title: Conditional deletion of caspase-8 in macrophages alters macrophage activation in a RIPK-dependent manner.
abstract: BACKGROUND
  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 Ly6C(high)CD11b(+)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.
authors.affiliation: Division of Rheumatology, Department of Medicine, Feinberg School of Medicine, Northwestern University, 240 East Huron Street, Room M300, Chicago, IL, 60611, USA. c-cuda@northwestern.edu.
authors.forename: Carla M
authors.initials: CM
authors.lastname: Cuda
authors.affiliation: Division of Rheumatology, Department of Medicine, Feinberg School of Medicine, Northwestern University, 240 East Huron Street, Room M300, Chicago, IL, 60611, USA. a-misharin@northwestern.edu.
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Misharin
authors.affiliation: Division of Rheumatology, Department of Medicine, Feinberg School of Medicine, Northwestern University, 240 East Huron Street, Room M300, Chicago, IL, 60611, USA. sonalkhare@northwestern.edu.
authors.forename: Sonal
authors.initials: S
authors.lastname: Khare
authors.affiliation: Division of Rheumatology, Department of Medicine, Feinberg School of Medicine, Northwestern University, 240 East Huron Street, Room M300, Chicago, IL, 60611, USA. r-saber@nlaw.northwestern.edu.
authors.forename: Rana
authors.initials: R
authors.lastname: Saber
authors.affiliation: Division of Rheumatology, Department of Medicine, Feinberg School of Medicine, Northwestern University, 240 East Huron Street, Room M300, Chicago, IL, 60611, USA. fntsai@u.northwestern.edu.
authors.forename: FuNien
authors.initials: F
authors.lastname: Tsai
authors.affiliation: Division of Rheumatology, Department of Medicine, Feinberg School of Medicine, Northwestern University, 240 East Huron Street, Room M300, Chicago, IL, 60611, USA. amy.archer@northwestern.edu.
authors.forename: Amy M
authors.initials: AM
authors.lastname: Archer
authors.affiliation: Division of Rheumatology, Department of Medicine, Feinberg School of Medicine, Northwestern University, 240 East Huron Street, Room M300, Chicago, IL, 60611, USA. philip.homan@northwestern.edu.
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Homan
authors.affiliation: Department of Pathology, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. kenneth.haines@mountsinai.org.
authors.forename: G Kenneth
authors.initials: GK
authors.lastname: Haines
authors.affiliation: Division of Rheumatology, Department of Medicine, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA. jack.hutcheson@utsouthwestern.edu.
authors.forename: Jack
authors.initials: J
authors.lastname: Hutcheson
authors.affiliation: Division of Rheumatology, Department of Medicine, Feinberg School of Medicine, Northwestern University, 240 East Huron Street, Room M300, Chicago, IL, 60611, USA. a-dorfleutner@northwestern.edu.
authors.forename: Andrea
authors.initials: A
authors.lastname: Dorfleutner
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL, 60611, USA. s-buding@northwestern.edu.
authors.forename: G R Scott
authors.initials: GR
authors.lastname: Budinger
authors.affiliation: Division of Rheumatology, Department of Medicine, Feinberg School of Medicine, Northwestern University, 240 East Huron Street, Room M300, Chicago, IL, 60611, USA. c-stehlik@northwestern.edu.
authors.forename: Christian
authors.initials: C
authors.lastname: Stehlik
authors.affiliation: Division of Rheumatology, Department of Medicine, Feinberg School of Medicine, Northwestern University, 240 East Huron Street, Room M300, Chicago, IL, 60611, USA. h-perlman@northwestern.edu.
authors.forename: Harris
authors.initials: H
authors.lastname: Perlman
mesh_terms: D000818:Animals; D000943:Antigens, Differentiation / Q000276:immunology / Q000378:metabolism; D000950:Antigens, Ly / Q000276:immunology / Q000378:metabolism; D051940:B7-2 Antigen / Q000276:immunology / Q000378:metabolism; D015153:Blotting, Western; D039481:CD11b Antigen / Q000276:immunology / Q000378:metabolism; D053181:Caspase 8 / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D002478:Cells, Cultured; D005260:Female; D005434:Flow Cytometry; D007249:Inflammation / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D008262:Macrophage Activation / Q000276:immunology*; D008264:Macrophages / Q000201:enzymology / Q000276:immunology* / Q000378:metabolism; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008822:Mice, Transgenic; D009113:Muramidase / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D022423:Myeloid Cells / Q000201:enzymology / Q000276:immunology / Q000378:metabolism; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D013154:Spleen / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D051193:Toll-Like Receptors / Q000276:immunology / Q000378:metabolism
chemical_list: D000943:Antigens, Differentiation; D000950:Antigens, Ly; D051940:B7-2 Antigen; D039481:CD11b Antigen; C494192:Ly-6C antigen, mouse; D051193:Toll-Like Receptors; C036763:monocyte-macrophage differentiation antigen; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; C485615:Ripk3 protein, mouse; D009113:Muramidase; C472450:lysozyme M, mouse; D053181:Caspase 8