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. Author manuscript; available in PMC: 2025 Jul 3.
Published in final edited form as: J Immunol. 2023 Oct 1;211(7):1073–1081. doi: 10.4049/jimmunol.2300182

End Stage IPF lung microenvironment promotes impaired NK activity

Tamara Cruz *,1, Paula A Agudelo †,1, Julián A Chamucero-Millares †, Anna Bondonese ‡, Nilay Mitash ‡, John Sembrat ‡, Tracy Tabib ||, Wenping Zhang ‡, Nouraie Seyed ‡, Victor Peters †, Sean Stacey †,§,, Dario Vignali #,**,††, Ana L Mora †, Robert Lafyatis ||, Mauricio Rojas †,2
PMCID: PMC12225614  NIHMSID: NIHMS1919961  PMID: 37566492

Abstract

Idiopathic Pulmonary Fibrosis (IPF) is a fibrotic age-related chronic lung disease characterized by the accumulation of senescent cells. Whether impaired immune response is responsible for the accumulation of senescent cells in the IPF lung remains unknown. Here we characterized the NK phenotype in IPF lungs via flow cytometry using C12FDG, markers of tissue residence, and chemokine receptors. The effect of the lung microenvironment was evaluated using lung fibroblast (LF) conditioned media and the bleomycin induced pulmonary fibrosis mouse model was used to assess the in vivo relationship between NK cells and the accumulation of senescent cells. We found that NK cells from the lower lobe of IPF patients (IPF-LL) exhibited immune-senescent and impaired CD57-NKG2A+ phenotype. We also observed that culture of NK cells from healthy donors in conditioned media (CM) from IPF-LL-LF induced a senescent like phenotype and impaired cytotoxic capacity. There is an impaired NK recruitment by LF and NKs presented decreased migration toward their CM. Additionally, NK cell-depleted mice treated with bleomycin showed increased collagen deposition, and accumulation of different populations of senescent cells compared to controls. The IPF lung microenvironment induces a dysfunctional NK phenotype limiting the clearance of lung senescent cells and the resolution of lung fibrosis. We propose that impaired NK activity could be one of the mechanisms responsible for perpetuating the accumulation of senescent cells in IPF lungs.

Introduction

Idiopathic pulmonary fibrosis (IPF) is an age-related disease characterized by the accumulation of scar tissue in the lung interstitium, mainly affecting the lower lobes1. We and others have reported accumulation of senescent fibroblasts in the lungs of IPF patients2–5 as one of the main factors inducing the extracellular matrix remodeling characteristic of the pathology. One of the hallmarks of senescence is the secretion of high levels of inflammatory cytokines, proteases and factors that promote senescence in neighboring cells and collagen deposition6, also known as SASP (senescence-associated secretory phenotype). Although the mechanism leading to the accumulation of senescent cells has not been completely defined, several lines of evidence suggest that impaired immune clearance is one of the main culprits.

NK cells are innate cytotoxic lymphocytes that kill target cells without any prior exposure and are the host’s first line of defense. In humans, NK cells are defined as CD3-CD56+ cells and can be divided into two major subsets based on the level of expression of CD56 and CD16. CD56dimCD16+ are circulating cells with a potent cytotoxic capacity. In contrast, CD56brightCD16- NK cells secrete pro-inflammatory cytokines and a small subset co-expressing tissue resident markers6; 7. Immuno-senescence is the progressive deterioration of the immune system during aging8. Aged NK cells exhibit a reduction in cytotoxic activity and cytokine production9. As an age-related disease, NK cells present in the IPF lung may be subjected to changes in their composition, phenotype, and function, due to the proinflammatory and profibrotic microenvironment where they reside.

We have recently reported a reduced percentage of NK cells in IPF lungs exhibiting impaired gene expression10. Here we show that IPF NK cells, especially the tissue-resident subset in the IPF lower lobe (IPF-LL), exhibit immuno-senescence features and decrease expression of activation and functional markers. Our data suggest that the local microenvironment in the IPF lung promotes this altered NK cell phenotype. Pre-treating blood NK cells with conditioned media (CM) from IPF-LF resulted in decreased NK cytotoxicity and induction of senescence. Our data also suggests that the reduced proportion of NK cells in IPF lungs is due to the impaired ability of the lung to recruit them. Furthermore, using a mouse model of lung fibrosis, we show NK cells activity is important to clear senescent cells and to efficiently resolve fibrosis. Altogether our data shows that NK cells are key mediators of the immune clearance of senescent cells during IPF progression and sets them as an attractive target for the development of immunotherapies.

Materials and Methods

Patient samples

Lung tissue samples were collected from explanted IPF patients and healthy donors after rejection as organ donors. Blood samples were obtained from healthy volunteers and IPF patients during their clinical controls. All protocols were approved by the Institutional Review Board and the Committee for Oversight of Research at the Ohio State University and the University of Pittsburgh, and Clinical Training Involved Decedents of the University of Pittsburgh. Signed informed consent forms were collected before organ procurement.

Isolation and preparation of single cell suspensions for flow cytometry

Tissue samples were processed after organ procurement with less than 20 min of warm ischemia and within 12–24 hours of cold ischemia. Lung samples were processed using enzymatic and mechanical digestion, as previously described11 to obtain single cell suspensions. Lung tissue homogenates and PBMCs were stained following the same protocol. A minimum of 106 cells of lung tissue homogenates or PBMCs were analyzed by flow cytometry. To determine the senescent fate, we measured the β-galactosidase activity by flow cytometry using the fluorogenic substrate C12FDG (5-Dodecanoylaminofluorescein Di-β-D-Galactopyranoside) (0.5 μM) (Molecular Probes, USA) for 75 mins at 37°C and 5% CO2. Staining conditions were optimized with proliferative and senescent fibroblast to have a clear increase in the intensity with a specific histogram signaling. This compound is a membrane-permeable non-fluorescent substrate of the β-galactosidase that, after being process, emits fluorescence and remains inside the cells. To induce lysosomal alkalization, cells were pre-treated with 100nM Bafilomycin A (Sigma, USA) for 1h in DMEM no phenol red at 37°C and 5% CO2. At the end of the incubation, cells were stained 5 mins with the viability staining (Fixable viability-Alexa600, BD, USA) and stained for extracellular markers 30 mins at 4ºC with CD3-PECy5.5, CD45-Alexa700, CD16-BV605, CD56-PE, CD57-APC, NKG2A-AF700, CD103-PE, Dazzle, CD49a-PE, CD69-PE.CY7, CCR2-BV711, CCR5-PECF594, CXCR1-BV650 and CXCR3-PECy7 (BD, USA). For intracellular staining, cells were fixed after surface staining using fixation/permeabilization solution (BD cat# 51–2090KZ), and then staining in perm/wash buffer (BD cat# 51–2091KZ) at 4°C for 30 min. Data were acquired on a BD LSRFortessa and on a Cytek Northern Lights and analyzed using FlowJo v10.8.1. NK cells were selected as CD45+, CD3- and CD56+. From the NK cells the subpopulations were determined based on the expression of CD56 and CD16, detailed gating strategy in Supplementary Figure 2. The percentage of NK cells expressing the characterization markers were obtained using a Fluorescent Minus One (FMO) that allowed us to set the positive gate for each marker (Supplementary Figure 3).

NK cells isolation

NK cells were purified from human PBMCs using EasySep™ Human NK Cell Isolation Kit (Stem Cell cat#17955). NK cell purity was confirmed by flow cytometry.

Flow cytometry based NK cytotoxic assay

NK cells were cultured overnight in CM in the presence or not of 25 ng/ml of IL-15. The following day, fresh AIM-V media supplemented with 10% HS was added and a cell-killing assay over the K562 cell line (ATCC, USA) was performed. K562 cells were pre-stained with the cell tracker deep red dye (Invitrogen cat# C34565). The killing assay was performed for 3 hours at a 5:1 effector to target ratio. The percentage of apoptotic and dead cells was determined using annexin V-Alexa488 and PI (Invitrogen, USA).

LDH-based NK cytotoxicity assay

5,0×10^5 proliferating or BLM–induced senescent hLF/well were seeded in a 96 well plate and left overnight to allow adherence. hLF were coculture with activated NK cells from an allogeneic donor at different effector to target ratios. Samples (10 μl) were collected from the same wells at 6 hours and frozen in LDH Storage Buffer at a 1:25 dilution. After thawed, experimental samples and internal controls were incubated with LDH detection reagent (Promega cat# J2381) at room temperature (RT) for 60 minutes and luminescence was recorded after that. Data was normalized and the percentage of cytotoxicity was calculated using the following formula, % Cytotoxicity = (Experimental LDH release-Medium background / Max LDH release control-Medium background) ×100

NK cell migration assay

5×10^5 freshly isolated NK cells were seeded in the upper well of a transwell filter in a final volume of 100 μL (Corning 24 well, 5 μm pore). RPMI 1640 (Gibco cat# 11875093) supplemented with FBS, or CM generated from old and IPF lung fibroblasts, was added to the bottom wells and incubated at 37°C and 5% CO2 for 3h. Migrated cells were counted using a cellometer (Nexcelom, Bioscience USA), and the migration index was calculated by dividing the number of cells migrated by the number of randomly migrated cells to RPMI.

To evaluate the effect of the CM over healthy innate immune cells, NK cells were cultured overnight in the presence of AIM-V (Gibco cat# 12055083) supplemented with 10% human serum (HS) or (3:4) CM. C12FDG and intracellular IL-6 detection was performed by flow cytometry the next day as described above.

Lung fibroblast isolation and generation of conditioned media

Human lung fibroblasts (hLF) were isolated and grown, as described previously2. hLF were expanded in-vitro until 60–70% confluence. Fresh media was added and collected after two days as the conditioned media (CM). FBS used for the generation of the CM was previously ultracentrifuged to eliminate microparticles.

BLM-induced senescence in human lung fibroblast.

hLF from healthy and young donors were treated with 15 ug/ml BLM for 48h hours at 37°C in completed media. Treatment was removed and fresh completed media was added every 48h hours until day 7. Senescence was confirmed by the expression of the senescent associated (SA) – beta galactosidase activity in a colorimetric assay.

Determination of plasma cytokines

Blood plasma cytokines were measured using a customized 12-plex Human Magnetic Luminex Assay (R&D, USA) for the following analytes CCL2, CXCL1, CXCL2, CXCL8, CXCL14, IFNα, IL-12/IL-23 p40, IL-15, IL-18, MICA, MICB, and ULBP2–5.

Transcriptomic expression analysis

scRNA-seq data come from our previous publication (GSE 128033, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE12803312. scRNA-seq raw count matrices were generated by the Cell Ranger pipeline with human reference genome annotation GRCh38. We inputted dropout events in gene expression using Single-cell Analysis Via Expression Recovery (Saver)13. Seurat (versions 2.3.4), an R package developed for single-cell analysis, was used to normalize gene expression, perform differentially expressed gene analysis, dimensionality reduction, clustering, and visualize clusters graphically14; 15. Clustering was performed based on Harmony embedding and visualized through UMAP and each cluster was identified by differentially expressed genomic signatures16.

RT-PCR analysis

hLF were lysed and collected in RLT buffer (Qiagen, US), mouse lung was flash frozen and cryo pulverized. Subsequently, RNA was extracted using RNeasy mini kit (Qiagen cat# 74106) and RNA concentration was quantified by Nanodrop. Taqman primers for HLA-E (human) and NKp46 (mouse) (IDT, US) were used, and real time PCR was run using RNA to CT one-step (Applied Biosystems, US).

Bleomycin-induced pulmonary fibrosis mouse model and NK cell depletion.

All animals were maintained in the Division of Laboratory Animal Resources at the University of Pittsburgh. All animal protocols were reviewed and approved by the IACUC committee. Young C57Bl/6 mice (8 weeks) were divided into control group (n=5) and NK cell-depleted group (n=12). Pulmonary fibrosis was induced by intratracheal instillation of 1.5 U/kg of bleomycin (Fresenius Kabi, Germany). On day 14 after bleomycin, the corresponding group was NK cell depleted by intraperitoneal administration twice a week of the mononuclear antibody anti-mouse NK1.1 (BioXCell BE0036). Both groups of mice were sacrificed at week 7 for the specific analysis. Mouse lung was digested with an adaptation of the human protocol using the specific mouse lung program on the Miltenyi Dissociator. The NK cells were determined by flow cytometry as CD3-NK1.1+ and the percentage of senescent cells was determined by C12FDG staining as previously described for human samples. Histology, beta-galactosidase staining, and OCT blocks were performed as described previously17. Images were taken with a light microscope over tissue sections and quantification was performed using ImageJ. The threshold was adjusted to automatically measure the total area of the tissue and the area stained in blue to calculate the proportion of the senescent area. Ashcroft fibrosis score was determined using the reference publication18. Tissue analyses were performed by two independent researchers with similar results to assure the reproducibility of the technique.

Statistics

Results are presented as mean ± standard deviation. Differences between groups were assessed using the Kruskal-Wallis with a post-hoc comparison with correction for multiple tests (Dunn’s) and Mann-Whitney tests as appropriate using GraphPad software. Significance was calculated when P<0.05. The specific samples and statistical tests employed in each experiment are specified in the figure legends and the patient characteristics table, Table 1.

Table 1. Patient’s characteristics.

Due to tissue availability, the in-depth NK characterization experiments could only be performed in some samples. The features of the subset of samples are specified accordingly.

n Gender (%F) Age (mean ± S.D) Smoking (Pack per year)
Lung samples Young controls 14 27.27 30.82 ± 8.98 0
Old controls 22 54.54 62.73 ± 6.56 0
Smokers 12 41.67 47.67 ± 19.11 21.90 ± 14.00
IPF Never smokers 8 50.00 63.88 ± 6.88 0
IPF Former smokers 6 66.67 66.33 ± 4.97 13.30 ± 11.8
Lung NK cells senescence and chemokine receptors Young controls 5 40 31.4 ± 2.88 0
Old controls 5 40 63.8 ± 7.53 0
IPF 4 50 65.7 ± 2.99 0
Lung NK cells in deep phenotypic characterization Young controls 5 0 26.3 ± 2.08 n/a
Old controls 5 100 66.0 ± 2.64 n/a
IPF 6 33.3 65.0 ± 1.00 n/a
Blood samples Controls 9 33.3 61.72 ± 3.98 0
IPF 8 37.5 71.37 ± 9.33 0
Lung fibroblast CM Controls 3 66.6 62.66 ± 2.08 0
IPF 3 33.3 64 ± 2.65 0
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Results

Lung NK cells exhibit a senescent phenotype and increased expression of impaired function markers in IPF-LL.

Tissues were obtained from healthy and IPF donors and classified by age and gender (Table 1). Total lung cell suspensions were analyzed by flow cytometry. NK cells were defined based on gating on the CD45+CD3-CD56+ population and further dividing into two major subsets, CD56brightCD16- and CD56dimCD16+ (Supplementary Figure 1). Increased β-galactosidase (β-Gal) activity is associated with cell senescence; we used C12FDG a fluorescent β-Gal substrate to identify senescent cells by flow cytometry. NK cells from IPF lungs exhibited a higher percentage of C12FDG+ cells compared to the control groups and matching the severity of the disease, this proportion was higher in IPF-LL than IPF-UL (Figure 1A). Noteworthy, these significant changes were only observed in the CD56brightCD16- subpopulation (Figure 1B). We also examined the production of IL-6, as an important component of the SASP. We observed a significant increase in the proportion of IL-6 producing senescent NK cells in the IPF-LL compared to the healthy controls (Figure 1C). To determine if these immune-senescence markers are also increased in the systemic response, we interrogated the status of circulating NK cells. Strikingly, we found no significant differences in the percentage of C12FDG+ cells between controls and IPF samples (data not shown), and as expected these cells did not produce IL-6 (data not shown).

Figure 1: Lung NK cells have a senescent phenotype.

Figure 1:

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(A) An increase in the percentage of senescent NK cells in the lower lobe of IPF patients is observed. (B) IPF-LL also presents an increased proportion of the CD56brightCD16- subpopulation but not the CD56dimCD16+ NK subpopulation. (C) Senescent NK cells express higher levels of IL-6. Healthy young (n=5), healthy old (n=5), IPF upper and lower lobes (n=4). *p<0.05, by Kruskal Wallis with a post-hoc comparison with correction for multiple test (Dunn’s), IPF-UL (IPF Upper Lobe) and IPF-LL (IPF Lower Lobe).

To gain insight into the phenotype and function of NK cells in the lungs of IPF patients, we evaluated the expression of distinct maturation and functional markers. IPF-LL CD56brightCD16- cells have increased expression of the inhibitory receptor NKG2A, pointing toward a more immature or exhausted phenotype (Figure 2A) and a reduction of the CD57 marker associated with terminally differentiated NK cells (Figure 2B). Being described as senescent, we evaluated the population of CD57-NKG2A+ cells19–21 and observed a significant increase in the proportion of the IPF-LL NK cells affecting both subpopulations (Figure 2C). Our data suggest that IPF-LL NK cells exhibit an abnormal differentiation pattern with high levels of senescence markers. A phenomenon that has been observed in other cell populations in IPF lungs 46.

Figure 2: Only the IPF-LL lung CD56brightCD16- NK cells have an impaired and a lung resident phenotype.

Figure 2:

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(A) IPF-LL CD56brightCD16- NK cells have an increased expression of NKG2A. (B) IPF-LL CD56brightCD16- NK cells have reduced expression of CD57. (C) The NKG2A+CD57- population is increased in the IPF-LL CD56brightCD16- NK cells. (D) Only the IPF-LL CD56brightCD16- NK cells express the 3 tissue residence markers simultaneously, CD49a, CD103 and CD69. Healthy young (n=5), healthy old (n=6), IPF upper and lower lobes (n=6). *p<0.05, by Kruskal Wallis with a post-hoc comparison with correction for multiple test (Dunn’s), IPF-UL (IPF Upper Lobe) and IPF-LL (IPF Lower Lobe).

The IPF lung microenvironment induces an impaired phenotype in IPF-LL NK cells.

The described senescent features such as C12FDG and IL-6. As well as the increased NKG2A expression concomitant with reduced expression of CD57 defines an impaired phenotype with an abnormal differentiation process, affecting mainly the CD56brightCD16- NK subset in the IPF-LL. To determine if this population is formed by lung resident cells or represents transiently recruited circulating cells, the expression of tissue-residency markers CD49a, CD69 and CD103 was measured. Interestingly, only the IPF-LL CD56brightCD16- cells showed high expression of the 3 markers combined (Figure 2D) as no significant changes were observed in CD56dimCD16+ cells. The individual expression of CD49a and CD103 was increased in the LL-IPF CD56dimCD16+ NK cells, but the expression of CD69 was similar across groups (Supplementary Figure 2A-2C).

The observation that only the CD56brightCD16- subpopulation exhibited a senescent and abnormal differentiation phenotype with high expression of lung-resident markers in the IPF-LL suggests that the lung microenvironment may be influencing NK phenotype and function. We hypothesized that the secretome of IPF senescent cells could modulate NK function and differentiation. To test this hypothesis, we evaluated the effect of conditioned media (CM) from IPF-LL-LF and age-matched healthy controls on blood healthy NK cells. Remarkably, NK cells cultured in CM from IPF-LF, exhibited a senescent phenotype with a higher percentage of C12FDG+ cells (Figure 3A) and increased IL-6 production (Figure 3B).

Figure 3: Lung fibroblast conditioned media (CM) alters NK senescent fate.

Figure 3:

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Blood NK cells from a healthy donor were incubated overnight in the presence of CM from IPF and age-matched fibroblast (n=3 per group, experiment run in triplicate). Preconditioned NK cells in the presence of IPF fibroblast CM showed increase proportion of senescent (A) and IL-6 producing NK cells (B). *p<0.05, **p<0.01, the effect of the CM was analyzed by Mann-Whitney test. Control CM (conditioned media from healthy age-matched control human lung fibroblast) and IPF CM (conditioned media from IPF human lung fibroblast).

NK cytotoxic capacity is reduced in the IPF-LL and is affected by the lung microenvironment

We evaluated the protein expression of granzyme B and perforin, two key molecules that mediate apoptosis of target cells. As expected, the CD56dimCD16+ subpopulation exhibited the highest proportion of positive cells, and no differences were observed among conditions. Previous studies have suggested that the CD56brightCD16- subpopulation can acquire cytotoxic activity under certain conditions47. We found that the CD56brightCD16- subpopulation had a lower expression of granzyme B+ perforin+ cells (Figure 4A).

Figure 4: NK cytotoxic capacity towards senescent cells is reduced in the IPF-LL and is affected by the lung microenvironment.

Figure 4:

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(A) The expression of granzyme b and perforin is reduced in the IPF-LL CD56brightCD16- NK subpopulation. Healthy young (n=5), healthy old (n=6), IPF upper and lower lobes (n=6). *p<0.05, by Kruskal Wallis with a post-hoc comparison with correction for multiple test (Dunn’s). (B) NK cells have increased killing activity toward senescent cells. (C) NK cells have the tendency to increase killing activity toward IPF lung fibroblast than to age-matched controls, although it didn’t reach statistical significant difference. (D) Conditioned media preconditioned NK cells in the presence or absence of IL-15 were co-cultured with the K562 cell line for 3 hours at a (1:5) ratio. The percentage of apoptotic and dead cells were evaluated with annexin V and PI by flow cytometry. NK cells incubated with the IPF fibroblast CM have reduced capacity to induce apoptosis and cell death. IL-15 addition in the preconditioning incubation competes with the effect of the CM (n=3 per group, experiment run in triplicate). *p<0.05, **p<0.01, the effect of the CM was analyzed by a Mann-Whitney test between groups and inside each group to determine the effect of the IL-15. IPF-UL (IPF Upper Lobe) and IPF-LL (IPF Lower Lobe), control CM (conditioned media from healthy age-matched control human lung fibroblast) and IPF CM (conditioned media from IPF human lung fibroblast).

To validate the NK cells cytotoxic capacity against senescent LF, we performed an LDH-cytotoxicity assay using bleomycin-induced senescent LF and proliferating fibroblasts as control of the possible cytotoxic activity due to HLA-mismatched. We found that NKs had increased killing activity over senescent fibroblasts (Figure 4B). Although not significant, when comparing young, old and IPF fibroblasts, IPF and aged fibroblasts also seemed more sensitive to the cytotoxic activity of NK cells (Figure 4C).

We interrogated whether the secretome from IPF-LF can also influence the cytotoxic activity of NK cells. We performed an in-vitro co-culture cytotoxic assay using the common NK target cell line K562. We found a reduction in apoptosis of K562 when co-cultured with NK cells cultured in IPF-CM (Figure 4D). To further explore the influence of the IPF microenvironment on NK cells, we tested the impact of one of the main NK-activating cytokines, IL-15, known to enhance cytotoxic activity47. NK cells preconditioned with the IPF-CM in the presence of IL-15 showed a similar killing capacity to control NK cells in media without IL-15, suggesting that IL-15 supplementation can boost cytotoxic activity and attenuates the effect of the IPF-CM (Figure 5D).

Figure 5: The CCR2-CCL2 axis for the NK recruitment to the lung and to the senescent foci is impaired.

Figure 5:

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(A) Flow cytometry characterization of the expression of chemokine receptors in lung NK cells in healthy young (n=5), healthy old (n=5), IPF upper and lower lobes (n=4) and in (B) blood NK cells in controls (n=7) and IPF (n=7). (C) There is a reduction in the expression of CCR2 in the IPF-LL CD56dimCD16- NK subpopulation in the lung and (D) also in the blood. *p<0.05, by Kruskal Wallis with a post-hoc comparison with correction for multiple test (Dunn’s).

Impaired recruitment of NK cells to the IPF lung leads to their accumulation in circulation.

We recently described a diminished proportion of NK cells in the lung of IPF patients and an increased frequency of these cells in the blood compared to age-matched controls10. Then we studied the expression of components of the CCL2-CCR2 axis, an essential signaling pathway required to recruit NK cells into the lung and for the clearance of senescent cells22; 23. The IPF-LL and IPF blood NK cells showed a reduced expression of CCR2 but not other receptors implicated in NK cell migration, such as CCR5, CXCR1, and CXCR3 (Figure 5A-B). Additionally, CD56dimCD16+ NK cells are the subset exhibiting this diminished expression of CCR2 in both lung and blood compartments (Figure 5C-D). Taking advantage of our previously published scRNA-seq data set12, we confirmed that CCR2 exhibited a reduced expression in IPF-NK cells and CCL2 was reduced in IPF fibroblasts compared to controls (Supplementary Figure 2D-E). The other essential chemokine in NK recruitment, CCL324, was also reduced (Table 2). Interestingly, CXCL12, recently associated with reducing NK cells in liver fibrosis and metastasis25, was increased (Table 2). There was a dramatic shift in the chemokines expressed by LF in favor of recruiting the CD56brightCD16- subpopulation26–29. The expression of CXCL10 and CCL5 involved in the recruitment of CD56brighCD16- NKs was slightly increased. While CXCL8, CXCL1, CXCL3, and CXCL2, chemo-attractants of CD56dimCD16+ cells, were strikingly downregulated in IPF-LL fibroblasts, concomitant with decreased expression of CXCL8 and CXCL3 in the plasma of IPF patients (Figure 6A-B).

Table 2: Differentially expressed genes of chemokines in the fibroblast cluster.

Fold change (FC) was calculated with the expression of the IPF-UL or IPF-LL Vs controls and a Wilcoxon rank sum test was used to determine the p-value.

NK subtype NK receptor Chemokine Upper Lobe Lower Lobe
LgFC p-value LgFC p-value
All CCR2 CCL2 −0.5616 3.08E-25 −0.8735 3.72E-69
All CCR1 CCL3 −0.7587 0.6534 −0.5528 0.7592
All CXCR4 CXCL12 0.6165 4.02E-24 0.7653 1.27E-67
CD56dim CD16+ CXCR1 CXCL8 −0.9784 2.65E-34 −1.4140 2.14E-46
CD56dim CD16+ CXCR2 CXCL1 −1.3147 8.66E-36 −1.2145 1.18E-48
CD56dim CD16+ CXCR2 CXCL3 −0.9672 1.49E-26 −1.0559 1.09E-41
CD56dim CD16+ CXCR2 CXCL2 −1.1071 2.19E-47 −0.9600 4.24E-72
CD56dim CD16+ CXCR1 CXCL5 −1.1786 0.0167 −0.9139 0.0085
CD56dim CD16+ CX3CR1 CX3CL1 1.4356 0.0007 1.2377 4.06E-05
CD56brighyt CD16- CCR5 CCL5 0.1733 0.0132 0.4059 6.30E-05
CD56brighyt CD16- CXCR3 CXCL10 0.6106 0.0099 0.6076 0.0568
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Figure 6: IPF patients have an impaired NK recruitment capacity.

Figure 6:

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(A-B) Plasma from IPF patients have a lower concentration of NK chemoattractant CXCL3 and CXCL8 (controls n=6 and IPF n=9). (C) Conditioned media (CM) from IPF fibroblasts (yellow) induces a despaired migration capacity of healthy NK cells than the CM from age-matched controls (grey) in a transwell migration assay (n=3 per group, experiment run in triplicate). The migration index was normalized to the control group (RPMI without FBS). (D) IPF hLF have more expression of HLA-E than controls by RT-PCR. *p<0.05 by Kruskal Wallis or Mann-Whitney test, IPF-UL (IPF Upper Lobe) and IPF-LL (IPF Lower Lobe).

Since we saw a decreased expression of NK chemo-attractant molecules in IPF-LF, we performed migration assays to investigate further the effect of the SASP produced by IPF-LF on NK cell recruitment. In line with our previous data, we found that NK cells exhibited reduced migration towards IPF-CM (Figure 6C). This data suggests that IPF-LF fails to recruit NK cells to the lung compartment, exacerbating NK cells accumulation in the blood. This impaired trafficking to the lung seems to affect other innate lymphocytes, like NKT-like cells that showed a similar pattern of reduced lung infiltration (Supplementary Figure 3) but not other lymphocyte populations (Supplementary Figure 3).

HLA-E signaling has been described as mechanism senescent cells use to evade their clearance by the immune system30. We found that isolated IPF-LF had increased expression of HLA-E compared to healthy controls (Figure 6D). The downregulation of NK chemo-attractants while up-regulating the expression of HLA-E may be an important mechanism for senescent fibroblasts in the IPF lung to evade the immune-clearance.

NK cell depletion impairs resolution of fibrosis in the bleomycin-induced pulmonary fibrosis model.

To determine the importance of lung NK cells in the clearance of senescent cells and the resolution of fibrosis, we tested the consequences of NK cells depletion during the resolution phase of the bleomycin-induced pulmonary fibrosis mouse model. NK cell depletion was performed twice a week from day 14 after the exposure to bleomycin and after the peak of fibrosis was reached, using an anti-NK1.1 antibody. NK cell depletion was confirmed by assessing the number of NK cells in the lung by flow cytometry (Figure 7A) and the expression of the NKp46 gene (Supplementary Figure 4A). NK cell-depleted mice showed increased collagen deposition, determined by hydroxyproline content (Figure 7B), and a trend toward increased fibrosis by Ashcroft score over trichrome sections (Supplementary Figure 4B). Flow cytometry quantification of C12FDG showed an increased percentage of senescent endothelial cells, epithelial cells, and fibroblasts in the NK-depleted group (Figure 7C), and the quantification of senescence by β-Gal staining on tissue sections showed a similar trend (Figure 7D).

Figure 7: NK depletion in a bleomycin mouse model aggravates lung fibrosis and senescent cells accumulation.

Figure 7:

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Bleomycin was used to induce lung fibrosis in mice. At day 14 of bleomycin treatment, NK1.1 antibody was administrated to deplete the NK cells (A) The elimination of the NK cells was evaluated by flow cytometry. (B) There was an increased deposition of collagen, measure by the hydroxyproline content. (C) NK cell-depleted mice also show an increase in the percentage of endothelial, epithelial and fibroblast senescent cells by flow cytometry (C12FDG+ cells). (D) Quantification of the senescent area in lung tissue by traditional B-Gal staining. Control (n=5) and NK depleted group (n=12) *p<0.05, **p<0.01, ***p<0.001 by a Mann-Whitney test.

Representative images of tissue trichrome histology and B-Gal determination are shown in Supplementary Figure 4C-D. Together, these results corroborate the importance of NK cells in the clearance of senescent cells and the resolution of lung fibrosis in vivo using a mouse model.

Discussion

This study demonstrates that the function and maturation of NK cells is profoundly compromised in IPF lungs. This altered NK response is more critical in the lower lobes, correlating with the severity of the pathology. IPF NK cells exhibited a decreased expression of the cytokine receptor CCR2 concomitant with a decreased capacity of the IPF-LF to generate recruitment signals, potentially resulting in their blood accumulation. Additionally, recruited NK cells in IPF lungs have an altered phenotype skewed towards immune-senescence, impaired activity and compromised maturation. The fibrotic lung microenvironment determines this impaired phenotype as only the CD56brightCD16- subpopulation with tissue-resident markers is affected. In a bleomycin mouse model, depletion of NK cells demonstrated a direct relationship between the lack of NK cells and increased accumulation of senescent cells, leading to impaired fibrosis resolution.

Our group has previously described the accumulation of senescent cells in the IPF lung2, which has been pointed out as a possible mechanism behind the development of lung fibrosis5; 31. Impaired lung immunosurveillance has been hypothesized to allow senescent cells to accumulate in the IPF lung. However, the mechanism behind senescent cell accumulation or the exact role that the immune system plays during IPF progression remains to be elucidated. NK activity has been identified as a leading mechanism targeting senescent cells in the liver32 and preventing liver fibrosis33. Consistent with our data, a recent study has described an altered NK phenotype in liver fibrosis34. In addition, a relationship between NK function and age-related diseases has been described in cancer, Alzheimer’s, cardiovascular and heart diseases, atherosclerosis, and others8; 32; 35. In IPF, previous studies have shown an increased number of NK cells in the blood36; 37. However, studies linking NK function and lung fibrosis development were lacking. Our studies using the bleomycin mouse model of fibrosis highlight the importance of NK cells for the efficient resolution of lung fibrosis, similar to what had been previously described in models of liver fibrosis38.

Like most cell types, NK cells exhibit aging-related changes, like decreased cytokine production and decreased killing activity on senescent or damaged cells8; 9; 35. A complete characterization of senescence-associated markers in NK cells is still needed. Recently, the expression of the inhibitory marker NKG2A and the loss of the maturation marker CD57 was described in advanced passages of prolonged cultures associated with loss of proliferative capacity20. We have found that the IPF-LL exhibit this impaired maturation phenotype (NKG2A+ and CD57-) concomitant with increased expression of the senescence marker C12FDG and IL-6 production. IL-6 is a potent inhibitor of NK cell cytotoxicity that reduces the expression of granzyme B and perforin39. Recent studies have described IL-6 as an immune evasion mechanism used by tumors, increasing the expression of PD-L1, which induces lymphocyte anergy and reduces NKG2D ligands40; 41. It is important to note that only the senescent IPF lung NK cells produce IL-6 and that this may represent an essential autoregulatory mechanism by inducing immunosuppression of surrounding NK cells. Further studies are needed to elucidate the role that IL-6 plays in NK cell function during IPF development.

In the tumor microenvironment, the SASP exhibits immunosuppressive activity42; 43. The effect that SASP elicits on immune cells in the IPF lungs remains highly unexplored. We found in vivo that the fibrotic lower lobes provide an environment that induces immuno-senescence in NK cells. Our findings were further supported by in vitro assays where IPF CM reduced killing capacity and increased β-galactosidase activity in NK cells. The CM is a proof of concept of the effect that SASP from IPF fibroblasts can exert over the NK cells, and we are aware that this does not represent the whole lung microenvironment. More profound studies on the characterization of the secretome of IPF lung cell types are currently being conducted. The effect of IL-15 on competing with the immunosuppressive IPF CM was remarkable, and it opens a window of opportunity for immunological therapies aiming to boost NK cell numbers and activity in IPF patients.

The reduced expression of CCR2 in lung NK cells and the impaired chemokine production by IPF-LF suggest a compromised recruitment to the lung, most likely contributing to the decreased proportion of lung NK cells in IPF patients. In addition, the chemokines are polarized towards the recruitment of the CD56brightCD16- with little cytotoxic capacity and recently described as regulatory cells in hepatocellular carcinoma44. Interestingly, only this subpopulation in the IPF-LL, expressed markers of tissue residency. Further work is needed to determine if the IPF lung microenvironment is selectively recruiting CD56brightCD16- NKs and inducing specific tissue-resident NK populations with impaired activity and regulatory function.

NK isolation was performed using positive selection beads due to lung heterogeneity and the poor purity obtained with negative selection kits. A possible NK activation by the interaction with the CD56 receptor was studied with appropriate controls, and any undesired effect on NK cells would be uniform. A significant limitation is that we could not obtain NK cells from the IPF lungs for functional studies due to their small proportion and low viability after purification.

The role of the immune response in IPF pathology has been controversial, partly because trials using anti-inflammatory or immunomodulatory drugs have failed. Here, we have shown a pattern of immuno-senescence and impaired NK activity in the IPF lung that may be responsible for the excessive accumulation of senescent cells. A deficiency in the lung NK recruitment capacity likely leads to their accumulation in the blood. Our study opens a new frame to target the immune system and improve the clearance of senescent cells alleviating the symptoms of fibrotic disorders.

Supplementary Material

1

Key Points.

Senescent IPF lung microenvironment impairs differentiation and function of NK cells.

NK cells are pivotal for senescent cells clearance and the resolution of fibrosis.

ACKNOWLEDGMENTS:

The authors would like to thank Lazar Vujanovic (University of Pittsburgh Hillman Cancer Center) for technical assistance in designing the in-vitro experiments and members of the University of Pittsburgh Flow Cytometry Core Facility for their support. We are incredibly grateful to Melinda J Klesen, Yingze Zhang, and the Simmons Center for Interstitial Lung Disease personnel at the University of Pittsburgh for assisting with patient records and sample collection. Finally, we acknowledge the the Lifeline of Ohio Organ Procurement Organization and The Ohio State University Wexner Medical Center DHLRI CTC Human Tissue Biorepository for providing the explanted donor organs, as well as the generous donors and their families whose selfless donations make lifesaving research possible.

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