NLRP10 Affects the Stability of Abin-1 to Control Inflammatory Responses Nora Mirza, Anna S

NLRP10 Affects the Stability of Abin-1 To Control Inflammatory Responses Nora Mirza, Anna S. Sowa, Katja Lautz and Thomas A. Kufer This information is current as of October 1, 2021. J Immunol published online 3 December 2018 http://www.jimmunol.org/content/early/2018/11/30/jimmun ol.1800334 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2018/11/30/jimmunol.180033 Material 4.DCSupplemental

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published December 3, 2018, doi:10.4049/jimmunol.1800334 The Journal of Immunology

NLRP10 Affects the Stability of Abin-1 To Control Inﬂammatory Responses

Nora Mirza,*,1 Anna S. Sowa,*,1 Katja Lautz,† and Thomas A. Kufer*

NOD-like receptors (NLR) are critical regulators of innate immune signaling. The NLR family consists of 22 human proteins with a conserved structure containing a central oligomerization NACHT domain, an N-terminal interaction domain, and a variable number of C-terminal leucine-rich repeats. Most NLR proteins function as cytosolic pattern recognition receptors with activation of downstream inflammasome signaling, NF-kB, or MAPK activation. Although NLRP10 is the only NLR protein lacking the leucine rich repeats, it has been implicated in multiple immune pathways, including the regulation of inflammatory responses toward Leishmania major and Shigella flexneri infection. In this study, we identify Abin-1, a negative regulator of NF-kB, as an interaction partner of NLRP10 that binds to the NACHT domain of NLRP10. Using S. flexneri as an infection model in human epithelial cells, our work reveals a novel function of NLRP10 in destabilizing Abin-1, resulting in enhanced proinflammatory signaling. Our data give insight into the molecular

mechanism underlying the function of NLRP10 in innate immune responses. The Journal of Immunology, 2019, 202: 000–000. Downloaded from

he field of innate immunity is rapidly expanding with activation of procaspase-1. Caspase-1 subsequently processes the understanding of the molecular details underlying the the proinflammatory cytokines pro-ILb and pro–IL-18, which are T function of cytosolic pattern recognition receptors known as involved in a range of cellular processes (2, 3). In contrast to NOD-like receptors (NLRs). NLR proteins have a tripartite domain the inflammasome-forming NLRs, the NF-kB/MAPK–signaling

organization comprising an N-terminal effector domain, typically a NLRs, exemplified by NOD1 and NOD2, contain a CARD http://www.jimmunol.org/ pyrin domain (PYD) or the N-terminal caspase recruitment domain N-terminal domain. They each bind different moieties of bacterial (CARD). A central nucleotide-binding domain, NACHT, mediates peptidoglycan (PGN) and can form oligomers interacting through protein oligomerization, and a series of leucine-rich repeats (LRRs) at their CARD domain with the receptor-interacting serine/threonine the carboxyl-terminus are involvedinligandsensing.NLRsare protein kinase 2 (RIPK2). The E3 ligase X-linked inhibitor of evolutionarily conserved between plants and mammals and have been apoptosis protein (XIAP) is then recruited to the NOD-RIPK2 implicated in sensing a wide range of cellular signals that most likely complex where it mediates the polyubiquitylation of RIPK2 converge into a limited number of cellular pathways (1). (4–8). It further promotes the recruitment of the linear ubiquitin NLR proteins can be assigned to three functional categories: assembly complex (LUBAC), which forms linear ubiquitin chains. inflammasome-forming NLRs, NF-kB and MAPK (NF-kB/ This allows for the activation of NF-kB as well as the MAPKs by guest on October 1, 2021 MAPK)-activating NLRs, and transcriptional regulators. NLRP1, ERK, p38 and JNK, and subsequent production of inflammatory NLRP3, and NLRC4 are among the best-characterized NLRs that cytokines (9, 10). Many members of the NLR family have been activate inflammasome assembly by their oligomerization and connected to chronic inflammatory diseases, including Crohn recruitment of the adaptor protein apoptosis-associated speck-like disease, fever syndromes, cancer, and diabetes (11–13). protein containing a CARD (ASC) via their PYD, resulting in the Although the better-described NLRs get a lot of attention, the majority of the 22 human NLR proteins are lacking character- *Institute of Nutritional Medicine, University of Hohenheim, 70593 Stuttgart, ization or a unifying underlying mechanism. One such poorly Germany; and †Institute for Medical Microbiology, Immunology and Hygiene, understood NLR is the NACHT, LRR, and PYD domain-containing University of Cologne, 50931 Cologne, Germany protein 10 (NLRP10), which is unique, as it lacks the LRRs present 1 N.M. and A.S.S. contributed equally to this work. on all other identified NLR proteins and are considered to be in- ORCIDs: 0000-0003-0741-7477 (A.S.S.); 0000-0003-4563-0412 (T.A.K.). volved in ligand binding (14, 15). NLRP10 was first described Received for publication March 8, 2018. Accepted for publication October 29, 2018. as a protein with function in ASC and caspase-1 inhibition. It This work was supported in part by German Research Foundation Grant SFB670-TP27 was subsequently shown that human NLRP10 can inhibit to T.A.K. ASC-mediated NF-kB activation and IL-1b release in epithelial Address correspondence and reprint requests to Prof. Thomas A. Kufer, Department cells (14). Although Imamura et al. (16) could confirm inhibition of Immunology, Institute of Nutritional Medicine, University of Hohenheim, Fruwirthstrasse 12, 70593 Stuttgart, Germany. E-mail address: Thomas.kufer@uni- of the IL-1b release in macrophages of NLRP10 transgenic mice, hohenheim.de the murine Nlrp10 failed to inhibit ASC aggregation in knockin The online version of this article contains supplemental material. mice overexpressing Nlrp10. The physiological role of NLRP10 Abbreviations used in this article: Abin-1, TNFAIP3-interacting protein 1; ASC, in inflammasome formation or activation, however, remains con- apoptosis-associated speck-like protein containing a CARD; BMDC, bone mar- troversial, as Nlrp10 knockout mice have no obvious defect in row–derived dendritic cell; BMDM, bone marrow–derived macrophage; CARD, caspase recruitment domain; CHX, cycloheximide; LRR, leucine-rich repeat; MEF, NLRP3-mediated responses (17). By contrast, inflammasome- mouse embryonic fibroblast; NLR, NOD-like receptor; NLRP10, NACHT, LRR, independent pro- and anti-inflammatory roles of NLRP10 in and PYD domain-containing protein 10; p.i., postinfection; Pen/Strep, penicillin/ streptomycin; PYD, pyrin domain; RAR, retinoic acid receptor; RIPK2, receptor- Shigella flexneri and Leishmania major infection were also de- interacting serine/threonine protein kinase 2; siRNA, small interfering RNA; UBAN, scribed (18, 19), combined with work suggesting a role of UBD in ABIN proteins and NEMO; UBD, ubiquitin-binding domain; WT, wild-type; NLRP10 in enhancing T cell responses by increasing IL-12 pro- Y2H, yeast two-hybrid. duction by dendritic cells (20). NLRP10 is highly expressed in the Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$37.50 epidermis of the skin, where keratinocytes express several pattern

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800334 2 NLRP10 INTERACTS WITH Abin-1 recognition receptors important for host defense against bacterial, SDS-PAGE, immunoprecipitation, and immunofluorescence micro- viral, and fungal pathogens (18, 21–23). Genome-wide association scopyHEK293T cells were transiently transfected using Lipofectamin 2000 studies suggest a link between NLRP10 and allergic and inflamma- (Thermo Fisher Scientific) according to the manufacturer’s conditions, with the indicated plasmids (3 mg of plasmid per 6-cm dish). Cells were in- tory pathways in atopic dermatitis, food allergies, and contact hy- cubated overnight and subsequently lysed in NP-40 buffer (150 mM NaCl, persensitivity. Most recently, NLRP10 was found upregulated in 1% NP-40, 50 mM Tris-HCl, pH 7.5) containing phosphatase inhibitors psoriasis, which is an immune-mediated genetic disease manifested in (20 mM b-glycerophosphate, 5 mM NaF, 100 mMNa3VO4) and complete the skin and joints and shown to contribute to delayed-type hyper- protease inhibitor mixture (Roche). Lysates were cleared for 20 min at 14,000 3 g at 4˚C. Subsequently, samples were either run on an sensitivity response in a mouse model of atopic dermatitis (24–27). SDS-PAGE gel or analyzed by immunoprecipitation. To elucidate further functions of NLRP10, we set out to identify Immunoprecipitation was carried out for 4 h at 4˚C by adding anti-FLAG new interaction partners of NLRP10. In the current study, we report beads (M2 gel; Sigma-Aldrich), GFP-Trap_MA agarose beads (Chromotek), on a yeast two-hybrid (Y2H) screen that identified the TNFAIP3- anti-HA probe (Y11, sc-805; Santa Cruz Biotechnology) bound to protein G interacting protein 1 (Abin-1) as a novel interaction partner of Dynabeads or anti-Myc beads (9E10 agarose; Santa Cruz Biotechnology) to the cell extracts. The beads were precipitated by centrifugation steps and human NLRP10. Abin-1 is a regulatory protein that was first washed three times in NP-40 buffer before SDS loading buffer was added. discovered to have an inhibitory role on the transmembrane Proteins were separated by Laemmli SDS-PAGE and transferred to a ni- TNFR1 (28). Since then it was also found to affect the signaling of trocellulose membrane (Bio-Rad). Proteins were detected by incubation of epidermal growth factor receptor (EGF-R), TLRs, and nuclear the membrane consecutively with primary and secondary Abs and finally with SuperSignal West Femto Maximum Sensitivity Substrate (Pierce). receptors such as peroxisome proliferator–activated receptors and Signals were recorded on an electronic camera system (Vilbert Fusion FX). the retinoic acid receptor (RAR) (29). Abin-1 is well known to Primary Abs were mouse anti-FLAG M2 (F3165; Sigma-Aldrich), rabbit inhibit NF-kB via binding to the ubiquitin-editing and deubiqui- anti-Myc (A-14; Santa Cruz Biotechnology), rabbit anti–Abin-1 (4664; Cell tylating enzyme TNF-a–induced protein 3 (TNFAIP3 or Zinc Signaling Technology), mouse anti–Abin-1 (1A11E3; Thermo Fisher Sci- Downloaded from finger protein A20) (30) either by physical interaction, linking entific), anti-GFP (Roche), anti-HA (Y11, sc-805; Santa Cruz Biotechnol- ogy), anti-GAPDH (FL-335; Santa Cruz Biotechnology), anti-Actin (A2066; A20 to ubiquitylated NEMO/IKKg, or competing with NEMO Sigma-Aldrich), and anti-NLRP10 monoclonal rat Ab 8H2 as published in for binding to polyubiquitinylated signaling proteins (30–32). (18). Secondary Abs were HRP-conjugated goat anti-mouse IgG (170-6616; Although Abin-1 was demonstrated to have effects on the sig- Bio-Rad) and HRP-conjugated goat anti-rabbit IgG (170-6515; Bio-Rad). naling cascades of multiple receptors, interactions that control the For indirect immunofluorescence microscopy, cells were seeded on

coverslips, fixed in 4% paraformaldehyde in PBS, and permeabilized with http://www.jimmunol.org/ cellular levels of Abin-1 itself have not been described until now. 0.5% Triton X-100 for 5 min. Cells were incubated in 3% BSA in PBS. In this paper, we demonstrate that NLRP10 forms a complex Staining was done by consecutive incubation with primary and secondary with Abin-1, involving ubiquitylation of NLRP10 and reduction of Abs in 3% BSA. The secondary Abs were Alexa 546–conjugated goat cellular levels of Abin-1 in a dose-dependent manner upon infection anti-mouse IgG or Alexa 546–conjugated goat anti-rabbit IgG (Molec- with invasive NOD1-activating bacteria. This provides a mechanistic ular Probes). DNA was stained with Hoechst 33258 (Carl Roth; Sigma- Aldrich). The images wereacquiredonaLeicaDMi8microscopeusing insight into how NLRP10 contributes to the enhanced NOD1- high numerical aperture 403 and 633 objectives and processed using mediated proinflammatory responses reported by us previously (18). ImageJ (National Institutes of Health, Bethesda, MD) and the LasX software (Leica). Materials and Methods Bacterial infections and cell stimulation by guest on October 1, 2021 Identification of proteins that interact with NLRP10 via Y2H screening Bacterial infection of cells was performed using the S. flexneri strains BS176 afaE and M90T afaE as described previously (37). M90T afaE is a wild-type Y2H screening and data analysis were performed by Hybrigenics SA, Paris, (WT) invasive strain of S. flexneri serotype 5a harboring the plasmid pIL22, France, essentially as described (33). The full NLRP10 was cloned into a which encodes the afimbrial adhesion afaE from uropathogenic Escherichia Y2H vector optimized by Hybrigenics SA. NLRP10 was screened against coli, whereas the BS176 is a virulence plasmid–cured, noninvasive isogenic a highly complex human placenta library (34). strain of M90T (38). Briefly, logarithmically growing bacteria were added to the cells, which were transferred to antibiotic-free medium and incubated for Generation and culture of mouse embryonic and primary 15 min at room temperature prior to transfer to 37˚C. S. flexneri M90T afaE skin fibroblasts was added at a multiplicity of infection of 10 to cells, and the medium was replaced with DMEM containing 100 mg/ml gentamicin 30 min after Mouse embryonic fibroblasts (MEF) were isolated as described (35). incubation at 37˚C. Briefly, embryos were dissected on embryonic day 11.5 and sacrificed by Cells were stimulated with TNF (10 or 20 ng/ml; InvivoGen) or E. coli decapitation. Samples were genotyped, and the remaining tissue was K12 LPS (10 ng/ml; InvivoGen) or MDP (10 mg/ml; InvivoGen). rinsed, dissected, and incubated in DMEM with 10% FBS plus 1% penicillin/streptomycin (Pen/Strep) at 100 IU/ml and 100 mg/ml, respec- Small interfering RNA knockdown tively. Nonadherent cells were removed after subsequent incubation. Cells were immortalized by transduction with SV40. Knockdown of Abin-1 and NLRP10 in HeLa cells was performed for 72 h 2/2 Primary skin fibroblasts were isolated from Nlrp10 knockout and using the Viromer Blue transfection reagent (Lipocalyx). For each reac- flox/flox Nlrp10 mice (24) essentially as described (36). Briefly, mouse ears tion, 25 nM of the following small interfering RNAs (siRNAs) were used: were dissected and incubated in RPMI 1640 medium with 30% FBS, 3% Abin-1 59-CAAGAAGTTGTTGATGAGCAA-39 (SI00748727; Qiagen); Pen/Strep, and 1% amphotericin B with collagenase type 1A (C2674; NLRP10 (NLRP10A): 59-CAGCTCCTATTTCACGGATGA-39 (SI04328254; Sigma-Aldrich). After overnight incubation, cells and tissue were resus- Qiagen); NLRP10 (NLRP10B): 59-AAGGAGGGCAAAGATAATATA-39 pended and medium changed until cells reached confluence. (SI00654423; Qiagen); and a nontargeting control siRNA (AllStars Neg- ative Control; Qiagen) as described (39). Generation of bone marrow–derived dendritic cells Bone marrow cells were isolated from the respective animals and cultured in NF-kB reporter assay RPMI 1640 with 2 mM L-glutamine, 50 mM b-mercaptoethanol, 1% Pen/ Gene reporter assays were performed as described (40). Briefly, HEK293T cells Strep, and 10% FBS supplemented with 20 ng/ml GM-CSF (PeproTech). transfected with a b-galactosidase–encoding plasmid and the NF-kB Fresh GM-CSF was added two times (day 3 and 5), and immature bone luciferase reporter plasmid and increasing amounts of an Abin-1 expres- marrow–derived dendritic cells (BMDC) were harvested after 6 d. sion plasmid normalized with cDNA using FuGENE6 (Roche). Cells were Cell lines induced with 10 ng/ml TNF and incubated 16–24 h at 37˚C. The next day, cells were lysed in 100 ml of NF-kB luciferase lysis buffer, and the HEK293T and HeLa cells were obtained from the American Type Culture luciferase activity was measured with a luminometer. The normalized Collection. Cells were cultivated at 37˚C with 5% CO2 in DMEM luciferase activity was calculated by dividing luciferase activity through supplemented with 10% FBS and 1% Pen/Strep. b-galactosidase activity. The Journal of Immunology 3

Plasmids (Fig. 2A). To exclude that this was due to a particularity of the pcDNA3.1 (Invitrogen), pcDNA-NLRP10-myc, pCMV-NLRP10-FLAG, expression constructs, we confirmed this effect using alternatively pcDNA-NLRP10DPYD-myc, and pcDNA-NLRP10D-FLAG are described tagged expression vectors (Supplemental Fig. 1C). Abin-1 protein (18). pcDNA-NOD1 (41) pCMV–Abin-1–FLAG was generated by levels inversely correlated with the amount of NLRP10 expressed PCR-based cloning in pCMV-Tag2B. The Abin-1 E476AA mutation was during S. flexneri M90T infection in both HEK293T and HeLa generated using the quick-site mutagenesis protocol. pCMV-NLRP10, pCMV- cells; however, noninvasive S. flexneri BS176, which are not able NLRP10DPYD, pCMV-NLRP10K179A-GFP, pCMV-NLRP10PYD-GFP were generated by PCR-based cloning in a pCMVTag2B vector where to trigger NOD1 signaling, did not induce reduction of Abin-1 the FLAG was replaced by eGFP. Mouse HA–Abin-1 plasmid was a levels (Fig. 2B). gift from H. Ha¨cker (St. Jude Children’s Research Hospital) and NOD1 overexpression, which is known to lead to autoactivation mouse FLAG-NLRP10 was obtained by cloning Mm.37991 cDNA clone of NOD1 and to a lesser extent TNF activation, also increased IRCLp5011A1123D (Source Bioscience) into pCMV-Tag2B (Agilent Technologies). All cloned inserts were sequenced. protein levels of Abin-1 (Fig. 2C). This effect of NOD1 was counteracted by NLRP10 expression (Fig. 2C). In line with the Detection of cytokines results obtained with bacterial infection, an increase in NLRP10/ The levels of human IL-8 and msCXCL1 (keratinocyte-derived chemokines) Abin-1 complex formation was also observed upon NOD1 over- were determined by ELISA using the DuoSet kits DY208 and DY453 (R&D, expression (Fig. 2D), suggesting that NOD1 activation, rather than Biotechne) according to the manufacturer’s instructions. effects of the bacterial infection are mediating this response. As Abin-1 was expressed from the highly active CMV promotor, Data analysis we assumed that changes in Abin-1 protein levels were due to Western blots were quantified by Vilber Imaging software and normalized to differential regulation of protein stability. Accordingly, we did a loading control. Subsequent analysis was performed on GraphPad Prism 7 not observe any effect of overexpression of NLRP10 on the mRNA Downloaded from (GraphPad Software). Student two-sided t test was used where appropriate. levels of neither endogenous nor ectopically expressed Abin-1 (Supplemental Fig. 2A, 2B). We thus tested if NLRP10 contributed Results to proteasomal degradation of Abin-1. To this end, HEK293T cells NLRP10 physically interacts with Abin-1 expressing Abin-1 were treated with cycloheximide (CHX) to block To find new interaction partners for human NLRP10, we conducted protein neosynthesis, and protein levels were analyzed over time. a Y2H screen using full-length human NLRP10 as bait. The screen First, although NLRP10 was readily degraded in a time-dependent http://www.jimmunol.org/ identified Abin-1 (TNIP1) (Fig. 1A) as a promising interaction manner (Fig. 2E), the Abin-1 protein appeared to be more stable candidate. The identified prey encoded the ubiquitin-binding do- (Fig. 2E). Overexpression of NLRP10 did not have an effect on the main (UBD) of Abin-1, which also interacts with the poly-UBD of rate of Abin-1 degradation (the decrease in protein over 4 h of CHX NEMO (30) (Fig. 1A), a previously published interaction partner treatment), although it had an effect on the total Abin-1 protein of NLRP10 (18). levels (for quantification see Supplemental Fig. 2C). In contrast to The physical interaction of Abin-1 and NLRP10 was subsequently nontreated or TNF-treated cells, overexpression of NOD1 led to a confirmed in human cells by coimmunoprecipitations from tran- more pronounced stabilization of NLRP10 and degradation of Abin-1 (Fig. 2E, lower panel), suggesting a contribution of NOD1 and/or

siently transfected HEK293T cells (Fig. 1B). We also found that the by guest on October 1, 2021 mouse homologs of Abin-1 and NLRP10 showed interaction when NOD1 signaling. overexpressed in human HEK293T cells (Supplemental Fig. 1A). To visualize the levels of endogenous Abin-1, we conducted To establish the protein domains in NLRP10 responsible for the indirect immunofluorescence studies in HeLa cells transiently interaction with Abin-1, coimmunoprecipitations with myc–Abin-1 transfected with GFP-NLRP10. Consistent with previous reports, and various deletion constructs of GFP-NLRP10 were performed the Abin-1 Ab detected dot-like structures (42), whereas NLRP10 (Fig. 1B) using transiently transfected HEK293T cells. Myc–Abin-1 was homogenously distributed throughout the cell (18). In the not only bound to the full-length GFP-NLRP10 but also to the course of infection, cells overexpressing GFP-NLRP10 showed Walker-A mutant and PYD deletion construct. By contrast, binding lower signals for endogenous Abin-1 compared with neighboring to the PYD domain could not be demonstrated. The NLRP10 in- cells without NLRP10 overexpression (Fig. 2F, white arrows; teraction site on Abin-1 was also reported to mediate binding of Fig. 2G for quantification). In some cells overexpressing NLRP10, NEMO (30), which we showed before to bind also NLRP10 (18). endogenous Abin-1 formed more pronounced foci, which colo- We subsequently mapped the interaction domain of NEMO and calized with NLRP10 over the time of infection (Fig. 2F, 4 h time NLRP10 and show that NEMO binds to the PYD deletion construct point). Data generated with primary cells from mice corroborated and to full-length NLRP10, suggesting that NEMO might be able to these findings. We observed higher cytosolic signals upon Shigella compete for binding of Abin-1 to NLRP10 (Fig. 1C). infection with an anti–Abin-1 Ab when staining MEF derived from Nlrp102/2 mice but not in MEF derived from Nlrp10fl/fl mice Stability of Abin-1 is altered by NLRP10, whereas complex by indirect immunofluorescence (Supplemental Fig. 3A, 3B). formation is strengthened Although we could not obtain robust detection of endogenous Recently, we showed that NLRP10 enhances proinflammatory Abin-1 in immunoblot to validate the immunofluorescence data, responses toward infection with the invasive Gram-negative similar results were obtained with isolated primary skin fibroblasts bacterial pathogen S. flexneri. Next, we asked if infection with from Nlrp10fl/fl and Nlrp102/2 mice, corroborating these results S. flexneri affects the interaction between Abin-1 and NLRP10. (Supplemental Fig. 3C). Coimmunoprecipitation experiments in transiently transfected To functionally link Abin-1 to Shigella-induced proin- HEK293T cells showed that interaction of the two proteins inflammatory signaling, we conducted siRNA-mediated knockdown creased with time of infection (Fig. 2A). This was also confirmed of NLRP10 and Abin-1 in HeLa cells, which were subsequently in a reverse pulldown (Supplemental Fig. 1B). During the coim- infected with S. flexneri. Bacterial-induced IL-8 levels, which are munoprecipitation experiments described above, we observed that mainly dependent on NOD1 (43), as well as basal IL-8 secretion the levels of ectopically expressed Abin-1 protein increased over were significantly increased upon Abin-1 knockdown (Fig. 3A). In time upon S. flexneri infection and that NLRP10 overexpression line with our earlier observation (18), reduction of NLRP10 led to counteracted this effect and led to overall lower levels of Abin-1 decreased IL-8 release from the cells (Fig. 3A). Accordingly, 4 NLRP10 INTERACTS WITH Abin-1 Downloaded from

FIGURE 1. Abin-1 is an interaction partner of NLRP10. (A) Identification of Abin-1 in a Y2H screen. Schematic representations of NLRP10 and Abin-1. Pyrin and NACHT domains of NLRP10 are depicted. Black bar in Abin-1 depicts the fragment of Abin-1 identified in the screen. Numbers refer to amino acid positions. Lower panel: National Institutes of Health NCBI Conserved Domain Search (CDD) identified the interacting domain to have similarity to the http://www.jimmunol.org/ polyubiquitin-binding domain of NEMO and the leucine zipper of domain CC2 of NEMO (a known interaction partner of Abin-1). DUF4407, domain of unknown function found in bacteria; SMC, chromosome segregation protein; MreC, rod-shaped determining protein; UBAN; CC2-LZ, leucine zipper of domain CC2 of NEMO. (B) Mapping of the NLRP10 interaction domain. Myc-tagged Abin-1 together with either empty GFP control or GFP-tagged NLRP10 constructs (full-length [fl], WalkerA K179A [wA] mutant, NLRP10 lacking the PYD domain [DPYD], and the PYD domain alone) were expressed in HEK293T cells. Lysates were immunoprecipitated using anti-GFP Ab. Precipitated proteins were detected using anti-myc or anti-GFP Ab. Schematic drawings of the deletion constructs used are in the lower panel. (C) Mapping of the NEMO and NLRP10 interaction domain. GFP-tagged NEMO and FLAG-tagged NLRP10 were coexpressed in HEK293T cells. Lysates were immunoprecipitated using anti-GFP Ab. Schematic representation of the domains that interacted with either NEMO or Abin-1. One representative experiment out of three is shown for (B) and (C). NCBI, National Center for Biotechnology Information. by guest on October 1, 2021

NLRP10 depletion resulted in enhanced Abin-1 protein levels Abin-1 (Fig. 4A). Infection of GM-CSF–differentiated Nlrp102/2 independent of stimulation (Fig. 3B). When combining knock- BMDC with S. flexneri led to a trend toward lower CXCL1 down of Abin-1 and NLRP10, we observed a reduction of and IL-6 release upon 6 h of infection compared with WT cells, both, Shigella- and TNF-induced IL-8 responses. However, the which was significant for CXCL1 after 6 h of Shigella infec- TNF-induced IL-8 release was only marginally affected by tion (Fig. 4B). The proinflammatory cytokine release of BMDC NLRP10 knockdown compared with the Shigella-mediated re- upon MDP and LPS stimulation was not significantly different sponse (Fig. 3A). siRNA treatment thereby significantly reduced between the genotypes (Fig. 4B). In BMDM, no significant effect expression of the target proteins (Fig. 3B). Notably, we observed of NLRP10 on cytokine release was observed (data not shown). that Abin-1 depletion led to enhanced NLRP10 levels (Fig. 3B). Lack of a pronounced phenotype of NLRP10 depletion in mouse This showed that changes in Abin-1 levels correlate with proin- myeloid cells might be explained by a differential regulation of flammatory responses induced by S. flexneri and that NLRP10 Abin-1 levels in these cells upon infection or the differences in can counteract Abin-1 function. In addition, we measured IL-1b Abin-1 expression in these cells (Fig. 4A). Indeed, we observed but did not detect any release of IL-1b from these cells in this that in contrast to our data on human epithelial cells and murine experiment (data not shown). MEF and keratinocytes, endogenous Abin-1 levels were highly Taken together, our data showed that activation of NOD1 in- reduced upon infection in dendritic cells and were not affected by creased the complex formation between NLRP10 and Abin-1. NLRP10 expression, suggesting differences in Abin-1 regulation NLRP10 expression led to degradation of Abin-1, which was in these cell types (Supplemental Fig. 3D). enhanced by NOD1. Combined with previously reported data, this To conclude, depletion of endogenous Nlrp10 led to slight re- suggests that NLRP10 supports proinflammatory signaling by duction of CXCL1 and IL-6 responses toward Shigella infection in decreasing Abin-1 levels. BMDC. However, mouse myeloid cells showed a differential regulation of Abin-1 levels, suggesting that Abin-1 might not be a Effect of knockout of Nlrp10 on innate immune responses in target of NLRP10 in these cells. mouse immune cells To expand on our in vitro data, we next analyzed NLRP10 and Ubiquitin-binding activity of Abin-1 affects binding to NLRP10 Abin-1 expression in primary cells derived from Nlrp10 knockout Abin-1 is a ubiquitin sensor, and modulation of ubiquitylation is an animals. We observed that keratinocytes showed the highest ex- important strategy of Shigella pathogenesis (30). To examine if pression of Nlrp10, albeit low expression of Abin-1. However, ubiquitin binding of Abin-1 is involved in its regulation by immune cells such as bone marrow–derived macrophages NLRP10, we tested if a UBD in ABIN proteins and NEMO (BMDM) and BMDC showed good expression of both Nlrp10 and (UBAN)-domain mutant of Abin-1 (E476AA) showed the same The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 2. S. flexneri infection and NOD1 activation affects Abin-1/NLRP10 complex formation and Abin-1 levels. (A and D) Coimmunoprecipitations were conducted using transiently transfected HEK293T cells. Signals obtained in immunoblots from the immunoprecipitants (IP) and total lysates (Input), using the indicated Abs, are shown. (A) FLAG-NLRP10 and myc–Abin-1 were ectopically expressed in HEK293T cells as indicated and cells infected with S. flexneri M90T afaE. Cells were lysed at the indicated time p.i. and the lysates precipitated using anti-FLAG Ab. Coprecipitated myc–Abin-1 was detected using anti-myc Ab. One representative experiment out of three is shown. (B) Increasing amounts (+: 0.125 mg, ++: 0.25 mg, +++: 0.5 mg plasmid per six-well) of myc-NLRP10 were transfected together with 0.25 mg of Abin-1 plasmid in both HEK293T and HeLa cells, and cells were infected with S. flexneri M90T afaE or S. flexneri BS176 afaE for 4 h. Probing for GAPDH served as loading control. * indicates a bacterial protein detected by the anti-GAPDH Ab. (C) Myc–Abin-1 and FLAG-NLRP10 were expressed in HEK293T cells as indicated. Cells were activated by cotransfection with NOD1, cotransfection of NOD1 together with 10 mg/ml TriDAP, or 10 ng/ml TNF treatment for 4 h. Probing for GAPDH as loading control is shown. (D) FLAG-NLRP10 and Myc–Abin-1 together with NOD1 were ectopically expressed in HEK293T cells as indicated. Lysates were precipitated using anti- FLAG Ab, and coprecipitated myc–Abin-1 was detected using anti-myc Ab. One representative experiment out of two is shown. (E) CHX chase experiment in HEK293T cells expressing myc–Abin-1 together with empty vector or two concentrations (+: 0.125 mg, ++: 0.25 mg per six-well) of FLAG-NLRP10. Indicated cells coexpressed NOD1 or were treated with 10 ng/ml TNF for 4 h. Cells were treated with 50 mg/ml CHX and lysed at the indicated time. Immunoblots of total cell lysates were performed using the indicated Abs. GAPDH served as loading control. (F) Indirect immunofluorescence micrographs of HeLa cells, which were transiently transfected with GFP-NLRP10 and infected with S. flexneri M90T afaE for the indicated time. Pictures show the signals obtained with an anti–Abin-1 Ab (red), GFP-NLRP10 (green), and DNA staining (blue), as well as the merge. Solid white arrows point to cells with GFP-NLRP10 overexpression and lower levels of Abin-1, whereas cells in open arrows show cells without GFP-NLRP10 expression and higher Abin-1 endogenous levels. Scale bar, 20 mm. Signal intensities along the depicted lines are shown on the right. (G) Quantification of (F) 100 cells were counted and scored for NLRP10 expression and a high Abin-1 signal. kinetics and effects upon infection and NLRP10 overexpression. infection with S. flexneri M90T, WT Abin-1 levels decreased more The E376AA mutation lies in the highly conserved DFxxER motif in response to NLRP10 overexpression (4 h postinfection [p.i.] of the ABIN homology domain 2 (AHD2) in the UBAN of Abin- ∼17-fold reduced) than the levels of Abin-1 E476AA (4 h p.i. ∼1. 1 that confers binding of Abin-1 to ubiquitin. The introduction 5-fold reduced) (Fig. 5A). In general, Abin-1 E476AA levels were of this mutation has been shown to render Abin-1 unable to higher compared with the WT protein and less regulated upon downregulate NF-kB (31) (Supplemental Fig. 1D). Side-by-side infection (Fig. 5A). Accordingly, we observed a reduced binding comparison of WT Abin-1 and Abin-1 E476AA showed that upon of Abin-1 E476AA to NLRP10 in the presence of NOD1, 6 NLRP10 INTERACTS WITH Abin-1

FIGURE 3. NLRP10 functionally counteracts Abin- 1. (A) Knockdown of NLRP10 (siNLRP10A and siNLRP10B) and Abin-1 (siAbin-1) and a combination of both in HeLa cells. After 72 h of siRNA treatment, cells were infected with S. ﬂexneri M90T afaE or stimulated with 20 ng/ml TNF. Six hours posttreat- ment, IL-8 was determined in the supernatant. A nontargeting siRNA duplex (siCTRL) was used as control. (B) Immunoblot of noninfected cells used in (A) detecting endogenous NLRP10 and Abin-1. Prob- ing for GAPDH served as loading control.

suggesting that ubiquitylation of NLRP10 might be involved (Fig. NOD1 stimulation. The UBAN mutant of Abin-1 did not show the 5B). We then asked if Abin-1 affects the ubiquitylation status of same binding capacity to NLRP10, and the levels of this Abin-1 NLRP10. We could detect that NLRP10 is ubiquitylated under mutant were not affected by NLRP10 overexpression. As Abin-1 steady-state conditions and that this ubiquitylation is reduced E476AA is unable to downregulate NF-kB signaling, this supports Downloaded from upon the overexpression of Abin-1, whereas Abin-1 E476AA was the functional role of the NLRP10/Abin-1 complex in regulating slightly less efficient to drive deubiquitylation of NLRP10 (Fig. inflammatory pathways. 5C). We also analyzed the effect of NLRP10 and NOD1 on Abin-1 ubiquitylation. This revealed increased ubiquitylation of WT Discussion but not of Abin-1 E476AA, whereas NLRP10 overexpression re- Our current understanding of NLRP10 is limited, and its function in duced Abin-1 levels and accordingly the ubiquitin signal. NOD1 immune signaling pathways require further clarification. NLRP10 expression increased Abin-1 ubiquitylation and led to higher has been proposed to have both anti-inflammatory and proin- http://www.jimmunol.org/ molecular complexes (Fig. 5D). flammatory properties. Some groups suggested an inhibitory role These data highlight the importance of the ubiquitin-binding for NLRP10 in ASC-mediated NF-kB activation as well as caspase- property of Abin-1 in downstream regulation of NF-kB upon 1–mediated IL-1b secretion (14, 16). However, Krishnaswamy by guest on October 1, 2021

FIGURE 4. Effect of Nlrp10 depletion on cytokine responses in myeloid cells. (A) Quantitative real-time PCR detection of basal mRNA levels of mouse Abin-1 and NLRP10 in different primary cells (fibrobl, fibroblasts; kera, keratinocytes; BMDM; BMDC). Mean expression normalized to b-actin expression (+SD n = 2) is shown. (B) BMDC derived from Nlrp102/2 (open circles) and Nlrp10fl/fl (filled circles) were infected with S. flexneri M90T afaE for 3 or 6 h or treated with MDP (10 mg/ml) or LPS (10 ng/ml) for 6 h, and release of CXCL1 and IL-6 into the medium was determined by ELISA. Means 6 SD of three to five mice (circles) are shown. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 5. Abin-1 regulation by NLRP10 depends on the ubiquitin-binding capacity of Abin-1. (A) Immunoblot of HEK293T cells expressing FLAG–Abin-1 or FLAG–Abin-1 E476AA together with myc-NLRP10 as indicated. Cells were infected with S. flexneri M90T afaE and lysed at the indicated time p.i. Probing with anti-FLAG (Abin-1) and anti-myc (NLRP10) together with anti-GAPDH, which served as loading control, are shown. Quantification of Abin-1 levels relative to Abin-1 in uninfected controls is shown in the graph below. (B) Myc-NLRP10 was coexpressed with FLAG–Abin-1 or FLAG–Abin-1 E476AA and NOD1 in HEK293T cells as indicated. Lysates were precipitated using anti-FLAG Ab. Coprecipitated myc-NLRP10 was detected using anti-myc Ab. (C) Ubiquitin status of NLRP10 is altered upon Abin-1 overexpression. Myc-NLRP10 was expressed together with FLAG–Abin-1 or FLAG–Abin-1 E476AA in HEK293T cells as indicated. Lysates were precipitated using anti-myc Ab. Bound HA-ubiquitin was detected using an anti-HA Ab, and NLRP10 was detected with a rat monoclonal NLRP10 Ab. One representative experiment out of three is shown. (D) Reverse immunoprecipitation of (C). Myc-NLRP10 was expressed together with FLAG– Abin-1 or FLAG–Abin-1 E476AA in HEK293T cells as indicated. Lysates were precipitated using anti-FLAG Ab. Bound HA-ubiquitin was detected using an anti-HA Ab, and NLRP10 was detected with a rat monoclonal NLRP10 Ab. (E) Schematic drawing showing interaction between Abin-1 and NLRP10. NLRP10 is stabilized upon NOD1 activation. NLRP10 is poly-ubiquitylated and can interact with NEMO. Upon NOD1 activation, Abin-1 levels begin to increase, andthe interaction of Abin-1 with NLRP10 strengthens. This interaction targets Abin-1 for degradation. Loss of Abin-1 leads to increased NF-kB levels and IL-8 release. 8 NLRP10 INTERACTS WITH Abin-1 et al. (44) did not find any effect of NLRP10 on the inflammasome occupying the PYD domain might regulate NEMO binding and activation in macrophages and dendritic cells of NLRP10 knockout raises the possibility that NEMO and Abin-1 might compete for mice. Recent studies showed a role of NLRP10 in the resolution binding to NLRP10. We assume that this competition could serve of local inflammatory responses during L. major infection (19) and as a switch mechanism to direct NLRP10-mediated inflammatory a positive effect of NLRP10 on the IL-12 release by dendritic response to various triggers. cells (20). The pathways underlying these diverse functions of We also demonstrate that NLRP10 is able to control cytosolic NLRP10 remain to be uncovered. levels of Abin-1. This is pathologically relevant as GWAS and array In this study, we identified the A20-binding inhibitor of NF-kB studies show a correlation of Abin-1 levels with multiple pathol- (Abin-1) as a novel interaction partner of human NLRP10. This ogies such as systemic lupus erythematosus, systemic sclerosis, interaction was verified in coimmunoprecipitation experiments rheumatoid arthritis, and leukemia/lymphoma (29, 49, 50). with human cells. Domain mapping identified the PYD to be Germline knockout of Abin-1 results in fatality and liver apoptosis dispensable for binding. Although a somewhat better binding with anemia (28), whereas knockin of an Abin-1 form which lacks of Abin-1 to a Walker A box mutation in NLRP10 was seen, the ability to interact with NEMO leads to a lupus-like autoim- this might be nonphysiological, as mutations in the ATP binding munity in the spleen and lymph nodes (47). Levels of Abin-1 site can cause conformational instability in NLRs. In HeLa are also critical in psoriasis development as blocking of Abin-1 cells, the two proteins partially colocalized during infection with increased proliferation of keratinocytes in psoriasis, whereas over- the invasive Gram-negative bacterial pathogen S. flexneri, whereas expression decreased keratinocyte proliferation and had a protective the interaction between the two proteins increased with time effect. This is especially interesting as NLRP10 was recently shown to of infection. be upregulated in psoriasis (27). Thus, although transcription of TNIP1 In previous work, we showed that NLRP10 contributes to skin is regulated in part by all-trans retinoic acid (ATRA) and RAR (51), Downloaded from contact hypersensitivity responses (24) and positively regulates we now show that cellular protein levels of Abin-1 are posttransla- innate immune responses mediated by NOD1 upon S. flexneri in- tionally regulated by NLRP10. The binding of NLRP10 that regulates fection in both epithelial cells and dermal fibroblasts via modulation available Abin-1 levels could be a contributing aspect of disease of p38 and NF-kB signaling pathways (18). NOD1 participates progression. A notable point is the comorbidity of psoriasis with in sensing PGN moieties and has been previously implicated in Crohn disease (a NOD1/2–linked condition) (reviewed in Ref. 52), Shigella infection (43). NOD1 signals via downstream interactions further strengthening the importance of the NOD/NLRP10/Abin-1 http://www.jimmunol.org/ with CARD-containing kinase RIPK2. Of note, a co-occurrence of pathway. NOD1, RIPK2, NLRP10, and Abin-1 is found across genomes Exactly how NLRP10 decreased the stability of Abin-1 at the (Supplemental Fig. 4), suggesting a conserved function of the molecular detail and why there is only a certain degree of speci- complex described in this paper. Of note, ties of Abin-1/NEMO ficity for NOD1 activation remains elusive. Of note, in HeLa cells, to S. flexneri response have been reported, whereby IpaH9.8, a Abin-1 reduction also led to higher endogenous NLRP10 protein Shigella effector possessing E3 ligase activity, interacts with Abin-1 levels, suggesting a more complex interplay. Polyubiquitylation of to promote NEMO polyubiquitylation and subsequent degradation NLRP10 might play a role here, as we could show that Abin-1 (45). Our work reveals a novel aspect of Abin-1 function, as it affects the ubiquitination status of NLRP10. The UBD (UBAN) by guest on October 1, 2021 shows an upregulation of Abin-1 protein levels, which is in some of Abin-1 is known to be specifically responsible for binding to cells dependent on NLRP10 and NOD1 activation. Why Abin-1 polyubiquitin and polyubiquitinylated proteins (28), and disruption levels are not regulated in the same manner in different cell types of polyubiquitin binding in Abin-1 promotes NF-kB and proin- and why there is only partial dependency on NOD1 signaling needs flammatory signaling, which in vivo is associated with glomeru- to be addressed in future research. lonephritis and lupus-like disease (31, 53, 54). We consequently Abin-1 is known to modulate different cellular signaling path- observed that the interaction of the UBAN mutant Abin-1E476AA ways, and its list of interacting partners includes a range of cellular has a weaker binding affinity for NLRP10 upon NOD1 over- and nuclear receptors (reviewed in Ref. 29). It is involved in a expression and that the levels of this mutant were not affected by number of cell-wide signaling events, including reducing activa- NLRP10 overexpression upon S. flexneri infection. It is plausible tion of transcription factors Elk2 and C/EBPb, coexpressing per- that other E3 ligases acting downstream of NOD1, such as XIAP oxisome proliferator–activated receptor and RAR, binding the (4, 55), might be involved in this process. Given the involvement HIV protein Nef or matrix. Its specific interaction partners include of Abin-1 in proinflammatory responses, these data could be a NEMO, A20, RIP-1, TRAF-1, FADD, p105, Nef, and ERK2 stepping stone in clarifying the role of NLRP10 in inflammatory (reviewed in Ref. 46). It acts downstream of the TNF-a receptor reactions in vivo. Of note, mouse myeloid cells exhibited a dif- via an interaction with NEMO (IKKg), reducing IKK complex ferent regulation of Abin-1 upon infection compared with fibro- activation and decreasing NF-kB–dependent gene expression, blasts and human epithelial cell lines. Whereas upon infection thereby inhibiting NF-kB, JNK, ERK, and p38 MAPK signaling with S. flexneri Abin-1 protein levels decreased in dendritic cells, and acting as an important regulator of homeostasis (47, 48). Abin-1 protein levels were enhanced in fibroblasts, human HeLa, This study expands on these previous observations and identifies and HEK293T cells. Furthermore, depletion of NLRP10 had only a novel Abin-1/NLRP10 complex formation during bacterial mild effects on cytokine responses in myeloid murine cells. S. flexneri infection. A change in the stoichiometry of the Compared to the clear effects in human and mouse epithelial cells, NLRP10/Abin-1 complex was observed after activation of NOD1, this suggests that NLRP10 has a primary function in the epithe- as the complex formation increased. This effect was also observed lium. It remains to be established if there might be different by immunofluorescence whereupon Abin-1 seemed to be differ- regulatory networks triggered by NLRP10 in human and mouse ently distributed in cells that overexpress NLRP10 and both cells. A predominant role of NLRP10 in epithelial versus myeloid Abin-1 and NLRP10 showed a more pronounced colocalization cells is also in line with the rather subtle phenotypes observed in p.i. The interaction between Abin-1 and NLRP10 was mediated by NLRP10 knockout mice upon infection, as these responses are the NACHT domain of NLRP10, which also binds NEMO, mainly governed by myeloid immune cells. whereas the binding of NEMO was negatively affected by the In summary, we provide mechanistic insight into the signaling presence of the PYD domain. This might suggest that proteins cascade of NLRP10 and how it impacts NOD1-mediated pathways. The Journal of Immunology 9

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NLRP10 affects the stability of Abin-1 to control inflammatory responses Nora Mirza*, Anna S. Sowa*, Katja Lautz†, Thomas A. Kufer*,‡

†Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Cologne, Germany *Institute of Nutritional Medicine, University Hohenheim, Stuttgart, Germany

Supplemental Figures 1-4

Mirza and Sowa et al., Supplemental Figure 1

A B FLAG-Abin-1 myc-NLRP10

msNLRP10msNLRP10 S. ﬂexneri (h): 1 2 4 + msAbin-1 α FLAG (ms NLRP10) αmyc (NLRP10) IP: α HA IP:Fα LAG αFLAG (Abin-1) α HA (ms Abin-1) αFLAG (Abin-1) α FLAG (ms NLRP10) Input αmyc (NLRP10) α HA (ms Abin-1) Input αActin

C D myc-NLRP10 FLAG-Abin-1 / / 1 2 4 / 1 2 4 S. ﬂexneri (h) αmyc (NLRP10)

αFLAG (Abin-1)

α NEMO αActin

Supplemental Figure 1. (A) Co-immunoprecipitation of mouse HA-Abin-1 and mouse FLAG-NLRP10 using anti HA antibody. (B, C) Myc-NLRP10 and FLAG-Abin-1 were ectopically expressed in HEK293T cells as indicated and cells infected with S. flexneri M90T afaE. Cells were lysed at the indicated time post infection and the lysates and immunoblots performed. (B) Co-immunoprecipitation of FLAG-Abin-1 using anti FLAG antibody. Co-precipitated myc-NLRP10 was detected with anti-myc antibody. (C) Cells expressing FLAG-Abin-1 and myc-NLRP10 were used and probed for Abin-1, NLRP10 and NEMO levels using the respective antibodies. Probing for actin served as loading control. (D) Functionality of the Abin-1 constructs. Expression of Abin-1 decreased NFκB levels in a dose-dependent manner. HEK293T cells were transiently transfected with NFκB luciferase reporter plasmid, a β-galactosidase expression plasmid and FLAG-Abin-1 or Abin-1 E476AA-FLAG. The cells were stimulated with TNF for 16 h and cell lysates were prepared. NFκB luciferase activity was measured in relative light units normalized with β-galactosidase enzyme activity (nRLU). Mean of three n=3 +/- SD is shown. Mirza and Sowa et al., Supplemental Figure 2

A B 0.015 1.5 ] ] Cq Cq ∆∆ ∆∆ [ [ 0.010 1.0 mRNA mRNA

0.005 0.5 Abin-1 Abin-1 expression expression rel. rel. 0.000 0.0 1 ck ck - 10 in P Mo RP10 Mo R Ab NL NL

-1 + in Ab

C Cells Cells + NLRP10 TNF TNF + NLRP10 1.5 NOD1 NOD1 + NLRP10

1.0

Abin-1

0.5

Levels of

Relative to Control 0.0 12 3 4 CHX treatment (h)

Supplemental Figure 2. (A,B) mRNA expression level of Abin-1 in HEK293T cells transiently transfected with the indicated plasmid. RT-qPCR data normalized to GAPDH expression (mean +S.D., n=3) is shown (C) Densitometric analysis of the blot shown in Fig.2 panel E. Mirza and Sowa et al., Supplemental Figure 3

A C

123

ﬂ

ﬂ/

Nlrp10

ﬂ

ﬂ/

Mock infected

S. ﬂexneri 0.5 h S. ﬂexneri 2 h 123

Nlrp10

(4h)

-/-

ﬂexneri

Nlrp10

20x S.

αAbin-1 (ms) 4 5 6 B

-/-

Mock infected

4 5 6

Nlrp10

(4h)

ﬂexneri

20x S. αAbin-1 (ms)

D NLRP10 fl/fl NLRP10 -/- 1 1 2 2 3 3 4 4 mouse # - + - + - + - + S. flexneri (3 h)

α Abin-1

α β-actin

Supplemental Figure 3. -/- fl/fl (A) Indirect immunofluorescence micrographs of MEF derived from Nlrp10 and Nlrp10 infected for the indicated time with S. flexneri M90T afaE. Pictures show the signals obtained with an anti-Abin-1 antibody (red) and DNA staining (blue). Scale bar: 20 µm. (B) mRNA expression of Nlrp10 in MEF shown by endpoint RT-PCR. -/- fl/fl (C) Indirect immunofluorescence micrographs of primary skin fibroblasts from Nlrp10 and Nlrp10 mice. Cells were mock-treated or infected for 4 h with S. flexneri M90T afaE. Pictures show the signals obtained with an anti-Abin-1 antibody (red) and DNA staining (blue). Scale bar: 20 µm. Numbers indicate animals from which cells were derived. n=3 mice per genotype. (D) Immunoblot BMDC infected with S. flexneri for 3 h, detecting endogenous Abin-1. Probing for alpha actin served as loading control. Results are shown for two mice per genotype (#1-4). Relates to figure 4B. Mirza and Sowa et al., Supplemental Figure 4

Supplemental Figure 4. STRING interaction prediction software was used to analyze the co-occurrence of genes across genomes. The color denotes, for each gene of interest, the similarity of its best hit in a given genome. Similarities in the presence/absence profiles can predict interactions. For groups of genomes which are collapsed in the viewer, two distinct colors indicate the lowest and highest similarity observed in that clade.