key: cord-104030-eb29t38n
authors: Morales-Nebreda, Luisa; Helmin, Kathryn A.; Markov, Nikolay S.; Piseaux, Raul; Acosta, Manuel A. Torres; Abdala-Valencia, Hiam; Politanska, Yuliya; Singer, Benjamin D.
title: Aging imparts cell-autonomous dysfunction to regulatory T cells during recovery from influenza pneumonia
date: 2020-06-05
journal: bioRxiv
DOI: 10.1101/2020.06.05.135194
sha: 
doc_id: 104030
cord_uid: eb29t38n

Regulatory T (Treg) cells orchestrate resolution and repair of acute lung inflammation and injury following viral pneumonia. Compared with younger patients, older individuals experience impaired recovery and worse clinical outcomes after severe viral infections, including influenza and the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Whether age is a key determinant of Treg cell pro-repair function following lung injury remains unknown. Here, we show that aging results in a cell-autonomous impairment of reparative Treg cell function following experimental influenza pneumonia. Transcriptional and DNA methylation profiling of sorted Treg cells provide insight into the mechanisms underlying their age-related dysfunction, with Treg cells from aged mice demonstrating both loss of reparative programs and gain of maladaptive programs. Novel strategies that restore youthful Treg cell functional programs could be leveraged as therapies to improve outcomes among older individuals with severe viral pneumonia.

Age is the most important risk factor determining mortality and disease severity in patients infected with influenza virus or the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (1, 2). Global estimates of seasonal influenza-associated mortality range from 300,000-650,000 deaths per year, with the highest at-risk group comprised of individuals over age 75 (3) .

In the United States, influenza-associated morbidity and mortality have steadily increased, an observation linked to an expansion of the aging population. Pneumonia related to both severe influenza A virus and SARS-CoV-2 infection results in an initial acute exudative phase characterized by release of pro-inflammatory mediators that damage the alveolar epithelial and capillary barrier to cause refractory hypoxemia and the acute respiratory distress syndrome (ARDS) (4) . If a patient survives this first stage, activation of resolution and repair programs during the ensuing recovery phase is crucial for restoration of lung architecture and function, which promotes liberation from mechanical ventilation, decreases intensive care unit length-of-stay and extends survival.

Foxp3 dampen inflammatory responses to both endogenous and exogenous antigens. Aside from their role in maintaining immune homeostasis through their capacity to suppress over-exuberant immune system activation, Treg cells reside in healthy tissues and accumulate in the lung in response to viral injury to promote tissue repair (5, 6) . Our group and others have shown that in murine models of lung injury, Treg cells are master orchestrators of recovery (7) (8) (9) (10) . Treg cells are capable of promoting tissue regeneration and repair, at least in part through release of reparative mediators such as the epidermal growth factor receptor ligand amphiregulin (Areg), which induces cell proliferation and differentiation of the injured tissue (11) .

Epigenetic phenomena, including DNA methylation, modify the architecture of the genome to control gene expression and regulate cellular identity and function throughout the lifespan (12) .

Aside from being one of the best predictive biomarkers of chronological aging and age-related disease onset, DNA methylation regulates Treg cell identity through tight epigenetic control of Foxp3 and Foxp3-dependent programs (13) . Biological aging is associated with a progressive loss of molecular and cellular homeostatic mechanisms that maintain normal organ function, rendering individuals susceptible to disease (14, 15) . Because of their tissue-reparative functions, Treg cells are important modulators of the immune response that promotes tissue regeneration following injury (16) . Whether age plays a key role in determining the pro-repair function of Treg cells in the injured lung during recovery from viral pneumonia remains unknown. If aging indeed impacts Treg cell-mediated recovery, is it a Treg cell-autonomous phenomenon or is it because the aging lung microenvironment is resistant to Treg cell-mediated repair? Using heterochronic (age-mismatched) adoptive Treg cell transfer experiments and molecular profiling in mice, we sought to determine whether the age-related impairment in repair following influenza-induced lung injury is intrinsic to Treg cells. Our data support a paradigm in which aged Treg cells fail to upregulate youthful reparative programs, activate maladaptive responses and consequently exhibit a cell-autonomous impairment in pro-recovery function, which delays resolution from viralinduced lung injury in aged hosts.

To evaluate the age-related susceptibility to influenza-induced lung injury, we administered influenza A/WSN/33 (H1N1) virus via the intratracheal route to young (2 months) and aged (18 months) wild-type mice. Aged mice exhibited > 50% mortality when compared with young animals ( Figure 1A) , impaired recovery of total body weight following a similar nadir ( Figure 1B ) and more severe lung injury by histopathology at a late recovery time point, day 60 post-infection ( Figure   1C ). At this same time point, aged mice also displayed an increase in the total number of cells per lung (Figure 1D ), which were mainly comprised of immune cells identified by the panhematopoietic marker CD45 (Figure 1E) , suggesting non-resolving tissue inflammation during recovery in older mice. We next wanted to determine whether the age-related susceptibility to influenza-induced lung injury was due to a differential inflammatory response during the initial acute injury phase. Accordingly, we examined a different group of young and aged mice at a time point when viral clearance was complete (17) and weight nadir was observed in both groups, 14 days post-infection. Aged mice demonstrated increased mortality when compared with young animals at this time point (Supplemental Figure 1A) , but other markers of acute inflammation, including weight loss (Supplemental Figure 1B) , total lung cells (Supplemental Figure 1C ) and total lung CD45 + cells in surviving animals were not significantly different between groups (Supplemental Figure 1D) . Collectively, these results suggest that aging results in similar early injury but persistent lung inflammatory pathology during the recovery phase of influenza-induced lung injury. 

Having established that aging results in an increased susceptibility to persistent lung injury after influenza infection, we explored whether the impaired recovery in aged mice was linked to a persistent failure to repopulate the structural components of the alveolar-capillary barrier (i.e., failure to repair). Flow cytometry analysis (Supplemental In previous studies, investigators demonstrated that following influenza-induced lung injury, a population of cytokeratin 5 + (Krt5 + ) basal-like cells expand and migrate to the distal airspaces in an attempt to repair the injured epithelial barrier (18) . These cells lack the capacity to transdifferentiate into functional AT2 cells, resulting in a dysplastic response that contributes to a dysregulated and incomplete repair phenotype following injury (19) . Using a flow cytometry quantitative approach, we found that at 60 days post-infection, aged mice showed a significant increase in Krt5 + cells compared with young animals (Figure 2G-H) . In summary, older mice failed to repair the injured lung during the recovery phase of influenza-induced lung injury. 

We and others have identified an essential role for regulatory T (Treg) cells in orchestrating resolution and repair of acute lung injury (7) (8) (9) (10) (11) . Having established that aged mice fail to repair the injured lung, we next sought to determine whether this finding is due to age-related features altering the lung microenvironment or is driven by cell-autonomous, age-associated Treg cell factors. Thus, we performed heterochronic (age-mismatched) adoptive transfer of 1x10 6 splenic young or aged Treg cells via retro-orbital injection into aged or young mice 24 hours post-infection ( Figure 3A) . Notably, adoptive transfer of young Treg cells into aged hosts resulted in improved survival when compared with aged mice that received PBS (control), while adoptive transfer of aged Treg cells into young hosts worsened their survival when compared with their respective controls ( Figure 3B ). We next turned to an inducible Treg cell depletion system using Foxp3 DTR mice in order to eliminate Treg cells from recipients and specifically determine the age-related effect of donor Treg cells on the susceptibility to influenza-induced lung injury ( Figure 3C ).

Adoptive transfer of aged Treg cells into Treg cell-depleted Foxp3 DTR mice 5 days post-infection resulted in increased mortality when compared with adoptive transfer of young Treg cells ( Figure   3D ). Combined, our findings demonstrate that the loss of the Treg cell-associated pro-repair function in aged hosts is dominated by intrinsic, age-related changes in Treg cells and not conferred extrinsically by the aging lung microenvironment. K-means clustering of these differentially expressed genes demonstrated that Cluster II was both the largest cluster and the one that defined the differential response to influenza infection between naïve and influenza-treated mice ( Figure 4C) . Notably, genes from this cluster were significantly upregulated among young Treg cells when compared with aged Treg cells following influenza infection ( Figure 4D ). Functional enrichment analysis revealed that this cluster was enriched for processes related to tissue and vasculature development and extracellular matrix formation Figure 6A-B) .

We found that during the Treg cell response to influenza, there were 1,678 upregulated genes in young mice and only 445 upregulated genes in aged mice when compared with their respective naïve state (FDR q-value < 0.05). Gene set enrichment analysis revealed upregulation of prorepair hallmark processes in both young and aged Treg cells during recovery from influenza infection (Supplemental Figure 6C-D) . We next compared the age-related transcriptional response to influenza infection and found 342 shared genes between both age groups that were associated with pro-repair processes (Supplemental Figure 6E) 

In addition to representing one of the hallmarks of aging, epigenetic phenomena such as DNA methylation regulate the development, differentiation and functional specialization of T cell lineages, including Treg cells (12) (13) (14) . Therefore, we reasoned that age-related changes to the Treg cell DNA methylome could inform the divergent pro-repair transcriptional response seen between young and aged Treg cells following influenza infection. We performed genome-wide (5'-cytosine-phosphate-guanine-3') CpG methylation profiling with modified reduced representation bisulfite sequencing (mRRBS) of sorted lung Treg cells during the naïve state or recovery phase following influenza infection (day 60) (Figure 6A) . PCA of ~70,000 differentially methylated cytosines (DMCs, FDR q-value < 0.05) revealed tight clustering according to group assignment with the main variance across the dataset (PC1) reflecting methylation changes due to age (Figure 6B) , consistent with prior studies (15, 24) . We next identified genes that were both differentially expressed and had differentially methylated cytosines within their gene promoters (ANOVA, FDR q-value <0.05), and found 1,319 genes meeting this parameter threshold ( Figure   6C ). K-means clustering of gene expression levels revealed a substantial similarity to the DEG heat map shown in Figure 4C . Gene set enrichment analysis of these genes demonstrated that this methylation-regulated gene expression program was associated with pro-recovery processes and was significantly skewed toward young Treg cells ( Figure 6C) . Combined, these results show that age-related DNA methylation regulates the pro-reparative transcriptional regulatory network during recovery from influenza-induced lung injury. 

We sought to unambiguously address the paradigm of how aging affects Treg cell function during recovery from influenza pneumonia. We used heterochronic (age-mismatched) Treg cell adoptive transfer following influenza infection to establish that the age-related pro-repair function of these 1, 25, 26) . Here, we found that similar to human epidemiologic data and previous pre-clinical murine studies, aged mice exhibit increased susceptibility and impaired recovery following influenza infection. Injury to alveolar epithelial type I, II and endothelial cells disrupts the tight gas exchange barrier causing accumulation of fluid and pro-inflammatory mediators in the alveolar space, a hallmark of ARDS pathophysiology (4) . Notably, we found that during late recovery from influenza infection, aged hosts demonstrated a decreased number of alveolar epithelial type II cells and endothelial cells when compared with young animals, suggesting that failure to repopulate the alveolar lining contributes to the observed age-related impairment in recovery. Severe influenza infection leads to a robust expansion of Krt5 + cells, which migrate distally to form cystic-like structures or pods intended to cover the damaged alveolar wall (19) . These pods persist long after the initial infection, lack the capacity to generate a functional alveolar epithelium and therefore constitute an insufficient reparative response to injury (19) . Here, we showed that aged animals display an increased percentage of Krt5 + cells during the recovery phase of influenza-induced lung injury, which reflects the dysregulated repair response in aged hosts.

Over the past decade, regulatory T cells have emerged as key mediators of wound healing and tissue regeneration (6, 16, 27) . This group of specialized cells has been primarily known for their ability to suppress effector immune cell subsets leading to resolution of inflammation, but they are also capable of directly affecting tissue regeneration through production of pro-repair mediators such as amphiregulin and keratinocyte growth factor (11, 28, 29) . Investigators have demonstrated that aging can negatively impact the composition and function of the Treg cell pool throughout the lifespan, rendering them inefficient as facilitators of tissue repair (30) . This decline might occur through cell-autonomous mechanisms resulting in T cell maladaptive responses that lead to increased susceptibility to disease. For instance, loss of stemness accompanied by differentiation into pro-inflammatory Th1/Th17 phenotypes, activation of DNA damage responses and the senescence secretome are among some of the T cell maladaptations that result from the mounting challenges to which the T cell repertoire is exposed over a lifetime (20) . These T cell maladaptive changes could also result from an age-related loss in stromal signals and circulating factors from the tissue microenvironment that either affect T cell function directly or render the microenvironment resistant to T cell responses. Our heterochronic adoptive Treg cell transfer experiments definitively address this paradigm, showing that the observed age-related Treg cell dysfunction is due to cell-autonomous mechanisms and dominant over the aged pulmonary microenvironment. Our data demonstrate that aging not only imparts a loss of pro-recovery Treg cell function, but also a gain of some of these maladaptive features when compared with young hosts.

What are the molecular mechanisms underpinning the age-associated Treg cell gain or loss-of pro-reparative function in the lung following influenza infection? Gene expression profiling of lung Treg cells during the recovery phase of influenza infection showed that young Treg cells significantly upregulated genes (when compared with aged Treg cells) linked to biologic processes associated with a robust pro-repair signature, including extracellular matrix organization, alveologenesis and vasculogenesis. Here, we demonstrate that the young Treg cell pro-repair program is dominated by Areg expression, accompanied by upregulation of IL-18 and IL-33 receptors and other genes related to the above-mentioned reparative processes.

Interestingly, we found no difference when comparing the suppressive phenotype of young versus aged Treg cells, suggesting that following influenza-induced lung injury, the reparative program of Treg cells is separable and distinct from their suppressive program. This is an important observation that informs the development of novel Treg cell-based immunotherapies to specifically target molecular pathways regulating their reparative function. In regard to aged Treg cells, we found that although capable of upregulating a pro-repair program following influenza infection, it is less robust when compared with the youthful reparative response. Moreover, aged Treg cells displayed increased expression of genes associated with an effector phenotype. Accordingly, we found increased expression of Th1 canonical markers, Tbet and Ifn-γ. Whether this finding represents an age-related functional adaptability of Treg cells following influenza infection or it is the result of Treg cell lineage instability leading to effector differentiation remains unknown.

Establishment of a Treg cell specific DNA hypomethylation pattern at key genomic loci is necessary to maintain the lineage stability and immunosuppressive function of Treg cells (13) .

Epigenomic profiling has revealed that Treg cell-specific alterations in methylation patterning modulate Treg cell transcriptional programs and increase susceptibility to human autoimmune diseases (31) . Whether epigenetic phenomena have a similar regulatory role in modulating the Treg cell reparative gene expression program remains unknown. Here, we used an unsupervised bioinformatics analysis to uncover a Treg cell-specific methylation-regulated transcriptional program enriched for reparative processes during recovery from influenza infection. Our computational integrative approach provides inferential evidence that age-related DNA methylation can modify the expression of genes linked to pro-repair processes in Treg cells but does not prove causality and therefore represents a limitation of our study. Future research could focus on leveraging epigenome editing technologies to establish the causality of age-related, Treg cell-specific DNA methylation patterns in controlling their regenerative function.

In conclusion, our study establishes that aging imparts cell-autonomous dysfunction to the 

Young (2-4-month-old) and aged (18-22- 

Wild-type C57BL/6 mice were anesthetized with isoflurane and intubated using a 20-gauge angiocatheter cut to a length that placed the tip of the catheter above the carina. Mice were instilled with a mouse-adapted influenza A virus (A/WSN/33 [H1N1]) (3 pfu/mouse or 2 pfu/mouse for Foxp3 DTR mice, in 50 µL of sterile PBS) as previously described (32) .

To prepare organ tissues for histopathology, the inferior vena cava was cut and the right ventricle was perfused in situ with 10 mL of sterile PBS and then sutured a 20-gauge angiocatheter into the trachea via a tracheostomy. The lungs were removed en bloc and inflated to 15 cm H2O with 4% paraformaldehyde. 5-µm sections from paraffin-embedded lungs were stained with hematoxylin-eosin and examined using light microscopy with the high-throughput, automated, slide imaging system, TissueGnostics (TissueGnostics GmbH).

Single-cell suspensions from harvested mouse lungs were prepared and stained for flow cytometry analysis and fluorescence-activated cell sorting as previously described using the reagents shown in Supplemental Table 1 (33, 34) . The CD4 + T Cell Isolation Kit, mouse (Miltenyi) was used to enrich CD4 + T cells in single-cell suspensions prior to flow cytometry sorting. Cell counts of single-cell suspensions were obtained using a Cellometer with AO/PI staining (Nexcelom Bioscience) before preparation for flow cytometry. Data acquisition for analysis was performed using a BD Symphony A5 instrument with FACSDiva software (BD). Cell sorting was performed using the 4-way purity setting on BD FACSAria SORP instruments with FACSDiva software. Analysis was performed with FlowJo v10.6.1 software.

Lungs were harvested from young and aged mice and a single-cell suspension was obtained.

Red blood cells were removed with ACK Lysis Buffer (Thermo Fisher) following the manufacturer's instructions. Single-cell suspensions were plated on 12-well cell culture plates (Thermo Fisher) at a concentration of 1 x 10 6 cells/mL with RPMI plus 2 µL/mL Leukocyte Activation Cocktail with GolgiPlug (BD) and incubated for 4 hours at 37 °C. After incubation, cells were resuspended in PBS and stained with a viability dye and subsequently with fluorochromeconjugated antibodies directed at IFN-γ (clone XMG1.2), IL-17 (clone TC11-18H1) and IL-4 (clone 11B11). Data acquisition and analysis was performed as described above.

Splenic CD4 + CD25 + Treg cells were isolated from euthanized young (2-4-month-old) and aged 

Flow cytometry sorted lung Treg cells were pelleted in RLT plus buffer with 2-mercaptoethanol and stored at -80 °C until RNA extraction was performed. The Qiagen AllPrep DNA/RNA Micro Kit was used for RNA and DNA simultaneous isolation (35) . RNA quality was assessed with the 4200 TapeStation System (Agilent Technologies). mRNA was isolated from purified 1 ng total RNA using oligo-dT beads (New England Biolabs, Inc). NEBNext Ultra™ RNA kit was used for full-length cDNA synthesis and library preparation. Libraries were pooled, denatured and diluted, resulting in a 2.0 pM DNA solution. PhiX control was spiked at 1%. Libraries were sequenced on an Illumina NextSeq 500 instrument (Illumina Inc) using NextSeq 500 High Output reagent kit (1x75 cycles). For RNA-seq analysis, FASTQ reads were demultiplexed with bcl2fastq v2. 17 A ranked gene list from young and aged phenotypes was ordered by log2(fold-change) in average expression, using 1,000 permutations and the Hallmark gene set database (37) .

Genomic DNA was isolated from sorted lung Treg cells using Qiagen AllPrep DNA/RNA Micro Kit. Endonuclease digestion, fragment size selection, bisulfite conversion and library preparation were performed as previously described (36, (38) (39) (40) . Sequencing was performed on NextSeq 500 instrument (Illumina). DNA methylation analysis and quantification were performed using Trim 

The raw and processed next-generation sequencing data sets have been uploaded to the GEO database (https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE151543, which will be made public upon peer-reviewed publication. 

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We thank the Northwestern University Flow Cytometry Core Facility supported by Cancer Center Support Grant (NCI CA060553). Flow cytometry Cell Sorting was performed using BD FACSAria SORP systems purchased through the support of NIH 1S10OD011996-01 and 1S10OD026815-01. Histology services were provided by the Northwestern University Mouse Histology and