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

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 CreLysMCasp8fl/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. 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 CreLysMCasp8fl/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. Electronic supplementary material The online version of this article (doi:10.1186/s13075-015-0794-z) contains supplementary material, which is available to authorized users.

We previously showed that conditional deletion of Fas or caspase-8 specifically in innate immune cells supports a role for these signaling components in cell activation. An aggressive systemic lupus erythematosus (SLE)-like disease develops in mice following myeloid cell-specific deletion of Fas (Cre LysM Fas fl/fl ) [9] or dendritic cell (DC)-specific deletion of caspase-8 (Cre CD11c Casp8 fl/fl ) [10]. Cre LysM Fas fl/fl are also more susceptible than control mice to lipopolysaccharide (LPS)-induced shock [9]. In addition, caspase-8-deficient bone marrow-derived dendritic cells (BMDCs) are hyperresponsive to TLR activation in an RIPK1-dependent manner, and Cre CD11c Casp8 fl/fl splenic DCs upregulate costimulatory molecules in response to in vivo administration of TLR agonists [10]. Further, deletion of MyD88 in Cre CD11c Casp8 fl/fl mice prevents SLE-like disease, although oral antibiotic treatment is ineffective at diminishing inflammatory phenotypes [10]. Because these data reveal a cell-specific role for Fas in myeloid cells and caspase-8 in DCs in the regulation of TLR signaling, we evaluated the consequences of caspase-8 deletion in the myeloid cell compartment.
We now document that caspase-8 functions in myeloid cells to maintain macrophage activation, in part through RIPK1 and RIPK3. Specific deletion of caspase-8 in myeloid cells (Cre LysM Casp8 fl/fl ) leads to the development of a mild systemic inflammation characterized by splenomegaly, lymphadenopathy, immune complex deposition in the kidney, proteinuria, hypergammaglobulinemia, and elevated amounts of serum cytokines that is preventable by RIPK3 deletion. Although Ly6C high and Ly6C low CD11b + F4/80 + splenic populations are increased in Cre LysM Casp8 fl/fl mice, these cells are insufficient inducers of antigen-specific T-cell proliferation. In vitro cell culture studies reveal that caspase-8-deficient macrophages are prone to a caspase-independent death in response to 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. Despite the relatively mild inflammatory phenotype of Cre LysM Casp8 fl/fl mice, caspase-8-deficient bone marrow-derived macrophages (BMDMs) are hyperresponsive to TLR activation in an RIPK1-dependent manner. Further, myeloid cell-specific caspase-8 deficiency appears to dictate the in vivo response to TLR activation, as injection of TLR ligands into Cre LysM Casp8 fl/fl mice upregulates the expression of costimulatory CD86 on the Ly6C high CD11b + F4/80 + splenic population. In addition, oral antibiotic treatment prevents inflammatory disease phenotypes in young Cre LysM Casp8 fl/fl mice. Moreover, caspase-8 controls the polarization of macrophages in response to M1-skewing media in an RIPK1-dependent manner. These data document a role for caspase-8 as a regulator of both the TLR response and macrophage polarization via limiting RIPK in macrophages.

Histopathologic studies
Paraffin-embedded kidney sections (5 μm) were treated with periodic acid-Schiff stain, and a pathologist blinded to the study scored kidney sections using an Olympus BS40 microscope (Olympus Life Science, Center Valley, PA, USA) as previously described [12]. Frozen kidney sections (10 μm) were stained with anti-IgG-fluorescein isothiocyanate [12]. All images were photographed at × 40, ×200, ×400, or × 600 magnification on an Olympus BX41 microscope equipped with an Olympus DP20 camera.

Flow cytometry
Surface staining of cell suspensions and gating strategies were carried out as previously described [9,10,13]. At least 100,000 events were captured on a BD LSR II flow cytometer (BD Biosciences, San Jose, CA, USA). Data were analyzed with FlowJo software (Tree Star, Ashland, OR, USA). Dead cells were excluded using the Molecular Probes LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Life Technologies, Carlsbad, CA, USA). For cellsorting studies, splenocytes preincubated with Fc block antibody were stained with fluorescent antibodies (information available upon request). Splenocyte populations sorted on a BD FACSAria II instrument (BD Biosciences) at the University of Chicago and Northwestern University Cancer Center Flow Core had an average purity of 97 %.

In vitro assays
For mixed leukocyte reactions, splenocytes were incubated with anti-CD19 beads and negative fractions were incubated with anti-CD11b magnetic-activated cell sorting beads (Miltenyi Biotec, Bergisch Gladbach, Germany) to purify antigen-presenting cells (APCs). Purified APCs were pulsed with 10 μg/ml ovalbumin (OVA) peptide (amino acids 323-339) for 60 minutes at 37°C. OVA-specific splenic CD4 + T cells were isolated from B6.CD45.1/OT-II/RAG −/− mice using CD4 + T-cell isolation kits (Miltenyi Biotec) according to the manufacturer's instructions. Purity of APCs and T cells was 90 %. T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) (500 nM for 12 minutes at 37°C; Invitrogen, Carlsbad, CA, USA). Pulsed APCs at various ratios were incubated with 2 × 10 5 CFSElabeled T cells with or without 5 μg/ml class B CpG (ODN 1668; InvivoGen, San Diego, CA, USA) in triplicate in 96-well flat-bottomed plates at 37°C for 3 days. Cell clusters were dissociated with 7.5 mM ethylenediaminetetraacetic acid for 15 minutes, and stained with anti-CD4 (BD Biosciences). 7-Aminoactinomycin D (0.25 mg/test; BD Biosciences) was used to exclude dead cells. A constant number of CaliBRITE beads (BD Biosciences) were added for acquisition of equal parts in each culture. Live T cells were gated, and the number of divided cells showing less than maximal CFSE fluorescence intensity was determined.

Data analysis
For analysis of macrophage polarization, data were normalized to expression of housekeeping genes and imported into Partek Genomics Suite V6.6 software (Partek, St. Louis, MO, USA). Differentially expressed genes between the different groups of stimulated macrophages, as well as transcripts with variable expression within the data set, were calculated using one-way analysis of variance (ANOVA). Differentially expressed genes between two analyzed macrophage populations were defined by a Bonferroni-corrected p value <0.05 unless stated otherwise. Principal component analysis (PCA) using all transcripts was performed for visualization of sample relationships. Hierarchical clustering of the differentially expressed genes was performed based on a Euclidean algorithm for dissimilarity and average linkage method to determine distance between clusters. All other data are shown as mean ± SD and were compared by Mann-Whitney U test using GraphPad Prism 5.0 software (GraphPad Software, San Diego, CA, USA).

Mice with conditional deletion of caspase-8 in myeloid cells develop a mild systemic inflammatory disease
The authors of a previous report demonstrated that Cre LysM Casp8 fl/− mice failed to generate caspase-8 deficiency in myeloid cells or an observable phenotype [14]. However, in the present study, Cre LysM Casp8 fl/fl mice exhibited caspase-8 deletion specifically in myeloid populations. PCR and immunoblot analysis of FACS-sorted splenic neutrophils (CD11b + Ly6G + ) and Ly6C low and Ly6C high CD11b + F4/80 + cells (Additional file 1: Figure S1a, b) and M-CSFgenerated BMDMs (Additional file 1: Figure S2,a, b) from Cre LysM Casp8 fl/fl mice showed caspase-8 deletion, whereas lymphocytes and DC populations retained caspase-8. Further, there was no defect in the generation of BMDMs from mice with myeloid cell-specific caspase-8 deficiency (Additional file 1: Figure S2,c, d). Loss of caspase-8 in myeloid cells led to splenomegaly in both young and aged mice (Fig. 1a, b). However, there was no increase in total splenocyte numbers (Fig. 1c). In addition, lymphadenopathy was present in young and aged Cre LysM Casp8 fl/fl mice (Fig. 1d). Cre LysM Casp8 fl/fl mice failed to develop glomerulonephritis (Fig. 1e, f), although mild IgG deposition in the kidney (Fig. 1e) and higher proteinuria levels ( Fig. 1g) were detected compared with control mice. Further, Cre LysM Casp8 fl/fl mice displayed elevated levels of IgG2b and total IgM antibodies ( Fig. 1i) but did not show evidence of circulating autoreactive antibodies (Fig. 1h). Moreover, serum levels of proinflammatory molecules soluble receptor activator of nuclear factor κB ligand (sRANKL) and keratinocyte chemoattractant/growth-regulated oncogene-α (KC/Gro-α) (Fig. 1j) were elevated compared with levels in control mice. Mortality was similar between Cre LysM Casp8 fl/fl and control mice (Fig. 1k). These data indicate that myeloid cell-specific deletion of caspase-8 induces a mild systemic inflammatory disease that may be mediated by enhanced circulating cytokines and chemokines.

Myeloid cell-specific loss of caspase-8 increases splenic CD11b + F4/80 + populations
To determine the cellular mechanism responsible for the mild inflammatory phenotype that arises in Fig. 1 Mice with conditionally deleted caspase-8 exhibit mild systemic inflammation. We evaluated 2-3-month old (young) and 6-8-month-old (aged) female Casp8 fl/fl (control) and Cre LysM Casp8 fl/fl mice (n ≥ 4) for systemic autoimmune disease phenotypes. a Representative spleens and cervical lymph nodes from aged mice. b Spleen weights of young and aged mice. c Total numbers of live splenocytes from young and aged mice. d Cervical lymph node weights of young and aged mice. e Formalin-fixed kidney sections (5 μm) of aged mice stained with periodic acid-Schiff (PAS) and frozen kidney sections (10 μm) stained with IgGα-fluorescein isothiocyanate. f Kidney score of aged mice. g Proteinuria of aged mice assessed using Uristix reagent strips. Serum from aged mice was evaluated for levels of h ssDNA-, dsDNA-, histone-, and chromatin-reactive IgG antibodies; i total IgM and IgG isotypes; and j cytokines and chemokines. k Survival study. Data are represented as mean ± SD and were compared by Mann-Whitney U test: *p < 0.05; ***p < 0.0005. Casp8 caspase-8, dsDNA double-stranded DNA, Gro-α growth-regulated oncogene-α, IFN interferon, Ig immunoglobulin, IL interleukin, KC keratinocyte chemoattractant, LN lymph node, MCP monocyte chemoattractant protein, OD optical density, sRANKL soluble receptor activator of nuclear factor κB ligand, ssDNA single-stranded DNA, TNF tumor necrosis factor Cre LysM Casp8 fl/fl mice, multiparameter flow cytometry was used. Cre LysM Casp8 fl/fl mice exhibited increased numbers of Ly6C high and Ly6C low splenic CD11b + F4/80 + cells, whereas the CD11b + F4/80 − Ly6G + neutrophils and CD11b − F4/80 + red pulp macrophage populations were not statistically altered compared with control mice (Fig. 2a). Although increased in numbers, no significant alterations in surface expression of activation markers were observed on caspase-8-deficient Ly6C high and Ly6C low splenic CD11b + F4/80 + cells compared with control populations (Fig. 2b). Cre LysM-Casp8 fl/fl mice also showed no statistical difference in conventional and plasmacytoid DC numbers (Fig. 2c) or activation status (Fig. 2d) compared with control mice. The lymphocytic populations were then evaluated to determine whether the increased presence of Cre LysM Casp8 fl/fl Ly6C high and Ly6C low splenic CD11b + F4/80 + cells affected B-and T-cell functionality. Loss of caspase-8 in myeloid cells had no observable effect on total B-cell numbers (Fig. 2e), subset distribution (Fig. 2f), or activation and maturation (Fig. 2g). In addition, Cre LysM Casp8 fl/fl mice did not exhibit any alterations in CD4 + or CD8 + total, naïve (CD44 − CD62L + ), or activated (CD44 + CD62L − ) T cells or in the numbers of double-negative T cells (CD4 − CD8 − CD3 + B220 + ), which are associated with deficiencies in Fas [15][16][17] (Fig. 2h). Activated CD4 + and CD8 + T cells expressed similar levels of CD69 and PD-1 (Fig. 2i), and regulatory T-cell numbers were unchanged (Fig. 2j), between Cre LysM Casp8 fl/ fl and control mice. Additionally, the capacity for antigen presentation to T cells was assessed. Caspase-8-deficient CD11b + cells incubated with OVA peptide suppressed OT-II-specific (C57BL/6-Tg(TcraTcrb)425Cbn/Crl) CD4 + T-cell proliferation in the presence or absence of TLR9 activation compared with control CD11b + cells (Fig. 2k). Taken together, these results suggest that, although there are increased numbers of Ly6C high and Ly6C low splenic CD11b + F4/80 + cells in Cre LysM Casp8 fl/fl mice, these cells do not promote heightened activation and functionality of surrounding cells and in fact are capable of suppressing CD4 + T-cell proliferation on a per-cell basis.
Caspase-8 deficiency alters macrophage TLR responses in vivo but has a minimal effect on myeloid cell homeostasis As TLRs play a central role in macrophage activation, we assessed the in vivo expression pattern of TLRs and response to TLR agonists of macrophages in the absence of caspase-8. TLR2, TLR4, TLR7, and TLR9 expression was similar between Cre LysM Casp8 fl/fl and control Ly6C high and Ly6C low splenic CD11b + F4/80 + cells (Fig. 3a). To determine the functional response of these TLRs in caspase-8-deficient populations, LPS, imiquimod, or CpG was intraperitoneally injected into Cre LysM Casp8 fl/fl and control mice. Both TLR4 and TLR9 in vivo activation induced increased CD86 expression on caspase-8-deficient Ly6C high splenic CD11b + F4/80 + cells compared with control cells (Fig. 3b). These data suggest that, although this population expressed CD86 at normal levels under steady-state conditions, caspase-8 deficiency confers the capacity to upregulate this costimulatory marker upon TLR activation. Further, TLR4 and TLR9 in vivo activation induced elevated levels of circulating anti-and proinflammatory cytokines in Cre LysM Casp8 fl/fl and control mice ( Fig. 3c and Additional file 1: Figure S3), but to a much greater extent in control mice in response to TLR4 activation (Fig. 3c). Over the past several years, gut microflora have been suggested to be a reservoir for endogenous TLR ligands [18,19]. To reduce the potential for endogenous TLR ligands from gut microflora, young Cre LysM Casp8 fl/fl mice were treated with oral antibiotics, which prevented both splenomegaly (Fig. 3d) and lymphadenopathy (Fig. 3e) compared with untreated Cre LysM Casp8 fl/fl mice.
Further, the survival of myeloid cell subsets was examined using mixed bone marrow chimeric mice (Fig. 3f ) maintained on low-dose oral antibiotics. As expected on the basis of our prior results with oral antibiotic treatment, mixed bone marrow chimeric mice (wild-type [WT] + Cre LysM Casp8 fl/fl ) did not display splenomegaly or lymphadenopathy (Fig. 3g, h). However, proteinuria persisted in these mice despite low-dose oral antibiotic treatment (Fig. 3i). Consistent with our observations in aged Cre LysM Casp8 fl/fl mice, serum levels of sRANKL were elevated in WT + Cre LysM Casp8 fl/fl mice compared with WT + Casp8 fl/fl mice (Fig. 3j). Loss of caspase-8 in myeloid cells did not result in enhanced survival, as splenic myeloid cell numbers remained unchanged in mixed bone marrow chimeric mice (Fig. 3k). Further, there was no preferential expansion of CD11b + Cre LysM Casp8 fl/fl -derived splenic myeloid populations in mixed bone marrow chimeric mice (Fig. 3l). These data indicate that loss of caspase-8 in vivo does not confer a survival advantage to splenic myeloid cells. However, we observed that the Cre LysM Casp8 fl/fl -derived CD11b − F4/80 + red pulp macrophage population exhibited reduced expansion in mixed bone marrow chimeric mice. Taken together, these data suggest that caspase-8 in myeloid cells controls the TLR response to the gut microflora and plays a only minor role in myeloid cell survival.
Because caspase-8 is a downstream signaling component of the DR Fas, the responses to cell death stimuli were evaluated in caspase-8-deficient myeloid cells. Total splenocytes from Cre LysM Casp8 fl/fl and control mice were incubated with either Fas ligand (FasL) or etoposide. Whereas ex vivo splenic Ly6C high and Ly6C low CD11b + F4/80 + cells were susceptible to both FasLinduced (Additional file 1: Figure S4a, b) and etoposideinduced (Additional file 1: Figure S4a, c) death, splenic neutrophils appeared responsive only to FasL. Thus, the availability of caspase-8 had no effect on the level of cell death in response to either treatment.
Previous studies in lymphocytes showed that loss of caspase-8 results in necroptosis, which is mediated by RIPK1/RIPK3 signaling. Therefore, the role that caspase-8 plays in macrophage death was evaluated in vitro. M-CSF-generated caspase-8-deficient BMDMs expressed Fas under steady-state conditions, which was upregulated with TLR activation (Additional file 1: Figure S5). Without stimulation, BMDMs deficient in caspase-8, RIPK3, or both caspase-8 and RIPK3 displayed increased LDH activity, indicating an increase in cell death. Similar to ex vivo caspase-8-deficient myeloid populations, caspase-8-deficient BMDMs responded like control BMDMs to FasL and etoposide at 48 h (Additional file 1: Figure S6) posttreatment, despite the overall higher cell death in caspase-8-deficient BMDM cultures. However, unlike in control BMDMs, the addition of Z-VAD did not reverse the death induced by FasL in caspase-8-deficient cells (Additional file 1: Figure S6), indicating that these caspase-8-deficient BMDMs underwent a caspaseindependent cell death. Deletion of RIPK3 in caspase-8deficient BMDMs restored the response to that of control BMDMs, as the addition of Z-VAD blocked death in RIPK3 −/− Cre LysM Casp8 fl/fl BMDMs. Taken together, these results suggest that caspase-8 deficiency predisposes macrophages to caspase-independent cell death upon DR ligation.
Whereas IL-6 and TNF-α transcription was increased with TLR4 and TLR7 activation in caspase-8-deficient BMDMs, these BMDMs produced higher levels of IL-6 and TNF-α in response to TLR4, TLR7, and TLR9 ligation compared with control BMDMs (Fig. 5a and Additional file 1: Figure S7c). Transcription of IL-6 was reduced with Nec-1 in response to TLR4, TLR7, and TLR9 ligation and with RIPK3 deletion in response to TLR4 activation (Fig. 5a). In addition, RIPK1 blockade reduced transcription of TNF-α in caspase-8-deficient BMDMs following stimulation with TLR4 and TLR7 (Additional file 1: Figure S7c). Similarly to transcription, blockade of RIPK1 reduced production of TNF-α by Cre LysM Casp8 fl/fl BMDMs in response to TLR4, TLR7, and TLR9 activation. However, suppression of RIPK1 affected only IL-6 following TLR7 and TLR9 activation. In contrast, deletion of RIPK3 reduced TNF-α production by Cre LysM Casp8 fl/fl BMDMs in response to TLR7 and TLR9 activation and IL-6 in response to TLR7 activation ( Fig. 5a and Additional file 1: Figure S7C). Elevated transcription and hypersecretion of IL-1β without the requirement for ATP was observed with TLR4, TLR7, and TLR9 ligation in Cre LysM Casp8 fl/fl BMDMs and was also blocked by the addition of Nec-1 or by RIPK3 deletion (Additional file 1: Figure S7D). Further, IL-1β was secreted at increased levels, and abrogated by Nec-1, in Cre LysM Casp8 fl/fl BMDMs compared with control BMDMs following stimulation with TLR4, TLR7, and TLR9 and addition of ATP (Additional file 1: Figure S7E).
The environmental milieu has been shown to influence macrophages to assume alternate phenotypes. We examined whether caspase-8 was involved in the polarization of BMDMs to classically activated M1 macrophages that are proinflammatory or to alternatively activated M2 macrophages that are necessary for repair. Polarization of BMDMs was analyzed via gene expression using a custom QuantiGene 2.0 panel [20] (see Additional file 1: Table S1) following stimulation with IFN-γ/LPS (M1skewing media) and IL-4 (M2-skewing media). Among the 52 genes included in the panel, those genes that were differentially expressed between populations of BMDMs according to selected criteria (differentially expressed in at least one two-group comparison with Bonferronicorrected p value <0.05) can be found in Additional file 2. Further, heat maps were generated to visualize differential gene expression between unstimulated control and caspase-8-deficient BMDMs (Additional file 1: Figure S8A), Cre LysM Casp8 fl/fl BMDMs with and without Nec-1 (Additional file 1: Figure S8B), M1-polarized control and Cre LysM Casp8 fl/fl BMDMs (Additional file 1: Figure S8C), unstimulated Cre LysM Casp8 fl/fl and M1polarized Cre LysM Casp8 fl/fl BMDMs (Additional file 1: Figure S8D), M1-polarized Cre LysM Casp8 fl/fl with and without Nec-1 (Additional file 1: Figure S8E), M2-polarized control and Cre LysM Casp8 fl/fl BMDMs (Additional file 1: Figure S8F), unstimulated Cre LysM Casp8 fl/fl and M2polarized Cre LysM Casp8 fl/fl BMDMs (Additional file 1: Figure S8G), and M2-polarized Cre LysM Casp8 fl/fl with and without Nec-1 (Additional file 1: Figure S8H). PCA revealed that unstimulated caspase-8-deficient BMDMs cluster separately from control BMDMs, whereas BMDMs (See figure on previous page.) Fig. 3 Caspase-8 deficiency alters the macrophage TLR response in vivo but does not affect cell survival. a Splenocytes from 6-8-month-old (aged) female Casp8 fl/fl (control) and Cre LysM Casp8 fl/fl mice (n ≥ 7) were analyzed by flow cytometry. Shown are representative fluorescence-activated cell sorting (FACS) plots of splenic CD11b + F4/80 + Ly6C high and CD11b + F4/80 + Ly6C low populations displaying relative levels of TLR expression. b and c Representative FACS plots and quantitative graphs of results representing the fold change in CD86 expression over PBS injection alone. b 3-monthold control and Cre LysM Casp8 fl/fl mice (n = 4) that received LPS or CpG injection (200 μg/mouse) were evaluated 4 h later for splenic CD11b + F4/80 + Ly6C high cell expression of CD86. c Serum levels of cytokines and chemokines from TLR agonist-injected mice. d and e 3-week-old control and Cre LysM Casp8 fl/fl mice (n = 4) treated with oral antibiotics (ampicillin, vancomycin, neomycin sulfate, metronidazole) for 8 weeks were evaluated for d spleen weight and e cervical lymph node weight. f-l Mice reconstituted with equal portions of B6.CD45.1 (wild-type [WT]) and either control or Cre LysM Casp8 fl/fl FACS-sorted LSK populations (n = 5) were maintained on low-dose oral antibiotics. f Representation of chimera generation. Chimeric mice were evaluated 8 months posttransfer for g splenomegaly, h lymphadenopathy, i proteinuria, j serum cytokine and chemokine levels, k myeloid cell subset numbers, and l distribution of WT (45.1)-derived and control or Cre LysM Casp8 fl/fl (45.2)-derived myeloid populations. Data are represented as mean ± SD and were compared by Mann-Whitney U test: *p < 0.05; **p < 0.005; ***p < 0.0005. TLR Toll-like receptor, Casp8 caspase-8, LPS lipopolysaccharide, PBS phosphate-buffered saline, IL interleukin, Gro-α growth-regulated oncogene-α, IFN interferon, Ig immunoglobulin, KC keratinocyte chemoattractant, TNF tumor necrosis factor, sRANKL soluble receptor activator of nuclear factor κB ligand, LN lymph node, WT wild type, MCP monocyte chemoattractant protein, LSK lineage-negative, Sca-1 + , c-kit + incubated with the inhibitor of caspase-8 enzymatic activity, Z-IETD-FMK, cluster more closely with control BMDMs (Fig. 5b). Further, Casp8 fl/fl , Casp8 fl/fl + Z-IETD-FMK, and Cre LysM Casp8 fl/fl BMDMs behaved similarly in response to M2-skewing media (Fig. 5b). However, Cre LysM Casp8 fl/fl BMDMs clustered independently from both Casp8 fl/fl and Casp8 fl/fl + Z-IETD-FMK BMDMs in response to M1-skewing media (Fig. 5b). In all instances, the addition of Nec-1 restored caspase-8-deficient BMDM populations to those of control BMDMs (Fig. 5c). These data suggest not only that caspase-8 is involved in the suppression of macrophage responses to TLR activation in an RIPK1-and RIPK3-dependent manner, but also that caspase-8 controls macrophage polarization in response to M1-skewing media in an RIPK1-dependent fashion.

Discussion
Our data suggest that caspase-8 controls the response of macrophages to TLR activation and M1-skewing media by dampening RIPK activity. Cre LysM Casp8 fl/fl mice develop a mild systemic inflammatory disease characterized by splenomegaly, lymphadenopathy, immune complex deposition in the kidney, proteinuria, and elevated amounts of serum cytokines and antibodies. The observed splenomegaly is not attributable to increased numbers of splenocytes, indicating that the spleen may be enlarged for other reasons, such as increased red blood cell numbers or elevated collagen deposition. Although we observed increased Ly6C high and Ly6C low CD11b + F4/80 + splenic populations, analysis of mixed bone marrow chimeric mice reveals that these caspase-8-deficient populations are not preferentially expanded, indicating that perhaps disease progression, rather than lack of caspase-8, increases these populations, potentially through increased migration, decreased egress, and/or enhanced proliferation of progenitor populations. Further, the systemic inflammatory phenotypes in Cre LysM Casp8 fl/fl mice may arise independently of the role of caspase-8 in survival, as mixed bone marrow chimeric mice are reconstituted with equal proportions of normal and caspase-8-deficient splenic populations. Moreover, deletion of RIPK3 is sufficient to prevent inflammation in Cre LysM Casp8 fl/fl mice and restore the CD11b + F4/80 + splenic populations to those of control mice. The increased CD11b + F4/80 + splenic populations indicate that the absence of caspase-8 does not lead to rampant necroptosis due to RIPK3 action, as is the case with T-cell caspase-8 deficiency [21]. Further, if insufficient apoptosis due to the lack of caspase-8 was the cause of increased CD11b + F4/80 + splenic populations, deletion of RIPK3 would exacerbate rather than correct this phenotype, as necroptosis would also be prevented. Rather, these data suggest that caspase-8 functions to suppress RIPK activity in myeloid populations independent of their roles in cell death.
Conditional deletion has revealed roles for caspase-8 in a number of cell death-independent activities. TLR4, TLR7, and TLR9 activation induces hyperproduction of proinflammatory cytokines in caspase-8-deficient BMDMs. Similar to caspase-8-deficient DCs [10], blocking RIPK1 kinase activity dampens the TLR-induced secretion of proinflammatory cytokines in caspase-8-deficient macrophages. TLR engagement can induce RIPK signaling independent of DR activation, thereby leading to formation of a ripoptosome, a complex containing proteins that participate in necroptosis, including RIPK1, caspase-8, and cFLIP [8,23]. Recent evidence suggests that ripoptosome activity and RIPK3 signaling in BMDMs can induce production of proinflammatory cytokine IL-1β in a caspase-8-dependent manner [6] independent of cell death. Both caspase-8-deficient BMDCs [10] and BMDMs secrete elevated IL-1β in response to TLR activation with the addition of ATP. Nec-1 decreases this IL-1β secretion in both control and casapse-8-deficient BMDCs [10] and BMDMs, albeit to a lesser extent, indicating that RIPK1 is potentially involved in IL-1β release independent of caspase-8. In addition, we and others have shown that caspase-8-deficient BMDCs activated by LPS secrete IL-1β without the requirement for secondary ATP stimulation [5,10]. Further, we previously reported that this secretion of IL-1β by BMDCs also occurs via TLR7 and TLR9 activation [10]. Similar to our previous findings with BMDCs, we show that caspase-8-deficient BMDMs secrete IL-1β without the requirement for secondary ATP stimulation in response to TLR4, TLR7, and TLR9 activation and that this secretion is abrogated by the addition of Nec-1 and deletion of RIPK3. Thus, our data implicate caspase-8 in the suppression of the macrophage TLR response in a manner that may require the components of the ripoptosome.
SLE is a severe multisystem autoimmune disease characterized by an immune response mounted against nuclear self-antigens that results in multiple tissue and/or organ damage. Loss of Fas in myeloid cells [9] or loss of caspase-8 in DCs [10] results in a SLE-like disease. Although Cre LysM Casp8 fl/fl mice present with hallmarks of SLE-like disease, these phenotypes are markedly less robust than those observed in Cre LysM Fas fl/fl or Cre CD11c Casp8 fl/fl mice. Similar to Cre LysM Casp8 fl/fl mice, Cre LysM FADD fl/fl mice develop a mild systemic inflammation that is reversed by RIPK3 deletion [24]. These results indicate that Fas, FADD, and caspase-8 play a cell-type-specific role in suppressing autoimmune phenotypes. Although Cre LysM Fas fl/fl, Cre LysM FADD fl/fl and Cre LysM Casp8 fl/fl myeloid populations are numerically dysregulated, their activation status is not altered. In contrast, Cre CD11c Casp8 fl/fl DC populations display an elevated expression of costimulatory molecules. Further, the splenomegaly observed in Cre LysM Casp8 fl/fl does not result in increased lymphocyte populations, which is different from the situation in Cre LysM Fas fl/fl and Cre LysM FADD fl/fl mice [9,24]. However, both caspase-8-and Fas-deficient macrophages are unable to directly induce T-cell proliferation in mixed leukocyte reactions. Thus, these data suggest that, in myeloid cell-specific caspase-8-or Fas-deficient mice, the effect on lymphocytes is indirect and may be mediated by cytokines or by a macrophage-specific paracrine effect on nonlymphocytic cells such as DCs.
Investigators in previous studies have examined the effect of myeloid cell-specific caspase-8 deletion [5,14]. In contrast to the data presented here, there was a failure to produce M-CSF-stimulated BMDMs from myeloid cell-specific caspase-8-deficient bone marrow. Additionally, there was no in vivo dysregulation of macrophage populations. These previous findings conflict with the present studies in that Cre LysM Casp8 fl/fl mice present increased numbers of both Ly6C high and Ly6C low splenic CD11b + F4/80 + cells. Further, previous studies did not show caspase-8 deletion in peritoneal neutrophils, which is in contrast to the complete deletion of caspase-8 in Cre LysM Casp8 fl/fl splenic neutrophils. The conflicting results observed between the Cre LysM-Casp8 fl/fl mice and previously published strains presumably stem from differences in the generation of in vitro bone marrow-derived populations and the cell-specific caspase-8 deletion constructs. In the present study, BMDMs were generated with M-CSF, whereas researchers in previous studies relied on the use of L929 media, which can contain unpredictable levels of M-CSF as well as other unknown components. Additionally, in the present study, both alleles of caspase-8 are floxed, whereas previous studies floxed only one allele of caspase-8 and the other was deleted [14]. Because caspase-8 has been shown to be necessary for early myeloid progenitor formation [25,26], deletion of caspase-8 in mice that have only one allele of caspase-8 may increase selective pressure on these cells. Thus, in this scenario, the cells that have complete deletion will die before becoming monocytes or macrophages, whereas others that fail to delete caspase-8 live and are not selected. However, in mice with two alleles of floxed caspase-8, progenitor deletion of caspase-8 may not be complete but caspase-8 is fully deleted as cells mature (Additional file 1: Figure S1), thereby allowing for cell survival throughout the differentiation process.

Conclusions
In Cre LysM Casp8 fl/fl mice, activation of TLRs, potentially via PAMPs derived from gut microflora, may lead to increased RIPK1 and RIPK3 activity, induction of costimulatory molecules, and increased production of proinflammatory cytokines, ultimately culminating in a mild systemic inflammatory disease. Our data suggest that, under steady-state conditions, following TLR activation of myeloid cells by the gut microflora, caspase-8 associates with RIPK1 and RIPK3 and limits their downstream signaling, thereby preventing the continued activation of these cells to keep systemic inflammation in check. These data provide a macrophagespecific link between caspase-8 and the heightened TLR responses to endogenous ligands leading to inflammation and show, for the first time to our knowledge, that caspase-8 controls the macrophage response to TLR activation and polarization.