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

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

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

Introduction: Although 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. Methods: Cre LysM Casp8 fl/fl mice were bred via a cross between Casp8 fl/fl mice and Cre LysM mice, and RIPK3 -/- Cre LysM Casp8 fl/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. Cre LysM Casp8 fl/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. Results: Loss 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 Cre LysM Casp8 fl/fl mice. Further, caspase-8-deficient macrophages are hyperresponsive to TLR activation and exhibit aberrant M1 macrophage polarization due to RIPK activity. Conclusions: These data demonstrate that caspase-8 functions uniquely in macrophages by controlling the response to TLR activation and macrophage polarization in an RIPK-dependent manner.

Original languageEnglish (US)
Article number291
JournalArthritis Research and Therapy
Volume17
Issue number1
DOIs
StatePublished - Oct 16 2015

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Macrophage Activation
Caspase 8
Toll-Like Receptors
Macrophages
Cell Culture Techniques
Autoimmune Lymphoproliferative Syndrome
Cell Death
Cytokines
Anti-Bacterial Agents
Death Domain Receptors
Protein-Serine-Threonine Kinases
Microbiota
Splenomegaly
Survival Analysis
Nonparametric Statistics
Caspases
Chemokines
Population
Autoimmune Diseases
Fluorescent Antibody Technique

ASJC Scopus subject areas

  • Rheumatology
  • Immunology
  • Immunology and Allergy

Cite this

Cuda, C. M., Misharin, A. V., Khare, S., Saber, R., Tsai, F. N., Archer, A. M., ... Perlman, H. (2015). Conditional deletion of caspase-8 in macrophages alters macrophage activation in a RIPK-dependent manner. Arthritis Research and Therapy, 17(1), [291]. https://doi.org/10.1186/s13075-015-0794-z

Conditional deletion of caspase-8 in macrophages alters macrophage activation in a RIPK-dependent manner. / Cuda, Carla M.; Misharin, Alexander V.; Khare, Sonal; Saber, Rana; Tsai, FuNien N.; Archer, Amy M.; Homan, Philip J.; Haines, G. Kenneth; Hutcheson, Jack; Dorfleutner, Andrea; Budinger, G. R Scott; Stehlik, Christian; Perlman, Harris.

In: Arthritis Research and Therapy, Vol. 17, No. 1, 291, 16.10.2015.

Research output: Contribution to journalArticle

Cuda, CM, Misharin, AV, Khare, S, Saber, R, Tsai, FN, Archer, AM, Homan, PJ, Haines, GK, Hutcheson, J, Dorfleutner, A, Budinger, GRS, Stehlik, C & Perlman, H 2015, 'Conditional deletion of caspase-8 in macrophages alters macrophage activation in a RIPK-dependent manner', Arthritis Research and Therapy, vol. 17, no. 1, 291. https://doi.org/10.1186/s13075-015-0794-z
Cuda, Carla M. ; Misharin, Alexander V. ; Khare, Sonal ; Saber, Rana ; Tsai, FuNien N. ; Archer, Amy M. ; Homan, Philip J. ; Haines, G. Kenneth ; Hutcheson, Jack ; Dorfleutner, Andrea ; Budinger, G. R Scott ; Stehlik, Christian ; Perlman, Harris. / Conditional deletion of caspase-8 in macrophages alters macrophage activation in a RIPK-dependent manner. In: Arthritis Research and Therapy. 2015 ; Vol. 17, No. 1.
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AU - Tsai, FuNien N.

AU - Archer, Amy M.

AU - Homan, Philip J.

AU - Haines, G. Kenneth

AU - Hutcheson, Jack

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N2 - Introduction: Although 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. Methods: Cre LysM Casp8 fl/fl mice were bred via a cross between Casp8 fl/fl mice and Cre LysM mice, and RIPK3 -/- Cre LysM Casp8 fl/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. Cre LysM Casp8 fl/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. Results: Loss 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 Cre LysM Casp8 fl/fl mice. Further, caspase-8-deficient macrophages are hyperresponsive to TLR activation and exhibit aberrant M1 macrophage polarization due to RIPK activity. Conclusions: These data demonstrate that caspase-8 functions uniquely in macrophages by controlling the response to TLR activation and macrophage polarization in an RIPK-dependent manner.

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