Compartmentalized effects of aging on group 2 innate lymphoid cell development and function

Abstract The effects of aging on innate immunity and the resulting impacts on immunosenescence remain poorly understood. Here, we report that aging induces compartmentalized changes to the development and function of group 2 innate lymphoid cells (ILC2), an ILC subset implicated in pulmonary homeostasis and tissue repair. Aging enhances bone marrow early ILC2 development through Notch signaling, but the newly generated circulating ILC2 are unable to settle in the lungs to replenish the concomitantly declining mature lung ILC2 pool in aged mice. Aged lung ILC2 are transcriptomically heterogeneous and functionally compromised, failing to produce cytokines at homeostasis and during influenza infection. They have reduced expression of Cyp2e1, a cytochrome P450 oxidase required for optimal ILC2 function. Transfer of lung ILC2 from young mice enhances resistance to influenza infection in old mice. These data highlight compartmentalized effects of aging on ILC and indicate that targeting tissue‐resident ILCs might unlock therapies to enhance resistance to infections and diseases in the elderly.

. Aging-induced BM stromal cell failure and thymus involution result in diminished production of naïve T and B lymphocytes (Chinn et al., 2012). The maintenance and maturation of adaptive lymphocytes are further impaired by age-related structural and functional changes to the lymph nodes, leading to increased vulnerability to pathogens and poor responses to vaccination (Thompson, Smithey, Surh, & Nikolich-Zugich, 2017;Turner & Mabbott, 2017). Aging is also associated with changes to nonlymphoid tissues such as the lungs (Lowery, Brubaker, Kuhlmann, & Kovacs, 2013); however, how aging influences tissue immunity remains very poorly understood.
Innate lymphoid cells (ILCs) are a unique family of innate lymphocytes that lack clonally distributed antigen receptors but functionally and molecularly resemble T cells (Vivier et al., 2018). GATA3-expressing group 2 innate lymphoid cells (ILC2) are the predominant ILC subset in the lung. Lung-resident ILC2 are implicated in pulmonary homeostasis, epithelial repair, and lung remodeling (Vivier et al., 2018). ILC2 are seeded into the lungs during prenatal or perinatal stages (Gasteiger, Fan, Dikiy, Lee, & Rudensky, 2015;Nussbaum et al., 2013). Mature lung ILC2 in adult young mice are noncirculating tissue-resident cells, and their local proliferation, but not recruitment from the BM, is a signature of activation (Gasteiger et al., 2015). Nevertheless, BM lLC lymphopoiesis remains active in adult mice (Yang & Bhandoola, 2016). Various ILC and ILC2 precursors have been identified in the BM of adult mice (Yang & Bhandoola, 2016). When transferred to irradiated recipients, these BM ILC/ILC2 precursors efficiently give rise to mature ILC2 in the lungs of recipient mice (Yang & Bhandoola, 2016). However, it remains unknown whether BM precursors may home into the lungs to replenish mature lung ILC2 in physiologic and pathologic conditions other than irradiation. Very little is known about whether and how aging may impact ILC development or function.
In this study, we have explored the effects of aging on ILC2 development and function. Interestingly, our data reveal highly compartmentalized effects of aging on ILC2. Specifically, aging is paradoxically associated with increased early ILC2 development in the BM, but reduced maintenance and function of mature ILC2 in the lungs. Aging leads to a drastic increase in the numbers of BM ILC2 precursors (ILC2P) through Notch signaling-dependent mechanisms. However, this increase is not sufficient to replenish the concomitant decline in mature ILC2 in the lungs of aged mice. Mature lung ILC2 in aged mice are numerically and functionally compromised, failing to produce cytokines both at homeostasis and during influenza infection. Aged lung ILC2 have reduced fatty acid uptake and decreased expression of peroxisomal and cytochrome p450 (CYP) enzymes that are required for optimal ILC2 function. Transfer of activated ILC2 purified from the lungs of young mice enhances resistance to influenza infection in old mice. Together, these data highlight highly tissue-specific effects of aging on innate lymphoid cell development and function, and indicate that targeting tissueresident ILC might unlock new therapies to enhance resistance to infectious diseases in the elderly.

| Aging enhances early ILC2 development in the bone marrow
A recognized mechanism of immunosenescence is diminished adaptive lymphopoiesis leading to declined production of naïve T/B cells (Nikolich-Zugich, 2018). However, it remains unclear whether such a decline also occurs in early innate lymphoid cell development. Thus, we compared known ILC progenitor numbers in the BM of young (2-3 months) versus old (19-24 months) mice. The average number of PLZF + common helper innate lymphoid progenitors (CHILP) (Constantinides, McDonald, Verhoef, & Bendelac, 2014;Klose et al., 2014) showed a trend toward increasing with age, although the increase was not statistically significant due to heterogeneity in the aged population (Figure 1a,b). In contrast, the number of BM ILC2P (Hoyler et al., 2012) was increased more than fivefold in old mice, suggesting enhanced differentiation toward the ILC2 lineage ( Figure 1c,d). The expression of proliferation marker Ki67 was comparable between young and aged BM ILC2P, suggesting that other mechanisms might contribute to the accumulation of ILC2P with aging ( Figure S1a). Together, in contrast to declining adaptive lymphopoiesis with aging, aging is associated with increased ILC2 development in the BM.
As a clue to ascertain the signals through which aging increases ILC2 numbers in the BM, we noted that aging correlated with reduced IL-33R expression in BM ILC2P (Figure 1e). Because Notch signaling can repress IL-33R expression in ILC2 (Zhang et al., 2017) and may also promote early ILC2 development (Yang et al., 2015(Yang et al., , 2013, we hypothesized that aged BM precursors might be driven to the ILC2 lineage through enhanced Notch signaling. Using receptor-specific blocking antibodies, we confirmed that inhibition of Notch1 and Notch2 partially restored IL-33R expression in old BM ILC2P (Figure 1f). More importantly, we discovered that antibody treatment also reversed the aging-induced accumulation of BM ILC2P quickly within three days of treatment ( Figure 1g). This rapid decrease in BM ILC2P numbers in aged mice demonstrates that maintaining the higher numbers of BM ILC2 that are observed with aging requires a tonic Notch signal ( Figure 1g). Of note, inhibition of Notch1 and Notch2 did not affect BM ILC2P numbers in young mice, suggesting that Notch signaling specifically promotes aging-associated accumulation of BM ILC2P in old mice ( Figure S1b).

| Mature ILC2 in the aged lungs are numerically and functionally compromised
We then examined whether the enhanced BM ILC2 development might lead to increased cellularity of mature ILC2 in the lungs of old mice. We compared the number of mature ILC2, defined by Lin -GATA3 + Thy1 + CD45 + cells, in the lungs of young and old mice. To our surprise, we did not observe an increase in the numbers of lungresident ILC2 with aging. In contrast, the average numbers of mature ILC2 in the lungs were reduced with aging (Figure 2a,b). In addition, remarkable cellular heterogeneity was noted among individual old mice (Figure 2a,b). Some old mice have moderate reduction in the cellularity of lung-resident ILC2, whereas others were nearly depleted of mature ILC2 in the lung (Figure 2a,b). Together, aging is paradoxically associated with increased early ILC2 development in the BM but reduced maintenance of mature ILC2 in the lung.
We next compared the molecular and functional characteristics of mature ILC2 in the lungs of young and old mice. Lung ILC2 in aged mice expressed GATA3, but at a lower level than young lung ILC2 (Figure 2c,d). Like young ILC2, aged lung ILC2 lacked the expression of molecules characteristic of other ILC lineages, such as T-bet and Rorγt, indicating that they were committed to the ILC2 lineage ( Figure 2c). Both aged and young lung ILC2 expressed KLRG1, verifying that they are mature ILC2 ( Figure S2a). Intravenous labeling of anti-CD45.2 antibody indicated that lung ILC2 from both young and aged mice are noncirculating, tissue-resident cells ( Figure S2b).
Previous work has established that mature ILC2 utilize fatty acid metabolism for maintenance and function (Wilhelm et al., 2016).
Interestingly, the cell size of aged lung ILC2 was smaller than those of young lung ILC2 (Figure 2e,f), and their fatty acid uptake was markedly reduced (Figure 2g,h), indicating reduced cellular metabolism. Neither young nor aged lung ILC2 took up significant amounts of glucose, arguing against a switch from fatty acid metabolism to glucose metabolism ( Figure S2c). Previous studies indicate that mature lung ILC2 in healthy young mice constantly produce IL-5 and amphiregulin that restrict airway inflammation and protect epithelial barrier integrity (Califano et al., 2018;Guo et al., 2018;Monticelli et al., 2011). We detected homeostatic production of IL-5 and amphiregulin by lung ILC2 in young mice using intracellular staining, without PMA/ionomycin re-stimulation (Figure 2i-l). Such homeostatic cytokine production was abolished in aged lung ILC2, indicating a loss of physiologic function of lung ILC2 with aging (Figure 2i-l).
Production of IL-13, another known ILC2 product, was undetectable in either young or aged lung ILC2 (not shown). Together, aging leads to reduced cellularity and functional activity of mature lung ILC2.

| BM ILC2P are unable to replenish mature ILC2 in the lungs of old mice
We sought to understand why aging is paradoxically associated with increased early ILC2 development in the BM but reduced cellularity of mature ILC2 in the lung. We first examined the maturation capability of BM ILC2P in aged mice using established OP9 co-culture assay (Hoyler et al., 2012). ILC2P sorted from the BM of aged mice differentiated into KLRG1 + mature ILC2 on OP9 stroma at a comparable efficiency as young ILC2P, indicating that the maturation capability of BM ILC2P does not decline with aging ( Figure 3a,b).
In addition, we detected both KLRGimmature ILC2P and KLRG1 + mature ILC2 in the peripheral blood of young and old mice, suggesting that active ILC2 maturation occurs in the circulation (Figure 3c).
F I G U R E 1 Aging enhances early ILC2 development in the bone marrow. (a) Representative flow cytometry profile of common helper innate lymphoid cells (CHILP, Lin -IL7R + α4β7 + Thy1 + PLZF + ) in the bone marrow of young (2-3 months) and old (19-24 months) mice. Plots were pregated on Lin -IL7R + α4β7 + cells. (b) Number of CHILP from bone marrow of young and old mice. (c) Representative flow cytometry profile of ILC2P (Lin -CD25 + Sca-1 + ) from the bone marrow of young and old mice. Plots were pregated on Lincells. (d) Number of ILC2P cell from bone marrow of young and old mice. (e) Mean fluorescence intensity (MFI) of IL-33R of ILC2P cells. (f) MFI of IL-33R from old mice treated with isotype controls or αNotch1 and αNotch2 antibodies. (g) Number of ILC2P from young and old mice treated with isotype controls or αNotch1 and αNotch2 for 3 days. Data are from 3 to 6 mice per group; *, p < 0.05; **, p < 0.01; ***, p < 0.001 The numbers of both immature KLRG -ILC2P and mature KLRG1 + ILC2 were elevated with aging, which might reflect increased output from the BM (Figure 3c). The ratio of circulating KLRG1 + mature ILC2 versus KLRG1immature ILC2P was comparable between young and old mice, verifying that the in vivo maturation of ILC2P is not impaired with aging ( Figure 3d). Together, these data indicate that the ILC2P in aged mice do not exhibit maturation defects and that circulating mature ILC2 might be constantly replenished by immature precursors.
We further explored potential destination of aged BM ILC2P.

| Aged lung ILC2 display remarkable transcriptomic heterogeneity and have reduced expression of peroxisomal and cytochrome P450 enzyme genes that are required for optimal ILC2 function
We next compared the transcriptomes of lung-resident ILC2 in Of note, treatment with anti-Notch1 and anti-Notch2 neutralizing antibodies did not affect the numbers of mature ILC2 in the lungs of old mice ( Figure S4). Thus, Notch signaling specifically promotes aging-associated accumulation of BM ILC2P, but does not affect the maintenance of mature ILC2 in the lungs of aged mice. In addition, the expressions of p16 and p16-interacting proteins were undetectable in either young or aged lung ILC2, suggesting that p16driven cellular senescence pathways might be repressed in lung ILC2 ( Figure 4d). Aged lung ILC2 also had reduced expression of fatty acid transport gene Fabp4, which might explain the decreased fatty acid uptake by ILC2 with aging ( Figure 4d). The expression of known F I G U R E 3 Bone marrow (BM) and circulating ILC2 maturation do not decline with aging. (a) Sort-purified BM ILC2P were co-cultured with OP9 stroma in the presence of 10 ng/ml IL-2, IL-7, SCF, and IL-33. Shown are representative flow cytometry plots of KLRG1 + mature ILC2 that emerged at day 10 after culture. (b) Percentage of KLRG1 + ILC2 that appeared was examined at day 10 after OP9-coculture. (c) Number of immature ILC2 precursor (Lin -Thy1 + T1/ST2 + KLRG1 -) and mature ILC2 (Lin -Thy1 + T1/ST2 + KLRG1 + ) in the peripheral blood of young and old mice. (d) Percentage of KLRG1 + and KLRG1cells among total ILC2 in the peripheral blood of young and old mice. (e) Number of ILC2 in the small intestinal lamina propria (SILP) of young and old mice. (f) ILC2P from old mice were injected intravenously into Rag2 -/-Il2rg -/mice, and recipient mice were sacrificed 4 weeks later. Representative flow cytometry plot of mature ILC2 in lungs and SILP of recipient mice with or without ILC2P transfer. (g) Number of ILC2 in the SILP or lung of recipient mice. Data are from 3 to 5 mice per group; *, p < 0.05; **, p < 0.01 transcriptional regulators of ILC2, such as Tcf7, Bcl11b, and Gfi1, was not reduced with aging ( Figure 4d).

| Increased levels of IL-18 and IL-12 in the aged lungs inhibit Cyp2e1 expression and repress mature ILC2 function
Innate lymphoid cells are long-lived cells, and thus, cell-intrinsic mechanisms likely play a significant role in ILC aging (Lowery et al., 2013;Nussbaum et al., 2013). Nevertheless, aging also induces complex changes to the lung microenvironment (Lowery et al., 2013). We thus determined whether microenvironmental factors in the aged lungs might also contribute to ILC2 aging. development and maintenance is likely due to hematopoietic cellintrinsic mechanisms or irradiation-sensitive factors (Figure 5a).
Donor-derived mature lung ILC2 expressed comparable levels of GATA3 in young and old recipient, suggesting that the reduced GATA3 expression in aged lung ILC2 might also be due to cell-intrinsic mechanisms (Figure 5c). However, donor young ILC2 still failed to efficiently produce IL-5 in the lungs of aged recipient mice, indicating potential involvement of irradiation-resistant microenvironmental factors (Figure 5d). IL-33, TSLP, and IL-25 are important activators of ILC2 (Artis & Spits, 2015), but their concentrations in the lungs were not reduced with aging ( Figure   S5). Using ELISA and multiplex assays, we noted aged lungs have increased concentrations of IL-18 and IL-12, two pro-inflammatory cytokines that may regulate mature ILC2 activity (Figure 5e). The levels of IFNγ, another known regulator of ILC2, remained comparable between young and aged lungs at homeostasis (Figure 5e).
Notably, inhibition of IL-18 and IL-12 by neutralizing antibodies enhanced IL-5 production by donor-derived lung ILC2 in old recipient mice, indicating that increased amounts of IL-12 and IL-18 contribute to decreased functional activity of mature lung ILC2 with aging ( Figure 5f). Anti-IL-18 and anti-IL-12 treatment did not affect the expression of GATA3 (Figure 5g), but increased the expression of the cytochrome P450 oxidase Cyp2e1 (Figure 5h). Because Cyp2e1 is required for optimal ILC2 function (Figure 4f), increased IL-18

| Transfer of activated young ILC2 enhanced resistance to influenza infection in old mice
Influenza infection in the elderly remains a severe public health threat (Wilhelm, 2018). We thus examined the effects of aging on ILC2 responses during influenza infection. Infection with 340 PFU of influenza A H1N1 Cal/04/09 (H1N1 CA04) virus resulted in 100% lethality in old mice, while all young mice survived this dose (Figure 6a).
Hence, old mice were more susceptible to influenza A virus (IAV) infection, mimicking human responses to influenza. The number of lung ILC2 in infected old mice was fewer than those in infected young mice, at 6 days after H1N1 CA04 infection (Figure 6b). Fatty acid uptake and IL-5 production remained lower in aged lung ILC2 after influenza infection than those in young lung ILC2 (Figure 6c,d).
Thus, lung ILC2 in aged mice remain numerically and functionally compromised during influenza infection.
We next determined whether transfer of young ILC2 into old mice might enhance their resistance to influenza infection. We obtained activated ILC2 from the lungs of IL-33-treated young mice and transferred 2 × 10 5 cells into old mice intravenously. As we de- Viral burdens, however, remained similar in the lungs of old mice with or without young ILC2 transfer, indicating that the protective function of ILC2 might be independent of viral clearance (Figure 6j).
Lung inflammation was markedly alleviated in mice that received young ILC2 transfer, suggesting that the improved survival might be associated with reduced inflammation (Figure 6k). Together, transfer of activated young lung ILC2 enhanced resistance to influenza in old mice.

| D ISCUSS I ON
The present work reveals compartmentalized effects of aging on precursors might lead to a strong bias toward the ILC2 lineages during later developmental stages. Nevertheless, the newly generated ILC2 and immature precursors might not be able to settle in the lungs to replenish the concomitantly declining mature lung ILC2 pool, resulting in futile developmental efforts. Thus, strategies to enhance precursor homing into the lungs might hold promises to enhance resistance to infections and diseases in the aged population, by allowing replenishment of newly generated "young" ILC2 in the aged lungs.
Not only do aging-related changes in early ILC2 development differ from those in adaptive lymphocyte development, but aged mature lung ILC2 also exhibit many unique characteristics that differ from the aging of other cell types. Aging or senescence has been associated with enlarged cell size, enhanced lipid peroxidation, and senescence-associated secretory phenotype in other cell types (Biran et al., 2017;Coppe, Desprez, Krtolica, & Campisi, 2010;Flor, Wolfgeher, Wu, & Kron, 2017;Petursdottir, Farr, Morley, Banks, & Skuladottir, 2007). However, these phenotypes have not been observed in aged lung ILC2. In contrast, aged lung ILC2 are smaller in size and have reduced cytokine secretion capability. In addition, aged lung ILC2 have reduced fatty acid uptake and decreased expression of peroxisomal and cytochrome p450 enzymes. Finally, the expression of p16 is not upregulated in aged lung ILC2, suggesting that cellular senescence pathways are not activated with aging in mature lung ILC2. Because lung ILC2 are long-lived slow-cycling cells, it is possible that p16-driven cellular senescence pathways have been repressed in these cells and that other aging-related mechanisms might play predominant roles. Together, our data indicate that unique cellular and molecular pathways underlie the aging of long-lived tissue-resident innate lymphocytes, likely due to the combined effects of multiple cell-intrinsic mechanisms and microenvironmental factors.
Our data also identify a novel role for the cytochrome oxidase Cyp2e1 in promoting lung ILC2 function. Cyp2e1 is a major liver cytochrome P450 oxidase involved in drug metabolism and ω-1 hydroxylation of fatty acid (Porubsky, Meneely, & Scott, 2008). The Th2 cytokine IL-4 can induce the expression of Cyp2e1 in hepatocytes during acute liver injury (Abdel-Razzak et al., 1993). Cyp2e1 is also expressed in lymphocytes of human diabetes patients (Hannon-Fletcher, O'Kane, Moles, Barnett, & Barnett, 2001). Cyp2e1 may promote the proliferation of CD4 T-cell proliferation and the activity of CD8 + CD57 + cytotoxic T cells (Griffin, Gilbert, & Pumford, 2000;Seth et al., 2014). But the precise role of Cyp2e1 in lymphocyte function remains unknown. Interestingly, our RNA-seq data indicate that lung ILC2 in young mice express high amounts of Cyp2e1 and other cytochrome P450 genes, indicating high activity of cytochrome P450. CRISPR-mediated gene knockout technique revealed that Cyp2e1 is required for optimal ILC2 function. More importantly, aging increases the levels of pro-inflammatory cytokines IL-12 and IL-18 that repress Cyp2e1 expression in tissue-resident ILC2, leading to reduced ILC2 function. Together, these data indicate that altered cytochrome P450 activity is associated with the aging of tissue-resident innate lymphocytes.
Respiratory and other infections remain severe threats in the aged population. More than 70% of influenza-related deaths occur among people over 65 years old (Wilhelm, 2018). ILC2, the predominant ILC subset in the lung, have been implicated in host defense against influenza infection in several previous studies (Califano et al., 2018;Gorski, Hahn, & Braciale, 2013;Li et al., 2018;Monticelli et al., 2011). But the precise role of ILC2 in pulmonary homeostasis and airway infections remains to be delineated. In this study, we have provided direct evidence that transfer of activated young ILC2 can enhance resistance to influenza infection in old mice. The protective function of ILC2 is independent of viral clearance, but is likely associated with alleviated airway inflammation and injury. Of note, aging is also associated with well-documented changes in other immune cell types. In particular, naive T and B cells are diminished with aged mice and humans, whereas memory and memory-like T and B lymphocytes with dysregulated function accumulate with aging (Fukushima, Minato, & Hattori, 2018;Hao, O'Neill, Naradikian, Scholz, & Cancro, 2011). Whether and how the aging-associated changes in adaptive immunity may influence ILC2 activity warrants future investigation. Understanding mechanisms of innate lymphoid cell aging might suggest new opportunities to enhance resistance to infections and diseases in the elderly.

| ILC2P adoptive transfer
For ILC2P adoptive transfer, 10,000 ILC2P were sorted from bone marrow of old mice and injected intravenously into nonirradiated Rag2 -/-Il2rg -/mice. ILC2 reconstitution in lung and SILP was examined at 4 weeks post-transfer.

| Isolation of hematopoietic cells from the lungs and small intestinal lamina propria
Lungs were harvested after perfusion with 10 ml of cold PBS.
Tissues were minced using scissors and digested in Hank's balanced salt solution containing 0.2 mg/ml Liberase TM (Roche) and 0.1 mg/ml DNase I (Roche) for 30 min at 37°C. Single-cell suspension was prepared by passing the tissue through an 70-μM cell strainer. SILP lymphocytes were isolated as we described previously (Spencer et al., 2014;Yang et al., 2015). Briefly, tissues were incubated with an epithelial strip buffer (alpha-MEM medium with 1 mM EDTA, 1 mM DTT, and 5% FCS) for 30 min at 37°C, followed by digestion with 10 ml liberase TM solution for another 20 min.

| Flow cytometry and cell sorting
Antibodies (

| CRISPR-mediated gene knockout
LentiCRISPRv2GFP was a gift from David Feldser (Addgene plasmid # 82416). Guide RNA (gRNA)-encoding sequences were cloned into LentiCRISPRv2GFP vectors to achieve gene knockout effects as described (Sanjana, Shalem, & Zhang, 2014 pM2.G. Mature ILC2 were cultured for 7 days with 10 ng/ml of IL-2, IL-7, and IL-33, and transduced with the indicated CRISPRv2GFP lentivirus by spin infection at 1,000 g for 1.5 hr as we previously described (Yang et al., 2013). Cytokine production was examined by intracellular staining after 7 days post-transduction.
Cells were isolated and stained with surface markers followed by flow cytometric analysis for examination of BODIPY and 2-NBDG uptake.

| Reverse transcription-quantitative PCR (RT-qPCR)
For RT-qPCR analysis, mRNA was purified from sorted young and old lung ILC2 using TRIzol LS (Thermo) or RNeasy Plus Mini Kit (Qiagen) according to manufacturer's protocol. cDNA was generated using SuperScript II Reverse Transcriptase (Thermo).

| Statistics
Difference between two groups was examined by two-tailed Student's t tests. Difference between more than two groups was examined by one-way ANOVA. Survival rates between groups were assessed by log-rank (Mantel-Cox) test. p < 0.05 was considered significant.

ACK N OWLED G EM ENTS
This work was supported by the U.S. National Institutes of Health Grants R01HL137813, R01AG057782, K22AI116728 (to Q. Yang), and HL140496 (to D.W.Metzger), and the Alexandrine and Alexander L. Sinsheimer Scholar Award (to Q. Yang).

CO N FLI C T O F I NTE R E S T S
The authors declare no competing financial interest.