Title : Role of Innate Immune System Signals in Acute Sterile Inflammation and Its Resolution

Keywords : Organ Ischemy Reperfusion, Immunoregulation, T lymphocytes, alarmins, amphiregulin, cytokines, lactate shuttle, tissue repair

Thesis director : André Herbelin et Alice Barbarin

Subject :
Organ Ischemia Reperfusion (IR) syndrome is being studied in our laboratory as a model of acute sterile inflammation, as part of both basic and medical research in organ transplantation.
Organ IR is characterized by a large leukocyte infiltrate and severe tissue damage whose initiator and resolution signals remain poorly described to date. Assuming that the early release of alarmins by the suffering cells is critical to this process, we have demonstrated in mouse models that a key mechanism responsible for tissue damage associated with an IR sequence in the kidney (Ferhrat et al, 2018) and liver (Barbier et al, 2021) is the biological axis consisting of the alarmin IL-33 and innate T lymphocytes (ITL) called iNKT (invariant killer T cells). Based on the literature, IL-33 is known to have modulatory properties that direct immune responses to their restoration stage. We will investigate whether IL-33 in our mouse model of hepatic IR involves a restoration axis involving Treg, ITL (iNKT, MAIT and LT-γδ), ILC (for “innate lymphoid cells”) and M2
macrophages.
As underlying mechanisms, we will pay particular attention to the place of: 1) AREG, as a growth factor described as a direct mediator of tissue repair and as a positive regulator of immune system-mediated restoration activity (particularly Treg and ITL) in response to IL-33; 2) lactate and its MCT1 transporter, whose interaction would control the inflammatory response and its resolution, promoting the restoration functions of LTIs and Treg or the suppressive/protective effects of ILC2 and M2
macrophages.

Methods:
To analyze the consequences of deletion of IL-33, AREG, and MCT1 in our mouse model, during successive phases of inflammation and repair, we will use transgenic mice from the cross between Creert 2 tamoxifen (tamoxifen-dependent conditional Cre recombinase) mice and mice with loxP sites flanking one or more exons of the genes encoding IL-33, AREG, and MCT1 (IL-33fl/fl, AREGfl/fl, and MCT1fl/fl). In these different situations, we will perform clinico-biological analyses (measurement of hepatic suffering: ALT/ASAT dosage; measurement of tissue injury and fibrosis and of the regeneration/proliferation status of hepatocytes by immuno-histochemistry). In parallel, we will evaluate by spectral flow cytometry the number, phenotype
(membrane/intracytoplasmic expression of MCT1 and IL-33 ST2 receptor), activation status (membrane expression of CD69 marker), and associated regulatory/repair functions
(membrane/intracytoplasmic expression of AREG, IL-10, TGF-β, and IL-4) of immune cells (neutrophils monocytes, Treg, ILC, ITL) both locally (liver) and in periphery (spleen)

As a complementary approach, the effects alone or in combination of IL-33 and lactate on Treg and ITL in terms of proliferation, IFN-γ/TNF-α production, AREG, ST2, and MCT1 expression modulation, and M1/M2 differentiation will be modelled in vitro from isolated mononuclear cell sources of mouse spleen and liver and human peripheral blood.

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