Older adults are at greater risk of severe respiratory infection. This is likely to be a result of multiple factors including a decline in adaptive immunity, co-morbidities and age-related changes to the lung structure, microenvironment and innate immune response [3, 4]. The age-related changes to the innate response of resident lung cells are incompletely understood. Overall, our data demonstrate that aged mice produce a greater inflammatory response to lung LPS exposure than young mice, consisting of greater chemokine production by lung cells and enhanced neutrophil recruitment into the lung and airways. Resident cells from the lungs of aged mice, including AMs, epithelial and endothelial cells exhibited a more pro-inflammatory response following LPS challenge.

Consistent with our observation of a greater recruitment of neutrophils to the lungs in aged mice, higher neutrophil recruitment and chemokine expression has been observed in murine models of Influenza, Acinetobacter baumannii and Streptococcus pneumoniae infection [19, 28, 29]. However, age-related differences in pathogen load in these studies make interpretation of the results more complex, and none of these studies assess the early four-hour time point we study here. The use of a non-replicating stimulus, LPS, and the study of an early time point, allowed us to determine the response of resident cells of young and aged mice with an equal dose of LPS. There were higher levels of CXCL1, CXCL2 and CCL2 in the airways of aged compared to young mice in response to LPS at the time point studied. This suggests that aged mice produce these cytokines more rapidly and / or in greater quantities than young mice, however a time course study of the in vivo response would be needed to fully determine this. Although the chemokines CXCL1 and CXCL2 are important mediators of neutrophil recruitment to the lung, factors other than these chemokines could also promote neutrophil recruitment to the lungs in the aged mice.

The use of precision cut lung slices allowed us to study the dose response of resident lung cells from aged mice to LPS stimulation, without the potential contribution of recruited cells to the response. This method has the further advantages that it allows the retention of the structural organisation of lung tissue and the response of resident cells from the same sample can be determined at multiple time points and concentrations of stimulus. These studies demonstrated higher levels of expression and production of inflammatory cytokines from the resident lung cells of aged mice than those of the young. Interestingly, in response to LPS exposure, the lung slices from aged mice did not demonstrate a significantly higher production of CCL2, which was higher in the lungs of in vivo challenged mice at 4 h. This may be due the differences in the concentration of LPS used, the time point, or alternatively, recruited cells may be required for the exacerbated production of CCL2 in the aged lung. Similarly, Il1b and Tnf expression were significantly different ex vivo following LPS exposure of the PCLS, but measured levels of the proteins were not significantly different between young and aged mice in vivo. Again, this may reflect the concentration of LPS used and the time point, the regulation of the response by circulating cells or factors that are not fully represented ex vivo. Furthermore, the effects of the process of producing PCLS on cellular activity, and loss of barrier function, may also influence the nature of the response.

Individual lung resident populations were sorted by flow cytometry four hours after challenge with LPS to determine their differential responses in vivo with age. A higher expression of chemokines in AMs, epithelial and endothelial cells from aged mice was found. This demonstrates that multiple resident lung cells exhibit an enhanced inflammatory phenotype in vivo in aged mice. The differences in responses of resident cells could not be accounted for by increased Tlr4 or TLR4 signalling component expression in lung cells prior to stimulation with LPS, however, the levels of these proteins would need to be directly measured to rule this possibility out. This differs from reports of higher levels of A20 (Tnfaip3) in the aged lung [30].

We found no age-related differences in AM or epithelial cell cytokine expression at baseline. We further compared our results to published gene expression analyses in the unchallenged, aged lung and specifically assessed the expression of our genes of interest and extended this to other genes that could influence inflammatory responses in the lung, including TLR3, the pro-inflammatory cytokines IL-6, IL-18 and CCL3, and the regulatory cytokine Il-10 (Tnf, Il1b, Ccl2, Cxcl2, Cxcl1, Tlr3, Tlr4, Irak3, Irak4, Tnfaip3, Il6, Il18, Ccl3, Il10). In agreement with our findings, analysis by Angelidis et al. showed no significant differences in expression of these genes in resident cell populations in the lung of aged compared to young mice, with the exception of Tnf and Ccl3 expression which were modestly elevated in in silico identified aged AMs, analysed using scRNAseq, but not in FACS sorted macrophages analysed by bulk RNAseq (6). Of our genes of interest, others report higher Tnf, Il6, Cxcl1, Cxcl2, Ccl3 and Il1b, and lower Tlr4 and Il18 expression in bulk RNAseq analysis of FACS sorted aged AMs [8], higher Ccl2, Cxcl2, Il1b, Cxcl1 and Ccl3 in microarray analysis of FACS purified aged AMs [9] and higher Ccl2, Tnf and Il10 expression in PCR analysis of aged AMs isolated by lavage compared to AMs from young mice [14]. Differences between studies may derive from the age and housing conditions of the mice, cell isolation processes and gene expression methodology.

We find reduced numbers of AMs in the lungs with age which is consistent with reports of reduced self-renewal capacity in these cells with age [8, 9]. Ex vivo studies of responses of AMs from aged mice have been contradictory, with some reporting enhanced cytokine responses in these cells and others finding a reduced response with age [11,12,13]. To the best of our knowledge, this is the first study to investigate age-associated functional alterations in AMs following in vivo LPS challenge, and we find enhanced inflammatory cytokine production from these cells.

We report a greater inflammatory cytokine gene expression in lung epithelial cells 4 h following LPS challenge. Others have reported higher expression of cytokines and chemokines in AEC2 from aged mice challenged with LPS at 24 h, and 6 days after influenza virus infection [18, 19]. However, a lower response to house dust mite extract in tracheal epithelial cells from older mice stimulated ex vivo was also reported [31]. Age-related differences in the inflammatory response in epithelial cells may be influenced by the time point, the nature of the stimulant and the source and composition of epithelial cells studied [3]. We sorted all lung epithelial cells for this analysis, and there is potential heterogeneity within that population that may be altered with age [6]. Furthermore, during ageing, senescent cells accumulate in tissues. These arrested cells are characterised by secretion of the senescence associated secretory phenotype, which includes production of pro-inflammatory cytokines. They may contribute to the low-level chronic inflammation seen with age known as ‘inflammaging’ that may drive age-associated conditions, including those of the lung [3, 32,33,34,35]. The presence of a higher frequency of senescent epithelial cells in aged mice than in young [19, 36] may contribute to inflammation, either directly or indirectly by influencing the responses or phenotype of other resident cells and this warrants further study.

We also observe age-related differences in the response of lung endothelial cells in the aged mice. To our knowledge, this is the first study of how the cytokine and chemokine responses of lung endothelial cells are influenced by age and we find that they are more pro-inflammatory than the young. An impaired ability of aged lung endothelial cells to regenerate and promote resolution of inflammation in the lung was reported in mouse sepsis models [22]. As lung endothelial cells can regulate inflammatory responses in the lung during infection, alteration in their immune function with age is an area that merits further study [20, 21].

Although we have used LPS as a stimulant to mimic acute respiratory infection in our model, its receptor TLR4 and its ligands have been implicated in the chronic inflammation associated with ageing and with age-related diseases in other tissues [37, 38]. For example, signals from the microbiota may promote inflammation that increases myeloid cell differentiation of haematopoietic stem cells, which in turn promotes immunosenescence [39, 40]. TLR4 has also been implicated in age-related neurodegenerative diseases [38], adipose inflammation and poor glucose tolerance, and cardiovascular disease [41, 42]. Therefore, the changes to TLR4 mediated responses with age may have wider implications for lung ageing and age-related disease. Limitations of our study include the use of only a single sex, male, and single strain of mice, C57BL/6. Therefore, further work will be needed to determine the wider relevance of these results to aging and age-related diseases.