IRF8 Regulates Gram-Negative Bacteria− Mediated NLRP3 Inflammasome Activation and Cell Death

This information is current as Rajendra Karki, Ein Lee, Bhesh R. Sharma, Balaji Banoth of September 25, 2021. and Thirumala-Devi Kanneganti J Immunol published online 23 March 2020 http://www.jimmunol.org/content/early/2020/03/20/jimmun ol.1901508 Downloaded from

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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 © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 23, 2020, doi:10.4049/jimmunol.1901508 The Journal of Immunology

IRF8 Regulates Gram-Negative Bacteria–Mediated NLRP3 Inflammasome Activation and Cell Death

Rajendra Karki,*,1 Ein Lee,*,†,1 Bhesh R. Sharma,*,1 Balaji Banoth,* and Thirumala-Devi Kanneganti*

Inflammasomes are intracellular signaling complexes that are assembled in response to a variety of pathogenic or physiologic stimuli to initiate inflammatory responses. Ubiquitously present LPS in Gram-negative bacteria induces NLRP3 inflammasome activation that requires caspase-11. We have recently demonstrated that IFN regulatory factor (IRF) 8 was dispensable for caspase-11–mediated NLRP3 inflammasome activation during LPS transfection; however, its role in Gram-negative bacteria– mediated NLRP3 inflammasome activation remains unknown. In this study, we found that IRF8 promotes NLRP3 inflammasome activation in murine bone marrow–derived macrophages (BMDMs) infected with Gram-negative bacteria such as Citrobacter

rodentium, Escherichia coli,orPseudomonas aeruginosa mutant strain DpopB. Moreover, BMDMs deficient in IRF8 showed Downloaded from substantially reduced caspase-11 activation and gasdermin D cleavage, which are required for NLRP3 inflammasome activation. Mechanistically, IRF8-mediated phosphorylation of IRF3 was required for Ifnb transcription, which in turn triggered the caspase- 11–dependent NLRP3 inflammasome activation in the infected BMDMs. Overall, our findings suggest that IRF8 promotes NLRP3 inflammasome activation during infection with Gram-negative bacteria. The Journal of Immunology, 2020, 204: 000–000.

nflammasomes are molecular platforms that are assembled caspase 4/5 (in humans) and occurs strictly in response to the http://www.jimmunol.org/ in response to a variety of pathogenic or physiologic stimuli Gram-negative bacteria–derived PAMP LPS (3, 4). Cytosolic LPS I to initiate activation of inflammatory caspases, resulting in activates caspase-11, leading to the cleavage of gasdermin D production and cell death. Although some inflammasomes (GSDMD), which is sufficient to induce pyroptosis (5, 6). Processing have defined ligands, the NLRP3 inflammasome serves as a global of the proinflammatory IL-1b and IL-18 is still dependent sensor of pathogen-associated molecular patterns (PAMPs) and on subsequent NLRP3 inflammasome activation downstream of damage-associated molecular patterns (1). The recognition of caspase-11 activation. Thus, successful cytoplasmic delivery of PAMPs and damage-associated molecular patterns by NLRP3 LPS via intracellular infection with Gram-negative bacteria, de- requires two steps: priming and activation. The priming step leads livery of bacterial outer membrane vesicles, or direct LPS trans- to the transcriptional upregulation of NLRP3 and pro–IL-1b;the fection is a fundamental requirement for noncanonical NLRP3 by guest on September 25, 2021 activation step then leads to NLRP3 oligomerization and initiates inflammasome activation (7). the assembly of the NLRP3 inflammasome (2). However, non- IFN signaling has been recognized as a central regulator of canonical NLRP3 activation requires caspase-11 (in mice) or inflammasome activation during bacterial infections. In particular, type I IFN priming is required for noncanonical NLRP3 inflam- masome activation during infection with Gram-negative bacteria (8). *Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN We and others have previously discovered that the TLR4– axis 38105; and †Integrated Biomedical Sciences Program, University of Tennessee regulates caspase-11 expression and noncanonical NLRP3 inflam- Health Science Center, Memphis, TN 38163 masome activation during infection with enteropathogens such as 1 R.K., E.L., and B.R.S. contributed equally. Escherichia coli and Citrobacter rodentium (9–11). However, bone ORCIDs: 0000-0003-3609-833X (B.R.S.); 0000-0002-6395-6443 (T.-D.K.). marrow–derived macrophages (BMDMs) deficient in TRIF or the Received for publication December 20, 2019. Accepted for publication February 24, IFN-a/b (IFNAR) undergo pyroptosis at a rate similar to 2020. that of wild-type (WT) BMDMs transfected with LPS, suggesting This work was supported by funding from the National Institutes of Health Grants that TRIF/IFN signaling is dispensable for noncanonical NLRP3 CA163507, AR056296, AI124346, and AI101935 and by American Lebanese Syrian Associated Charities (to T.‐D.K.). inflammasome activation by LPS transfection (4). IFN-inducible R.K. and T.-D.K. conceptualized the study; R.K., E.L., and B.R.S. designed the guanylate-binding proteins (GBPs) and immunity-related GTPase methodology; R.K., E.L., B.R.S., and B.B. performed the experiments; R.K., E.L., family member b10 (IRGB10) also contribute to noncanonical B.R.S., and B.B. conducted the analysis; and R.K. and T.-D.K. wrote the manuscript NLRP3 inflammasome activation by liberating Gram-negative with input from all authors. T.-D.K. acquired the funding and provided overall supervision. bacteria from pathogen-containing vacuoles and disrupting the Address correspondence and reprint requests to Dr. Thirumala-Devi Kanneganti, structural integrity of the bacteria. This process ultimately St. Jude Children’s Research Hospital, MS #351, 570 St. Jude Place, Suite E7004, releases LPS into the cytoplasm, allowing detection by caspase-11 Memphis, TN 38105-2794. E-mail address: [email protected] (12–14). The online version of this article contains supplemental material. Type I IFN induction in dendritic cells is greatly enhanced by Abbreviations used in this article: ASC, apoptosis-associated speck-like protein con- IFN regulatory factor (IRF) 8 via the prolongation of recruitment taining a caspase activation and recruitment domain; BMDM, bone marrow–derived macrophage; CST, Technology; GBP, guanylate-binding protein; of basal transcription machinery to the IFN promoters during viral GSDMD, gasdermin D; IRF, IFN regulatory factor; IRGB10, immunity-related infection (15). Similarly, the concerted activation of IRF8 and GTPase family member b10; LB, Luria-Bertani; MOI, multiplicity of infection; IRF3 in human monocytes regulates IFN-b production in response PAMP, pathogen-associated molecular pattern; t, total; WT, wild-type. to LPS or viral infection (16), suggesting IRF8 is a critical Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 contributor to the rapid and abundant type I IFN production in

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1901508 2 REGULATION OF CASPASE-11–MEDIATED NLRP3 INFLAMMASOME BY IRF8 immune cells. Our recent study found that IRF8 is not required (catalog no. 4302; CST), anti–p-IKKε (catalog no. 8766; CST), anti– for noncanonical NLRP3 inflammasome activation during LPS t-IKKε (catalog no. 3416; CST), anti-GAPDH (catalog no. 5174; CST), transfection (17). However, given that IRF8 plays an impor- anti-IRF1 (catalog no. 8478; CST), anti-GBP2 (catalog no. 27299-1-AP; b Proteintech), anti-GBP5 (catalog no. 13220-1-AP; Proteintech), anti–p-STAT1 tant role in IFN- production, we hypothesized that IRF8 (catalog no. 7649; CST), anti–t-STAT1 (catalog no. 14994; CST), anti–caspase-3 would be required for Gram-negative bacteria–mediated NLRP3 (catalog no. 9662; CST), anti–cleaved caspase-3 (catalog no. 9661; CST), inflammasome activation. anti–caspase-7 (catalog no. 9492; CST), anti–cleaved caspase-7 (catalog no. 9491; CST), anti–caspase-8 (AG-20T-0138-C100; AdipoGen Life Sciences), anti–cleaved caspase-8 (catalog no. 8592; CST), anti-IRGB10 Materials and Methods rabbit serum raised against recombinant full-length IRGB10 (1:10,000 Mice dilution) (21), and anti-IRF8 (catalog no. A5798; ABclonal). Membranes 2/2 2/2 2/2 2/2 2/2 were then washed and incubated with the appropriate HRP-conjugated Irf8 (17), Stat1 (18), Casp11 (14), Irgb10 (14), and Nlrp3 secondary Abs (1:5000 dilution; Jackson ImmunoResearch Laboratories, (14) mice have been described previously. Six-to-eight-week-old male and anti-rabbit [111-035-047], anti-mouse [315-035-047], and anti-rat [112-035-003]) female mice were used in this study. Mice were bred at St. Jude Children’s for 1 h. Proteins were visualized by using Luminata Forte Western HRP Research Hospital. Animal studies were conducted according to the proto- Substrate (WBLUF0500; Millipore). cols approved by the St. Jude Institutional Animal Care and Use committee. Real-time cell death analysis Bacterial culture Real-time cell death assays were performed using a two-color IncuCyte C. rodentium (51549; American Type Culture Collection), E. coli (11775; ZOOM incubator imaging system (Essence Biosciences). BMDMs were American Type Culture Collection), and the isogenic mutant of Pseudomonas D seeded into 12-well plates and incubated at 37˚C overnight. On the next aeruginosa strain popB (providedbyDr.B.Berwin,Departmentof day, following bacterial infection, 20 nM SYTOX Green (S7020; Life Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Technologies), a cell-impermeable DNA-binding fluorescent dye, was Downloaded from Lebanon, NH), were inoculated into Luria-Bertani (LB) media (3002-031; added, and the resulting images were analyzed using the software package MP Biomedicals) and incubated overnight under aerobic conditions at 37˚C. supplied with the IncuCyte imager, which allows precise analysis of the Bacteria were subcultured (1:25) into fresh LB media for 3 h at 37˚C to number of SYTOX Green+ cells present in each image. Experiments were generate log phase bacteria for infection. conducted using a minimum of three separate wells for each experimental Cytokine analysis condition and a minimum of six image fields per well. Dead cell events were counted based on SYTOX Green staining and plotted using GraphPad

Cytokines were measured by performing multiplex ELISA (MCYTOMAG-70K; Prism v6.0 software. http://www.jimmunol.org/ Millipore), ELISA for IL-18 (BMS618-3; Invitrogen) according to the manufacturer’s instructions (19), or ELISA for IFN-b (439407; BioLegend) Real-time quantitative PCR analysis according to the manufacturer’s instructions. For bacterial stimulation, the following conditions were used: E. coli, D Cell culture and stimulation C. rodentium,and popB at 10 MOI for 2, 4, and 6 h. RNA was extracted using TRIzol (15596026; Thermo Fisher Scientific) according to the BMDMs were cultured for 6 d in DMEM (11995-073; Thermo Fisher manufacturer’s instructions. The isolated RNA was reverse-transcribed Scientific) supplemented with 1% nonessential amino acids (11140-050; into cDNA by using a First-Strand cDNA Synthesis Kit (4368814; Thermo Fisher Scientific), 10% FBS (S1620; Biowest), 30% medium Applied Biosystems). Real-time quantitative PCR was performed conditioned by L929 mouse fibroblasts, and 1% penicillin and streptomycin on an Applied Biosystems 7500 real-time PCR instrument by using 3 (15070-063; Thermo Fisher Scientific). BMDMs in antibiotic-free medium 2 SYBR Green (4368706; Applied Biosystems) and the appropriate by guest on September 25, 2021 were seeded onto 12-well plates at a density of 1 3 106 cells per well, primers. Real-time quantitative PCR primer sequences are detailed in followed by overnight incubation. For bacterial infection, the following Table I. conditions were used: E. coli and C. rodentium at 1, 10, or 20 multiplicity of infection (MOI) for 16 h of incubation (for activation of caspases); Statistical analysis D popB at 10, 20, or 50 MOI for 16 h of incubation (for activation of GraphPad Prism v6.0 software was used for data analysis. Data are shown as D caspases); and E. coli, C. rodentium, and popB at 10 MOI for indicated mean 6 SEM. Statistical significance was determined by t tests (two-tailed) time of incubation (for signaling assessment). Cell culture supernatants for two groups and ANOVA (with Dunnett multiple comparisons test, Sidak were collected for ELISAs. multiple comparisons test, or Tukey multiple comparisons test) for three or more groups. The p values , 0.05 were considered to be statistically Bacterial phagocytic uptake significant. BMDMs were grown in 12-well plates and infected with 20 MOI of E. coli, C. rodentium, and DpopB at 37˚C. Two hours postinfection, cells were washed three times with PBS. Cells were then lysed in 0.1% Triton X-100. Results The lysates were then serially diluted and plated on LB agar plates. The IRF8 promotes NLRP3 inflammasome activation by colonies were counted after 18 h of growth at 37˚C. Gram-negative bacteria Immunoblot analysis Noncanonical NLRP3 inflammasome activation is observed BMDM cell lysates and culture supernatants were combined in caspase lysis in response to a number of Gram-negative bacteria because of buffer (containing protease inhibitors, phosphatase inhibitors, 10% NP-40, the ubiquitous presence of LPS in their outer membrane (3). and 25 mM DTT) and sample loading buffer (containing SDS and 2-ME) Both C. rodentium and E. coli are considered classical triggers for immunoblot analysis of caspase-1 (20). For immunoblot analysis of of caspase-11–mediated NLRP3 inflammasome activation (9). signaling, supernatants were removed, and BMDMs were washed once with PBS, then lysed in RIPA buffer and sample loading buffer (containing Additionally, our recent findings show that the P. aeruginosa SDS and 2-ME). Proteins were separated by electrophoresis through mutant strain DpopB can also engage the noncanonical NLRP3 8–12% polyacrylamide gels. Following electrophoretic transfer of proteins inflammasome (22). To investigate the role of IRF8 in regulating onto PVDF membranes (IPVH00010; Millipore), nonspecific binding was Gram-negative bacteria–mediated noncanonical NLRP3 inflamma- blocked by incubation with 5% skim milk, then membranes were incubated some activation, we first infected WT and Irf82/2 BMDMs with with primary Abs: anti–caspase-1 (catalog no. AG-20B-0042; AdipoGen Life Sciences), anti–caspase-11 (catalog no. NB120-10454; Novus Biologicals), E. coli and analyzed caspase-1 activation. We observed reduced 2/2 anti-GSDMD (catalog no. Ab209845; Abcam), anti-NLRP3 (catalog no. caspase-1 activation in Irf8 BMDMs compared with WT BMDMs AG-20B-0014; AdipoGen Life Sciences), anti–apoptosis-associated (Fig. 1A, Supplemental Fig. 1A). The decreased production of speck-like protein containing a caspase activation and recruitment do- IL-18 and IL-1b in E. coli–infected Irf82/2 BMDMs provided main (ASC) (catalog no. AG-25B-006-C100; AdipoGen Life Sciences), 2/2 anti–pro-IL-1b (catalog no. 12507; Cell Signaling Technology [CST]), further evidence for reduced caspase-1 activation in Irf8 BMDMs anti–p-TBK1 (catalog no. 5483; CST), anti–total (t)-TBK1 (catalog (Fig. 1B). In line with previous reports (9), caspase-1 activation no. 3504; CST), anti–p-IRF3 (catalog no. 37829S; CST), anti–t-IRF3 andcytokineproductioninE. coli–infected WT BMDMs were The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 1. IRF8 promotes NLRP3 inflammasome activation by Gram-negative bacteria. (A) Immunoblot analysis of pro–caspase-1 (CASP1) (p45) and cleaved CASP1 (p20) in WT or Irf82/2 BMDMs infected with E. coli (MOI 20) for the indicated time. (B) Assessment of IL-18 and IL-1b release by ELISA in BMDMs 16 h after E. coli infection. (C) Real-time cell death analysis by IncuCyte and SYTOX Green staining in BMDMs infected with E. coli for the indicated time. (D) Representative images of BMDMs under light microscopy after E. coli infection for 16 h. The arrows indicate pyroptotic cells. Original magnification 320. (E) Immunoblot analysis of pro-CASP1 (p45) and cleaved CASP1 (p20) in WT or Irf82/2 BMDMs infected with C. rodentium (MOI 20) for the indicated time. (F) Assessment of IL-18 and IL-1b release by ELISA in BMDMs 16 h after C. rodentium infection. (G) Immunoblot analysis of pro-CASP1 (p45) and cleaved CASP1 (p20) in WT or Irf82/2 BMDMs infected with P. aeruginosa DpopB (MOI 50) for the indicated time. (H) Assessment of IL-18 and IL-1b release by ELISA in BMDMs 16 h after P. aeruginosa DpopB infection. Data are representative of three experiments (A, D, E, and G) or are from three independent experiments (B, C, F, and H) (mean 6 SEM). *p , 0.05, **p , 0.01, ***p , 0.001 by one-way ANOVAwith Dunnett multiple comparisons test. dependent on both NLRP3 and caspase-11 (Fig. 1B, Supplemental requiring downstream NLRP3 (23). Cell death analysis showed Fig. 1A). Another feature of noncanonical NLRP3 inflammasome reduced cell death in BMDMs lacking IRF8 compared with WT signaling is caspase-11 activation inducing pyroptosis without BMDMs postinfection with E. coli (Fig. 1C, 1D). Casp112/2 4 REGULATION OF CASPASE-11–MEDIATED NLRP3 INFLAMMASOME BY IRF8

BMDMs but not Nlrp32/2 BMDMs were protected from pyroptotic IRF8 promotes inflammasome activation without affecting cell death (Fig. 1C, 1D). Similar phenomena were observed in bacterial uptake (Supplemental Fig. 3). Overall, our results Irf82/2 BMDMs following infection with C. rodentium and DpopB indicate that IRF8 is required for optimal noncanonical NLRP3 (Fig. 1E–H, Supplemental Figs. 1B, 1C, 2). However, release of inflammasome activation in cells infected with Gram-negative inflammasome-independent cytokines KC, TNF, and IL-10 fol- bacteria. lowing bacterial infections was not impaired in the absence of IRF8 (Supplemental Fig. 3). Altogether, these results suggest IRF8 regulates GSDMD activation but not other components of that IRF8 contributes to caspase-11–mediated NLRP3 inflamma- the NLRP3 inflammasome some activation and pyroptosis during infection with Gram-negative Bacterial infection activates TLR signaling to upregulate the ex- bacteria. pression of NLRP3 and pro–IL-1b, which potentiates NLRP3 Changes in bacterial uptake can affect inflammasome acti- inflammasome activation (2). Failure to upregulate NLRP3 in the vation (24). To understand whether the reduced inflammasome absence of IRF8 may explain the reduced NLRP3 inflammasome activation observed in Irf82/2 BMDMs during infection could activation observed in Irf82/2 BMDMs during bacterial infection. be because of decreased bacterial uptake, cells were infected To investigate whether there is a priming defect in Irf82/2 with E. coli, C. rodentium,orDpopB, and bacterial uptake BMDMs, we analyzed the protein expression of inflammasome was quantified at 2 h postinfection. We found similar bacte- components during E. coli, C. rodentium,orDpopB infection. rial uptake in BMDMs from both genotypes, suggesting that The protein expression of NLRP3, ASC, and pro–IL-1b was not Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 2. IRF8 regulates GSDMD activation. (A) Immunoblot analysis of NLRP3, ASC, pro–IL-1b, caspase-11 (CASP11), and GSDMD in WT or Irf82/2 BMDMs infected with E. coli (MOI 10) for the indicated time. (B) Immunoblot analysis of NLRP3, ASC, pro–IL-1b, CASP11, and GSDMD in WT or Irf82/2 BMDMs infected with C. rodentium (MOI 10) for the indicated time. (C) Immunoblot analysis of NLRP3, ASC, pro–IL-1b, CASP11, and GSDMD in WT or Irf82/2 BMDMs infected with P. aeruginosa DpopB (MOI 10) for the indicated time. (D) Immunoblot analysis of p-IkBa and IkBa in WT or Irf82/2 BMDMs infected with E. coli (MOI 10) for the indicated time. GAPDH was used as an internal control. Data are representative of three independent experiments. The Journal of Immunology 5 impaired in Irf82/2 BMDMs, demonstrating that the priming When caspase-11 recognizes bacterial LPS, it oligomerizes and signal required for NLRP3 inflammasome activation was not becomes activated, which in turn cleaves GSDMD to generate the compromisedintheabsenceofIRF8(Fig.2A–C).Thesimilar pyroptogenic 30 kDa N-terminal fragment of GSDMD (5, 6, 25). induction of NLRP3 was further supported by comparable Therefore, reduced caspase-11 activation can result in reduced activation of NF-kB between WT and Irf82/2 BMDMs during GSDMD cleavage. We observed reduced caspase-11 activation infection with E. coli (Fig. 2D). Additionally, upregulation and in Irf82/2 BMDMs compared with WT BMDMs during E. coli activation of caspase-11 during infection are crucial for non- infection (Fig. 3A). Recently caspase-8 has also been shown canonical NLRP3 inflammasome activation. We observed similar to induce GSDMD cleavage during Yersinia infection (26). We ob- upregulation of pro–caspase-11 expression in WT and Irf82/2 served reduced activation of caspase-8 and downstream executioner BMDMs (Fig. 2A–C). Although the expression of GSDMD was caspases, caspase-3 and -7, in Irf82/2 BMDMs compared with WT similar between WT and Irf82/2 BMDMs, there was a reduction BMDMs postinfection with E. coli (Fig. 3A). Similar findings were in the cleavage of GSDMD in Irf82/2 BMDMs during the bac- observed upon infection with C. rodentium and DpopB (Fig. 3B, terial infection (Fig. 2A–C), suggesting that IRF8 acts upstream of 3C). Altogether, these results suggest that IRF8 enhances caspase-11 GSDMD activation. and caspase-8 cleavage to induce GSDMD activation. Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 3. IRF8 regulates both CASP11 and CASP8 activation. (A) Immunoblot analysis of pro– and cleaved caspase-11 (CASP11), caspase-8 (CASP8), caspase-3 (CASP3), and caspase-7 (CASP7) in WT or Irf82/2 BMDMs infected with E. coli (MOI 20) for the indicated time. (B) Immunoblot analysis of pro- and cleaved CASP11, CASP8, CASP3, and CASP7 in WT or Irf82/2 BMDMs infected with C. rodentium (MOI 20) for the indicated time. (C) Immunoblot analysis of pro- and cleaved CASP11, CASP8, and CASP7 in WT or Irf82/2 BMDMs infected with P. aeruginosa DpopB (MOI 50) for the indicated time. Data are representative of three independent experiments. 6 REGULATION OF CASPASE-11–MEDIATED NLRP3 INFLAMMASOME BY IRF8

FIGURE 4. IRF8 enhances IFN-b production, and IFN-b supplementa- tion rescues inflammasome activation in Irf82/2 BMDMs. (A–C) The ex- pression of Ifnb in WT or Irf82/2 BMDMsat2,4,and6hpostinfection with E. coli (MOI 10), C. rodentium (MOI 10), or P. aeruginosa DpopB (MOI 10). (D) Assessment of IFN-b release by ELISA in WT or Irf82/2 BMDMs 12 h after E. coli (MOI 10), C. rodentium (MOI 10), or P. aeruginosa DpopB (MOI 10) in- E b fection. ( ) Assessment of IFN- Downloaded from release by ELISA in WT or Irf82/2 BMDMs9hafterstimulationwithLPS (100 ng/ml) or poly(I:C) (10 mg/ml) in the media. (F and G) Immunoblot analysis of pro– and cleaved caspase-1 (CASP1) and GSDMD and the as-

sessment of IL-18 release in BMDMs http://www.jimmunol.org/ pretreated with IFN-b (400 U/ml) for 4hfollowedbyinfectionwithE. coli (MOI 20) for an additional 16 h. Data are representative of three experiments (F) or are from three independent ex- periments (A–E and G)(mean6 SEM). ***p , 0.001, ****p , 0.0001 by one-way ANOVAwith Sidak multiple comparisons test (D and E) and two- way ANOVA with Tukey multiple by guest on September 25, 2021 comparisons test (G).

IRF8 promotes IFN-b production for NLRP3 inflammasome rIFN-b and infected them with E. coli, C. rodentium,orDpopB. activation by Gram-negative bacteria We observed that IFN-b supplementation restored caspase-1 and b 2/2 Type I IFN production and signaling through the IFNR are re- GSDMD cleavage and IL-18 and IL-1 production in Irf8 quired for noncanonical NLRP3 inflammasome activation by BMDMs to levels similar to those in WT BMDMs (Fig. 4F, 4G, b Gram-negative bacteria (10). Given that IRF8 regulates rapid and Supplemental Fig. 4), suggesting that IRF8-regulated IFN- pro- abundant type I IFN production in immune cells (15, 16), we duction facilitates noncanonical inflammasome activation during hypothesized that IRF8-mediated IFN-b production during in- Gram-negative bacterial infection. b fection with Gram-negative bacteria could regulate noncanonical The TLR4/TRIF signaling axis triggers upregulation of IFN- ε inflammasome activation. To test this, we infected WT and Irf82/2 via the IKK and TBK1 signaling network, which induces BMDMs with E. coli, C. rodentium, and DpopB and determined phosphorylation of IRF3 (27). This causes IRF3 dimerization and b the expression and protein production levels of IFN-b.We translocation to the nucleus, where it binds to the IFN- promoter b to stimulate IFN-b transcription (27). The reduced IFN-b tran- observed reduced IFN- transcription and protein production 2/2 in Irf82/2 BMDMs compared with WT BMDMs, demonstrat- scription and protein production in Irf8 BMDMs could be the ing that IRF8 regulates IFN-b production during infection with Gram-negative bacteria (Fig. 4A–D, Table I). Likewise, we stimu- Table I. Real-time quantitative PCR primer sequences lated WT and Irf82/2 BMDMs with LPS or poly(I:C) and measured the amount of IFN-b released in cell supernatants. As expected, Gene Primer Sequence 2/2 b Irf8 BMDMs produced less IFN- compared with WT BMDMs Ifnb Forward: 59-GCCTTTGCCATCCAAGAGATGC-39 (Fig. 4E). To confirm whether impaired noncanonical inflamma- Reverse: 59-ACACTGTCTGCTGGTGGAGTTC-39 some activation in Irf82/2 BMDMs was a consequence of reduced Hprt Forward: 59-CTCATGGACTGATTATGGACAGGAC-39 IFN-b production, we supplemented WT and Irf82/2 BMDMs with Reverse: 59-GCAGGTCAGCAAAGAACTTATAGCC-39 The Journal of Immunology 7 result of reduced activation of the molecules in the signaling Discussion cascade for IFN-b production. To test whether IRF8 regulates IRF8 plays a key role in NLRC4 inflammasome activation by signaling steps upstream of IFN-b transcription during infection regulating the transcription of encoding NAIPs and NLRC4 2/2 with Gram-negative bacteria, we infected WT and Irf8 BMDMs (17). However, IRF8 is dispensable for AIM2, pyrin, and NLRP3 with E. coli or C. rodentium and measured the kinetics of IKKε, inflammasome activation in response to classical triggers and LPS TBK1, and IRF3 phosphorylation. We observed reduced phos- transfection. In this study, we identified an additional role for 2 2 phorylation of IRF3 but not IKKε and TBK1 in Irf8 / BMDMs IRF8 in promoting Gram-negative bacteria–mediated NLRP3 compared with WT BMDMs (Fig. 5), demonstrating that IRF8 inflammasome activation. Deficiency of IRF8 in BMDMs resulted mediated the activation of IRF3 to trigger IFN-b gene upregula- in reduced NLRP3 inflammasome activation, caspase-11 and GSDMD tion during infection with Gram-negative bacteria. Overall, these cleavage, and cell death. However, the expression of major com- data suggest that IRF8 controls IFN-b production during infection ponents of the noncanonical NLRP3 inflammasome, including with Gram-negative bacteria to allow noncanonical inflammasome NLRP3, ASC, caspase-11, and GSDMD, was not impaired in activation. IRF8-deficient BMDMs. The supplementation of IFN-b in Irf82/2 BMDMs rescued the inflammasome activation, suggesting that IRF8 promotes NLRP3 inflammasome activation independently reduced production of IFN-b in Irf82/2 BMDMs was the driving of GBPs and IRGB10 force for the attenuated inflammasome activation. We also found IFN-inducible proteins, especially GBPs and IRGB10, are known that IRF8 regulated IRF3 phosphorylation to control the production to activate caspase-11 by targeting and destroying pathogen- of IFN-b. containing vacuoles to release bacterial LPS, thereby assembling The inflammatory caspases caspase-1 and -11 and the apoptotic Downloaded from the NLRP3 inflammasome (14). Given that IRF8 regulates IFN-b caspase caspase-8 are activated during microbial infections and are production (Fig. 4), we hypothesized that IRF8-regulated GBPs and known to cleave GSDMD, a key executioner of pyroptosis (6, 26). IRGB10 induction contribute to NLRP3 inflammasome activation. The precise molecular mechanism for the activation of the To test this, we determined the kinetics of IRF1, GBP2, GBP5, NLRP3 inflammasome by caspase-11 is still unknown. Further- and IRGB10 induction in WT and Irf82/2 BMDMs during in- more, caspase-8 also acts as an effector and regulator of NLRP3 fection with E. coli and C. rodentium. We found that IRF1, GBP2, inflammasome signaling (28). The decreased GSDMD cleavage http://www.jimmunol.org/ 2 2 GBP5, and IRGB10 were similarly induced in both genotypes and NLRP3 inflammasome activation in Irf8 / BMDMs could (Fig. 6A, 6B), suggesting that the reduced inflammasome acti- be explained by the reduced activation of both caspase-11 and vation observed in Irf82/2 BMDMs was not associated with caspase-8. However, the cross-talk between these caspases in the GBPs or IRGB10. Reduced IFN-b production in Irf82/2 BMDMs regulation of the NLRP3 inflammasome during bacterial infections would affect activation of STAT1, which is a central hub for the needs further investigation. Caspase-3 activation during bacterial IFN signaling pathway. As expected, we observed defective infection is one of the consequences of caspase-8 activation. STAT1 activation in Irf82/2 BMDMs (Fig. 6C). STAT1 has pre- Caspase-3 can cleave GSDME, another member of the gasdermin viously been shown to be required for GBP2 and GBP5 induction family, which has been shown to induce pyroptosis in certain cancer to activate the AIM2 inflammasome during Francisella infection cells (29). Therefore, the reduced caspase-3 activation observed in by guest on September 25, 2021 2 2 (18). However, we observed similar kinetics of GBP2 and GBP5 Irf8 / BMDMs could also be contributing to decreased pyroptotic induction in Stat12/2 BMDMs during Gram-negative bacterial cell death executed by GSDME. infection (Fig. 6D). Despite having similar GBP2 and GBP5 The molecules involved in type I IFN production and IFN expression, Stat12/2 and Irf82/2 BMDMs showed reduced signaling promote NLRP3 inflammasome activation during caspase-1 cleavage during C. rodentium infection (Fig. 6E), Gram-negative bacterial infection but not in response to LPS trans- 2 2 suggesting that IRF8 and STAT1 regulate NLRP3 inflammasome fection (4). The supplementation of IFN-b in Trif / BMDMs activation by Gram-negative bacteria independently of GBPs and rescues the reduced inflammasome activation in those cells. IRGB10. However, the detailed molecular mechanisms behind IFN-b that

FIGURE 5. IRF8 activates IRF3 for IFN-b production. (A and B) Immunoblot analysis of p-IRF3, IRF3, p-TBK1, TBK1, p-IKKε, and IKKε in WT or Irf82/2 BMDMs infected with E. coli (MOI 10) and C. rodentium (MOI 10) for the indicated time. GAPDH was used as an internal control. Data are representative of three experiments. 8 REGULATION OF CASPASE-11–MEDIATED NLRP3 INFLAMMASOME BY IRF8

FIGURE 6. IRF8 does not reg- ulate GBPs and IRGB10 for NLRP3 inflammasome activation. (A) Im- munoblot analysis of IRF1, GBP2, andGBP5inWTorIrf82/2 BMDMs after E. coli (MOI 10) or C. rodentium (MOI 10) infection for the indicated time. (B) Immunoblot analysis of IRGB10 and IRF8 in WT or Irf82/2 E. coli

BMDMs after (MOI 10) in- Downloaded from fection for the indicated time. (C) Immunoblot analysis of p-STAT1 and STAT1inWTorIrf82/2 BMDMs after E. coli (MOI 10) or C. rodentium (MOI 10) infection for the indicated time. (D) Immunoblot analysis of 2/2

GBP2 and GBP5 in WT, Irf8 ,or http://www.jimmunol.org/ Stat12/2 BMDMs after E. coli (MOI 10) or C. rodentium (MOI 10) in- fection for the indicated time. (E) Immunoblot analysis of pro–caspase-1 (CASP1) (p45) and cleaved CASP1 (p20) in WT, Irf82/2, Stat12/2,or Casp112/2 BMDMs infected with C. rodentium (MOI 20) for 16 h. Data are representative of three inde- pendent experiments. by guest on September 25, 2021

enhance NLRP3 inflammasome activation during bacterial infec- IRGB10 in the WT and Irf82/2 BMDMs infected with Gram- tion are obscure. Several studies have suggested that members of negative bacteria is, therefore, possibly a result of the similar the GBP and IRG families, which are induced during bacterial activation of NF-kB signaling. Alternatively, the low level of IFN-b infection in an IFN-b–dependent manner, target and destroy in Irf82/2 BMDMs may be sufficient to induce GBPs to the extent pathogen-containing vacuoles to release bacterial LPS, resulting observed in WT BMDMs. Indeed, constitutive IFN has been shown in caspase-11 activation and assembly of the NLRP3 inflamma- to maintain the level of GBP expression required for the release of some (14, 18). Despite reduced IFN-b production in Irf82/2 bacterial ligands upstream of pyroptosis (35). Therefore, it is BMDMs, we did not observe differential induction of caspase-11, likely that other inducible genes that are unique to the IFN sig- GBP2, GBP5, or IRGB10 expression between WT and Irf82/2 naling pathway promote NLRP3 inflammasome activation, which BMDMs. The pro–caspase-11 and GBP5 promoters contain is further supported by the attenuated but not abolished caspase-11 NF-kB and STAT binding sites, and their expression is induced and caspase-1 cleavage observed in Gbpchr3 cells (12, 14) during by NF-kB–activating ligands such as LPS and TNF (30–32). bacterial infection. Similarly, both NF-kB and TRIF signaling contribute to GBP2 and Previous studies have shown that IRF8 contributes to IFN-b IRGB10 induction (33). The Gram-negative bacteria not only in- production in murine dendritic cells and human monocytes (16). duce type I IFN but also robustly activate NF-kB signaling (14, 34). The scaffold complex consisting of IRF8 and PU.1 on the IFN-b The similar IkBa phosphorylation, along with the similar expres- promoter recruits IRF3 for the rapid induction of IFN-b tran- sion of NLRP3, observed in WT and Irf82/2 BMDMs suggests that scription in human monocytes (16). Upon stimulation or infection, IRF8 does not regulate NF-kB signaling during the bacterial in- cytosolic IRF3 undergoes phosphorylation and dimerization and fection. The similar expression of caspase-11, GBP2, GBP5, and then translocates into the nucleus to carry out its transcriptional The Journal of Immunology 9 activity. In this study, we found reduced phosphorylation of IRF3 15. Tailor, P., T. Tamura, H. J. Kong, T. Kubota, M. Kubota, P. Borghi, L. Gabriele, 2/2 b and K. Ozato. 2007. The feedback phase of type I induction in den- in Irf8 BMDMs, which accounted for the decreased IFN- dritic cells requires interferon regulatory factor 8. Immunity 27: 228–239. production during bacterial infection. IRF3 phosphorylation relies 16. Li, P., J. J. Wong, C. Sum, W. X. Sin, K. Q. Ng, M. B. Koh, and K. C. Chin. on TBK1 activation. However, TBK1 was similarly phosphorylated 2011. IRF8 and IRF3 cooperatively regulate rapid interferon-b induction in 2/2 human blood monocytes. Blood 117: 2847–2854. in WT and Irf8 BMDMs, suggesting that IRF8 activates IRF3 17. Karki, R., E. Lee, D. Place, P. Samir, J. Mavuluri, B. R. Sharma, A. Balakrishnan, without impairing the activation of TBK1. It is possible that IRF8 R. K. S. Malireddi, R. Geiger, Q. Zhu, et al. 2018. IRF8 regulates transcription of interacts with both TBK1 and IRF3, promoting the phosphoryla- naips for NLRC4 inflammasome activation. Cell 173: 920–933.e13. 18. Man, S. M., R. Karki, R. K. Malireddi, G. Neale, P. Vogel, M. Yamamoto, tion of IRF3 by TBK1. Indeed, IRF8 has been shown to be an M. Lamkanfi, and T. D. Kanneganti. 2015. The IRF1 and interacting partner of IRF3 in human monocytes (16). Furthermore, a guanylate-binding proteins target activation of the AIM2 inflammasome by Francisella infection. Nat. Immunol. 16: 467–475. similar bridging concept has been demonstrated with STING and 19. Sharma, B. R., R. Karki, E. Lee, Q. Zhu, P. Gurung, and T. D. Kanneganti. 2019. RIOK3 in the activation of the type I IFN pathway (36). The increased Innate immune adaptor MyD88 deficiency prevents skin inflammation in production of KC and expression of pro–IL-1b in Irf82/2 BMDMs SHARPIN-deficient mice. Cell Death Differ. 26: 741–750. 20. Karki, R., S. M. Man, R. K. S. Malireddi, P. Gurung, P. Vogel, M. Lamkanfi, and could be a consequence of decreased production of type I IFN in those T. D. Kanneganti. 2015. Concerted activation of the AIM2 and NLRP3 cells. Type I IFNs produced during Streptococcus pyogenes infection inflammasomes orchestrates host protection against Aspergillus infection. Cell inhibit the production of IL-1b to prevent lethal systemic hyper- Host Microbe 17: 357–368. 21. Steinfeldt, T., S. Ko¨nen-Waisman, L. Tong, N. Pawlowski, T. Lamkemeyer, inflammation of soft tissue (37). Similarly, KC is upregulated in L. D. Sibley, J. P. Hunn, and J. C. Howard. 2015. Correction: phosphorylation of peritoneal macrophages from IFN-b null mice upon bacterial in- mouse immunity-related GTPase (IRG) resistance proteins is an evasion strategy for virulent Toxoplasma gondii. PLoS Biol. 13: e1002199. fection (38). Altogether, our findings provide insights into the role 22. Balakrishnan, A., R. Karki, B. Berwin, M. Yamamoto, and T. D. Kanneganti. of IRF8 in mediating noncanonical inflammasome activation. 2018. Guanylate binding proteins facilitate caspase-11-dependent pyroptosis in Downloaded from response to type 3 secretion system-negative Pseudomonas aeruginosa.[Pub- lished erratum appears in 2019 Cell Death Discov. 5: 116.] Cell Death Discov. 4: 3. Acknowledgments 23. He, Y., H. Hara, and G. Nu´n˜ez. 2016. Mechanism and regulation of NLRP3 We thank A. Burton (St. Jude Children’s Research Hospital) for technical inflammasome activation. Trends Biochem. Sci. 41: 1012–1021. 24. Patankar, Y. R., R. R. Lovewell, M. E. Poynter, J. Jyot, B. I. Kazmierczak, and support and Dr. R. Tweedell for scientific editing. B. Berwin. 2013. Flagellar motility is a key determinant of the magnitude of the inflammasome response to Pseudomonas aeruginosa. Infect. Immun. 81:

2043–2052. http://www.jimmunol.org/ Disclosures 25. Liu, X., Z. Zhang, J. Ruan, Y. Pan, V. G. Magupalli, H. Wu, and J. Lieberman. The authors have no financial conflicts of interest. 2016. Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores. Nature 535: 153–158. 26. Orning, P., D. Weng, K. Starheim, D. Ratner, Z. Best, B. Lee, A. Brooks, S. Xia, H. Wu, M. A. Kelliher, et al. 2018. Pathogen blockade of TAK1 References triggers caspase-8-dependent cleavage of gasdermin D and cell death. Science 1. Kanneganti, T. D. 2018. The inflammasome starts rolling. Nat. Rev. Immunol. 18: 362: 1064–1069. 483. 27. Fitzgerald, K. A., S. M. McWhirter, K. L. Faia, D. C. Rowe, E. Latz, 2. Karki, R., and T. D. Kanneganti. 2019. Diverging inflammasome signals in D. T. Golenbock, A. J. Coyle, S. M. Liao, and T. Maniatis. 2003. IKKepsilon and tumorigenesis and potential targeting. Nat. Rev. Cancer 19: 197–214. TBK1 are essential components of the IRF3 signaling pathway. Nat. Immunol. 4: 3. Kayagaki, N., S. Warming, M. Lamkanfi, L. Vande Walle, S. Louie, J. Dong, 491–496.

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Immunol. 195: 2461–2471. 1 SUPPLEMENTARY FIGURES AND LEGENDS

/– – /– A – /– – E. coli WT Irf8 Casp11Nlrp3

MOI 0 1 10 20 0 1 10 20 20 20 p45 CASP1 p20

/– – /– – B /– – C. rodentium WT Irf8 Casp11Nlrp3

MOI 0 1 10 20 0 1 10 20 20 20 p45 CASP1 p20

/– – /– – /– C – ΔpopB WT Irf8 Casp11Nlrp3

MOI 0 10 20 50 0 10 20 50 50 50 p45

CASP1 p20

2

3 Supplementary Figure 1. Caspase-1 cleavage by Gram-negative bacteria is enhanced by

4 IRF8.

5 (A–C) Immunoblot analysis of pro-caspase-1 (CASP1) (p45) and cleaved CASP1 (p20) in wild-

6 type (WT), Irf8–/–, Casp11–/–, or Nlrp3–/– bone marrow-derived macrophages (BMDMs) after

7 infection with the indicated multiplicity of infection (MOI) of E. coli,Supplemental C. rodentium, or FigureP. aeruginosa 1

8 ΔpopB for 16 h. Data are representative of 3 independent experiments.

1 A B WT Irf8–/–

) Media C. rodentium 5 15 WT WT –/– –/– 10 Irf8 Irf8 Casp11–/– Casp11–/– Casp11–/– Nlrp3–/– 5 Nlrp3–/– Nlrp3–/– rodentium

C. C. 0 Green objects/well (10 0 4 8 12 16 Time (h)

–/– C WT Irf8 D ) ) MediaMedia ΔpopBE. coli 5 155 15 ) WTWT Media WTΔpopBWT 5 15 Irf8–/–WT –/– Irf8–/–WT –/– 1010 Irf8 Irf8 –/– –/– –/– –/– 10 Casp11Casp11Irf8 Casp11Casp11Irf8 Casp11–/– –/– –/– Casp11–/– –/– –/– Casp11–/– Nlrp3–/– 5 5 Nlrp3Nlrp3 Nlrp3Nlrp3 popB 5 Nlrp3–/– Nlrp3–/– Δ 0 0 Green objects/well (10 Green objects/well (10 0 0 4 4 8 8 0 12 1216 16 Green objects/well (10 0 4 8 TimeTime (h) (h) 12 16 Time (h) 9

10 Supplementary Figure 2. IRF8 regulates cell death by Gram-negative bacteria

11 (A,C) Images of bone marrow-derived macrophages (BMDMs) under light microscopy after C.

12 rodentium (multiplicity of infection [MOI], 20) or P. aeruginosa ΔpopB (MOI, 50) infection for 16 h.

13 (B,D) Real time cell death analysis by Incucyte and SYTOX green staining after C. rodentium

14 (MOI, 20) or P. aeruginosa ΔpopB (MOI, 50) infection. Data are representative of 3 independent

15 experiments.

Supplemental Figure 2

2 A

E. coli NS NS NS 20 * 2.0 15 3

15 1.5 )/ml

10 7 2 10 1.0 5 1 KC (ng/ml) KC

5 TNF (ng/ml)

0.5 (ng/ml) IL-10 CFU (x 10 (x CFU 0 0.0 0 0 /– /– /– /– – – – – WT WT WT WT B Irf8 Irf8 Irf8 Irf8

C. rodentium NS NS NS 15 * 1.5 15 1.0 0.8 )/ml

10 1.0 10 7 0.6 0.4 5 0.5 5 KC (ng/ml) KC TNF (ng/ml) IL-10 (ng/ml) IL-10 0.2 CFU (x 10 (x CFU 0 0.0 0 0.0 /– /– /– /– – – – – WT WT WT WT Irf8 Irf8 Irf8 Irf8 C

ΔpopB NS NS NS 50 * 1.5 40 2.0 40

30 )/ml 1.5 30 1.0 7 20 1.0 20 0.5 KC (ng/ml) KC TNF (ng/ml) 10 (ng/ml) IL-10 10 0.5 CFU (x 10 (x CFU 0 0.0 0 0.0 /– /– /– /– – – – – WT WT WT WT Irf8 Irf8 Irf8 Irf8 16

17 Supplementary Figure 3. Release of inflammasome-independent cytokines and

18 phagocytosis of Gram-negative bacteria are not impaired in Irf8–/– BMDMs

19 (A–C) The production of the inflammasome-independent cytokines KC, TNF, and IL-10 and the Supplemental Figure 3 20 intracellular bacterial numbers in wild-type (WT) or Irf8–/– bone marrow-derived macrophages

21 (BMDMs) 16 h after infection with E. coli (multiplicity of infection [MOI], 20), C. rodentium (MOI,

22 20), or P. aeruginosa ΔpopB (MOI, 50). NS, not significant; *P < 0.05 (two-tailed t test). Data are

23 representative of three independent experiments (mean ± SEM).

3 A B C WT Irf8–/– Casp11–/–

NS NS NS 6 ** 2.0 ** 10 **

1.5 8 4 6 (ng/ml) (ng/ml)

(ng/ml) 1.0 β β β 4 2 IL-1 IL-1 IL-1 0.5 2 0 0.0 0 + + + β β β Media Media Media popB IFN E. coli IFN IFN Δ popB E. coli Δ C. rodentium C. rodentium

24

25 Supplementary Figure 4. IFN-b supplementation rescues IL-1b release in Irf8–/– BMDMs

26 (A–C) The assessment of IL-1b released from bone marrow-derived macrophages (BMDMs)

27 pretreated with IFN-b (400 U/mL) for 4 h followed by infection with E. coli (multiplicity of infection

28 [MOI], 10), C. rodentium (MOI, 10), or P. aeruginosa ΔpopB (MOI, 10) for an additional 16 h. NS,

29 not significant; **P < 0.01 (two-way ANOVA with Tukey’s multiple comparisons test). Data are

30 representative of three independent experiments (mean ± SEM).

Supplemental Figure 4

5