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
Authors : Iris Chang1,2†, Abhinav Kaushik,
PhD1,2†, Pattraporn Satitsuksanoa
PhD1, Minglin
Yang1, Laura Buergi Msc1, Stephan R.
Schneider Msc1, Cezmi A. Akdis, MD1,
Kari Nadeau MD, PhD2, Willem van de Veen,
PhD1, Mübeccel Akdis, MD,
PhD1*
1 Swiss Institute of Allergy and Asthma Research
(SIAF), University of Zürich, Davos, Switzerland.
2 Sean N. Parker Center for Allergy and Asthma
Research, Department of Medicine, Stanford University, Palo Alto, CA,
USA.
† 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 accumulation1. 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 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 (Figure 1A and Online Supplementary
Methods) . Interestingly, we observed B-cell clones with mutually
exclusive expression profile of HRH1 and HRH2 genes
(Figure 1B), with more HRH1+ B-cell clones in
BCR-stimulated samples than unstimulated samples. The subsequentHRH1+ vs HRH2+ differential gene expression analysis
(Figure 1C ), reveal 27 differentially expressed (DE) GPCRs in
unstimulated samples, with up-regulated P2RY13 and C5AR1genes in HRH2 + B-cell clones (Figure 2A) , which
are associated with the cAMP signaling and suppressive
pathway3,4. 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 HRH1+ vs HRH2+ clones. The analysis
reveals that the purinergic receptor family of GPCRs
(i.e. P2RY1 , P2RY13 ) and complement component 5a
receptor family of genes (i.e. C5AR1 and C5AR2 ) share
highest degree of interactions. These genes are up-regulated inHRH2+ samples and are well-known to affect cAMP signaling
pathway3,4 (Figure S1A ). Intriguingly, we
also observed upregulation of GPR35 in HRH2 + B cells,
which is associated in maintaining a low baseline Ca2+
level5. Similarly, we also observed up-regulation
of GPR68 and GPR171 in HRH1 + B cells; both are
known to stimulate Ca2+ flux (Online Supplementary Discussion) .
Similarly, 28 GPCRs were differentially expressed in BCR-stimulated
samples (Figure 2B ), including higher expression of serotonin
receptor type 1A (HTR1A ) and HCAR1 (or GPR81 ) inHRH2+ samples, with a cAMP-linked suppressive function. In
addition, we also observed upregulation of complement component 5a
receptor family of genes (i.e., C5AR1 and C5AR2 )
and GPR35 , in agreement with the trend observed in unstimulatedHRH2 + B-cell clones. Surprisingly, we observed a higher
expression of prostaglandin E2 receptor subtype EP4 (PTGER4) and
adenosine A2A receptor
(ADORA2A ) in HRH2+ samples3,6, which are
known to be associated with activation of cAMP production and share the
highest strength of interactions with the cAMP signaling sub-network
(Figure S1B ). Among the up-regulated genes in HRH1 +
samples, we found three Ca2+ mobilizing genes, i.e., GPR34 ,P2RY10 and 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.