Identification of an Increased Alveolar Macrophage Subpopulation in Old Mice That Displays Unique Inflammatory Characteristics and Is Permissive to This information is current as Mycobacterium tuberculosis Infection of September 25, 2021. William P. Lafuse, Murugesan V. S. Rajaram, Qian Wu, Juan I. Moliva, Jordi B. Torrelles, Joanne Turner and Larry S. Schlesinger J Immunol published online 11 September 2019 Downloaded from http://www.jimmunol.org/content/early/2019/09/06/jimmun ol.1900495 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2019/09/06/jimmunol.190049 Material 5.DCSupplemental

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published September 11, 2019, doi:10.4049/jimmunol.1900495 The Journal of Immunology

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,* Juan I. Moliva,*,† Jordi B. Torrelles,*,† Joanne Turner,*,† and Larry S. Schlesinger*,†

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 Downloaded from expressed higher mRNA levels of CCL2, IFN-b, IL-10, IL-12p40, TNF-a, and MIF than young mice, and old mice contained higher levels of CCL2, IL-1b, IFN-b, and MIF in their alveolar lining fluid. We identified two distinct AM subpopulations, a major CD11c+ CD11b2 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 b + 2 mice expressed higher mRNA levels of CCL2, IL-1 , and IL-6, whereas CD11c CD11b AMs expressed higher mRNA levels of http://www.jimmunol.org/ immune-regulatory cytokines IFN-b 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 inflam- matory signature, and enhanced M. tuberculosis phagocytosis and survival when compared with resident CD11c+ CD11b2 AMs, which are more immune regulatory in nature. The Journal of Immunology, 2019, 203: 000–000.

he global elderly human population is predicted to in- developing and developed countries due to mobility of the human crease 3-fold to ∼2.1 billion by 2050 (1). The elderly are population. T more susceptible to pulmonary infections, such as influ- Aging is associated with low-grade chronic inflammation, by guest on September 25, 2021 enza, pneumococcal pneumonia, and tuberculosis (TB) (2–5). termed inflammaging (8). In humans, inflammaging is man- United States data indicate that the risk of TB is 50% higher ifested by increased levels of circulating proinflammatory cy- in older adults than that of persons aged 21–64 (6). It is also tokines (9–11) and increased risk of chronic diseases, including estimated that 57% of TB deaths globally occurred in individu- cancer, type 2 diabetes, cardiovascular diseases, and infectious als older than 50, with most of the deaths in individuals older than diseases (8, 12–15). Inflammation with aging is implicated in 65 (7). The largest increase in the elderly population is projected TB susceptibility (reviewed in Ref. 5). Mycobacterium tuber- to occur in developing countries where TB is endemic, which culosis, the causative agent of TB, is transmitted by inhaled will lead to a significant burden on health care systems in aerosol droplets into the lung where it is phagocytosed by al- veolar macrophages (AMs). M. tuberculosis delivered to adult mice by the aerosol route grows relatively unrestricted for the *Department of Microbial Infection and Immunity, The Ohio State University, first 2–3 wk. Once the adaptive immune system is activated, a Columbus, OH 43210; and †Texas Biomedical Research Institute, San Antonio, TX 78227 chronic stable infection is established with M. tuberculosis con- 1 tained within granulomas that can last up to 1 y (16). Old mice W.P.L. and M.V.S.R. contributed equally to this work. demonstrate enhanced early resistance to M. tuberculosis because ORCIDs: 0000-0003-1056-3862 (W.P.L.); 0000-0002-1515-1122 (M.V.S.R.); 0000- + 0002-8939-7280 (Q.W.); 0000-0001-7702-5941 (J.B.T.). of production of IFN-g by CD8 T cells (17–23). However, this Received for publication May 2, 2019. Accepted for publication August 8, 2019. resistance to M. tuberculosis does not last as chronic stable M. tuberculosis infection is established at a higher level in old This work was supported by National Institutes of Health Grants P01-AG051428 and P30- CA016068. mice, which also succumb earlier to the infection. Address correspondence and reprint requests to Dr. William P. Lafuse and Dr. Murugesan The initial interaction of M. tuberculosis with AMs occurs V. S. Rajaram, Department of Microbial Infection and Immunity, The Ohio State Univer- within alveolar lining fluid (ALF) that contains several innate sity, 718 Biomedical Research Tower, 460 West 12th Avenue, Columbus, OH 43210. immune determinants that have a significant impact in con- E-mail addresses: [email protected] (W.P.L.) and [email protected] (M.V.S.R.) trolling microbial infections (24–29) and are affected by an The online version of this article contains supplemental material. increasingly oxidized environment during aging (30, 31). These Abbreviations used in this article: ALF, alveolar lining fluid; AM, alveolar macro- studies in both mouse and humans indicate that aging greatly phage; BAL, bronchoalveolar lavage; COX2, cyclooxygenase 2; MFI, mean fluores- alters the lung environment and impacts the function of mac- cence intensity; MR, mannose receptor; qRT-PCR, quantitative RT-PCR; TB, rophages that reside in the lung. We previously showed that the tuberculosis. lungs of old mice contain increased levels of IFN-g,TNF-a, Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 and IL-12 mRNA compared with young mice (32) as well as

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900495 2 ANALYSIS OF ALVEOLAR MACROPHAGE POPULATIONS IN OLD MICE increased protein (30). Purified populations of lung macrophages of protein aliquots were lyophilized in a SpeedVac concentrator, resus- from old mice contain higher mRNA levels of IFN-g–induced pended in 100 ml of sterile water, and added to individual ELISA plate genes and inflammatory cytokines in response to M. tuberculosis wells (Nunc MaxiSorp plates; Thermo Fisher Scientific). ELISA kits were purchased from R&D Systems (CCL2 [DY429], IL-1b [DY421], MIF (32, 33), indicating that they are more activated. Lung macro- [DY1978], TNF-a [DY410], and IL-6 [Dy406]), MABTECH (IL-10 phages from old mice phagocytose more M. tuberculosis and have [34320]), and PBL Assay Sciences (IFN-b [4210 Verikine-HS kit]). higher percentages of phagosome-lysosome fusion events (32), yet Turbo TMB-ELISA Substrate Solution (Thermo Fisher Scientific) was over time, the growth of M. tuberculosis in lung macrophages used for detection. from old mice is greater than in those from young mice. Confocal microscopy In the current study, we examined the effect of aging on the AMs (1 3 105) were cultured on coverslips, fixed with 2% paraformal- phenotype and function of isolated AMs and the local environment dehyde, and permeabilized with 100% methanol for 5 min at room tem- in which they exist. We extended our previous observations of perature (34). The cells were blocked overnight at 4˚C in blocking buffer increased TNF and IL-6 in ALF of old mice (30, 32) to additional (PBS + 5 mg/ml BSA + 10% heat-inactivated FBS) and incubated with the cytokines and chemokines and observed that the ALF of old mice indicated Ag-specific Abs or isotype control Abs, followed by incubation contains higher levels of the chemokine CCL2 and cytokines with Alexa 488– and Alexa 594–labeled secondary Abs. Coverslips were then dried, mounted onto slides using ProLong Gold Anti-Fade, and ex- IL-1b, IFN-b, and MIF. Further, we show that AMs isolated from amined by confocal microscopy (Flow View 1000 Laser Scanning Con- old mice express higher mRNA levels of CCL2, IFN-b, IL-10, focal microscope; Olympus). Mean fluorescence intensity (MFI) was IL-12p40, and TNF-a compared with AMs isolated from young determined for 40–60 individual cells per group on duplicate slides mice and conjecture that during homeostasis, AMs are important using ImageJ software. contributors to the levels of these cytokines and chemokines in Flow cytometry Downloaded from the ALF. Thus, our current observations and previous observations 3 5 from our group indicate that the alveolar cytokine environment of AMs (1–2 10 ) were aliquoted into FACS tubes and centrifuged at 250 3 g for 10 min. Cell pellets were resuspended in 60 ml of FACS buffer old mice contains a specific mixture of cytokines, with increased (PBS buffer with 2% BSA and 0.10% sodium azide) and 40 ml of mouse levels of both proinflammatory cytokines/chemokines (CCL2, serum. After 30 min of incubation on ice, cells were stained with Abs for IL-1b,MIF,TNF-a, IL-12) and immune-regulatory cytokines 30 min in the dark at 4˚C. Cells were washed twice, fixed in 2% para- formaldehyde (Electron Microscopy Sciences) in FACS buffer for 10 min, (IL-10, IFN-b). When AMs from old mice were infected with http://www.jimmunol.org/ centrifuged, and resuspended in FACS buffer. AMs were processed on a M. tuberculosis, they produce more CCL2, IFN-b, and IL-10 BD LSRII flow cytometer and analyzed using FlowJo software (Tree Star). Abs than AMs from young mice but less IL-1b and TNF-a.Further, (purchased from BioLegend) were BV421 anti-CD11b (clone M1/70), BV785 we found that AMs consist of two populations: a major pop- anti-CD11c (clone N418), BV605 anti-CD206 MMR (clone C06872), ulation that is CD11c+ CD11b2 and a minor CD11c+ CD11b+ Alexa 488 anti–MHC class II (clone M5/114.15.2), allophycocyanin anti- population that is monocyte derived and increased 4-fold in old CD36 (clone HM36), PERCP/Cy5.5 anti-CD16/CD32 (clone 93), Alexa + 2 488 anti-TLR2 (clone T2.5), allophycocyanin anti-CD80 (clone 16-10A1), mice. Whereas the major CD11c CD11b population in old mice PERCP/Cy5.5 anti-CD64 (clone X54.5/7.1), BV605 anti-Ly6C (clone has immune-regulatory properties with higher mRNA levels HK1.4), Alexa 488 anti-CX3CR1 (clone SA011511), and PERCP/Cy5.5 of IFN-b and IL-10, the CD11c+ CD11b+ AM subpopulation anti-CD115 (clone mAFS98). Isotype controls were BV421 rat IgG2b, displays more inflammatory properties with higher CCL2, IL-1b, BV785 hamster IgG, BV605 rat IgG2a, Alexa 488 rat IgG2a, allophycocyanin by guest on September 25, 2021 + hamster IgG, PERCP/Cy5.5 mouse IgG1, Alexa 488 mouse IgG2a, and and cyclooxygenase 2 (COX2) mRNA levels. Further, the CD11c PERCP/Cy5.5 rat IgG2a. Specific MFI was calculated by subtracting the + CD11b AM population of old mice phagocytosed more M. tu- isotype MFI from the Ab-specific MFI. berculosis and was more permissive for M. tuberculosis growth. AMs from old mice were sorted into CD11c+ CD11b2 and CD11c+ + Our findings provide evidence that inflammaging has a significant CD11b AM populations by flow sorting using the BD FACSAria II flow impact on the alveolar compartment of lungs, which impacts host cytometer and allophycocyanin anti-CD11b (clone M1/70) and PE anti- CD11c (clone N418) Abs. Flow-sorted populations were pelleted by response to M. tuberculosis and likely other airborne pathogens. centrifugation at 250 3 g for 10 min, and RNA was isolated using TRIzol reagent (Invitrogen). Flow-sorted CD11c+ CD11b2 and CD11c+ CD11b+ Materials and Methods AM populations were also cultured onto coverslips (4 per each pop- Mice ulation) and incubated with mCherry–M. tuberculosis H37Rv at the ra- tio of 5:1 (M. tuberculosis bacillus/AM). Phagocytosis and growth of Specific pathogen-free C57BL/6 female mice were purchased from Charles M. tuberculosis in AM populations were determined by confocal mi- River Laboratories (Wilmington, MA) at an age of 3 mo (young) or 18 mo croscopy at 2 and 24 h, respectively, analyzing two coverslips of each (old) through a contract to Charles Rivers Laboratory from the National population. Institute on Aging. Mice were housed in microisolator cages and acclimated to the facility for 1 wk prior to use. All procedures were approved by The Ohio M. tuberculosis infection State University Institutional Laboratory Animal Care and Use Committee. AMs were plated in 48-well plates at 50,000 AMs per well in RPM 1640 Isolation of AMs and ALF media containing 2 mM glutamine and 10% heat-inactivated FBS. After 2 h, nonadherent macrophages were removed by washing, and the AMs were

Young and old mice were euthanized by CO2 following a protocol approved incubated with M. tuberculosis H37Rv at the ratio of 5:1 (M. tuberculosis by The Ohio State University Institutional Laboratory Animal Care and Use bacillus/AM). RNA was isolated at various times from 0 to 72 h using 2 Committee. AMs and ALF were obtained by bronchoalveolar lavage (BAL) TRIzol reagent. Flow cytometry–sorted CD11c+ CD11b and CD11c+ of mice by washing the lungs 10 times with 0.50 ml of sterile endotoxin-free CD11b+ AM populations were also plated in 48-well plates and incubated saline (0.90% NaCl). AMs were collected by centrifugation at 300 3 g for with M. tuberculosis. RNA was isolated at 24 h postinfection using TRIzol 10 min. In each experiment, AMs from 10 mice were pooled (a total of ∼1–2 reagent. million pooled AMs). Three aliquots of the pooled AMs (each ∼50,000 cells) were pelleted, and RNA was isolated using TRIzol reagent (Invitrogen) RNA isolation and qRT-PCR for basal RNA measurements by quantitative RT-PCR (qRT-PCR). The RNA from TRIzol lysates was extracted with chloroform and precipitated remaining AMs were used in confocal microscopy, flow cytometry, and with isopropanol. The RNA pellet was washed once with 75% ethanol, and M. tuberculosis infection experiments. The supernatant fraction containing the 2 RNA was reconstituted with DNase/RNase-free water. RNA was reversed ALF from individual mice was quickly frozen and stored at 80˚C until use. transcribed using random primers by the Promega Reverse Transcription ELISA system. mRNA expression was analyzed by qRT-PCR using IQ SYBR Green Supermix (BioRad). The amplification conditions were 95˚C for ALF cytokine levels were determined by ELISA, according to manufac- 10 min, followed by 45 cycles of 95˚C for 15 s, 60˚C for 30 s, and 72˚C for turer’s instructions. ALF protein concentration was determined, and 20 mg 30 s. Validated mouse primers listed on PrimerBank (35) were used, and The Journal of Immunology 3 sequences are listed in Supplemental Table 1. Relative expression was We next compared the mRNA expression of CD11b, CD11c, calculated by the D threshold cycle method using b-actin as the normalizer MR (CD206, Mcr1), and TLRs in AMs immediately after BAL (36). Expression levels of basal mRNA in AMs from old mice were de- isolation without adherence. As expected, AMs from old mice termined relative to the level of mRNA expressed in AMs from young mice. Expression levels of mRNA present in flow-sorted populations were expressed higher mRNA levels of CD11b than AMs from young determined relative to levels in the CD11c+ CD11b2 AM population. mice (Fig. 2D). There was no difference in mRNA levels of Expression levels of mRNA in the M. tuberculosis infection experiments CD11c, MR, TLR2, TLR4, and TLR6 (Fig. 2E). TLR1 mRNA were determined relative to control resting samples at each time point. expression was higher in AMs from old mice (Fig. 2E). This is of Expression levels of M. tuberculosis gene expression in M. tuberculosis– infected AMs were normalized to b-actin, and the level of expression interest in that TLR1 forms a heterodimer with TLR2 and binds relative to M. tuberculosis 16s RNA was determined. triacetylated lipoproteins and lipoglycans, such as lipomannan and lipoarabinomannan, which are major TLR2 ligands of M. tubercu- Statistical analysis losis (38–40). AMs from old mice also expressed higher mRNA All data are presented as mean 6 SEM. Statistical significance was cal- levels of COX2, a key enzyme in the biosynthesis of PGs produced culated by two-tailed Student t test with Welch correction. Differences during inflammation (Fig. 2F). Likewise, AMs from old mice were considered to be statistically significant when p values were *p , 0.05, **p , 0.01, ***p , 0.001, and ****p , 0.0001. expressed higher mRNA levels of IFN-g–inducible genes MHC class II Aa, CIITA, and IRF-1 (Fig. 2F), which is consistent with our previous report (32) of increased levels of IFN-g mRNA in the Results lungs of old mice and higher levels of IRF1 and CIITA mRNA in Proinflammatory and immune-regulatory cytokine expression lung macrophages isolated by lung digestion. in alveolar environment of old mice Our confocal microscopy experiments suggested that there are Downloaded from To better define the alveolar cytokine environment in which AMs two AM populations in old mice expressing either low or high exist and their contribution to the cytokines produced during ho- levels of CD11b (Fig. 2A). To further explore this possibility, meostasis, we measured cytokine levels in the ALF from young we examined AMs harvested from young and old mice by flow (3 mo) and old (18 mo) mice by ELISA and cytokine mRNA levels cytometry for CD11b and CD11c expression. Flow cytometry of isolated AMs by qRT-PCR. ALF was isolated by BAL and confirmed the presence of two AM populations: a major pop- + 2 normalized by protein content. Previously, we reported that cy- ulation that is CD11c CD11b and a minor population that is http://www.jimmunol.org/ tokine expression of TNF-a and IL-6 was significantly higher in CD11c+ CD11b+. Fig. 3A and 3B are representative flow cytometry old mice compared with young mice, whereas IL-10 did not differ plots of AMs from young and old mice, respectively. In young mice, between young and old mice (30). In the current study, we ex- the percentage of AMs that were CD11c+ CD11b+ was 1.29 6 0.50% tended these studies to include additional cytokines and chemo- (n = 6 BAL isolations) (Fig. 3C). In the old mice, the percentage of kines. We found that the levels of chemokine CCL2 and cytokines CD11c+ CD11b+ AMs was 4-fold higher (4.92 6 0.36%, n =6BAL IL-1b, IFN-b, and MIF were higher in old mice compared with isolations). A corresponding small decrease in the percentage of 2 young mice (Fig. 1A–C). To determine if AMs contribute to the CD11c+ CD11b AMs was also observed (Fig. 3D). cytokine production in ALF, we measured cytokine mRNA levels + + + CD11c CD11b AMs are in a more-activated state than by guest on September 25, 2021 of BAL cells (which are 90–95% CD11c AMs) by flow cytom- 2 CD11c+ CD11b AMs etry (37), immediately after isolation without culturing. This was necessitated by the observation that gene expression in AMs We next examined the expression of cell surface markers in these changes in culture, even to some degree during a 2-h adher- two AM populations by flow cytometry (Fig. 4). We found that the ence. By immediately isolating RNA, we are able to determine MFIs of CD206 (MR), CD86, CD36, and TLR2 were significantly + + + 2 basal cytokine mRNA levels of AMs. Consistent with the ALF higher in CD11c CD11b AMs compared with CD11c CD11b cytokine levels, CCL2, IFN-b,andTNF-a mRNA levels were AMs, independent of age. When comparing the two subsets in + higher in AMs from old mice, but IL-1b mRNA levels were not young mice, MHC class II and CD16/32 expression in CD11c + + 2 significantly different (Fig. 1D, 1E). However, mRNA levels of CD11b AMs was significantly higher than CD11c CD11b IL-12p40 and IL-10 were higher in AMs from old mice (Fig. 1E). AMs. When comparing old versus young mice, only CD64 ex- + + mRNA levels of the growth factor M-CSF were higher in AMs pression was significantly higher in CD11c CD11b AMs from + + from old mice, but there was no significant difference in GM-CSF old mice compared with CD11c CD11b AMs from young mice. + 2 mRNA levels (Fig. 1F). Together, these results indicate that the The MFI of MHC class II was higher in CD11c CD11b AMs + 2 lung cytokine environment of old mice represents a specific array from old mice than in CD11c CD11b AMs from young mice. + 2 of both proinflammatory and immunoregulatory cytokines, mostly Because CD11c CD11b AMs are the major AM population produced by AMs, which can impact macrophage function and in the lung, increased expression of cell surface MHC in this innate immunity in the lung. AM population is consistent with increased MHC class II Aa mRNA expression in AMs from old mice (Fig. 2B). Overall, the Characterization of AMs in old mice: an increased data indicate that the subpopulation of double-positive CD11c+ subpopulation with high expression of CD11b CD11b+ AMs from both young and old mice is in a more-activated To assess how AMs from old mice differ from AMs from young state, with increased expression of key surface molecules involved mice, we examined the phenotype of AMs by confocal micros- in macrophage inflammatory functions when compared with the copy and flow cytometry using specific membrane markers CD11c, major AM population (CD11c+ CD11b2).

CD11b, and CD206 (the mannose receptor [MR]) as the primary + + screen. AMs from young and old mice were adhered to coverslips CD11c CD11b AMs are monocyte derived and examined by confocal microscopy. Representative images are We also examined the expression of monocyte markers Ly6C, shown in Supplemental Fig. 1. The MFI was determined for in- CX3CR1, and CD115 (41–43) by flow cytometry (Fig. 4B). dividual cells. We observed that AMs from old mice had signifi- CD11c+ CD11b+ AMs expressed increased levels of Ly6C, cant higher surface expression of CD11b and CD11c than young CX3CR1, and CD115 in both young and old mice, suggesting that mice (Fig. 2A, 2B). Although the surface expression of MR in old these AMs are derived from monocytes (44). These cell markers mice is higher, this was not statistically significant (Fig. 2C). were absent in the CD11c+ CD11b2 AMs, consistent with these 4 ANALYSIS OF ALVEOLAR MACROPHAGE POPULATIONS IN OLD MICE

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–C). ALF was isolated from individual mice by BAL. Protein concentration was normalized to 0.20 mg/ml, and cytokine levels in 100 ml were determined Downloaded from by ELISA (n = 30 young mice and 28 old mice). (D–F) mRNA levels in AMs from young and old mice. RNA was isolated from AMs immediately after BAL, and cytokine mRNA levels were determined by qRT-PCR. mRNA levels were normalized to b-actin levels, and cytokine and chemokine expression levels were determined relative to levels in AMs from young mice. Data represent three to five experiments from young mice or old mice. For each experiment, BAL-obtained AMs were pooled from 10 mice. *p , 0.05, **p , 0.01.

dim high AMs being the resident AMs derived from fetal origin (45, 46). of old mice into CD11b and CD11b populations (Supplemental http://www.jimmunol.org/ We also considered that the double-positive CD11c+ CD11b+ AMs Fig. 2A). The majority of the CD11c+ CD11b+ AMs are CD11bdim might be CD11b+ dendritic cells. However, the CD11c+ CD11b+ cells. We compared the expression of macrophage markers in these AMs from old mice expressed CD64 (FcgR1) (Fig. 4A), which is two populations with the CD11c+ CD11b2 AMs (Supplemental absent in dendritic cells (47), suggesting that these cells are not Fig. 2B). Both of the CD11c+ CD11b+ AM populations express dendritic cells. higher levels of macrophage markers than the CD11c+ CD11b2 cells, indicating that both populations are in a more-activated state Heterogeneity in CD11b expression in the + 2 + + than the CD11c CD11b AMs. Only MHC class II and CD86 CD11c CD11b AMs from old mice were significantly higher in the CD11bhigh AMs compared with CD11b expression varies in intensity among the CD11c+ CD11b+ the CD11bdim AMs. Both the CD11bdim and CD11bhigh AMs by guest on September 25, 2021 AM population of old mice. CD11b dim cells appear as an ex- expressed higher levels of monocytic markers than the CD11c+ tension of the CD11c+ CD11b2 population, whereas CD11b CD11b2 AMs (Supplemental Fig. 2C), although the CD11bdim bright cells appear to be separate (Fig. 3B). Thus, it is possible AMs tended to express higher levels of the monocytic markers, that the CD11c+ CD1b+ AMs represent phenotypically distinct which suggests that the CD11bdim cells are the more immature cell populations. Therefore, we gated the CD11c+ CD11b+ AMs macrophage population.

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 MR (C) was determined by confocal microscopy. 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 (MR), TLR2, TLR4, TLR1, TLR6, COX2, MHC class II Aa, CIITA, and IRF1 was determined by qRT-PCR (D–F). mRNA levels were determined relative to levels in AMs from young mice. Data represent three to five 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. The Journal of Immunology 5

FIGURE 3. Percentage of C11c+ CD11b+ AMs is higher in old mice. Representative flow cytometry plots showing the major CD11c+ CD11b2 and minor CD11c+ CD11b+ AM populations in young (A) and old mice (B). (C and D) Graphs showing the percentage of AM populations from six young and six old mouse ex- periments. For each experiment, BAL-obtained AMs were pooled from 10 mice. *p , 0.05, ****p , 0.0001. Downloaded from http://www.jimmunol.org/

AMs from young and old mice differ in cytokine mRNA expressed significantly higher levels of CD11c, TLR1, and CD86 production in response to M. tuberculosis infection mRNA and a trend toward higher expression of CD80 and CD64 (FcgR1) (Fig. 6B, 6C). Neither the CD11c+ CD11b2 or To study the innate immune response of AMs, adherent AMs from + + young and old mice were infected with M. tuberculosis. After 6, the CD11c CD11b AMs expressed mRNA for CCR2, the 24, 48, and 72 h postinfection, RNA was isolated and gene ex- chemokine receptor for CCL2, which regulates entry of mono- pression was determined by real-time RT-PCR. M. tuberculosis cytes into the lung. Next, we determined the mRNA expression infection of AMs from old mice increased expression of CCL2 of cytokines and COX2 in the sorted cell populations. Notably,

+ + by guest on September 25, 2021 and IL-10 mRNA at 24 h compared with young mice (Fig. 5A). CD11c CD11b AMs expressed significantly higher mRNA levels + Expression levels of IFN-b mRNA were higher in old mice at 6 h of cytokines CCL2, IL-1b, IL-6, and COX2 (Fig. 6D) than CD11c 2 + 2 and also tended to be higher in M. tuberculosis–infected AMs CD11b AMs, whereas CD11c CD11b expressed higher levels at 24 h, but the difference did not reach statistical significance. In of IFN-b and IL-10 (Fig. 6E). There was no difference in TNF-a contrast, M. tuberculosis infection of AMs from old mice induced expression between the two cell populations (Fig. 6D). TLR2, lower levels of IL1-b and TNF-a mRNA at early time points TLR4, TLR6, and MHC class II Aa were also equally expressed (6 and 24 h) compared with AMs from young mice. To confirm (Fig. 6F). Overall, these results are consistent with the major + 2 that differences in mRNA levels are reflected in cytokine secre- CD11c CD11b AM population playing a more immunoreg- + + tion, we measured the cytokine levels in the 24-h culture super- ulatory role and the CD11c CD11b AMs being more activated natants. AMs from old mice secreted significantly higher levels of in a proinflammatory state. CCL2 in response to M. tuberculosis infection but had lower levels The CD11c+CD11b+AM subpopulation contains more of TNF-a (Fig. 5B). Overall, these studies indicate that AMs from M. tuberculosis old mice produce higher mRNA levels of a unique subset of Adherent flow cytometry–sorted CD11c+ CD11b2 and CD11c+ inflammatory cytokines/chemokines, specifically CCL2, IL-10, + and IFN-b, upon M. tuberculosis infection than AMs from young CD11b AM populations from old mice were infected with mice, while producing lower levels of IL-1b and TNF-a. mCherry–M. tuberculosis for 2 and 24 h and examined for of the number of M. tuberculosis bacteria per cell by confocal mi- + Gene expression profiles of flow cytometry–sorted CD11c croscopy. CD11c+ CD11b+ AMs phagocytosed significantly more + + 2 CD11b and CD11c CD11b AMs from old mice M. tuberculosis bacteria at 2 h than CD11c+ CD11b2 AMs Our flow cytometry experiments showed that old mice contain (Fig. 7). Although the number of M. tuberculosis bacteria per higher numbers of CD11c+ CD11b+ AMs than young mice. Also, macrophage increased for both populations at 24 h, 49% of the these CD11c+CD11b+ AMs express higher surface expression of CD11c+CD11b+ AMs contained more than seven M. tuberculosis proteins involved in macrophage activation. To characterize gene bacteria per macrophage, whereas only 4.8% of the CD11c+ 2 expression of the two AM populations, we next flow cytometry– CD11b AMs contained seven or more M. tuberculosis bacteria, sorted the two populations from old mice. Purity of two populations suggesting that the CD11c+ CD11b+ AMs are more permissive for was 98% for CD11c+ CD11b2 AMs and 89% for CD11c+ CD11b+ the growth and survival of M. tuberculosis. The small number of AMs (Supplemental Fig. 3). RNA was isolated immediately after AMs obtained precluded our ability to confirm this by CFUs. cell sorting, and gene expression in the two populations was com- However, we wished to pursue the issue of permissibility and pared by real-time RT-PCR. As expected, the CD11c+ CD11b+ elected in a pilot study to isolate M. tuberculosis RNA from in- AMs expressed significantly higher mRNA levels of CD11b than fected, flow-sorted AM populations at 24 h to examine for genes the CD11c+ CD11b2 AMs (Fig. 6A). CD11c+ CD11b+ AMs also associated with M. tuberculosis growth in macrophages. In this 6 ANALYSIS OF ALVEOLAR MACROPHAGE POPULATIONS IN OLD MICE Downloaded from http://www.jimmunol.org/

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 Ab MFI 2 isotype MFI). (B) CD11c+ CD11b+ AMs express monocyte markers that are absent on CD11c+ CD11b2 AMs. Data in (A) and (B) represent six young and six old mouse experiments. For each experiment, BAL-obtained AMs were pooled from 10 mice. *p , 0.05, **p , 0.01, ***p , 0.001. by guest on September 25, 2021 study, we obtained sufficient CD11c+ CD11b+ AMs for one well in macrophages (48–53), were higher in CD11c+ CD11b+ AMs of infected AMs. M. tuberculosis 16s rRNA and other selected compared with CD11c+ CD11b2 AMs. RNA levels were examined by qRT-PCR, and expression of M. tuberculosis genes was determined relative to the level of Discussion M. tuberculosis 16s rRNA (Fig. 8). RNA levels of M. tuberculosis The focus of this study was on determining the effect of aging genes dnaA, icl, sodC, sigA, and sigE, which are required for growth on the phenotype and function of AMs and further defining the

FIGURE 5. AMs from young and old mice differ in cytokine mRNA expression postinfection with M. tuberculosis. AMs isolated from young and old mice were cultured in 48-well culture plates for 2 h and then infected with M. tuberculosis (5:1 bacteria/macrophage) for 6, 24, 48, and 72 h. (A) RNA was isolated, and cy- tokine mRNA expression was determined by qRT-PCR. mRNA levels were normalized to b-actin levels, and cytokine expression levels at each time point were de- termined relative to control nontreated AMs at each time point. (B) Expression of CCL2 and TNF-a in culture fluid from 24-h cultures of control and M. tuberculosis– infected AMs. Data represent three to seven 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. The Journal of Immunology 7

FIGURE 6. CD11c+ CD11b+ and CD11c+ CD11b2 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+ CD11b2 and CD11c+ CD11b+ AM subpopulations. RNA was isolated Downloaded from from the flow-sorted populations, and gene expression was determined by qRT-PCR. Expression levels were normalized to b-actin, and expression levels in the CD11c+ CD11b+ AMs were determined relative to the CD11c+ CD11b2 AMs. Relative expression levels in CD11c+ CD11b+ AMs .1 indicate preferential expression in the CD11c+ CD11b+ AMs, whereas relative expression levels in CD11c+ CD11b+ AMs ,1 indicate preferential expression in the CD11c+ CD11b2 AMs. mRNA levels of CD11b (A), CD11c, CD80, CD86 (B), TLR1, FcgR1 (C), CCL2, IL-1b, IL-6, COX2, TNF-a (D), IFN-b, IL-10 (E), TLR2, TLR4, TLR6, and MHC class II Aa (F) are shown. Cumulative data from three experiments are shown. *p , 0.05, **p , 0.01. http://www.jimmunol.org/ cytokine environment in which the AMs are located. Our prior upon LPS stimulation (55). We recognize that T lymphocytes studies established that inflammaging occurs in the lungs of old may also be a source for ALF cytokines. However, we have mice and impacts M. tuberculosis interactions by altering the found that CD4+ and CD8+ T cells make up ,1% of the BAL cytokine environment (30, 32) and creating a proinflammatory and cells during homeostasis. This does not preclude T cells in the pro-oxidative state in the lung mucosa that increases susceptibility interstitium contributing to ALF cytokines, but the cytokines to M. tuberculosis via dysfunctional soluble innate components, would have to translocate across the epithelial layer. Overall, such as surfactant proteins (31). Cytokine mRNA levels of total our studies indicate that aging in the lung creates a unique lung homogenates showed increased levels of IFN-g, TNF-a, and cytokine environment with increases in both proinflammatory IL-12 mRNA (32), whereas levels of IL-6 and TNF-a were in- (CCL2, IL-1b,MIF,TNF-a, IL-12) and immune-regulatory by guest on September 25, 2021 creased in the ALF (30). However, the nature of the specific cell (IFN-b, IL-10) cytokines. types (e.g., lung macrophages versus AMs or other potential cell Several studies have reported that aging induces alterations in types) involved in the differential production of these cytokines TLR expression and signaling with varying results. Studies by was not known. Renshaw et al. (56) reported that splenic and peritoneal macro- In this study, we focused on the alveolar space, examining both phages from old C57BL/6 mice expressed lower levels of all TLRs AMs and ALF. We expanded our previous findings, showing that and secreted less TNF-a and IL-6 when stimulated with TLR li- ALF from old mice contained higher levels of the chemokine CCL2 gands. However, other studies have yielded different results, likely and cytokines IFN-b, IL-1b, and MIF compared with ALF from as a result of different culture conditions. TLR2 and TLR4 ex- young mice. However, during the course of our studies, we de- pression on macrophages from aged BALB/c mice was found not termined that the nature of the inflammatory state of AMs differed to differ, but the TNF-a and IL-6 responses were lower than those depending on whether we studied these cells after the standard of young mice (57). In another study using AMs from aged procedure of 2-h cell culture adherence versus measuring cytokine BALB/c mice, TLR1 and TLR2 expression was reported to be mRNA levels in AMs directly isolated without culturing, consis- unchanged, but TLR2 signaling and cytokine production in re- tent with our recent study of human AMs (54). Thus, we conclude sponse to Streptococcus pneumoniae declined with aging (58). In that measurement of cytokine RNA levels from freshly obtained, the current study, mRNA levels of TLR2, TLR4, and TLR6 did nonadherent AMs represents the most accurate assessment of not differ between freshly isolated AMs from young and old mice. the vivo state of these cells. As expected, AMs represent 90–95% Interestingly, we found that TLR1 mRNA was significantly higher of the cells obtained by BAL, and thus we infer that the ALF in old mice. In terms of M. tuberculosis infection, TLR1 upreg- cytokine mRNA examined in the basal state of the mice was ulation with old age may be significant because TLR1 associates predominantly from AMs. We found that AMs from old mice with TLR2 and recognizes mycobacterial 19-kDa lipoprotein (59) contained higher levels of CCL2 and IFN-b mRNA, which cor- and other M. tuberculosis lipoproteins, lipoglycans, and glyco- related with their increased production in ALF, consistent with lipids (38–40). We found that M. tuberculosis infection of AMs AMs being their source. AMs also had higher levels of IL-10, from old mice induces higher levels of CCL2 and IL-10 mRNA TNF-a, M-CSF, and IL-12p40 mRNA. Interestingly, IL-1b at 24 h. Thus, TLR signaling pathways in AMs from old mice mRNA levels did not differ between AMs from young and old involved in induction of these cytokines appear to be intact. mice, whereas the IL-1b levels in ALF differed substantially. This Proinflammatory cytokines IL-1b and TNF-a were induced by suggests that AMs from old mice have higher levels of inflamma- M. tuberculosis infection to high mRNA levels at early time points some activation and processing of pro-IL-1b into its mature form. (6 and 24 h) in AMs from young and old mice, but the levels in Alternatively, there may be a second cell source for ALF IL-1b, AMs from old mice were significantly lower. Secretion of cyto- such as alveolar type II epithelial cells, which can produce IL-1b kines by infected AMs was consistent with mRNA levels, with 8 ANALYSIS OF ALVEOLAR MACROPHAGE POPULATIONS IN OLD MICE Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 7. CD11c+ CD11b+ AMs phagocytose significantly more M. tuberculosis and contain higher numbers of M. tuberculosis at 24 h than CD11c+ CD11b2 AMs. AMs were isolated from 10 old mice by BAL and flow sorted into CD11c+ CD11b2 and CD11c+ CD11b+ AM populations. The flow-sorted populations were cultured on coverslips and infected with mCherry–M. tuberculosis H37Rv at 5:1 bacteria/macrophage. M. tuberculosis present in AM populations was determined by confocal microscopy. Results are representative of n = 2 experiments. (A) Mean number of M. tuberculosis per macrophage at 2 and 24 h. (B) Distribution of M. tuberculosis per macrophage. (C) Representative confocal images with M. tuberculosis detected by mCherry and macrophages by DAPI staining. Original magnification 360. Between 144 and 284 macrophages were analyzed in (A) and (B). ***p , 0.001.

AMs from old mice secreting more CCL2 and less TNF-a Interestingly, flow cytometry revealed a CD11c+ CD11b+ AM than AMs from young mice. These results suggest that the effect subpopulation that is present in higher numbers in old mice. This of old age on AM cytokine gene expression is gene specific and AM population expressed higher levels of cell surface markers may involve differences in transcription factor activation at dif- than the resident AMs and contained higher mRNA levels of ferent promoters as well as age-related transcriptional repression CD11b, CD11c, TLR1, CD86, and FcgR1. The CD11c+ CD11b+ mechanisms. AMs also expressed several markers, CX3CR1, Ly6C, and The major AM population is resident CD11c+ CD11b2 AMs, CD115, normally associated with monocytes (41, 64), that are which are considered to be of fetal liver monocyte origin and self- absent in the resident CD11c+ CD11b2 AMs, suggesting that this maintained locally (60–62). These AMs function to maintain lung subpopulation is of monocytic origin. However, they are not mucosa (surfactant and ALF) homeostasis and gas exchange while monocytes because they express high levels of CD11c and MHC limiting inflammation to minimize lung injury. The phenotype and class II, whereas monocytes express lower levels of CD11c and homeostatic function of these AMs is maintained by the cyto- MHC class II (65, 66). The CD11c+ CD11b+ AMs also did not kine environment, in particular, GM-CSF produced by the lung express CCR2 mRNA (data not shown), which is expressed by epithelium (45, 63). We examined the phenotype of the resident monocytes but declines after entry of monocytes into tissues. Nor CD11c+ CD11b2 AMs by flow cytometry analysis. However, with are they dendritic cells because they express FCgR1, which is the exception of MHC class II expression, which was higher in absent on dendritic cells (47), and they do not have a dendritic AMs from old mice, there were no differences in the expression morphology when plated on coverslips. The CD11c+ CD11b+ levels of cell surface markers between CD11c+ CD11b2 AMs AMs preferentially expressed more CCL2, IL-1b, IL-6, and COX2 from young and old mice. mRNA compared with the resident CD11c+ CD11b2 AMs. Thus, The Journal of Immunology 9 Downloaded from FIGURE 8. M. tuberculosis gene expression is substantially higher in CD11c+ CD11b+ AMs infected with M. tuberculosis for 24 h. AMs were isolated from 10 old mice by BAL and flow sorted into CD11c+ CD11b2 and CD11c+ CD11b+ AM populations. The flow-sorted populations were infected with M. tuberculosis at 5:1 bacteria/macrophage in 48-well culture plates for 24 h. RNA was isolated, and M. tuberculosis 16S rRNA and M. tuberculosis mRNA levels were determined by qRT-PCR. Expression levels of M. tuberculosis genes were determined relative to M. tuberculosis 16S rRNA. The results are representative of two experiments in which two wells of CD11c+ CD11b2 and one well of CD11c+ CD11b+ AMs were infected with M. tuberculosis. http://www.jimmunol.org/ we conjecture that the CD11c+ CD11b+ AMs are either inflam- the CD11c+ cells were the predominant cells producing IL-12 p40 matory macrophages differentiated from monocytes that have and TNF-a but with equal production by these cells from both entered the lung environment from circulation, or alternatively they young and old mice. This study differs from the current study in could be of fetal liver monocyte origin, and the increased numbers which we show that AMs from old mice produce less TNF-a of these macrophages in old mice are due to increased replica- mRNA and protein than AMs from young mice. The difference tion resulting from changes in the cytokine environment with aging. between the two studies is likely due to the source of macrophages: A recent single-cell RNA sequencing study by Mould et al. (67) Rottinghaus et al. (33) examined CD11c+ pulmonary macrophages identified a minor AM population in young (10–12 wk) C57BL/6 containing a mixture of myeloid cells, including AMs, interstitial mice during homeostasis and inflammation that represents a pro- macrophages, monocytes, and dendritic cells, whereas the current by guest on September 25, 2021 liferative pool of resident AMs. Thus, this population could be a study examined AMs isolated by BAL. precursor population that replicates and differentiates in response to Previous studies have reported little or no CD11b+ CD11c+ AMs the lung cytokine environment into the CD11c+ CD11b2 AMs that in the lungs of healthy adult mice, consistent with the low num- are immune regulatory with preferential expression of IFN-b and bers of CD11b+ CD11c+ cells we have found in young mice. IL-10 and inflammatory CD11c+CD11b+AMs. Fate mapping and Sterile inflammation induced by LPS (69, 70), organic dust and adoptive transfer experiments are planned in future experiments to peptidoglycan (71), allergic lung inflammation induced by host determine the origin of the CD11c+ CD11b+AMs. dust mites and OVA (72), influenza A virus (69, 70), and pathogens A prior study by Rottinghaus et al. (33) evaluated macrophage S. pneumoniae (73), Coxiella burneti (74), and M. tuberculosis (75) populations isolated from lung tissue digests in which two mac- have been shown to induce the appearance of CD11c+ CD11b+ rophage populations were identified: a major population that is AMs in the lung. Our studies differ in that higher numbers of these CD11c+ CD11blow-neg and a minor population that is CD11b+ with AMs are present in the alveolar space of naive old mice. We pro- intermediate to low levels of CD11c expression and increased pose that these AMs accumulate as the result of multiple rounds of numbers in old mice. This minor population differs to some de- low-grade inflammatory inciting events or a persistent low-grade gree from the CD11c+ CD11b+ AMs isolated by BAL described in baseline inflammation that occurs with aging. LPS-induced in- this article because the latter have high expression of CD11c. This flammation can recruit CD11c2 CD11b+ macrophages into the lung suggests that the minor population described by Rottinghaus et al. alveolar space, which peaks at 3 d after LPS instillation (69). As represents an interstitial macrophage population. Three interstitial inflammation resolves, the recruited AMs begin to acquire CD11c lung macrophage populations have recently been described by expression. Return to homeostasis occurs as the recruited macro- Gibbings et al. (68) in young mice that are CD11b+ with varying phages progressively decline in number because of FAS-mediated levels of CD11c expression. The percentage of lung myeloid cells apoptosis. However, at resolution of the inflammation at 12 d, that are interstitial macrophages (9%) is also comparable to the recruited AMs with high expression of CD11c and CD11b are still percentage of CD11b+ CD11c1ow-interm. macrophages (10% in present. Thus, inflammation with aging could represent an inability young mice and 20% in old mice) described by Rottinghaus et al. to return to homeostasis, resulting in more CD11c+ CD11b+ AMs in The observation that this population is higher in old mice may the lung. indicate that interstitial macrophages increase with age. The Several studies have examined the in vivo uptake and transport of Rottinghaus et al. (33) study also investigated the response of M. tuberculosis by immune cells during the early innate immune CD11c+ pulmonary cells isolated by positive selection after lung phase (76, 77) and emphasize the important role of AMs in this tissue digestion. They showed that CD11c+ pulmonary cells from process. Macrophages of different lineages have divergent re- old mice had higher TLR2 expression levels compared with young sponses to M. tuberculosis. For example, Huang et al. (77) have mice. In response to M. tuberculosis infection, they showed that shown that AMs are more permissive for M. tuberculosis growth 10 ANALYSIS OF ALVEOLAR MACROPHAGE POPULATIONS IN OLD MICE than recruited monocyte-derived interstitial macrophages, whereas was heat inactivated; thus, AM uptake assessed was largely non- the interstitial macrophages are responsible for the strong proin- opsonic. CD11c+ CD11b2 AMs express CR4 (CD11c/CD18) and flammatory response. In the current study, CD11c+ CD11b+ AMs MR to which M. tuberculosis canbindintheabsenceofop- from old mice are more permissive for M. tuberculosis than sonins (88, 89). The CD11c+ CD11b+ AMs also expressed CR3 CD11c+CD11b2 AMs. Thus, we suggest that CD11c+ CD11b+ (CD11b/CD18). Because CR3 mediates ∼40–50% of non- AMs provide a unique replicative niche for M. tuberculosis ex- opsonic binding to CD11b+ macrophages (90), it is likely that pansion in the alveoli and possibly translocate M. tuberculosis to expression of CR3 on the CD11c+ CD11b+ AMs accounts for the interstitium. Cohen et al. (76) reported that translocation of the increased M. tuberculosis phagocytosis. The CD11c+ infected AMs requires IL-1R signaling by nonhematopoietic cells, CD11b+ AMs also express higher levels of TLR1, which pairs most likely epithelial cells. Because we have found that aging with TLR2, to bind triacetylated lipoproteins, lipoglycans, increases IL-1b levels, it will be interesting to determine if aging and glycolipids expressed by M. tuberculosis. Because TLR2 increases the translocation of infected cells into the interstitium. signaling can increase the avidity of CR3 (91), the increased Previous studies (37, 78, 79) have designated lung CD11c+ expression of TLR1 may further enhance phagocytosis. The CD11b+ cells as dendritic cells. However, Cohen et al. (76) increased numbers of M. tuberculosis seen in infected CD11c+ reported that M. tuberculosis was not present in conventional CD11b+ AMs at 24 h may directly relate to the increased dendritic cells. This difference can be explained by studies phagocytosis but may also indicate that these cells enable in- (80–82) showing that monocyte-derived CD11c+ CD11b+ cells creased growth, a focus of ongoing studies. previously defined as conventional dendritic cells express CD64, a Cell transfer studies (92, 93) have shown that the lung envi- marker that is exclusively expressed on macrophages and absent ronment maintains the phenotype of AMs. For example, cell Downloaded from on dendritic cells (44), resulting in these cells being reclassified as transfer of yolk sac macrophages, fetal liver, and adult monocytes monocyte-derived macrophages. Our studies show that CD11c+ into mice lacking AMs generates AMs with identical phenotype CD11b+ AMs from old mice also express CD64 and are positive (93). However, our knowledge of how the cytokine environment for Ly6C, indicating these cells are also monocyte-derived mac- induces the phenotype of the two AM subpopulations is mostly rophages. Therefore, we speculate that the CD11c+ CD11b+ AMs limited to studies of growth factors GM-CSF and M-CSF.

not only transport M. tuberculosis to the interstitium but also GM-CSF is required for the differentiation of fetal monocytes http://www.jimmunol.org/ transport M. tuberculosis to lung draining lymph nodes where into AMs (45) and acts through transcription factor PU.1 and M. tuberculosis is disseminated to lymph node dendritic cells. In nuclear receptor PPAR-g (63, 94) to regulate surfactant homeo- support of this, we have found that CD11c+ CD11b+ AMs pref- stasis and innate immune functions of AMs (95), including the erentially express CCR7, which is required for migration to lymph ability to phagocytose and kill M. tuberculosis (96). M-CSF is nodes (83). Alternatively, the M. tuberculosis–infected CD11c+ responsible for survival and differentiation of monocytes into CD11b+ AMs could disseminate the M. tuberculosis to infil- macrophages. Both GM-CSF and M-CSF are capable of inducing trating monocyte-derived (CD64+ CD11c+ MHCII+) cells after AM proliferation (97). We found that CD11c+ CD11b+ AMs ex- entering the interstitium [which Cohen et al. (76) reported to press CD115, the receptor for M-CSF, and preferentially express contain M. tuberculosis at 11 d postinfection] that then transport higher mRNA levels of M-CSF than the CD11c+ CD11b2 AMs, by guest on September 25, 2021 the M. tuberculosis to the draining lymph nodes. suggesting that CD11c+ CD11b+ AMs are the source of M-CSF We also examined the in vitro phagocytosis of M. tuberculosis and are responsive to M-CSF. During the course of M. tuberculosis by the two AM subpopulations isolated by cell sorting AMs from infection in mice, there is a progressive decrease in the levels of old mice. Our prior study (32) showed that phagocytosis and M-CSF in the lung (98). Intratracheal delivery of M-CSF into phagocyte-lysosome fusion were higher in lung macrophages lungs of M. tuberculosis–infected mice reduced the number of from old mice but that M. tuberculosis growth was also higher at foamy macrophages and increased expression of MHC class II, 24 h postinfection. In the current study, we determined that CD11b, and CCR7 in AMs. Further, there was an increase in the CD11c+ CD11b+ AMs phagocytosed more M. tuberculosis than T cell stimulating activity of M-CSF–treated AMs. In another CD11c+ CD11b2 AMs at 2 h and contained more M. tuberculosis study, intratracheal delivery of M-CSF increased CCL2 in BALF at 24 h. Further, in a pilot experiment, we found that expression of and in vitro M-CSF induced production of CCL2 by bone mar- M. tuberculosis genes dnaA, icl, sodC, sigA, and sigE was sig- row–derived macrophages (99). Together, these two studies are nificantly higher in CD11c+ CD11b+ AMs. The gene product indicative of M-CSF potentially maintaining the phenotype of encoded by dnaA is involved in the initiation of M. tuberculosis CD11c+CD11b+ AMs we described in this article. replication (48), and thus upregulation of dnaA in CD11c+ In summary, our studies show that old mice possess a unique CD11b+ AMs is an indication that M. tuberculosis is replicating. baseline alveolar cytokine environment consisting of both in- The icl gene encodes the isocitrate lysate enzyme, which is es- flammatory and immune-regulatory cytokines, largely generated sential for the metabolism of fatty acids and is required for per- by AMs. Aging also alters the phenotype of AMs with a newly sistence of M. tuberculosis in macrophages (49). sodC encodes identified increase in a subpopulation of CD11c+ CD11b+ AMs superoxide dismutase, which is a strong superoxide radical scav- that are more inflammatory than the resident CD11c+ CD11b2 enger and is involved in the resistance of M. tuberculosis to oxi- AMs, phagocytose more M. tuberculosis, and allow for more dative stress (50). sigA and sigE genes encode s factors SigA and bacterial growth. Our studies raise the possibility that CD11c+ SigE. SigA is the principal s factor and mediates enhanced growth CD11b+ AMs transport M. tuberculosis into the lung interstitium in macrophages and in vivo (51). SigE is induced by various ox- and draining lymph node, enabling bacterial dissemination. We idative stressors, and sigE mutant M. tuberculosis is defective in propose that the changes in cytokine levels and AM phenotype the ability to grow in human and mouse macrophages and is un- account in part for the eventual loss of M. tuberculosis control that able to arrest phagosome maturation (52, 53). These experiments occurs with aging. We posit that increased levels of CCL2 will support our premise that CD11c+ CD11b+ AMs are more per- increase migration of CD11b+ macrophages into the lung and missive for M. tuberculosis than CD11c+CD11b2 AMs. maintain CD11b expression in response to M. tuberculosis in- Macrophages phagocytose M. tuberculosis following opsonic fection (100), whereas increased IFN-b and IL-10 are expected and nonopsonic binding (27, 84–88). In the current study, serum to counteract IFN-g by inhibiting production of IL-12 and The Journal of Immunology 11 downregulating the IFN-gR (101–104). In this milieu, IFN-b 28. Ferguson, J. S., J. J. Weis, J. L. Martin, and L. S. Schlesinger. 2004. Com- plement protein C3 binding to Mycobacterium tuberculosis is initiated by the may also act to sustain CCL2 production (105). classical pathway in human bronchoalveolar lavage fluid. Infect. Immun. 72: 2564–2573. 29. Arcos, J., S. J. Sasindran, N. Fujiwara, J. Turner, L. S. Schlesinger, and Disclosures J. B. 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Supplemental Figure. S1 Representative confocal images of CD11b, CD11c and Man R expression in AMs from young and old mice. Representative of N = 3 experiments with 10-15 images/Ab in each experiment.

Supplemental Figure S2. Flow cytometry analysis of CD11b sub-populations in old mice. A.

Representative gating strategy. B. MFI of macrophage markers in CD11b negative, CD11b dim, and CD11b high populations. C. MFI of monocytic markers. N=4 BAL isolations of 10 mice.

Supplemental Figure S3. Representative flow cytometry CD11c CD11b plots of AMs from old mice showing the pre-sort AM populations and Post-sort CD11c+ CD11b- and CD11c+ CD11b+ AM subpopulations. Representative of N = 10 flow sorting experiments.