Age-induced prostaglandin E2 impairs mitochondrial fitness and increases mortality to influenza infection

Type II AECs are a primary source of PGE₂ in the aged lung

Prior studies, including our own, have shown that PGE₂ is increased in the aged lung, specifically within the bronchoalveolar lavage fluid (BALF), in non-infected mice^11,12. However, cellular sources driving the age-associated increase of PGE₂ remain unknown. To identify which lung cell type(s) secrete(s) PGE₂ with aging, we initially utilized a scRNA-seq dataset (Tabula Muris, GEO GSE109774)²³ to identify the cell types that highly express the enzymes COX1 and COX2, both critical, rate-limiting enzymes of PGE₂ production^24,25. COX1 is encoded by the gene Ptgs1 and COX2 is encoded by the gene Ptgs2. Our initial query showed that epithelial cells exhibited high expression of both genes (Supplementary Fig. 1a). To determine if type I or type II alveolar epithelial cells (AECs) are potent PGE₂ producers, we re-analyzed publicly available scRNA-seq data of AECs (GEO GSE113049) and found that type II AECs express ~5.5-fold higher transcript levels of Ptgs1 and ~8.5-fold higher transcript levels of Ptgs2, relative to type I AECs (Supplementary Fig. 1b). Therefore, we considered type II, rather than type I, AECs as potential contributors to PGE₂ levels with aging. Given that myeloid immune cells are known secretors of PGE₂^26,27,28_, we also considered that AMs, lung resident myeloid cells, as candidate contributors to the overproduction of PGE₂ with aging. The localization of both type II AECs and AMs at the alveolar surface make them prime candidates to account for the excess PGE₂ levels found in the BALF of aged mice.

We isolated and cultured both AMs and type II AECs from non-infected young (2–4 months of age) and aged (18–22 months of age) female C57BL/6 mice. Following 2 days of culture, we collected the cell culture medium and measured PGE₂ by ELISA. We found that type II AECs produced PGE₂ at 3 orders of magnitude higher than AMs regardless of host age (Fig. 1a). Importantly, type II AECs from aged mice produced ~2-fold more PGE₂ than type II AECs from young mice (Fig. 1a). Type II AECs isolated from young and aged mice had similar rates of viability and apoptosis following cell culture (Supplementary Fig. 1c, d), indicating that the differences in PGE₂ levels are not due to culture viability differences. These results suggest that type II AECs, but not AMs, are a major source of PGE₂ in the BALF at steady state, and aging increases the production of PGE₂ by type II AECs.

We next determined if IAV infection increases PGE₂ production by type II AECs and whether aging exacerbates the phenotype. Hence, we cultured type II AECs isolated from either young or aged mice, infected the AECs ex vivo with IAV at a multiplicity of infection (MOI) of 0.1 for 2 days and subsequently measured PGE₂ in the culture medium via ELISA. Importantly, IAV led to increased PGE₂ production by ~1.5-fold in aged type II AECs, and infected type II AECs from aged mice producing ~2-fold higher levels of PGE₂ as compared to infected type II AECs isolated from young mice (Fig. 1b).

We next examined how aging and IAV infection impacts PGE₂ levels within the BALF in vivo. Hence, we infected young and aged female C57BL/6 mice with 400 plaque forming units (pfu) of H1N1 A/PR/8/34 IAV intranasally (i.n.) as 400 pfu is the lethal dose 70% (LD₇₀) in aged mice (Supplementary Fig. 1e, survival tracked up to 15 days post infection (dpi)) and collected the BALF at 0, 1, 3, 6, and 8 dpi for the PGE₂ ELISA. Prior to infection (i.e., 0 dpi), aged mice exhibited ~2-fold higher levels of PGE₂ as compared to young mice (Fig. 1c) and at 8 dpi aged mice exhibited a ~3-fold increase of PGE₂ in the BALF as compared with infected young mice (Fig. 1c). Overall, these ex vivo and in vivo results demonstrate that aging upregulates PGE₂ secretion within the lung both in the presence and absence of IAV infection.

We next determined whether the age-enhanced levels of PGE₂ in the BALF is a genotype- or sex- specific phenomenon, by measuring PGE₂ in the BALF of young (6 months) and aged (22 months) male non-infected UM-HET3 mice, a 4-way crossed outbred mouse strain used by the National Institute on Aging Interventions Testing Program²⁹. Similar to the female C57BL/6 mice, the aged male UM-HET3 exhibited a ~3-fold increase of PGE₂ within the BALF compared to the young mice (Supplementary Fig. 1f).

To determine if BALF PGE₂ levels increase with aging in humans, we analyzed PGE₂ levels in BALF collected from 22 healthy human patients (Supplementary Table 1). Using simple linear regression, we found a positive correlation between age and PGE₂ levels in the BALF (Fig. 1d). Taken together, these results indicate that increasing PGE₂ levels in the BALF with aging is a conserved finding across sex, genotype, and species.

Senescence increases PGE₂ by type II AECs

Next, we sought to determine a cellular mechanism by which aging increases PGE₂ production by type II AECs, and focused our attention on senescence as a possible mechanism. To determine if senescence increases PGE₂ secretion by type II AECs and if PGE₂ is a SASP factor within the lung, we first characterized whether type II AECs from aged mice exhibit features of senescence. We found that type II AECs from aged mice exhibited a ~2–3-fold increased secretion of two SASP factors, IL-6 and TNF-α, as compared to type II AECs from non-infected young mice (Fig. 2a, b). Type II AECs from young mice had undetectable levels of the senescence marker p21, whereas type II AECs from aged mice exhibited high expression of p21 (Fig. 2c). Overall, these results show that type II AECs isolated from aged mice exhibit evidence of senescence.

To determine if senescence increases PGE₂ secretion, we irradiated type II AECs isolated from young mice with either 0, 5, 10, or 15 Gy of radiation to induce a senescent phenotype within the cells³⁰. The levels of p21 in type II AECs increased with increasing doses of radiation, confirming the induction of a senescence phenotype (Supplementary Fig. 2a). We then measured PGE₂ in the cell culture medium of the radiated type II AECs by ELISA and found increasing PGE₂ production with increasing irradiation, correlating with p21 expression (Fig. 2d and Supplementary Fig. 2b).

To determine the necessity of senescence for PGE₂ overproduction in type II AECs with aging, we cultured type II AECs from young and aged mice with dasatinib and quercetin (D&Q), a combination senolytic treatment that depletes senescent cells^31,32. D&Q treatment reduced p21 expression in type II AECs from aged mice as measured by western blotting, validating our approach (Supplementary Fig. 2c). We then measured secreted PGE₂ levels in the culture medium and normalized the results based on total protein levels of live, adherent type II AECs. The senolytic D&Q treatment reduced PGE₂ secretion of type II AECs from aged mice by ~5.5-fold (Fig. 2e). However, the D&Q treatment did not significantly alter PGE₂ secretion of type II AECs from young mice, as expected.

Taken together, these results suggest that PGE₂ is a SASP factor produced by senescent type II AECs, and that senescent type II AECs are a major producer of PGE₂ in the lung with aging.

Blocking PGE₂ signaling via the EP2 receptor increases AM numbers in aged mice

We next examined the in vivo consequences of age-elevated PGE₂ levels in the lung. Of the four PGE₂ receptors, EP2 and EP4 have been shown to be upregulated on AMs during IAV infection¹⁵. In particular, PGE₂ signaling through the EP2 receptor regulates AM functions such as phagocytosis of bacteria³³, toll-like receptor expression³⁴, and production of suppressor of cytokine signaling 3 (SOCS3)³⁵ and in vitro proliferation capacity¹¹. Based on this and our results thus far, we hypothesized that age-elevated PGE₂ levels reduce AM numbers and function in a way that primes aged animals to IAV infection. It has been previously shown that aging reduces the number of AMs in female C57BL/6 mice⁸, and we first confirmed that this phenotype is generalizable to young and aged male UM-HET3 mice (Supplementary Fig. 3a).

We next investigated whether PGE₂ signaling affects AM numbers in vivo, by blocking PGE₂ signaling via intraperitoneal (i.p.) injections of an antagonist against the EP2 receptor³⁶ daily for 7 days, followed by the enumeration of AMs (defined as CD45⁺ CD11c⁺ SiglecF⁺) from the BALF by flow cytometry. This treatment reduced PGE₂-induced cAMP, a downstream molecule of the PGE₂ receptors¹⁴, in the lungs (Supplementary Fig. 3b), validating our approach. Blocking PGE₂ signaling through the EP2 receptor significantly increased total AM numbers by ~1.3-fold in aged mice (Fig. 3a). In contrast, blocking the EP2 receptor in young C57BL/6 mice did not alter total AM numbers (Supplementary Fig. 3c). These results show that excessive PGE₂-EP2 signaling is a major factor that limits AM numbers in the aging lung.

Increased proliferation and/or reduced apoptosis could explain the increase in AM numbers seen with the EP2 receptor blockade. Using BrdU to label proliferating cells³⁷, we found that blocking the EP2 receptor in aged mice led to a ~2-fold increase in the percent of BrdU-positive AMs compared to the vehicle control (Fig. 3b). On the other hand, Annexin V staining for apoptotic cells revealed that EP2 receptor blockade does not affect AM apoptosis in aged mice (Fig. 3c).

To determine if the EP2 antagonist, which was given systemically, increased AM proliferation in an AM-dependent manner, we gave aged mice clodronate-loaded liposomes i.n. to deplete resident AMs and then performed an adoptive transfer of CSFE-labeled C57BL/6 WT AMs or AMs deficient of the EP2 receptor (EP2 KO) (Fig. 3d). Four weeks following the transfer, we analyzed the proliferation of the transferred AMs as measured by CFSE dilution. We observed that the EP2 KO AMs had higher rates of proliferation compared to the WT AMs within aged mice (Fig. 3e, f). Overall, these results show that PGE₂ limits AM numbers with aging, via the EP2 receptor, by reducing AM proliferation and without altering apoptosis.

To determine if aging alters the expression of the EP2 receptor on AMs, we re-analyzed a publicly available RNA-seq dataset (GEO GSE134397) of sorted AMs from young and aged mice¹⁰. The trimmed mean of M-values (TMM) normalized counts from the RNA-seq dataset showed no age-associated differences in expression of the EP2 receptors on AMs on a transcriptomic level (Supplementary Fig. 3d), indicating that the differential response to EP2 antagonism in young versus aged mice is independent of altered expression of the EP2 receptor and is instead likely to reflect the higher levels of PGE₂.

PGE₂ signaling impairs the mitochondrial fitness of AMs

Reduced mitochondrial function, specifically the inhibition of the electron transport chain and oxidative phosphorylation, has been shown to limit proliferation in a variety of cell types such as intestinal stem cells³⁸, Jurkat cells³⁹, vascular smooth muscle cells⁴⁰, and human colon cancer cells HCT116⁴¹. In addition, PGE₂ has been shown to limit oxidative phosphorylation in human monocyte-derived macrophages²⁷. Hence, we hypothesized that a plausible mechanism underlying the effect of PGE₂ on AM proliferation might be reduction of mitochondrial fitness and energetic output. To test this, we collected AMs from EP2 antagonist or vehicle-treated aged mice and measured their mitochondrial mass via MitoTracker, mitochondrial reactive oxygen species (ROS) via MitoSOX, and mitochondrial membrane potential via tetramethylrhodamine methyl ester (TMRM) staining. Our results indicate that blocking the EP2 receptor reduced mitochondrial mass (Fig. 4a), mitochondrial ROS (Fig. 4b), and mitochondrial membrane potential (Fig. 4c) of AMs in aged mice. Similarly, in ex vivo culture of AMs isolated from young C57BL/6 mice, PGE₂ treatment increased MitoTracker staining for mitochondrial mass by ~1.3-fold (Fig. 4d). Analyzing the mitochondria of AMs isolated from young and aged mice by transmission electron microscopy (TEM) also showed that the area per mitochondria in AMs also increases with age (Fig. 4e, f).

A possible avenue by which PGE₂ could increase mitochondrial mass of AMs is by decreased mitophagy, leading to the accumulation of damaged mitochondria. Mitophagy is the autophagic recycling of damaged mitochondria⁴², and is known to be dysregulated with aging⁴³. However, how aging affects mitophagy in AMs is unknown. To determine if aging alters mitophagy of AMs, we utilized a mitophagy reporter mouse model, known as the MitoQC mice. MitoQC mice express a pH-sensitive GFP and mCherry mitochondrial marker. Under neutral pH, both the mCherry and GFP fluoresce, but within the acidic environment of the autolysosome the GFP fluorescence is quenched, allowing for the in vivo detection of mitophagy at a single-cell level⁴⁴. We analyzed AMs obtained from young (2–3 months) and aged (22–25 months) C57BL/6 MitoQC mice by flow cytometry. The mitophagy index was then calculated based on the mean fluorescence intensity (MFI) of mCherry divided by the MFI of GFP, and the results normalized to control conditions (i.e., AMs from young mice, or AMs from vehicle control treated mice). We found that AMs from aged MitoQC mice exhibited a reduced mitophagy index compared to AMs from young MitoQC mice (Fig. 4g). To determine if PGE₂ is an age-associated factor that limits mitophagy in AMs, we treated middle-aged (i.e., 9–10 months) non-infected MitoQC mice with seven daily doses of the EP2 antagonist to block PGE₂ signaling and detected increased mitophagy in AMs in vivo (Fig. 4h). Overall, these results indicate that aging, via elevated PGE₂, restricts mitophagy in AMs, which could potentially lead to an accumulation of damaged mitochondria, hence increased mitochondrial mass.

The accumulation of damaged mitochondria could lead to dysregulated cellular metabolism and impaired mitochondrial oxidative phosphorylation has been shown to limit proliferation in a variety of cell types^38,39,40,41. To understand how PGE₂ affects cellular metabolism in AMs, we measured the oxygen consumption rate (OCR), a readout of oxidative phosphorylation, and extracellular acidification rate (ECAR), a readout of glycolysis, in AMs isolated from young mice and cultured with PGE₂ via a Seahorse assay (Agilent)⁴⁵. The addition of PGE₂ reduced the OCR over the course of the assay (Fig. 4i), including reductions in both the basal OCR (Fig. 4j) and the maximal OCR (Fig. 4k). In addition, PGE₂ also restricts the ECAR of AMs (Fig. 4l). Overall, these results suggest that PGE₂ restricts both oxidative phosphorylation and glycolysis of AMs, to impair mitochondrial homeostasis and energy generation in AMs, and ultimately reduce AM proliferation.

We also employed an immortalized murine AM cell line, MH-S^46,47 which showed similar mitochondrial changes as primary AMs in response to PGE₂. We first noted that MH-S cells increased mitochondrial ROS (Supplementary Fig. 4a, b), increased mitochondrial membrane potential (Supplementary Fig. 4c, d), and reduced proliferation when cultured with PGE₂ (Supplementary Fig. 4e). These findings are compatible with our in vivo results in which EP2 antagonism decreased mitochondrial ROS, decreased mitochondrial membrane potential in AMs (Fig. 4b, c), and increased AM cell numbers (Fig. 3a). We then employed the MH-S cell line in the Seahorse assay, which revealed that PGE₂ lowered OCR and ECAR of MH-S cells, which is similar to our findings with the primary AMs (Supplementary Fig. 4f–i).

Prophylactic blockade of PGE₂ signaling through the EP2 receptor improves survival to IAV infection in aged mice

AMs are crucial for host defense against respiratory viruses such as IAV^8,9. Our results thus far suggest that PGE₂ limits AM numbers in non-infected aged mice. Therefore, we hypothesized that blocking PGE₂ signaling using a 7-day prophylactic course of the EP2 antagonist in aged mice prior to infection, thereby boosting AM numbers (Fig. 3a), would improve survival to lethal IAV infection (Fig. 5a). Indeed, we found that prophylactic EP2 antagonist treatment significantly increased survival in aged IAV-infected mice from ~15% (vehicle control) to ~50% (EP2 antagonist) (Fig. 5b, survival tracked up to 16 dpi).

IAV infection upregulates both the EP2 and EP4 receptors on AMs¹⁵. However, a 7-day prophylactic blockade of the EP4 receptor, unlike the 7-day prophylactic blockade of the EP2 receptor, did not increase survival in aged mice infected with IAV (Supplementary Fig. 5a, b, survival tracked up to 15 dpi). To investigate potential synergy between the EP2 and EP4 receptor blockades, we administered both the EP2 and EP4 receptor antagonists prophylactically from 7 days pre-infection and to 4 dpi (days −7 to +4) to aged mice and infected the mice intranasally with IAV (Fig. 5a). Under this dosing regimen, aged mice treated with the dual EP2/EP4 antagonists exhibited a significant increase in survival versus those treated with vehicle control; however, there were no survival differences between the aged mice given the EP2 antagonist treatment alone versus the dual EP2/EP4 receptor antagonists (Fig. 5b). Thus, dual blockade treatment does not provide additional therapeutic advantages compared to the single EP2 blockade treatment.

To investigate if the EP2 antagonism improved aged mice survival to IAV infection in an AM-dependent manner, we employed an AM adoptive transfer model in which aged mice, following clodronate-liposome depletion of resident AMs, were given either WT or EP2 KO AMs. Following AM transfer, the mice were given either the EP2 antagonist or vehicle control prior to lethal IAV infection (Fig. 5c). The EP2 antagonism did not improve survival in aged mice transferred with EP2 KO AMs compared to those given vehicle control (Fig. 5d). However, EP2 antagonism improved survival in aged mice transferred with WT AMs, suggesting that the EP2 antagonism improves survival in an AM-expressed EP2-dependent manner (Fig. 5d). In addition, vehicle-treated aged mice given EP2 KO AMs showed a 2-fold increased survival compared to vehicle-treated aged mice given WT AMs, again indicating that the lack of PGE₂ signaling through the EP2 receptor on AMs is sufficient to improve aged mice survival to IAV (Fig. 5d)

In contrast to aged mice, young C57BL/6 mice treated prophylactically with the EP2/EP4 antagonist (Supplementary Fig. 3c) and infected with a lethal dose of IAV (i.e., 800 pfu, doubled the dose given to aged mice) failed to exhibit improved survival (Supplementary Fig. 5d, survival tracked up to 13 dpi). These results are compatible with our observation that EP2 blockade failed to increase AM numbers in non-infected young mice (Supplementary Fig. 3c). Therefore, excessive PGE₂ signaling via EP2 compromises survival to IAV in an age-dependent manner.

We next examined if EP2 blockade could alter the clinical response to IAV in aged mice when the blockade is administered during active infection to model a therapeutic solution rather than a prophylactic one. To this end, we began administration of 7 daily i.p. injections of the EP2 antagonist, or vehicle control, starting on the day of infection in aged C57BL/6 mice (Supplementary Fig. 5e). We found no differences in survival between the treated and control aged mice in this scenario (Supplementary Fig. 5f, survival tracked up to 16 dpi).

Prophylactic blockade of PGE₂ signaling through the EP2 receptor reduces influenza viral load and disease severity in aged mice

Given that prophylactic EP2 receptor blockade increased survival of aged mice following IAV infection, we next examined if EP2 antagonism affected viral load. To monitor viral load, we treated aged C57BL/6 mice prophylactically with the EP2 antagonist or vehicle for 7 days, infected them with IAV, and measured the viral protein hemagglutinin (HA) in lung homogenates at various time points throughout the infection. EP2 antagonism significantly reduced viral HA protein levels by ~2-fold compared to the vehicle control at both 4 and 6 dpi (Fig. 6a). Similar to our observations that EP2 antagonism increased total AM cell numbers in non-infected aged mice, we observed an increase in AM cell numbers in BALF of EP2 antagonist-treated mice over the course of infection (Fig. 6b). Linear regression analysis showed that the viral load, as measured by HA protein levels, negatively correlated with AM numbers in the BALF during IAV infection (Supplementary Fig. 6a).

As PGE₂ is a lipid known to regulate several immune cell types^48,49, we characterized how prophylactic blockade of the EP2 receptor affects immune cell accumulation into the lung and BALF following IAV infection using flow cytometry (gating strategy shown in Supplementary Fig. 6b). Within the lung tissue, we observed no differences in the number of CD45⁺ hematopoietic (Supplementary Fig. 6c), CD4⁺ T cells (CD3⁺CD4⁺CD8^–) (Supplementary Fig. 6D), CD8^{+ T} cells (CD3⁺CD4^–CD8⁺) (Supplementary Fig. 6e), B cells (CD45⁺ B220⁺) (Supplementary Fig. 6f) and AEC II cells (CD45^–EpCAM⁺) (Supplementary Fig. 6g). We also found no differences in neutrophil numbers in the BALF between EP2 antagonist vs. vehicle control treated groups (Supplementary Fig. 6h).

EP2 blockade reduced lung damage, inferred from a reduced level of albumin in the BALF, a marker of lung damage, at 4 dpi as compared to control (Fig. 6c). Aged mice treated with the EP2 antagonist exhibited ~2–3-fold reductions in the levels of the proinflammatory cytokines IL-6 (6 and 9 dpi) and TNF-α (6 dpi) (Fig. 6d, e). We also observed higher levels of the anti-viral cytokine interferon (IFN)-β in the EP2 antagonist-treated mice at 6 dpi (Fig. 6f). Mice begin to recover from infection and resolve the inflammation in the lungs at 9 dpi, and we found that the EP2 antagonist-treated mice exhibited a ~1.75-fold increase in the immunosuppressive cytokine IL-10 at 9 dpi (Fig. 6g), indicating that the EP2 antagonist-treated mice are better able to resolve the inflammation within the lungs than vehicle-treated counterparts. Mice treated with the EP2 antagonist also showed reduced histological evidence of inflammation at 4 dpi (Fig. 6h). Overall, these data indicated that the prophylactic EP2 antagonist treatment in aged mice leads to reduced viral load, inflammatory cytokines, and subsequent lung damage following infection.