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# A human homolog of SIR2 antiphage proteins mediates immunity via the Toll-like receptor pathway

Delphine Bonhomme, Hugo Vaysset, Eirene Marie Q. Ednacot, Vasco Rodrigues, Yazan Salloum, Jean Cury, Anny Wang, Axel Benchetrit, Pierre Affaticati, Veronica Hernandez Trejo, et al.

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## To cite this version:

Delphine Bonhomme, Hugo Vaysset, Eirene Marie Q. Ednacot, Vasco Rodrigues, Yazan Salloum, et al.. A human homolog of SIR2 antiphage proteins mediates immunity via the Toll-like receptor pathway. Science, 2025, 389 (6758), pp.eadr8536. 10.1126/science.adr8536. hal-05230522

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A human homolog of SIR2 antiphage proteins mediates immunity via the TLR pathway

Delphine Bonhomme¹*, Hugo Vaysset²,³*, Eirene Marie Q. Ednacot⁴,⁵, Vasco Rodrigues⁶, Yazan Saloum⁷, Jean Cury², Axel Benchetrit⁸, Pierre Affaticati⁸, Veronica Hernandez Trejo¹, Paul Vittot¹, Charlie Bories¹, Alexis Cornec¹, Jean-Pierre Levraud⁸,⁹, Pedro P. Hernández⁷, Philippe Benaroch⁶, Benjamin R. Morehouse⁴,⁵,¹⁰,¹¹, Aude Bernheim²* & Enzo Z. Poirier¹*

¹Innate Immunity in Physiology and Cancer team, Institut Curie, PSL Research University, INSERM U932, Paris, France

²Institut Pasteur, Université Paris-Cité, CNRS UMR2535, Molecular Diversity of Microbes, Paris, France

³AgroParisTech, Université Paris Saclay, Paris, France

⁴Department of Molecular Biology and Biochemistry, School of Biological Sciences, University of California Irvine, Irvine, CA 92697, USA

⁵Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University of California Irvine, Irvine, CA 92697, USA

⁶Myeloid Cells and Immunity team, Institut Curie, PSL Research University, INSERM U932, Paris, France

⁷Institut Curie, PSL Research University, CNRS UMR 3215, INSERM U934, Paris, France

⁸TEFOR Paris-Saclay, CNRS UAR2010, Paris-Saclay University, Saclay, France

⁹Université Paris-Saclay, CNRS, Institut Pasteur, Université Paris-Cité, Institut des Neurosciences Paris-Saclay, Saclay, France

¹⁰Institute for Immunology, University of California Irvine, Irvine, CA 92697, USA

¹¹Center for Virus Research, University of California Irvine, Irvine, CA 92697, USA

*These authors contributed equally to this work

Correspondence: enzo.poirier@curie.fr; aude.bernheim@pasteur.fr
**Abstract**

The full extent of immune system conservation between bacteria and eukaryotes is unknown. However, recent research supports that a subset of bacterial antiphage proteins is conserved in eukaryotes and likely gave rise to key actors of mammalian immunity. Here, we show that the SIR2 protein domain, present in bacterial antiphage systems, plays a role in eukaryotic innate immunity. Through phylogenetic analysis, we identify SIRal, a human protein with a SIRim domain (subtype of SIR2). We demonstrate that SIRal plays a pivotal role in the animal Toll-like receptor (TLR) pathway of innate immunity by mediating the transcriptional upregulation of proinflammatory genes downstream of TLR stimulation, and protecting against bacterial and viral infections. We demonstrate that proteins containing a SIRim domain are diverse and widespread, found in 5 distinct lineages across 19% of eukaryotic genomes. This includes zebrafish, where SIRal plays a role in inflammation. This work opens avenues of research on the potential role of eukaryotic SIRim proteins in immunity, as well as on the involvement of SIRal in human pathology.
Introduction

The arms race between hosts and pathogens drives continuous diversification of immune
mechanisms. As bacterial, archaeal and eukaryotic organisms face distinct threats, research thus
far has largely focused on clade-specific immunity. However, recent work uncovered immune
mechanisms shared between bacteria and eukaryotes, including humans, revealing an
overlooked aspect of immunity: conservation (1–3). This conservation includes the cGAS-STING
pathway, which is pivotal in the detection of DNA viruses, as well as caspases/gasdermins, which
regulate cell death, and viperins, central antiviral effectors (4–9). Similarly, Toll-interleukin-1
receptor (TIR) domains are involved in antiphage systems as well as in animal immune pathways
(10–12). In prokaryotes and plants, TIR domains perform an enzymatic function, degrading NAD+
to trigger cell demise or to produce immune secondary messengers (10, 12–14). In mammals,
Toll-like receptors (TLRs), which are crucial in detecting pathogen cues upon infection, transduce
immune signals via a cytosolic TIR domain (11). The breadth of conservation of immune actors
across domains of life remains to be investigated.

Prokaryotes and eukaryotes encode proteins bearing a Silent Information Regulator 2 (SIR2) domain, originally described in the sirtuin family of enzymes (15). By performing NAD-dependent deacetylation, sirtuins play an important role in regulating gene expression via the modification of histones in eukaryotes, or through the regulation of metabolism in bacteria (15, 16). Recently, a novel function was uncovered for the SIR2 domain in bacteria: a role in antiphage defence (12, 17–20). Multiple antiphage systems encode a SIR2 domain that provides immunity through degradation of cellular NAD+. For example, phage-mediated activation of the protein Thoeris A (ThsA) leads to SIR2-dependent depletion of NAD+ and growth arrest of the infected cell (12, 19, 20). SIR2 domain-containing proteins thus perform immune and non-immune functions in bacteria. Whether SIR2 domains from antiphage systems are conserved in eukaryotes and contribute to human immunity is currently unknown.
# Results

## Two human proteins are homologs of SIRim antiphage proteins

We set out to identify all prokaryotic and eukaryotic proteins containing a SIR2 domain encoded in publicly available sequenced genomes. We first implemented homology-based detection of SIR2 proteins that are unrelated to bacterial, archaeal, or eukaryotic immunity, and of SIR2 proteins involved in bacterial antiphage systems. To do so, we built a general protein profile for the SIR2 family using 4,302 SIR2 homologs from across the tree of life (see Methods) and used it to search a database of 30,254 bacterial, archaeal and eukaryotic genomes. This homology search identified 57,911 SIR2 proteins across kingdoms (29,634 in bacteria, 2,094 in archaea, and 26,183 in eukaryotes, Table S1). To better characterise the diversity within the SIR2 family, we constructed a phylogenetic tree (Fig 1a, see Methods). In this tree, SIR2 proteins are clustered into two monophyletic groups (Fig 1a, beige and brown), with each group composed of bacterial, archaeal, and eukaryotic proteins (inner circle, blue, grey and green, respectively). This indicates that the family of SIR2 proteins can be partitioned into two subfamilies of ancient origin.

To better characterise the two subfamilies, we mapped the proteins involved in bacterial immunity on the phylogeny. We first identified bacterial SIR2 proteins involved in documented antiphage systems with the program DefenseFinder (Fig 1a, outer circle) (21). All bacterial antiphage SIR2 proteins are located in one out of the two clades, suggesting that the entire clade may be involved in immunity (Fig 1a, brown clade). In contrast, all the bacterial and eukaryotic non-immune sirtuins are found in the other clade (beige clade) (22). Since DefenseFinder only identifies homologs of defence systems whose antiphage activity has been experimentally validated, these predictions likely represent only a fraction of all existing antiphage systems. We thus sought to identify additional putative SIR2-containing antiphage proteins. To do this, we leveraged the tendency of antiphage genes to cluster within bacterial genomes, and analysed the frequency with which known antiphage systems are found in the vicinity (+/-20 genes) of SIR2 proteins (18, 23, 24). The genomic neighbourhood of bacterial SIR2 proteins from the first, brown clade shows a 12-fold enrichment in antiphage systems compared to the neighbourhood of SIR2 proteins from the second, beige clade (Fisher exact test, F=12.40, p<10⁻³, Fig 1a & S1). Such bipartite distribution strongly suggests that the majority of the bacterial proteins from the first clade, but not from the second clade, have an immune function (18, 23, 24). This phylogenetic analysis led us to divide the SIR2 family of proteins into two subfamilies with seemingly distinct
functions: i) sirtuins, which have a classical SIR2 domain and are involved in non-immune functions (Fig 1a, beige clade) and ii) sirims, equipped with a SIRim domain (for SIR and immunity, described hereafter), present in validated and predicted antiphage proteins, as well as in some eukaryotic proteins (Fig 1a, brown).

Analysing the experimental and predicted structures of SIR2 proteins sampled across the phylogeny reveals an overall conservation of the SIR2 domain topology, including the presence of a Rossmann fold with a 3-layer sandwich. The central β-sheet contains a core of six β-strands, with accessory β-strands in a subset of clades (Fig 1b, blue). A variable α-helical loop is located downstream of the first β-strand (Fig 1b, green) (25). Sirtuins and sirims from bacteria, archaea and eukaryotes show conservation of the NAD⁺ binding pocket, present at the junction between the Rossmann fold and the variable loop, as well as of catalytic residues involved in NAD⁺ degradation (Fig S2a) (16, 17, 20, 26). Structural differences however exist between sirtuins and sirims. The Rossmann fold of sirtuins, bearing a classical SIR2 domain, contains a β-sheet with exactly six β-strands, while seven or more strands are usually observed in sirim proteins. Sirtuins utilise a Zn²⁺ coordination site that allows binding of substrate amino acids of target proteins. Such a site is systematically absent from sirims, as is the tetrad of cysteines responsible for interactions with Zn²⁺ (Fig 1b, orange & Fig S2b). This suggests that proteins of the sirim clade are unable to bind acetylated amino acids, contrary to classical sirtuins (16, 26). Overall, this structural and sequence analysis is congruent with the phylogenetic partition and suggests that the subfamilies of SIR2 proteins could have different functions.

We assessed the diversity of SIR2 proteins in humans by performing homology detection and identified 9 proteins. Seven of these are sirtuins, well-documented to regulate histone deacetylation (15, 22, 27). The two others, FAM118A and FAM118B, belong to the SIRim subfamily and have poorly-documented functions, with no known immune role. Variations of FAM118A transcripts levels have been observed in epithelium infected with respiratory syncytial virus, whereas FAM118B has been documented to play a role in Cajal bodies of the nucleus (28, 29). The evolutionary relationship between the FAM118 family of proteins and antiphage systems of bacteria led us to hypothesise that they may be involved in immunity. FAM118B was renamed SIRal (for SIR antiphage-like) and FAM118A SIRal2.
SIRal is involved in the mammalian TLR pathway

To gain insights into the function of SIRal, we analysed the genomic localisation of the *SIRal* gene. In bacteria, genes coding for proteins of a given pathway tend to colocalise within genomes, displaying functional synteny. Although less common, multiple occurrences of a functional syntenic organisation have been documented in eukaryotes (7–9, 30). In 73% of metazoans, including humans, *SIRal* is located two genes away from the gene encoding for TIRAP (Fig S3a). TIRAP carries a TIR domain and contributes to TLR2 and TLR4 signalling. Upon ligand-induced TLR activation, TIRAP’s homotypic interaction with the cytosolic TIR domain of the receptor allows for immune signal transduction and production of proinflammatory cytokines (Fig 2a) (31, 32). Synteny between the *SIRal* and *TIRAP* genes suggests that SIRal may be involved in the TLR pathway.

Analysis of publicly available datasets indicate that *SIRal* is broadly expressed in tissues and is enriched in macrophages (Fig S3b). The latter are essential immune cells that detect pathogens through pattern recognition receptors, including TLRs. TLR activation leads to inflammatory and antimicrobial responses, as well as the priming of adaptive immunity. Cytokines such as tumor necrosis factor (TNF), interleukin-6 (IL6) and IL8, as well as interferons (IFN), are produced upon TLR stimulation. IFN signaling through the IFN-α/β receptor (IFNAR) leads to the transcriptional upregulation of antipathogen effectors named interferon-stimulated genes (ISGs). TLR signaling also results in the production of the antimicrobial agent nitric oxide (NO), synthesized by the enzyme inducible nitric oxide synthase (iNOS). To address the role of mouse SIRal (mSIRal) in the TLR pathway, we implemented a loss-of-function approach by knock-out in RAW264.7 murine macrophage-like cells (Δ*mSIRal* cells, Fig S3c,d). The TLR response was assessed by stimulating cells with purified lipopolysaccharide (LPS) from *E. coli*, an agonist of TLR4, and measuring the inflammatory response (Fig 2b,c). In wild type macrophages, LPS stimulation leads to upregulation of iNOS and the production of NO, which is abrogated in Δ*mSIRal* macrophages (Fig 2b,c & S3e). Δ*mSIRal* macrophages also display an impaired production of IL6, IFNβ and of the ISG Rsad2 upon LPS stimulation, indicating that mSIRal is a central mediator of TLR4-driven inflammation (Fig 2b,c). In contrast, the production of TNF appears mostly independent of mSIRal (Fig 2b,c). No defect in viability was observed for Δ*mSIRal* macrophages (Fig S3f,g). Δ*mSIRal* cells express wild type levels of TLRs, including TLR4, indicating that decreased NO production cannot be explained by receptor expression deficiency (Fig S3h). We additionally excluded LPS degradation in the absence of mSIRal by showing that
170 both wild type and Δ*mSIRal* cells trigger pyroptosis in response to cytosolic LPS sensing through inflammatory caspases (Fig S3g) (33). We further assessed the importance of SIRal by knock-down in mouse and human primary macrophages (Fig 2d,e). Reducing mSIRal levels in mouse bone marrow-derived macrophages decreases IL6—but not TNF—production upon TLR4 stimulation (Fig 2d and S3i,j). Human SIRal (hSIRal) knock-down in macrophages derived from blood translates into reduced expression of the ISGs Rsad2, IFI44L and Mx1 upon LPS stimulation, while TNF and IL8 productions remain at control levels (Fig 2e and S3k). Altogether, these data demonstrate that SIRal plays a pivotal role in the induction of some inflammatory targets and ISGs downstream of TLR4 stimulation.

180 In addition to TLR4, it is well documented that other TLRs trigger the production of cytokines, IFN and NO (34–36). To interrogate if mSIRal is also involved in signalling downstream of these receptors, we stimulated wild type and Δ*mSIRal* RAW264.7 cells with agonists of TLR1/2, TLR3, TLR7/8 and TLR9. In every case, lack of mSIRal translates into impaired NO, IL6 and IFNβ production (Fig 2f & S3l). As observed with TLR4, lack of mSIRal does not alter TNF production upon stimulation of the other members of the TLR family (Fig 2f). Human primary macrophages knocked-down for hSIRal similarly show impaired cytokine and ISG upregulation downstream of TLR7/8 stimulation (Fig 2g and S3k,m). Transcription of inflammatory genes depends on the activation of the transcription factors NFκB and IRFs by phosphorylation (Fig 2a). LPS-driven NFκB and IRF3 phosphorylation is not significantly impacted in murine Δ*mSIRal* macrophages, suggesting that SIRal acts downstream of the nuclear translocation of these transcription factors (Fig S3n-o). Innate immune activation not only depends on TLRs, but also on other receptors such as cGAS that detects cytosolic DNA from viral or bacterial origin (37). cGAS produces the second messenger cGAMP that activates STING, leading to pro-inflammatory gene transcription. To assess if hSIRal plays a part in other signaling pathways of innate immunity, we stimulated human primary macrophages with cGAMP and measured ISG expression (Fig 2h). Knock-down of hSIRal results in a 20% decrease in Mx1 transcript, while levels of IL8 and IFI44L transcripts remain unaffected (Fig 2h). SIRal is thus not central to STING signaling. Similar results were obtained upon IFN signalling through the IFNAR receptor, indicating that SIRal's role in inflammation seems rather restricted to the TLR pathway (Fig 2i). Altogether, these results document that SIRal is a central mediator of TLR-driven inflammation, demonstrating its role in mammalian immunity.
SIRal contributes to the control of HSV-1 and *Salmonella* in macrophages

TLRs protect against a variety of pathogens, including herpes simplex virus 1 (HSV-1), detected by TLR9, and the bacteria *Salmonella*, recognized by TLR4 (11). To test the importance of mSIRal in controlling pathogens that activate TLRs, we compared infection between control and *ΔmSIRal* RAW264.7 cells. By using an herpes virus strain engineered to express GFP fused to a viral capsid protein, it is possible to discriminate viral entry from productive viral replication. HSV-1 entry in cells results in intermediate GFP levels, while viral replication translates into the production of additional GFP molecules and cells with high GFP fluorescence (Fig 3a,b). Acyclovir, an inhibitor of viral replication, prevents the appearance of GFP+ high cells containing replicative virus (Fig 3a,b). Viral entry is equivalent in wild type and *ΔmSIRal* cells (Fig 3b and S4a,b). However, absence of mSIRal translates into an increase in the percentage of productively infected cells and a 2-fold increase in GFP mean fluorescence intensity (MFI), a measure of viral replication (Fig 3b,c and S4a-c). These results demonstrate that mSIRal plays a role in the control of HSV-1 infection in macrophages.

To interrogate the role of mSIRal in TLR-driven antibacterial immunity, RAW264.7 cells were infected with *Salmonella enterica* strain SL1344 and intracellular bacterial replication was measured by colony forming unit (CFU) after gentamicin protection assay (Fig 3d). A 2-fold increase in *S. enterica* accumulation is observed in *ΔmSIRal* cells, compared to wild type cells (Fig 3d). Additionally, infected *ΔmSIRal* macrophages show an abrogation of the production of the ISG Rsad2, whereas TNF transcript levels appear unmodified (Fig S4d). To address if TLR4-signaling is accountable for the difference of bacterial loads in wild type and *ΔmSIRal* cells, we antagonized the receptor with LPS of *Rhodobacter sphaeroides* (Fig 3e and S4e) (38). Blocking TLR4 does not affect the accumulation of *S. enterica* in *ΔmSIRal* macrophages. On the other hand, blocking TLR4 in wild type cells translates into higher intracellular replication, reaching the levels measured in *ΔmSIRal* cells (Fig 3e). Abrogating the difference between wild type and *ΔmSIRal* cells by antagonizing TLR4 demonstrates that mSIRal acts within the TLR4 pathway in this infectious model (Fig 3e). Altogether, these results demonstrate that mSIRal plays a role in TLR-driven immune protection against viruses and bacteria.
Sirims from bacteria and animals are evolutionarily related proteins that participate in immunity. We investigated the evolutionary history of the SIRal lineage. To do so, we queried EukProt, a database containing protein sets from 985 species spanning all major documented eukaryotic lineages, using the SIRim protein profile HMM constructed in Fig 1 (39). SIRal homologs are detected in a total of 35 diverse species including the freshwater protist *P. lacustris*, the zebrafish (*D. rerio*), the slime mold (*D. discoideum*), the Australian ghost shark (*C. milii*), and the mallard (*A. platyrhynchos*, Table S4). SIRal homologs are distributed in a wide range of eukaryotic taxa suggesting that the acquisition in eukaryotic organisms is very ancient (Fig 4a & S5, Clade A). More precisely, the presence of SIRal homologs in many genomes of opisthokonts, a eukaryotic taxon that includes fungi, choanoflagellates and animals (metazoa) suggests that the acquisition event occurred prior to their last common ancestor, more than 1.4 billion years ago, and that major classes of opisthokonta such as insects have lost the gene (Fig 4a, S5-S8; Table S4). Beyond the lineage of SIRal homologs (named Clade A), our phylogenetic analysis uncovers four additional lineages of eukaryotic sirims (Clades B to E) in 189 different species. In total, 19.2% of the eukaryotic species we queried encode a SIRim protein (Fig 4a & S5-S7, Table S4). Clades A to C are distributed among a wide diversity of eukaryotes, with for example clade C, encompassing 83 species. Conversely, clade D and E are limited to a small number of eukaryotes, with clade E detected only in choanoflagellates (Fig 4a & S5, Table S4). The interspersed distribution of the five eukaryotic lineages within bacterial and archaeal clades in the phylogenetic tree strongly suggests that the five clades of eukaryotic sirims arose independently during the history of eukaryotes. Interestingly, these lineages branch with bacterial sirims belonging to different antiphage systems (clades B and C with ThsA-like sirims, and clades D and E in pAgo-like sirims). These multiple acquisition events were followed by frequent gene losses, explaining the patchy distribution of sirims across eukaryotes (Fig S5-S7).

We sought to gain insight regarding the biological function of eukaryotic sirims from the five identified clades. Strikingly, we observed that the SIRim domain is frequently fused to additional domains which are often observed in immune proteins. Clade A, for example, contains fungal and animal sirims with an N-terminal SIRim domain, a central nucleotide-binding domain (such as NACHT or NB-ARC), and C-terminal repeats (e.g., ankyrin or tetratricopeptide). This tripartite architecture is typical of nucleotide-binding domain, leucine-rich repeat-containing (NLR) proteins, which are immune receptors key to detecting a variety of infection cues in mammals and plants, as well as to inducing programmed cell death upon mating of incompatible germlings in
fungi (Fig 4b, Clade A) (40–43). In clade C, sirims are typically composed of a SIRim domain fused to a Macro domain (Fig 4b). This mirrors the domain architecture of ThsA from the type II Thoeris antiphage system in which a Macro domain is fused to an effector of cell death, a role that is fulfilled by a SIRim domain in type I Thoeris systems (44, 45). The genetic architectures of sirims thus suggest that they play an immune role in a wide diversity of eukaryotes.

To further probe this notion, we focused on the unique sirim homolog encoded in zebrafish (zSIRal, previously termed fam118b). Zebrafish encode for TLRs, and display innate immune pathways that are evolutionary related to pathways present in mammals, including the production of ILs, IFNs, and the transcriptional upregulation of ISGs upon inflammation (46). To interrogate if zSIRal is involved in innate immunity in vivo, we created crispants of the gene in zebrafish larvae (knock-out approach) and performed infections with the *Edwardsiella tarda* strain FL60. No difference in survival was observed between wild type and ΔzSIRal larva upon infection (Fig S9a). We then addressed the inflammatory response upon infection by measuring ILs and IFNs transcripts 3 days post-infection. Infection triggered the upregulation of ILs (il26) in both wild-type and ΔzSIRal larvae (Fig 4c). However, the IFN response (*ifng* and *ifnphi1*) upon infection with *E. tarda* was visible in wild-type larva but abrogated in ΔzSIRal larvae (Fig 4d), demonstrating that zSIRal is involved in regulating IFNs *in vivo*. Interestingly, ΔzSIRal larvae showed higher constitutive levels of *ifnphi1* compared to wild-type (Fig 4d).

Our results show that sirim function in both bacterial and eukaryotic immunity. We next investigated whether the underlying biochemical activity of sirims is conserved between bacteria and eukaryotes. Bacterial sirims perform an antiphage function by processing cellular NAD⁺ (12, 20). To investigate if SIRal similarly degrades NAD⁺, recombinant hSIRal was purified and mixed *in vitro* with ε-NAD, an NAD⁺ analog whose degradation can be monitored using a fluorescence-based assay. In the presence of ε-NAD alone, recombinant hSIRal did not demonstrate detectable ε-NAD degradation activity (Fig 4e and S9b). This finding, however, does not exclude the possibility that SIRal requires an activation trigger, such as a protein-protein interaction, to initiate NADase activity. In bacteria, activation of a homologous sirim, ThsA, depends on oligomerization and formation of stable, enzymatically active protein filaments (20). We therefore hypothesised that hSIRal may be similarly activated through oligomerization. To assess this, we introduced the molecular crowding agent polyethylene glycol (PEG) as a means of artificially stimulating protein-protein interactions *in vitro*. In the presence of PEG, hSIRal demonstrated degradation of ε-NAD, albeit at a low rate (Fig 4e). hSIRal enzymatic activity was further confirmed with the natural NAD⁺ substrate and HPLC analysis revealing reduced detection of NAD⁺ and the presence of
compounds whose elution/retention profiles are compatible with those of ADPR and NAM (Fig S9c). The NADase activity of bacterial ThsA can be abolished by introducing alanine substitutions for the conserved catalytic residues N112 and H152 (12, 20). To generate a catalytically inactive hSIRal, we mapped these conserved residues on the human sequence, confirming the residues' putative homologous positions in an Alphafold2 model, and constructed N163A and H206A single and double mutants (Fig S9d-e). All predicted catalytically inactive hSIRal mutants had no detectable NADase activity, irrespective of the presence of PEG (Fig 4e). To probe if the NADase activity is conserved in diverse eukaryotic sirims, representative members of each clade were subjected to an HPLC-based NAD⁺ *in vitro* cleavage assay. Sirims from clade A, B, and D showed interaction with NAD⁺ (Fig 4f and S9f,g). Sirims from mouse and zebrafish (clade A, mSIRal and zSIRal) as well as from the algae *Paragymnodinium shiwhaense* (clade B) cleaved NAD⁺ into ADPR and NAM. The sirim from the amoeba *Paulinella microspora* (clade D) also led to a reduction in NAD⁺ signal, without producing major ADPR or NAM. NADase activity was not detected for two sirims of clades C and E (Fig 4f and S9f,g). In all cases, absence of the native activating signal or truncation of the protein to the SIRim domain alone may have also influenced NADase activity, and still warrant further study. Overall, this demonstrates that the ability to hydrolyze NAD⁺ is conserved in bacteria and eukaryotes and could be key for the immune function of sirims across domains of life.

We next evaluated if the NADase activity is necessary for the immune function of eukaryotic sirims, by investigating if the role of hSIRal in the TLR pathway depends on its NADase activity. Human embryonic kidney cells (HEK293T) engineered to express TLR4 and co-receptors, as well as a colorimetric reporter for NFκB-driven inflammation, can be used to monitor TLR4 signalling after LPS stimulation (Fig S9h,i). Expression of wild type hSIRal induces an average two-fold increase in LPS-driven TLR4 inflammation, which is absent when the catalytically inactive version of hSIRal (N163A/H206A) is expressed instead (Fig S9h,i). These results demonstrate that hSIRal participates in TLR signalling through its NADase activity. To interrogate if the activation of SIRal is potent enough to deplete cellular NAD⁺, we monitored intracellular NAD⁺ concentration in murine macrophages upon LPS stimulation. ΔmSIRal macrophages did not show significant changes in NAD⁺ concentration compared to control cells, suggesting that, as measured *in vitro*, SIRal is a weak NADase (Fig S9j). Our results demonstrate that, akin to their bacterial homologs, eukaryotic sirims are NADases involved in immunity.
Discussion

In this work, we demonstrate that the widespread SIRim domain involved in antiphage defence also participates in eukaryotic immunity. We identify SIRal, a mammalian innate immune protein with a SIRim domain involved in signalling downstream of TLRs.

Given the vast body of literature on TLR pathways, one may be surprised that such a pivotal protein was not discovered sooner, through the implementation of a CRISPR screen or otherwise. One such CRISPR-Cas screen was performed to identify members of the TLR cascade, but measured TNF levels as a read-out of TLR4 activation (47). Our data indicate that its production is mostly independent of SIRal, explaining why it was not identified in that study. A recent publication also mapped members of the myddosome, a MyD88-dependent supramolecular structure responsible for signalling downstream of TLR activation (48). SIRal was not identified in this work, suggesting that its mechanism of activation may be independent of interactions with myddosome components. SIRal was originally depicted as a protein involved in the Cajal bodies of the nucleus (28). Our data place it instead in the cytosol, in which it participates in the TLR pathway. Its molecular mechanism is in the process of being deciphered.

The SIRim domain is involved in multiple antiphage systems, protecting bacteria against viral infection. In mammals, it is integrated within the TLR pathway, and participates in the protection against viral and bacterial infections. There is thus a diversification of this domain's protective role in eukaryotes. Conserved antiphage domains appear tailored to clade-specific immunity, in link with selective pressures arising from eukaryote and/or clade-specific pathogens. Such adaptability was observed for additional conserved immune proteins, such as cGAS/cGLRS and viperins (8, 9, 49). At the molecular level, the adaptation of the SIRim domain to the context of eukaryotic innate immunity translates into a shift in its biochemical activity: from a strong NADase in bacteria that depletes NAD⁺ and triggers cell death, to a weaker NADase activity in humans or zebrafish, that does not trigger cell demise but probably participates in signal transduction.

Beyond humans and animals, we uncovered hundreds of sirims in a wide diversity of eukaryotes, including fungi, amoeba, alveolata, and diatoms. Other domains of antiphage origin also show conservation across a wide range of eukaryotes. TIR domains participate in immune signal transduction within the Toll receptor of insects and as part of mammalian TLRs (11).
Homologs of cGAS, coined cGAS-like receptors (cGLRs), are conserved across metazoans and participate in immunity in species such as coral and oyster (49–51). Based on SIRim-containing proteins being frequently fused with domains involved in immunity, and the ability of certain sirims to degrade NAD+, we predict that sirims could participate in host defence in many of these – often non-model – organisms. We have, for example, identified sirim homologs in *Chaetoceros* diatoms, which are keystone organisms in marine ecosystems. Studying immunity in these organisms could improve our understanding of how host-pathogen interactions shape ecosystems.

To account for the ever-growing number of immune members conserved across domains of life, we recently proposed the concept of ancestral immunity (52). Ancestral immune modules are proteins or protein domains evolutionarily conserved between prokaryotes and eukaryotes, involved in immunity in both kingdoms. We show in this work that, akin to PNP, NACHT or TIR, SIRim corresponds to an ancestral immune domain. Conservation across kingdoms can be harnessed for mechanistic studies, by interrogating the module's molecular features in multiple species in parallel (e.g., bacteria and humans). Whether SIRal plays a role in human pathology, and can be harnessed for therapy, will be the topic of future investigation.
<figure><img src="image_4.png" /><figcaption>**Figure 1.** Human proteins belonging to the monophyletic group of SIRim proteins are associated with bacterial antiphage defence proteins.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Figure 2. SIRal contributes to TLR signaling in macrophages.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Figure 3.** SIRal contributes to the control of HSV-1 & *Salmonella* in macrophages.</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Figure 4.** SIRal is an NADase and is part of a large family of sirims in Eukaryotes.</figcaption></figure>
**Figure 1. Human proteins belonging to the monophyletic group of SIRim proteins are associated with bacterial antiphage defence proteins.**

a. Phylogenetic tree of SIR2 family proteins across the tree of life. A total of 2,723 sequences representative of all the SIR2 domain-containing proteins identified through homology search were used to build the tree (midpoint rooted). SIR2 proteins cluster into two subfamilies based on amino acid sequences, namely SIR2 (inner ring, beige) and SIRim (inner ring, brown). The domain of life from which each protein originates is provided in the central ring (blue: bacteria, grey: archaea and green: eukaryotes). For proteins identified in bacterial organisms, we assessed if the protein is present in a known antiphage defence system and indicate its name (outer ring) (21). The location of human sirtuins (SIRT1-7), of SIRal and SIRal2, as well as of other proteins exemplified in Fig 1b are indicated. Tree branch support values are assessed using UltraFast Bootstrap and are indicated in shades of green. Support is also specified on the branch delimiting the two subfamilies. b. Structures of selected SIR2 proteins from the SIR2 (top, beige) and the SIRim subfamilies (bottom, brown). Three sirtuins (CobB (PDB: 2H2F), HST2 (PDB: 1SZC), SIRT6 (AFDB: Q8N6T7)) and six sirims (Thoeris A (AFDB: J8G6Z1, residues 1-279), Dsr2 (AFDB: R8X3J8, residues 1-281), SIRal (AFDB: Q9BPY3), PD-T7-2 (AFDB: I3Z638), pAgo (AFDB: AIW90156), AbiH (AFDB: A0A1V0M776)) are provided (PDB: Protein DataBank, AFDB: AlphaFold Protein Structure Database). For bacterial sirims, the name of the protein is coloured according to the antiphage system in which the protein is found. In ThsA, Dsr2, and pAgo, only the SIRim domain is shown while it is fused to additional domains in the original proteins. The central β-sheet of the Rossmann fold (blue) and the variable loop (green) are highlighted to underline the structural similarities between proteins. For each protein, the species of origin is indicated. A * after a protein name indicates that it is found in a eukaryotic organism.

390

395

400

405

410

**Figure 2. SIRal contributes to TLR signaling in macrophages**

a. Schematic of TLR4 signalling in macrophages. b. Wild type or *ΔmSIRal* RAW264.7 cells were stimulated with LPS from *E. coli* (100ng/mL). Levels of TNF, iNOS and Rsad2 transcripts were measured by RT-qPCR at 2h and 8h. c. Wild type or *ΔmSIRal* RAW264.7 cells were stimulated with LPS from *E. coli* (10-100ng/mL). Production of TNF, IL6, IFNβ and nitric oxide (NO) was measured at 24h. d. Bone-marrow derived macrophages (BMDMs) transfected with a control siRNA, or a siRNA targeting *mSIRal*, were stimulated with LPS from *E. coli* (100ng/mL). Production of TNF and IL6 was measured at 24h. e. Human monocyte-derived macrophages (huMDMs) transduced with a control shRNA, or a shRNA targeting *hSIRal*, were stimulated with LPS from *E. coli* (100ng/mL). Levels of TNF, IL8, Rsad2, IFI44L and Mx1 transcripts were measured by RT-qPCR at 8h. f. Wild type or *ΔmSIRal* RAW264.7 cells were stimulated with Pam3CysSK4 (500ng/mL), Poly(I:C) (1µg/mL), LPS (100ng/mL), R848 (1µg/mL) or CpG

415

420
(1µg/mL), agonists of TLR1/2, TLR3, TLR4, TLR7/8 and TLR9, respectively. Production of TNF and NO was measured at 24h. g. huMDMs transduced with a control shRNA, or a shRNA targeting *hSIRal*, were stimulated with LPS (100ng/mL), flagellin (500ng/mL) or R848 (1µg/mL), agonists of TLR4, TLR5 and TLR7/8, respectively. Levels of IL8 and IFI44L transcripts were measured by RT-qPCR at 8h. h,i. huMDMs transduced with a control shRNA, or a shRNA targeting *hSIRal*, were stimulated with either cGAMP (the agonist of STING, 4µg/mL) (h) or universal type-I IFN (activator of IFNAR, 100U/mL) (i). Levels of IL8, IFI44L and Mx1 transcripts were measured by RT-qPCR at 8h.

Each dot represents the mean of technical replicates for each independent experiment (b,c,f), independent mouse BMDMs isolation (d) or independent human donor (h). Bars correspond to the mean and error bars to the standard deviation of all the independent experiments/mouse/donors. Statistical analyses were performed using two-ways ANOVA and p-values were classified as * p<0.05; ** p<0.01; *** p<0.001. Grey stars correspond to comparisons within one group and dark stars correspond to comparisons between groups.

<caption>Figure 3. mSIRal contributes to the control of HSV-1 and *Salmonella* in macrophages.</caption>

a. Schematic of HSV-1-GFP infection. Viral particles contain GFP fused to the capsid protein VP26, hence viral entry is detected in cells by intermediate GFP levels, measured by flow cytometry. Productive viral replication results in GFP production and high levels of fluorescence. Viral replication, but not entry, is inhibited by the drug acyclovir. b. Wild type or *ΔmSIRal* RAW264.7 cells were infected or not with HSV-1 (MOI 7.5), in the presence or absence of acyclovir. Levels of GFP were measured by flow cytometry at 24h. c. Wild type or *ΔmSIRal* RAW264.7 cells were infected or not with HSV-1 (MOI 5-10). Levels of GFP were measured by flow cytometry at 24h and the percentage of productively infected cells (GFP high) was determined. The geometric mean fluorescence intensity (geoMFI) of the GFP high population is indicated as a surrogate of viral replication. d,e. Wild type or *ΔmSIRal* RAW264.7 cells were infected with *Salmonella enterica* serovar Typhimurium strain SL1344 (MOI 10). Intracellular replication of bacteria was quantified by colony forming unit (CFU) after gentamicin protection assay. In (e), LPS from *Rhodobacter sphaeroides* (1µg/mL) is used as a TLR4 antagonist.

Each dot represents the mean of technical replicates for each independent experiment (c,d,e). Bars correspond to the mean and error bars to the standard deviation of all the independent experiments. Statistical analyses were performed using two-ways ANOVA and p-values were classified as * p<0.05; ** p<0.01; *** p<0.001. Grey stars correspond to comparisons within one group and dark stars correspond to comparisons between groups.
**Figure 4. SIRal is part of a large family of sirims in Eukaryotes, including NADases with immune functions.**

a. **(Left)**. Phylogenetic tree of all identified SIRim domain-containing proteins across the tree of life. Representative sequences of all detected bacterial, archaeal, and eukaryotic sirims (n=2,266) are aligned on their SIRim domain and a phylogenetic tree was built from the multiple sequence alignment. The domain of life of each protein is indicated in the central ring (blue: bacteria, grey: archaea, green: eukaryotes) and the specific taxon is indicated for eukaryotic sequences in the outer ring. Antiphage systems are annotated using DefenseFinder for bacterial sirims (inner ring) (21). The five major eukaryotic clades of sirims (clades A to E) are coloured in shades of purple (inner ring) and within each clade, specific sequences are exemplified in Fig 4c. Branch support was assessed using UltraFast Bootstraps and is indicated in shades of green. Support values are indicated on key branches. **(Right)**. Taxonomic distribution of SIRim proteins in Eukaryotes. The phylogenetic tree of The Comparative Set (TCS) from Eukprot includes 196 species from all major known eukaryotic phyla (39). The taxonomic distribution of each of the five eukaryotic SIRim protein clades is displayed. SIRim proteins detected in species present in the Eukprot v3 database (985 species) but not in the TCS (196 species) are assigned to their closest relative present in the TCS tree. Sirim presence in a specific clade is reported only when at least two distinct species of the clade are identified as encoding a sirim gene in their genome. The tree is midpoint rooted. The tree is also pruned, and branch lengths are ignored for visualisation purposes (see Fig S6). For each major eukaryotic taxon, a logo of one example species encoding a *sirim* gene is provided. **b.** Examples of sirims found in eukaryotic organisms. Sirims are sampled from each of the five major eukaryotic sirim clades and are exemplified (Fig 4a & Fig S7-S8). For each sequence, the domain architecture as well as the AlphaFold2 model is provided. The SIRim domain is coloured in pink and additional, fused, domains are indicated in grey. Whenever the specified domain could be characterised using HHpred, its name is provided. NACHT (PF05729), SNAP (PF14938), n.a.: domain not identified. **c-d.** Wild type or ΔzSIRal zebrafish larvae were infected with *Edwardsiella tarda*. Levels of *il22*, *il26*, *il1b*, *ifng*, and *ifnphi1* transcripts were measured by RT-qPCR at 3 days post-infection. Each symbol represents a larva and bars correspond to the median of all the larva. The three independent experiments are represented with dots, squares and triangles. Statistical analyses were performed using two-ways ANOVA and p-values were classified as * p<0.05; ** p<0.01; *** p<0.001. **e.** Enzymatic NADase activity assay of purified recombinant hSIRal (WT, N163A mutant, H206A mutant, N163/H206A double-mutant), incubated with ε-NAD, with or without PEG 400 over time. **f.** Purified recombinant zSIRal from *D. rerio* (Opisthokonta, clade A), sirim from *P. shiwhaense* (Alveolata, clade B),
*P. violaceum* (Amoebozoa, clade C), *P. micropora* (Rhizaria, clade D) or *Acanthoecidae* sp. (Opisthokonta, clade E) were incubated with NAD+, with or without PEG 400. NADase activity was addressed by measuring NAD+ degradation by HPLC analysis after 16h incubation with NAD+. Shades of red relate to statistical significant (p-value).

## Acknowledgments

We are grateful to members of the MDM lab, U1284, U932 as well as mentors and friends for their useful comments on earlier versions of the manuscript. We thank C.W. for the kind gift of cell lines, A.R. for the kind gift of bacterial strain and S.H.L., A.W., and E.H. for cloning assistance. Some of the bioinformatic analyses were performed on the Core Cluster of the Institut Français de Bioinformatique (IFB) (ANR-11-INBS-0013). We also thank the team managing the Phylopic database of logos as well as all the contributors (https://www.phylopic.org/); the freely shared content helped us design certain figures of the paper.

## Funding

H.V. and A.B are supported by ERC Starting Grant (PECAN 101040529) and funding from Institut Pasteur. E.Z.P, V.H.T and D.B. are supported by an ERC Starting Grant (STEMGUARD 101075865), funding from the Fondation pour la Recherche Médicale (Amorçage Jeune Equipe), from Paris Science & Lettres University, from the Fondation Chercher Trouver and funding from Institut Curie. This work is funded by a grant "Impact Santé" supported by Agence Nationale de la Recherche under France 2030 (ANR-24-RRII-0005) on funds administered by Inserm, as well as under the program France 2030 launched by the French Government (ANR-10-IDEX-0001-02 PSL). P.B. and V.R. are supported by the Fondation Chercher Trouver. P.P.H and Y.S. are supported by ERC-StG Cytok-Gut 101041422, ATIP-Avenir Starting Grant R21045DS, Laboratoire d'Excellence (Labex) DEEP (ANR-11-LBX-0044, ANR-10-IDEX-0001-02 PSL), Ville de Paris Emergence Program (2020 DAE 78), and funding from the Institut Curie, INSERM, and CNRS. A.B., P.A. and J.P. are supported by ANR-TEFOR-"Investissement d'avenir" (ANR-II-INBS-0014)

## Author contributions

A.B and E.Z.P conceptualised the project. All the authors designed experiments and analysed data. D.B., H.V., E.M.Q.E., V.R., Y.S., J.C. and V.H.T. performed data curation and formal analysis. D.B., E.M.Q.E., B.R.M., V.R., Y.S., Ax.B., P.A., P.V., A.C. and V.H.T. performed
experiments. H.V. ran all the bioinformatic analysis. All authors contributed to writing of the manuscript. A.B., E.Z.P., P.B., P.P.H. and B.R.M. acquired funding.

## Competing interests

The authors declare no competing interests.

## Data and materials availability

All the data, including phylogenetic trees, HMM profiles and protein structures generated with AlphaFold, are available at https://gitlab.pasteur.fr/mdm-lab/siral.

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## Database of eukaryotic, archaeal and bacterial genomes

We downloaded 4,616 Eukaryotic genomes from 2,407 species and 22,803 Bacterial complete genomes representing 7,231 species from the Genbank database (https://ftp.ncbi.nlm.nih.gov/genomes/all/GCA/) in January 2022 (53). 2,339 Archaeal genomes were also downloaded from the Genome Taxonomy DataBase (GTDB) in January 2023 and concatenated to them 496 inhouse Asgard Archaea genomes resulting in a database of 2,835 Archaeal genomes (54). This resulted in a database of 30,254 genomes spanning across the three domains of life.

## Systematic detection of SIR2 and SIR2-like proteins using iterative profile HMM search

An initial, loose, detection of SIR2 family proteins was performed by building a general profile HMM modelling the SIR2 family of proteins. This was done by aligning known SIR2 homologs including (i) known eukaryotic homologs of sirtuins (e.g. orthologs of *H. sapiens* SIRT1 to SIRT7) as well as (ii) homologs of bacterial antiphage proteins containing a SIR2 domain and identified by running DefenseFinder (v1.2.3, default parameters) on our database of genomes (21). SIR2 genes from the following systems were included in the selection of antiphage SIR2 proteins: Thoeris, Dsr1, Dsr2, PD-T7-2, Nezha, Avs, AbiH, Lamassu and prokaryotic Argonautes. This yielded a set of *n*=4,302 sequences which were aligned using Muscle v5.1 (option -super5) and a profile HMM was built using the hmmbuild command from HMMER (version 3.3.2) (55, 56). This initial SIR2 family profile was queried against our database of genomes using hmmsearch (HMMER package) which yielded *n*=57,911 hits satisfying the inclusion criteria (e-value < 1e-5, bit score > 20, query coverage > 60% and 100 < hit length < 2500 amino acids) in bacteria, archaea and eukaryotes (Table S1). These proteins were clustered using MMseqs2 v13.45111 easy-cluster (30% identity, 80% coverage), representative sequences were aligned using Muscle v5.1 and the alignment was trimmed using Clipkit v1.3.0 (options -m gappy -g 0.99) (57). An initial maximum likelihood phylogenetic tree was built from the trimmed alignment using IQTree v2.2.0 using the LG+G4 substitution model (58). Maximum likelihood optimization was performed for 1000 iterations (data not shown).

A refined detection of SIR2 proteins was then performed per SIR2 subfamily (SIR2 and SIRim separately) using an iterative profile HMM search against our database of bacterial, archaeal and eukaryotic genomes. This detection was performed to get a curated set of proteins
per subfamily, to be used in all the remaining analyses. First, FAM118A and FAM118B orthologs were fetched from the NCBI Orthologs database (https://www.ncbi.nlm.nih.gov/gene/79607/ortholog), by filtering out proteins designated as "PREDICTED" or "LOW QUALITY" leading to a set of n=453 FAM118 orthologs from Vertebrate organisms. Proteins were aligned using Muscle v5.1 (option -super5 ) and a profile HMM of FAM118A and FAM118B orthologs was constructed from the Multiple Sequence Alignment (MSA) using hmmbuild from the HMMER package (55, 56). This initial profile HMM was queried against the database of Eukaryotic proteins only in order to identify FAM118 homologs in this domain of life. This search ("Round 1") led to obtaining n=751 hits among Metazoa and Fungi satisfying the including criteria (e-value < 1e-05, 200 < length < 600 aminoacids). Hits were aligned using Muscle v5.1 (option -super5) and a new profile HMM was built to search the database of eukaryotic genomes for three additional round ("Round 2" to "Round 4") using HMMER, until no additional SIR2-domain containing proteins was identified in eukaryotes (Table S3) (55, 56). At each round, among all the profile HMM hits, only those satisfying the inclusion criteria were included in the list of valid hits and were included in the profile HMM for the subsequent profile HMM search (Table S3). FAM118 homology search was then expanded in Bacteria and Archaea using four additional rounds of profile HMM search ("Round 5" to "Round 8") against our database bacterial, archaeal and eukaryotic genomes. At each round, only hits satisfying the inclusion criteria were included to the list of valid hits (Table S3). Iterative search was stopped once no additional hits satisfying the including criteria were retrieved from the database, yielding n=8,697 of sirims across domains of life. To this set of proteins were added the list of abiH homologs (n=1,208) which were retrieved by querying the PF14253 Pfam profile against our database of genomes using hmmsearch (–cut_ga option) (56). This led to a total of 9,905 hits including SIRal/SIRal2 (FAM118A/B) homologs in eukaryotes as well as proteins involved in known bacterial antiphage defence systems.

All deacetylating sirtuins were also retrieved from the database of genomes using a similar procedure of iterative profile HMM search. Initially, orthologs of all human sirtuins (SIRT1 to 6) were fetched on the NCBI Orthologs database and hits with "LOW QUALITY" or "PREDICTED" headers were filtered out. The set 3,861 human sirtuin orthologs was clustered using MMseqs2 easy-cluster at 98% identity and 98% coverage, representative sequences were aligned using Muscle v5.1 and a profile HMM of human sirtuin orthologs was built using hmmbuild (55, 56, 59). The obtained profile was iteratively queried against our database of eukaryotic genomes for three rounds ("Round 1" to "Round 3") until no novel eukaryotic sirtuins were detected (Table S2). After round 3, hits satisfying the inclusion criteria (e-value < 1e-5, bit score > 110, query coverage >
80% and 200 < protein length < 1500 amino acids, n=23,192) were clustered at 50% identity / 80% coverage using MMseqs2 easy-cluster. Representative sequences (n=1,944) were aligned using Muscle v5.1, a profile HMM was built using hmmbuild and the profile was queried against bacterial, archaeal and eukaryotic genomes. Iterative homology search was performed across the tree of life for two additional iterations until no additional sirtuin homologs were detected, leading to a set of n=46,623 detected sirtuin homologs satisfying the inclusion criteria (e-value < 1e-5, bit score > 80, query coverage > 60% and 200 < protein length < 2500 amino acids, (Table S2).

## Annotation of defence systems in bacterial genomes

To assess which SIR2 family proteins are involved in known antiphage systems, we extracted the genomic neighbourhood (twenty genes upstream, twenty genes downstream) surrounding each sirtuin and sirim identified in the refined detection and ran DefenseFinder v 1.2.3 with default parameters to identify which defence systems contains a sirtuin or a sirim (Fig 1a) (21). We also identified whether the genomic neighbourhood of bacterial sirtuins and sirims is enriched in defence systems as antiphage defence systems tend to cluster together in bacterial genomes (23, 24). We measured whether at least one defence system occurs (Fig S1a) and as well as how many defence systems (Fig S1b) can be found in the genomic neighbourhood of sirtuins and sirims (20 genes upstream and downstream). The difference in the frequency of neighbouring defence systems between sirims and sirtuins was assessed using a two-sided Fisher exact test (Fig S1a) and the difference in the number of neighbouring defence systems was assessed using a two-sided Mann-Whitney test (Fig S1b), as implemented in the scipy python package (v1.8.0) (60).

## Phylogenetic inference

We first built a phylogenetic tree for the whole SIR2 family (including both SIR2 and SIRim subfamily proteins) using the proteins identified in the refined detection (Fig 1a). All identified sirtuins (n=46,623) and sirims (n=8,697) were clustered differentially in order to increase the representation of eukaryotic and archaeal proteins compared to their bacterial counterparts (59). Precisely, sirtuins were at 50% identity-80% coverage, bacterial sirims were clustered at 50% identity-80% coverage whereas archaeal and eukaryotic sirims were clustered at 90% identity-90% coverage. A total of 2,723 representative sequences (n=947 sirtuins and n=1,776 sirims) were aligned on their SIR2 domain using Muscle v5.1 (option -super5 -permut none), the alignment was trimmed using Clipkit v1.3.0 (options -m gappy -g 0.99) and was used to build a maximum likelihood phylogenetic tree using IQTree (55, 57, 61). The Q.pfam+G4 amino acid
substitution model was selected using ModelFinder and the Bayesian Information Criterion (62).
Maximum likelihood tree search was performed until convergence and branch support was
assessed using 1,000 UltraFast Bootstrap trees (63).

We then built phylogenetic trees of all sirims identified in our database of genomes (Fig 4a, Fig S7 & S8). Prior to building a phylogenetic tree, in order to increase the representation of undersampled eukaryotic clades (e.g. rhizaria or alveolata), representative sequences of all identified sirims in the refined detection (n=8,687) were used to build a profile HMM that was queried against the Eukprot v3 database using hmmsearch (39). Hit satisfying the inclusion criteria (e-value < 1e-5, bit score > 55, query coverage > 60% and 100 < protein length < 2500 amino acids, n=482 taking diverse isoforms of the same protein into account) among 205 genomes (over a total of 985 in the Eukprot v3 database) were further filtered to remove putative bacterial contaminations. To do so, all n=482 Eukprot v3 hits were used as queries with Diamond v1.2.6 (option --fast) against the NCBI nr database of proteins (64). Eukprot v3 proteins having more than sequence 70% identity with any bacterial protein in nr were filtered out. This led to a curated set of n=443 sirims detected in n=189 different genomes from the Eukprot v3 database (Table S4). These curated hits were included in the set of all sirims used to build the tree. All sirims were clustered differentially depending on their domain of life using MMseqs2 easy-cluster : bacterial sirims were clustered at 50% identity-80% coverage whereas eukaryotic and archaeal sirims were clustered at 90% identity-90% coverage in order to increase their abundance in the tree compared to their bacterial counterparts (57). The set of representative sequences was manually cured by removing proteins lacking key amino acids in the GxG motif and in the xxNxD motif and gap-inducing proteins at these loci. Sirims found in the *Homo sapiens* genome were explicitly added to the set of representative sequences. This step led to a total number of n=2,101 cured representative sequences of sirims. We also included a set of sirtuins from across the three domains of life to the tree of sirims to serve as an outgroup. To do so, n=165 sequences were sampled from the set of representative deacetylating sirtuin sequences (clustered at 50% identity/80% coverage using MMseqs2 easy-cluster) were sampled from all three of domains of life and added to set of proteins included in the tree of sirims (57). The obtained representative sirtuins and sirims (n=2,266) were then aligned on their SIR2/SIRim domain using Muscle v5.1 (option -super5), the MSA was trimmed using clipkit (option gappy, 99%) and a maximum likelihood phylogenetic tree was inferred using IQTree v3 (58). The Q.pfam+G4 substitution model was selected via ModelFinder using the Bayesian Information Criterion (BIC), and the tree was optimised until convergence and branch support was assessed using 1,000 UltraFast Bootstraps
(Fig 4a) (62, 63). To assess if the topology of the phylogenetic tree of sirims is to perturbations of the input MSA, additional SIR2/SIRim domain MSA were generated using (i) MAFFT v7.505 L-INS-i algorithm (option --localpair) and (ii) Muscle v5.1 by permuting the guide tree when building the alignment (option -super5 -perm all). This procedure yielded five additional MSA of sirims which were trimmed using clipkit (-m gappy -g 0.99) and were used to build five phylogenetic trees with IQTree (same procedure as before) (55, 58, 61–63, 65) (Fig S7). Another phylogenetic tree containing only eukaryotic sirims was built (Fig S8). All detected eukaryotic sirims were clustered at 90% identity and 90% coverage using MMseqs2 leading to a set of *n*=485 representative sequences which were aligned using Muscle v5.1 (option -super5) together with *n*=165 sirtuins from across the tree of life serving as an outgroup to root the tree. The alignment was trimmed using Clipkit (-m gappy -g 0.995) and the tree was built using the Q.pfam+G4 substitution model and maximum likelihood search was performed until convergence (55, 58, 59). Tree branch support was assessed using 1,000 generated UltraFast Bootstrap.

In all the phylogenetic trees of sirims, we looked for clades of eukaryotic sirims (Fig 4a-4b & Fig S6-8). The criteria to consider that a eukaryotic clade is valid are the following (i) the clade of eukaryotic proteins is well supported, (ii) the clade contains sequences from at least two distinct eukaryotic species, (iii) the genomes in which sirims are detected were produced in at least two distinct studies and (iv) the clades are stable to perturbations in the phylogenetic analysis (66). In the case where a clade of bacterial proteins forms a well-supported clade with the eukaryotic proteins and is basal to it, we consider that a putative Horizontal Gene Transfer (HGT) event from bacteria to eukaryotes is likely to have occurred (66).

To compute the taxonomic distribution of each lineage of sirims in eukaryotes, we analysed the set of sirims detected in the Eukprot v3 database (genomes from *n*=985 species). As no phylogenetic tree was available for the full Eukprot v3 species, we plotted the distribution on the phylogenetic tree of The Comparative Set (TCS) from Eukprot (genomes from *n*=196 species) (39). For each species in Eukprot v3, either the species is also present in TCS, or it is not. In the latter case, the species is assigned to its closest relative in Eukprot TCS using TaxonKit (67). For each species in TCS, we consider that sirims are detected if (i) sirims are detected in at least two Eukprot v3 different species represented by this species or (ii) the clade represented by the TCS species contains only one Eukprot v3 species and sirims are detected within it (Fig 4b & Fig S6). Divergence time between eukaryotic taxa was taken from the TimeTree 5 (http://www.timetree.org/) (68).

Domain annotation and folding of eukaryotic sirims
We sought to annotate the protein domains of all detected eukaryotic sirims (Clades A to E, Fig 4c). Domain annotation was performed using the HHpred server (default parameters) against Pfam-A v36 and the Protein DataBank (PDB) mmCIF70 (69). Hits satisfying inclusion criteria (probability > 0.90) were considered valid. Proteins were also folded using AlphaFold2/ColabFold as implemented in the localcolabfold package v1.5.2 (https://github.com/YoshitakaMo/localcolabfold, single prediction using 12 optimization recycles) (70, 71).

## RAW264.7 cells culture and agonist stimulation

RAW264.7 macrophage-like cells (initially purchased from ATTC) were kindly provided by Dr. Catherine Werts (Institut Pasteur, Paris, France). KO cell lines were generated using transduction with Lenti-CRIPSR-v2 containing sgRNA targeting *mSI/Ral/fam118b* or non-targeting control guides (Fig S3b & Table S5). Transduction was followed by puromycin selection, single cell cloning, and knock-out phenotype was confirmed using both PCR showing genomic deletions, and western blot confirming the absence of protein expression (Fig S3c-d).

RAW264.7 cells (control and Δ*mSI/Ral/fam118b*) were cultivated in complete RPMI medium (Gibco) (10% FCS + Penicillin-Streptomycin) and were plated at 0.5 x 10⁶ cells/mL in 200µL/96-well plate or 1mL/24-well plate (TPP) the day before stimulation. Stimulation with TLRs agonists was performed the day after plating. Cells were stimulated for 2 hours, 8 hours or 24 hours with LPS of *Escherichia coli* strain O111:B4 (LPS-EB Ultrapure, Invivogen), synthetic triacylated lipopetide (Pam3CysSK4, Invivogen), dsRNA analog poly(I:C) (PIC-HMW, Invivogen), flagellin of *Salmonella typhimurium* (FLA-ST Ultrapure, Invivogen), resiquimod (R848, Invivogen) or short synthetic single-stranded DNA (CpG ODN 1018, Invivogen). After stimulation, cells were frozen immediately for subsequent RNA extraction and supernatants were recovered for dosage of nitric oxide.

## Primary mouse bone-marrow derived macrophages (BMDMs)

Bone-marrow cells were obtained after cervical dislocation of female C57BL/6J mice (7-20 weeks old), extraction of tibias, femurs and iliac bones and bone marrow recovery by flushing with 22 G needle and syringe. Bone-marrow cells were differentiated during 7 days in complete RPMI medium (Gibco) (10% FCS + Penicillin-Streptomycin) supplemented with 10% L929 supernatant. After differentiation, bone-marrow derived macrophages (BMDMs) were scrapped in PBS and plated at 1 x 10⁶ cells/mL in 200µL/96-well plate or 1mL/24-well plate (TPP) the day before transfection.
965

siRNA transfections of BMDMs

siRNA transfections were performed on differentiated BMDMs using lipofectamine reagent (Lipofectamine-3000, ThermoFisher) in serum-free medium (OptiMEM, Gibco). Briefly, pools of siRNA targeting *mSIRal/fam118B* (#GS109229) (FlexiTube, Qiagen), or scrambled control siRNA (AllStar Negative Control, Qiagen) were incubated with lipofectamine in OptiMEM for 30 minutes at room temperature before drop-to-drop addition on the BMDMs at 100mM final concentration (4 x 25nM of each siRNA). After 6 hours incubation with siRNA in OptiMEM, equivalent volume of complete medium was added and cell were incubated overnight for recovery and stimulated with agonists as described above.

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975

Primary human monocyte-derived macrophages (huMDMs)

Plasmapheresis residues from healthy adult donors were obtained from the Établissement Français du Sang (Paris, France) in accordance with the Institut National de la Santé et de la Recherche Médicale (France) (INSERM) ethical guidelines. According to French Public Health Law (art L 1121-1-1, art L 1121-1-2), written consent and Institutional Review Board approval are not required for human non interventional studies.

980

Human peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors using Ficoll-Paque (GE Healthcare) and CD14⁺ monocytes were then positively selected using magnetic beads (Miltenyi). Differentiation of the cells into macrophages was induced in RPMI medium (Gibco) with 5% FCS + 5% human serum AB (Sigma), penicillin-streptomycin (Gibco) with 50 ng/mL human macrophage colony-stimulating factor (M-CSF, Miltenyi) for 7 days.

985

For cell transduction, 10 x 10⁶ monocytes were plated in 10-cm non-treated cell culture dishes and treated with 5 mL of freshly-collected lentiviral particles (see below), in the presence of 8 mg/mL of protamine sulphate (Sigma Aldrich). Selection of the transduced cells was performed with 2 µg/mL of puromycin 48 hours post-transduction. Seven days post differentiation and transduction, human monocyte-derived macrophages (MDMs) were collected, plated and stimulated with agonists as described above.

990

995

shRNA-lentiviral particles production for huMDMs

HEK293-LTV cells were maintained in DMEM supplemented with 10% FCS, 100 U/mL penicillin/streptomycin, and 1% non-essential amino acids (Gibco). Cells were routinely sub-cultured. For lentiviral production, 7 x 10⁶ cells were seeded in T75 cm² flasks and transfected the
next day. Transfection mixes consisted of 4.1 mg of lentiviral genome, 3 mg of the packaging plasmid PSPAX2 and 1.25 mg of VSV-G. DNA was complexed with 7.5 mM of polyethylenimine (PEI) for 20 min and added to cells. Sixteen hours later, culture media was renewed, and the lentiviral particles allowed to produce and accumulate for an additional 30 hours, after which they were collected, filtered on 0.45 µm, and immediately used to transduce monocytes. The lentiviral vectors employed were; pLKO.1-puro Non-Mammalian shRNA Control Plasmid DNA (SHC002, Sigma) and pLKO.1-Puro shRNA hSIRal/fam118b (TRCN0000130258, Sigma).

## Cells RNA extraction and RT-qPCR analysis of cytokines and ISGs

RNA extraction was performed on frozen cells from p24-well plates using NucleoSpin RNA (Machery-Nagel), according to the supplier's instructions. Reverse transcription was performed with SuperScript RT (Invitrogen). RT-qPCR was performed with Taqman Universal Master Mix (Applied Biosystems), according to the manufacturer's instructions (references of primers in Table S6). Fold change was calculated using the 2^(-ΔΔCt) method, with normalisation by internal control (GAPDH for mouse cells and HPRT for human cells) followed by normalisation by the non-stimulated condition.

## Nitric oxide dosage & cytokine quantification by ELISA

Nitric oxide (NO) production by macrophages was measured in fresh macrophages supernatants from p96-well plates by the Griess reaction (Griess reagent system, Promega), according to the supplier's recommendations. Cytokine quantifications were performed on frozen cell culture supernatants from p96-well plates, using DuoSet ELISA kits (R&D) according to the manufacturer's instructions.

## Propidium iodide integration and LDH release assay

RAW264.7 cells (control and ΔmSIRal/fam118b) were plated at 0.25 x 10⁶ cells/mL in 200µL/96-well plate the day before transfection. Transfection with LPS of *Escherichia coli* strain O111:B4 (LPS-EB, Invivogen) was performed using a transfection reagent (FuGENE, Promega) in OptiMEM (Gibco). Cells were then incubated overnight and LDH release in the supernatant was measured (CyQUANT cytotoxicity assay, Thermofisher) according to the manufacturer's instructions. In parallel, the integration of 1 µg/mL propidium iodide was measured by fluorescence on a plate reader (SpectraMax, MolecularDevices).
### HSV-1 and *Salmonella* infections of RAW264.7 cells

RAW264.7 cells (control and Δ*mSIRal* / fam118b) were cultivated in complete RPMI medium (Gibco) (10% FCS + Penicillin-Streptomycin) and were plated at 0.5 x 10⁶ cells/mL in 200µL/96-well plate or 1mL/24-well plate (TPP) the day before infection.

Viral infections were performed in 24-well plates, by addition of HSV-1 (GFP) in the cell culture medium at a multiplicity of infection (MOI) of 5, 7.5 or 10, with or without pre-incubation with 35 µM acyclovir. Cells were incubated for 24 hours, were detached using trypsin (TripLE, Gibco) for 5 minutes at 37°C, were then stained for 5min with live/dead fixable viability dye in PBS (eFluor 450nm, eBioscience) before washing and 4% PFA-fixation during 10min. Cells were then resuspended in cytometry buffer (PBS, 3% FCS, 5mM EDTA, 0.02% sodium azide) and analysed by flow cytometry (CytoFLEX, Beckman Coulter). Analyses were performed on live cells (eFluor<sup>negative</sup>) and both the percentage of GFP<sup>positive</sup> (high and intermediate) cells and the geometric mean fluorescence intensity (gMFI) of the respective populations were analysed (FlowJo v10, BD).

*Salmonella enterica* serovar Typhimurium strain SL1344 used for bacterial infections was kindly provided by Dr. Aline Rifflet (Institut Pasteur, Paris, France). Bacteria were grown overnight in liquid antibiotic-free LB medium for a maximum of 12 hours. Stationary bacterial culture was centrifuged at 4000 rpm for 10 minutes and resuspended in PBS before OD₆₀₀ measurement. Bacterial concentration was calculated based on standard curve with 1 OD₆₀₀ = 8 x 10⁸ CFUs/mL. Infections of RAW264.7 cells were performed in 96-well plates by addition of bacteria at MOI 10 in antibiotic-free cell culture medium with or without 30 minutes pre-incubation with 1 µg/mL of LPS from *Rhodobacter sphaeroides* (LPS-RS Ultrapure, Invivogen). After 30 minutes of infection, the cells were washed gently 3 times with PBS and incubated for 30 minutes in medium with 50 µg/mL gentamicin to kill all the extracellular bacteria. After gentamicin protection assay, cells were washed again 3 times with PBS and incubated in medium with 10 µg/mL gentamicin to prevent any extracellular replication of the residual bacteria. At the indicated time points (1-9 hours), media was removed and RAW264.7 cells were lysed with 0.2% sodium deoxycholate in PBS for 30 seconds. Intracellular bacteria were then enumerated by colony-forming unit (CFU) after plating on antibiotic-free LB-agar plates and overnight incubation at 37°C.

### Plasmids

Plasmids were ordered from GenScript: pcDNA3.1 backbone (neomycin resistance) with either human hSIRal / FAM118B, with a N-term FLAG-tag. Alanine substitution of hSIRal / FAM118B (N163A & H206A) was performed by site directed mutagenesis (Quick change XL, Agilent),
according to the manufacturer's instructions. All Minipreps and Midipreps were performed using endotoxin-free kits (Macherey-Nagel).

TLR4-NF-κB reporter systems

HEK293T-Blue-Null2 and HEK293T-Blue-hTLR4 (initially purchased from Invivogen) were kindly provided by Dr. Catherine Werts (Institut Pasteur, Paris, France). NF-κB reporter HEK293T-Blue-Null2 and HEK293T-Blue-hTLR4 cells (stably transfected with human TLR4, MD2 and CD14) were cultivated in complete DMEM medium (Gibco) (10% FCS + Penicillin-Streptomycin). hSIRal / FAM118B overexpression was achieved by transfection of both cell lines with 10 ng/well pcDNA3.1-hSIRal / FAM118B (or catalytic mutant) in 12-wells plates (TPP), using lipofectamine (Lipofectamine-3000, ThermoFisher) in serum-free medium (OptiMEM, Gibco). Transfection was performed for 48 hours, and cells were then collected by gentle flushing in PBS, enumerated and resuspended in commercial detection medium (HEK-Blue Detection, Invivogen), containing the substrate of the NF-κB-reporter secreted embryonic alkaline phosphatase (SEAP). LPS stimulation was performed by incubating 50 000 transfected cells/well with 0.1-100 ng/mL LPS of *Escherichia coli* strain O111:B4 (LPS-EB, Invivogen) overnight in detection media in 96-wells plate (TPP). Activity of the NF-κB reporter SEAP was measured by colorimetry at OD<sub>655</sub> nm.

Recombinant protein expression and purification

A synthetic gene construct (Integrated DNA Technologies) for hSIRal / FAM118B was cloned into a custom pET16 vector with an N-terminal 6xHis-hSUMO2 fusion tag, codon optimized for recombinant expression in *E. coli*, using Gibson assembly. N163A, H206A, and N163A/H206A double mutant hSIRal / FAM118B were all generated using 'round the horn mutagenesis and the wild-type sequence as template. Sequencing results for plasmid preparations were confirmed before transformation into BL21-CodonPlus(DE3)-RIL *E. coli* (Agilent) for protein expression and subsequent purification as described below.

Transformant *E. coli* were plated on MDG-agar plates (0.5% glucose, 25 mM Na₂HPO₄, 25 mM KH₂PO₄, 50 mM NH₄Cl, 5 mM Na₂SO₄, 2 mM MgSO₄, 0.25% aspartic acid, 100 mg/mL ampicillin, 34 mg/mL chloramphenicol, and trace metals) and grown overnight at 37 °C. Single colonies were then inoculated into liquid MDG media and grown overnight at 37 °C, 230 rpm shaking. Overnight MDG cultures were used as inoculum for 1 L M9ZB minimal media liquid cultures (0.5% glycerol, 1% Cas-amino Acids, 47.8 mM Na₂HPO₄, 22 mM KH₂PO₄, 18.7 mM NH₄Cl, 85.6 mM NaCl, 2 mM MgSO₄, 100 mg/mL ampicillin, 34 mg/mL chloramphenicol, and trace metals). M9ZB cultures were grown at 37 °C, 230 rpm until OD600nm >2.5, rested on ice
1100 for 20 minutes and then induced with 0.5 mM IPTG and set to shake at 230 rpm overnight at 16 °C. Cells were pelleted by centrifugation, washed with PBS, and flash frozen in liquid N₂ for storage at -80 °C.

Frozen cell pellets were resuspended in lysis buffer (20 mM HEPES-KOH pH 7.5 measured at room temperature, 400 mM NaCl, 10% glycerol, 30 mM imidazole, 1 mM DTT) and subjected to sonication to extract soluble protein. Cell lysates were clarified with centrifugation and purified using conventional gravity-flow Ni-NTA affinity chromatography. Briefly, lysate was loaded on Ni-NTA resin (Qiagen) equilibrated with lysis buffer and the flow-through was added back over the column once to increase chances of binding. Columns were then washed with lysis buffer supplemented to 1M NaCl, washed with lysis buffer, and protein was ultimately eluted in lysis buffer supplemented to 300 mM imidazole. Eluted protein was dialyzed overnight (20 mM HEPES-KOH pH 7.5, 250 mM KCl, 5% glycerol, 1 mM DTT) in the presence of human SENP2 protease (D364-L589, M497A) to remove the N-terminal 6xHis-SUMO2 solubility tag. Protein was concentrated post-dialysis using 30 kDa cutoff Amicon centrifuge filters and loaded onto a Superose-6 16/600 (Cytiva) gel filtration column (equilibrated in 20 mM HEPES-KOH pH 7.5, 250 mM KCl, 1 mM TCEP) connected to an NGC FPLC (Bio-Rad). Fractions were verified for protein content and purity via SDS-PAGE analysis and pooled fractions were further concentrated >10 mg/mL and flash frozen with liquid N₂ for long-term storage at -80 °C.

## Fluorescence plate reader analysis of NADase enzymatic activity

50 µL reactions were assembled in PCR tubes before transfer to white-walled clear bottom 96-well plates. Reactions included hSIRal / FAM118B at 15 µM, nicotinamide 1,N(6)-ethenoadenine dinucleotide (ε-NAD) at 500 µM, 20 mM HEPES-KOH pH 7.5 at room temperature, 100 mM NaCl, and -/+ 10% PEG 400. All components were thawed on ice and reactions were incubated for 15 minutes at 37 °C prior to addition of fluorescent NAD⁺ analog and transferred to a Synergy H1 plate reader (BioTek). Fluorescence recordings at 300 nm excitation/410 nm emission were measured in kinetic mode every minute for 1 hour.

## HPLC analysis of SIRal & sirims enzymatic activities

Reactions included an individual sirim protein ranging from 3 µM to 20 µM, nicotinamide adenine dinucleotide (NAD⁺) at 500 uM, 20mM ammonium acetate pH 8.0 at room temperature, and +/- 10% PEG 400. All components were thawed on ice. 100 uL reactions were assembled on ice and incubated 16 hours at 37°C. Samples were centrifuged at 13000 x g for 5 minutes at 20°C, and supernatants applied to 30 kDa cutoff Amicon centrifuge filters. Filtered flowthrough as well as
adenine diphosphate ribose (ADPR) and nicotinamide (NAM) synthetic standards were then
injected on the HPLC (Agilent 1200 Series) equipped with a 4.6 x × 150 mm and 5 µm particle-
size Zorbax Bonus-RP C18 column, with absorbance at 254 nm collected over time. The HPLC
program method used 0.1% (w/v) ammonium acetate running buffer with a methanol elution
gradient of 0-100% over 10 mL, 100% methanol for 2 mL, followed by reequilibration in the running
buffer. Elution times and peak integration corresponding to NAD⁺ were compared to standards to
determine molecule identity and NAD⁺ depletion.

Zebrafish Housing and Husbandry

Zebrafish were maintained at a density of five adults per liter under controlled conditions, following
European guidelines for zebrafish housing and care. The environmental parameters were as
follows: photoperiod of 14 h light / 10 h dark, temperature of 26.5 ± 1 °C, pH of 7.8 ± 0.1,
conductivity of 240 ± 30 µS/cm, and undetectable levels of NH₄⁺ and NO₂⁻, with NO₃⁻
concentrations below 50 mg/L.

Embryos were incubated at 28 °C until 5 days post-fertilization (dpf). During the first two
weeks of feeding, larvae were provided with rotifers (Brachionus plicatilis, > 500 per fish per day),
followed by dry food (Skretting, Gemma Micro) to apparent satiation, twice daily.

The AB zebrafish line used in this study was obtained from the European Zebrafish Resource
Center (Germany). All procedures were conducted in accordance with European Union Directive
2011/63/EU and were approved by the local ethics committee (approval no. 59 CEEA).

**Generation of SIRim crispants zebrafish**

gRNA Selection and Preparation

Three guide RNAs (gRNAs) were designed to target the third exon of the SIRim gene for the generation of crispant larvae, following a protocol adapted from Shah et al. (2015). Initially, seven candidate CRISPR RNAs (crRNAs) were selected using CRISPOR (http://crispor.tefor.net/) against the Danio rerio UCSC May 2017 (GRCz11/danRer11) reference genome, using the 20bp-NGG - SpCas9, SpCas9-HF1, and eSpCas9 1.1 enzyme. Selection criteria included target locus proximity, specificity score, minimal off-target effects, and the absence of sequence motifs that could impair cleavage efficiency or specificity.

To generate gRNAs, 100 µM of crRNA was annealed with 100 µM of tracrRNA (Integrated
DNA Technologies, IDT) by heating at 95°C for 5 minutes, followed by gradual cooling to 20°C.
The gRNAs were then diluted in IDT duplex buffer to a final concentration of 20 µM.
## RNP Complex Formation and Injection

Ribonucleoprotein (RNP) complexes were assembled using a mutated SpCas9 protein (R221K-N394K), generously provided by Jean-Paul Concordet (TACGene, MNHN). Each gRNA was combined with Cas9 at final concentrations of 13.3 µM and 10 µM, respectively. Individual gRNA-Cas9 complexes were injected separately into one-cell-stage zebrafish embryos to evaluate in vivo cleavage efficiency.

1170

To assess editing efficiency, pools of 10 larvae at 5 days post-fertilization (dpf) per gRNA injection were sequenced using Sanger sequencing (Eurofins Genomics, https://eurofinsgenomics.eu/). Sequence data were analyzed with the ICE online software (Synthego, https://ice.editco.bio/#/) to determine cleavage efficiency and predicted frameshift induction. Three gRNAs demonstrating >95% cutting efficiency and inducing 50-90% frameshift mutations through non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ) were selected for crispant generation.

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1180

## Crispant Generation

For the crispant experiments, each gRNA was assembled into RNP complexes separately to prevent preferential hybridization. The three RNP complexes were subsequently pooled at equimolar concentrations to yield a final concentration of 4.4 µM per gRNA. A total of 2 nL of the final RNP mix was injected into one-cell-stage embryos.

1185

Control injections were performed using a mixture of the three negative control crRNAs (Alt-R™ Cas9 Negative Control crRNA #1, 2, and 3; IDT), following the same assembly and injection protocol as the experimental groups.

1190

## crRNA Sequences

The crRNA sequences targeting SIRim were obtained from IDT (SIRim crRNA 1: TCTGCAAGAGGACAAGAACC, SIRim crRNA 2: GACCAAGCGAGCTCCTGAGC, SIRim crRNA 3: CGCTGGCACCTGAGGAGCTA). This experimental setup ensured robust gene disruption for subsequent phenotypic analyses of SIRim crispants.

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## *Edwarsiella tarda* infection of wild type and ΔzSIRal zebrafish larva

*Edwarsiella tarda* FL60, provided by Dr. Phillip Klesius (USDA, Agricultural Research Service, Aquatic Animal Health Research Unit), were grown in LB medium + 15 µg/mL tetracycline at 28 °C overnight. A 1:100 dilution was performed, and the bacteria were grown to reach OD<sub>600</sub> = 0.250.

1200
The bacteria were then centrifuged at 4000 rcf for 10 minutes and resuspended in E3 water to achieve OD₆₀₀ = 0.250. 40 mL of bacterial suspension in E3 were added to 40 larvae, 4 days post-fertilization (dpf), for 5 hours at 28 °C. Larvae were washed three times with E3 water. 3 days post-infection (dpi), infected larvae were used for qPCR.

## Larva RNA extraction and RT-qPCR analysis of ILs and ISGs

Single larvae were used for RNA extraction. RNA was extracted using the Single Cell RNA Purification Kit (Norgen, #51800) following the manufacturer's instructions. Synthesis of cDNA was performed using the M-MLV Reverse Transcriptase Kit (Invitrogen, #28025013). qPCR was carried out using the Takyon™ Kit (Takyon, #UF-LPMT-C0701) on QuantStudio™ 5 System (sequences of primers in Table S7). Fold change was calculated using the 2⁻⁰⁻⁰ Ct method, with normalisation by internal control (2f1a) followed by normalisation by the wild type non-infected condition.
# Supplementary Material

## List of supplementary figures

**Figure S1.**

The genomic context around bacterial proteins from the SIRim subfamily is significantly enriched in known antiphage systems compared with the genomic context nearby bacterial SIR2 subfamily proteins.

**Figure S2.**

Multiple sequence alignment of twenty SIR2 family proteins sampled from bacteria and eukaryotes.

**Figure S3.**

Characterization of the role of SIRal in the TLR pathway in macrophages.

**Figure S4.**

SIRal participates in TLR-driven control of infection.

**Figure S5.**

Distribution of the five major eukaryotic SIRim clades identified in eukaryotes.

**Figure S6.**

The five major clades of eukaryotic sirims are stably identified regardless of the sequence alignment method.

**Figure S7.**

Phylogenetic tree of the eukaryotic SIRim.

**Figure S8.**

Proposed evolutionary scenario explaining the appearance of the SIRal lineage through horizontal gene transfer from bacteria to eukaryotes.

**Figure S9.**

Characterization of the NADase activities of SIRal and various sirims.
Supplementary Figures

**Figure S1.** The genomic context around bacterial proteins from the SIRim subfamily is significantly enriched in known antiphage systems compared to the genomic context nearby bacterial SIR2 subfamily proteins.

a. Number of bacterial proteins which have at least one defense system in their genomic neighborhood (20 genes upstream and 20 genes downstream). Proteins are grouped according to their SIR2 subfamily: SIR2 (beige) and SIRim (brown). Two sided Fisher exact test is used to assess statistical significance of the difference between the two distributions (***: *p*<10<sup>-3</sup>). b. Distribution of the number of defense genes in the genomic context of bacterial SIR2 family proteins per subfamily: SIR2 (beige) and SIRim (brown). The x-axis ("Number of defense genes nearby") is duplicated across the two subfamilies for visualization purposes, but the y-axis ("Percentage of proteins within each subfamily") is shared. Two-sided Mann-Whitney test is used to assess statistical significance of the difference between the two distributions (***: *p*<10<sup>-3</sup>).

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>
**Figure S2. Multiple sequence alignment of twenty SIR2 family proteins sampled from bacteria and eukaryotes.**

Sequences are grouped by subfamily (SIR2: beige box, SIRim: brown box). Conserved residues are indicated by an arrow. **a.** Selected regions of the multiple sequence alignment which exhibit strong conservation both within subfamilies and among subfamilies. All the conserved motifs (G.G, ..N.D and .HG) are located within the NAD+ binding site of the proteins (1-4). **b.** Selected regions of the multiple sequence alignment which exhibit strong conservation within one of the two subfamilies only. The four cysteine (C) residues conserved within the SIR2 subfamily (beige) form a tetrad which coordinates a Zn²⁺ ion and forms the binding site of deacetylated amino acid residue (Fig 1b) (1). This tetrad is absent from all SIRim proteins (brown).

a

<figure><img src="image_10.png" /></figure>

b

<figure><img src="image_11.png" /></figure>

**Conserved residues**

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>
<caption>Figure S3. Characterization of the role of SIRal in the TLR pathway in macrophages.</caption>

a. Examples of *SIRal* genomic loci in animal genomes (metazoa). *SIRal* (pink) colocalizes with *TIRAP* (blue) in 73% of metazoa. In *Xenopus laevis*, the African clawed frog, *TIRAP and SIRal* are located on the same chromosome, but encoded at different loci. b. Heat maps of *SIRal* RNA levels (number of transcripts per million, nTPM) in immune cells and enrichment ratio of *SIRal* RNA in macrophages of various tissues (Genotype-Tissue Expression, GTEx, Broad Institute). c. Knock-out strategy for *mSIRal* in mouse RAW264.7 cells. d. PCR on genomic DNA of wild type or *ΔmSIRal* (two clones) RAW264.7 cells (left). Western blot & stain free (WB & SF) of wild type or *ΔmSIRal* (two clones) RAW264.7 cells (right). e. Wild type or *ΔmSIRal* (two clones) RAW264.7 cells were stimulated with Pam3CysSK4 (500ng/mL), Poly(I:C) (1µg/mL), LPS from *E. coli* (100ng/mL), R848 (1µg/mL) or CpG (1µg/mL), agonists of TLR1/2, TLR3, TLR4, TLR7/8 and TLR9, respectively. Production of nitric oxide (NO) was measured at 24h. f. Wild type or *ΔmSIRal* (two clones) RAW264.7 cells were stimulated with LPS from *E. coli* (100ng/mL). Viability was measured by Alamar Blue assay at 24h. g. Wild type or *ΔmSIRal* (two clones) RAW264.7 cells were transfected with LPS from *E. coli* (1-10µg/mL). Release of lactate dehydrogenase (LDH) and propidium iodide (PI) integration were measured at 24h. h. Levels of *GAPDH* and *TLR* transcripts in wild type or *ΔmSIRal* RAW264.7 cells measured by RT-qPCR. i. Experimental setup using mouse bone-marrow derived macrophages (BMDMs) and human monocyte-derived macrophages (huMDMs) to probe the activation of the TLR pathway. j. Levels of *mSIRal* mRNA measured by RT-qPCR in mouse BMDMs transfected with a control siRNA, or a siRNA targeting *mSIRal*. k. Levels of *hSIRal* mRNA measured by RT-qPCR in huMDMs transduced with a control shRNA, or a shRNA targeting *hSIRal* and stimulated with the indicated TLR agonists. l. Wild type or *ΔmSIRal* RAW264.7 cells were stimulated with Pam3CysSK4 (500ng/mL), Poly(I:C) (1µg/mL), LPS from *E. coli* (100ng/mL), R848 (1µg/mL) or CpG (1µg/mL), agonists of TLR1/2, TLR3, TLR4, TLR7/8 and TLR9, respectively. Production of TNF and NO was measured at 24h. m. huMDMs transduced with a control shRNA, or a shRNA targeting *hSIRal*, were stimulated with LPS (100ng/mL), flagelin (500ng/mL) and R848 (1µg/mL), agonists of TLR4, TLR5 and TLR7/8, respectively. Levels of *Mx1* and *RsaD2* transcripts were measured RT-qPCR at 8h. n-o. Wild type or *ΔmSIRal* RAW264.7 cells were stimulated with LPS from *E. coli* (100ng/mL). Both total and phosphorylated p65 (NFκB) and IRF3 were monitored by Western blots (n) at 30min and 3h and quantification of the (phosphorylated/total) ratio was performed (o).

Each dot represents the mean of technical replicates for each independent experiment (e,f,g,h,i,o), independent mouse BMDMs isolation (j) or independent human donor (k,m). Bars correspond to the mean and error bars to the standard deviation of all the independent experiments/mouse/donors. Statistical analyses were performed using two-ways ANOVA and p-values were classified as * p<0.05; ** p<0.01; *** p<0.001. Grey stars correspond to comparisons within one group and dark stars correspond to comparisons between groups.
<figure><img src="image_14.png" /><figcaption>a</figcaption></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /><figcaption>b</figcaption></figure>

SIRal RNA (nTPM)
in immune cells

<figure><img src="image_23.png" /></figure>

Enrichment of SIRal
RNA in macrophages

<figure><img src="image_24.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_25.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_26.png" /><figcaption>e</figcaption></figure>

<figure><img src="image_27.png" /><figcaption>f</figcaption></figure>

<figure><img src="image_28.png" /><figcaption>g</figcaption></figure>

<figure><img src="image_29.png" /><figcaption>h
PI integration</figcaption></figure>

<figure><img src="image_30.png" /><figcaption>h</figcaption></figure>

<figure><img src="image_31.png" /><figcaption>i</figcaption></figure>

<figure><img src="image_32.png" /><figcaption>j</figcaption></figure>

<figure><img src="image_33.png" /><figcaption>k</figcaption></figure>

<figure><img src="image_34.png" /><figcaption>l</figcaption></figure>

<figure><img src="image_35.png" /><figcaption>m</figcaption></figure>

<figure><img src="image_36.png" /><figcaption>n</figcaption></figure>

<figure><img src="image_37.png" /><figcaption>o</figcaption></figure>

<figure><img src="image_38.png" /></figure>
<caption>**Figure S4. SIRal participates in TLR-driven control of infection.**</caption>

a. Wild type or *ΔmSIRal* RAW264.7 cells were infected or not with HSV-1 (MOI 5-10). Viability was measured by flow cytometry at 24h. b. Wild type or *ΔmSIRal* RAW264.7 cells were infected or not with HSV-1 (MOI 5-10), in the presence or absence of acyclovir. Levels of GFP were measured by flow cytometry at 24h and the percentage of entry (GFP intermediate) was determined. c. Wild type or *ΔmSIRal* RAW264.7 cells were infected or not with HSV-1 (MOI 10). Viral loads in the supernatant were quantified by plaque assay at 24h. d. Wild type or *ΔmSIRal* RAW264.7 cells were infected with *Salmonella enterica* (MOI 10). Levels of *TNF* and *RsaD2* transcripts were measured by RT-qPCR at 9h. e. HEK293T-hTLR4 NF-κB reporter cells were stimulated with PFA-fixed *Salmonella enterica*, in the presence or absence of LPS from *Rhodobacter sphaeroides* (1µg/mL), used as a TLR4 antagonist. NF-κB activation was measured at 24h.

Each dot represents the mean of technical replicates for each independent experiment (a,b,d). Bars correspond to the mean and error bars to the standard deviation of all the independent experiments. Statistical analyses were performed using two-ways ANOVA and p-values were classified as * p<0.05; ** p<0.01; *** p<0.001. Grey stars correspond to comparisons within one group and dark stars correspond to comparisons between groups.

<figure><img src="image_39.png" /></figure>
<caption>**Figure S5.** Distribution of the five major eukaryotic SIRim clades identified in eukaryotes.</caption>

The phylogenetic tree from The Comparative Set (TCS) of Eukprot (196 species) is used to draw the taxonomic distribution of the SIRim proteins identified in the Eukprot v3 database of eukaryotic genomes (985 species) (5). SIRim proteins found in species present in the Eukprot v3 database (985 species) but not in the TCS (196 species) are assigned to their closest relative present in the TCS tree. We consider that a sirim protein is detected in the taxon represented by a given leaf of the tree if (i) sirims are detected in at least two distinct species represented by this leaf or (ii) the leaf represents a single eukaryotic species in Eukprot v3 and sirims are detected within this species. Eukaryotic sirims are grouped into five distinct clades based on our phylogenetic analysis (Fig 4a, Fig S6 & S7). The tree is midpoint rooted. A pruned version of this tree can be found in Fig 4a.

<figure><img src="image_40.png" /></figure>
**Figure S6.** The five major clades of eukaryotic sirims are stably identified regardless of the sequence alignment method.

The same representative sequences of bacterial, archaeal and eukaryotic sirims are used as in Fig 4a. Representative sequences are aligned on their SIRim domain using either Muscle super5 (first four trees) or MAFFT (last tree, bottom). The Multiple Sequence Alignment (MSA) generated using Muscle are also built by permuting the guide tree (permutations: "none", "abc", "acb", "bca"). The domain of life from which each protein originates (inner ring, blue, gray, green), the taxon for eukaryotic sirims (middle ring) and the corresponding antiphage system type for bacterial sirims (outer ring) are also indicated on each tree. One phylogenetic tree is built from each MSA using IQTree. This procedure allows us to test the stability of the identified eukaryotic clades of sirims (clades A to E) to perturbations of the MSA used to build the phylogenetic tree. We observed that the five clades of eukaryotic sirims defined from the phylogenetic tree in Fig 4a are stably formed in all the obtained trees, indicating that they are significant. We also observe that the clades of eukaryotic sirims tend to associate with the same clades of bacterial antiphage sirims in all the trees.
Tree scale 1:

<figure><img src="image_41.png" /></figure>

Tree scale 1:

<figure><img src="image_42.png" /></figure>

Tree scale 1:

<figure><img src="image_43.png" /></figure>

Tree scale 1:

<figure><img src="image_44.png" /></figure>

Tree scale 1:

<figure><img src="image_45.png" /></figure>

SIRim Clade (Eukaryote)

Clade A (SIRal)

Clade B

Clade C

Clade D

Clade E

Support

≤80

90

100

Taxon (Eukaryote)

Alveolata

Stramenopiles

Rhizaria

Euglenozoa

Haptophyta

Chloroplastida

Centroplasthelida

Amoebozoa

Opisthokonta

Fungi

Eumetazoa

Other

Domain of life

Bacteria

Archaea

Eukaryote

Antiphage system (Bacteria)

pAgo

Lamassu

Dsr 1

AbiH

Nezha

PD-T7-2

Thoeris

Dsr 2

Avs

Other
Figure S7. Phylogenetic tree of the eukaryotic SIRim.

The tree only includes SIRim proteins from eukaryotic organisms and is rooted using the SIR2 subfamily of proteins (sirtuins) from across the tree of life as an outgroup. The five clades of eukaryotic sirims defined from the tree in Fig 4a are represented (inner ring) and are also retrieved in this tree with good support. Eukaryotic sirim clades left in white branch are unstable when rebuilding the trees, so are left apart from the classification of eukaryotic sirims. The domain of life (middle ring) and the eukaryotic taxon (outer ring) are also indicated. Shades of green on branches indicate UltraFast Bootstrap support values. Support values are explicitly provided for key branches defining the major eukaryotic sirim clades.

<figure><img src="image_46.png" /></figure>
<caption>**Figure S8.** Proposed evolutionary scenario explaining the appearance of the SIRal lineage through horizontal gene transfer from bacteria to eukaryotes and its subsequent diversification.</caption>

The evolutionary scenario presented above exemplifies four major eukaryotic taxa: amniota, teleostei (bony fishes), insecta and fungi, even though SIRal lineage proteins are found in other taxa. For each taxon, example organisms are provided at the bottom of the figure. The depicted time scale is arbitrary.

In bacteria, the set of extant SIRim proteins present today originates from a pool of ancient SIRim proteins. In eukaryotes, homology detection and phylogenetic trees reveal several independent lineages of SIRim proteins, including the SIRal lineage (Fig 4a, Fig S5-7, Clade A). This lineage is present in diverse eukaryotes including fungi and animals (opisthokonta), suggesting that it was present in eukaryotes prior to the last common ancestor of opisthokonta more than 1,400 million years ago (Fig 4a, Fig S5-7, Clade A). Based on the trees, the ancestor to all SIRal lineage proteins was probably acquired from an ancient bacterial sirim through horizontal gene transfer, and then diversified with the appearance of the diverse eukaryotic taxa. In some taxa of opisthokonts, the gene was lost (e.g. in insecta). In the rest of the taxa, the gene was maintained and can be found in extant biological organisms. In the genome of some deuterostomia (a taxon that notably contains mammals and birds), two copies of the gene can be found (*SIRal*, originally named *FAM118B*, and *SIRal2*, named *FAM118A*). This suggests that the *SIRal* gene underwent a duplication event that led to two paralogs.

<figure><img src="image_47.png" /></figure>
<caption>**Figure S9. Characterization of the NADase activities of SIRal and various SIRims.**</caption>

a. Wild type or ΔzSIRal zebrafish larvae were infected with *Edwardsiella tarda* and survival was monitored up to 3 days post-infection. b. SDS-PAGE of recombinant purified hSIRal WT, N163A mutant, H206A mutant or N163A/H206A double-mutant. c. High performance liquid chromatography (HPLC) analysis of NAD⁺ with or without PEG after a 16h incubation with recombinant purified hSIRal. Standard elutions volumes for ADPR and NAM are indicated. d. Local alignment of the amino acid sequences of the SIRim domains of hSIRal and bacterial ThsA. Identical and similar residues are highlighted (red and grey, respectively), as well as the catalytic residues of ThsA (from (12), yellow). e. AlphaFold prediction of hSIRal (red) interacting with NAD⁺ (grey), with predicted interacting residues N163 and H206 (yellow). f. SDS-PAGE of recombinant purified zSIRal from *D. rerio* (clade A, 41 kDa) or sirims from *P. shiwhaense* (clade B, 32 kDa), *P. violaceum* (clade C, 128 kDa), *P. micropora* (clade D, 108 kDa) or *Acanthoecidae* sp. (clade E, 49 kDa). g. HPLC analysis of NAD⁺ with or without 8h incubation with recombinant purified zSIRal from *D. rerio* (clade A) or sirims from *P. shiwhaense* (clade B), *P. violaceum* (clade C), *P. micropora* (clade D) or *Acanthoecidae* sp. (clade E). Standard elution volumes for ADPR and NAM are indicated. h-i. HEK293T-hTLR4 NFκB reporter cells were transiently transfected with plasmids coding hSIRal WT or hSIRal N163A/H206A double-mutant and were stimulated with LPS from *E. coli* (0,1-10ng/mL). Levels of *hSIRal* transcripts were measured by RT-qPCR and NFκB activation was measured at 24h. Each dot represents the mean of technical replicates for each independent experiment. Bars correspond to the mean and error bars to the standard deviation of all the independent experiments. j. Wild type or Δ*mSIRal* RAW264.7 cells were stimulated with LPS from *E. coli* (100ng/mL). Endogenous NAD⁺ levels were analyzed by mass spectrometry at 4h.
<figure><img src="image_48.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_49.png" /></figure>

Stainfree

1: hSIRal

WT

2: hSIRal

N163A

3: hSIRal

H206A

4: hSIRal

N163A/H206A

<figure><img src="image_50.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_51.png" /><figcaption>e Predicted interaction hSIRal/NAD</figcaption></figure>

<figure><img src="image_52.png" /><figcaption>f</figcaption></figure>

Stainfree

SIRim

A: *D. rerio* (41kDa)

B: *P. shiwhaense* (32kDa)

C: *P. violaceum* (128kDa)

D: *P. micropora* (108kDa)

E: *Acanthoecidae* sp. (49kDa)

<figure><img src="image_53.png" /><figcaption>h hSIRal mRNA</figcaption></figure>

<figure><img src="image_54.png" /><figcaption>i NFκB reporter</figcaption></figure>

<figure><img src="image_55.png" /><figcaption>j Endogenous NAD</figcaption></figure>

<figure><img src="image_56.png" /></figure>
References

1. D. A. Gold, D. A. Sinclair, Sirtuin Evolution at the Dawn of Animal Life. *Molecular Biology and Evolution* **39**, msac192 (2022).

2. G. Ofir, E. Herbst, M. Baroz, D. Cohen, A. Millman, S. Doron, N. Tal, D. B. A. Malheiro, S. Malitsky, G. Amitai, R. Sorek, Antiviral activity of bacterial TIR domains via immune signalling molecules. *Nature* **600**, 116–120 (2021).

3. J. Garb, A. Lopatina, A. Bernheim, M. Zaremba, V. Siksnys, S. Melamed, A. Leavitt, A. Millman, G. Amitai, R. Sorek, Multiple phage resistance systems inhibit infection via SIR2-dependent NAD+ depletion. *Nat Microbiol*, doi: 10.1038/s41564-022-01207-8 (2022).

4. G. Tamulaitiene, D. Sabonis, G. Sasnauskas, A. Ruksenaite, A. Silanskas, C. Avraham, G. Ofir, R. Sorek, M. Zaremba, V. Siksnys, Activation of Thoeris antiviral system via SIR2 effector filament assembly. *Nature*, doi: 10.1038/s41586-024-07092-x (2024).

5. D. J. Richter, C. Berney, J. F. H. Strassert, Y.-P. Poh, E. K. Herman, S. A. Muñoz-Gómez, J. G. Wideman, F. Burki, C. De Vargas, EukProt: A database of genome-scale predicted proteins across the diversity of eukaryotes. *Peer Community Journal* **2**, e56 (2022).
# Supplementary Material

## List of Supplementary Tables

**Table S1.**
Initial detection of proteins of the SIR2 family.

**Table S2.**
Curated detection of proteins of the SIR2 subfamily.

**Table S3.**
Curated detection of proteins of the SIRim subfamily.

**Table S4.**
Detection of sirims in the Eukprot v3 database of eukaryotic genomes.

**Table S5.**
Primers and sgRNA used to construct Δ*mSIRal* / *FAM118B* RAW264.7 cells.

**Table S6.**
References of Taqman primers and probes used for RT-qPCR on RAW264.7 cells, mouse BMDMs and human monocyte-derived macrophages.

**Table S7.**
Sequences of primers used for RT-qPCR on zebrafish larvae.
Supplementary Tables

<table><caption>Table S5. Primers and sgRNA used to construct Δ*mSIRal* / *FAM118B* RAW264.7 cells.</caption><thead><tr><td>TYPE</td><td>SEQUENCE</td></tr></thead><tbody><tr><td>sgRNA</td><td>forward CACCGGAAGTTATGGTAAGTAGTAC</td></tr><tr><td>sgRNA</td><td>reverse AAACGTACTACTTACCATAACTTCC</td></tr><tr><td>Primer</td><td>forward AAGCGGAAGCTAAGTGTGCTAC</td></tr><tr><td>Primer</td><td>reverse AGAGACAACGAAGGAAGGTCTG</td></tr></tbody></table>

<table><caption>Table S6. References of Taqman primers and probes used for RT-qPCR on RAW264.7 cells, mouse BMDMs and human monocyte-derived macrophages.</caption><thead><tr><td>SPECIES</td><td>TARGET</td><td>REFERENCE</td></tr></thead><tbody><tr><td>Mouse</td><td>GAPDH</td><td>Mm99999915_g15</td></tr><tr><td>Mouse</td><td>FAM118B</td><td>Mm00615099_m1</td></tr><tr><td>Mouse</td><td>TNF</td><td>Mm00443258_m1</td></tr><tr><td>Mouse</td><td>iNOS (Nos2)</td><td>Mm00440502_m1</td></tr><tr><td>Mouse</td><td>Rsad2</td><td>Mm00491265_m1</td></tr><tr><td>Human</td><td>HPRT</td><td>Hs02800695_m1</td></tr><tr><td>Human</td><td>FAM118B</td><td>Hs01090503_m1</td></tr><tr><td>Human</td><td>IL8</td><td>Hs00174103_m1</td></tr><tr><td>Human</td><td>IL6</td><td>Hs00174131_m1</td></tr><tr><td>Human</td><td>TNF</td><td>Hs00174128_m1</td></tr><tr><td>Human</td><td>IFI44L</td><td>Hs00915292_m1</td></tr><tr><td>Human</td><td>Rsad2</td><td>Hs00369813_m1</td></tr><tr><td>Human</td><td>Mx1</td><td>Hs00895608_m1</td></tr></tbody></table>

<table><thead><tr><td>TARGET</td><td>FORWARD</td><td>REVERSE</td></tr></thead><tbody><tr><td>2F1A</td><td>ACCTACCCTCCTCTTGGTCG</td><td>GGAACGGTGTGATTGAGGGAA</td></tr><tr><td>IL22</td><td>TGCAGAATCACTGTAAACACGA</td><td>CTCCCGATTGCTTTGTTAC</td></tr><tr><td>IL26</td><td>ACTGCCAACTCAGTTCATTGAC</td><td>TTGCCTTCAGTCGCACAGTT</td></tr></tbody></table>
<table><caption>Table S7. Sequences of primers used for RT-qPCR on zebrafish larvae.</caption><tr><td>IL1β</td><td>GAGACAGACGGTGCTGTTTA</td><td>GTAAGACGGCAGTGAATCCA</td></tr><tr><td>IFNγ</td><td></td><td></td></tr><tr><td>IFNφ1</td><td>AATGGCTTGGCCGATACAGG</td><td>CACCTGGTCCTCCACCTTTG</td></tr></table>