Type I Interferon signaling controls the accumulation and transcriptomes of monocytes in the aged lung

Abstract Aging is paradoxically associated with a deteriorated immune defense (immunosenescence) and increased basal levels of tissue inflammation (inflammaging). The lung is particularly sensitive to the effects of aging. The immune cell mechanisms underlying physiological lung aging remain poorly understood. Here we reveal that aging leads to increased interferon signaling and elevated concentrations of chemokines in the lung, which is associated with infiltration of monocytes into the lung parenchyma. scRNA‐seq identified a novel Type‐1 interferon signaling dependent monocyte subset (MO‐ifn) that upregulated IFNAR1 expression and exhibited greater transcriptomal changes with aging than the other monocytes. Blockade of type‐1 interferon signaling by treatment with anti‐IFNAR1 neutralizing antibodies rapidly ablated MO‐ifn cells. Treatment with anti‐IFNAR1 antibodies also reduced airway chemokine concentrations and repressed the accumulation of the overall monocyte population in the parenchyma of the aged lung. Together, our work suggests that physiological aging is associated with increased basal level of airway monocyte infiltration and inflammation in part due to elevated type‐1 interferon signaling.

Interferons (IFN) are important drivers of immune defenses. IFNs are best known for their pivotal role in anti-viral responses (Makris et al., 2017;McNab et al., 2015;Platanias, 2005). Viral infections induce activation of IFN signaling which stimulates expression of interferonstimulated genes with anti-viral functions (Makris et al., 2017;McNab et al., 2015;Schneider et al., 2014;Schoggins, 2014). IFNs play important roles in initiating inflammatory responses during airway infections and inflammatory disorders (Goritzka et al., 2014(Goritzka et al., , 2015Lee et al., 2017;Lin et al., 2014;Stetson & Medzhitov, 2006). Despite its potent role in host defense, activation of IFN signaling is also associated with collateral tissue damage and susceptibility to tissue inflammation (Baruch et al., 2014). Previous reports indicate that Type 1 interferon may control monocyte abundance and activity in systemic and tissue infection and inflammation (Channappanavar et al., 2016;Lee et al., 2009Lee et al., , 2017Seo et al., 2011). The role of Type 1 IFN signaling in the aging of homeostatic lungs, however, remains largely unknown.
In this study, we examined the effects of physiological aging on the abundance and gene expression profiles of monocytes in lungs during homeostasis, as well as the role of IFNs in controlling monocyte abundance and transcriptomes in the aged lung. We reveal that aging leads to monocyte infiltration into the lung parenchyma, which is associated with elevated concentrations of chemokines and a global increase in IFN signaling in the lung. A novel subset of monocytes expressed very high levels of interferon-stimulated genes, exhibited the greatest transcriptomal changes with aging, and was nearly ablated by in vivo blockade of type 1 interferon. Blockade of Type 1 interferon signaling also reduced the concentrations of chemokines and repressed the accumulation of the overall monocyte population in the lung parenchyma of aged mice. Together, our data suggest that monocyte infiltration and inflammation is a hallmark of physiological lung aging and that type-1 IFN might control the abundance and transcriptomes of monocytes in the aged lung via multiple layers of direct and indirect mechanisms.

| Aging is associated with increased interferon signaling, elevated concentrations of chemokines in the lung
To explore mechanisms of lung aging, we performed genome-wide microarray analyses of the whole lung tissue from young (2-3 months old) and aged (20-22 months old) mice ( Figure 1a). Mice were obtained from NIH via Charles River and rested in the SPF facility of Albany Medical College (AMC) for at least two weeks before experiments. Female mice were housed in regular Allentown cages, with 4-5 mice per cage. Aged male mice exhibited excessive aggressiveness and were individually housed. Female mice were used for the majority of this study due to concerns about potential confounding factors induced by stress and individual housing, but major findings were verified with male mice.
Interestingly, Gene Set Enrichment Analysis (GSEA) revealed that genes that were upregulated with aging were enriched for interferon response genes and chemokines ( Figure 1b). Specifically, aging was associated with elevated expression of many interferon-stimulated genes (ISG) in the lung (Figure 1c). Thus, aging is associated with global increase of interferon signaling in the lung.
The expression of a variety of chemokine genes in the lung was also significantly increased with aging ( Figure 1c). Multiplex cytokine assays and ELISAs verified that the concentration of many chemokines, including CCL2, CCL5, CCL20, CXCL10, CCL3, CCL4, Eotaxin, and CXCL9, were extensively increased in the lung homogenate with aging ( Figure 1d). Serum chemokine levels were also increased with aging ( Figure S1). However, the concentrations of serum chemokines were generally lower than those in the lung ( Figure S1). Together, aging is associated with elevated concentrations of chemokines in the lung. F I G U R E 1 Increased Type I Interferon signaling and chemokines in lungs of aged mice. (a) Principle component analysis of microarray data from total lung cells of young (gray) and aged (orange) female mice. (b) Significant gene sets based on Gene Set Enrichment Analyses (GSEA) of microarray data from lungs of young and aged mice. (c) Heatmap depicting the expression of representative genes in young and aged mouse lungs. p value <0.05 for all gene shown in the heatmap. (d) Chemokine concentrations from the lung homogenates of naïve young and aged mice. Data are from 4-5 mice per group. (e) Representative flow cytometry plot of monocytes (CD45 + Gr1 + CD11b + Ly6C + ) and neutrophils (CD45 + Gr1 + CD11b + Ly6G + ) from lungs of young and aged mice. (f) Number of monocytes and neutrophils in lungs of young and aged mice. (g) Number of monocytes in lungs of young and aged mice. (h) Geometric mean fluorescence intensity of Ki67 staining of monocytes and neutrophils in lungs of young and aged mice. (i) Percentage of activated caspase + monocytes and neutrophils in lungs of young and aged female mice. (j) Representative immunofluorescence staining of monocytes (Ly6C + ) and vascular endothelial cells (CD31 + ) in the lungs of young and aged mice. (i) Distance between monocytes and vascular endothelium. Data are from female mice (a-k). Data are from 3 mice per group (a-c), or are from 4-6 mice per group, two independent experiments (d-k). Error bars are mean ± SD. *, p < 0.05; **, p < 0.01

| Aging leads to monocyte infiltration in the lung parenchyma
Many of these chemokines have established roles in inducing tissue infiltration of myeloid cells such as monocytes or neutrophils (Charo & Ransohoff, 2006;Ichikawa et al., 2013;Keophiphath et al., 2010;Lin et al., 2014;Schaller et al., 2017;Zhao et al., 2017). We thus examined whether aging is associated increased infiltration of monocytes or neutrophils in the lung. Specifically, we examined the numbers of Ly6C + monocytes and Ly6G + neutrophils, two major subsets of airway infiltrating myeloid cells, in the lungs of young and aged mice. To remove blood cells from lungs, mice were extensively perfused with 50 ml of PBS and the lungs were visibly white before collection. Notably, the numbers of pulmonary Ly6C + monocytes, but not Ly6G + neutrophils, were significantly increased with aging ( Figure 2a, b). We did not observe a significant difference in the numbers of monocytes in the spleen between young and aged mice, suggesting that aging-associated accumulation of monocytes is tissue specific ( Figure 2c).
Of note, Ki67 staining and activated caspase assays revealed that monocytes in aged lungs exhibited comparable proliferation and apoptosis rates as those in young mice (Figure 2d, e). Thus, other mechanisms, such as altered cell migration, might contribute to monocyte accumulation in the aged lung.
While the above studies were performed with female mice, we observed similarly increased abundance of monocytes with aging in male mice ( Figure S2a). Thus, aging leads to monocyte accumulation in the lung in both sexes.
We next performed immunofluorescence staining to examine the micro-anatomic locations of monocytes in the lung. In young mice, monocytes were largely restricted to the luminal side of vessels in the lung (Figure 1j, k). Strikingly, the majority of monocytes infiltrated to the lung parenchyma in aged mice (Figure 1j, k). Thus, aging is associated with monocyte infiltration into the lung parenchyma.

| scRNA-seq identified a novel monocyte subset that expressed high levels of interferonstimulated genes and exhibited greatest transcriptomal changes with aging
We then performed single-cell RNA sequencing (scRNA-seq) to examine the effects of aging on the gene expression profiles of pulmonary Ly6C hi monocytes and Ly6G + neutrophils ( Figure S3a). scRNA-seq detected 25 genes that were differentially expressed in pulmonary monocytes between young and aged mice, and 17 genes that were differentially expressed in neutrophils with age ( Figure   S3b). Of note, the majority (22/25) of differentially expressed genes (DEGs) in monocytes was upregulated with aging ( Figure S3b). The genes upregulated with aging in monocytes were enriched for genes associated with monocyte activation and inflammation. They include the monocyte activation marker Wfdc17, proinflammatory cytotoxic molecules Ctsc and Ctsd, proinflammatory cytokine Il1b and other proinflammatory secreted proteins Prtn3, Plin2 and Cfh ( Figure S3c, d) (Calippe et al., 2017;Dey et al., 2018;Dinarello, 1996;Hamon et al., 2016;Karlstetter et al., 2010;Kessenbrock et al., 2008;Korkmaz et al., 2018;Norman et al., 2018). The few genes down-

| Blockade of type 1 Interferon signaling reduced monocyte accumulation in the lung parenchyma of aged mice
We next examined whether increased interferon signaling contributed to aging-associated monocyte changes in the aged lung. We treated aged mice with anti-IFNAR1 or anti-IFNγ neutralizing antibodies every other day for 1 week. Anti-IFNAR1 blocks Type1 interferon signaling, whereas anti-IFNγ blocks IFNγ signaling. Notably, treatment with anti-IFNAR1, but not anti-IFNγ, repressed monocyte accumulation in the aged lung (Figure 3a, b). Neutrophil numbers remained unaffected by anti-IFNAR1 or anti-IFNγ treatment ( Figure   S4a). Notably, the number of pulmonary monocytes exhibited a decreasing trend as early as 24 hrs after a single dose of anti-IFNAR1, and the decrease became more evident after 1 week of treatment ( Figure S4b). Of note, anti-IFNAR1 treatment reduced monocyte accumulation in the lung parenchyma, but did not significantly alter the micro-anatomic location of monocytes (Figure 3c-e). While the above experiments were performed with female mice, we observed that anti-IFNAR1 treatment similarly reduced monocyte numbers in the lung of aged male mice ( Figure S2b). Together, Type 1 interferon signaling promotes monocyte accumulation in the lung parenchyma.
We next explored the mechanisms that contribute to enhanced interferon signaling in the aged lung. The concentrations of IFNα and IFNβ in the lung were not significantly increased with age, suggesting that other mechanisms might lead to increased interferon signaling in the aged lung ( Figure 3g). Because T cells are a significant source of chemokines such as CCL4, we examined whether aging might lead to increased expression of type-1 interferon receptors in pulmonary T cells (Castellino et al., 2006). Notably, mRNA expression of both Ifnar1 and Ifnar2 increased in lung CD4 + cells, but not CD8 + T cells, with aging ( Figure 3h). Pulmonary CD4 + T cells dramatically upregulated Ccl4 expression with age ( Figure 3i). Pulmonary CD8 + T cells also upregulated Ccl4 expression with age; however, the expression of Ccl4 was much higher in CD4 + cells than in CD8 + T cells in aged mice ( Figure 3i). Treatment with anti-IFNAR1 markedly reduced the expression of Ccl4 in CD4 + cells, but not in CD8 + T cells in the aged lung ( Figure 3j). Together, our results suggest a model in which aging results in increased expression of type 1 interferon receptors that enhances chemokine production by pulmonary CD4 + T cells, which may in turns promote monocyte infiltration in the lung.

| The effects of type 1 Interferon signaling blockade on the transcriptomes of monocytes in the aged lung
We examined whether neutralization of Type 1 interferon may alter the gene expression profile of monocytes in the aged lung.
We treated aged mice with anti-IFNAR1 antibody and performed scRNA-seq with FACS-sorted Ly6C + monocytes and Ly6G + neutrophils after 24 hrs or 1 week of treatment ( Figure 4a). We used the anti-IFNAR1 antibody clone MAR1-5A3, because previous work establishes that MR1-5A3 specifically neutralizes IFNAR1 bioactivity without inducing antibody-dependent cellular cytotoxicity (ADCC) effects (Channappanavar et al., 2016;Diamond et al., 2011;Sheehan et al., 2006). We included the time point of 24 hours post-treatment here, because cells most sensitive to anti-IFNAR1 treatment were

| The specific chemokines regulated by Type 1 interferon during influenza infection differ from the chemokines regulated by Type 1 interferon in physiological aging
We next examined monocyte responses to influenza infection in aged mice and the specific roles of Type 1 interferon in regulating influenza-induced monocyte responses in the aged lung. Massive monocyte infiltration was observed in aged mice at day 6 postinfection of the CA/04 Influenza A Virus (IAV) strain (Figure 6a, b).
The numbers of monocytes in aged infected mice were comparable to those in young infected mice (Figure 6a, b). Anti-IFNAR1 treatment repressed monocyte infiltration in the lungs of IAV-infected aged mice (Figure 6b). Consistent with previous reports, anti-IFNAR1 treatment also inhibited monocyte infiltration in the lungs of IAVinfected young mice (Figure 6b) (Lee et al., 2009;Seo et al., 2011).
Of note, anti-IFNAR1 treatment appeared to induce even greater effects on pulmonary monocytes in IAV-infected aged mice than those in infected young mice, because the numbers of monocytes in the lungs of anti-IFNAR1 treated and IAV-infected aged mice were even lower than those in young mice (Figure 6b). Neutrophils also infiltrated in both young and aged mice during influenza infection, but their numbers were unaltered by anti-IFNAR1 treatment (Figure 6c).
We then examined chemokine concentrations in IAV-infected mice. Influenza infection leads to drastically elevated CCL2 concentration in the lungs of both young and aged mice, and anti-IFNAR1 treatment repressed lung CCL2 concentrations in both young and aged mice (Figure 6d). Of note, CCL2 concentration was also increased with aging at homeostasis in naïve mice, but its concentration was not altered by anti-IFNAR1 treatment at homeostasis (Figure 3f). In addition, the chemokines that were regulated by Type-1 interferon at homeostasis, such as CCL3, CCL4 and CCL20, were not significantly changed by anti-IFNAR1 treatment in IAV-infected mice (Figure 6d). Thus, although Type 1 interferon promoted both IAV and aging-induced pulmonary monocyte infiltration, the specific chemokines regulated by Type 1 interferon during influenza infection differ from the chemokines regulated by Type 1 interferon in physiological aging.
Treatment with anti-IFNAR1 antibodies did not significantly change the survival or weight loss of aged mice in IAV infection ( Figure S6). Of note, type-1 interferon affects many cell types and molecular pathways. Future research with aged transgenic mice that specifically lack interferon signaling in monocytes might be needed, in order to understand the precise role of monocyte-intrinsic interferon signaling in influencing tissue inflammation and host defense during IAV infection.

| DISCUSS ION
Our results together reveal that aging is associated with increased basal levels of monocyte inflammation in the lung partly via Type 1 interferon dependent mechanisms. Our data indicate that type

| Mice
Young (2-3 month) and aged (20-22 month) female C57BL/6 mice were obtained from the National Institute of Aging via Charles River.
All animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee at Albany Medical Center. 3.1.7 | Single-cell RNA-sequencing and microarray analysis

| Antibody treatments
For single-cell RNA sequencing, monocytes and neutrophils of young and aged mice, Ly6C + monocytes and Ly6G + neutrophils were sorted from lungs of mice using TotalSeq C0951 antibody (Biolegend) to label cells from young mice or TotalSeq-C0952 (Biolegend) to label cells from aged mice. For scRNA-seq of monocytes and neutrophils from aged mice treated with anti-IFNAR1, aged mice were treated with isotype control once or with anti-IFNAR1 antibody (500 μg, intraperitoneally) once or every other day for a week (a total of 4 treatments). Mice were sacrificed the day after the last injection. Ly6C + monocytes and Ly6G + neutrophils were sorted by FACS. scRNA-seq was performed as we previously described Harly et al., 2019;Zhang et al., 2020). scRNA-seq libraries were generated using the chromium 5' single-cell gene expression kit (10x genomics) according to the manufacturer's instructions. Sequencing was carried out using a NextSeq 500 (Illumina). Preliminary data analysis was carried out using Cellranger V3.0.2. Data were normalized and scaled using Seurat (Stuart et al., 2019). Cells were clustered by Uniform Manifold Approximation and Projection (UMAP) function in Seurat.
Differentially expressed genes were identified based on Seurat's nor-

| Influenza infection
For Influenza infection, mice were infected with 340 PFU of Influenza A virus (CA04 strain), administered via the intranasal route.
Mice were treated with anti-IFNAR1 or isotype control antibodies (500 μg i.p.) every other day, starting from the day before infection, up to day 5 post-infection. Monocyte responses were measured at day 6 post-infection.

| Statistical analysis
Differences between two groups was examined using two-tailed Student's t tests. Differences between more than two groups was analyzed using one-way ANOVA. p<0.05 was considered significant.
Wilcoxon rank sum test was used to test the statistical significance of differentially expressed genes by scRNA-seq.

ACK N OWLED G EM ENTS
This work was supported by the U.S. National Institutes of Health Grants R01HL137813, R01AG057782, and R01HL155021 (to Q. Yang).

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

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available in GEO at https://www.ncbi.nlm.nih.gov/geo/ reference number GSE16 7236 and GSE16 7170 and GSE16 7215.