In vitro sepsis up-regulates Nociceptin/Orphanin FQ receptor expression and function on human T- but not B-cells

Mark F Bird; Christopher P Hebbes; Anushuya Tamang; Jonathon Mark Willets; Jonathan P Thompson; Remo Guerrini; Girolamo Calo; David G Lambert

doi:10.1111/bph.16088

In vitro sepsis up-regulates Nociceptin/Orphanin FQ receptor expression and function on human T- but not B-cells

Br J Pharmacol. 2023 Sep;180(17):2298-2314. doi: 10.1111/bph.16088. Epub 2023 May 11.

Authors

Mark F Bird¹, Christopher P Hebbes¹, Anushuya Tamang², Jonathon Mark Willets³, Jonathan P Thompson¹, Remo Guerrini⁴, Girolamo Calo⁵, David G Lambert¹

Affiliations

¹ Department of Cardiovascular Sciences, Anaesthesia, Critical Care and Pain Management, University of Leicester, Leicester, UK.
² Cellomatics Biosciences Ltd, Nottingham, UK.
³ Molecular and Cell Biology, University of Leicester, Leicester, UK.
⁴ Department of Chemical, Pharmaceutical and Agricultural Sciences, University of Ferrara, Ferrara, Italy.
⁵ Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padua, Italy.

Abstract

Background and purpose: In animal models of sepsis, increased activation of the Nociceptin/Orphanin FQ (N/OFQ) receptor NOP is associated with mortality and NOP antagonists improved survival. We have explored the role of the N/OFQ-NOP system in freshly isolated volunteer human B- and T-cells incubated with lipopolysaccharide (LPS) and peptidoglycan G (PepG) as a model of in vitro sepsis.

Experimental approach: B- and T-cell NOP expression was measured using the NOP fluorescent probe N/OFQ_ATTO594 , N/OFQ content was measured using immunofluorescence, N/OFQ release was tracked using a CHO_hNOPGαiq5 biosensor assay and NOP function was measured using transwell migration and cytokine/chemokine release using a 25-plex assay format. Cells were challenged with LPS/PepG.

Key results: CD19-positive B-cells bound N/OFQ_ATTO594 ; they also contain N/OFQ. Stimulation with CXCL13/IL-4 increased N/OFQ release. N/OFQ trended to reduced migration to CXCL13/IL-4. Surface NOP expression was unaffected by LPS/PepG, but this treatment increased GM-CSF release in an N/OFQ sensitive manner. CD3-positive T-cells did not bind N/OFQ_ATTO594 ; they did contain N/OFQ. Stimulation with CXCL12/IL-6 increased N/OFQ release. When incubated with LPS/PepG, NOP surface expression was induced leading to N/OFQ_ATTO594 binding. In LPS/PepG-treated cells, N/OFQ reduced migration to CXCL12/IL-6. LPS/PepG increased GM-CSF release in an N/OFQ sensitive manner.

Conclusions and implications: We suggest both a constitutive and sepsis-inducible N/OFQ-NOP receptor autocrine regulation of B- and T-cell function, respectively. These NOP receptors variably inhibit migration and reduce GM-CSF release. These data provide mechanistic insights to the detrimental role for increased N/OFQ signalling in sepsis and suggest a potential role for NOP antagonists as treatments.

Keywords: B-cells; NOP receptor; Nociceptin/Orphanin FQ; T-cells; biosensor; confocal microscopy; sepsis.

MeSH terms

Animals
Granulocyte-Macrophage Colony-Stimulating Factor
Humans
Interleukin-4
Interleukin-6
Lipopolysaccharides / pharmacology
Nociceptin
Nociceptin Receptor
Opioid Peptides / physiology
Receptors, Opioid* / metabolism
Sepsis* / drug therapy

Substances

Receptors, Opioid
Nociceptin Receptor
Granulocyte-Macrophage Colony-Stimulating Factor
Lipopolysaccharides
Interleukin-4
Interleukin-6
Opioid Peptides