\author[1]{Mubeccel Akdis}%
\affil[1]{Universitat Zurich Schweizerisches Institut fur Allergie- und Asthmaforschung}%
\affil[2]{Stanford University Sean N Parker Center for Allergy and Asthma Research}%


\vspace{-1em}



  
  \date{May 25, 2024}


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\textbf{Title} : Distinct and mutually exclusive Ca\textsuperscript{++}
flux- and adenyl cyclase-inducing gene expression profiles of
G-Protein-Coupled Receptors on human antigen-specific B cells

\textbf{Authors} : Iris Chang\textsuperscript{1,2+}, Abhinav Kaushik,
PhD\textsuperscript{1,2+}, Pattraporn Satitsuksanoa
PhD\textsuperscript{1}, Minglin Yang\textsuperscript{1}, Laura Buergi
Msc\textsuperscript{1}, Stephan R. Schneider Msc\textsuperscript{1},
Cezmi A. Akdis, MD\textsuperscript{1}, Kari Nadeau MD,
PhD\textsuperscript{2}, Willem van de Veen,
PhD\textsuperscript{1}\textsubscript{,} M\selectlanguage{ngerman}übeccel Akdis, MD,
PhD\textsuperscript{1*}

\textsuperscript{1~}Swiss Institute of Allergy and Asthma Research
(SIAF), University of Zürich, Davos, Switzerland.

\textsuperscript{2}~Sean N. Parker Center for Allergy and Asthma
Research, Department of Medicine, Stanford University, Palo Alto, CA,
USA.

\selectlanguage{english}+ Contributed equally

* Corresponding author

B cells play an essential role in allergies by producing
allergen-specific IgE, which is a prerequisite for allergen-induced
degranulation of mast cells (MCs) and basophils. MCs, basophils,
dendritic cells and bacteria are capable of releasing inflammatory
mediators including histamine. Histamine is a bioactive amine that
exerts its function through binding to histamine receptors (HRs), which
are 7-transmembrane G-protein-coupled receptors (GPCRs). There are four
types of HRs (HR1-4), wherein HR1 ligation triggers Ca2+ mobilization,
HR2 stimulates and increases cAMP concentrations, and HR3 and HR4
inhibit cAMP accumulation\textsuperscript{1}. In the presence of
histamine in the environment, high affinity HR1is triggered causing
cellular activation, followed by expression of 10 times lower affinity
HR2 to regulate the over-inflammatory events. These HRs trigger
different intracellular events upon activation, with HR1~as a Ca2+
flux-inducing activating receptor and~HR2~as an adenyl
cyclase-stimulating suppressive receptor \textsuperscript{1,2}.
Therefore, to explore the response of B-cells in allergic diseases, we
analyzed the expression profile of HRs and other GPCRs in B cell clones.
We hypothesized that the expression profile of HRs (HR1+ vs HR2+ B cell
clones) is associated with significant changes in the expression profile
of other GPCRs that govern the downstream cascade of pathways associated
with cAMP signaling or Ca2+ mobilization.

A total of 27 IgG1 and IgG4 expressing B cell clones were isolated for
gene expression analysis under BCR stimulated and unstimulated
conditions \textbf{(Figure 1A} and \textbf{Online Supplementary
Methods)} . Interestingly, we observed B-cell clones with mutually
exclusive expression profile of \emph{HRH1} and \emph{HRH2} genes
(\textbf{Figure 1B),} with more \emph{HRH1+} B-cell clones in
BCR-stimulated samples than unstimulated samples. The
subsequent\emph{HRH1+} vs \emph{HRH2+} differential gene expression
analysis (\textbf{Figure 1C} ), reveal 27 differentially expressed (DE)
GPCRs in unstimulated samples, with up-regulated~\emph{P2RY13~}
and\emph{~C5AR1}genes\emph{~} in \emph{HRH2} + B-cell clones
(\textbf{Figure 2A)} , which are associated with the cAMP signaling and
suppressive pathway\textsuperscript{3,4}. To further prioritize the DE
GPCRs specifically associated with Ca2+ and cAMP signaling pathways, we
reconstructed the co-expression networks and performed the weighted
degree analysis across \emph{HRH1+} vs \emph{HRH2+} clones. The analysis
reveals that the purinergic receptor family of GPCRs (i.e.~\emph{P2RY1}
,~\emph{P2RY13} )\emph{~} and complement component 5a receptor family of
genes (i.e.~\emph{C5AR1~} and\emph{~C5AR2} ) share highest degree of
interactions. These genes are up-regulated in\emph{HRH2+} samples and
are well-known to affect cAMP signaling pathway\textsuperscript{3,4}
(\textbf{Figure S1A} ). Intriguingly, we also observed upregulation of
\emph{GPR35} ~in \emph{HRH2} + B cells, which is associated in
maintaining a low baseline Ca2+ level\textsuperscript{5}. Similarly, we
also observed up-regulation of~\emph{GPR68} and \emph{GPR171} in
\emph{HRH1} + B cells; both are known to stimulate Ca2+ flux
(\textbf{Online Supplementary Discussion)} .

Similarly, 28 GPCRs were differentially expressed in BCR-stimulated
samples (\textbf{Figure 2B} ), including higher expression of serotonin
receptor type 1A (\emph{HTR1A} ) and \emph{HCAR1} ~(or~\emph{GPR81} )
in\emph{HRH2+} samples, with a cAMP-linked suppressive function. In
addition, we also observed upregulation of complement component 5a
receptor family of genes (i.e.,~\emph{C5AR1~} and\emph{~C5AR2} )
and~\emph{GPR35} , in agreement with the trend observed in
unstimulated\emph{HRH2} + B-cell clones. Surprisingly, we observed a
higher expression of prostaglandin E2 receptor subtype EP4
(\emph{PTGER4)~} and adenosine A2A receptor (\emph{ADORA2A} ) in
\emph{HRH2+} samples\textsuperscript{3,6}, which are known to be
associated with activation of cAMP production and share the highest
strength of interactions with the cAMP signaling sub-network
(\textbf{Figure S1B} ). Among the up-regulated genes in \emph{HRH1} +
samples, we found three Ca2+ mobilizing genes, i.e.,~\emph{GPR34}
,\emph{P2RY10} ~and \emph{PTAFR} .

The results reported in this study provides data for a novel hypothesis
suggesting investigation of co-expressed genes that may play important
synergistic or antagonistic regulatory roles in B-cell function.

\textbf{References}

1. Jutel M, Akdis M, Akdis C. Histamine, histamine receptors and their
role in immune pathology. \emph{Clinical \& Experimental
Allergy.}2009;39(12):1786-1800.

2. Jutel M, Watanabe T, Klunker S, et al. Histamine regulates T-cell and
antibody responses by differential expression of H1 and H2
receptors.\emph{Nature.} 2001;413(6854):420-425.

3. Thompson RJ, Sayers I, Kuokkanen K, Hall IP. Purinergic Receptors in
the Airways: Potential Therapeutic Targets for Asthma? \emph{Frontiers
in allergy.} 2021:16.

4. Li XX, Lee JD, Massey NL, et al. Pharmacological characterisation of
small molecule C5aR1 inhibitors in human cells reveals biased activities
for signalling and function. \emph{Biochem Pharmacol.} 2020;180:114156.

5. Schneditz G, Elias JE, Pagano E, et al. GPR35 promotes glycolysis,
proliferation, and oncogenic signaling by engaging with the sodium
potassium pump. \emph{Sci Signal.} 2019;12(562).

6. Kim HJ, Kim SH, Kim M, et al. Inhibition of 15-PGDH prevents ischemic
renal injury by the PGE(2)/EP(4) signaling pathway mediating
vasodilation, increased renal blood flow, and increased adenosine/A(2A)
receptors. \emph{Am J Physiol Renal Physiol.} 2020;319(6):F1054-F1066.

\textbf{Figure Legends}

\textbf{Figure 1} . Study design and differential gene expression of
histamine receptors. \textbf{a.} The study design used for isolation and
sorting of B-cells clones for gene expression analysis and the data
analysis workplan for identification and prioritization of
GPCRs.\textbf{b.} Mutually exclusive expression profile of \emph{HRH1}
and\emph{HRH2} in both unstimulated and BCR-stimulated samples. The
dotted box represents the threshold within which samples were considered
as double negative, i.e., \emph{HRH1-} /\emph{HRH2-} . \textbf{c} .
Volcano plots to highlight differentially expressed GPCR genes in
\emph{HRH1+}vs \emph{HRH2+} samples in both unstimulated and
BCR-stimulated samples. The red dots represent significantly
differentially expressed genes (p \textless{} 0.05 \& log-foldchange
\textgreater{} 0.5).

\textbf{Figure 2} . Differentially expressed GPCRs in \emph{HRH1+}
vs\emph{HRH2+} samples. \textbf{a.} Heatmap showing expression profile
of differentially expressed GPCR genes in all the BCR-stimulated
samples, including double negative samples. The top two panels represent
the\emph{HRH1+} and \emph{HRH2+} status of each sample. \textbf{b.} The
boxplot showing topmost significantly differentially expressed GPCR
genes in (p \textless{} 0.001) in BCR-stimulated samples.
\textbf{c.}Heatmap showing expression profile of differentially
expressed GPCR genes in all the unstimulated samples. The annotations
are same as Figure2a. \textbf{d.} The boxplot showing topmost
significantly differentially expressed GPCR genes in (p \textless{}
0.001) in unstimulated samples.

\textbf{Conflict of Interest:} Authors declare no conflict of interest.

\textbf{Author Contribution:} IC: Writing, review \& editing, formal
analysis; AK: Writing, review \&editing, software and formal analysis;
PS, LB, SRS: review \& editing; MY: formal analysis; KN: supervision,
review \& editing; WvdV: review \& editing, conceptualization, method,
provide data; CAA and MA: review \& editing, conceptualization,
supervision\selectlanguage{english}
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