Monocytes from Older Adults Maintain Capacity for Metabolic Compensation during Glucose Deprivation and Lipopolysaccharide Stimulation

Inflammaging is the chronic low-grade inflammation that occurs with age that contributes to the pathology of age-related diseases. Monocytes are innate immune cells that become dysregulated with age and which can contribute to inflammaging. Metabolism plays a key role in determining immune cell functions, with anti-inflammatory cells primarily relying on fatty acid oxidation and pro-inflammatory cells primarily relying on glycolysis. It was recently shown that lipopolysaccharide (LPS)-stimulated monocytes can compensate for a lack of glucose by utilizing fatty acid oxidation. Given that mitochondrial function decreases with age, we hypothesized that monocytes taken from aged individuals would have an impaired ability to upregulate oxidative metabolism along with impaired effector functions. Aging did not impair LPS-induced oxygen consumption rate during glucose deprivation as measured on a Seahorse XFp system. Additionally, aged monocytes maintained inflammatory gene expression responses and phagocytic capacity during LPS stimulation in the absence of glucose. In conclusion, aged monocytes maintain effector and metabolic functions during glucose deprivation, at least in an ex vivo context.

anti-inflammatory phenotypes as in quiescent memory T-cells, regulatory T-cells, and M2 macrophages 18 . The metabolic inflexibility of an immune cell can lead to increased inflammation. Macrophages are able to convert from one form to another and can switch their metabolism during inflammation from relying on glycolysis in the M1 state to relying on OXPHOS in the M2 state 19 . Inhibition of OXPHOS in macrophages inhibits the expression of the M2 anti-inflammatory phenotype 20 . Therefore, diminished mitochondrial function is presumed to affect the function and phenotype of certain immune cells.
Aging has been shown to cause decreased mitochondrial function with an estimated decline of 8% in ATP producing capacity per decade 21 . This decrease in function is thought to be largely caused by mitochondrial DNA (mtDNA) mutations, which occur at an estimated rate of 15x that of the nuclear genome 21 . Since mitochondrial dysfunction is impaired it is reasonable to assume that many cell types in older individuals must produce more energy through non-oxidative metabolism. Aged mice have been shown to have increased lactate and reduced glycolytic intermediates in muscle and liver tissue which suggest an increased reliance on anaerobic glycolysis 22 . Whether or not mitochondrial dysregulation is a primary cause of age-related monocyte dysfunction has yet to be determined, though we've recently provided evidence that aging impairs maximal and spare mitochondrial respiratory capacity in classical monocytes isolated from humans 13 .
Raulien and colleagues recently showed that in the deprivation of glucose (as is often the case in tissues with inflammation) 23, 24 , LPS activated monocytes are able to switch from aerobic glycolysis to OXPHOS while maintaining their effector functions 14 . This switch is regulated by AMP-activated protein kinase (AMPK) which stimulates catabolic pathways including fatty acid oxidation, autophagy, and mitochondrial biogenesis 14 . Since we have previously demonstrated mitochondrial dysfunction in monocytes 13 , we hypothesized that aging would result in a reduced ability to upregulate oxidative metabolism when glucose is unavailable following LPS-stimulation and that this would be associated with reduced inflammation and phagocytic capacity.

Data availability
The datasets and analytical scripts supporting the conclusions of this article are available in the FigShare repository 25 .

Subject characteristics
Subject demographics and anthropometric data are shown in Table 1. There was a total of 9 subjects in the aged group and 11 subjects in the young group. Besides age, the two groups did not differ significantly on other demographic or anthropometric data. All subjects are the same individuals as those reported in our previous paper as cohort 2 26 .

Characteristics
Aged (

Metabolic flexibility assay, cytokine expression, and phagocytic capacity
Aging had no significant effects on any of the calculated measures for metabolic flexibility, cytokine expression, or phagocytic capacity in CD14+CD16-monocytes. Oxygen consumption rate (OCR) response to LPS in glucose-deprived monocytes was slightly higher across all timepoints in the aged group, though this difference was not significant (Fig. 1a). Extracellular acidification rate (ECAR) response to LPS in glucose-deprived monocytes also showed no difference between groups (Fig. 1b).
Aging had no effect on any of the calculated oxidative metabolic parameters ( Fig.1c & d) including maximum OCR (p=0.3312), minimum OCR (p=0.3702), the difference between them (p=0.2206), and OCR kinetics as measured by area under the curve (AUC) (p=0.3312). Aging also had no significant effects on measures of cytokine expression in monocytes in response to LPS (Fig. 1e) for IL-10 (p=0.5675), IL-6 (p=0.8421), TNFα (p =0.9048), or IL-1β (p=0.7802). Phagocytic capacity, as measured by percentage in the positive gate, was tested in glucose-deprived classical monocytes (Fig. 2). An example of the gating strategies used to isolate classical monocytes (Fig.2a) and to calculate percentage positive for beads (Fig.2b) are shown. Aging had no effect on the mean fluorescence intensity (MFI) in LPS-stimulated (p=0.748, Fig.  2c) or unstimulated (p=0.1396, Fig. 2c) glucose-deprived classical monocytes. Similarly, aging had no effect on percentage of cells in the positive gate ( Fig. 2d) in LPS-stimulated (p=0.2340) or unstimulated (p = 0.6738) glucose-deprived classical monocytes. There was a significant main effect whereby LPS stimulation increased MFI compared to unstimulated (p=0.00301, not shown). Likewise, LPS stimulation caused a near significant increase in percentage of cells in the positive gate compared to unstimulated (p=0.0531, not shown).

Discussion
Monocytes switch from oxidative phosphorylation to aerobic glycolysis when activated by LPS to carry out their effector functions, which is dependent on the availability of glucose and glutamine. Monocytes routinely experience a variety of different microenvironments and must be metabolically flexible to retain their functions. In circulation glucose is readily available, but in areas with inflammation glucose is often drastically reduced, and monocytes must compensate through catabolic processes such as fatty acid oxidation and autophagy 14 . AMPK is the critical regulator orchestrating this metabolic switch, which provides fatty acids to the mitochondria through downstream effects 19 . Decreased mitochondrial function is known to occur in many cell types with age 21 , and we have previously provided evidence for reduced mitochondrial function in classical monocytes with aging-namely that aging impairs mitochondrial maximal respiration and spare capacity 13 .
We hypothesized that classical monocytes from aged individuals would have reduced ability to upregulate oxidative metabolism when glucose is unavailable due to decreased mitochondrial function, and that this inflexibility would impair their inflammatory responses and hamper their ability to perform phagocytosis. In this study, we show that aging has no effect on the ability of glucose-deprived classical monocytes to upregulate oxidative metabolism, and has no effect on the expression of IL-10, IL-6, TNFα, or IL-1β during acute LPS activation ex vivo. Additionally, we found aging has no effect on ex vivo phagocytic capacity in classical monocytes, regardless of the availability of glucose or whether they were activated by LPS. The differences in mitochondrial respiration in aged monocytes that we previously reported 13 were only under maximizing conditions (FCCP stimulation), and thus normal conditions are likely not severe enough to cause any noticeable impairments in mitochondrial metabolism.
While there is an ever-growing body of evidence supporting age-related changes in monocyte and macrophage functions, it has not been clear whether this is due to intrinsic changes in the monocytes themselves, or due to changes in circulating factors that alter gene expression. This study gives more credibility to the assumption that classical monocytes are not experiencing intrinsic changes to a degree which affect their functions. Aging may instead result in reductions in monocyte effector functions due to alterations in the in vivo environment, including exposure to inflammatory cytokines, damage-associated molecular patterns, and bacterial products which are increased in the circulation in older adults 8 .
There are several other potential reasons why we observed no differences in this study, which is likely not translatable to monocyte function in vivo. We previously corroborated evidence that alterations in monocyte subset proportions occur with age 13 . CD14+CD16-classical monocytes are reduced, while CD14+CD16+ intermediate and CD14dimCD16+ nonclassical monocytes are increased with age. Our monocyte isolation technique removes CD16+ monocytes and thus only included the classical monocyte subset. This was done to prevent biased results, as CD16+ monocytes have generally shown to be more pro-inflammatory and are more prone to senescence 27 . Furthermore, monocyte subsets have been shown to display varying responses to LPS 28 . Isolating all monocyte subsets therefore may have yielded different results, and it would be interesting to see a similar experiment with respect to metabolic flexibility performed with CD16+ monocytes only, to see if there are intrinsic changes in CD16+ monocytes with age.
Monocytes also have varying responses to different types of pattern recognition receptors (PRR). Activation by Pam3CysSK4, a TLR2 agonist, shows significant differences in TCA cycle, OXPHOS, and lipid metabolism compared to activation by LPS in glucose-fueled monocytes 29 . Therefore, activation of PRRs other than TLR4 may have given different results.
In summary, we showed that aging has no effect on the ability of ex vivo LPS-stimulated classical monocytes to compensate for a lack of glucose by upregulating oxidative metabolism. Furthermore, aging has no effect on cytokine expression in ex vivo LPS-stimulated glucose-deprived classical monocytes for IL-10, IL-6, TNFα, or IL-1β . Aging also had no effect on the phagocytic ability of classical monocytes under various conditions.

Participants
Males and females between the ages of 18-35 were recruited for the young group, which consisted of 11 subjects total. Males and females between the ages of 60-80 were recruited for the aged group, which consisted of 9 subjects total. Demographic and Anthropometric data of the subjects can be seen in Table 1. All subjects were recruited from the surrounding Memphis area via word-of-mouth, email, or flyers. Exclusion criteria included subjects who had diagnosed conditions that can affect metabolic or immune function. This includes obesity (BMI >30), cardiovascular disease, diabetes, hypertension, chronic fatigue syndrome, mitochondrial diseases, autoimmune diseases, etc. All subjects completed a questionnaire to determine eligibility, which asked for health history, list of medications and supplements, major illnesses or hospitalizations within the last two years, and exercise type/frequency. Subjects visited the lab in a fasted state, up to 6 times, and had 8-16 mL of blood taken by venipuncture into 1-2 EDTA vacutainer tubes per visit for monocyte isolation.
Monocyte Isolation and Metabolic Flexibility Assay CD14+ monocytes were isolated from whole blood via magnetic sorting by negative selection using Stemcell Technologies' EasySep Direct Human Monocyte Isolation Kit. CD16+ monocytes were excluded to prevent bias as monocyte subset proportions change with age 13 . Therefore, in this study we only looked at classical monocytes. After isolation the monocytes were counted using EMD Millipore's Scepter 2.0 cell counter for use in downstream assays. The Agilent Seahorse XFp Analyzer was used to test the metabolic flexibility of the isolated classical monocytes. The Seahorse analyzer measures the oxygen consumption rate (OCR) and extra-cellular acidification rate (ECAR) of the sample. OCR is an indicator of OXPHOS and ECAR is an indicator of glycolysis. Monocytes were seeded into the Seahorse plates at 1.5 x 10 5 cells per well (B-G) along with XF media (DMEM, pyruvate, glutamine), and either glucose or no glucose. 20µL of 100ng/mL LPS was added to the injection port of three wells containing glucose (B-D), three wells without glucose (E-G), and two wells (A, H) consisting of no cells, but XF media, which are used as blanks. The Seahorse plates were incubated for 60 minutes at 37 o C in a non-CO2 incubator to de-gas the plate. The Seahorse machine was then run for 160 minutes to measure the acute response in OCR and ECAR of the monocytes activated by LPS in media with or without glucose. This data was used to compare the metabolic flexibility of isolated monocytes between the aged and young groups. Wells B-G of the Seahorse plate were imaged using a microscope at 10x magnification to confirm cell adherence and for use in cell number normalization when doing data analysis. After completion, wells B-G received 100µg of TRIzol, pooled by group (+ or -glucose) and were stored in a -80 o C freezer for later RNA quantification using Real-Time polymerase chain reaction (qPCR).

Phagocytosis Assay
To test potential differences in monocyte effector function between young and aged CD14+ CD16-monocytes, a phagocytosis assay was used in the presence and absence of glucose. The assay uses latex beads coated with fluorescently labeled IgG to quantify phagocytosis in vitro and was measured using the Attune Nxt flow cytometer. Monocytes were isolated as above and added to 2mL tubes at a concentration of 5 x 10 5 cells. The 2 groups consisted of XF media without glucose, and LPS + XF media without glucose. After 30 minutes of incubation at 37 o C with 5% CO2, 1µL phagocytotic beads was added. After another hour of incubation 20µL anti-CD14-PE antibody was added. After another 30 minutes of incubation the cells were washed with PBS and resuspended in 400µL PBS then analyzed with the Attune Nxt flow cytometer to determine the mean fluorescence intensity (MFI, the average amount of beads phagocytosed) and percentage of beads (% gated) phagocytosed by CD14+CD16-monocytes.

Cytokine Expression Quantification using qPCR
To test differences in gene expression of 4 cytokines (IL-1β , IL-6, TNFα, IL-10), qPCR was performed on monocyte lysates from the Seahorse assay as we have previously described 26 . Relative expression levels were calculated using the comparative CT method using β 2 microglobulin (B2M) as a control. B2M was picked as a control as there is evidence suggesting it's the most stable reference gene in LPS-stimulated monocytes 30 .

Statistical Analysis
Statistical analysis was performed using R software (R v. 3.5.1). Categorical demographic data was analyzed by chi-square test (sex, race). Continuous demographic and anthropometric data (age, height, weight, body mass index) were analyzed by independent-samples t-test between young and older subjects. For metabolic parameters all data followed a normal distribution according to Shaprio-Wilk tests. However, only difference in OCR (max-min) between groups had equal variances according to Levene's test. Therefore independent-samples t-test was only used to calculate difference in OCR for metabolic parameters. Due to the unequal variances for the remaining parameters (max OCR, min OCR, area under the curve (AUC) kinetic OCR response) Mann-Whitney U tests were performed to test for significance between groups. For cytokine gene expression data at least 1 group for all genes did not meet the criteria for approximating normal distribution according to Shaprio-Wilk test, although all genes displayed equal variance between groups according to Levene's test. Therefore, Mann-Whitney U tests were performed to test for significance between groups. For phagocytosis, all data met criteria for normal distribution and equality of variances according to Shapiro-Wilk and Levene's test. Between group differences were determined by 2x2 (Condition x Group) ANOVA. As is standard, a p value < 0.05 was considered significant. Reported results are mean ± SEM.