Zα2 domain of ZBP1 is a molecular switch regulating Influenza-induced PANoptosis and perinatal lethality during development

Z-DNA-binding protein 1 (ZBP1) is an innate nucleic acid sensor which regulates host defense responses and development. ZBP1 activation triggers inflammation and pyroptosis, necroptosis, and apoptosis (PANoptosis) by activating RIPK3, caspase-8, and the NLRP3 inflammasome. ZBP1 is unique among innate sensors because of its N-terminal Zα1 and Zα2 domains, which bind to nucleic acids in the Z-conformation. However, the specific role of these Zα domains in orchestrating ZBP1 activation and subsequent inflammation and cell death is not clear. Here we have generated Zbp1ΔZα2/ΔZα2 mice that lack the Zα2 domain of ZBP1 and demonstrate that this domain is critical for influenza A virus (IAV)-induced PANoptosis and perinatal lethality in RIPK1-RHIM mutated (Ripk1RHIM/RHIM) mice. Deletion of the Zα2 domain in ZBP1 abolished IAV-induced PANoptosis and NLRP3 inflammasome activation. Furthermore, deletion of the Zα2 domain of ZBP1 was sufficient to rescue Ripk1RHIM/RHIM mice from the perinatal lethality which is caused by ZBP1-driven cell death and inflammation. Our findings identify the essential role of the Zα2 domain of ZBP1 in physiological functions and establish a link between sensing of Z-RNAs via the Zα2 domain and the promotion of influenza-induced PANoptosis and perinatal lethality.


INTRODUCTION
Activation of inflammation and cell death are interconnected cellular events which regulate host defense and immune responses. RIP homotypic interaction motif (RHIM)family proteins play a major role in orchestrating inflammation and cell death to profoundly shape immune responses and host defense (1,2). Z-DNA-binding protein 1 (ZBP1) is one such RHIM protein that interacts with the RHIM proteins receptor-interacting protein kinase 3 (RIPK3) and receptor-interacting protein kinase 1 (RIPK1) by forming homotypic interactions (3). ZBP1 and RIPK3 were also shown to participate in virus-induced necroptosis (4). We recently demonstrated that ZBP1 recognizes infection with influenza A virus (IAV), an RNA virus, and triggers NLRP3 inflammasome activation and pyroptosis, necroptosis, and apoptosis (PANoptosis) pathways (5). ZBP1 activation assembles RIPK3 and caspase-8 signaling complexes which mediate activation of the NLRP3 inflammasome and pyroptosis, RIPK3-MLKL-driven necroptosis, and FADD-caspase-8driven apoptosis (5)(6)(7)(8). These studies unraveled specific cellular functions that are regulated by ZBP1 in response to influenza infection. Further studies identified an endogenous physiological function of ZBP1 by demonstrating its role in perinatal lethality in mice (9,10). These studies found that mice with mutations that abolish the RHIM activity of RIPK1 have perinatal lethality and skin inflammation, and activation of ZBP1-driven necroptosis is responsible for the pathological phenotypes seen in these mice (9,10). ZBP1 consists of two N-terminal Z-nucleic acid-binding domains (Za1 and Za2) followed by RHIM domains and a functionally undefined C-terminal region (11). The Za1 and Za2 domains of ZBP1 bind to DNA/RNA in the Z-conformation (12)(13)(14)(15). Recognition of endogenous RNA, viral RNAs, and vRNPs that might exist in the Z-nucleic acid conformation is thought to activate ZBP1-mediated inflammation and cell death (6,8,16).
The Za domains of ZBP1 show high affinity for Z-nucleic acids in vitro (12)(13)(14)17); however, whether the recognition of Z-nucleic acids regulates cellular functions is not studied. In addition, the role of the Z-nucleic acid-binding Za domains of ZBP1 in regulating cell death and inflammation remains poorly investigated. Here we identify the critical role of the Za2 domain of ZBP1 in regulating IAV-induced NLRP3 inflammasome activation, cell death, and perinatal lethality during mouse development. Our observations define a pivotal role for the Za2 domain of ZBP1 in vivo and provide evidence for the activation of ZBP1 by endogenous Z-RNAs.

Generation of C57BL/6 mice that lack the Za2 domain of ZBP1
Experiments using ZBP1-knockout mice, which show complete loss of ZBP1 expression throughout the body (Zbp1 -/-), have delineated the role of ZBP1 during in vivo infections and in mouse development (11). ZBP1 is a critical innate immune sensor for activating inflammatory signaling and PANoptosis in both immune and non-immune cells in response to IAV infection (5,18). In addition, ZBP1 also activates necroptosis and inflammation during RIPK1-RHIM mutant mouse development, leading to perinatal lethality (6,9,10). Both the Za1 and Za2 domains of ZBP1 are capable of binding to Znucleic acids, and specific mutations in either of these Za domains block their Z-nucleic acid-binding activity (12,13,19). Although several recent studies established the role of ZBP1 in innate immunity and development, specific contributions from the Z-nucleic acid-recognizing Za domains of ZBP1 in vivo are not clear. To investigate this, we generated ZBP1 mutant mice with deletion of the Za2 domain in the C57BL/6 background using CRISPR/Cas9 technology (hereafter referred as Zbp1 DZa2/DZa2 mice) ( Fig. 1A and Suppl Table 1). At the amino acid sequence level, the CRISPR/Cas9 approach generated a Za2 domain deletion in ZBP1 by joining residues at positions 76 and 151 and deleting residues at positions 77-150 (Fig. 1A). Zbp1 DZa2/DZa2 mice were viable and did not show visible phenotypic defects.
To confirm the deletion of the Za2 domain at the protein level, we generated bone marrow-derived macrophages (BMDMs). Zbp1 DZa2/DZa2 BMDMs showed similar expression of the myeloid markers CD11b and F4/80 compared with BMDMs generated from WT littermates, suggesting the deletion caused no defects in the ability of bone marrow progenitors to differentiate into macrophages (Suppl Fig. 1). Lipopolysaccharide BMDMs, indicating the Za2 domain deletion did not interfere with the expression of ZBP1 protein (Fig. 1B). A shift in ZBP1 protein towards a lower molecular weight was observed in Zbp1 DZa2/DZa2 BMDMs, confirming the deletion of the Za2 domain (ZBP1-DZa2) in these mice (Fig. 1B). Isoform-2 of ZBP1, which consists of only the Za1-Za2 domains, was not seen in Zbp1 DZa2/DZa2 BMDMs (Fig. 1B). These results established the deletion of the Za2 domain of ZBP1 in mice with no detectable phenotypic differences in the mice and macrophages. H1N1]). Similar to LPS stimulation, IAV infection upregulated the expression of ZBP1-DZa2 to a similar extent that ZBP1 was upregulated ( Fig. 2A). In addition, there was no defect in IAV replication in Zbp1 DZa2/DZa2 BMDMs as indicated by NS1 protein expression ( Fig. 2A). IAV infection triggers specific activation of the NLRP3 inflammasome (5,6,20,21). Zbp1 DZa2/DZa2 BMDMs showed a complete lack of caspase-1 (CASP1) activation compared with WT BMDMs, suggesting inhibition of IAV-induced NLRP3 inflammasome activation in these BMDMs ( Fig. 2A). Cleavage of gasdermin D (GSDMD) and release of the GSDMD N-terminal domain (p30), which is a measure of GSDMD activation, was also inhibited in Zbp1 DZa2/DZa2 BMDMs in response to IAV infection ( Fig.   2A). These observations suggest that the Za2 domain is critical for ZBP1-mediated activation of NLRP3 inflammasome and GSDMD-induced pyroptosis.

Za2 domain of ZBP1 triggers inflammasome activation and
We further investigated the role of the Za2 domain of ZBP1 in regulating IAV-induced PANoptosis in BMDMs. We found that IAV infection induced robust cell death in WT BMDMs compared with mock treatment (Fig. 2B). This IAV-induced cell death was completely abolished in Zbp1 DZa2/DZa2 BMDMs, suggesting a lack of ZBP1-mediated signaling (Fig. 2B). Consistent with previous reports (5,6,8), Zbp1 -/-BMDMs showed complete inhibition of IAV-induced cell death (Fig. 2C). These results suggest that the Za2 domain of ZBP1 is necessary and sufficient for ZBP1 sensing of IAV infection to engage PANoptosis. Deletion of the Za2 domain of ZBP1 showed defects in cellular functions similar to those seen with the loss of complete ZBP1 expression.

RIPK1-RHIM mutant mice
Previous studies unraveled an important role for ZBP1 by studying RIPK1-RHIM mutant mice (9,10) To understand this, we tested whether deletion of the Za2 domain of ZBP1 is sufficient to rescue Ripk1 RHIM/RHIM mice from perinatal lethality. Consistent with recent studies (9,10), deletion of ZBP1 rescued Ripk1 RHIM/RHIM mice from lethality, and viable mice were only produced when both the alleles of Zbp1 were absent (Suppl Fig. 2A). We found that deletion of the Za2 domain of ZBP1 also rescued perinatal lethality in Ripk1 RHIM/RHIM mice, and the mice became viable (Fig. 3A & 3B). Similar to WT and Ripk1 RHIM/RHIM Zbp1 -/mice, Ripk1 RHIM/RHIM Zbp1 DZa2/DZa2 mice also survived and developed normally into adulthood (Fig. 3C). Deletion of the Za2 domain in both the alleles of Zbp1 was necessary to make Ripk1 RHIM/RHIM mice viable (Fig. 3A-3C). This suggests that complete inhibition of the Z-RNA sensing activity of ZBP1 through deletion of the Za2 domain is necessary to inhibit ZBP1-driven necroptosis and perinatal lethality in Ripk1 RHIM/RHIM mice. These observations identify that the Za2 domain of ZBP1 is critical for driving cell death during development in Ripk1 RHIM/RHIM mice which in turn indicates potential sensing of endogenous RNA ligands by ZBP1.
To further investigate the impact of deleting the Za2 domain of ZBP1, we studied the immune cell population in circulation. The numbers and percentages of circulating lymphocytes, monocytes, and red blood cells were unaltered in viable Ripk1 RHIM/RHIM Zbp1 DZa2/DZa2 mice in comparison to WT mice (Suppl Fig. 2B). We observed a higher percentage of circulating neutrophils in both Ripk1 RHIM/RHIM Zbp1 DZa2/DZa2 mice and

DISCUSSION
Several recent studies unraveled an important role for ZBP1 in regulating cell death and inflammation and uncovered its role as an innate immune sensor. ZBP1 was first described as a tumor-associated protein that was upregulated in macrophages (22).
Identification of a RHIM domain in ZBP1 facilitated its characterization in cell death and inflammation (3,4,11). The RHIM-containing proteins regulate inflammatory signaling, apoptosis, and inflammatory cell death (1). Initial reports on ZBP1 suggested it plays a role in virus-induced necroptosis via its RHIM-mediated interaction with RIPK3 (3,4). We identified that IAV infection and its RNA genome products activate the NLRP3 inflammasome to mount inflammation and lung repair responses in vivo (20,21,23).
Identification of ZBP1's role in sensing IAV and activating the inflammasome and multiple programmed cell death pathways expanded its functions in diverse cellular activities and illustrated potential regulation of inflammation in vivo (5,11). The N-terminal Za domains of ZBP1 bind to nucleic acids in the Z-conformation. The Za domains bind to both Z-DNA or Z-RNA because of their conformational similarity once they attain the Z-conformation (12,13,15,17,19). Although Za domains interact with Z-nucleic acids in vitro, activation and cellular functions of these Za domains of ZBP1 and in vivo relevance were not studied.
Our findings here suggest that the Za2 domain is critical for the activation of ZBP1's functions. The Za2 domain is required for activation of ZBP1 to drive NLRP3 inflammasome activation, PANoptosis, and perinatal lethality in mice. It is interesting to find that deletion of the Za2 domain is sufficient to block ZBP1's functions. Our observations also suggest that the Za1 domain might be dispensable for ZBP1 regulation in vivo. The Za2 domain of ZBP1 shows a distinct way of binding to Z-nucleic acids unlike other Za domains from various other proteins (12). This suggests that the Za2 domain of ZBP1 might have evolved to control cellular functions by executing distinct mechanisms, and our findings showing its critical role in regulating host innate responses support this hypothesis. Earlier in vitro studies also demonstrated that deletion of the Za1 domain was not sufficient to inhibit ZBP1-induced cell death (8). Our results also suggest that the Za2 domain of ZBP1 may recognize viral or endogenous RNAs which attain Z-conformation to activate ZBP1-driven cell death and perinatal lethality. The N122 and Y126 residues in the Za2 domain of ZBP1 are essential for the Z-nucleic acid interaction, and mutations in these positions were shown to abolish the Z-nucleic acid-binding potential of ZBP1 (12).

BMDM cultures and IAV infection
Primary BMDMs from mouse bone marrow were grown for 6 days in IMDM (Gibco) media supplemented with 30% L929-conditioned media, 10% FBS (Atlanta Biologicals), 1% penicillin and streptomycin, and 1% non-essential amino acids (Gibco). BMDMs were seeded and cultured overnight before using them for stimulations or infections. For lipopolysaccharide (LPS) stimulation experiments, BMDMs were mock treated (media only) or stimulated with 100 ng/mL of LPS (InvivoGen) for 4 h. The IAV-PR8 strain was generated as described previously (5,6). BMDMs were infected with IAV (MOI, 10) for 2 h in serum-free DMEM media (Sigma). After 2 h of infection, 10% FBS was added. Whole cell lysates were collected at indicated time points for immunoblotting analysis or cell death was monitored.

Immunoblotting analysis
Immunoblotting and detection was performed as described previously (6). BMDMs were washed with PBS once, cells were lysed in RIPA buffer, and sample loading buffer containing SDS and 2-mercaptoethanol was added. Proteins were separated on 8%-12% polyacrylamide gels and transferred onto PVDF membranes (Millipore). Membranes were blocked in 5% skim milk followed by incubation with primary antibodies and secondary

SYTOX Green staining and cell death analysis
Real-time cell death assays of IAV-infected BMDMs were performed using an IncuCyte Zoom incubator imaging system (Essen Biosciences). BMDMs were seeded in 12-well tissue culture plates in the presence of 100 nM SYTOX Green (Thermo Fisher Scientific, S7020), which is a cell-impermeable DNA-binding fluorescent dye that enters dying cells upon membrane permeabilization. Analysis was done using the software package supplied with the IncuCyte imager. Using this software, a precise analysis of the number of SYTOX Green-positive (SYTOX Green + ) cells present in each image can be performed. The number of dead cells was acquired by counting SYTOX Green + cells and plotted using GraphPad Prism v8 software.