Open Access

Acute Lipopolysaccharide-Induced Inflammation Lowers IL-2R Signaling and the Proliferative Potential of Regulatory T Cells

Sunnie Hsiung, Alejandro Moro, Yuguang Ban, Xi Chen, Alicia Santos Savio, Rosmely Hernandez and Thomas R. Malek
ImmunoHorizons December 1, 2020, 4 (12) 809-824; DOI: https://doi.org/10.4049/immunohorizons.2000099

Abstract

IL-2R signaling is essential for the development and homeostasis of CD4+Foxp3+ regulatory T cells (Tregs). Low-dose IL-2 is being advanced as a therapy for autoimmune diseases because of its ability to expand Tregs. Although Treg stability and function is diminished by chronic inflammation, the impact of inflammation on proximal IL-2R signaling and/or responsiveness to low-dose IL-2 is poorly understood. In this study, we show that acute inflammation induced by LPS, analogous to responses to acute bacterial infection, led to decreased endogenous STAT5 signaling and proliferative potential as measured by Ki67 in mouse Tregs. This impaired Treg activity was transient, did not lead to a reduction in Treg numbers or function, and was due to TLR signaling by non-Tregs. Although acute LPS induced high levels of IL-1 and IL-6, these cytokines did not solely mediate dysregulated Treg activity. Global gene expression analyses demonstrated that acute LPS-induced inflammation substantially and rapidly altered the Treg transcriptome. In the presence of an IL-2R agonist, the mouse IL-2/CD25 fusion protein (mIL-2/CD25), this type of inflammatory response tempered the transcription of IL-2R–dependent genes in vivo. Gene enrichment and pathway analyses are consistent with LPS attenuating mIL-2/CD25–dependent genes related to the cell cycle, DNA replication, and cholesterol biosynthesis while enhancing mRNAs that mediated Treg suppression in vivo. Acute LPS-induced inflammation diminished some responses by Tregs to mIL-2/CD25 treatment in vivo. Together, these results suggest a role for persistent IL-2R signaling in mitigating some but not all of the deleterious effects of inflammation on Treg proliferation while supporting their function.

Introduction

Regulatory T cells (Tregs) are essential for maintaining self-tolerance by suppressing autoreactive effector cells that escape into the periphery. IL-2 critically controls thymic Treg development (1, 2), Treg peripheral homeostasis (3, 4), and Treg identity (5, 6). Correspondingly, when IL-2 or IL-2R subunits are ablated in the germline or conditionally in Tregs, the resulting mice die of lethal autoimmunity (7, 8). When IL-2Rα (CD25) ablation is induced in peripheral Tregs, these cells and their progeny only persist for several months because of impaired IL-2–dependent survival (9, 10). Many immune cell types, including CD4+ conventional T cells, CD8+ T cells, and NK cells, can upregulate CD25 upon Ag- and/or cytokine-dependent activation, but Tregs are unique in that Foxp3, the master transcriptional regulator for Tregs, directly contributes to CD25 expression, and consequently, Tregs constitutively express elevated levels of high-affinity IL-2R (11, 12). Proximal IL-2R signaling in Tregs primarily induces tyrosine phosphorylation of STAT5 (p-STAT5), which leads to upregulation of Foxp3 and Cd25 in a positive feedback loop to maintain Treg identity (13–15).

Immune homeostasis may become dysregulated by increased frequency of autoreactive T cells or by decreased frequency of Tregs. This type of imbalance may result in the development of autoimmune diseases, which are characterized by inflammation that leads to tissue damage inflicted by autoreactive cells. Although Tregs are integral to maintaining immune tolerance, inflammatory cytokines can also negatively affect Tregs (16), potentially furthering the imbalance between T effector (Teff) and Treg function in autoimmunity. Monocyte-derived proinflammatory cytokines, such as IL-1β (17), TNF, (18), and IL-6 (19–21), limit Treg function and destabilize their identity by downregulating Foxp3 expression. These cytokines can also promote STAT3 and RORɣt activation in Tregs to potentially adopt properties of Teff cells, especially Th17 (22–24). Additionally, proinflammatory cytokines and LPS induce STUB1, a ubiquitin ligase, which downregulates Foxp3 expression and impairs Treg function (25). Increased production of IL-1β and IL-6 and pathogenic Th17 cells are associated with many autoimmune diseases, including multiple sclerosis (26), rheumatoid arthritis (27), psoriasis (19, 28, 29), systemic lupus erythematosus (30), and inflammatory bowel disease (31). IFN-ɣ production by autoreactive Th1 cells and other cell types also limit Treg expansion (32) and promote Treg fragility (33) (i.e., Tregs retain Foxp3 expression but their function is impaired). Therefore, identifying pathways that can stabilize Treg identity and improve Treg function in the presence of inflammation is important for the development of effective therapies for autoimmune diseases.

Treg development and homeostasis but not Teff activity are supported by low-dose IL-2R signaling (34). Administering low-dose IL-2 in several preclinical models prevented autoimmunity through selective expansion of Tregs (35–37). Although inflammation destabilizes Tregs to limit their suppressive capacity and is associated with autoimmune diseases, the extent to which inflammation affects the response of Tregs to both endogenous IL-2 and to low-dose IL-2 therapy is unknown. These points are relevant to the potential efficacy of physiologic and therapeutic IL-2R signaling in Tregs to suppress autoreactive T cells.

In the current study, these questions were investigated by determining the effects of acute inflammation induced by LPS on IL-2R signaling in Tregs. We reasoned that LPS-induced inflammation models a bacterial infection and/or inflammation that may be associated with some autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, Kawasaki syndrome, Guillain-Barre syndrome, and Graves disease (38, 39). We show that LPS-induced inflammation resulted in a decrease in IL-2R signaling in vivo that was at least partially cell intrinsic. Administering an agonist, the mouse IL-2/CD25 fusion protein (mIL-2/CD25), during this inflammatory response caused globally impaired expression of genes involved in cellular proliferation. Correspondingly, Tregs showed an attenuated response to mIL-2/CD25. The implications of these findings are discussed in relationship to low-dose IL-2 therapy.

Materials and Methods

Mice

C57BL/6, IL-6−/− (B6.129S2-Il6tm1Kopf/J), IL-1R−/− (B6.129S7-Il1r1tm1Imx/J), TLR4−/− (B6(Cg)-Tlr4tm1.2Karp/J), Foxp3YFP/Cre, Foxp3-RFP (C57BL/6-Foxp3tm1Flv/J), and TLR4flox (B6(Cg)-Tlr4tm1.1Karp/J) mice were purchased from The Jackson Laboratory. All strains were bred in a specific pathogen–free animal facility at the University of Miami. For TLR4flox × Foxp3-YFP/Cre Treg-conditional TLR4 knockout mice, genotyping was performed with primers and protocols from The Jackson Laboratory (forward primer, 5′-TGACCACCCATATTGCCTATAC-3′ and reverse primer, 5′-TGATGGTGTGAGCAGGAGAG-3′). Both male and female mice were 6–12 wk of age when the experiments were conducted. Animal studies were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Miami.

Mice were injected with LPS (Escherichia coli O55:B5; Sigma-Aldrich) i.v. LPS was reconstituted according to the manufacturer’s instructions. Mice were injected i.p. with mouse IL-2 and mIL-2/CD25. Mouse IL-2 (PeproTech) was reconstituted according to the manufacturer’s instructions. mIL-2/CD25 was prepared as previously described (40).

Luminex assay

Mouse-specific Luminex reagents were ordered from R&D Systems. Cytokine measurements were performed using the manufacturer’s instructions in a 96-well plate. Cytokine standard mixtures were also prepared per manufacturer’s instructions. Mouse serum samples were kept at −20°C prior to analysis. The serum was thawed and diluted 1:2 in the Luminex Calibrator Diluent RD6-52. Fifty microliters of diluted serum or standard was transferred to a 96-well assay plate and mixed with 50 μl of the provided Microparticle Cocktail. Plates were securely covered with a foil plate sealer and incubated for 2 h at room temperature on a horizontal microplate shaker set at 800 rpm. Using a magnet designed for a microplate, the plate was washed three times with 100 μl of the provided wash buffer. Fifty microliters of diluted biotin–Ab mixture was added to each well, securely covered with a foil plate sealer, and incubated for 1 h at room temperature on the shaker set at 800 rpm. The plate was washed three times, and 50 μl of diluted streptavidin-PE was added to each well, securely covered with a foil plate sealer, and incubated for 30 min at room temperature on the shaker set at 800 rpm. The plate was washed three times, and the microparticles were resuspended by adding 100 μl of wash buffer to each well. The plate was then incubated for 2 min on the shaker at 800 rpm. The plate was read using a Luminex MAGPIX analyzer and analyzed using xPONENT software. The raw data, collected as mean fluorescence intensity (MFI), was converted into picograms per milliliter from the standard curve of each analyte.

Cell preparation and purification

Single-cell suspensions from the spleens, mesenteric lymph nodes, inguinal lymph nodes (ILN), bone marrow, and lungs were prepared by mechanical disruption. Liver was first mechanically disrupted against a mesh screen, and RBCs and lymphocytes were separated out using 33% Percoll. RBCs were then lysed with ACK to prepare the single-cell suspension. To purify Tregs (RFP+) and T conventional cells (RFP−) from Foxp3/RFP reporter mice, total CD4+ T cells were obtained using anti-CD4 magnetic MicroBeads (Miltenyi Biotec). The cells were then stained with FITC anti-CD4 (GK1.5), and the CD4+ T cells were sorted based on the expression of the reporter using a BD FACSAria II system. Cells were >95% pure.

Abs and flow cytometry

The mAbs used in this study are listed in Supplemental Table I. For surface staining, cells were first incubated with 2.4G2 to block Fc receptors for 1 min, then stained with mAbs in HBSS containing 2% BSA and 0.1% sodium azide staining buffer, as previously described (9). Intracellular staining of Bcl-2, CTLA4, Foxp3, and Ki67 was performed using the Foxp3 Transcription Factor Staining Buffer Set (eBioscience) according to the manufacturer’s instructions. Typically, between 200,000 and 500,000 events were collected for each sample on BD LSR II or BD LSRFortessa machines. Data were analyzed using FlowJo software (FlowJo) and BD FACSDiva software (BD Biosciences).

Ex vivo and in vitro p-STAT5 assays

For ex vivo p-STAT5 assays, single-cell suspensions of spleens or lymph nodes were immediately prepared in ice-cold HBSS. A 200 μl aliquot was rapidly transferred to a tube (10 × 75 mm), and cells were immediately fixed in 1.6% paraformaldehyde in complete media (CM) consisting of RPMI 1640 (VWR International) supplemented with 5% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM l-glutamine, and 0.05 mM 2-ME at 37°C for 10 min. The fixed cells were then permeabilized in 100% methanol at 4°C for 30 min and subsequently washed twice with staining buffer and stained with mAbs. For in vitro p-STAT5 assays, single-cell suspensions of 2 × 106 cells/ml were rested at 37°C for 30 min, then stimulated with IL-2 at 37°C for 15 min. The cells were then fixed, permeabilized, and stained as described above.

IL-2 bioassay

Splenocytes (2 × 106) were cultured in CM containing anti-CD3 (145-2C11, 5% culture supernatant) in 24-well flat-bottom plates for 24 h at 37°C. IL-2 activity in the culture supernatants from test splenocytes was determined using the CTLL-2 bioassay, as previously described (40).

In vitro Treg suppression assay

Responder (CD4+Foxp3−) and suppressor (CD4+Foxp3+) cells were purified from the spleen of Foxp3/RFP mice by cell sorting after CD4+ enrichment with magnetic MicroBeads (Miltenyi Biotec). CD11c+ dendritic cells (DCs) were used as APCs and were also sorted from Foxp3/RFP mice. Responders (2.5 × 104 cells per well) and Tregs were cultured in a U-bottom 96-well plate with 5 × 103 DCs and 0.25 μg/ml soluble anti-CD3 Ab (clone 145-2C11). Cells were pulsed with 1 μCi [3H]thymidine per well for the last 4 h of the 72-h culture period. Data were collected as mean [3H]thymidine incorporation from triplicate cultures and used to determine the percentage suppression in reference to cultures without Tregs.

RNA sequencing

FACS-purified Tregs were resuspended in TRIzol (Thermo Fisher Scientific). Separation of the RNA phase was performed according to the manufacturer’s instructions. RNA was isolated using the RNeasy Micro Kit (QIAGEN), according to the manufacturer’s instructions. Quality control analysis, library generation, and RNA sequencing (RNA-seq) were carried out by the Oncogenomics Core Facility at the University of Miami. Quality control analysis of RNA samples was performed using the Bioanalyzer 2100 platform (Agilent Technologies). Libraries were prepared using KAPA’s RNA HyperPrep protocol and sequenced on a 75-bp paired-end run using the Illumina NextSeq 500 High Output Kit (150 cycle; 400 M flow cell).

Reads from the RNA-seq were mapped to the Mus musculus genome GRCm38 using STAR (ver.2.5.0) aligner (41). Raw counts were generated based on the Ensembl genes (GENCODE M13) with featureCounts (ver.1.5.0) (42). Differentially expressed (DE) genes were identified using DESeq2 (43) and determined by a threshold of false discovery rate (FDR) <0.01. Pathway analysis was performed on all the DE genes, and DE genes of each clade were identified by clustering using QIAGEN’s Ingenuity Pathway Analysis (IPA). Significant pathways were selected with cutoffs with an FDR <0.01.

Apoptosis and oxidative stress assays

For Annexin V staining, splenocytes were first stained with BV510 anti-CD4 (RM4-5) and subsequently stained with an FITC Annexin V Apoptosis Detection Kit (BioLegend), according to manufacturer instructions, in CM. For caspase-3/7 staining, splenocytes were first stained with Alexa Fluor700 anti-CD4 (RM4-5) and subsequently stained with CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit (Thermo Fisher Scientific) and SYTOX AAdvanced Dead Cell Stain, according to manufacturer instructions. For oxidative stress detection, splenocytes were first plated in CM at a density of 2 × 106/ml and subsequently stained with 500 nM of CellROX Deep Red Reagent for oxidative stress (Thermo Fisher Scientific) diluted in DMSO at 37°C for 1 h. Cells were then surface stained with FITC anti-CD4 (GK1.5), resuspended in 1 ml of staining buffer, and stained with SYTOX Blue Dead Cell Stain for flow cytometry.

Metabolic assays

Extracellular acidification rate (ECAR), oxygen consumption rate (OCR), and proton production rate (PPR) were measured using the Seahorse XFp Analyzer. Sorted Tregs (2 × 105) were seeded in each well of Seahorse XF Cell Culture Microplate and incubated at 37°C for 30 min in the absence of CO2 in RPMI supplemented with 10 mM pyruvate, 2 mM glutamine, and 10 mM glucose. ECAR, OCR, and PPR were measured under basal conditions and after the addition of 1 μM oligomycin, 8 μM carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone, and 0.5 μM rotenone plus antimycin A (Agilent Technologies).

Real-time quantitative PCR

RNA was extracted using a miRNeasy Mini Kit (QIAGEN) according to the manufacturer’s instructions. cDNA was generated using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific). Quantitative PCR was performed on the StepOnePlus Real-Time PCR System using the Power SYBR Green Master Mix (Thermo Fisher Scientific) and the following primers: Il1β forward, 5′-TGGAAAAGCGGTTTGTCT-3′and reverse, 5′-ATAAATAGGTAAGTGGTTGCC-3′; Il6 forward, 5′-CCTCTGGTCTTCTGGAGTACC-3′ and reverse, 5′-ACTCCTTCTGTGACTCCAGC-3′; Tnf forward, 5′-ATGAGCACAGAAAGCATGA-3′ and reverse, 5′-AGTAGACAGAAGAGCGTGGT-3′. PCR conditions were 95°C for 10 min for one cycle followed by 40 cycles of 95°C for 15 s and 60°C for 20 s. Results were analyzed by 2−ΔΔCt method, where Ct is cycle threshold, using Gapdh as a housekeeping gene.

Statistical analyses

Data were analyzed using GraphPad Prism Software. All data are shown as mean ± SD. The specific statistical analyses are stated in the figure legends.

Data availability

The RNA-seq data presented in this study have been submitted to the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov) database under accession number PRJNA661309.

Results

Acute and transient inflammation lowers p-STAT5 and other markers in Tregs

Acute administration of LPS was used as a model to study the immediate effects of inflammation on IL-2R signaling and Treg homeostasis. C57BL/6 mice were injected with a single high-dose of LPS (100 μg), and serum was collected over 3 d to assess the duration that inflammatory cytokines (IL-1α, IL-1β, IL-6, IL-12, and TNF) increased. The cytokine concentrations in the blood increased dramatically at 4 h, but these rapidly declined to low or undetectable levels by 24 h (Fig. 1A). Additionally, mRNAs for IL-1β, IL-6, and TNF were readily detected in spleen cells of mice that received LPS 4 h previously (Fig. 1B). These data indicate that a single high-dose of LPS induces a strong but transient inflammatory response.

FIGURE 1.
FIGURE 1.

A single injection of high-dose LPS causes transient increases in cytokine production and significantly alters Treg phenotype.

C57BL/6 mice were injected with PBS or LPS (100 μg) and assessed over time. (A) The indicated inflammatory cytokines in the serum were evaluated by Luminex (n = 3 per group, except at 48 h, n = 2). (B) Real-time PCR of several inflammatory cytokines of splenocytes 4 h after LPS injection (n = 3–5 per group). Data (A and B) were analyzed by a one-sided unpaired t test, in which significance was assessed for the response at 4 h relative to 0 h. (C and D) Representative histograms and contour plots (C) and scatter plots (D) of quantitative data for the indicated parameters. The percentage positive cells are shown within each histogram, and the MFIs for CD25 and Foxp3 are shown within the contour plots, in which the MFI is normalized to PBS controls. Data (n = 4–8 per group, except PBS, for which n = 20) were analyzed by Brown–Forsythe and Welch ANOVA tests and Dunnet T3 multiple comparisons test. Significance was assessed relative to PBS control mice shown at time 0. Data are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Approximately 10–20% of normal splenic Tregs show tyrosine phosphorylation of STAT5. Past studies established that essentially all p-STAT5 expression is due to IL-2 (44, 45). To determine the extent that this type of acute inflammation might affect IL-2R signaling in Tregs, we initially profiled Tregs from C57BL/6 mice for 5 d after receiving a single high-dose of LPS (100 μg). Shown in Fig. 1 are representative histograms and contour plots for key readouts (Ki67, p-STAT5, Foxp3, CD69, and CD25) at 8 and/or 24 h postinjection (Fig. 1C) and quantitative data from all mice (Fig. 1D). Initially, a rapid reduction in the frequency of Ki67 expression by Tregs was observed, reaching its lowest point 16 h post-LPS administration, suggesting a reduction in splenic Treg proliferation. This effect was closely followed by an ∼3-fold reduction in the frequency of p-STAT5+ Tregs in the spleen that was also most pronounced at 16 h post-LPS administration. The reduction in p-STAT5 may reflect decreased signaling of endogenous IL-2 in the Tregs. In contrast, the frequency of CD4+Foxp3+ Tregs remained initially stable, but increased slightly with a peak at day 3 post-LPS administration. This may reflect a compensatory mechanism to the apparent decrease in proximal IL-2R signaling and proliferation by Tregs. The frequency of Tregs that expressed CD69 rapidly increased early. Although expression of CD69 is usually taken as a measure of recent TCR stimulation (46), this effect may be related to the rapid LPS-induced production of type I IFNs, which also upregulate CD69 (47, 48). Most of these effects slowly returned to basal levels by 3–5 d post-LPS administration. Surprisingly, considering the reduction of p-STAT5+ Tregs, the level of CD25 expression, as reflected by the MFI, in the total population of Tregs rapidly but transiently increased with a peak at 8 h post-LPS administration. Additionally, the MFI of Foxp3 expression in Tregs slowly decreased by ∼25% at day 5. Similar to the spleen, when other tissues (ILN, bone marrow, blood, lung, and liver) were examined at 24 h post-LPS injection, the number of Tregs was similar to the control, but expression of Ki67 was reduced and CD69 was increased (Supplemental Fig. 1A). These data are consistent with a systemic effect of LPS-induced inflammation on Tregs.

LPS-induced inflammation did not markedly alter the frequency of major Treg subsets, based on expression of CD62L and CD44 that define resting or central Tregs (CD62LhiCD44lo) and activated or effector Tregs (eTregs; CD62LloCD44hi) (Supplemental Fig. 1B). The fraction of Tregs that expressed Klrg1 and CD103, other markers associated with eTregs, was also unaffected by LPS administration. Collectively, these data indicate that LPS-induced inflammation causes the production of inflammatory cytokines in a rapid but transient manner that has longer-lived downstream effects on Tregs, including lower proximal IL-2R signaling and reduced proliferation as measured by Ki67, but does not alter the distribution of Treg subsets.

LPS-induced Treg dysregulation is independent of TLR4 signaling by Tregs

The inflammatory response associated with LPS is largely mediated by cytokines secreted by macrophages and DCs (49), which express TLR4, the receptor for LPS. However, Tregs also express TLR4 (50), which raised the possibility that LPS-dysregulated Treg p-STAT5 and Ki67 and CD69 expression in Tregs may be due to a direct effect of LPS on Tregs. To test this question, the effects of LPS were evaluated on a Treg phenotype in germline and Treg-conditional TLR4 knockout mice. The latter mice were generated by crossing TLR4-flox mice with Foxp3-YFP/Cre mice. Germline but not Treg-specific deletion of TLR4 abrogated LPS-dependent dysregulation of p-STAT5, Ki67, CD25, and CD69 (Fig. 2A). Thus, these data indicate that LPS regulation of Tregs is indirect and likely due to LPS-dependent cytokines likely produced by macrophages and DCs.

FIGURE 2.
FIGURE 2.

Impaired p-STAT5 and Ki67 expression are independent of TLR4 signaling by Tregs and IL-1 and IL-6.

Role of TLR4 (n = 4–7 per group) (A) and IL-1 and IL-6 (n = 4–9 per group) (B) in altered p-STAT5 and Ki67 responses by splenic Tregs. The indicated mice were injected with PBS or LPS (100 μg), and 24 h later, the expression of the indicated marker was determined for splenic Tregs (n = 4–7 per group). The MFI is normalized to PBS controls. Data were analyzed by unpaired two-sided t test with Welch correction and are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

IL-1β and IL-6 are a few of the cytokines highly induced by LPS (Fig. 1A, 1B), and these cytokines have been implicated in destabilizing Tregs (17, 19–21). Therefore, we assessed the extent either of these LPS-induced cytokines individually accounted for abnormal IL-2R signaling or Ki67 and CD69 expression in Tregs. For these experiments, IL-6 and IL-1R germline knockout mice and littermate mice were treated with LPS, and the effects of LPS on Tregs were assessed 24 h later. In each of these knockout mice, p-STAT5, Ki67, CD25, and CD69 expression in Tregs remained dysregulated, with phenotypes found for LPS-treated C57BL/6 mice (Fig. 2B). Therefore, these data indicate that these selected cytokines are not individually and independently responsible for LPS-dependent dysregulation of Tregs, but rather, these effects may be the result of multiple factors induced by LPS that in turn act on Tregs.

Persistent IL-2R signaling in vivo does not optimally induce p-STAT5 during acute LPS-induced inflammation

The increase in the levels of CD25, as reflected by the MFI, in combination with lowered p-STAT5 levels (Fig. 1C, 1D) raised the possibility that LPS might have intrinsically altered proximal IL-2R signaling in Tregs. To interrogate this point, IL-2–dependent p-STAT5 activation was evaluated in Tregs from control and LPS-treated mice by restimulation with IL-2 in vitro at 4 and 24 h post-LPS injection. Dose-response experiments and nonlinear regression analysis of the data revealed a similar EC50 (Fig. 3A). Thus, this inflammatory response does not intrinsically alter proximal IL-2R signaling when assessed in vitro.

FIGURE 3.
FIGURE 3.

Persistent IL-2R signaling only partially overcomes LPS-impaired proximal IL-2R signaling in Tregs.

C57BL/6 mice received PBS or LPS (100 μg), and proximal IL-2R signaling by splenic Tregs (A) and IL-2 production by Teff cells (B) were determined. Dose response of Tregs to IL-2 was determined by measuring p-STAT5 at 4 and 24 h postinjection. Data (n = 2–8 per group) were analyzed by nonlinear regression analysis, and EC50s were determined. (B) Spleen cells were stimulated with anti-CD3; IL-2 in the culture supernatants was measured by CTLL-2 assay 24 h later. Data (n = 4 per group) were analyzed by two-sided unpaired t test with Welch correction. (C and D) C57BL/6 mice were injected with PBS or LPS (100 μg) followed by a subsequent injection of mIL-2/CD25 (4 μg) 4 h later (n = 8–10 per group). Twenty hours later, Tregs from the spleen were assessed for expression of the indicated markers. Representative histograms (C) and scatter plots (D) of quantitative data for the indicated parameters are shown. Data were analyzed by two-sided unpaired t test with Welch correction (B) and Brown–Forsythe and Welch ANOVA tests and Dunnett T3 multiple comparisons test (D). Significance was assessed in relation to PBS control mice, unless otherwise indicated. MFI is normalized to PBS controls. (A–D) Data are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

The source of IL-2 for Tregs is conventional T cells (13, 51). Another potential explanation for the low fraction of p-STAT5+ Tregs after LPS-induced inflammation is insufficient IL-2 in vivo to support IL-2R signaling. To probe this question, we assessed the extent to which T cells from LPS-treated mice secreted IL-2 after stimulation with anti-CD3 in vitro. Indeed, when compared with splenocytes from control mice, the splenocytes from LPS-treated mice secreted ∼2-fold lower amount of IL-2 (Fig. 3B).

If sufficient IL-2 was not available in vivo to support IL-2R signaling and proliferation of Tregs, we reasoned that providing IL-2 to the LPS-treated mice might overcome these low responses. To directly address this point, we determined whether the administration of IL-2 to LPS-treated mice might support normal p-STAT5 and Ki67 expression in Tregs. IL-2 did not promote expression of p-STAT5+ and Ki67+ Tregs or lower the frequency of CD69+ Tregs when coadministered with LPS, although CD25 levels were slightly increased (Supplemental Fig. 2). As Cd25 transcription is upregulated by IL-2 (52), this finding suggests that Tregs received a productive IL-2 signal.

One caveat that might limit the ability of this experimental design to accurately assess the response of LPS-conditioned Tregs to IL-2 surrounds its short half-life, which is around 10 min. IL-2–dependent p-STAT5 might not persist for the 20-h period between the administration of IL-2 and the measurement of p-STAT5+ cells in vivo. To test whether this might influence our results, we also assessed the capacity of a new long-acting IL-2 biologic, a fusion protein of mouse IL-2 covalently linked to mouse CD25 (mIL-2/CD25) to support p-STAT5 activation and Ki67 expression in vivo (37). mIL-2/CD25 persistently stimulates Tregs because of its long half-life of 16–18 h and its selectivity toward the high-affinity IL-2R, which is constitutively and abundantly expressed on Tregs. Past studies revealed that a single injection of a low dose (4 μg) of mIL-2/CD25 readily activated p-STAT5 for several days, leading to proliferation and expansion of Tregs in C57BL/6 and NOD mice (37, 53). In contrast to IL-2, mIL-2/CD25 induced p-STAT5 on Tregs to percentages much higher than found in control mice and nearly as high as found in mice treated with mIL-2/CD25 in the absence of LPS (Fig. 3C, 3D). This elevated p-STAT5 expression in Tregs is characteristic of persistent IL-2R signaling by mIL-2/CD25 (37). However, mIL-2/CD25 was less effective in promoting p-STAT5 when the mice were treated with LPS (Fig. 3C, 3D). Thus, the low levels of p-STAT5 after LPS-induced inflammation may be in part due to a lack of IL-2, but the low levels also suggests that Tregs from LPS-injected mice may have some intrinsic defect in proximal IL-2R signaling that depends on the in vivo environment. Importantly, activation of proximal IL-2R signaling in Tregs by mIL-2/CD25 did not increase expression of Ki67 either alone or in combination with LPS, nor did it lower expression of CD69 associated with LPS-induced inflammation. These data suggest that some of the effects of this type of inflammatory response might also have effects on downstream IL-2R signaling. Alternatively, these effects on Tregs may be independent of IL-2R signaling entirely.

Acute LPS-induced inflammation affects caspase-3/7–mediated apoptosis and reactive oxygen species production in Tregs but does not lower function or alter metabolism

We further assessed by flow cytometry whether other properties of Tregs were affected by LPS-induced inflammation. The levels of CTLA4 and Bcl-2 expression on Tregs were increased ∼2.8- and 1.4-fold, respectively, whereas those of ICOS, CD39, and CD73 were unaffected (Fig. 4A, 4B). These results suggest that contact-dependent suppression by CTLA4 and survival through Bcl-2 may increase in Tregs by LPS-induced inflammation, whereas adenosine-mediated suppression through CD39 and CD73 remained unaffected. However, when tested in vitro, LPS did not alter Treg-suppressive function when assessed at 24 h posttreatment (Fig. 4C), the time at peak dysregulation of p-STAT5 and Ki67 expression (Fig. 1D).

FIGURE 4.
FIGURE 4.

Acute inflammation by LPS broadly affects molecules related to Treg-suppressive function, survival, and migration.

C57BL/6 mice were injected with LPS (100 μg) and assessed 24 h later. Representative FACS histograms (A) and scatter plots (B) of the indicated markers in Tregs are shown. Data (n = 4–13 per group) were analyzed by two-sided unpaired t test with Welch correction. MFI is normalized to PBS controls. (C) In vitro suppression of CD4+Foxp3− T cells (effectors) by FACS-sorted CD4+Foxp3+ Tregs (suppressors, at the indicated ratios) isolated from Foxp3-RFP mice 24 h after PBS or LPS (100 μg) administration (n = 4). (D–G) Foxp3-RFP mice were injected with PBS or LPS (100 μg), and Tregs were isolated from the spleens by FACS for RNA-seq analysis. (D) Pathways most altered by LPS administration as assessed by IPA. Red bars indicate pathway upregulation. Blue bars indicate pathway downregulation. Gray bars indicate no direction of pathway dysregulation. Most differentially regulated genes related to (E) superpathway of cholesterol biosynthesis and (F) Th1/Th2 activation pathways are shown. (G) LPS-dependent expression of select immune-related genes that have FDR <0.05 are shown. Fold change represents expression in LPS-treated mice relative to PBS-treated controls. (A–C and E–G) Data are shown as mean ± SD. *p < 0.05, ****p < 0.0001.

We next performed RNA-seq of sorted Tregs 24 h after LPS administration, and 875 DE genes (488 upregulated, 386 downregulated; FDR <0.01) were identified. IPA of these DE genes revealed that LPS administration decreased expression of genes related to cholesterol biosynthesis while upregulating expression of genes related to IFN signaling, IFN regulatory factor activation, and Th1 pathways (Fig. 4D). The DE genes identified by IPA related to cholesterol biosynthesis were all substantially downregulated (Fig. 4E). The DE genes related to Th1/Th2 activation did not contain upregulated signature genes (e.g., Ifng, Il4, and Tbx21) of Th1 and Th2 Teff cells but rather reflected changes in the activation state of the Tregs (Fig. 4F). To further evaluate the immunological consequences of these LPS-dependent changes in gene expression in Tregs, the DE expressed genes (FDR < 0.05) were compared with a list of 488 genes by NanoString that were annotated in their immunology panel. Of the 80 DE genes identified, some involved in Treg-suppressive function, such as Il10 and Lag3, were upregulated, whereas Tgfb1 was downregulated by LPS. Prdm1, which promotes Il10 transcription (54, 55); Tigit, a surface molecule associated with eTregs; and Il7ra also increased in Tregs after LPS administration (Fig. 4G). However, other transcripts associated with eTregs (i.e., Cd44, Irf4, and Gata3) and two transcripts associated with TCR and IL-2R signaling (i.e., Cd3e and Il2ra) were downregulated. However, other transcripts encoding key molecules (Il2rb, Il2rg, Jak1, Jak3, Stat5a, and Stat5b) associated with proximal IL-2R signaling were unaffected (data not shown). Several chemokines (Cxcl9, Cxcl10) and a chemokine receptor (Ccr5) were upregulated, which may promote trafficking of Tregs and other immune cells to inflamed tissue sites. Moreover, Socs3 was substantially upregulated by LPS, which may act as negative feedback for IL-6 signaling in Tregs (56). These data indicate that an acute inflammatory response leads to substantial changes in global transcriptional regulation in Tregs that impacts Treg activation, function, and responsiveness to homeostatic signals.

To directly address the effect of LPS-induced inflammation on Treg survival and oxidative stress, the frequency of Annexin V+, Caspase-3/7+, and CellROX+ Tregs were evaluated directly ex vivo. In the proportions of Annexin V+ Tregs in the spleen, mesenteric lymph nodes, and ILN, the large majority that were 7-aminoactinomycin D− and indicative of apoptosis were unaffected by LPS (Fig. 5A, 5D). However, we more readily observed an increase of Caspase-3/7+ 7-aminoactinomycin D− cells after they were LPS treated (Fig. 5B, 5D). The percentage of CellROX+ Tregs decreased in mice injected with LPS compared with PBS (Fig. 5C, 5D), suggesting this type of acute inflammation decreases the amount of reactive oxygen species (ROS). Thus, although the overall proportion of Annexin V+ apoptotic Tregs did not differ after LPS treatment, the pathways contributing to cell death may vary in Tregs, as acute LPS-mediated inflammation increased caspase-3/7 activity that may have counteracted higher levels of Bcl-2 (Fig. 4A) and lower levels of ROS.

FIGURE 5.
FIGURE 5.

Acute inflammation induced by LPS increases apoptosis by caspase-3/7 but decreases ROS levels.

C57BL/6 mice were injected with LPS (100 μg) and assessed ex vivo 24 h later. Representative contour plots showing (A) Annexin V staining (n = 4–5 per group) and (B) caspase-3/7 staining (n = 3–7 per group). (C) Representative histograms showing CellROX staining (n = 10 per group). (D) Scatter plots showing quantitative data from Annexin V staining, caspase-3/7 staining, and CellROX staining. Data (mean ± SD) were analyzed by two-sided unpaired t test with Welch correction. *p < 0.05, **p < 0.01, ***p < 0.001.

To assess whether LPS-induced inflammation translated to altered Treg metabolic activity, Seahorse analyses were performed on FACS-purified Tregs from spleens after LPS injection. The OCR, ECAR, and PPR were similar in Tregs from both PBS- and LPS-treated mice (Fig. 6A). Further comparison of these data revealed no differences in metabolic fitness (Fig. 6B). Consequently, these data indicate that the immediate Treg response to LPS-induced inflammation is broader than downregulation of IL-2R signaling and proliferation and may be accompanied by enhanced suppression mediated by CTLA4 and an increase in caspase 3/7–dependent apoptosis, whereas metabolism is unaffected.

FIGURE 6.
FIGURE 6.

Largely normal Treg respiration after acute inflammation induced by LPS.

Foxp3-RFP mice were injected with PBS or LPS (100 μg). Tregs from these mice were isolated from spleens by FACS for Seahorse analysis. (A) OCR measurement, ECAR, and PPR measured in Tregs after LPS-induced inflammation. (B) Relative values of metabolic parameters. Data (n = 4 independent experiments) are shown as mean ± SD and analyzed by two-sided unpaired t test with Welch correction. p-STAT5.

LPS-induced inflammation broadly dysregulates IL-2–dependent gene transcription in Tregs

To more broadly understand the consequences of acute inflammation on Tregs, RNA-seq was performed on FACS-purified Tregs (Fig. 7A) from spleens of mice that had been treated with LPS in the presence or absence of mIL-2/CD25 administered 4 h after LPS injection. This timing was chosen to assess how increasing IL-2R signaling in Tregs might affect an ongoing inflammatory response, as LPS-induced serum cytokines were highest at this time (Fig. 1A). The transcriptomes of Tregs from mice treated with LPS or mIL-2/CD25 individually or in combination was performed at the 24 h time point, when dysregulation of p-STAT5 and Ki67 expression was the greatest (Fig. 1D), to broadly determine the consequences of LPS-induced inflammation and IL-2R signaling.

FIGURE 7.
FIGURE 7.

Acute LPS-induced inflammation and sustained IL-2R signaling induces global changes and distinct transcriptional profiles in Tregs.

Foxp3-RFP mice were injected with PBS or LPS (100 μg) followed by a subsequent injection of PBS or mIL-2/CD25 (4 μg). Tregs from these mice were isolated from spleens by FACS for RNA-seq analysis. (A) Sorting strategy and purity of Foxp3+ Tregs FACS-purified from the spleen. (B) Principal component analysis of different groups showing distinct clustering of genes affected. (C) Cluster analysis of genes with FDR <0.0001 with samples that had more than 100 reads in all triplicates in each group, made with Morpheus (https://software.broadinstitute.org/morpheus). (D) IPA revealing the top regulated pathways of each clade. (E) Heatmaps of representative genes of select clades. (F) Heatmap showing effects of LPS and mIL-2/CD25 on genes that mediate suppressive function in vivo.

In comparison with PBS-treated mice, 874, 3,444, and 3039 DE transcripts (FDR < 0.01) were associated with Tregs from mice treated with LPS, mIL-2/CD25, and the combination of LPS and mIL-2/CD25, respectively. Principal component analysis revealed that the four groups clustered distinctly from each other, indicating distinctive gene signatures in each experimental group (Fig. 7B). Thus, each mode of stimulation substantially and distinctively affected the Treg transcriptome.

Next, a list of DE genes (FDR < 0.0001) was stringently selected and then clustered using hierarchical clustering with Euclidean distance, from which seven dominant clades were identified (Fig. 7C). IPA pathway analyses were performed for the genes within each clade, and the three most significant pathways for each clade are shown (Fig. 7D). Clades 1–3 and 6 represent genes upregulated or downregulated, respectively, when mice were treated with mIL-2/CD25 (Fig. 7C). Genes in clades 1 and 2 were largely unaffected by LPS alone but were substantially upregulated by mIL-2/CD25. These clades were highly enriched in genes that are involved in cell cycle and DNA replication (Fig. 7D), consistent with the ability of mIL-2/CD25 to support Treg expansion (37). Selected genes enriched in this pathway are shown in Fig. 7F, and a complete list of genes associated with this pathway and the others in Fig. 7F are shown in Supplemental Table II. The combination treatment of LPS and mIL-2/CD25 resulted in lower expression of mRNAs in clade 1 but not clade 2. Clade 3 was highly enriched in genes that are involved in cholesterol biosynthesis (Fig. 7D, 7E). These genes were downregulated by LPS, substantially upregulated by mIL-2/CD25, and dominantly downregulated in Tregs from mice that received both LPS and mIL-2/CD25. Thus, acute inflammation associated with LPS has a substantial effect on downstream activation of mIL-2/CD25–dependent transcription; the large majority of genes upregulated by mIL-2/CD25 were either partially (clade 1) or substantially (clade 3) inhibited, and those downregulated by mIL-2/CD25 (clade 6) were partially activated by LPS.

The genes in clades 4, 5, and 7 represent a somewhat distinctive pattern of gene regulation in Tregs after treatment of mice with LPS and/or mIL-2/CD25 (Fig. 7C). Clade 4 is characterized by genes that are dominantly induced by LPS and that are related to pathways associated with TLR, IFN, and death receptor signaling (Fig. 7D, 7E). The genes associated with clades 5 and 7 were largely unaffected by sole application of LPS or mIL-2/CD25, but the combined action of these agents led to substantial upregulation of genes associated with the Th1/Th2 pathways (clade 5) or downregulation of genes associated with TCR and costimulatory signaling (clade 7). Collectively, these data indicate that a substantial acute inflammatory response rapidly acts on Tregs to downregulate a subset of genes associated with IL-2R, TCR, and costimulatory signaling while promoting gene expression more classically associated with Teff cells.

The expression of 10 mRNAs encoding proteins that mediate Treg suppression in vivo were also compared (Fig. 7F). Treatment with LPS supported a slight increase in Ctla4 and larger increases in Il10, Lag3, and Ebi3 (subunit of IL-35), whereas Tgfb1 decreased. The cotreatment with LPS and mIL-2/CD25 supported substantial upregulation of Ctla4, Nt5e (CD73), Entpd1 (CD39), Il10, Fgl2, and Gzmb. These data suggest that Treg-suppressive activity may increase in vivo. Thus, LPS-induced inflammation is associated with lower proliferative potential alone or after IL-2R agonism, but this effect may be compensated for by increased suppressive function as indicated by this transcriptome analysis.

Acute LPS-induced inflammation leads to an extended reduction in IL-2R signaling and function in Tregs

To test whether LPS-dependent acute inflammation affected Tregs at their peak of expansion by mIL-2/CD25, splenic Tregs were assessed 3 d postinjection with LPS and low-dose (4 μg) mIL-2/CD25. When comparing the response of Tregs to mIL-2/CD25 versus the combination of LPS and mIL-2/CD25, a nonstatistical reduction in Treg numbers was observed under the latter condition, which was accompanied by lower expression of p-STAT5, Foxp3, CD25, Ki67, and CTLA4 (Fig. 8A, 8B). This result indicates that this acute LPS-induced inflammatory response, with its associated transient production of inflammatory cytokines (Fig. 1A), leads to an attenuation in the Treg response to mIL-2/CD25 that extends to 72 h postinjection.

FIGURE 8.
FIGURE 8.

Acute LPS-induced inflammation has long-lasting effects on Treg IL-2R signaling.

C57BL/6 mice were injected with PBS or LPS (100 μg) followed by a subsequent injection of PBS or mIL-2/CD25 (4 μg) 4 h later. Spleens were assessed by flow cytometry 3 d later. Representative histograms (A) and scatter plots (B) of quantitative data for the indicated parameters are shown. Data (n = 5–6 per group) were analyzed by Brown–Forsythe and Welch ANOVA tests and Dunnett T3 multiple comparisons test. Significance was assessed in relation to PBS control mice, unless otherwise indicated. MFI is normalized to PBS controls. Data are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Discussion

To maintain immune homeostasis, Tregs must limit self-reactivity during situations of heightened inflammation (e.g., immune responses to infectious agents). Past work has established that inflammation dysregulates Treg stability and increases their plasticity to acquire properties of Th1 and Th17 cells (22, 57). IL-2R signaling through direct action on Foxp3 transcription reinforces Treg stability (13–15), raising the question as to whether impaired IL-2R signaling may accompany inflammation. Thus, the current study was designed to directly assess the extent that acute inflammation induced by LPS, which models inflammation that accompanies an immune response to a bacterial pathogen or may be associated with some autoimmune diseases, affects IL-2R–dependent signaling and responsiveness in Tregs. Our results are consistent with a mechanism by which LPS acts on cells, such as macrophages and DCs, but not Tregs to transiently secrete inflammatory cytokines, and in turn, these cytokines act on Tregs to lower IL-2R signaling and proliferation as measured by p-STAT5 and Ki67, respectively, and to cause other immune-related and transcriptional changes. These effects on Tregs are not solely accounted for by IL-1 or IL-6, which are known to destabilize Tregs (17, 19–21). These acute effects of LPS-induced inflammation were not associated with deceased Treg function in vitro, and with the exception of Tgfb1 mRNA, other mediators of Treg function in vivo remained unchanged or increased. Thus, the immediate response to this type of acute inflammation may be to reprogram Tregs to lower their proliferative potential without destabilizing their functional activity.

Previous work has shown that endogenous p-STAT5 in Tregs is the result of IL-2R signaling (44, 45). Thus, the lower IL-2R signaling that we observed after LPS administration is likely a consequence of inflammatory effects on IL-2R signaling. The decrease in p-STAT5 in Tregs was not coincident with the peak of inflammatory cytokine secretion (4 h post-LPS injection) but rather was a sequential consequence that was most obvious at 16 h post-LPS injection. This phenotype did not return to normal levels until 5 d post-LPS injection, indicating this type of short-term inflammatory response has an extended effect on Tregs. The mechanism by which LPS-induced acute inflammatory response impairs IL-2R signaling in Tregs appears to be complex. Following the injection of mice with LPS, their T cells produced lower IL-2 after in vitro stimulation with anti-CD3. The Tregs from these mice also expressed lower p-STAT5 when challenged with mIL-2/CD25 in vivo, supporting persistent IL-2R agonism in vivo. Thus, lower IL-2R signaling associated with an acute inflammatory response appears to be the result of lower IL-2 production and a potential Treg-intrinsic deficit. We reached this conclusion because, if impaired IL-2 production was the responsible mechanism, mIL-2/CD25 was expected to fully restore IL-2R–dependent p-STAT5. One caveat to this latter conclusion is that when Tregs from LPS-injected mice were stimulated with IL-2 in vitro, these Tregs responded identically to control Tregs when measuring STAT5 activation. Thus, a cell-intrinsic impaired response of Tregs to mIL-2/CD25 after LPS injection in vivo may require other in vivo environmental cues.

Other immunological changes in Tregs were also noted after acute LPS-induced inflammation. Phenotypic and RNA-seq analyses of control versus LPS-treated Tregs indicate more wide-ranging effects on Tregs. Pathway analysis indicated that Tregs showed gene signatures of responsiveness to IFNs, a link to the inflammatory response, and properties of Th1/Th2 cells, which likely reflect changes in their activation rather than acquisition of Th1 and Th2 effector activity. Our data indicate that Tregs rapidly reprogram their transcriptome to adapt to this inflammatory response. These phenotypic and genetic changes are consistent with decreased Treg proliferation (decreased Ki67), but they enhanced survival (increased Bcl-2) that is accompanied by altered migration (increased Cxcl9, Cxcl10, Ccr5) and activation (increased Tigit, Prdm1; decreased Gata3, Irf4, Cd44, Cd3e, Il2ra). Acute LPS-induced inflammation did not affect Treg-suppressive function in vitro. However, several mediators of Treg-suppressive function in vivo were affected (increased CTLA4, Lag3, and Il10 and decreased Tgfb). These results suggest some aspects of Treg suppression are variably altered and enhanced in vivo. In the context of an infection, these types of transient changes in Tregs are likely beneficial, as they permit a heightened effector response to clear infections without resulting in autoimmunity. As these changes are transient, immune homeostasis would be reset upon clearing infection. These data also raise the possibility that chronic inflammation might lead to more prolonged alteration in these and other parameters of Tregs that could interfere with their ability to maintain self-tolerance.

LPS-induced inflammation decreases expression of many genes related to cholesterol metabolism. This result suggests that lipid metabolism and fatty acid oxidation might be lower in Tregs. However, Seahorse analyses revealed that this inflammatory response did not affect Treg metabolism. Cholesterol is used in many different types of crucial cellular processes, including but not limited to TCR and CD28 activation (58–60). In addition, impaired cholesterol biosynthesis is associated with diminished Treg stability and function (61, 62). Consequently, the lower gene expression related to cholesterol biosynthesis may affect Treg activation and aspects of their function by changing the spatial distribution of lipid rafts on the surface of their membranes, as lipid rafts depend heavily on the levels of cholesterol in Tregs (63, 64). Indeed, RNA-seq analyses also revealed changes related to the activation status of Tregs, as discussed above, although the frequency of Tregs with phenotypic properties of central Tregs and eTregs were unaffected.

Given the striking effect of LPS-induced inflammation on the reduction of Ki67 expression in Tregs in the spleen, we were somewhat surprised that we did not detect a reduction in Treg proportions and numbers. There are several nonmutually exclusive explanations for this result. First, only a fraction of Tregs are Ki67+ (10–20%) in normal control mice, and a reduction of only Ki67+ Tregs would not substantially affect the total pool of Tregs in the spleen. Second, any reduction in Tregs through lower proliferation might be counteracted by enhanced survival of the remaining Tregs. Indeed, LPS-induced inflammation increased Bcl-2 levels in Tregs, which may lessen any consequences of decreased proliferation. Third, although caspase-3/7+ and ROS+ cells often characterize apoptotic cells, LPS injection slightly increased the former and decreased the latter. These effects were not accompanied by an increase in dying Annexin V+ Tregs, consistent with the notion that acute LPS-induced inflammation did not substantially lower Treg survival. Moreover, similar results related to Treg numbers and reduced Ki67 expression were noted for other lymphoid and nonlymphoid tissues. These findings are consistent with a systemic effect of LPS-induced inflammation on Tregs and suggest that the reduction of Ki67+ Tregs in the spleen is not due to redistribution of these Tregs to other tissues. Thus, our data indicate that, during this acute inflammatory response, too few Tregs are actively proliferating or dying to alter their numbers.

RNA-seq data showed that LPS-induced inflammation substantially affects gene pathways in Tregs related to cell cycle progression and DNA replication. This was revealed when we compared the transcriptomes of Tregs stimulated with mIL-2/CD25 to transcriptomes of Tregs stimulated with the combination of mIL-2/CD25 and LPS. Many genes related to cellular replication were upregulated in Tregs stimulated with mIL-2/CD25, and these were reduced, but not to baseline levels, after costimulation with LPS. Our data reveal that LPS-induced acute inflammation has a substantial effect not only on proximal IL-2R signaling but also IL-2R–dependent gene transcription. Three days after stimulation with both mIL-2/CD25 and LPS, p-STAT5 expression was decreased, which was accompanied by decreased levels of Ki67, Foxp3, and CD25 and a trend for lower Treg numbers as well. Thus, these data are consistent with a model in which an acute LPS-induced inflammatory response broadly alters the IL-2R–dependent gene program with a potential to limit Treg proliferation.

The findings in this study have several considerations concerning the use of low-dose IL-2 therapy for autoimmune diseases. First, inflammation lowers markers of endogenous IL-2R–dependent signaling and a subset of downstream gene activation after application of an exogenous IL-2R agonist. Thus, a strong inflammatory response that often accompanies an infection may temper some of the increased activities in Tregs due to low-dose IL-2 therapy with mIL-2/CD25. Such a decrease might facilitate an immune response to an infection and may be beneficial for patients that are chronically treated with an agent, such as low-dose IL-2, that aims to enhance immunosuppressive mechanisms. Second, low-dose IL-2 in the form of mIL-2/CD25 nevertheless overcame some of the reduced activities associated with inflammation on Tregs. mIL-2/CD25 increased IL-2R signaling, gene expression, and Treg proportions over the level observed in Tregs after administration of LPS alone. The latter result is perhaps the most critical because the ratio of Treg/Teff dictates the effective suppression of autoreactive T cells (65). Third, another beneficial consequence of mIL-2/CD25 is that it increased RNAs encoding 6 of 10 mediators (Ctla4, Nt5e or CD7, Entpd1 or CD39, IL10, Fgl2, or Gzmb) of Treg-suppressive function in the context of LPS-induced inflammation. LPS alone either did not affect or only modestly increased these same suppressive mediators. This finding further raises the possibility that this form of low-dose IL-2 therapy may overcome some of the negative impact of inflammation on Tregs. Indeed, our past results showed that persistent IL-2R signaling using the same dose of low-dose mIL-2/CD25 prevents diabetes in asymptomatic young and hyperglycemic NOD mice (37, 53). However, chronic autoimmune inflammation in the NOD pancreas and other autoimmune diseases, which are often T cell mediated, is likely qualitatively and quantitatively different from that due to infectious agents and LPS, which are mediated primarily by macrophages and DCs. The extent that inflammation induced by autoimmunity resembles that induced by LPS and mediates similar effects on Tregs directly or in the context of low-dose IL-2 therapy remains to be determined. Last, these findings raise the possibility that dampening inflammatory responses may optimally enhance Treg responsiveness to low-dose IL-2 therapy.

Disclosures

The University of Miami, T.R.M., and R.H. have patents pending on IL-2/CD25 fusion proteins (Wo2016022671A1; T.R.M.) and their use (PCT/US20/13152; T.R.M., R.H.) that have been licensed exclusively to Bristol Myers Squibb, and some research on IL-2/CD25 fusion proteins has been supported in part by a collaboration and sponsored research and licensing agreement with Bristol Myers Squibb. The other authors have no financial conflicts of interest.

Acknowledgments

We thank Patricia Guevara, Jay Enten, and Shannon Saigh from the Flow Cytometry Shared Resource at the Sylvester Comprehensive Cancer Center, Oliver Umland from the Diabetes Research Institute Flow Core, and Margaret Roach for assistance with the Luminex assay.

Footnotes

  • This work was supported by funding to T.R.M. from the National Institutes of Health (R01DK093866 and R01AI148671) and the Diabetes Research Institute at the University of Miami.

  • The sequencing data presented in this article have been submitted to the National Center for Biotechnology Information database (https://www.ncbi.nlm.nih.gov) under accession number PRJNA661309.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    CM
    complete media
    DC
    dendritic cell
    DE
    differentially expressed
    ECAR
    extracellular acidification rate
    eTreg
    effector Treg
    FDR
    false discovery rate
    ILN
    inguinal lymph node
    IPA
    ingenuity pathway analysis
    MFI
    mean fluorescence intensity
    mIL-2/CD25
    mouse IL-2/CD25 fusion protein
    OCR
    oxygen consumption rate
    PPR
    proton production rate
    RNA-seq
    RNA sequencing
    ROS
    reactive oxygen species
    Teff
    T effector
    Treg
    regulatory T cell.

  • Received November 17, 2020.
  • Accepted November 20, 2020.

This article is distributed under the terms of the CC BY-NC 4.0 Unported license.

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