IMMUNOLOGY

ORIGINAL ARTICLE

Relevance of Foxp3+ regulatory T cells for early and late phases of murine sepsis Roman Tatura,1 Michael Zeschnigk,2 Wiebke Hansen,1 Joerg Steinmann,1 Pedrina Goncalves Vidigal,1 Marina Hutzler,1 Eva Pastille,1 Astrid M. Westendorf,1 Jan Buer1 and Jan Kehrmann1 1

Institute of Medical Microbiology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany and 2Institute of Human Genetics, University Hospital Essen, University of Duisburg-Essen, Essen, Germany

doi:10.1111/imm.12490 Received 8 January 2015; revised 8 May 2015; accepted 2 June 2015. Correspondence: Jan Kehrmann, Institute of Medical Microbiology, University Hospital Essen, University of Duisburg-Essen, Hufelandstrasse 55, 45122 Essen, Germany. Email: [email protected] Senior author: Jan Kehrmann

Summary The role of Foxp3+ regulatory T (Treg) cells in the course of the early hyper-inflammatory and subsequent hypo-inflammatory phases of sepsis is ambiguous. Whereas Nrp1 expression has been reported to discriminate natural Treg cells from induced Treg cells, the Treg cell stability depends on the methylation status of foxp3-TSDR. To specifically evaluate the role of Foxp3+ Treg cells in the early and late phases of sepsis, we induced sepsis by caecal ligation and puncture and subsequent Pseudomonas aeruginosa lung infection in a DEREG (DEpletion of REGulatory T cells) mouse model. We found an increase of Foxp3+ Treg cells to all CD4+ T cells during murine sepsis. Using a new methylation-sensitive quantitative RT-PCR method and deep amplicon sequencing, we demonstrated that natural (Nrp1+ Foxp3+) Treg cells and most induced (Nrp1 Foxp3+) Treg cells are stable and exhibit unmethylated foxp3-TSDR, and that both Treg populations are functionally suppressive in healthy and septic mice. DEREG mice depleted of Foxp3+ Treg cells exhibit higher disease scores, mortality rates and interleukin-6 expression levels than do non-depleted DEREG mice in early-phase sepsis, a finding indicating that Foxp3+ Treg cells limit the hyper-inflammatory response and accelerate recovery. Treg cell depletion before secondary infection with P. aeruginosa 1 week after caecal ligation and puncture does not influence cytokine levels or the course of secondary infection. However, a moderate Treg cell recurrence, which we observed in DEREG mice during secondary infection, may interfere with these results. In summary, Treg cells contribute to a positive outcome after early-phase sepsis, but the data do not support a significant role of Treg cells in immune paralysis during late-phase sepsis. Keywords: foxp3-TSDR; methylation; Nrp1; Pseudomonas aeruginosa; regulatory T cells.

Introduction Sepsis is one of the leading causes of death among patients in intensive care units. A recently developed model of sepsis divides the disease into two phases.1,2 The early phase is characterized by shock and severe inflammation, including the release of pro-inflammatory cytokines,1 whereas the late phase is characterized by an impairment in the function of the immune system.1,3,4 During this anti-inflammatory late phase of sepsis, patients are at risk of death from secondarily acquired bacterial infections. Pneumonia complicates the disease course of 10–30% of patients in intensive care units who 144

are treated with mechanical ventilation because of septic shock.5 It has been assumed that the immune function does not recover in those patients, who die in the late phase of sepsis.6 Regulatory T (Treg) cells are specialized immune cells that play an important role in immune homeostasis. During infection, Treg cells limit inflammation and collateral tissue damage but may also weaken bacterial clearance.7 The late phase of sepsis in particular is characterized by immune-suppressive conditions.8 We and others have shown that the percentage of Treg cells is higher in septic patients than in patients without the disease.9–11 It seems natural to assume that Treg cells contribute to ª 2015 John Wiley & Sons Ltd, Immunology, 146, 144–156

Treg cells in sepsis immune-suppressive conditions during the course of sepsis, but the relevance of these cells during the hyperinflammatory phase and the later course of the disease is still unclear, and the results of existing studies are contradictory. On the one hand, it has been reported that the relative increase in the percentage of Treg cells contributes to lymphocyte anergy and impairs survival11 and that a reduction in the percentage of Treg cells is accompanied by an improvement in survival rates among septic mice.12 On the other hand, it has also been shown that an adoptive transfer of Treg cells improves sepsis-related mortality rates13 and that Treg cells are necessary for recovery from sepsis.14 However, the depletion of CD4+ CD25+ Treg cells in mice does not alter survival rates.15–17 Previous studies did not sufficiently differentiate the role of Treg cells during the early hyper-inflammatory phase of sepsis from that during the subsequent hypoinflammatory phase. Ours is the first study to differentiate the early hyper-inflammatory phase and the later hypo-inflammatory phase of sepsis to specifically study the specific role of Foxp3+ Treg cells with the DEREG (DEpletion of REGulatory T cells) mouse model. This method is a more specific approach for depleting Treg cells than is depletion by CD25 antibodies, the method that was often used in previous studies and that also affects activated conventional T cells.18 We performed caecal ligation and puncture (CLP), an established model of murine polymicrobial sepsis.19 To study the effect of Treg cells after the hyper-inflammatory phase of sepsis, we induced a secondary infection 1 week after CLP with Pseudomonas aeruginosa, a Gram-negative rod and a typical cause of nosocomial pneumonia among mechanically ventilated patients in ICUs. Foxp3+ Treg cells comprise two separate cell populations on the basis of their origin: natural Treg cells are derived from the thymus, whereas induced Treg cells are derived from naive T cells in the periphery.20 Natural Treg cells are characterized by their stable suppressive function and their constitutive expression of Foxp3,21 whereas many peripherally induced Treg cells exhibit unstable Foxp3 expression, may lose their suppressive function, and exhibit the plastic potential to differentiate into cells with an effector function.22 The proportions and the stability of natural and induced Treg cells during sepsis are still unclear. Immunotherapy for sepsis is a promising approach for improving disease outcome, and Treg cells may be a potential target for such therapy.2 Knowledge about the origin and stability of Treg cells during sepsis may be relevant for establishing and improving targeted immunotherapies. Nrp1, a transmembrane molecule that is highly expressed by Treg cells,23 has recently been reported to be sufficient for discriminating natural Treg cells from induced murine Treg cells.24,25 Nrp1 has been proposed to be involved in interactions between Treg cells and ª 2015 John Wiley & Sons Ltd, Immunology, 146, 144–156

dendritic cells;26 it is preferentially expressed by natural Treg cells in wild-type mice and not by induced Treg cells in the gut.24 The stability of Treg cells, including the stability of Foxp3 expression and their suppressive function, is crucially dependent on the methylation status of the foxp3TSDR (Treg-specific demethylated region). This region specifically is completely demethylated in stable Treg cells committed to the Treg cell lineage, but it is heavily methylated in all other blood cells.27,28 Demethylation of the foxp3-TSDR ensures the stability of Foxp3 expression and suppressive function of Treg cells.21 Natural Treg cells are completely demethylated within the foxp3-TSDR, whereas murine induced Treg cells may either exhibit a methylated foxp3-TSDR or differentiate into fully stable Treg cells with a demethylated foxp3-TSDR under particular conditions, e.g. by antigen-specific signals through tolHence, this erogenic DEC205 vaccination.29–31 methylation is a valid marker characterizing stable committed Treg cells regardless of the Treg cell type (natural or induced).29 Because of the reported plasticity of induced Foxp3+ murine Treg cells with a methylated foxp3-TSDR, a further discrimination between foxp3-TSDR-methylated and -unmethylated induced Treg cells may be relevant for disease progression. Therefore, in the study reported here we established a single-tube methylation-sensitive quantitative RT-PCR assay for analysing the methylation status of the foxp3-TSDR and characterizing the stability of the various Foxp3+ Treg populations during sepsis.

Materials and methods Mice All animal experiments were performed in accordance with institutional, state and federal guidelines and were approved by the local ethics committee of the State Government of the Landesamt f€ ur Natur, Umwelt, und Verbraucherschutz Nordrhein-Westfalen (LANUV NRW; Az: 84-02.04.2012.A262). All animals used in this study were 8- to 12-week-old female or male mice bred on a BALB/c background and housed under specific pathogen-free conditions in the Laboratory Animal Facility of the University Hospital Essen. Wild-type BALB/c mice were obtained from Harlan Winkelmann GmbH (Borchen, Germany). DEREG mice were established as previously described32 bred on a BALB/c background. They express a diphtheria toxin receptor (DTR)-enhanced green fluorescent protein (GFP) fusion protein under the control of the foxp3 locus; this expression allows the detection and the inducible depletion of Foxp3+ Treg cells.32 This protein is highly specific and allows us to study the role of Foxp3+ Treg cells by applying diphtheria toxin (DT) at any desired time point during the immune response.33 145

R. Tatura et al. This model is more specific than the model of depleting Treg cells with CD25 antibodies, the method that was frequently used in the past. Foxp3-GFP mice, which express both Foxp3 and GFP under the endogenous regulatory sequence of the foxp3 locus, were obtained from the Jackson Laboratory (Bar Harbor, ME).

Peritoneal sepsis model To induce sepsis, we used the CLP model.19 Mice were anaesthetized with intraperitoneal injections of ketamine (CEVA, Duesseldorf, Germany) and xylazine (CEVA, 100 lg/5 lg per g bodyweight). After a midline skin incision, the distal third of the caecum was ligated. The ligated section was punctured once with a 27-gauge needle, and a small amount of caecal content was extruded. After the caecum had been returned to the abdominal cavity, 1 ml of sterile isotonic saline was injected into the abdominal cavity for volume substitution. Finally, the peritoneum and the skin were sutured. As a control, the sham procedure resembled CLP but without injury to the caecum. Disease severity was monitored and documented with a scoring system using a four-point scale (0, no disease burden; 1, light burden; 2, strong burden; 3, heaviest burden, requiring euthanasia of the mouse) to assess the following variables: weight loss, appearance, activity, breathing, wound healing and excretions. Disease severity was rated as the sum of the scores for all variables.

Depletion of Treg cells We depleted Treg cells in DEREG mice with intraperitoneal injections of DT (30 ng per g bodyweight; Merck, Darmstadt, Germany). The initial injection was performed 2 days before the desired Treg depletion and was followed by additional injections administered every other day. To study the relevance of Treg cells during the early hyper-inflammatory phase, we applied DT for the first time 2 days before the CLP procedure. To study the relevance of Treg cells during the hypo-inflammatory phase, we injected DT for the first time 5 days after the CLP procedure (2 days before intratracheal infection) and subsequently every other day.

sels were flushed with sterile PBS, after which lung cells were isolated. Subsequently, the lung tissue was digested in Iscove’s modified Dulbecco’s medium supplemented with 5% FCS, 80 lg/ml collagenase D and 10 lg/ml DNAse at 37° for 45 min; it was then meshed through a 70-lM strainer and rinsed with PBS. After centrifugation, the supernatant was aspirated, and the remaining erythrocytes were lysed with erythrocyte lysis buffer. Cells were washed and resuspended in PBS containing 2 mM EDTA and 2% FCS.

Isolation of Treg cells, T effector cells and antigen-presenting cells To isolate CD11c+ antigen-presenting cells, we rinsed spleens with collagenase/DNAse solution and cut them into small pieces. Antigen-presenting cells were isolated with CD11c MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s recommendations. Treg cells were characterized as CD4+ CD25+ Foxp3+, and conventional T cells were characterized as CD4+ CD25 . Both Treg cells and conventional T cells were isolated with FACS.

Antibodies and flow cytometry Cells were stained with fluorochrome-labelled anti-mouse CD4-Pacific Blue (RM4-5), CD25-phycoerythrin (PE) (PC61) and GATA3-PE-Cy7 (L50-823) antibodies, all from BD Biosciences (Heidelberg, Germany); RorcT-allophycocyanin (APC) (AFKJS-9) and T-bet-PE (eBio4B10), from eBioscience (Frankfurt, Germany); and Nrp1-APC (R&D Systems, Minneapolis, MN). Foxp3 was detected with anti-Foxp3 FITC (FJK-16s) and the Foxp3 staining kit from eBioscience, according to the manufacturer’s recommendations.

DNA extraction and bisulphite modification DNA was isolated from splenocytes with the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), and bisulphite modification of DNA was performed with the BisulFlash DNA Modification Kit (Epigentek, Farmingdale, NY) according to the manufacturer’s guidelines.

Isolation of murine splenocytes, mesenteric lymph node cells, and blood and lung leucocytes

Next-generation amplicon sequencing

After spleens had been rinsed with an erythrocyte lysis buffer, spleens or mesenteric lymph nodes (mLNs) were meshed through 100-lM cell strainers and washed with PBS containing 2 mM EDTA and 2% fetal calf serum (FCS). Heparinized murine blood was washed, incubated with erythrocyte lysis buffer, and centrifuged three times. Cells were resuspended in PBS containing 2 mM EDTA and 2% FCS. The aorta was cut, and the pulmonary ves-

For deep amplicon analysis of the foxp3-TSDR, we amplified bisulphite-treated murine DNA with tagged primers (mFoxp3_Amp2-fw CTT GCT TCC TGG CAC GAG ATT TGA ATT GGA TAT GGT TTG T and mFoxp3_Amp2rev CAG GAA ACA GCT ATG ACA ACC TTA AAC CCC TCT AAC ATC) by using the AmpliTaq Polymerase kit (Life Technologies, Carlsbad, CA) and the following settings: 5-min denaturation at 95°; first 14 touchdown

146

ª 2015 John Wiley & Sons Ltd, Immunology, 146, 144–156

Treg cells in sepsis cycles from 63° to 56°; 40 cycles at 95° for 20 seconds, at 56° for 1 min, and at 72° for 1 min; and a final elongation cycle at 72° for 5 min. The PCR products were separated on a 1% agarose gel and were purified with the QIAEX II Gel Extraction Kit (Qiagen) according to the standard protocol. After this, sample-specific sequences (multiplex identifiers, MIDs) and universal linker tags (454 adaptor sequences, A- or B-primer) were added in a second PCR, with the same settings as described above. The PCR products were purified with the QIAEX II Gel Extraction Kit (Qiagen), and DNA concentration was measured with a NanoDrop ND-1000 Spectrophotometer (peqLAB, Erlangen, Germany). Amplicons were purified with the Agencourt AMPure XP Beads system (Beckman Coulter, Krefeld, Germany) according to the protocol recommended by the manufacturer (Roche Amplicon Library Preparation Method Manual, Roche Diagnostics, Basel, Switzerland) and were quantified with a NanoDrop ND-1000 Spectrophotometer (paqLAB). The bisulphite amplicons were diluted, pooled, clonally amplified in an emulsion PCR (emPCR), and sequenced on the Roche/ 454 GS junior system according to the manufacturer’s protocol (Roche emPCR Amplification Method ManualLib-A and Roche Sequencing Method Manual).

Methylation-sensitive real-time PCR The quantitative analysis of methylated alleles (QAMA) method is a methylation-sensitive real-time PCR, allowing the relative quantification of methylated and unmethylated DNA from cell mixtures in one reaction tube with the use of two TaqMan MGB probes (Life Technologies). This method was previously established for quantification of human Treg cells.9 For the murine QAMA assay, the two differentially labelled TaqMan probes were designed to bind specifically to either the methylated or the unmethylated foxp3-TSDR target sequence. A covering of three CpGs guaranteed high specificity of the TaqMan probes. For the QAMA assay, the difference between the cycle threshold values is determined for quantification. The methylation ratio is calculated with a standard containing defined ratios of methylated and unmethylated DNA. To generate the standards, we amplified the foxp3 target region from BALB/c splenocyte DNA in a reaction volume of 25 ll containing 5 ll of GoTaq reaction buffer, 0
125 ll GoTaq polymerase (Promega, Fitchburg, WI), and 0
5 lM each of the following primers: mFoxp3Seqfw CTT GCT TCC TGG CAC GAG AAA ATC CGT TGG CTT TGA GA and mFoxp3Seqrev CAG GAA ACA GCT ATG ACG GCG TTC CTG TTT GAC TGT T. The deoxynucleotide triphosphates (dNTPs) were adjusted to a final concentration of 200 lM, and MgCl2 was adjusted to a final concentration of 1
5 mM. PCR products were purified with the QIAquick Gel Extraction Kit (Qiagen). The ª 2015 John Wiley & Sons Ltd, Immunology, 146, 144–156

concentration of isolated DNA was determined with a NanoDrop ND-1000 spectrophotometer (peqLAB). A fraction of the Foxp3-PCR product was methylated with CpG methyltransferase (M. SssI) (New England Biolabs, Ipswich, MA), and defined DNA mixtures were treated with bisulphite. For validation, we quantified defined amounts of methylated and unmethylated target DNA and various mixtures of FACS-sorted CD4+ CD25+ Foxp3+ T cells from male Foxp3-GFP mice and CD4+ CD25 Foxp3 T cells. Because foxp3 is located on the X chromosome and one of both chromosomes is methylated as a result of X inactivation in female mice, we used male mice for these analyses because their use enables a more precise quantification of the methylation status. Polymerase chain reaction was performed in 96-well optical trays with a LightCycler 480 system (Roche) to a final reaction volume of 20 ll, containing 10 ll twofold Roche TaqMan Probe Master 480, 2 ll bisulphite-treated DNA, and 1 lM of each primer (mFoxp3qPCRfw, AAA TTT GTG GGG TAG ATT ATT TGT TTT TT; mFoxp3qPCRrev, ATC ACA ACC TAA ACT TAA CCA AAT TTT TCT). The probes (VIC-labelled methylated Foxp3, ATT CGG TCG TTA TGA CGT T; FAM-labelled unmethylated Foxp3, ATT TGG TTG TTA TGA TGT TAA T) were added to a final concentration of 166 nM. All samples were analysed in duplicate. After initial denaturation at 95° for 10 min, the samples were subjected to 40 cycles at 95° for 15 seconds and at 60° for 1 min. Primers were designed with the METHPRIMER software (LiLab, UCSF, San Francisco, CA; http://www.urogene.org), and probes were designed with PRIMER EXPRESS software V2.0 (ABI, Carlsbad, CA).

Cytokine expression Serum samples were obtained from venous blood collected 24 hr after the initiation of infection. Serum was stored at 80°. interleukin-6 (IL-6), IL-10 and tumour necrosis-factor-a (TNF-a), were quantified with the Procara Cytokine Assay Kit (Panomics, Fremont, CA) according to the manufacturer’s recommendations. The assay was performed on a Luminex 200 instrument (Luminex Corporation, Austin, TX).

Functional assays Proliferation of various T-cell populations was measured after 2 9 105 cells had been labelled with PKH26 and cultivated in Iscove’s modified Dulbecco’s medium supplemented with 10% FCS, 100 U/ml penicillin, 0
1 mg/ml streptomycin and 25 lM 2-mercaptoethanol. The cells were co-cultured in the presence of anti-CD3 (1 lg/ml) and 2 9 105 CD11c+ cells from naive BALB/c mice; these cells served as antigen-presenting cells. 147

R. Tatura et al. The suppressive function of various T-cell populations was measured by co-cultivating them with 2 9 105 PKHlabelled CD4+ CD25 T cells from wild-type mice at a ratio of 1 : 1. The cells were cultured in the presence of anti-CD3 (1 lg/ml) and 2 9 105 CD11c+ cells. Proliferation and suppressive capability were assessed by flow cytometry after 72 hr.

Pseudomonas aeruginosa lung infection Secondary infection with P. aeruginosa was performed 7 days after CLP or sham operation. Pseudomonas aeruginosa (strain PAO1) was cultured on Columbia Blood Agar for 16 hr at 37°. We adjusted 40 ml of tryptic soy broth to an optical density at 600 nm (OD600) of 0
2– 0
25 with grown P. aeruginosa. The broth was shaken for 1 hr at 37° and 125 rpm. After centrifugation, the pellet was resuspended in RPMI-1640 medium supplemented with HEPES (Life Technologies, Carlsbad, CA). The OD600 of a 10-fold dilution was determined, and the number of colony-forming units was extrapolated from a standard growth curve. Bacteria were washed in RPMI1640 medium and re-suspended at the desired volume. Mice were anaesthetized by intraperitoneal injection of ketamine and xylazine, and intratracheal infection was performed with a volume of 20 ll bacterial suspension (7
5 9 106 to 1 9 107 colony-forming units) with the help of a laryngoscope, as previously described.34,35

Statistical analysis Statistical analysis was performed with Graphpadprism 5.02 software (Graph Pad Software, La Jolla, CA). Student’s t-test was used to determine statistical significance when two groups were compared. For more than two groups, multiple adjusted group-wise comparisons were performed with one-way analysis of variance and the Bonferroni post hoc test. Results were expressed as means  SEM. Statistical significance was assigned at the level of P < 0
05 (*P < 0
05, **P < 0
01, ***P < 0
001). Survival was shown graphically with Kaplan–Meier curves and was evaluated statistically with the log-rank test.

Results Frequencies of Foxp3+ Treg cells increase during sepsis, and these Treg cells largely exhibit unmethylated foxp3-TSDR Foxp3-GFP reporter mice were subjected to CLP. We then monitored the course of sepsis and studied the frequencies of Treg cells and their DNA methylation status within the foxp3-TSDR. CLP caused a sepsis of medium severity, defined by a mortality rate of 20–25% after 1 week. Approximately 23% of the mice died within the 148

first week; of these, 90% died within the first 3 days after CLP (Fig. 1a). The maximal disease severity score was observed on day two; recovery from sepsis, characterized by a decrease in clinical score and weight gain, began after day 3 (Fig. 1b,c). The disease severity score for sham mice remained at zero for the entire observation period. Compared with healthy mice, diseased mice exhibited a decrease in the relative percentage of CD4+ T cells in blood, spleen, and mLNs as early as day 2; this decrease persisted for the first week (Fig. 1d–f). The percentage of Foxp3+ Treg cells was clearly higher in mLNs, spleen and blood at day 2 and became even higher in all three organs during the course of sepsis within the first week. Although the percentage of Treg cells in spleen and blood had increased by day 2 after sham operation, this percentage returned to normal by day 7 (Fig. 1g,h). Studies have shown that the foxp3-TSDR is demethylated in all murine natural Treg cells and in some induced Foxp3+ CD4+ CD25+ Treg cells with a stable phenotype, whereas it is highly methylated in CD4+ CD25 T cells and in unstable induced Foxp3+ Treg cells.28–31 To determine whether the increased percentage of Treg cells is due to stable Treg cells with a demethylated foxp3-TSDR or to unstable Treg cells with a methylated foxp3-TSDR, we confirmed the degree of methylation in the murine foxp3-TSDR in isolated CD4+ CD25+ Foxp3+ Treg cells and conventional CD4+ CD25 T cells of male mice by using next-generation deep amplicon sequencing and a QAMA assay. All 12 analysed CpGs within the foxp3TSDR were differentially methylated (Fig. 2a). In healthy male mice, 90–95% of CD4+ CD25+ Foxp3+ T cells exhibited a demethylated foxp3-TSDR, a finding indicating that most of the Treg cells are stable; 5–10% of these cells exhibited a methylated foxp3-TSDR. We found that 95–100% of the CD4+ CD25 T cells were completely methylated within the foxp3-TSDR in healthy mice. These percentages were not substantially different from the methylation status of septic mice 4 days after CLP. The results of QAMA assay and next-generation deep amplicon bisulphite sequencing showed concordant results (Fig. 2a,b).

Frequencies of Nrp1 Foxp3+ induced Treg cells increase during sepsis, and these Treg cells exhibit a methylation pattern and function similar to those of Nrp1+ Foxp3+ Treg cells To determine whether the percentage of induced Treg cells increases during sepsis, we analysed the Nrp1 expression of Foxp3+ Treg cells. Whereas the percentage of Nrp1-expressing Foxp3+ T cells in mLNs and spleens at day 7 after CLP was largely similar to that in the mLNs and spleens of healthy mice, it was significantly lower in blood (68% versus 82%; P < 0
01) and in lungs (67% versus 80%; P < 0
05; Fig. 3a), a finding suggesting a ª 2015 John Wiley & Sons Ltd, Immunology, 146, 144–156

Treg cells in sepsis

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Figure 1. Increase in the Foxp3+ regulatory T-cell ratio during sepsis. (a) Survival rate of mice after caecal ligation and puncture (CLP) and after sham operation. Mice were subjected to either sham (continuous line) or CLP (dotted), and survival rates were documented for 7 days. (b) Disease severity was determined by a scoring system based on weight, appearance, activity, breathing, wound healing and excretions; the score for sham mice was zero for the entire observation period. (c) Changes in body weight in CLP-treated mice (a–c, n = 35 for each group). The percentage of CD4+ lymphocytes within the lymphocyte gate was analysed by flow cytometry in (d) blood, (e) spleen and (f) mesenteric lymph nodes (mLNs) 2, 4 and 7 days after CLP or sham surgery. The expression of Foxp3 in CD4+ T cells is shown in (g) blood, (h) spleen and (i) mLNs (d–i, n = 3 to n = 7). Data are shown as means  SEM; *P < 0
05. **P < 0
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relative increase in the number of induced Nrp1 Treg cells in these organs during sepsis. These cells also exhibited a mainly demethylated foxp3-TSDR (Fig. 3b), a finding indicating limited plasticity, because the demethylation status within foxp3 is characteristic of Treg cells committed to the Treg cell lineage. Foxp3+Nrp1+ Treg cells were almost completely demethylated (98%) in healthy and septic mice (Fig. 3b). We were also interested in determining the functionality of the various Nrp1 populations. The proliferative ª 2015 John Wiley & Sons Ltd, Immunology, 146, 144–156

capability of sorted Foxp3+ Nrp1+ and Foxp3+ Nrp1 Treg cells was much lower than that of Foxp3 Nrp1+ and Foxp3 Nrp1 T cells. Interestingly, the proliferative capability of Foxp3 Nrp1+ T cells was lower than that of Foxp3 Nrp1 T cells (Fig. 3c). We also analysed the functionality of the various Nrp1 populations in an in vitro suppression assay. Foxp3+ Nrp1+ and Foxp3+ Nrp1 populations exhibited a strong in vitro suppressive capability, which was clearly higher than that of the Foxp3 Nrp1+/ populations (Fig. 3d). 149

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Figure 2. DNA-methylation of CD4+ CD25+ Foxp3+ regulatory T (Treg) cells and CD4+ CD25 T cells within the foxp3 Treg-specific demethylated region. (a) Schematic view of foxp3 locus illustrates the intron–exon (rectangles) structure, including the foxp3 Treg-specific demethylated region (TSDR). Enlargements of 12 CpGs of the foxp3TSDR are shown. Results of next-generation amplicon sequencing show the degree (%) of methylation of each of the 12 CpGs in CD4+ CD25 T cells (red) and CD4+ CD25+ Foxp3+ T cells (blue). The three CpGs, spanned by the TaqMan probes of a quantitative analysis of methylated alleles (QAMA) assay, are framed. (b) Results of QAMA assay in Foxp3+ Treg cells and CD4+ CD25 T cells of splenocytes from naive and septic mice 4 days after caecal ligation and puncture (CLP) displays the degree of methylation within the foxp3-TSDR (n = 4 to n = 7, summarized as mean  SEM). A representative gating strategy is illustrated in Fig. S2.

Depletion of Treg cells increases the severity and mortality rates of sepsis We used the DEREG mouse model to assess the role of Treg cells during the early phase of sepsis. After treatment with DT, the course of sepsis was more severe in mice lacking Treg cells (Fig. 4a–c). DT was applied for the first time 2 days before the CLP procedure. Although the mortality rate of Treg-depleted mice was 42% after 2 days and 66% after 7 days, only 8
3% of non-depleted DEREG mice had died 2 days after CLP, and 25% had died 1 week after CLP (Fig. 4a). The more severe course of disease in Treg-depleted mice also became evident through a greater loss of body weight. Treg-depleted mice did not regain weight significantly within 1 week after CLP; in contrast, septic DEREG mice without Treg cell depletion regained weight starting at day 3 (Fig. 4b). The disease severity score was clearly higher in Tregdepleted mice than in non-depleted mice during the entire observation period. The maximum severity score was higher in Treg-depleted mice, and recovery from disease began on day 4, whereas the severity score of 150

non-depleted mice began to decrease 2 days earlier (Fig. 4c). Seven days after CLP operation we verified the efficacy of DT in depleting Treg cells within mLNs, spleen and blood. DT treatment depleted 90–95% of Treg cells in all studied organs by 7 days after CLP (Fig. 4d–f). Concordant with the results obtained from Foxp3-GFP mice, the Foxp3+ Treg cell percentage to all CD4+ T cells increased in DEREG mice that did not receive DT (Fig. 4d–f). To determine whether the depletion of Treg cells affects pro-inflammatory and antiinflammatory serum cytokines, we measured the levels of IL-6, TNF-a and IL-10 24 hr after CLP. Although the IL-6 level was significantly higher in septic DT-treated mice than in septic mice not treated with DT, the levels of TNF-a and IL-10 were concordantly but not significantly higher in DT-treated mice than in PBS-treated mice (Fig. 4g–i).

The course of secondary infection with P. aeruginosa after CLP is not influenced by reductions in Treg percentages Dysfunction of the adaptive immune system has been reported to contribute to the suppression of the immune response during sepsis.36,37 After detecting increases in the percentages of Treg cells 1 week after CLP, we determined whether this increase in Treg frequencies contributes to immune dysfunction 7 days after CLP. We examined whether the induction of a secondary infection during this phase is less controlled in normal mice than in Treg-depleted mice. To do so, we induced P. aeruginosa lung infection in CLP mice that had or had not been subjected to the depletion of Treg cells. Mice were injected with DT for the first time 2 days before intratracheal application of P. aeruginosa. The mortality rate was 22% (Fig. 5a), and treatment with DT did not produce relevant differences in the disease severity score (Fig. 5b), changes in body weight (Fig. 5c), and mortality rates (Fig. 5a). The efficacy of Treg depletion by treatment with DT was evaluated 2 days and 7 days after infection with P. aeruginosa. Although the application of DT resulted in a 90–95% depletion of Treg cells by day 7 after CLP (Fig. 4d–f), only 78–89% of Treg cells were depleted 2 days after P. aeruginosa infection, and only 28–62% were depleted by day seven after infection, depending on the organ (Fig. 5d–g). These Treg cells were primarily insensitive to DT, as shown by the fact that they largely did not express GFP (Fig. 5h). Recurring Treg cells express Foxp3 through the endogenous foxp3 gene; therefore, these recurring Treg cells are not influenced by DT, in contrast to Treg cells in uninfected DEREG mice, which predominantly express Foxp3 through the additional BAC foxp3-DTR construct, the only such construct that is sensitive to DT. ª 2015 John Wiley & Sons Ltd, Immunology, 146, 144–156

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05; **P < 0
01; ***P < 0
001. A representative gating strategy is illustrated in Fig. S3.

Additionally, 7 days after secondary infection with P. aeruginosa we found no relevant difference between Treg-depleted mice and non-depleted mice in the expression of the master transcription factors of CD4+ T cells, such as RORcT or GATA3. Tbet expression was slightly higher in Treg-depleted mice than in non-depleted mice, a finding indicating a trend toward an increase in the type 1 helper (Th1) response (Fig. 5i–l). The levels of the cytokines IL-6, TNF-a and IL-10 did not differ significantly between DT-treated and PBS-treated mice 1 day after P. aeruginosa infection (Fig. 5m–o).

Discussion An imbalance in the ratio of Treg cells to conventional T cells has been reported to contribute to the development of autoimmune diseases and to influence pathogen clearance and host damage during viral and parasitic infections.38,39 A relative increase in the number of Treg cells during sepsis, an increase that is explained by the higher resistance of Treg cells to lymphocyte apoptosis, is a typical feature in mice and humans.9–11,14,16,40,41 In the study reported here, we confirmed that the percentage of CD4+ T cells decreases during sepsis and found that the relative increase in the number of Treg cells is largely attributed to Foxp3+ Treg cells with an unmethylated foxp3-TSDR. In many ª 2015 John Wiley & Sons Ltd, Immunology, 146, 144–156

previous murine studies, the relative increase in the number of Treg cells was attributed to CD4+ CD25+ T cells. Because CD25 expression is also up-regulated in CD4+ T cells after TCR activation,42 the use of CD25 may not correctly measure the Treg cell population, especially for diseases associated with a highly activated immune response, such as sepsis. Because Foxp3 is selectively expressed by murine Treg cells, Foxp3 expression describes murine Treg cells more specifically than does CD25 expression. In addition, we found that 95% of Foxp3+ Treg cells are demethylated in the foxp3-TSDR; this demethylation is a feature typical of stable Treg cells that are committed to the lineage.30,43 Although a demethylated FOXP3TSDR is highly specific for human natural/thymic Treg cells, some murine induced Treg cells may also exhibit a demethylated foxp3-TSDR.28,31 In the gut, induced murine Foxp3+ Treg cells have been reported to exhibit a largely unmethylated foxp3-TSDR.24 Therefore, the foxp3TSDR methylation status cannot reliably differentiate thymic from induced murine Treg cells, but determines Treg stability. We detected no difference between healthy and septic mice in the foxp3-TSDR methylation status of CD4+ CD25+ Foxp3+ cells, a finding indicating that the percentage of stable Foxp3+ Treg cells is unchanged during sepsis. In addition, our finding that 5% of Foxp3 CD4+ T cells are unmethylated within the 151

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Figure 4. Relevance of regulatory T (Treg) cells during the hyper-inflammatory phase of sepsis. (a) Survival rate of DEREG mice treated with diphtheria toxin (DT; dotted line) or PBS (continuous line) 7 days after caecal ligation and puncture (CLP). DT was administered 2 days before CLP and subsequently every other day. Course of (b) body weight and (c) severity index of septic mice after CLP (a–c: PBS, n = 12; DT, n = 12; summarized as mean  SEM). Percentage of Foxp3-expressing CD4+ T cells 7 days after operation in (d) blood, (e) spleen and (f) mesenteric lymph nodes (mLNs) of mice treated with DT or PBS (n = 3 to n = 8, summarized as mean  SEM). Levels of the cytokines (g) tumour necrosis factor-a (TNF-a), (h) interleukin-6 (IL-6) and (i) IL-10 in blood 24 hr after operation were determined by the Luminex platform from CLPtreated and PBS-treated septic mice (g–i: n = 3 to n = 14, shown as mean  SEM). *P < 0.05; **P < 0.01; ***P < 0.001. A representative gating strategy is illustrated in Fig. S1.

Figure 5. Relevance of regulatory T (Treg) cells in secondary infection after the hyper-inflammatory phase of sepsis. Disease severity was documented in a–c: (a) Secondary infection survival rate of DEREG mice treated with either diphtheria toxin (DT) or PBS beginning 5 days after caecal ligation and puncture (CLP) (2 days before Pseudomonas aeruginosa infection). (b) Disease severity score after CLP (0d) and P. aeruginosa lung infection (7d). (c) Changes in body weight after CLP (0d) and P. aeruginosa infection (7d) (a–c: PBS, n = 16; DT, n = 21; summarized as mean  SEM). Percentage of Foxp3 expression of CD4+ T cells in (d) blood, (e) spleen, (f) mesenteric lymph nodes (mLNs), and (g) lungs in PBS-treated or DTtreated naive mice, sham-operated mice (7 days after operation), CLP mice (7 days after operation), and mice after secondary infection with P. aeruginosa (2 days (sec. inf. 2d) or 7 days (sec. inf. 7d) after P. aeruginosa infection) as analysed by FACS) (d–g: n = 3 to n = 9, lung without data for CLP and sham, summarized as mean  SEM). (h) Percentage of GFP+ and GFP Foxp3-expressing T cells after secondary infection as determined by FACS. Expression of various master transcription factors in T cells 7 days after P. aeruginosa secondary infection. Tbet (Th1), GATA3 (Th2), and RORcT (Th17) from (i) blood, (j) spleen, (k) mLNs, and (l) lung were studied in DT-treated and PBS-treated mice as determined by FACS (h–l: n = 3 to n = 10, summarized as mean  SEM). Levels of the cytokines (m) tumour necrosis factor-a (TNFa), (n) interleukin-6 (IL-6) and (o) IL-10 in blood 24 hr after secondary infection were quantified with a Luminex system in DT-treated or PBS-treated mice (m–o: n = 3 to n = 10 with mean  SEM; each dot represents one mouse). *P < 0.05; **P < 0.01; ***P < 0.00.1 A gating strategy is illustrated in Figs S1 and S4.

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foxp3-TSDR suggests that some committed Treg cells have lost Foxp3 expression. Those Foxp3 CD4+ T cells with an unmethylated foxp3-TSDR were recently called ‘latent’ Treg cells and were shown to be functional Treg cells that ª 2015 John Wiley & Sons Ltd, Immunology, 146, 144–156

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reacquire Foxp3 expression upon activation and efficiently suppress T-cell proliferation.31 In the past, it was impossible to distinguish natural murine Treg cells from induced murine Treg cells on the basis 153

R. Tatura et al. of expressed protein markers because cell-type-specific marker molecules were not available. The results of TCR repertoire analysis have suggested that induced Treg cells constitute as much as 30% of total Treg cells in the periphery but that most Treg cells are natural Treg cells.44,45 Recently Nrp1 has been reported to distinguish natural from induced murine Treg cells,24,25 although it was also found that induced Treg cells from mice undergoing severe inflammatory reactions may express Nrp1.24 Using Nrp1 Foxp3+ expression to characterize induced Treg cells, we found that 20–30% of the Treg cells in the blood, spleen, mLNs and lungs of healthy mice are induced Treg cells. In spite of the notion that induced Treg cells may express Nrp1 during severe inflammation, we found that the percentage of Nrp1-expressing Foxp3+ T cells in blood and lungs is reduced during sepsis, a finding indicating that the ratio between natural and induced Treg cells shifts toward induced Treg cells in these organs. Our study has shown that Nrp1+ Foxp3+ Treg cells are completely demethylated within foxp3-TSDR, that they do not proliferate, and that they inhibit the proliferation of responder cells in healthy and septic mice. We also found that Nrp1– Foxp3+ Treg cells are mainly demethylated within the foxp3-TSDR, a finding indicating that most of them are stable Treg cells and exert a suppressive function similar to that of Nrp1+ Foxp3+ Treg cells. In the gut, induced Nrp1– Foxp3+ Treg cells have been reported to exhibit a largely unmethylated foxp3-TSDR.24 Additionally, we found a population of slowly proliferating Foxp3– Nrp1+ T cells that has no suppressive function but exhibits a reduction in the methylation of the foxp3TSDR. Given that the foxp3-TSDR is exclusively demethylated in committed Treg cells, most committed Foxp3– Treg cells hide within this Nrp1+ population. In this study, we examined the relevance of Foxp3+ Treg cells for the early and late phases of sepsis. We found that the depletion of Foxp3+ Treg cells leads to a more severe course of sepsis with a higher mortality rate and a significantly higher IL-6 level than in DEREG mice that have not undergone Treg cell depletion. This finding indicates that Treg cells attenuate the hyper-inflammatory immune response during the early phase of CLP sepsis. Earlier studies analysing the relevance of Treg cells during sepsis yielded contradictory results. Improved survival rates had been reported by the reduction of Treg cells12 as well as by an increase of Treg cells by adoptive transfer13 It has been suggested that Treg cells are necessary for recovery from sepsis14 while others reported that an increase in lymphocyte anergy is brought about by an increasing Treg ratio during sepsis.11 Other researchers have reported that the depletion of CD4+ CD25+ Treg cells in mice does not relevantly influence survival rates.15–17 CD25 antibodies have often been used to study the role of Treg cells in sepsis, because no more-specific 154

approaches were available. Because CD25 expression is up-regulated by non-regulatory CD4+ T cells after activation,42 CD25 antibodies also affect these cells, and this effect may result in lower pathogen clearance. The DEREG mouse model allows the specific depletion of T cells that express Foxp3 and is a more specific approach for studying the role of Treg cells in the early hyper-inflammatory phase of sepsis. Our finding of a significantly increased IL-6 level and a trend toward higher levels of TNF-a in Treg-depleted mice during sepsis supports the hypothesis that the hyper-inflammatory response during the early phase of sepsis is intensified in mice lacking Treg cells. We also studied the role of Treg cells in the late phase of sepsis, the phase that is associated with immune suppression. Sepsis can be divided into two phases: an initial hyper-inflammatory phase and a subsequent, more protracted immunosuppressive phase.1,18 This hyperinflammatory phase in mice that have undergone CLP has been previously described.46 In contrast to Murphey and Sherwood,46 who induced a secondary infection 5 days after CLP, we induced a secondary infection 7 days after CLP. In our setting, Treg cell frequencies had normalized in sham-operated mice by 7 days after CLP, but after 4 days they were still higher than those in unoperated control mice. For this reason it seemed most appropriate to study the relevance of Treg cells for a secondary infection 1 week after CLP, when any effects of the sham operation would no longer remain. The percentages of Treg cells 7 days after CLP were generally higher than those at earlier time-points (Fig. 1g–i). During a secondary P. aeruginosa pneumonia 1 week after CLP, when the frequencies of Foxp3 Treg cells were high, we found no changes in disease severity, mortality rates, or cytokine levels between Treg-depleted mice and nondepleted mice. Therefore, our findings do not suggest that higher Foxp3+ Treg cell frequencies during the late phase of sepsis are crucial for influencing the course of this secondary infection with P. aeruginosa or for immune paralysis at that time point. Treg cell depletion before P. aeruginosa infection without a previous CLP operation tends to result in a slightly higher loss of body weight and in higher disease severity scores and mortality rates (see Supplementary material, Fig. S5). Pseudomonas aeruginosa lung infection also increases the proportion of Foxp3+ Treg cells (Fig. S5). Nevertheless, our study of secondary infection with P. aeruginosa has one limitation that may be relevant for the unchanged course of disease in both Treg-depleted and undepleted mice. The depletion of Treg cells during secondary infection with P. aeruginosa was not as efficient as the depletion before CLP. While 90–95% of Treg cells were depleted before the induction of sepsis and throughout the entire observation period, the efficacy of Treg cell depletion was distinctly reduced during secondary ª 2015 John Wiley & Sons Ltd, Immunology, 146, 144–156

Treg cells in sepsis P. aeruginosa lung infection, even though DT was applied for the first time 2 days before P. aeruginosa infection. We detected a recurrence of Treg cells as early as 2 days after infection, and this recurrence was clearly increased at day 7. These Treg cells were GFP and DTR negative, a finding indicating that during secondary infection of DEREG mice the Treg niche is rapidly replaced by a Treg population that does not respond to DT treatment. The recurring Treg cells in DEREG mice during secondary infection with P. aeruginosa express Foxp3 through the endogenous foxp3 gene instead of through the additional BAC-transgene foxp3-DTR, which is expressed by most Treg cells in uninfected DEREG mice. Because of the absence of DTR expression, the application of DT is ineffective in depleting these recurring Treg cells. A recurrence of DT-insensitive Foxp3+ Treg cells in DEREG mice has recently been reported to limit the impact of Treg cell depletion by DT during mycobacterial infection.47 The reason for the increased expression of endogenous foxp3 in recurring Treg cells during secondary infection is so far unknown. However, we also cannot exclude the possibility that a more effective depletion of Treg cells or a depletion at other time-points may have improved the course of secondary infection and that the targeting of Treg cells during sepsis may nevertheless be a successful immunotherapy for sepsis. Our studies have shown that Treg cells limit the severity of early sepsis, but our findings do not support the essential relevance of Treg cells for immune paralysis during the late phase of sepsis. We have also demonstrated that during sepsis most Treg cells within the foxp3-TSDR are demethylated, including both natural Nrp1+ Foxp3+ Treg cells and most Nrp1– Foxp3+ induced Treg cells. Because the lineage plasticity of cells with an unmethylated foxp3-TSDR is limited, manipulating them by transdifferentiation into effector T-cell subsets with the aim of reducing immunosuppression may be challenging.

Acknowledgements We thank Mechthild Hemmler-Roloff for excellent technical assistance with luminex analyses and Witold Bartosik and Patrick Juszczak for cell sorting. We thank Nils Lehmann of the Institute of Medical Informatics, Biometry and Epidemiology, University of Duisburg-Essen, Essen, Germany, for assistance in performing statistical analyses. This work was supported in part by grants from the Stiftung Mercator.

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Disclosures

latory T cells enhances their interactions with dendritic cells during antigen recognition. Immunity 2008; 28:402–13. 27 Baron U, Floess S, Wieczorek G et al. DNA demethylation in the human FOXP3 locus discriminates regulatory T cells from activated FOXP3+ conventional T cells. Eur J Immunol 2007; 37:2378–89. 28 Floess S, Freyer J, Siewert C et al. Epigenetic control of the foxp3 locus in regulatory T

None of the authors has any potential financial conflict of interest related to this manuscript.

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Additional Supporting Information may be found in the online version of this article: Figure S1. Representative gating strategy for analysing CD4 expression on cells within lymphocyte gate and Foxp3 expression in CD4+ T cells, which is used in Figs 1, 4 and 5. Figure S2. Representative gating strategy for fluorescence-activated cell sorting (FACS) of CD4+ CD25+ Foxp3+ T cells and CD4+ CD25- T cells. Figure S3. Representative gating strategy for analyses of Foxp3+ Nrp1+, Foxp3+ Nrp1–, Foxp3– Nrp1+, and Foxp3– Nrp1– T cells. Figure S4. Representative gating strategies for analysis of Tbet, RORcT, GATA3 and Foxp3 expression of CD4+ T cells (Fig. 5). Figure S5. Relevance of regulatory T (Treg) cells in Pseudomonas aeruginosa lung infection.

ª 2015 John Wiley & Sons Ltd, Immunology, 146, 144–156