Zhx2 Accelerates Sepsis by Promoting Macrophage Glycolysis Via Pfkfb3

Zhx2 Accelerates Sepsis by Promoting Macrophage Glycolysis via Pfkfb3 Zehua Wang, Liang Kong, Siyu Tan, Yankun Zhang, Xiaojia Song, Tixiao Wang, Qinghai Lin, Zhuanchang Wu, Peng This information is current as Xiang, Chunyang Li, Lifen Gao, Xiaohong Liang and of September 29, 2021. Chunhong Ma J Immunol published online 16 March 2020 http://www.jimmunol.org/content/early/2020/03/13/jimmun

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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 16, 2020, doi:10.4049/jimmunol.1901246 The Journal of Immunology

Zhx2 Accelerates Sepsis by Promoting Macrophage Glycolysis via Pfkfb3

Zehua Wang,* Liang Kong,† Siyu Tan,* Yankun Zhang,* Xiaojia Song,* Tixiao Wang,* Qinghai Lin,* Zhuanchang Wu,* Peng Xiang,* Chunyang Li,‡ Lifen Gao,*,x Xiaohong Liang,*,x and Chunhong Ma*,x

Sepsis is a life-threatening condition with limited therapeutic options, characterized as excessive systemic inflammation and multiple organ failure. Macrophages play critical roles in sepsis pathogenesis. Metabolism orchestrates homeostasis of macrophages. However, the precise mechanism of macrophage metabolism during sepsis remains poorly elucidated. In this study, we identified the key role of zinc fingers and homeoboxes (Zhx2), a ubiquitous transcription factor, in macrophage glycolysis and sepsis by enhancing 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (Pfkfb3) expression. Mice with myeloid Zhx2-specific deletion (abbreviated as MKO) showed more resistance to cecal ligation and puncture and LPS-induced sepsis, exhibiting as Downloaded from prolonged survival, attenuated pulmonary injury, and reduced level of proinflammatory cytokines, such as TNF-a, IL-6, and IL- 1b. Interestingly, Zhx2 deletion conferred macrophage tolerance to LPS-induced glycolysis, accompanied by reduced proinflammatory cytokines and lactate. Consistently, treatment of glycolytic inhibitor 2-deoxyglucose almost completely abrogated the protection of mice from LPS-induced sepsis initiated by Zhx2 deletion in macrophages. RNA sequencing and chromatin immunoprecipitation assays confirmed that Zhx2 enhanced transcription of Pfkfb3, the glycolysis rate-limiting enzyme, via binding with Pfkfb3 promoter. Furthermore, Pfkfb3 overexpression not only rescued the reduction of macrophage glycolysis caused by Zhx2 http://www.jimmunol.org/ deficiency, displaying as extracellular acidification rates and lactate production but also destroyed the resistance of mice to LPS- induced sepsis initiated by transfer of bone marrow–derived macrophages from MKO mice. These findings highlight the novel role of transcription factor Zhx2 in sepsis via regulating Pfkfb3 expression and reprogramming macrophage metabolism, which would shed new insights into the potential strategy to intervene sepsis. The Journal of Immunology, 2020, 204: 000–000.

epsis is characterized as a life-threatening organ dysfunc- sepsis. Macrophages affect both immune homeostasis and inflam- tion caused by an uncontrolled host immune responses to matory processes (5–8) by releasing multiple proinflammatory and S disseminated infection (1). Clinical manifestations of sepsis anti-inflammatory cytokines (9). In vivo and ex vivo studies showed by guest on September 29, 2021 are driven from systemic exacerbated release of inflammatory cy- that macrophages from peritoneal, alveoli, and liver produced lots tokines, such as TNF-a, IL-6, and IL-1b and concomitant anti- of TNF-a, IL-6, and IL-1b under LPS stimulation (6). Modulating inflammatory processes (2). Although great advancements have functions and phenotypes of macrophages has become a useful tool been achieved in antimicrobial drugs and organ function support to improve septic mice survival rate by significantly ameliorating technologies, sepsis still remains the leading cause of death in the inflammation (10, 11). intensive care unit (3, 4). Accumulated evidence suggested that macrophages display During hyperinflammation stage in sepsis, a multitude of defects different metabolic profiles depending on their phenotypes. Under in immunity involved with adaptive and innate immunocytes are physiological conditions, macrophages use oxidative phosphory- activated. As one of the most important innate immunocytes, lation as the major metabolism for their energy requirements, which macrophage contributes greatly to hyperinflammation stage in triggers macrophages to appear anti-inflammatory and proresolution

*Key Laboratory for Experimental Teratology of Ministry of Education, Department C.M. designed and supervised the study and organized and revised the manuscript, of Immunology, Shandong University School of Basic Medical Sciences, Jinan, and acquired funding. All authors reviewed and approved the manuscript. Shandong 250012, People’s Republic of China; †Department of Clinical Laboratory, The sequences presented in this article have been submitted to the Sequence Read Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan, Archive (https://www.ncbi.nlm.nih.gov/sra/PRJNA598552) under accession number Shandong 250012, People’s Republic of China; ‡Key Laboratory for Experimental PRJNA598552. Teratology of Ministry of Education, Department of Histology and Embryology, Shandong University School of Basic Medical Science, Jinan, Shandong 250012, Address correspondence and reprint requests to Prof. Chunhong Ma, Key Laboratory People’s Republic of China; and xKey Laboratory of Infection and Immunity of for Experimental Teratology of Ministry of Education and Department of Immunol- Shandong Province, Shandong University School of Basic Medical Sciences, Jinan, ogy, Shandong University School of Basic Medical Sciences, Shandong University, Shandong 250012, People’s Republic of China 44 Wenhua Xi Road, Jinan, Shandong 250012, People’s Republic of China. E-mail address: [email protected] Received for publication October 16, 2019. Accepted for publication February 12, 2020. The online version of this article contains supplemental material. This work was supported by grants from the National Science Foundation of China Abbreviations used in this article: BMDM, bone marrow–derived macrophage; ChIP, (Key Project 81702647 and 81830017, 81902051, 81902443, and 81972819), the chromatin immunoprecipitation; CLP, cecal ligation and puncture; 2-DG, 2-deoxy-D- National Natural Science Foundation for Outstanding Youth Fund (81425012), the glucose; ECAR, extracellular acidification rate; F, forward; Hk2, hexokinase 2; Mup, National Key Research and Development Program (2018YFE0126500), the Taishan major urinary protein; Pfkfb3, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase Scholarship (tspd20181201) and the Shandong Provincial Key Innovation Project 3; PM, peritoneal macrophage; R, reverse; RNA-seq, RNA sequencing; WT, wild- (2018FYJH0503). type; Zhx2, zinc fingers and homeoboxes 2.

Z.W. designed and performed the study, analyzed data, and wrote the manuscript, Ó L.K. constructed lentivirus, and S.T., Y.Z., Q.L., T.W., and P.X. performed some Copyright 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 experiments, Z.W., X.S., C.L., L.G., and X.L. provided insightful suggestions.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1901246 2 REPROGRAMMING OF MACROPHAGES IN SEPSIS programs (12, 13). During sepsis, pathogen-related signals induce BMDM and peritoneal macrophages preparation and F4/80+ a shift of the macrophages metabolic profile from oxidative macrophages purification phosphorylation to glycolysis to adopt an inflammatory state. BMDM were derived by isolating bone marrow from mice aged around Enhanced glycolysis of macrophages results in increased lactate 6 wk old. Bone marrow cells were flushed from femurs and cultured in production, which has been recognized as a useful biomarker in DMEM with 10% FBS (Life Technologies BRL, Gaithersburg, MD) and septic patients (14). Likewise, LPS treatment promotes bone mar- 100 ng/ml M-CSF (Novus Biologicals, Littleton, CO) for 7 d. Peritoneal row–derived macrophages (BMDM) to increase the expression of macrophages (PM) were collected and cultured in DMEM with 10% FBS. Macrophages were purified using the MojoSort Streptavidin Nanobeads proglycolytic genes, including 6-phosphofructo-2-kinase/fructose- (BioLegend, San Diego, CA) according to the manufacturer’s instructions. 2,6-bisphosphatase 3 (Pfkfb3), which contributes to increased pro- Macrophages purity was determined using the anti-CD11b and anti-F4/80 duction of IL-1b and IL-6 (15). Blocking glycolytic flux with a mAb by flow cytometry analysis. competitive inhibitor of hexokinase 2 (Hk2), 2-deoxy-D-glucose Mice irradiation and BMDM transfer (2-DG), decreases IL-1b secretion in macrophages, reduces organ injury, and improves the outcome of sepsis (16). Pfkfb3-overexpression lentivirus was established in pUltra vector. BMDM were infected with lentivirus for 48 h. Mice were exposed to 5.5 Gy of Transcription factor is the most important driver of metabolic whole-body g-irradiation with an irradiator (X-Rad 225 OptiMAX) and reprogramming. Hypoxia inducible factor 1a (Hif-1a) induces then injected with 5 3 105 BMDM infected with lentivirus, followed with the glycolytic enzyme expression, including Hk2 and Pfkfb3, to treatment with LPS (10 mg/kg) to induce sepsis. promote the switch to glycolysis (17–19). Hif-1a deletion in Quantitative real-time RT-PCR macrophages attenuated secretion of TNF-a, IL-1b, and IL-6 and enhanced survival of septic mice (20). Given the promising results Total RNA from macrophages were extracted using TRIzol reagent Downloaded from of modulating immunometabolism in sepsis, identifying novel (TIANGEN Biotech, Beijing, China) and reverse (R) transcribed into cDNA with RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scien- transcription regulators for macrophages metabolism may provide tific). RT-PCR was carried out using a Bio-Rad Laboratories C1000 a promising strategy for treatment of sepsis. Thermal Cycler CFX96 Real-Time System with SuperReal PreMix Plus Zinc fingers and homeoboxes 2 (Zhx2) is a ubiquitous tran- (SYBR Green; TIANGEN Biotech). Primers used are shown below: mouse scription factor originally identified as a regulator of a–fetal b-actin forward (F): 59-TGCGTGACATCAAAGAGAAG-39,R:59-CCA- TACCCAAGAAGGAAGG-39; mouse Zhx2 F: 59-CCGGCTCCAGC- protein (21). Zhx2 is involved in many pathological conditions, TACCCCACTTCTC-39,R:59-TGGAAGCGAGGCGGCACATCAG-39; http://www.jimmunol.org/ including caner and metabolism-related disease such as athero- mouse Pfkfb3 F: 59-TCATCGAGTCGGTCTGTGACGA-39,R:59-CA- sclerosis (22–24). Recently, a computational network study indi- TGGCTTCTGCTGAGTTGCAG-39; mouse lactate dehydrogenase F: 59- cated Zhx2 as one of the most regulated transcription factors in GCAGACAAGGAGCAGTGGAAGGAG-39,R:59-ACACTGAGGAAG- myeloid cells to avoid an avalanche transcription event (25). ACATCCTCATTG-39; mouse Pfkl F: 59-CCATCAGCAACAATGTGC- CTGG-39,R:59-TGAGGCTGACTGCTTGATGCGA-39; mouse Pgam1 F: However, the role of Zhx2 in macrophage metabolic reprogram- 59-CTGGAATGAGGAGATCGCACCT-39,R:59-ATTCCAGTGGGCAG- ming during sepsis is largely unknown. GTTCAGCT-39; mouse Eno1 F: 59-ACCAACCCTAAGCGGATTGCC- In this study, we identified Zhx2 as a critical regulator that AAG-39,R:59-AGTCTTGATCTGCCCAGTGCAGAG-39; mouse Hk2 F: switches macrophages into glycolytic metabolism, leading to en- 59-CCCTGTGAAGATGTTGCCCACT-39,R:59-CCTTCGCTTGCCAT-

TACGCACG-39; mouse Pgkl F: 59-GATGCTTTCCGAGCCTCACTGT-39, by guest on September 29, 2021 hanced inflammation during sepsis. Mice with myeloid-specific R: 59-ACCAGCCTTCTGTGGCAGATTC-39; mouse TNF-a F: 59-AT- Zhx2 deletion were more resistant to LPS-induced and cecal li- GAGCACAGAAAGCATGATC-39,R:59-TACAGGCTTGTCACTCGA- gation and puncture (CLP)–induced sepsis. Mechanistically, Zhx2 ATT-39; mouse IL-6 F: 59-ACAACCACGGCCTTCCCTAC-39,R:59- preferred macrophages to produce proinflammatory cytokines via CATTTCCACGATTTCCCAGA-39; and mouse IL-1b F: 59-ACAACTG- switching to glycolysis in a Pfkfb3-dependent manner. CACTACAGGCTCC-39,R:59-GCTTGGGATCCACACTCTCC-39. Western blot Materials and Methods BMDM from wild-type (WT) and MKO mice were homogenized in cell Mice lysis buffer, and protein extracts were quantified by BCA protein assay (Beyotime Biotechnology, Shanghai, China). Equal of protein were loaded Male C57BL/6 mice (6–8 wk of age) were purchased from Animal Re- in SDS-PAGE, transferred to Immobilon-P Membranes (MilliporeSigma, cre search Center of Shandong University. LysM mice were obtained from Darmstadt, Germany), and incubated overnight at 4˚C with the following The Jackson Laboratory. Myeloid-specific Zhx2-deficient mice (abbrevi- primary Abs from Proteintech: mouse monoclonal anti-GAPDH (catalog cre f/f ated as MKO) were generated by crossing LysM and Zhx2 mice, which no. 60004, 1:10,000), rabbit polyclonal anti-ZHX2 (catalog no. 20136-1- were gifted by Prof. B. T. Spear from University of Kentucky. All mice AP, 1:4000), and rabbit polyclonal anti-PFKFB3 (catalog no. 13763-1-AP, were maintained under specific pathogen-free conditions, and experiments 1:4000). The membranes were washed with PBS with Tween 20 for three were carried out with under the approval of the Shandong University times and subsequently incubated with secondary HRP-conjugated anti- Laboratory Animal Center. mouse or anti-rabbit IgG secondary Abs (Proteintech, Chicago, IL). The signal was detected by enhanced chemiluminescence reagent using the Mice model of sepsis DNR Bio Imaging Systems (DNR Bio Imaging Systems, Neve Yamin, Sepsis was established by CLP and LPS. CLP-induced sepsis was prepared Israel). as described before (26, 27). Briefly, after induction of anesthesia, one third of the station of the cecum was ligated with the 4-0 suture line and ELISAs and lactate measurement punctured with 22-gauge needle. Fecal material was extruded and then Levels of IL-1b, IL-6, and TNF-a were measured by using a commercially returned to the peritoneal cavity. For LPS-induced sepsis model, mice were available ELISA Kit (Dakewe Biotech) according to the manufacturer’s i.p. injected with LPS (Sigma-Aldrich, St Louis, MO) in dosage of 10 mg/kg. instructions. Briefly, serum was added to plates with 37˚C for 90 min, and Glycolysis inhibitor 2-DG (2 g/kg) (Sigma-Aldrich) was i.p. injected 3 h the unbound materials were removed and washed. After incubation for before LPS treatment to inhibit glycolysis. Survival was monitored once 30 min with 100 ml streptavidin–HRP, plates were washed and followed per 24-h period. Serum was collected 24 h after CLP or LPS. with tetramethylbenzidine incubation. Finally, 100-ml/well stop solution Histological analysis was added and read at a wave of 450 nm and a reference wave of 630 nm with an ELISA reader. Lung tissues were fixed with 4% paraformaldehyde for 24 h and embedded Concentrations of lactate in the supernatant of BMDM and PM was in paraffin. The 4-mm paraffin sections were deparaffinized and mounted on measured by commercially available lactate assay (Nanjing Jiancheng slides. After placed in xylene and concentrations of ethanol (100–50%), Biology Engineering Institute, Jiangsu, China), following the manufac- the slides were stained with standard H&E procedure. A blinded observer turer’s recommendations. Briefly, 20 ml of cell-free culture supernatant was assigned to evaluate the histopathological pulmonary injury. were transferred into a tube and mixed with 1 ml of lactate dehydrogenase The Journal of Immunology 3 and 200 ml developer assay mixture. Two milliliters of stopping buffer was and Western blot confirmed the deletion of Zhx2 in macrophages added after incubation for 10 min at 37˚C; the OD reflecting lactate was (Supplemental Fig. 1A–C). determined at 530 nm using an ELISA reader. To mimic human sepsis process, MKO and WT mice were Extracellular acidification rate measurement treated with LPS, with PBS as control. As shown in Fig. 1A, LPS

5 treatment led to altered Zhx2 expression in several immune cell BMDM or PM was seeded at 2 3 10 cells per well into XF96-well plates andthenstimulatedwithLPSfor24hafteradherence.Beforemea- subsets, whereas macrophages displayed the most significantly surement, cells were washed three times with XF Base Medium con- increased Zhx2 expression in LPS-treated mice. Accordingly, LPS taining 2 mM glutamine (pH ∼7.4 6 0.05). For extracellular acidification enhanced Zhx2 expression in BMDM in a time-dependent manner rate (ECAR) experiment, the concentration of oligomycin was 20 mM. (Fig. 1B). We then compared survival between LPS-treated MKO Measurements were made using a XF96 Extracellular Flux Analyzer (Seahorse Bioscience) and results processed with Wave v.2.2.0 software. and WT. As shown in Fig. 1C, PBS-treated MKO and WT mice Glycolysis values represented maximum rate measurement before oligo- are all healthy during the process. In the contrast, 75% of WT mycin injection minus basic measurement, and glycolytic capacity values mice died of LPS injection at 4-d time point, and a significant meant maximum rate measurement after oligomycin injection minus basic increased survival rate was observed in MKO mice. Consistently, measurement. H&E staining of lung tissues 24 h after LPS treatment demon- Isolation of splenic lymphocytes strated obviously less inflammation in the lung from MKO mice than that from WT mice, indicating attenuated pathological injury The spleens were harvested and mashed through a nylon mesh to get primary cell suspension. The suspensions were centrifuged at 300 3 g for in MKO mice (Fig. 1D). ELISA results showed that, compared 5 min, and the pellets were mixed with RBC lysis buffer and incubated at with WT mice, MKO mice displayed a significant reduction of

4˚C for 10 min to get the collected lymphocyte. proinflammatory cytokines, such as TNF-a, IL-6, and IL-1b,in Downloaded from Flow cytometry analysis serum (Fig. 1E), accompanied with a reduction in serum lactate (Fig. 1F). Anti-mouse Abs used in this study included anti-CD45, anti-CD3, anti-CD4, To further verify the role of macrophage-specific depletion of anti-CD8, anti-CD19, anti-CD11b, anti-CD11c, anti–Ly-6C, anti–Ly-6G, anti-NK1.1, and anti-F4/80 (from BioLegend). Briefly, cell membrane Zhx2 in sepsis, CLP-induced sepsis was also performed with WT markers were directly labeled for 30 min in the dark at 4˚C. CD8 T cell, and MKO mice. As expected, deletion of Zhx2 in macrophage-

B cell, NK cell, macrophages, and dendritic cells were sorted using MoFlo protected mice from sepsis induced death and tissue injury. http://www.jimmunol.org/ Astrios EQ (Beckman Coulter, Brea, CA). For intracellular staining in MKO mice displayed significantly prolonged survival than WT m macrophages, cells were restimulated with ionomycin (1 g/ml) plus PMA mice (Fig. 1G) accompanied with attenuated pulmonary injury (50 nM) and brefeldin A for an additional 4 h before harvest in DMEM with 10% FBS. The cells were fixed, permeabilized (Cytofix/Cytoperm Kit), and (Fig. 1H). Concurrently, compared with WT mice, MKO mice stained with Abs including anti–TNF-a,anti–IL-1b,andanti–IL-6(from showed obviously reduced level of proinflammatory cytokines BioLegend). The cell count and percentage were detected by CytoFLEX S (Fig. 1I) as well as decreased lactate in serum (Fig. 1J). Collec- Flow Cytometer (Beckman Coulter). tively, these results reveal that Zhx2 deficiency in macrophages Chromatin immunoprecipitation assay protects mice from sepsis and inhibits systemic inflammation during sepsis.

RAW264.7 transfected with pcDNA3-Zhx2-HA, and BMDM from WT and by guest on September 29, 2021 MKO mice were harvested for chromatin immunoprecipitation (ChIP) Zhx2 deletion inhibits proinflammatory cytokines in assay (MilliporeSigma). Briefly, cells were fixed with 1% formaldehyde for 10 min at 37˚C. The protein–DNA complexes were immunoprecipitated macrophages during sepsis with anti-HA Ab, control IgG, or anti-ZHX2 Ab. PCR were performed using The onset of sepsis is accompanied by defective innate and adaptive 2 2 the primers shown below: promoter section 1 ( 1423 to 1253) F: 59- immune responses (6), and macrophages are responsible for sys- TGCATGATTTTTGCCACCGC-39,R:59-GCAGACCTT-CTTTGGAGC- CAGTT-39; promoter section 2 (2990 to 2875) F: 59-CATAGGGAGTC- temic inflammation of sepsis. ZHX2 in macrophages has been GGTCAGGCG-39,R:59-CCCCAGTGAAAGACGCGAGT-39;and reported to reduce apoptosis during atherosclerosis (24). We did promoter section 3 (2443 to 2364) F: 59-GATTCGAGCGTGTC- not detect change in the number and percentage of macrophages CCCTGTG-39,R:59-TCCCGAAGGACGAGACCTGA-39. in MKO septic mice, as well as other immunocytes, including RNA sequencing T cells, B cells, and NK cells in septic mice (Supplemental Fig. 2A). We further evaluated the production of proinflammatory BMDM from WT and MKO mice were harvested without stimulation (n = 3). RNA sequencing (RNA-seq) was carried out by the Beijing Ge- cytokines in macrophages from WT and MKO mice after LPS nomics Institute, following standard protocols. Libraries were sequenced treatment. As detected by flow cytometry (Fig. 2A), both the on the BGISEQ-500 platform. RNA-seq data were submitted to the Se- number and percentage of proinflammatory cytokines (TNF-a, quence Read Archive and are unrestrictedly accessible with accession IL-6, and IL-1b) producing CD11b+F4/80+ macrophages from number PRJNA598552 (https://www.ncbi.nlm.nih.gov/sra/PRJNA598552). MKO mice were significantly decreased. Furthermore, mean Statistical analysis fluorescence intensities showed that proinflammatory cytokines were reduced upon Zhx2 deletion in macrophages (Fig. 2B). This Statistical analysis was carried out with GraphPad Prism 6 software. Data + were presented as the mean 6 SD. Paired t tests were used to analyze was verified with purified F4/80 macrophages from spleen. RT- differences between two groups. The survival rate was analyzed using PCR demonstrated the obviously reduced mRNA expression of Kaplan–Meier log-rank test. Statistical significance was reported as highly TNF-a, IL-6, and IL-1b in macrophages from MKO septic mice , , , significant using *p 0.05, **p 0.01, or ***p 0.001. (Fig. 2C). Flow cytometry confirmed the success of macrophage separation (Supplemental Fig. 2B). We also examined the protein Results level of IL-6 and IL-1b with Western blot and found that the Deletion of Zhx2 in macrophages protects mice from sepsis and expression of IL-6 and IL-1b were decreased in PM from MKO inhibits inflammation septic mice (Fig. 2D). Above data suggest that Zhx2 promotes To determine the role of macrophage-specific Zhx2 deficiency in macrophages to produce proinflammatory cytokines during sepsis. sepsis, we crossed Zhx2-floxed mice (gifted from B. T. Spear from Kentucky University) with LysMcre mice to ablate Zhx2 in the Zhx2 enriches genes of glycolysis pathway in macrophages myeloid compartment (LysMcre+Zhx2f/f mice referred to as MKO We next sought to address the possible mechanism by which Zhx2 and LysMcre2Zhx2f/f littermates mice referred to as WT). RT-PCR regulates macrophage-mediated inflammation. Global RNA-seq 4 REPROGRAMMING OF MACROPHAGES IN SEPSIS Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. Zhx2 protects mice form sepsis shock and inhibits inflammation. (A) C57BL/6 mice were challenged with PBS and LPS (10 mg/kg, i.p.) for 24 h. CD8 T cell (CD3+ CD8+), B cell (CD32 CD19+), macrophage (CD11b+ F4/80+), and dendritic cell (CD11b+CD11c+) from spleen were sorted by MoFlo Astrios EQ. Zhx2 mRNA was detected by RT-PCR. (B) BMDM from WT C57BL/6 were treated with 100 ng/ml LPS for the indicated duration of time. Levels of Zhx2 were determined by RT-PCR. (C–J) WT and MKO mice challenged with LPS (10 mg/kg, i.p.) (C–F) or CLP treatment (G–J) to induce sepsis. (C and G) Survival curves (PBS: n/WT = 10, n/MKO = 10, LPS: n/WT = 13, n/MKO = 10; sham operation (abbreviated as Sham): n/WT = 7, n/MKO = 7, CLP: n/WT = 7, n/MKO = 5). (D and H) H&E staining of lung tissues. Original magnification 3100. Scale bars, 20 mm. (E and I)Serum concentrations of IL-6, TNF-a, and IL-1b after 24 h (n = 4). (F and J) Serum concentrations of lactate (n = 6) were analyzed. Data were presented as mean 6 SD from three independent experiments and analyzed by unpaired, two-tailed t test with significance. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. analysis was performed with BMDM isolated from WT and MKO than that from WT mice (Fig. 3D). In accordance, BMDM from mice. Gene set enrichment analysis showed that signatures of MKO mice produced significantly lower level of lactate than that GLYCOLYSIS_GLUCONEDGENESIS were enriched in BMDM from WT mice after LPS treatment (Fig. 3E). Similar results were from WT mice other than MKO mice (Fig. 3A, 3B). Results of obtained with PM from LPS-induced mice. PM from septic MKO quantitative RT-PCR further validated the reduced expression of mice exhibited lower glycolytic capacity, basic glycolysis, and genes coding enzymes in the glycolysis pathway, including lactate secretion than that from WT mice (Fig. 3F, 3G). Taken Pfkfb3, Hk2, and lactate dehydrogenase in MKO BMDM with or together, these results suggest that Zhx2 promotes glycolysis without LPS stimulation (Fig. 3C). Therefore, we hypothesized metabolism in macrophages during sepsis. that Zhx2 might take roles in regulating glucose metabolism of macrophages. To address that, BMDM from WT and MKO mice Blocking glycolysis eliminates the protection initiated by were stimulated with LPS, and glucose consumption and changes macrophage-specific Zhx2 deletion in sepsis in ECAR were assessed. As expected, the glycolytic capacity and As reported, enhanced glycolysis has tightly connection with in- basic glycolysis in BMDM from MKO mice were much lower flammatory reactions in macrophages (28). To investigate the The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 2. Zhx2 aggravates proinflammatory factor secretion from macrophages in septic mice. WT and MKO mice were injected with i.p. LPS (10 mg/kg; n = 4). (A and B) Gated on CD11b+F4/80+ cells, intracellular TNF-a, IL-6, and IL-1b was detected by flow cytometry. (A) Representative dot plots were shown on the left panels and the percentage (top) and number (bottom) were shown on the right panels. (B) Representative histograms and mean fluorescence intensities (MFIs) are shown. (C) F4/80+ macrophages were isolated from the spleen of WT and MKO mice. RT-PCR detected the mRNA expression of TNF-a, IL-6, and IL-1b (n = 6). (D) PM was harvested from WT and MKO mice challenged with LPS for 24 h. Western blot detected the protein level of IL-6 and IL-1b. The data were presented as means 6 SD from two independent experiments and analyzed by unpaired, two-tailed t test with significance. *p , 0.05, **p , 0.01, ***p , 0.001. role of aerobic glycolysis in alleviated sepsis mediated by LPS stimulation to induce sepsis (Fig. 4E). Flow cytometry con- macrophage-specific deletion of Zhx2, 2-DG, a competitive in- firmed the irradiation-mediated depletion of immune cells in hibitor of glucose uptake and metabolism, was included in LPS- mice (Supplemental Fig. 3A). As shown in Fig. 4F–H, transfer induced sepsis with WT and MKO mice. As expected, compared of BMDM with enhanced Zhx2 promoted pulmonary damage with PBS group, 2-DG treatment significantly improved sepsis- and serum level of proinflammatory cytokines, together with induced survival in both WT and MKO mice. Moreover, 2-DG increased lactate in sepsis mice, which confirmed the specific abolished the difference in MKO and WT mice. Two groups of effect of Zhx2 in macrophage during sepsis. Moreover, 2-DG mice showed similar survival rate after LPS treatment (Fig. 4A). treatment greatly blocked this exacerbation mediated by In accordance, 2-DG led to reduced pulmonary damage and de- macrophage-specific overexpression of Zhx2 in sepsis. creased serum level of proinflammatory cytokines and lactate Collectively, these data suggest that Zhx2 aggravates systemic (Fig. 4B–D). Interestingly, 2-DG treatment destroyed the protec- inflammation in sepsis by promoting macrophage glycolysis. tion mediated by macrophage-specific deletion of Zhx2 in sepsis. WT and MKO mice displayed comparable levels of pulmonary Zhx2 promotes glycolysis of macrophages via transcriptional injury (Fig. 4B), proinflammatory cytokines, such as TNF-a, IL-6, upregulation of Pfkfb3 and IL-1b (Fig. 4C), and lactate (Fig. 4D) in serum after combined As shown in our RNA-seq and RT-PCR analysis above, Pfkfb3, the treatment of LPS with 2-DG. rate-limiting enzyme in glycolysis, is the most downregulated gene To further confirm above results, 2-DG was included in mice with in BMDM from MKO mice (Fig. 3C). This was further verified Zhx2-overexpression macrophages. Briefly, Zhx2 overexpression was with protein assays. Results of Western blot showed that Pfkfb3 in introduced into BMDM by lentivirus, with mock lentivirus as PM and BMDM from MKO mice were significantly reduced than control (Supplemental Fig. 3B). These BMDM was transferred into that from WT mice (Fig. 5A, Supplemental Fig. 3C). Similar re- irradiation-pretreated WT mice, followed with 2-DG treatment and sults were gotten with LPS-stimulated BMDM from WT and 6 REPROGRAMMING OF MACROPHAGES IN SEPSIS Downloaded from http://www.jimmunol.org/

FIGURE 3. Zhx2 deﬁciency inhibits glycolysis in macrophages. (A and B) BMDM from WT and MKO mice were collected for RNA-seq. (A) Gene set B A C enrichment analysis (GSEA) of different expressed genes (normalized enrichment score = 1.68, p value = 0.01). ( ) Heatmap of glycolytic genes in ( ). ( ) by guest on September 29, 2021 RT-PCR analysis of expression of glycolysis rate-limiting enzymes in BMDM from WT and MKO mice treated with LPS (100 ng/ml) for 24 h. (D and E) BMDM from WT and MKO mice was treated with or without LPS (100 ng/ml) for 24 h. ECAR analysis was shown in (n =4)(D). Lactate in supernatant was measured (n =3)(E). Similar results were obtained in four independent experiments. (F and G) PM were harvested from WT and MKO mice challenged with LPS for 24 h. ECAR analysis was shown in (F), and concentration of lactate in supernatant was measured (n =6)(G). Data were analyzed using two- tailed Student t test and presented as means 6 SD. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001.

MKO mice. LPS stimulation greatly increased Pfkfb3 expression the ECAR and concentration of lactate. As shown in Fig. 5D, in both WT and MKO macrophages, and MKO macrophages 5E, ectopic Pfkfb3 almost completely eliminated the reduc- with or without LPS stimulation all expressed significantly de- tion of ECAR and lactate secretion in BMDM from MKO creased Pfkfb3 (Supplemental Fig. 4A, 4B). Because Zhx2 is well mice. known as a transcription factor, ChIP assay was performed with RAW264.7 cells transfected with pcDNA3-Zhx2-HA to analyze Zhx2-specific deletion in macrophage alleviates sepsis the occupancy of Zhx2 in promoter of Pfkfb3. PCR with primers via Pfkfb3 targeting with different regions of Pfkfb3 promoter showed that To confirm the effect of Pfkfb3 in vivo, BMDM from WTand MKO Zhx2 significantly occupied with Pfkfb3 promoter and mainly mice, with absence or presence of Pfkfb3-overexpressing lentivi- bound with Pfkfb3 promoter at section 3 (2443 to 2364 nt) (Fig. rus, were transferred into irradiation-pretreated WT mice and then 5B). To confirm the result, we stimulated BMDM from WT and challenged with LPS to induce sepsis. As expected, transfer of MKO mice with LPS and performed ChIP assays with anti-Zhx2 macrophages with Zhx2 depletion protected mice from LPS- Ab–mediated pulldown to examine ZHX2 binding to the Pfkfb3 induced systemic inflammation, displayed as alleviated pulmo- promoter regions at section 3 (2443 to 2364 nt), as above. As nary injury and reduced secretion of proinflammatory cytokines shown in Fig. 5C, there was significant recruitment of Zhx2 to the (TNF-a, IL-1b, and IL-6), as well as decreased serum lactate promoter of Pfkfb3, and the Zhx2 binding was further enhanced (Fig. 6). However, Pfkfb3 overexpression significantly blocked the after LPS simulation. protection mediated by macrophage depletion of Zhx2. With ec- Pfkfb3 is a member of the PFKFB family of bifunctional iso- topic Pfkfb3, pulmonary injury (Fig. 6A), serum proinflammatory zymes, critically controlling the glycolytic rate under physical (Fig. 6B), and lactate (Fig. 6C) were similar in mice transferred and pathophysiological conditions (29). To validate the in- with BMDM from WT or MKO. Taken together, these data volvement of Pfkfb3 in Zhx2-mediated regulation of macro- demonstrate that Zhx2 promotes macrophages to skew toward phage metabolism, Pfkfb3 overexpression was performed by glycolysis metabolism in a Pfkfb3-dependent manner, facilitates lentivirus in BMDM from WT and MKO mice (Supplemental systemic inflammation, and results in an increased mortality in Fig. 3D) and then stimulated with LPS for 24 h to evaluate sepsis mice. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 4. 2-DG treatment eliminates the effect mediated by Zhx2 in macrophages during LPS-induced sepsis. (A–D) WT and MKO mice challenged with LPS (10 mg/kg, i.p.) in the presence/absence of 2-DG (i.p., 2 g/kg) 3 h before injection. (A) Survival rate of septic mice (n/WT = 16, n/MKO = 7, n/WT+2-DG = 9, n/MKO+2-DG = 10). (B) H&E staining of lung tissues. Original magnification 3100. Scale bars, 20 mm. (C) ELISA of serum proinflammatory cytokines (n = 3). (D) Serum level of lactate (n = 3). Similar results were obtained in two independent experiments. (E–H) Mice pre-exposed to 5.5 Gy of whole-body g-irradiation were i.v. injected with BMDM infected with mock lentivirus (Mock-lenti)/Zhx2 overexpression introduced into BMDM by lentivirus (lenti-ZHX2). These mice were then treated with 2-DG (i.p., 2 g/kg) followed with LPS challenge (E). (F) H&E staining of lung tissues. Original magnification 3100. Scale bars, 20 mm. (G) ELISA assays of serum proinflammatory cytokines (n = 4). (H) Serum level of lactate (n = 4). Data were analyzed using two-tailed Student t test and presented as means 6 SD. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001.

Discussion also reinforce the notion that targeting macrophage glycolysis has One major problem of sepsis is the strong storm of inflammatory potential for treating infectious diseases that lead to lethal sepsis. factors. Thus, how to abate inflammation is a critical issue in In addition to hyperinflammatory responses, another charac- developing new and effective therapies. In this work, we dem- teristic of sepsis is the severe metabolic abnormal condition in onstrate that MKO protects mice from lethal sepsis and improves which aerobic glycolysis is enhanced and becomes the hallmark of mouse survival. This effect is related to the decrease of systemic sepsis (30, 31). Accumulated studies demonstrated metabolism inflammation and pulmonary injury. Although LysMcre mediates alterations that occur during immune cells activation can directly Zhx2 knockout in myeloid cells, Zhx2-overexpressing macro- regulated functions of immune cells, especially innate immune phages transferred into irradiation-pretreated mice verified the cells such as macrophages (32). Increased glycolysis enables specific role of Zhx2-expressing macrophages in sepsis. We fur- macrophages to produce energy and generate biosynthetic inter- ther show that Zhx2 promotes glycolysis in macrophage via directly mediates to carry out its particular effector functions, including upregulating the expression of glycolysis rate-limiting enzyme producing inflammatory cytokines (33). Blocking glycolytic flux Pfkfb3. These findings (for the first time, to our knowledge) not only with 2-DG inhibits macrophage activation in vitro and suppresses identified a previously unrecognized glycolytic-promoting function inflammation in vivo (34, 35). Regulation of glycolysis in mac- of Zhx2 relating with enhanced inflammation in macrophage, but rophages becomes a potential strategy of sepsis intervention. 8 REPROGRAMMING OF MACROPHAGES IN SEPSIS Downloaded from http://www.jimmunol.org/ FIGURE 5. Zhx2 modulates macrophage glycolysis in Pfkfb3-dependent way. (A) PM and BMDM from WT and MKO mice were harvested, and the expression of Pfkfb3 was detected by Western blot. (B and C) ChIP analysis of Zhx2 occupancy on promoters of glycolytic gene Pfkfb3 using RAW264.7 cells (B) and BMDM (C). (B) RAW264.7 cells were transfected with plasmid encoding HA-tagged murine Zhx2. ChIP analysis was performed using anti- HA. Primers targeting Pfkfb3 promoter regions (sections 1–3 [S1–S3]) (low panel) were included for PCR. (C) BMDM from WT and MKO mice were treated with LPS (100 ng/ml) for 24 h and performed ChIP assays with anti-ZHX2 Ab. Pfkfb3 promoter region S3 was amplified by PCR. (D and E) BMDM from WT and MKO mice were infected with Pfkfb3 overexpressing lentivirus or mock lentivirus. (D) Real-time rates of ECAR were analyzed as an indicator of glucose metabolism (n = 4). (E) Lactate production in supernatant were detected (n = 4). Data were presented as means 6 SD from two independent experiments and analyzed using two-tailed Student t test. **p , 0.01, ***p , 0.001.

In our study, to our knowledge, we ﬁrst point out that Zhx2 Zhx2 in macrophages during sepsis. Interestingly, our RNA- by guest on September 29, 2021 potentiated the ability of glycolysis in macrophages during seq data also found that the genes related with both lipid and sepsis. Global RNA-seq analysis demonstrated the enrichment purine metabolism were differentially expressed in BMDM of genes associated with glycolysis pathway in WT macro- from WT and MKO mice (data not shown). Indeed, previous phage expressing Zhx2 compared with that with Zhx2 dele- reports suggested Zhx2 as a novel regulator of plasma lipid tion. Blocking glycolysis with 2-DG attenuated the effect of metabolism (36, 37). However, it still needs further research to

FIGURE 6. Zhx2-specific deletion in macrophage alleviates sepsis via Pfkfb3. Mice pre-exposed to 5.5 Gy of whole-body g-irradiation were i.v. injected with WT and MKO BMDM with absence or presence of Pfkfb3 overexpression and treated with LPS (10 mg/kg) to induce sepsis. (A) H&E analysis of lung tissue were performed 24 h after LPS injection. Original magnification 3100. Scale bars, 20 mm. (B and C) Concentration of proinflammatory factors, including TNF-a, IL-6, IL-1b (B), and lactate (C) were detected in serum (n = 4). Data were analyzed using two-tailed Student t test and presented as means 6 SD. *p , 0.05, **p , 0.01. The Journal of Immunology 9 determine whether Zhx2 is involved in regulating other metabolism Shandong. This work was technically assisted by Advanced Medical Re- pathways in macrophages during sepsis. search Institute, Shandong University. One important question is how Zhx2 enhance the glycolysis in macrophages. Identified as a ubiquitous transcription factor, Zhx2 Disclosures represses expression of many genes, including AFP, GPC3 as The authors have no financial conflicts of interest. reported (21, 38), and cyclin A/cyclin E and MDR1, which we found (23, 39). In this study, we added Pfkfb3 as a new target of Zhx2. As a member of the PFKFB family, Pfkfb3 critically con- References trols the glycolytic rate under normal and pathophysiological 1. Reinhart, K., R. Daniels, N. Kissoon, F. R. Machado, R. D. Schachter, and S. Finfer. 2017. Recognizing sepsis as a global health priority - a WHO reso- conditions (40–42). Recent studies reported that deletion of the lution. N. Engl. J. Med. 377: 414–417. Pfkfb3 gene reduced cancer cells glycolysis and tumor growth, 2. Delano, M. J., and P. A. Ward. 2016. Sepsis-induced immune dysfunction: can making it a promising target for anticancer therapy (43). Jiang immune therapies reduce mortality? J. Clin. Invest. 126: 23–31. +/2 3. Singer, M., C. S. 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Corrections

Wang, Z., L. Kong, S. Tan, Y. Zhang, X. Song, T. Wang, Q. Lin, Z. Wu, P. Xiang, C. Li, L. Gao, X. Liang, and C. Ma. 2020. Zhx2 accelerates sepsis by promoting macrophage glycolysis via Pfkfb3. J. Immunol. 204: 22322241.

The n error values were omitted from the legend for Fig. 1C and 1G. The corrected legend for Fig. 1 is shown below.

FIGURE 1. Zhx2 protects mice from sepsis shock and inhibits inflammation. (A) C57BL/6 mice were challenged with PBS and LPS (10 mg/kg, i.p.) for 24 h. CD8 T cell (CD31 CD81), B cell (CD3À CD191), macrophage (CD11b1 F4/801), and dendritic cell (CD11b1CD11c1) from spleen were sorted by MoFlo Astrios EQ. Zhx2 mRNA was detected by RT-PCR. (B) BMDM from WT C57BL/6 mice were treated with 100 ng/ml LPS for the indicated duration of time. Levels of Zhx2 were determined by RT-PCR. (CJ) WT and MKO mice challenged with LPS (10 mg/kg, i.p.) (CF)orCLPtreatment(GJ)toin- duce sepsis. (C and G) Survival curves (PBS: n/WT 5 10, n/MKO 5 10, LPS: n/WT 5 26, n/MKO 5 25; sham operation (abbreviated as Sham): n/WT 5 9, n/MKO 5 9, CLP: n/WT 5 14, n/MKO 5 15), ****p value for four groups and *p value for LPS or CLP-treated WT/MKO mice. (D and H) H&E staining of lung tissues. Original magnification Â100. Scale bars, 20 mm. (E and I) Serum concentrations of IL-6, TNF-a, and IL-1 b after 24 h (n 5 4). (F and J) Serum concentrations of lactate (n 5 6) were analyzed. Data were presented as mean ± SD from three independent experiments and analyzed by unpaired, two-tailed t test with significance. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

In addition, because of an error in the assembly of Fig. 2A, the flow cytometry result for the WT IL-6 group was mistakenly duplicated from that of the MKO TNF-a group. This inadvertent error does not influence the interpretations and conclusions for Fig. 2 and for the work as a whole. The corrected version of Fig. 2 is shown below. The figure legend was correct as published and is shown below for reference.

The online version of the article has been corrected and now differs from the print version as originally published.

FIGURE 2. Zhx2 aggravates proinflammatory factor secretion from macrophages in septic mice. WT and MKO mice were injected with i.p. LPS (10 mg/kg; n 5 4). (A and B) Gated on CD11b1F4/801 cells, intracellular TNF-a, IL-6, and IL-1b were detected by flow cytometry. (A) Representative dot plots were shown on the left panels and the percentage (top) and number (bottom) were shown on the right panels. (B) Representative histograms and mean fluorescence intensities (MFIs) were shown. (C) F4/801 macrophages were isolated from the spleen of WT and MKO mice. RT-PCR detected the mRNA expression of TNF-a, IL-6, and IL-1b (n 5 6). (D) PM was harvested from WT and MKO mice challenged with LPS for 24 h. Western blot detected the protein level of IL-6 and IL-1b. The data were presented as means ± SD from two independent experiments and analyzed by unpaired, two-tailed t test with significance. *p < 0.05, **p < 0.01, ***p < 0.001.