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. 2019 Oct 15;203(8):2252-2264.
doi: 10.4049/jimmunol.1900495. Epub 2019 Sep 11.

Identification of an Increased Alveolar Macrophage Subpopulation in Old Mice That Displays Unique Inflammatory Characteristics and Is Permissive to Mycobacterium tuberculosis Infection

William P Lafuse  1 , Murugesan V S Rajaram  1 , Qian Wu  2 , Juan I Moliva  2   3 , Jordi B Torrelles  2   3 , Joanne Turner  2   3 , Larry S Schlesinger  2   3
Affiliations

Identification of an Increased Alveolar Macrophage Subpopulation in Old Mice That Displays Unique Inflammatory Characteristics and Is Permissive to Mycobacterium tuberculosis Infection

William P Lafuse et al. J Immunol. .

Abstract

The elderly population is more susceptible to pulmonary infections, including tuberculosis. In this article, we characterize the impact of aging on the phenotype of mouse alveolar macrophages (AMs) and their response to Mycobacterium tuberculosis. Uninfected AMs were isolated from bronchoalveolar lavage of young (3 mo) and old (18 mo) C57BL/6 mice. AMs from old mice expressed higher mRNA levels of CCL2, IFN-β, IL-10, IL-12p40, TNF-α, and MIF than young mice, and old mice contained higher levels of CCL2, IL-1β, IFN-β, and MIF in their alveolar lining fluid. We identified two distinct AM subpopulations, a major CD11c+ CD11b- population and a minor CD11c+ CD11b+ population; the latter was significantly increased in old mice (4-fold). Expression of CD206, TLR2, CD16/CD32, MHC class II, and CD86 was higher in CD11c+ CD11b+ AMs, and these cells expressed monocytic markers Ly6C, CX3CR1, and CD115, suggesting monocytic origin. Sorted CD11c+ CD11b+ AMs from old mice expressed higher mRNA levels of CCL2, IL-1β, and IL-6, whereas CD11c+ CD11b- AMs expressed higher mRNA levels of immune-regulatory cytokines IFN-β and IL-10. CD11c+ CD11b+ AMs phagocytosed significantly more M. tuberculosis, which expressed higher RNA levels of genes required for M. tuberculosis survival. Our studies identify two distinct AM populations in old mice: a resident population and an increased CD11c+ CD11b+ AM subpopulation expressing monocytic markers, a unique inflammatory signature, and enhanced M. tuberculosis phagocytosis and survival when compared with resident CD11c+ CD11b- AMs, which are more immune regulatory in nature.

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Conflict of interest statement

Disclosures

The authors have no financial conflicts of interest

Figures

FIGURE 1.
FIGURE 1.
Cytokine and Chemokine expression levels in ALF and AMs from young and old mice. mRNA Levels in the ALF of young and old mice (A). ALF was isolated from individual mice by BAL. Protein concentration was normalized to 0.20μg/μl and cytokine levels in 100 μl determined by ELISA. N= 30 young mice and 28 old mice. mRNA levels in AMs from young and old mice (B-D). RNA was isolated from AMs immediately after BAL and cytokine mRNA levels determined by qRT-PCR. mRNA levels were normalized to β-actin levels and cytokine and chemokine expression levels determined relative to levels in AMs from young mice. Data represent 3-5 experiments from young mice or old mice. For each experiment BAL-obtained AMs were pooled from 10 mice. * p<0.05, **p<0.01.
FIGURE 2.
FIGURE 2.
Expression of CD11b and CD11c is higher in AMs from old mice. AMs from young and old mice were freshly adhered to coverslips and expression of CD11b (A), CD11c (B), and mannose R (MR) (C) determined by confocal microscopy. Mean fluorescence intensity (MFI) was determined for 40-60 individual cells. Data are representative of N = 3 separate experiments. RNA was also isolated immediately after BAL isolation and mRNA expression of CD11b, CD11c, Mrc1 (mannose receptor), TLR2, TLR4, TLR1, TLR6, COX2, MHC class II Aα, CIITA, and IRF1 determined by qRT-PCR (D-F). mRNA levels were determined relative to levels in AMs from young mice. Data represent 3-5 experiments from young or old mice. For each experiment BAL-obtained AMs were pooled from 10 mice. * p<0.05, **p<0.01, *** p<0.001.
FIGURE 3.
FIGURE 3.
Percentage of C11c+ CD11b+ AMs is higher in old mice. Representative flow cytometry plots showing the major CD11c+ CD11b− and minor CD11c+ CD11b+ AM populations in young (A) and old mice (B). C-D. Graphs showing the percentage of AM populations from 6 young and 6 old mouse experiments. For each experiment BAL-obtained AMs were pooled from 10 mice. * p<0.05, **** p<0.0001
FIGURE 4.
FIGURE 4.
The CD11c+ CD11b+ AM population is in a more activated state with increased expression of key macrophage surface molecules involved in macrophage functions. A. Expression of macrophage surface molecules by CD11c+ CD11b+ and CD11c+ CD11b+ AM populations determined by flow cytometry. Data are expressed as net MFI (MFI of antibody MFI - isotype MFI). B. CD11c+ CD11b+ AMs express monocyte markers that are absent on CD11c+ CD11b− AMs. Data in A and B represent 6 young and 6 old mouse experiments. For each experiment BAL-obtained AMs were pooled from 10 mice. * p<0.05, **p<0.01, *** p<0.001.
FIGURE 5.
FIGURE 5.
AMs from young and old mice differ in cytokine mRNA expression after infection with M.tb. AMs isolated from young and old mice were cultured in 48 well culture plates for 2 h and then infected with M.tb (5:1bacteria/macrophage) for 6, 24, 48, and 72 h. A. RNA was isolated and cytokine mRNA expression determined by qRT-PCR. mRNA levels were normalized to β-actin levels and cytokine expression levels at each time point were determined relative to control non-treated AMs at each time point. B. Expression of CCL2 and TNFα in culture fluid from 24 h cultures of control and M.tb-infected AMs. Data represent 3–7 experiments for each time point. For each experiment BAL-obtained AMs were pooled from 10 mice. * p<0.05, **p<0.01, *** p<0.001.
FIGURE 6.
FIGURE 6.
CD11c+ CD11b+ and CD11c+ CD11b− AM subpopulations from old mice differ in mRNA expression of cell surface proteins, cytokines and COX2. AMs were isolated from 10 old mice by BAL and flow-sorted into CD11c+ CD11b− and CD11c+ CD11b+ AM subpopulations. RNA was isolated from the flow-sorted populations and gene expression determined by qRT-PCR. Expression levels were normalized to β-actin and expression levels in the CD11c+ CD11b+ AMs were determined relative to the CD11c+ CD11b− AMs. Relative expression levels in CD11c+ CD11b+ AMs >1 indicate preferential expression in the CD11c+ CD11b+ AMs, while relative expression levels in CD11c+ CD11b+ AMs < 1 indicate preferential expression in the CD11c+ CD11b− AMs. mRNA levels of CD11b (A), CD11c, CD80 and CD86 (B), TLR1 and FcγR1 (C), CCL2, IL-1β, IL-6, COX2 and TNFα (D), IFN-β and IL-10 (E), TLR2, TLR4, TLR6, and MHC class II Aα (F) are shown. Cumulative data from 3 experiments are shown. * p<0.05, **p<0.01.
FIGURE 7.
FIGURE 7.
CD11c+ CD11b+ AMs phagocytose significantly more M.tb and contain higher numbers of M.tb at 24 h than CD11c+ CD11b− AMs. AMs were isolated from 10 old mice by BAL and flow-sorted into CD11c+ CD11b− and CD11c+ CD11b+ AM populations. The flow-sorted populations were cultured on coverslips and infected with mCherry-M.tb H37Rv at 5:1 bacteria/macrophage. M.tb present in AM populations was determined by confocal microscopy. Results are representative of N = 2 experiments. A. Mean number of M.tb/ macrophage at 2 and 24 h. B. Distribution of M.tb/macrophage. C. Representative confocal images; between 144–284 macrophages were analyzed in A and B. *** p<0.001.
FIGURE 8.
FIGURE 8.
M.tb gene expression is substantially higher in CD11c+ CD11b+ AMs infected with M.tb for 24 h. AMs were isolated from 10 old mice by BAL and flow-sorted into CD11c+ CD11b− and CD11c+ CD11b+ AM populations. The flow-sorted populations were infected with M.tb at 5:1 in 48 well culture plates for 24 h. RNA was isolated and M.tb 16S rRNA and M.tb mRNA levels determined by qRT-PCR. Expression levels of M.tb genes were determined relative to M.tb 16S rRNA was determined. The results are representative of two experiments in which two wells of CD11c+ CD11b− and one well of CD11c+ CD11b+ AMs were infected with M.tb.
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