Conditional deletion of caspase-8 in macrophages alters macrophage activation in a RIPK-dependent manner.

Carla M Cuda,Harris Perlman,Amy M Archer,Alexander V Misharin,Andrea Dorfleutner,Rana Saber,Funien Tsai,G R Scott Budinger,G Kenneth Haines,Philip J Homan,Christian Stehlik,Sonal Khare,Jack Hutcheson

doi:10.1186/s13075-015-0794-z

Abstract

IntroductionAlthough caspase-8 is a well-established initiator of apoptosis and suppressor of necroptosis, recent evidence suggests that this enzyme maintains functions beyond its role in cell death. As cells of the innate immune system, and in particular macrophages, are now at the forefront of autoimmune disease pathogenesis, we examined the potential involvement of caspase-8 within this population.MethodsCreLysMCasp8fl/fl mice were bred via a cross between Casp8fl/fl mice and CreLysM mice, and RIPK3−/−CreLysMCasp8fl/fl mice were generated to assess the contribution of receptor-interacting serine-threonine kinase (RIPK)3. Immunohistochemical and immunofluorescence analyses were used to examine renal damage. Flow cytometric analysis was employed to characterize splenocyte distribution and activation. CreLysMCasp8fl/fl mice were treated with either Toll-like receptor (TLR) agonists or oral antibiotics to assess their response to TLR activation or TLR agonist removal. Luminex-based assays and enzyme-linked immunosorbent assays were used to measure cytokine/chemokine and immunoglobulin levels in serum and cytokine levels in cell culture studies. In vitro cell culture was used to assess macrophage response to cell death stimuli, TLR activation, and M1/M2 polarization. Data were compared using the Mann–Whitney U test.ResultsLoss of caspase-8 expression in macrophages promotes onset of a mild systemic inflammatory disease, which is preventable by the deletion of RIPK3. In vitro cell culture studies reveal that caspase-8–deficient macrophages are prone to a caspase-independent death in response to death receptor ligation; yet, caspase-8–deficient macrophages are not predisposed to unchecked survival, as analysis of mixed bone marrow chimeric mice demonstrates that caspase-8 deficiency does not confer preferential expansion of myeloid populations. Loss of caspase-8 in macrophages dictates the response to TLR activation, as injection of TLR ligands upregulates expression of costimulatory CD86 on the Ly6ChighCD11b+F4/80+ splenic cells, and oral antibiotic treatment to remove microbiota prevents splenomegaly and lymphadenopathy in CreLysMCasp8fl/fl mice. Further, caspase-8–deficient macrophages are hyperresponsive to TLR activation and exhibit aberrant M1 macrophage polarization due to RIPK activity.ConclusionsThese data demonstrate that caspase-8 functions uniquely in macrophages by controlling the response to TLR activation and macrophage polarization in an RIPK-dependent manner.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0794-z) contains supplementary material, which is available to authorized users.

Highlights

Caspase-8 is a well-established initiator of apoptosis and suppressor of necroptosis, recent evidence suggests that this enzyme maintains functions beyond its role in cell death
In vitro cell culture studies reveal that caspase-8–deficient macrophages are prone to a caspase-independent death in response to death receptor ligation; yet, caspase-8–deficient macrophages are not predisposed to unchecked survival, as analysis of mixed bone marrow chimeric mice demonstrates that caspase-8 deficiency does not confer preferential expansion of myeloid populations
In vitro cell culture studies reveal that caspase-8–deficient macrophages are prone to a caspase-independent death in response to death receptor (DR) ligation; yet, caspase-8–deficient myeloid populations are not predisposed to unchecked survival, as analysis of mixed bone marrow chimeric mice demonstrates that caspase-8 deficiency does not confer preferential expansion of myeloid populations

Summary

Introduction

Caspase-8 is a well-established initiator of apoptosis and suppressor of necroptosis, recent evidence suggests that this enzyme maintains functions beyond its role in cell death. Stimulation of a death receptor (DR), such as Fas or tumor necrosis factor (TNF) receptor 1, by its ligand promotes recruitment of Fas-associated death domain protein (FADD) [1] This protein aggregation facilitates recruitment of the cysteine-aspartic acid enzyme pro-caspase-8, which becomes active upon dimerization. Active caspase-8 initiates the degradative phase of apoptosis through activation of caspases-3/7 or blocks necroptosis via suppression of receptor-interacting serine/threonine protein kinase (RIPK)1-RIPK3 signaling, depending upon the availability of cellular FADD-like interleukin (IL)-1β-converting enzyme-inhibitory protein (cFLIP) [1, 2]. Evidence is emerging that suggests that caspase-8 maintains cell type–specific, RIPK-dependent, cell death–independent functions, including interferon regulatory factor IRF3 processing for degradation [3, 4], IL-1β production [5, 6], inflammasome activation [5, 7], and Toll-like receptor (TLR) signaling [8]

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Conclusion