Identification of a Unique Hybrid -Polarization State following Recovery from Tolerance

This information is current as Christine O'Carroll, Ailís Fagan, Fergus Shanahan and of September 30, 2021. Ruaidhrí J. Carmody J Immunol 2014; 192:427-436; Prepublished online 11 December 2013; doi: 10.4049/jimmunol.1301722

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Supplementary http://www.jimmunol.org/content/suppl/2013/12/27/jimmunol.130172 Material 2.DC1 http://www.jimmunol.org/ References This article cites 62 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/192/1/427.full#ref-list-1

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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 © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Identification of a Unique Hybrid Macrophage-Polarization State following Recovery from Lipopolysaccharide Tolerance

Christine O’Carroll,* Ailı´s Fagan,† Fergus Shanahan,* and Ruaidhrı´ J. Carmody‡

LPS tolerance is an essential immune-homeostatic response to repeated exposure to LPS that prevents excessive inflammatory responses. LPS tolerance induces a state of altered responsiveness in , resulting in repression of proinflammatory gene expression and increased expression of factors that mediate the resolution of inflammation. In this study, we analyzed the transcriptional plasticity of macrophages following LPS tolerance using genome-wide transcriptional profiling. We demonstrate that LPS tolerance is a transient state and that the expression of proinflammatory genes is restored to levels comparable to the acute response to LPS. However, following recovery from LPS tolerance a number of genes remained locked in a tolerizable state, including IL-33, CD86, IL-10, and NFIL3. Furthermore, we identified of a number of genes uniquely induced following recovery

from LPS tolerance. Thus, macrophages adopt a unique transcriptional profile following recovery from LPS tolerance and have Downloaded from a distinct expression pattern of regulators of Ag presentation, antiviral responses, and transcription factors. Our data suggest that recovery from LPS tolerance leads to a hybrid macrophage activation state that is proinflammatory and microbicidal in nature but that possesses a regulatory anti-inflammatory profile distinct from that of LPS-tolerant and LPS-activated macrophages. The Journal of Immunology, 2014, 192: 427–436.

nflammation is a powerful, organized, and complex immu- LPS tolerance may lead to profound consequences, including death as http://www.jimmunol.org/ nological response to and tissue damage. TLRs are the result of excessive and uncontrolled levels (4, 7). I key sensors of microbial presence and host damage and are Recent transcriptional profiling of macrophages identified two central to innate inflammatory responses (1). TLR4 is the most classes of LPS-responsive genes: those that are suppressed during extensively studied TLR that recognizes LPS, an outer membrane LPS tolerance, so-called “tolerizable genes,” which include TNF-a cell wall component of Gram-negative bacteria. Ligation of TLR4 and IL-6, and those genes whose expression is maintained or even triggers intracellular-signaling pathways, culminating in the pro- increased during LPS tolerance, so-called “nontolerizable genes,” duction of proinflammatory , chemokines, and type 1 IFNs which include antimicrobial and anti-inflammatory genes. Chro- to effectively clear infection (1–3). Chronic or repeated exposure of matin modifications are associated with LPS tolerance, which macrophage to LPS leads to a phenomenon termed LPS tolerance, promotes a global transcriptional switch in macrophage gene ex- by guest on September 30, 2021 which represents a state of altered responsiveness to additional LPS pression (8). This unique transcriptional signature was proposed to challenge. The most notable feature of LPS tolerance is the sharp drive a phenotypic switch in macrophage polarization from a reduction in proinflammatory cytokine expression, including TNF-a proinflammatory to an anti-inflammatory proresolution state (8). and IL-6, in response to LPS stimulation. LPS tolerance is critical in A number of negative regulators of TLR-induced responses have limiting the innate response to infection to reduce host damage been identified that are critical in promoting LPS tolerance. These and promote the resolution of inflammation (4–6). Failure to enforce include regulators of TLR4-induced signal transduction, such as IRAK-M, TOLLIP, SHIP, and A20, and transcriptional regulators, such as Bcl-3 (9–16). Macrophages display a spectrum of activation states, which are *Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland; broadly described as classical (M1), alternative (M2), and regu- †Department of Surgery, Royal College of Surgeons Ireland, Dublin 2, Ireland; and ‡Centre for Immunobiology, Institute of Infection, Immunity, and Inflammation, latory (17, 18). M1 macrophages are proinflammatory cytotoxic College of Medicine, Veterinary, and Life Sciences, University of Glasgow, Glasgow phagocytes and promote Th1 adaptive-immune responses fol- G12 8TA, United Kingdom lowing TLR and IFN-g challenge (17, 18). In contrast, IL-4/IL-13 Received for publication July 1, 2013. Accepted for publication October 30, 2013. specifically polarizes macrophages to an alternatively activated This work was supported by Science Foundation Ireland Grant 08/IN.1/B1843, M2 state. These cells are anti-inflammatory, promote tissue remod- Centres for Science, Engineering and Technology Grant 07/CE/B1368, and the Marie Curie International Reintegration grant program. eling, and are important in mediating Th2 immunity to fungal and parasitic (17, 19, 20). Regulatory macrophages are Microarray profiling data have been deposited in the National Center for Biotech- nology Information Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/ immune regulatory cells that display both anti-inflammatory and geo/query/acc.cgi?acc=GSE47783) under series entry identifier GSE47783. proinflammatory features through suppression of some proin- Address correspondence and reprint requests to Dr. Ruaidhri J. Carmody, Centre for flammatory genes and increased production of IL-10, while main- Immunobiology, Institute of Infection, Immunity, and Inflammation, College of Med- taining Ag-presenting capacity. Immune complexes and FcR icine, Veterinary, and Life Sciences, University of Glasgow, Glasgow G61 1QH, U.K. E-mail address: [email protected] engagement in conjunction with TLR activation polarize macro- The online version of this article contains supplemental material. phages toward a regulatory state. In addition, other stimuli, in- Abbreviations used in this article: A, acute response; BMDM, bone marrow–derived cluding , PGs, and apoptotic cells, can polarize macrophage; COA, correspondence analysis; FMO, fluorescence minus one; GO, macrophages into a regulatory macrophage state (17). These three gene ontology; M1, classical; M2, alternative; N, naive; PC, principal component; activation states represent the main groups in the emerging spec- qRT-PCR, quantitative real-time PCR; RM, recovered macrophage; T, LPS tolerized. trum of macrophage polarization. However, because of the large Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 number of activation stimuli that macrophages encounter in vivo, www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301722 428 TRANSCRIPTION AND RECOVERY FROM TOLERANCE many hybrid activation states exist. These include tumor-associated Thioglycollate-elicited peritoneal macrophages macrophages, intestinal macrophages, and LPS-tolerized (T) mac- Peritoneal macrophages were isolated as previously described (31). rophages, all of which share overlapping features of M1, M2, and Thioglycollate-elicited peritoneal macrophages were isolated from 6–8- regulatory macrophages while displaying distinct markers of acti- wk-old female C57BL/6 mice by peritoneal lavage 3 d post-i.p. injection vation (21–26). with 1 ml sterile 4% thioglycollate. Briefly, 10 ml ice-cold sterile PBS was LPS tolerance polarizes macrophages into an anti-inflammatory injected into the peritoneal cavity, followed by gentle massaging of the cavity to allow cells to be collected and withdrawn by peritoneal lavage. activation state distinct from M2 macrophage polarization (21– Collected cells from three mice were pooled, passed through a 70-mM cell 23). Although transcriptomics analysis has been performed on LPS strainer (BD Biosciences), and centrifuged at 4˚C for 5 min at 300 3 g. tolerance (8, 22, 27), the plasticity of this state has not been ex- Isolated thioglycolate-elicited peritoneal macrophage were allowed to plored using the same approach. In this study, we analyzed the adhere to tissue culture dishes overnight in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, 1% L-glutamine, and 1% nones- persistence of LPS tolerance using transcriptional profiling and sential amino acids. Nonadherent cells were removed prior to experi- bioinformatics analysis. We used a model of LPS activation of mental use. murine bone marrow–derived macrophages (BMDMs) that gener- ated naive (N), LPS-activated/acute response (A = M1), and T Microarray profiling polarization states. Our data demonstrate that LPS tolerance is a All data processing was carried out using Bioconductor packages (32) in the transient state in macrophages and that, following LPS tolerance, R software environment. The data were background corrected using the macrophages adopt a previously undescribed hybrid activation Limma package (33). The data were then corrected for batch effects using state, which we call “recovered macrophages” (RMs) to reflect the ComBat script (34). The corrected data were then normalized between arrays using quantile normalization (33). Differentially expressed genes the recovery from LPS tolerance. These data support previous were found using the Limma package (33). These genes were then cor- Downloaded from in vivo and in vitro findings that LPS tolerance is transient (8, rected for multiple testing using the Benjamini–Hochberg method, with an 27–29). Using genome-wide transcriptional analysis, we demon- adjusted p value cut off of p , 0.05 and a fold change of 1.5. Gene on- strate that recovery from LPS tolerance, as defined by cytokine tology (GO) was performed using DAVID (35). Gene-expression changes and gene-expression trends were visualized by hierarchical and K-means expression, is associated with a global change in the transcrip- clustering using Genesis software (36). Gene-expression profiling was tional profile of macrophages. Thus, RM cells express tolerizable validated independently by quantitative real-time PCR (qRT-PCR). Micro- genes, such as TNF-a and IL-6, following LPS stimulation at array profiling data were deposited in the National Center for Biotechnology http://www.jimmunol.org/ levels equivalent to LPS-activated M1-polarized cells. However, Information Gene Expression Omnibus database with the series entry a number of genes remain locked in an LPS-tolerizable state and identifier GSE47783. Total RNA was isolated using the RNeasy mini kit (QIAGEN, Man- do not recover from LPS tolerance, distinguishing RM cells from chester, U.K.) with DNase treatment. RNA quantification and purity were M1 cells. Furthermore, RMs express a subset of inducible genes measured using the Nanodrop ND1000 spectrophotometer (Thermo Sci- not induced in M1 or tolerized M2-like states. In addition, RM entific), with triplicate biological replicate samples submitted for micro- cells have a distinct expression pattern of regulators of Ag pre- arrayprofiling(BeckmanCoulterGenomics, Morrisville, NC). Briefly, 200 ng total RNA was fluorescently labeled with Cy3 nucleotides. Labeled sentation and antiviral responses. Our data suggest that recovery RNA (cRNA) was hybridized to Agilent mouse 8 3 60K mouse microarrays from LPS tolerance leads to a novel hybrid macrophage activation (Agilent-028005). Hybridized arrays were washed and scanned, with data state that is proinflammatory and microbicidal in nature but that extrapolated for downstream bioinformatics analysis. Each experiment is by guest on September 30, 2021 possesses a regulatory anti-inflammatory profile distinct from that representative of BMDM derived from bone marrow pooled from three of T and LPS-activated (M1) macrophages. mice. Microarray gene-expression profiling was performed in triplicate. Gene-expression analysis Materials and Methods Gene expression was measured by qRT-PCR. A total of 1 mg total RNA was Reagents used to synthesize cDNA with random hexamer primers using Transcriptor Ultrapure LPS from K12 Escherichia coli was used at a final concentration Reverse Transcriptase (Roche Applied Sciences, West Sussex, U.K.). The of 100 ng/ml (InvivoGen, Toulouse, France). Flow cytometry Abs (F4/80, reaction mix was incubated for 10 min at 25˚C to allow efficient annealing CD80, CD86, CCR3, and isotype controls) were purchased from BD of random primers, followed by incubation at 55˚C for 30 min. The en- Biosciences (San Jose, CA). DMEM, FBS penicillin and streptomycin, zyme was inactivated at 85˚C for 5 min. Gene expression was measured by L-glutamine, and nonessential amino acids were all purchased from Sigma- qRT-PCR using the LightCycler 480 (Roche Applied Sciences). Primers Aldrich (Dublin, Ireland). were designed using the Universal Probe Library system (Roche Applied Sciences). Gene expression for individual genes was measured and nor- LPS tolerance–recovery model malized to 18S (forward = 592AAATCAGTTATGGTTCCTTTGGTC-39, reverse = 59-GCTCTAGAATTACCACAGTTATCCAA-39). Gene-expression BMDMs were differentiated from bone marrow from isolated femur and changes were calculated using the 22DDCT method (37). Data are repre- tibia bones of 6–8-wk-old female C57BL/6 mice. BMDMs were differ- sented as relative mRNA. All research was performed under nuclease-free entiated in culture media supplemented with 30% L929-conditioned media conditions. for 7 d, as previously described (30). BMDMs were assessed by flow + cytometry and were typically .95% F4/80 . BMDMs were cultured in Western blotting complete BMDM media (DMEM, 10% FBS, 1% penicillin/streptomycin, 1% L-glutamine, and 1% nonessential amino acids). LPS tolerance was BMDMs were removed from tissue culture dishes using a cell scraper, with induced by stimulation with LPS (100 ng/ml) for 8 h, followed by washout cells collected into 13 ice-cold PBS (30). BMDMs were wash twice in ice- of LPS with fresh medium. Cells were rested for an additional 16 h prior to cold PBS and centrifuged at 300 3 g for 5 min at 4˚C. Total protein was restimulation with LPS (100 ng/ml) for 4 h. Recovery from LPS tolerance isolated by lysing the cell pellet in RIPA buffer (150 mM NaCl, 50 mM (R) was analyzed following an additional resting period of up to 4 d prior Tris-Cl [pH 7.4], 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM to restimulation with LPS (100 ng/ml) for 4 h to determine the optimal Na3VO4, 1 mM EDTA) supplemented with a protease and phosphatase time point for recovery. All research, including transcriptional-profiling inhibitor mixture (Sigma-Aldrich). Cytoplasmic and nuclear proteins were experiments, were performed on macrophages rested for 3 d prior to isolated from BMDMs using a nuclear extract kit (Active Motif, Carlsbad, restimulation (R). Additional analysis of recovery from LPS tolerance on CA). Protein quantification was performed using a BSA standard curve and days 2 and 4 is specified within the text, where appropriate. The acute Bradford reagent. Protein concentration was measured at 595 nm using response to LPS was measured in cells that received no stimulation with an absorbance plate reader. Total protein was resolved by SDS-PAGE LPS prior to stimulation with 100 ng/ml LPS for 4 h. Naive macrophages gels, transferred to polyvinylidene difluoride membrane, and immune remained untreated. For microarray analysis, all groups, with the exception blotted for p65, c-Rel, p50, RelB, IRF4 (Santa Cruz, Dallas, TX), IRF5 of the N group, were restimulated for 4 h prior to RNA isolation and (Abcam, Cambridge, U.K.), p-p38, P38, p-ERK, and ERK (Cell Signaling, subsequent downstream experiments (Fig. 1A). Boston, MA). The Journal of Immunology 429

Flow cytometry across each macrophage group (Fig. 2A). Cluster 1 contains genes Macrophages were removed from tissue culture dishes and collected in ice- that are highly induced by LPS in the T group, but not the A or RM cold 13 staining buffer (13 PBS, 0.5% BSA, 1% FBS, 0.1% sodium azide group, illustrating that clusters of genes are specific to the induction [Sigma-Aldrich]) and washed three times in ice-cold staining buffer. Cells and maintenance of LPS tolerance in macrophages. In contrast, were incubated with CD16/CD32 Ab for 15 min, according to the man- clusters 3 and 5 contain genes that were suppressed in T group but ufacturer’s protocol, to block nonspecific binding of Abs to the FcRs on were inducible again in the RM group. Interestingly, cluster 5 genes cells. Cells were surfaced stained with Abs to F4/80, CD80, CD86, and CCR3 in the dark on ice for 30 min. The cells were washed in staining were induced to a greater level in the RM group relative to the A buffer and resuspended in running buffer (13 PBS, 1 mM EDTA, 0.5% group, whereas cluster 3 genes were induced to a lesser degree. This BSA) prior to analyzing on the Accuri C6 Flow cytometer (BD Biosci- suggests that the transcriptional profile of RMs is different from that ence). Isotype controls and fluorescence minus one (FMO) were used as of A group. In support of this finding, cluster 6 contains genes that controls. Data were analyzed using FCS express flow cytometry software (De Novo Software, Los Angeles, CA). are highly induced in the RM group compared with all other groups, thus identifying a unique transcriptional signature for this group Cell viability (Fig. 2A). Viability of BMDMs was measured by quantifying ATP levels from cells of We next performed correspondence analysis (COA) on the each group (N, A, T, and R) using the CellTiter-Glo Luminescent Cell microarray dataset to visualize and measure the correlation between Viability Assay (Promega) at the experimental end point. Briefly, cells were the global transcriptional profile of each group (N, A, T, and RM). allowed to reach room temperature, then an equal volume of CellTiter-Glo A 30% principal component 1 (PC1) measure of variance between reagent was added and the mixture was shaken for 10 min. ATP levels were measured using an ATP standard curve, with luminescence recorded using the different macrophage groups was identified, with the A and RM a luminometer. Data are represented as the percentage viability. populations clustered closely together. This demonstrated that the A Downloaded from and RM groups share overlapping transcriptional profiles distinct Statistical analysis Differences between groups were determined using one-way ANOVA, with Tukey post hoc analysis. qRT-PCR expression data were calculated using the 22DDCT methods (37) and are expressed as relative mRNA. Statistical analysis was performed using GraphPad Prism software (San Diego, CA).

Data are mean 6 SEM and a p value , 0.05 was considered statistically http://www.jimmunol.org/ significant.

Results Recovery from LPS tolerance involves a global shift in the transcriptional profile We designed a model of LPS tolerance in ex vivo BMDMs based on previous studies (8, 22, 27) of tolerance in human and mouse BMDMs. We generated a transcriptional profile of N, M1 by guest on September 30, 2021 (A), and T macrophages. We also analyzed LPS responses in RM cells (Fig. 1A). Briefly, LPS tolerance was induced by treatment with LPS (100 ng/ml) for 8 h, followed by washout of LPS with fresh medium. Cells were rested for an additional 16 h prior to restimulation with LPS (100 ng/ml) for 4 h. Recovery from LPS tolerance was analyzed following an additional resting period of 3 d prior to restimulation with LPS (100 ng/ml) for 4 h. The acute response to LPS was measured in cells that were not stimulated with LPS prior to stimulation with 100 ng/ml LPS for 4 h. N macrophages remained untreated (Fig. 1A). The induction of LPS tolerance under the conditions used in this study was confirmed by qRT-PCR of three previously described LPS-tolerizable proinflammatory genes: IL-6, CXCL10,andTNF-a (8) (Fig. 1B). LPS-inducible expression of these genes was sup- pressed in the T group compared with the A group. In contrast, cells in the RM group demonstrated inducible gene expression in all three genes at levels comparable to the A group. Similar ex- pression patterns of IL-6, CXCL10,andTNF-a were seen in thioglycolate-elicited peritoneal macrophages treated in the same wayas BMDMs (Fig. 1C). The viability of cells recovering from FIGURE 1. Recovery from LPS tolerance restored macrophage proin- tolerance was similar to that of tolerized cells (Fig. 1D). This flammatory gene expression. (A) Ex vivo model of recovery of LPS tol- confirms that LPS tolerance is a transient macrophage-activation erance using murine BMDMs, as described in Materials and Methods. state in both mouse and human macrophage/ cells (Fig. Four unique macrophage-polarization states were induced: N, A, T, and B 1B, 1C), in agreement with previous in vitro and in vivo studies RM (day 3). ( ) CXCL10, IL-6, and TNF-a gene expression was measured by qRT-PCR for each macrophage-activation state 4 h postrestimulation (8, 27–29, 38). (BMDMs). (C) Gene expression of CXCL10, IL-6, and TNF-a was mea- We next performed microarray analysis of LPS-stimulated suredinthioglycolate-elicited peritoneal macrophages. (D) BMDM viability BMDMs in each of the groups described above. Initial K-means (% viability) was measured 4 h post-LPS restimulation. Data are represen- clustering analysis revealed a unique transcriptional signature for tative of more than four independent experiments and are shown as relative each macrophage treatment group (N, A, T, and RM), with 10 mRNA. *p , 0.05, **p , 0.01, ***p , 0.001, one-way ANOVAwith Tukey unique clusters that grouped genes with similar expression profiles post hoc test. D0, Day 0; D-1, day 1; D-2, day 2; D-3, day 3. 430 TRANSCRIPTION AND RECOVERY FROM TOLERANCE

associated with macrophages of the A group compared with the N group (N) from this study (Supplemental Fig. 2, Supplemental Tables I, II). The GO terms “cell cycle,” “cell division,” and “mitosis” were the most significantly enriched biological pro- cesses found from the analysis of genes upregulated in the RM group relative to the T group (Supplemental Fig. 1B, Supple- mental Tables I, II). These data suggest that, following recovery from LPS tolerance, macrophages are proinflammatory cells with increased cellular proliferative capacity compared with anti- inflammatory tolerized cells. Although COA (PC1: 30%) revealed similar transcriptional profiles between the A group and the RM group (Fig. 2B), clear differences in the expression of distinct groups of genes were found. Following recovery from tolerance, restimulation of mac- rophages with LPS induced the expression of 310 genes not induced during the initial acute LPS response. Conversely, 332 genes were no longer induced by LPS following recovery from tolerance. This finding is supported by COA (PC4: 8%) (Fig. 2B).

The chemokine receptor CX3CR1, which is often used to distin- Downloaded from guish between inflammatory and resident monocyte/macrophage lineages (25, 39–42), was one of the most significantly induced genes following recovery from LPS tolerance but not during the acute LPS response. Similarly, the chemokine receptors CCR5 and FIGURE 2. Recovery from LPS tolerance mediates a global shift in the CCR3, the folate receptor FOLR2, and IFN-g were specifically A transcriptional profile of macrophages. ( ) Microarray gene-expression induced to high levels in the R group (Figs. 3A, 4, Supplemental http://www.jimmunol.org/ profiling was performed on N, A, T, and RM groups (n = 3/group). Tran- Table I). The proinflammatory gene IL-33 was the most signifi- scriptional profiling was analyzed using K-means clustering, which identified cantly suppressed differentially expressed gene in the R group 10 unique gene profile clusters (left panel). All genes/cluster are represented as relative to the A group. IL-33 is suppressed during LPS tolerance normalized expression-intensity values (log2), with high levels of induction and remains “locked” in a tolerizable gene-expression state fol- outlined in red (right panels). Statistical significance was determined using Benjamini–Hochberg multiple-correction testing. Data are represented as lowing recovery from LPS tolerance (Figs. 3B, 4B, Supplemental log2 fold change of 60.585 (1.5-fold change) and p , 0.05. Upregulated Table I). genes (Up) are represented in red, and downregulated genes (Down) are Unsurprisingly, we found that the 310 LPS-induced genes dif- illustrated in green. (B) COA was performed on all macrophage groups (N, ferentially expressed in the RM group relative to the A group were A, T, and RM). PC1 identified 30% variance between individual groups. enriched with numerous immune-related processes and pathways by guest on September 30, 2021 PC2 revealed 13% variance between individual microarray-replicate experi- by GO analysis. These included “,” “response to ments. PC3 (11%) identified differences between the N and T groups, with wounding,” and “inflammatory response” (Fig. 3C, Supplemental PC4 (8%) revealing differences between the A and RM groups. Tables I, II). Of note, the most significant biological terms iden- tified from the downregulated differentially expressed genes were from those of the N and T groups. Upon closer examination, PC4 “antiviral response” and “response to virus.” Genes identified in (8% variance between groups) identified transcriptional differ- these groups include IFNA4, IFNA12, IFNA13, IFNA2, IFNA5, ences between the A and R groups, further supporting a unique and IFNA7 (Fig. 4A, 4B). Other significantly enriched GO terms gene-expression profile in RMs (Fig. 2B). This finding indicates were “cytokine activity,” “immune system development,” and a high degree of macrophage plasticity following the induction of “cytosolic DNA-sensing pathways” (Fig. 3C, Supplemental Tables LPS tolerance. Taken together, these initial findings confirm that I, II). Genes associated with these terms include the anti- LPS tolerance is a transient macrophage state and that recovery inflammatory cytokine IL-10 and transcription factor NFIL3 from LPS tolerance involves a global change in the macrophage (Fig. 4A, 4B, 4D). Taken together, the transcriptional profile of transcriptional profile. Each macrophage group in our model rep- RM cells reflects many features of A cells; however; these cells resents a distinct macrophage state; however, the gene-expression have a unique transcriptional signature that identifies them as a profile of the RM group shares some similarities with that of the novel macrophage-activation state, with altered expression of A group. This suggests that recovery from tolerance polarizes regulators of the antiviral responses. macrophages toward a proinflammatory classically activated–like Recovery from LPS tolerance induces a hybrid (M1) state. macrophage-activation state Differentially expressed genes and GO enrichment Ag presentation is a classical feature of M1 macrophages, whereas To further characterize the gene-expression signature of RMs, we M2 anti-inflammatory macrophages display a reduced capacity to identified differentially expressed genes in the RM group relative present Ags to adaptive immune cells (17, 43). We identified a to the N, A, and T groups (61.5-fold change, p , 0.05). GO analysis significant increase in the surface expression of CD86 and CD80 was used to determine the biological significance of differentially cells following acute activation with LPS (Fig. 5A). CD86 surface expressed genes. Unsurprisingly, “immune response,” “TLR sig- expression was suppressed during LPS tolerance and was not naling,” “Ag presentation,” “cytokine regulation,” and “defense further inducible following recovery from LPS tolerance. Similarly, response” were the most significantly enriched GO terms from LPS-inducible CD80 surface expression was suppressed in the T the genes upregulated in the RM group relative to the N group group and was not inducible in the RM group (Fig. 5A). In addition, (Supplemental Fig. 1, Supplemental Tables I, II). These data cor- LPS-inducible CD86 mRNA levels were suppressed in both the T relate with differentially expressed genes and biological processes and RM groups, similar to IL-33, IL-10, NFIL3, and type 1 IFN The Journal of Immunology 431 Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 3. Differentially expressed genes and GO enrichment. The top 30 (largest to smallest fold change) differentially expressed upregulated (A) and downregulated (B) genes in the RM group relative to the A group. (C) GO and pathway analysis for upregulated differentially expressed genes identified between the RM group and the A group. The top 10 GO terms and associated adjusted p values are listed in descending order. Differentially expressed genes were identified by a log2 fold change of 60.585 (1.5-fold change) and adjusted p value , 0.05. genes (Figs. 4A, 4B, 4D, 5B). Interestingly, we measured no dif- expression suggests that these cells retain their cytotoxic micro- ference in the LPS-inducible levels of NOS2 mRNA between cells bicidal function. In addition, the expression of both M1- and M2- in the RM and A groups, as determined by both microarray and associated genes in LPS-treated cells following recovery from qRT-PCR gene-expression analysis (Fig. 5C). LPS tolerance induces a hybrid macrophage state with a unique To further define this inducible macrophage hybrid RM state, transcriptional signature. we analyzed M1- and M2-polarizing markers from the microarray dataset. Using hierarchical clustering analysis, we identified an Recovery from LPS tolerance is associated with a unique overlap in M1- and M2-associated genes expressed following re- signaling and transcription factor profile in macrophages covery from tolerance (Fig. 5D). The M1-polarization markers Macrophage polarization is regulated by a number of key tran- IL-6, NOS2, and IL-12B (17, 18, 43) were significantly induced by scription factors, including the NF-kB and IRF families. To assess LPS following recovery from tolerance, similar to the acute-LPS the role of NF-kB in shaping macrophage responses following response. A number of M2-associated polarization markers, such recovery from LPS tolerance, we initially assessed the expression as CCL17 and CCL24 (26), were also significantly induced fol- of NF-kB subunits by immunoblot. The levels of RelB, c-Rel, and lowing recovery from tolerance. Furthermore, there was an LPS- p65 subunits in the RM group were similar to those found in the A induced increase in the expression of the M2-polarization markers group (Fig. 6A). In contrast, the T group demonstrated elevated MRC1 and chi3l3 (17, 26) following recovery from tolerance (Fig. levels of RelB, c-Rel, and p50 protein (Fig. 6A). Interestingly, 5D). These data suggest that recovery from LPS tolerance may alter total p50 levels remained elevated in the RM group. However, in macrophage Ag-presentation capacity and subsequent activation contrast to tolerized cells (14), increased p50 protein is not found of the adaptive-immune response. Our finding that, following in the nucleus prior to restimulation, indicating that elevated p50 recovery from tolerance, LPS may robustly induce iNOS gene protein is not repressive in RMs (Fig. 6B). In addition, the 432 TRANSCRIPTION AND RECOVERY FROM TOLERANCE Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 4. RMs: validated markers of identification. (A) Hierarchical clustering analysis of activation markers of the RM group (log2 fold change 6 0.585 (1.5-fold change) and adjusted p value of 0.05 (p , 0.05). (B) qRT-PCR validation of unique gene-expression patterns in the RM group in BMDMs. (C) CCR3 mean fluorescence intensity (MFI) for all macrophage-polarization groups. Data are representative of three independent experiments with FMO and isotype controls. (D) qRT-PCR analysis of IFNg, CCR5, and IL-10 gene expression in thioglycolate-elicited macrophages. *p , 0.05, **p , 0.01, ***p , 0.001, one-way ANOVA with Tukey post hoc test. restimulation with LPS was important for translocation of NF-kB IRF4, IRF8, and NFIL3 may play a role in defining the tran- into the nucleus in all groups (Fig. 6B). We next measured IRF4 scriptional signature following recovery from LPS tolerance. and IRF5 protein levels, which were associated with macrophage polarization (44, 45). IRF4 protein levels were reduced following Discussion recovery from tolerance, whereas no differences in IRF5 protein The acute response to LPS or endotoxin is one of the most ex- levels were observed (Fig. 6C). To assess LPS-induced signal trans- tensively studied immune processes, because of its powerful effects duction following recovery from LPS tolerance, we used phospho- on host immunity and disease. In this study, we used a transcriptomics specific Abs to measure activation of the ERK1/2 and p38 kinases approach to characterize the transcriptional signature of macro- following LPS stimulation for 15, 30, or 60 min (Fig. 6D). p38 and phages post-LPS tolerance induction. We identified that recovery ERK phosphorylation was suppressed in the T group at 30 min, in from LPS tolerance induced a macrophage-activation state in- agreement with previous studies (46). Following recovery from volving a global transcriptional shift from an anti-inflammatory tolerance, p38 phosphorylation was increased relative to the acute profile to a hybrid macrophage state. Although a large proportion LPS response. ERK1/2 were activated to levels comparable to those of tolerizable proinflammatory gene expression (IL-6, TNF-a, of the acute LPS–activated cells (Fig. 6D). CXCL10) was restored in RM cells, a number of proinflammatory Hierarchical clustering analysis of transcription factor mRNA (IL-33, type 1 IFNs), anti-inflammatory (IL-10), and immune- levels revealed no difference in the expression of NF-kB and STAT regulatory genes (NFIL3, CD86) remained locked in a toleriz- mRNA in the RM group compared with the A group. However, we able state, and, thus, failed to recover from LPS tolerance. The observed differences in the levels of the transcription factors NFIL3 transcriptional profiles generated appear to reflect the individual cell and IRF8, a member of the IRF family of transcription factors. LPS- rather than a heterogeneous population, because flow cytometry inducible NFIL3 and IRF8 gene expression was suppressed following analysis of cell surface markers did not reveal subpopulations of recovery from tolerance in a pattern similar to IL-33, IL-10,andthe cells. In addition, RMs expressed a number of unique markers not type I IFN genes (Fig. 6E). These data suggest that, although no expressed in the other macrophage groups. Taken together, these differences were observed in NF-kB protein levels, suppression of data confirm that recovery from LPS tolerance does not polarize The Journal of Immunology 433 Downloaded from

FIGURE 5. Recovery from LPS tolerance induces a hybrid macrophage-activation state. (A) Mean fluorescence intensity (MFI) of CD80 and CD86 staining. Data are representative of three independent experiments, with data normalized to FMO and isotype controls. (B) qRT-PCR gene-expression validation of CD86 gene expression. (C) Microarray (left panel) and qRT-PCR (right panel) validation of NOS2 gene expression for each macrophage group. qRT-PCR gene expression is represented as relative mRNA. Data are representative of more than four independent experiments. (D) Hierarchical clustering with complete linkage for M1- and M2-associated genes. Statistical significance was determined by log2 fold change 6 0.585 (1.5-fold change) and an adjusted p value of 0.05 (p , 0.05). Upregulated (Up) and downregulated (Down) genes are shown in red and green, respectively. *p , 0.05, **p , 0.01, ***p , 0.001, one-way ANOVA with Tukey post hoc test. ns, Not significant. http://www.jimmunol.org/ macrophages back into a proinflammatory M1 activation state but express reduced levels of two associated M2-polarizing markers rather a hybrid macrophage state with a unique transcriptional (IL-33, IRF4), which may be critical to promoting this hybrid signature. state. The reduced levels of IRF4 protein observed are not re- Macrophages are key components of the innate immune re- flected in the mRNA levels, suggesting that IRF4 may be post- sponse and central mediators of inflammation and defense. These translationally regulated in RMs. IL-33 is a proinflammatory cells also play important physiological roles involving tissue repair cytokine, alarmin, and amplifier of LPS activation (50–54). The and remodeling and phagocytosis of cellular debris and apoptotic lack of IL-33 expression in RMs may be important in limiting by guest on September 30, 2021 cells, in addition to having important metabolic functions (17). This the amplification of LPS responses and M2 polarization through range of biological roles is reflected in an emerging spectrum of paracrine signaling. Reduced expression of the M2-polarizing macrophage-activation states largely focused around the M1 transcription factor IRF4 (44, 55) may play a role in facilitating proinflammatory/M2 anti-inflammatory classification system. M1 recovery from LPS tolerance by limiting the potential for main- macrophages are mainly recruited under inflammatory conditions, tenance of an M2 macrophage-polarization state. IRF4 was shown such as bacterial or viral infections. These cells are highly cyto- to be important for the induction of M2-associated genes in re- toxic, produce high levels of proinflammatory cytokines, and are sponse to chitin in vivo (44) and in vitro (45). Previous studies (45, powerful activators of Th1 adaptive-immune responses. In con- 56) demonstrated that macrophage polarization may be skewed trast, M2 macrophages are anti-inflammatory cells that are im- toward an M2 profile following differentiation with M-CSF but portant in , tissue remodeling, and parasitic infections not GM-CSF, suggesting that differentiating factors may influence (17, 18, 26, 43, 47), with regulatory macrophages sharing overlapping the role of specific transcription factors in promoting activation proinflammatory M1 and anti-inflammatory M2 features. Because states. Some M2-associated genes, including CCL17 and CCL22, of the wide range of activation stimuli that macrophage encounter also may be induced in response to TLR activation (8, 27). Thus, in vivo, a wide range of hybrid states exists. Although these pop- M2-associated genes that are LPS responsive may be regulated ulations display stimulus-specific markers of activation and biological independent of the IRF4–JMJD3 axis, as described by Satoh et al function, they also share many overlapping anti-inflammatory–like (44). properties (17, 24–26). In addition, many resident macrophage pop- We found increased expression of CX3CR1 in the RM and T ulations, such as liver Kupffer cells and intestinal, placental, LPS- groups. CX3CR1 is expressed on some resident macrophage pop- tolerant, and tumor-associated macrophages, adopt a hybrid state ulations and has been implicated in cellular survival. Thus, in- that is dependent on the local microenvironment (22, 23, 26, 48, 49). creased expression of CX3CR1 in RMs may regulate survival In this study, we identified a novel macrophage-activation state postinflammatory challenge (57, 58). Although the reduced ex- that shares many properties of M1 and M2 macrophage pop- pression of a number of type 1 IFNsinRMsmayincreasesus- ulations. RMs express highly inducible levels of proinflammatory ceptibility to viral infections, suppression of these genes may cytokines and have cytotoxic capacity, as illustrated by inducible be important to limit their unnecessary production postbacterial NO synthase expression, suggesting that these cells are more inflammatory challenge. Our data demonstrate that RMs are distinct proinflammatory M1-like. However, the expression of markers from LPS-tolerized macrophages and represent a unique macro- of M2 activation (CCL17, CCL22), in addition to the reduced phage population primed to respond to new inflammatory chal- expression of regulators of Ag presentation (CD80, CD86) fol- lenges under strict environmental conditions. lowing recovery from LPS tolerance, suggests that RMs display The plasticity of macrophages could potentially have both features of both M1 and M2 polarization states. Importantly, RMs positive and negative outcomes, depending on the local environ- 434 TRANSCRIPTION AND RECOVERY FROM TOLERANCE

FIGURE 6. Recovery from LPS toler- ance induces a unique signaling and transcription factor profile in macro- phage. Immunoblot of total (A) and nuclear (B)NF-kB members during recovery from LPS tolerance. Total protein was measured following 60 min of restim- ulation with LPS and normalized to b-actin protein. Nuclear protein was measured at 0 and 60 min of LPS restimulation and Downloaded from normalized to HDAC protein. (C)Im- munoblot analysis for IRF4 and IRF5 protein from whole-cell lysates. b-actin wasusedasaloadingcontrol.(D)Im- munoblot analysis of p-p38, p38, ERK, and p-ERK from whole-cell lysates at

0, 15, 30, and 60 min of restimulation. http://www.jimmunol.org/ b-actin was used as a loading control. (E) Hierarchical clustering analysis with complete linkage of transcription factor genes during LPS tolerance recovery. Statistical significance was determined by a log2-fold change 6 0.585 (1.5-fold change) and p , 0.05. Data are repre- sentative of more than three independent experiments. D2, Day 2; D3, day 3; D4, day 4. by guest on September 30, 2021

mental requirements and activation stimuli that macrophages en- environmental microbial community. This environment is rich in counter. Although an endotoxin/LPS-tolerant state may limit po- the polarizing cytokines IL-10 and TGF-b, which contribute to an tentially devastating consequences of excessive inflammation, anti-inflammatory–tolerant environment in the gut (25). In addition, prolonged tolerance can have devastating consequences. Mono- the resident Kupffer cell population in the liver adopts an anti- cytes isolated from patients with sepsis or cystic fibrosis display inflammatory tolerized-like phenotype specific to the liver microen- reduced responsiveness to ex vivo challenges with LPS, as de- vironment, where they are exposed to a wide array of gut-derived and termined by proinflammatory cytokine production (59). These environmental toxins under normal physiologic conditions (48, 61, cells remained locked in a tolerant state that coincided with patient 62). These two macrophage populations represent environments in mortality due to an inability to produce a robust inflammatory re- which prolonged tolerance is a physiological protection mecha- sponse to secondary infections. Interestingly, patients who recovered nism. The loss of this normal tolerance state is associated with from sepsis are responsive to LPS upon LPS challenge ex vivo (59, alcoholic liver disease, hepatitis C viral infections, and inflam- 60). This suggests that recovery from endotoxin tolerance may be a matory bowel disease (63, 64). Of note, deficiencies in IL-10 and critical physiological response to maintain host protection to invading the transcription factor NFIL3, two genes that are locked in a microbial challenges. In addition, the high levels of IFN-g expressed tolerized state in RMs, have been associated with chronic in- in LPS-stimulated RMs, but not in tolerized cells or during the acute flammation in the gut (65). In addition, CD14+ lamina propria response, may function to elicit a heightened state of immune read- mononuclear cells from patients with Crohn’s disease or ulcer- iness that prepares the host for additional or repeated infections. ative colitis showed reduced NFIL3 gene expression in compari- Under normal homeostatic conditions, intestinal macrophages son with noninflamed cells (65). NFIL3 is a regulator of IL-12B adopt an anti-inflammatory tolerant-like state toward the local expression and is induced by IL-10 in macrophages. Although we The Journal of Immunology 435 measured no difference in LPS-inducible IL-12B gene expression 17. Mosser, D. M., and J. P. Edwards. 2008. Exploring the full spectrum of mac- rophage activation. Nat. Rev. Immunol. 8: 958–969. in RMs, the suppression of both IL-10 and NFIL3 expression in 18. Lawrence, T., and G. Natoli. 2011. Transcriptional regulation of macrophage RMs may facilitate the recovery from LPS tolerance. 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