Deletion of the Casp8 gene in epithelial tissues of mice results in severe inﬂammatory pathologies. Its ubiquitous deletion, or its speciﬁc deletion in endothelial cells, results in intrauterine death associated with capillary damage. These pathologies are all preventable by co-deletion of Casp8 and the genes encoding either the RIPK1 or the RIPK3 protein kinase. Since activation of RIPK3 in Caspase-8-deﬁcient cells can trigger necroptotic cell death, and since RIPK1 can activate RIPK3, it is widely assumed that the inﬂammatory states resulting from Caspase-8 deﬁciency occur as a consequence of RIPK3-induced necroptosis. Here, we report that although on a Ripk3-null background Casp8 deletion in mice does not result in outright pathological changes, it triggers enhanced expression of a variety of inﬂammatory genes in utero, which gradually subsides after birth. Deletion of Ripk1, or even of only one of its two alleles, obliterates this activation. Resembling the embryonic pathology observed in RIPK3-expressing cells, the activation of inﬂammatory genes observed on a Ripk3-null background seems to be initiated in endothelial cells. Analysis of endothelial cells isolated from livers of Caspase-8-deﬁcient embryos revealed neither an increase in the amount of RIPK1 in these cells after Casp8 deletion, nor triggering of RIPK1 phosphorylation. These ﬁndings indicate that the triggering of inﬂammation by Casp8 deletion in mice occurs, in part, independently of necroptosis or other functions of RIPK3, and rather reﬂects enhanced RIPK1-dependent signaling for activation of inﬂammatory genes. Introduction functional consequences of its deﬁciency. While its acti- vation triggers apoptotic cell death through the extrinsic Caspase-8 is unique among members of the caspase cell-death pathway [1, 2], deletion of the Casp8 gene, or cysteine protease family with regard to the far-reaching that of FADD—the adapter protein to which Caspase-8 binds—results in circulatory failure and death of mice at mid-gestation, associated with damage to capillaries [3–6]. Edited by A. Ashkenazi. The same lethal effect in utero is observed when the Casp8 gene is speciﬁcally deleted in endothelial cells . On the These authors contributed equally: Tae-Bong Kang, Ju-Seong Jeong. other hand, its deletion in epithelial tissues such as the Electronic supplementary material The online version of this article epidermis or the intestinal epithelium triggers a severe (https://doi.org/10.1038/s41418-018-0104-9) contains supplementary chronic inﬂammatory state post-partum, associated with material, which is available to authorized users. massive tissue damage [8, 9]. * David Wallach The ﬁnding that certain pathogens have evolved firstname.lastname@example.org mechanisms to block the function of caspases, including that of Caspase-8, has drawn considerable attention to Department of Biotechnology, College of Biomedical and Health mechanisms accounting for the inﬂammatory states dictated Science, Konkuk University, Chung-Ju 27478, Korea by Casp8 deletion, and the possible real-life corollaries of Department of Biomolecular Sciences, The Weizmann Institute of these experimental pathologies . Assessment of the Science, 76100 Rehovot, Israel consequences of Caspase-8 deﬁciency in cultured cells Present address: Systems Biotechnology Research Center, Korea revealed that these cells, while resistant to apoptotic-death Institute of Science and Technology (KIST), Gangneung 25451, Korea induction by receptors of the TNF family, display 1234567890();,: 1234567890();,: 1108 T-B Kang et al. dramatically enhanced vulnerability to the induction of expression of all four of these genes in embryonic lung, necroptotic death [11–13]. Since necrotic cell death yields intestine and kidney, as well as in the yolk sac and was the release of pro-inﬂammatory cellular components (dan- discernible, to varying extents, in these tissues as early as ger-associated molecular patterns—DAMPs), it is widely E12.5. Analysis of the expression of these genes in whole −/− −/− assumed that the acute inﬂammatory pathologies observed Casp8 Ripk3 E10.5 embryos revealed that the when Casp8 is deleted in epithelial tissues, as well as the expression of Il1b, Cxcl10, and Marco is already increased fatal outcome of its ubiquitous deletion, result from the by that stage. Evidently, therefore, this increase had already triggering of necrotic cell death . Supporting this notion occurred before or at the time of the pathological changes was the ﬁnding that deletion of the genes encoding either inﬂicted by deletion of Casp8 from RIPK3-expressing the RIPK1 or the RIPK3 protein kinase, previously shown embryos, which occurs at about E10.5  (Fig. 1b). to participate in signaling for necroptotic death, or of the After birth, the basal levels of the examined inﬂamma- pseudokinase MLKL which, once phosphorylated by tory genes were found to increase in the liver and lung, RIPK3, mediates the cellular membrane rupture that triggers while remaining low in the intestine and kidney (Supple- this death, attenuates the pathological states inﬂicted by mental Fig. S1). In all four tissues, Caspase-8 deﬁciency Caspase-8, or FADD deﬁciency [13, 15–19]. resulted in some further increase in expression of the tested Here, we analyze the impacts of Casp8, Ripk1, and Ripk3 genes, for several days after birth. However, the extent of deletion on the intrauterine expression of inﬂammatory this increase was signiﬁcantly lower than that observed genes in mice. We show that although the outright patho- before birth (Fig. 1b). logical changes known to result from Casp8 deletion Deletion of just one of the Caspase-8 alleles did not depend on the function of RIPK3 [3–9], the expression of result in increased expression of any of the four examined some inﬂammatory genes is enhanced by Casp8 deletion genes in the embryonic livers (Fig. 1c). even on a Ripk3-null background. This enhanced expression Using antibodies against MARCO for immunoblotting, was found here to be strictly dependent on expression of we found that the amounts of MARCO protein in the livers −/− −/− RIPK1. These ﬁndings implicate a mechanism that is of E16.5 Casp8 Ripk3 embryos were much higher +/− −/− independent of RIPK3 or of death induction by it, yet than in Casp8 Ripk3 livers (Fig. 1d). Thus, upregu- depends on RIPK1, in the inﬂammatory processes triggered lation of at least part of the inﬂammatory genes found to be −/− −/− by Caspase-8 deﬁciency. activated in the Casp8 Ripk3 mice resulted in enhanced expression of their encoded proteins. Results Inﬂammatory-gene activation due to Caspase-8 deﬁciency seems to begin preferentially in Caspase-8 deﬁciency on a Ripk3-null background endothelial cells and then be transmitted to other triggers upregulation of inﬂammatory genes in cells various embryonic tissues To identify the liver cell type(s) in which deﬁciency of Caspase-8-deﬁcient mice on a Ripk3-null background Caspase-8 on a Ripk3-null background triggers inﬂamma- −/− −/− (Casp8 Ripk3 ) display no evident histological tory gene expression, we used mice with a “ﬂoxed“ ﬂ/ﬂ abnormalities in utero [13, 15] (data not shown). However, Caspase-8 allele (Casp8 )ona Ripk3-null background to −/− −/− on examining the livers of these Casp8 Ripk3 speciﬁcally delete Casp8 in each of the main cell types in ﬂ/ﬂ −/− embryos at E16.5 we found, serendipitously, that their the liver. Mating of the Casp8 Ripk3 mice with mice expression of the mRNA encoding the inﬂammatory med- expressing Cre under control of the albumin promoter iator IL-1β was signiﬁcantly higher than in age-matched allowed deletion of Casp8 speciﬁcally in hepatocytes. The +/− −/− Casp8 Ripk3 embryonic livers. By employing the use of mice expressing Cre under control of the LysM Nanostring technique to proﬁle the genes expressed in promoter allowed deletion of Casp8 in the myelomonocytic −/− −/− embryonic livers of those Casp8 Ripk3 embryos, cells of the liver, and the use of mice expressing Cre under using a panel of 547 mouse genes known to contribute to control of the Tie1 promoter allowed its deletion in the the immune response, we found that besides the increase in endothelial and hematopoietic progenitor cells. The effec- interleukin Il1b, Caspase-8 deﬁciency also resulted in tiveness of Casp8 deletion by all three Cre transgenes was upregulation of many other inﬂammatory genes (Fig. 1a). high (Supplemental Fig. S2). Surprisingly, despite the pre- The expression of four of these genes—Il1b, Ccl5, disposition of myelomonocytic cells to strongly express Cxcl10, and Marco—was then assessed in various tissues at inﬂammatory genes, deletion of Casp8 in these cells did not different embryonic stages as well as post-partum. As lead to upregulation of inﬂammatory genes in the liver. shown in Fig. 1b, Caspase-8 deﬁciency resulted in enhanced Deletion of Casp8 in hepatocytes (the predominant liver Caspase-8 deﬁciency in mouse embryos triggers chronic RIPK1-dependent activation of inﬂammatory. . . 1109 Fig. 1 Upregulation of inﬂammatory genes in the embryonic tissues of embryos at E10.5. Values are expression levels in the indicated organs −/− −/− −/− −/− Casp8 Ripk3 mice. a NanoString analysis of the upregulation of of Casp8 Ripk3 mice (red circles) normalized to those of Casp8 −/− −/− +/− −/− immunoregulatory genes in fetal livers of Casp8 Ripk3 mice at Ripk3 mice (blue circles), assessed by analyzing at least ﬁve E16.5. Shown are the relative mRNA expression values in the livers of embryos or mice from 3 litters. ***p < 0.001, **p < 0.01, and *p < −/− −/− +/− −/− three Casp8 Ripk3 and three Casp8 Ripk3 mice for genes 0.05 relate to differences between the mean values for −/− −/− +/− −/− that were upregulated by more than 2-fold (p < 0.05) in the Casp8 Casp8 Ripk3 and Casp8 Ripk3 mice. c Assessment of the −/− −/− Ripk3 samples. Red color indicates expression levels higher than effect of deletion of just one Casp8 allele on the expression of the average value of the particular gene in the six examined embryonic inﬂammatory genes. Shown is expression of the indicated genes in the +/+ −/− +/− −/− livers. b Comparison of the effects of Caspase-8 deﬁciency on levels livers of Casp8 Ripk3 and Casp8 Ripk3 E16.5 mouse of the indicated mRNAs in various mouse organs at different embryos (n = 4). No signiﬁcant differences were found. d Immunoblot embryonic ages (E12.5, E14.5, and E16.5) and at different times after analysis of MARCO protein in whole liver extracts from embryos at birth (PN1, PN3, PN5, and PN7), and on their overall levels in the E16.5. Data are representative of three independent experiments cells) resulted in only mild activation of just two genes. In then assessed gene expression in the isolated cells. contrast, deletion of Casp8 by expression of Cre under the Figure 2c shows that of the three types of cells isolated in − + Tie1 promoter resulted in strong upregulation of multiple this way, only the endothelia (CD45 CD31 cells) inﬂammatory genes, and in several of them to an extent expressed a variety of different inﬂammatory genes at very similar to that obtained when Casp8 was deleted ubiqui- high levels. The gene for MARCO, however, was strongly tously (Fig. 2a, b). upregulated in all cell types (Fig. 2c, d). As an alternative approach to identifying the liver cell Taken together, the ﬁndings reached by both of these type(s) in which Caspase-8 deﬁciency triggers inﬂammatory modes of analysis indicated that triggering of inﬂammatory gene expression, we applied antibodies to CD31 (a speciﬁc gene expression in the liver on a Ripk3-null background is cell-surface marker for endothelial cells) and to CD45 (a largely restricted to endothelial cells. Once activated, cell-surface marker for hematopoietic cells) in order to sort however, some of these genes are apparently able to upre- out, by FACS, distinct cell types from the livers of Casp8 gulate the expression of inﬂammatory genes in other types −/− −/− +/− −/− Ripk3 and Casp8 Ripk3 E16.5 embryos. We of liver cells. 1110 T-B Kang et al. Fig. 2 Cell-type speciﬁcity of the upregulation of inﬂammatory genes in (4 litters), 6 pairs of Alb-Cre (3 litters), 5 pairs of LysM-Cre mice Caspase-8-deﬁcient embryonic liver. a NanoString analysis of the (2 litters). The data about the expression of Marco, Ccl5,and Cxcl10 upregulation of immunoregulatory genes in embryonic livers of Ripk3 were obtained by analysis of three pairs of Tie1-Cre (2 litters), three −/− mice at E16.5, as a result of ubiquitous deletions of the Casp8 gene pairs of Alb-Cre (2 litters), and three pairs of LysM-Cre mice (1 litter). (left-most column; data are from the experiment described in Fig. 1a) or **p< 0.01 and *p < 0.05 relate to differences between the mean values ﬂ/ﬂ −/− ﬂ/ﬂ −/− its cell-type-speciﬁc deletion by the indicated Cre transgenes. Shown are for the Casp8 Ripk3 and Casp8 Ripk3 Cre mice. c Heat-map the averages of the increases in expression of the genes presented in analysis of the relative mRNA expression, as determined by NanoString +/− Fig. 1a for three pairs of embryos of the following genotypes: Casp8 analysis, in cells sorted by FACS from three pairs (3 litters) of Casp8 −/− −/− +/− −/− ﬂ/ﬂ −/− −/− −/− −/− Ripk3 vs. Casp Ripk3 from 3 litters; Casp8 Ripk3 Alb- Ripk3 and Casp8 Ripk3 fetal liver cells, using antibodies ﬂ/ﬂ −/− ﬂ/ﬂ Cre vs. Casp8 Ripk3 (2 litters); Casp8 against the indicated cell-surface markers. Data are presented as in a. d −/− ﬂ/ﬂ −/− ﬂ/ﬂ Ripk3 Tie1-Cre vs. Casp8 Ripk3 (1 litter); and Casp8 Real-time PCR analysis of the cellular levels of mRNAs of Il1b and −/− ﬂ/ﬂ −/− Ripk3 LysM-Cre vs. Casp8 Ripk3 (1 litter), compared to those of Marco in the indicated cell populations isolated as in c.Shown arethe −/− −/− mouse embryos that did not express the Cre transgenes. #, undetectable. expression levels in cells derived from Casp8 Ripk3 (red circles) +/− −/− Red color indicates an expression level higher than the average value of normalized to those from Casp8 Ripk3 mice (blue circles). Hor- the particular gene in the corresponding control samples. b Real-time izontal lines indicate mean values for each group. Data for Il1b were PCR validation of expression levels of the indicated mRNAs in the obtained by analysis of seven pairs (2 litters), and for Marco by analysis ﬂ/ﬂ livers of the E16.5 mice analyzed in a. Blue circles indicate Casp8 of four pairs (2 litter). ***p< 0.001, **p< 0.01, and *p < 0.05 relate to −/− −/− control samples; red circles indicate tissue-speciﬁc Caspase-8-deﬁcient differences between the mean values for the Casp8 Ripk3 and +/− −/− samples. Horizontal lines indicate mean values for each group. The Il1b Casp8 Ripk3 mice expression data were obtained by analysis of 10 pairs of Tie1-Cre Upregulation of inﬂammatory genes in cell-autonomous manner. To deﬁne the operative mechan- −/− −/− Casp8 Ripk3 embryos yields systemic ism, we applied ELISA to determine the extent to which expression of inﬂammatory mediators inﬂammatory mediators occur in the sera of the embryos. We found that expression levels of the chemokines The dissimilar ﬁndings obtained by the above two approa- CXCL10 and CCL5, the pro-inﬂammatory cytokine TNF ches used to identify the cell type in which Caspase-8 and the acute-phase protein SAA3 in the sera of −/− −/− deﬁciency triggers activation of inﬂammatory genes, as well Casp8 Ripk3 embryos were all increased to a sig- +/− −/− as the multiplicity of tissues in which such activation was niﬁcantly greater extent than in the sera of Casp8 Ripk3 found, suggested that the activation occurs in part in a non- embryos (Fig. 3a, b). These results are consistent with the Caspase-8 deﬁciency in mouse embryos triggers chronic RIPK1-dependent activation of inﬂammatory. . . 1111 −/− Fig. 3 Serum expression of inﬂammatory mediators in Casp8 samples collected from more than ﬁve pairs (from 5 litters) of −/− +/− −/− −/− −/− Ripk3 mice. Serum concentrations of the cytokines CXCL10, Casp8 Ripk3 and Casp8 Ripk3 mice, as determined (a, c) CCL5, TNF-α, and the acute-phase protein SAA3 (a, b) in fetal blood by multiplex ELISA and by conventional ELISA (b, d). ***p< 0.001, of E16.5 embryos and in blood withdrawn postnatally at the indicated **p< 0.01, and *p < 0.05 relate to differences between the mean −/− −/− +/− −/− ages (c, d). Shown are values of the indicated proteins in plasma values for the Casp8 Ripk3 and Casp8 Ripk3 mice observed upregulation of genes encoding soluble pro- using an immunoregulatory gene panel conﬁrmed that inﬂammatory mediators in the Caspase-8-deﬁcient endothe- deletion of just one of the two Ripk1 alleles obliterated the lium (Fig. 2), and may well account for the apparently non- upregulation of the inﬂammatory genes induced by cell-autonomous activation of inﬂammatory genes in all other Caspase-8 deﬁciency (Fig. 4b). cell types in the liver (Fig. 2c, d). Some increase in CXCL10, CCL5, and SAA3 was also Caspase-8 deﬁciency does not cause increased −/− −/− observed in the sera of Casp8 Ripk3 mice during the expression of RIPK1 in endothelial cells, nor does it ﬁrst week after birth, but had abated by the time the mice seem to trigger RIPK1 phosphorylation were 3 months old (Fig. 3c, d). This increase is thus tem- porally distinct from the increase in inﬂammation and Caspase-8 has been shown to respond to various stimuli by −/− −/− in serum cytokines in Casp8 Ripk3 mice that is cleaving RIPK1, thereby arresting its signaling activities found to occur rather late after birth, as a consequence [20–22]. Given our ﬁnding that the upregulation of of a lymphoproliferative syndrome that they develop inﬂammatory genes in our Caspase-8-deﬁcient embryos is [13, 15, 19]. strictly dependent on their adequate expression of RIPK1, it seemed possible that this upregulation reﬂects cessation of The increased expression of inﬂammatory genes the constitutive downregulation of RIPK1 expression due to Caspase-8 deﬁciency is dependent on RIPK1 through Caspase-8-mediated RIPK1 cleavage. In such a case, RIPK1 levels in the embryonic endothelial cells, in Having shown that Caspase-8 deﬁciency in mice dictates which the upregulation of inﬂammatory genes occurs, upregulation of some inﬂammatory genes independently of would be expected to increase. To examine this possibility, RIPK3, we proceeded to examine how this upregulation is we designed a two-step antibody-assisted enrichment pro- affected by deletion of the gene encoding RIPK1. We found tocol to isolate the endothelial cells from embryonic livers that whereas in mice expressing both alleles of Ripk1 the at E16.5. As shown in Fig. 5, western analysis using an anti- deletion of Casp8 on a Ripk3-null background resulted in RIPK1 antibody, enabling us to reliably assess a change in marked upregulation of Il1b, Ccl5, Cxcl10, and Marco RIPK1 expression resulting from deletion of one of the expression, no such increase was observed in the livers of Ripk1 alleles, revealed no difference between the amounts embryos that were also deﬁcient in RIPK1 or even in only of RIPK1 in endothelial cells isolated from the livers of +/− −/− −/− −/− one of the two Ripk1 alleles (Fig. 4a). Nanostring analysis Casp8 Ripk3 and of Casp8 Ripk3 embryos. 1112 T-B Kang et al. −/− +/− −/− −/− Fig. 4 RIPK1-dependence of inﬂammatory gene upregulation in the (2 litters) Casp8 Ripk1 Ripk3 mice to 2 (2 litters) Casp8 −/− −/− −/− −/− embryonic Casp8 Ripk3 liver. a Real-time PCR analysis of the Ripk1 Ripk3 mice. Horizontal lines indicate mean values for cellular mRNA levels of the indicated genes in fetal livers of mice with each group. ***p< 0.001, **p< 0.01, and *p < 0.05 relate to differ- the indicated genotypes at E16.5. Each circle corresponds to the ences between the mean values for the experimental and control average ratio of gene expression in the livers of embryos with the groups of mice. b Nanostring analysis of the effect of Casp8 deletion +/− +/+ −/− indicated genotypes to that of Casp8 Ripk1 Ripk3 embryos, on the expression of the genes presented in Fig. 1b in mice with two obtained in four independent experiments, as follows: 5 (3 litters) Ripk1 alleles (upper panel: data are from the experiment described in +/− +/+ −/− −/− +/+ Casp8 Ripk1 Ripk3 to 4 (3 litters) Casp8 Ripk1 Fig. 1b) or only one 1 Ripk1 allele (lower panel). Data are presented as −/− +/− +/+ −/− +/− +/− −/− Ripk3 mice; 1 (1 litter) Casp8 Ripk1 Ripk3 and 1 (1 litter) mean ± SEM of 2 (2 litters) Casp8 Ripk1 Ripk3 mice; 3 −/− +/+ −/− −/− +/− −/− +/− −/− Casp8 Ripk1 Ripk3 mice and 2 (1 litter) Casp8 Ripk1 (3 litters) Casp8 Ripk1 Ripk3 mice; 3 (3 litters) −/− −/− −/− −/− +/− +/+ −/− −/− +/+ Ripk3 to 1 (1 litter) Casp8 Ripk1 Ripk3 mice; 3 (2 litters) Casp8 Ripk1 Ripk3 mice; and 3 (3 litters) Casp8 Ripk1 −/− +/+ −/− −/− +/− −/− Casp8 Ripk1 Ripk3 mice and 5 (3 litters) Casp8 Ripk1 Ripk3 mice, normalized to the average levels in the corresponding −/− −/− −/− −/− +/− Ripk3 mice to 1 (1 litter) Casp8 Ripk1 Ripk3 mice; 4 Casp8 mice To this western analysis we then further applied an sequential activation of RIPK1 and RIPK3 results in antibody that speciﬁcally recognizes RIPK1 molecules phosphorylation of the Mixed Lineage Kinase Domain-Like phosphorylated at Serine 166. This antibody allowed easy (MLKL) pseudokinase. The latter mediates necroptotic detection of phosphorylated RIPK1 found in extracts of death of Caspase-8-deﬁcient cells, and then DAMPs wild-type mouse ﬁbroblasts that were stimulated by TNF in released by the dying cells trigger the inﬂammatory changes the presence of a caspase inhibitor and a cIAP antagonist. It associated with these various pathologies. Our present did not, however, interact with comparable amounts of ﬁndings imply that the inﬂammatory processes inﬂicted by −/− −/− RIPK1 found in the Casp8 Ripk3 embryonic endo- Caspase-8 deﬁciency also involve activation of inﬂamma- thelial cells (Fig. 5). tory genes by a mechanism that does not depend on the function of RIPK3 or on the death induction by it, but is nevertheless still dependent on RIPK1. Discussion These ﬁnding are consistent with the large amount of evidence for contributions of RIPK1 in a variety of ways to The prevailing conception of the various pathological the initiation of several signaling pathways that lead to changes resulting from Caspase-8 deﬁciency is that they are activation of inﬂammatory genes, including the canonical all mediated through a common mechanism in which NF-κB pathway and MAP-kinase cascades. Such Caspase-8 deﬁciency in mouse embryos triggers chronic RIPK1-dependent activation of inﬂammatory. . . 1113 not result from necroptotic death but rather reﬂects non- deadly pro-inﬂammatory functions of RIPK3 and MLKL. Our ﬁndings suggest that both the increase in expression of inﬂammatory proteins in RIPK3-deﬁcient mouse embryos and the fatal outcome of Caspase-8 deﬁciency in embryos that do express RIPK3 are initiated in the same type of cell, namely, the endothelial cells. That ﬁnding, together with the similar timing of those changes, raises the possibility that despite marked differences in their con- sequences, the two mechanisms are interlinked. There are several possible links between the two. One intriguing possibility is that the RIPK3-independent activation of genes in response to deﬁciency of Caspase-8 is a pre- condition for those RIPK3-dependent pathologies that develop spontaneously in response to Caspase-8 deﬁciency. A plausible example of such interdependence is provided by our ﬁnding that TNF is one of the genes upregulated by Caspase-8 deﬁciency on a Ripk3-null background. TNF expression is a precondition for triggering of the RIPK3- dependent damage in E10.5 mouse embryos . It is thus reasonable to hypothesize that the RIPK3-independent Fig. 5 Assessment of the effects of Casp8 deletion on RIPK1 expression and phosphorylation in embryonic endothelial cells. Wes- generation of TNF in embryos in response to Caspase-8 tern blot assessments of the total amounts of RIPK1 and the amounts deﬁciency is the trigger for the RIPK3-dependent effects. of RIPK1 phosphorylated at Serine 166 (p-RIPK1), in embryonic The spontaneous development of pathological RIPK3- livers of mice of the indicated genotypes at E16.5 and in endothelial dependent consequences of Caspase-8 deﬁciency in adult cells isolated from embryonic livers. Immortalized MEFs, in which RIPK1 phosphorylation was triggered by combined treatment with mice may likewise depend on prior RIPK3-independent TNF-α, zVAD and BV6 (TBZ) as described in “Materials and meth- upregulation of some inﬂammatory genes. ods”, served as a positive control for detection of p-RIPK1. Albumin An alternative possible cause of the apparent temporal and β-actin were blotted as loading controls of the amounts of hepa- and spatial co-occurrence of RIPK3-dependent and RIPK3- tocyte extracts and for total protein, respectively. Shown are repre- sentative results of three independent experiments. NS non-speciﬁc independent consequences of Caspase-8 deﬁciency in band embryos is the existence of a mechanistic step that is common to both sets of consequences. This possibility raises the need to reconsider the identity of the Caspase-8 molecular target that accounts for the pro-inﬂammatory participation occurs both in a way that depends on effect of deﬁciency in this caspase. In prior discussions, it the RIPK1 protein-kinase function and independently of was suggested that this consequence of Caspase-8 deﬁ- it [23–26]. Our ﬁndings are also consistent with several ciency reﬂects the arrest of Caspase-8-mediated cleavage of studies in which Caspase-8 deﬁciency was found to facil- one or more of three signaling proteins: RIPK1, RIPK3, and itate such pro-inﬂammatory functions of RIPK1 without the deubiquitinating enzyme CYLD [13, 21, 38]. Our leading to cell death [27–30]. ﬁnding that Caspase-8 deﬁciency results in activation of As expected from the fact that Capase-8 deﬁciency in pro-inﬂammatory genes even in the absence of RIPK3 RIPK3-deﬁcient embryos does not result in outright clearly rules out an exclusive role for arrest of RIPK3- pathological changes, the extent of upregulation of inﬂam- cleavage in this process. The contribution of CYLD to matory genes observed in these embryos was far lower than signaling mediated by RIPK1 + RIPK3 occurs through that observed after Casp8 deletion in mice that do express strengthened association of these two proteins and has to do this protein kinase. The marked increase in inﬂammation with enhancement of their kinase function . Our seen in the RIPK3-expressing mice might result in part from observation that inﬂammatory proteins became activated in the initiation of necroptosis. However, in view of growing the absence of RIPK3 and apparently without phosphor- evidence that RIPK3 can also facilitate inﬂammation in ylation of RIPK1 casts doubt on the possibility that CYLD ways that do not depend on cell death [26, 30–33], as well cleavage is a player in the regulation of this process. as some evidence for death-independent pro-inﬂammatory Finally, our ﬁnding that endothelial cells isolated from functions of the RIPK3 target protein MLKL [34–36], it Caspase-8-deﬁcient mice and from mice not lacking seems likely that part of this enhanced inﬂammation does Caspase-8 contain equal amounts of RIPK1 casts doubt on 1114 T-B Kang et al. the arrest of Caspase-8-mediated RIPK1 cleavage as a FACS sorting possible player in this eventuality. In fact, we cannot exclude the possibility that the observed upregulation of Fetal livers were dissected and incubated in RPMI medium inﬂammatory genes as a result of Caspase-8 deﬁciency does containing collagenase D (50 U/ml; Thermo Fisher Scien- not reﬂect the prevention of cleavage of any substrate of this tiﬁc) and 5% fetal bovine serum for 40 min at 37 °C. protease, but rather reﬂects the arrest of some non- Collagenase-treated tissues were dissociated into single enzymatic function of Caspase-8. It has indeed been cells by pipetting and ﬁltering through a 70-μm cell strainer shown that Caspase-8, besides acting as a protease, also (BD Biosciences). Red blood cells were removed by treat- contributes to signaling by serving as a scaffold for the ment with a buffered ammonium chloride solution and then TM assembly of other signaling proteins [36, 40–42]. with mouse BD Fc Block (BD Biosciences) for 5 min. The cellular expression of RIPK3 is subject to modula- For the experiment presented in Fig. 2c and d, the cells tion by inducing agents and is increased in various were stained with allophycocyanin (APC)-conjugated anti- inﬂammatory diseases [43–45]. Our ﬁnding that the initia- mCD45 antibody (17-0451, BD Biosciences) and ﬂuor- tion of inﬂammation in vivo as a consequence of Caspase-8 escein isothiocyanate (FITC)-conjugated anti-mCD31 anti- deﬁciency also occurs in the absence of RIPK3, albeit to a body (11-0311, BD Biosciences), allowing sorting for − + much lower degree than in the presence of RIPK3, implies endothelial cells (CD45 CD31 ), hematopoietic cells + − − − that our cells possess the ability to respond in mild or (CD45 CD31 ), and other liver cells (CD45 CD31 ). alternatively in intense manner to pathogens that block The effectiveness of Casp8 deletion in individual cell caspase action. Such a graded response would offer more types (Supplemental Fig. S2) was assessed by isolation of discriminatory assistance in overcoming infection. endothelial cells by FACS sorting after staining as above, Restraining the expression of RIPK3, or of proteins and isolation of macrophages after staining with APC- acting downstream of it, can allow Caspase-8-deﬁcient cells conjugated anti-F4/80 antibody (17-4801, Thermo Fisher to initiate a defensive response through inﬂammatory Scientiﬁc) and with phycoerythrin (PE)-conjugated anti- changes not associated with their own damage. Should CD45 antibody (12-0415, Thermo Fisher Scientiﬁc). more powerful means of defense become necessary, upre- For the experiment presented in Fig. 5, endothelial cells gulation of the expression of RIPK3 and of proteins from the dissociated fetal liver tissue were isolated using a downstream of it would allow these defense mechanisms to MACS Cell Separation LS column (Miltenyi Biotech), be supplemented by others that do lead to self-destruction of using anti-CD31 antibody (BD Pharmingen) and anti-rat cells and tissues. IgG MicroBeads (Miltenyi Biotech). They were then further enriched by FACS sorting using anti-CD31 antibody as described above. After sorting, purity testing conﬁrmed that Materials and methods more than 96% of the isolated cells were endothelial. In all cases, staining with antibodies was carried out for Mice 20 min on ice. The cells were then washed with cold FACS buffer (2% FBS, 0.01% sodium azide) and resuspended in All mice used in this study were on a C57BL/6 background. the same buffer for cell sorting using a FACSAria II (BD Mouse strains carrying a knocked-out Casp8 allele Biosciences). Immediately before FACS analysis, the DNA −/+ ﬂ/+ (Casp8 ) and a conditional Casp8 allele (Casp8 ) dye 7-amino-actinomycin D was added for staining of dead  were established in our laboratory. The use of mice cells. The isolated cells were harvested by centrifugation at expressing Cre under control of the albumin promoter (Alb- 300 × g for 5 min at 4 °C and were either frozen immedi- Cre), the lysozyme M gene promoter (LysM-Cre), ately and kept at −80 °C pending extraction of total RNA or and the Tie1 promoter (Tie1 Cre) for deletion of the genomic DNA, or extracted with detergent for western blot Casp8 gene speciﬁcally in hepatocytes, in liver myelomo- analysis. nocytic cells, and in both endothelial cells and hemato- poietic progenitor cells, respectively, has been described Assessing the extent of deletion of the ﬂoxed Casp8 . Mice deﬁcient in Ripk1 were obtained from Dr. allele Michelle Kelliher  and mice deﬁcient in Ripk3 from Dr. Vishva Dixit . Embryos and yolk sacs were isolated on The extent of deletion of ﬂoxed Casp8 alleles in tissues and the indicated days of timed pregnancies and genotyped by cells isolated from mice that express Cre under control of PCR using tail genomic DNA, as described . All animal various promoters was assessed by real-time PCR, using experiments were approved by the Institutional Animal DNA extracted with a QIAamp DNA Micro Kit (Qiagen), Care and Use Committees at The Weizmann Institute of according to the manufacturer’s protocol. The assay was Science. performed in a total reaction volume of 20 μl containing 30 Caspase-8 deﬁciency in mouse embryos triggers chronic RIPK1-dependent activation of inﬂammatory. . . 1115 ng DNA, 300 nM oligonucleotide primers, 100 nM oligo- Proﬁling of gene expression nucleotide 3′-Minor groove binder (MGB) probes, and 10 μl of TaqMan Universal PCR Master Mix (Applied Biosys- NanoString technology with nCounter Digital Analyzer and tems). The PCR procedure was done by subjecting the nSolver software (NanoString Technologies) was used to reaction mixture to incubation for 10 min at 95 °C, and then assess gene expression in the indicated cell types and tissue. to 40 cycles of 15 s at 95 °C and of 1 min at 60 °C on an Brieﬂy, RNAs were hybridized with the nCounter Mouse ABI Prism 7300 System (Applied Biosystems). Primers and Immunology Panel (GXA-MIM1-12) and data were ana- probes were designed using Primer Express software lyzed according to the recommendations of NanoString (Applied Biosystems). The oligonucleotide primers and Technology. Total RNA (100 ng) isolated from fetal liver probes used to hybridize the ﬂoxed region in the Casp8 and from FACS-sorted hematopoietic cells (CD45 CD31 gene were 5′-TCCTGTGCTTGGACTACATCC-3′ (sense), ), or total cell lysates of 6000 sorted endothelial cells − + − 5′-TTCCCGCAGCCTCAGAAATAG-3′ (antisense), and (CD45 CD31 ) and of the other fetal liver cells (CD45 5′-6-FAM- AGAAGCAGGAGACCATCGAGGATGC- CD31 ) in 1.5 μl of buffer RLT (Qiagen), were hybridized MGB-3′ (probe). Data were normalized on the basis of with capture and reporter probes. quantiﬁcation of the non-loxP-ﬂanked region (Exon8) in the Casp8 gene using 5′- GCGCAGACCACAAGAACAAAG- Chemokine and cytokine assays in the blood of 3′ (sense), 5′-CCTTCCCATCCGTTCCATAGAC-3′ (anti- embryonic and adult mice sense), and 5′-6-FAM-TGCTTCATCTGCTGTATCC- TATCCCA-MGB-3′ (probe). The comparative threshold Blood samples were collected from the umbilical cords of cycle method (ΔCT) was used to quantify the deletion of mouse embryos and from the orbital sinuses of adult mice Casp8. were collected into EDTA-coated tubes and spun at 1500 × g for 15 min at 4 °C. Plasma concentrations of CXCL10, RNA extraction from tissues or sorted cells CCL5, and TNF-α were measured using the Magnetic Luminex Screening Assay system (R&D Systems) and a Embryos were dissected on the indicated embryonic day MAGPIX system (Luminex). Plasma concentrations of and their organs were isolated and placed in RNAlater serum amyloid A3 were determined by ELISA (Merck Stabilization Solution (Ambion) until processed for RNA Millipore), according to the manufacturer’s instructions. extraction. RNA was extracted using the RNeasy Mini kit (Qiagen), according to the manufacturer’s instructions. Stimulation of RIPK1 phosphorylation in mouse RNA from FACS-sorted cells was extracted using the embryonic ﬁbroblasts (MEFs) RNeasy Micro Kit (Qiagen). The quality of the RNA was assessed on the Agilent 2100 Bioanalyzer (Agilent MEFs immortalized by expression of the SV40 large T Technologies). antigen were cultured in Dulbecco’s Modiﬁed Eagle’s Medium supplemented with 10% FBS, 100 U/ml penicillin, Real-time RT-PCR and 100 mg/ml streptomycin. RIPK1 phosphorylation was stimulated in the MEFs by their treatment for 3 h with TNF Total RNA from tissue or cells was reverse-transcribed (1000 U/ml), together with the bivalent IAP (inhibitor of using the SuperScript II First-Strand Synthesis System apoptosis protein) antagonist BV6  and the caspase (Invitrogen). Real-time PCR was performed using the fol- inhibitor z-VAD-fmk (both from WuXi AppTec) at con- lowing TaqMan primers and probe sets (Applied centrations of 1 and 20 μM, respectively. Biosystems). Immunoblotting Target gene Catalog number For the experiment presented in Fig. 1d, whole liver extracts Il1b Mm00434228_m1 were prepared by boiling fetal liver in 1% SDS-lysis buffer Cxcl10 Mm00445235_m1 (50 mM Tris, pH 8, 150 mM NaCl, 1% SDS) followed by Ccl5 Mm01302428_m1 ultrasonication for 10 s. For the experiment shown in Fig. 5, Marco Mm00440265_m1 whole liver extracts, extracts of FACS-puriﬁed endothelial cells, and extracts of MEFs were prepared in RIPA buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% TritonX- 100, 0.5% sodium deoxycholate and 0.1% SDS) containing RNA expression was normalized to the housekeeping a cocktail of protease inhibitors (Sigma). Protein con- gene Hprt1 and calculated using the ddCt algorithm. centration in the extracts was determined using the BCA 1116 T-B Kang et al. Protein Assay Kit (Thermo Fisher Scientiﬁc). Proteins were 2. Muzio M, Chinnaiyan AM, Kischkel FC, O’Rourke K, Shev- chenko A, Ni J, et al. 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