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. Author manuscript; available in PMC: 2025 Aug 1.
Published in final edited form as: J Immunol. 2024 Aug 1;213(3):317–327. doi: 10.4049/jimmunol.2400140

Obesity inhibits alveolar macrophage responses to Pseudomonas aeruginosa pneumonia via upregulation of PGE2 in male, but not female mice

Gabrielle P Entrup *, Aayush Unadkat †, Helen I Warheit-Niemi ‡, Brooke Thomas †, Stephen J Gurczynski ‡, Yuxiao Cui §, Andrew M Smith §, Katherine A Gallagher ‡, Bethany B Moore ‡,∥, Kanakadurga Singer ¶,∥
PMCID: PMC11250913  NIHMSID: NIHMS1995649  PMID: 38905107

Abstract

Obesity is associated with increased morbidity and mortality during bacterial pneumonia. COX-2 and PGE2 have been shown to be upregulated in patients who are obese. In this study, we investigated the role of obesity and PGE2 in bacterial pneumonia and how inhibition of PGE2 improves anti-bacterial functions of macrophages. C57Bl/6J male and female mice were fed either a normal diet (ND) or high fat diet (HFD) for 16 weeks. After this time, animals were infected with Pseudomonas aeruginosa in the lung. In uninfected animals, alveolar macrophages were extracted for either RNA analysis or to be cultured ex vivo for functional analysis. HFD resulted in changes in immune cell numbers in both non-infected and infected animals. HFD animals had increased bacterial burden compared to ND animals, however, male HFD animals had higher bacterial burden compared to HFD females. Alveolar macrophages from HFD males had decreased ability to phagocytize and kill bacteria and were shown to have increased COX-2 and PGE2. Treating male, but not female alveolar macrophages with PGE2 leads to increases in cAMP and decreased bacterial phagocytosis. Treatment with lumiracoxib-conjugated nanocarriers targeting alveolar macrophages improves bacterial phagocytosis and clearance in both ND and HFD male animals. Our study highlights that obesity leads to worse morbidity during bacterial pneumonia in male mice due to elevated PGE2. Additionally, we uncover a sex difference in both obesity and infection, as females produce high basal PGE2, but due to a failure to signal via cAMP do not display impaired phagocytosis.

Keywords: Monocytes/macrophages, diabetes, bacterial infection, phagocytosis, lung, rodent

Introduction

Obesity is a global health concern. Overweight and obesity status is defined by BMI (body mass index) with a BMI of 25 to 30 kg/m2 defined as overweight, and a BMI greater than 30 kg/m2 defining obesity status. It is estimated that 69% of adults in the United States are overweight or obese, and this number continues to rise1. In 2021, it was estimated that 529 million people were living with diabetes globally. Type 2 diabetes (T2D) makes up 96% of these cases and is increasing in prevalence worldwide2. It is estimated that the cost to care for patients with T2D in the United States will triple by 2034, and is estimated to reach $336 billion3. While there is a growing list of treatments for obesity and type 2 diabetes, individuals with elevated BMI are also at risk for non-metabolic complications such as infection, as seen during the recent COVID-19 pandemic4. Therefore, it is critical to understand why these patients are more susceptible to infection, which can lead to better targeted treatment approaches for this at-risk population. The goal of this study is to evaluate the impact of obesity on bacterial lung infection, with a focus on sex differences.

While obesity and T2D are associated with increased risk for infection, T2D is associated with chronic inflammation termed meta-inflammation. This meta-inflammation leads to chronic macrophage activation and production of pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6, and IL-84. This is a consequence of cell specific changes driving this inflammatory profile, which has both metabolic and non-metabolic consequences.

Despite these pro-inflammatory changes, it has been shown that patients who are obese have worse outcomes to bacterial and viral pneumonia5. Hyperglycemia stemming from obesity leads to increased glucose concentrations in the lung, which allows for bacteria to more easily replicate and establish an infection6. Additionally, increased fat mass results in decreased diaphragm excursion, which restricts ventilation and can lead to decreased ability to clear infections7. Most studies related to pneumonia in obesity have focused on viral infection or on the bacterium Klebsiella pneumoniae. Thus, there is a gap in knowledge regarding outcomes with other Gram-negative pathogens and the mechanisms underlying increased severity of infection. Furthermore, little is known about how sex differences influence obesity-induced inflammation and outcomes of bacterial pneumonia.

Prior studies evaluating infection in animal models have demonstrated increased bacterial burden in obese animals. In a study looking at K. pneumoniae infection in obese mice, it was found that macrophages from obese mice had impaired bacterial phagocytosis8. Additionally, with K. pneumoniae, it took fewer bacteria to establish infection in obese mice and chemokine-mediated neutrophil recruitment to the site of infection was impaired8. In a different study, obese mice had increased numbers of macrophages, but these macrophages were not functional, resulting in increases in bacterial burden following infection with Gram-positive Staphylococcus aureus9. Given the increased risk for Pseudomonas aeruginosa infections in diabetes10 and a paucity of studies on this cause of pneumonia in obesity, we chose to study the mechanisms which might account for increased susceptibility to this Gram-negative infection. Treatments for Pseudomonas infections are of particular interest to study as this bacterium employs several resistance mechanisms and is a leading cause of hospital-acquired and ventilator-acquired pneumonia.

Interestingly, most investigations of meta-inflammation and impaired host defense to bacterial infection occur in males. Total diabetes prevalence is higher in males than in females, and men and women have different responses to an obese state11. In a mouse model of high fat diet (HFD) induced T2D, males fed a HFD have an increase in CD11c+ adipose tissue macrophages, while females are protected from this12. A lipidomic analysis of the adipose tissue in male and female HFD mice show that there is a sexual dimorphism in how each sex stores lipids13.

It is also known that there is a sex difference in innate and adaptive immunity between males and females14. Studies have shown that females are less likely to develop pneumonia, and if they do, they have a more favorable outcome compared to men15,16. In mice treated with LPS intratracheally, males exhibited more severe hypothermia and an increase in total bronchoalveolar lavage cells and neutrophils17.

With the prior literature showing impairment in clearing bacterial infections in obesity, there are two gaps in knowledge: the first is understanding the sexual dimorphism in both obesity and bacterial pneumonia responses and the second is understanding the mechanism for impaired bacterial clearance. One class of lipid mediators implicated in myeloid functions against pathogens are prostaglandins. The prostaglandin E2 (PGE2) pathway has been associated with impaired host defense and chronic inflammation in a variety of conditions, notably in the failure to heal diabetic wounds and in the setting of impaired host defense post-stem cell transplant18–20. PGE2 is a lipid mediator derived from arachidonic acid metabolism. It has been shown to have functions such as enhancing vascular permeability, stimulating production of cAMP, and suppressing phagocytosis. In a bone marrow transplant model, increased PGE2 following transplant leads to altered expression of scavenger receptors, which leads to a decreased ability of alveolar macrophages to phagocytize Pseudomonas aeruginosa21.

Therefore, we hypothesized that alveolar macrophages from male mice in an obese state have defects in bacterial phagocytosis and clearance of P. aeruginosa due to an increase in PGE2. To study this, we employed a high fat diet mouse model, where male and female mice were fed a diet consisting of 60% calories from fat for 16 weeks. The goal of this project is to understand the functional changes in alveolar macrophages that contribute to increased morbidity during bacterial pneumonia in patients who are obese and to understand differences in how males and females respond to metabolic disease.

Materials and Methods

Animals

C57Bl/6J male and female mice were purchased from Jackson Laboratories (000664) at 5 weeks of age. Animals were housed in a pathogen-free facility and at 6 weeks were either maintained on normal diet (ND) chow (5L0D, 13.5% fat, Lab Diets) or started a HFD chow (D12492: 60% fat, Research Diets) for the specified length of time (16 weeks). For bone marrow transplant studies, CD45.1 (Jackson Laboratories, 002014) mice were used as donors and wildtype C57Nl/6J CD45.2 mice were used as recipients. Animal protocols were in compliance with the Institute of Laboratory Animal Research Guide for the Care and Use of Laboratory Animals and approved by the University Committee on Use and Care of Animals at the University of Michigan.

P. aeruginosa Infection

PA01 was obtained from ATCC. Mice were anesthetized with isoflourane and 50 μL of PBS containing 5×105 CFU of PA01 was injected into the back of the throat while the tongue is pulled forward and inoculum was aspirated into the lungs. Experiments were terminated 24 hours after infection and mice were euthanized by inhalation of CO2.

Colony forming unit assay to detect bacterial burden

After euthanasia, the lungs were perfused with 3 mL sterile PBS. Lungs were homogenized in 1 mL of sterile PBS with cOmplete protease inhibitor (Roche, MilliporeSigma). Lung homogenate was serially diluted and plated on Pseudomonas Isolation Agar (BD Difco) for 24 hours at 37°C to determine bacterial burden. Remaining lung homogenate was stored at −80°C.

Bronchoalveolar lavage (BALF)

After euthanasia, a catheter was placed intratracheally, and the lungs were flushed twice with 1 mL of PBS. BALF was centrifuged at 400 × g for 5 minutes to pellet cells. Supernatant was collected and stored at −80°C until use. To obtain alveolar macrophages for ex vivo experiments, lungs were flushed with 20 × 1 mL of complete DMEM. Red blood cells were lysed with ACK lysis buffer and macrophages were adherence purified for 1 hour. Cell count and viability was determined by trypan blue (Corning) exclusion on a hemocytometer.

Bacterial phagocytosis and killing assay

Alveolar macrophages or neutrophils were plated at 200,000 cells per well in a 96 well flat-bottomed plate and allowed to adhere overnight (or for 1 hour for one experiment). For serum opsonization, 1 × 108 PA01 was resuspended in 5% immune serum in PBS and incubated for 15 minutes on a rocker at 37°C. Cells were incubated with opsonized PA01 for 30 minutes to allow for bacterial phagocytosis, after which extracellular bacteria were washed away. Any remaining extracellular bacteria were killed with 100 mg/mL gentamicin (ThermoFisherScientific) for 10 minutes, which we confirmed was sufficient to kill all extracellular bacteria without changing macrophage viability. One set of infected cells were lysed with 0.5% saponin to release intracellular bacteria, and cell lysates were serially diluted and plated on Pseudomonas Isolation Agar (BD Difco) to determine intracellular bacterial burden. The second set of infected cells were incubated for 2 hours to allow time for intracellular bacterial killing, after which cells were lysed with 0.5% saponin and diluted to determine surviving bacteria. To calculate percentage survival, the intracellular bacterial CFU after 2 hours incubation was divided by the intracellular bacterial CFU quantified after 30 minutes.

Bacterial Growth Curve

PA01 was grown in TSB broth (BD Difco) overnight at 37°C. Culture was then diluted to 1×106 CFU/mL. BALF was collected from untreated male or female mice fed normal or HFD in a 2 mL volume. Then 100 μL of cell-free BALF was plated per well in a 96 well round bottom plate and inoculated with 10 μL of 1×106 CFU/mL culture of PA01 resulting in 1×104 CFU/ml PA01; uninoculated control wells were also included. Plate was incubated at 37°C and shaken at 90 rpm for 20 hours with OD600 readings taken every 20 minutes. OD600 readings for uninoculated wells were subtracted from inoculated wells.

Total lung macrophage preparation for flow cytometry

Total lung cells were isolated via collagenase digest as previously described22. Lungs were collected and minced with scissors and incubated in media containing DMEM, 10% fetal calf serum, 1 mg/mL collagenase (MilliporeSigma), and 30 μg/mL DNase for 35 minutes at 37°C. After mechanical dispersion, the cell suspension was strained through a 100 μM filter. The cell suspension went through a 20% Percoll (MilliporeSigma) gradient by being centrifuged at 2000g for 20 minutes at 4°C with deceleration set to 0. Total cell counts and viability were determined via trypan blue (Corning) exclusion on a hemocytometer. Gating strategies were described by Yu et. al23.

After total lung cells were quantified following collagenase digest, nonspecific Fc binding was blocked by incubation of anti-CD16/32 for 15 minutes on ice. After Fc-block, primary antibodies were added to the cells and incubated for 30 minutes in the dark. The antibodies used were the following: Ly6G-FITC (catalog 561105), SiglecF-APC/Cy7 (catalog 565527, Biolegend), MHC II-BV421 (catalog 562564), CD45-BV510 (catalog 563891) (BD Biosciences), Ly6C-PE/Cy7 (catalog 128018), CD24-PerCP/Cy5.5 (catalog 101824), CD64-BV605 (catalog 139323), CD11b-BV650 (catalog 101259), and CD11c-PE/dazzle (catalog 117348) (BioLegend). After staining, cells were washed and fixed in 4% paraformaldehyde. Cells were run on a BioRad Ze5. Data was analyzed via FlowJo.

Bone Marrow Transplant (BMT)

Recipient male C57BL/6 CD45.2 mice were irradiated with 2 doses of 600 rads using an x-ray source, spaced 3 hours apart. Immediately after the second irradiation, mice were transplanted with 1×107 cells from male C57BL/6 CD45.1 mice through retroorbital injection. Animals were analyzed 5 weeks post BMT.

Isolation of RNA for qRT-PCR

Total RNA was isolated from cells using TRIzol (Invitrogen, ThermoFisher Scientific) according to the manufacturer’s instructions. RNA concentration was normalized between samples. qRT-PCR was performed using an ABI StepOnePlus real-time thermocycler (Applied Biosystems, Thermo Fisher Scientific) and TaqMan RNA-to-Ct 1-Step Kit (Applied Biosystems, Thermo Fisher Scientific). Expression was normalized to GAPDH.

PGE2 ELISA

Macrophages were plated in serum-free media for 24 hours and supernatant was collected. For whole lung studies, lungs were homogenized in 1 mL of sterile PBS with cOmplete protease inhibitor (Roche, MilliporeSigma). Lipids were concentrated from whole lung with solid-phase extraction using Sep-Pak cartridges (Waters Corporation). ELISA was performed using a kit from Cayman Chemical (514010, Ann Arbor, MI) according to manufacturer’s instructions.

cAMP assay

To measure cAMP production, alveolar macrophages were plated at 2×105 cells per well and treated with 100 nM PGE2 for 30 minutes. Cell lysates were prepared using the cAMP Direct EIA Kit (Assay Designs) according to manufacturer’s instructions.

PGE2 treatment and COX-2 inhibition (in vitro and in vivo)

PGE2 (Cayman Chemical) was used in culture at a concentration of 100 nM for 6 hours. Indomethacin (Cayman Chemical) was given at a concentration of 5 μM for 6 hours.

Nanoparticle synthesis and instillation

Nanoparticles were created to deliver a COX-2 inhibitor to macrophages directly in vivo. Nanocarriers containing free dextran or dextran-conjugated lumiracoxib were synthesized in house as previously described19,24. Dextran or dextran-conjugated lumiracoxib were given 1 mg/kg via oropharyngeal aspiration at the same time as bacterial infection. Lungs were harvested 24 hours after infection as described above.

Statistics

Statistics were performed using GraphPad Prism software. Comparisons between two experimental groups were performed using the Student’s t test. Comparisons among 3 or more groups were performed with ANOVA, with post-hoc analysis. Experiments were performed at least twice. A p-value less than 0.05 was considered statistically significant.

Results

High fat diet mice have altered lung immune cell landscapes.

We have previously described a murine model of obesity, consisting of 16 weeks on HFD chow with 60% calories from fat25. In this model, both males and females gain weight as we demonstrated in our cohorts (Figure 1A); however, only the males have increased hyperglycemia and insulin resistance25. We first sought to investigate the distribution of alveolar macrophages and neutrophils after HFD in mice. We isolated total lung cells via collagenase digestion and the resulting single-cell suspension was stained for analysis by flow cytometry. Both male and female HFD mice had an accumulation of alveolar macrophages (CD45+CD11c+CD64+MHCII+) (Figure 1B) and neutrophils (CD45+Ly6G+) (Figure 1C). Interstitial macrophages, monocytes, and dendritic cells showed no difference (data not shown). Given the increase in innate immune cells in the lung, we next chose to probe the susceptibility of the HFD-fed mice to Pseudomonas aeruginosa.

Figure 1. HFD mice gain weight and have increased immune cells in the lungs.

Figure 1.

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(A) Male and female mice were given normal diet control chow (ND) or high fat diet chow (HFD) for 16 weeks to induce obesity. Body weight (g) was measured. (B) Collagenase digest was performed on the lungs of mice after 16 weeks of diet and flow cytometry was used to identify cell populations. Alveolar macrophages are described in (B) and neutrophils are described in (C). Alveolar macrophages were quantified by CD45+, CD11c+, CD64+, MHCII+. Neutrophils were quantified by CD45+, Ly6G+. Statistical analysis by ANOVA with Tukey’s multiple comparisons, and with 2 independent experiments combined (Panel A and B) and 3 experiments combined in panel C. For each individual experiment, n=3–5 mice per group were used. The apparent subgrouping in Figure 1B reflects the two different experiments, combined, but both individual experiments showed significant elevations in the male HFD mice relative to ND controls. For all, error bars represent the mean ± SE. *p < 0.05, **p < 0.01, ****p < 0.0001.

HFD mice have impaired anti-bacterial host defense.

We challenged male and female ND and HFD fed mice with 5×105 CFU of P. aeruginosa strain PA01 (PA01) for 24 hours (Figure 2A). We found that the HFD mice had no difference in percent weight loss compared to ND mice (Figure 2B). Both male and female HFD mice had increased bacterial burden in the lungs 24 hours after infection compared to the ND mice (Figure 2C). Strikingly, male mice had much higher bacterial burden than did female mice (Figure 2C). To determine if the bacteria are better able to grow in the lungs of the HFD mice, we performed a bacterial growth curve in BALF from ND and HFD mice. We found that PA01 grew better in the HFD BALF compared to ND BALF, regardless of sex (Figure 2D). To determine the immune cell profiles post-infection, we isolated total lung cells as previously described. Post-infection, there were no differences in the numbers of alveolar macrophages (Figure 2E), however, there was a significant decrease in neutrophil numbers in female HFD mice (Figure 2F).

Figure 2. HFD mice have impaired anti-bacterial host defense.

Figure 2.

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Schematic of mouse model is shown in (A). (B) Weight loss shown as a percentage of mice weight pre-infection. (C) Bacterial burden in the lungs from mice described in (A); representative of 3 independent experiments. (D) OD600 of P. aeruginosa grown in BALF for 20 hours, representative of 3 independent experiments. Each experiment contained 5 biological BALF replicates with mean calculated for each using 2 technical replicates of each. Data points on the graph represent the mean determined from the 5 biologic replicates. (E) Quantification of alveolar macrophages identified by flow cytometry from animals described in (A). (F) Quantification of neutrophils in the lung identified by flow cytometry from animals described in (A). For B, C, E, and F statistical analysis by ANOVA with Tukey’s multiple comparisons and with at least 2 independent experiments. For D, statistical analysis by student’s t test at the final time point, indicating significance of the difference in the equations for the slope of the growth curve for each group. For all, error bars represent the mean ± SE with n=4–5 mice per group. *p < 0.05, **p < 0.01, ****p < 0.0001.

Cell source and environment play a role in bacterial phagocytosis.

Based on these data, we sought to understand if alveolar macrophages in HFD mice had impairments in anti-bacterial functions. We measured PA01 phagocytosis and killing in alveolar macrophages cultured overnight from ND and HFD male and female mice and found that alveolar macrophages from male HFD mice had defects in both bacterial phagocytosis and killing (Figure 3A top and bottom panels). To confirm that the overnight culture did not change the macrophage phenotype, we repeated this assay in cells adhered for only 1 hour and got the same results (data not shown) suggesting the phenotypes were stable in culture for at least 24 h. Because this defect was only seen in male mice, we treated alveolar macrophages from male ND and HFD mice with BALF from male ND and HFD mice for 24 hours and then measured PA01 phagocytosis and killing. Treating ND alveolar macrophages with HFD BALF led to decreased bacterial phagocytosis, while treating HFD alveolar macrophages with ND BALF led to an increase in bacterial phagocytosis (Figure 3B, top). Bacterial killing was not altered (Figure 3B, bottom). To further explore the link between cell source, cell environment, and anti-bacterial functions, we employed a bone marrow transplant experiment using male mice. As described in Figure 3C, donor and recipient male mice were put on ND or HFD for 16 weeks. After 16 weeks, the recipient mice were irradiated and transplanted with donor bone marrow. The recipient mice remained on their same diet for 5 weeks to allow the bone marrow source to repopulate the lung compartment, as described previously26. After 5 weeks, alveolar macrophages were harvested, and we measured PA01 phagocytosis and killing. Only the HFD donor into a HFD recipient had defects in bacterial phagocytosis (Figure 3D left); however, killing was not different, likely reflecting altered regulation due to the bone marrow transplant procedure (Figure 3D, right). However, these data do suggest both cell source and environment play roles in anti-bacterial function of alveolar macrophages.

Figure 3. Cell source and environment have roles in impaired bacterial phagocytosis in male alveolar macrophages.

Figure 3.

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(A) Bacterial phagocytosis and killing of opsonized P. aeruginosa as described in materials and methods. Percentage survival is calculated by dividing the intracellular CFU quantified after 2 hours by the intracellular CFU quantified after 30 minutes and is representative of the bacterial killing by alveolar macrophages. Dots represent technical replicates of pooled cells from n=4–5 mice per group. (B) Alveolar macrophages treated with BALF from naïve male normal or high fat diet mice (n=4 per group) overnight before challenge with bacteria, represents technical replicates of pooled cells from one experiment, representative of 2 experiments with n=4–5 mice per group. Bacterial phagocytosis and killing described as before. (C) Schematic of bone marrow transplant mouse model. (D) Bacterial phagocytosis and killing from mice described in (C) with donors listed first and recipients listed second, representative of 3 independent experiments with n=4 mice per group. Statistical analysis by ANOVA with Tukey’s multiple comparisons and with at least 2 independent experiments. For all, error bars represent the mean ± SE. *p < 0.05, **p < 0.01, ****p < 0.0001.

Increased PGE2 in high fat diet males leads to impaired bacterial phagocytosis.

We have previously shown that wound macrophages in both HFD mice and patients with diabetes have increases in COX-2/PGE2, which leads to dysregulated wound healing19. Additionally, we have shown that increases in PGE2 following bone-marrow transplantation result in impaired host defense during bacterial pneumonia in male mice21. Therefore, we hypothesized that the male HFD alveolar macrophages had an increase in both COX-2 and PGE2. RNA was isolated from male and female ND and HFD alveolar macrophages and we found an increase in COX-2 gene expression in male HFD mice (Figure 4A). To measure PGE2 production, alveolar macrophages from male and female ND and HFD mice were plated in serum-free media overnight, and the supernatant was collected and PGE2 was measured via ELISA. We found that male HFD alveolar macrophages secreted more PGE2 than cells from ND male mice (Figure 4B). Interestingly however, cells from female mice expressed higher PGE2 levels on ND, and these levels were decreased somewhat on HFD (Figure 4B). Given this surprising sexual dimorphism, we wanted to assess the impact of PGE2 signaling in cells from each sex. To determine how exogenous PGE2 effects anti-bacterial functions, we treated male and female ND macrophages with PGE2 for 24 hours and then measured PA01 phagocytosis and killing. We found that male ND alveolar macrophages treated with PGE2 had defects in bacterial phagocytosis (Figure 4C, left), but not killing (Figure 4C, right). Females alveolar macrophages treated with PGE2. had no defects in either phagocytosis or killing, and in fact showed higher killing levels than did male mice. Previous research has shown that PGE2 treatment impairs bacterial phagocytosis via an increase in intracellular cAMP in males27. We treated male and female ND and HFD alveolar macrophages with PGE2 and measured intracellular cAMP with a fluorescence-based assay. We found that male ND and HFD alveolar macrophages treated with PGE2 showed increased amounts of intracellular cAMP whereas cells from female mice were unresponsive to addition of PGE2 (Figure 4D).

Figure 4. Increased PGE2 in HFD males leads to decreased bacterial phagocytosis.

Figure 4.

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(A) Measurement of COX-2 by qPCR from alveolar macrophages, combined from 3 independent experiments with n=3 mice per group (B) Measurement of PGE2 from alveolar macrophages via ELISA, combined from 2 independent experiments with n=4 mice per group. (C) Normal diet macrophages treated with medium control or PGE2 for 6 hours prior to bacterial phagocytosis and killing assay as described in materials and methods, combined from 2 experiments with n=3–4 mice per group. (D) Alveolar macrophages treated with PGE2 as in (C) before cAMP detection assay as described in materials and methods, representative of 2 independent experiments with n=4 mice per group. Statistical analysis by ANOVA with Tukey’s multiple comparisons and with at least 2 independent experiments. For all, error bars represent the mean ± SE. *p < 0.05, **p < 0.01, ****p < 0.0001.

High fat diet and PGE2 treatment led to decreased phagocytosis-associated receptors in male mice.

Previous research has shown that PGE2 administration leads to downregulation of MARCO, a macrophage scavenger receptor important for recognition of non-opsonized P. aeuriginosa in male mice fed ND21. We confirm here that alveolar macrophages from male mice on ND treated with PGE2 have decreases in MARCO, but this is not seen in female mice (Figure 5A). Additionally, HFD male alveolar macrophages have decreased MARCO gene expression (Figure 5B) which may be attributable to the PGE2 signaling in males. No doubt the decrease in MARCO may help explain the impaired host defense of the male HFD mice in vivo where some bacteria are likely encountered without opsonization. However, in our ex vivo phagocytosis and killing assays, we use opsonized P. aeruginosa, which would primarily be recognized by Fc receptors. We found that alveolar macrophages from male ND mice treated with PGE2 (Figure 5C) and alveolar macrophages from male HFD mice (Figure 5D) have decreased trends of FCγR1. PGE2 binds to prostaglandin E2 receptors (EP1–4), which are G-protein coupled receptors. To determine if altered signaling between males and females resulted from different levels of receptor expression, we measured the abundance of relevant EP receptors by mRNA expression as antibodies to these receptors are not available. Both EP2 (Figure 5E) and EP4 (Figure 5F) expression showed a trend towards decrease in macrophages from HFD male mice, however this did not reach statistical significance. We concluded that altered EP receptor expression likely could not explain the differential cAMP responsiveness between males and females.

Figure 5. HFD and PGE2 treatment lead to decreased phagocytosis-associated receptors in male mice.

Figure 5.

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(A) MARCO expression in male and female alveolar macrophages treated with PGE2 and (B) alveolar macrophages from normal and high fat diet mice. (C) FCγR1 expression in male and female alveolar macrophages treated with PGE2 and (D) alveolar macrophages from normal and HFD mice. (E) EP2 receptor expression and (F) EP4 receptor expression in male and female normal and HFD alveolar macrophages. Data are normalized to ND males. Statistical analysis by ANOVA with Tukey’s multiple comparisons, and combined from 2 independent experiments with n=4–5 mice per group for all panels. For all, error bars represent the mean ± SE. *p < 0.05, **p < 0.01, ****p < 0.0001.

Blocking PGE2 improves bacterial clearance in high fat diet male mice.

Previous research has shown that nanocarriers can be used to deliver COX-inhibitors to limit prostaglandin production in diabetic wound healing19. Therefore, we used these nanocarriers carrying a COX-2 inhibitor (lumiracoxib) in our model to test effects of COX inhibition19. We pre-treated macrophages from ND and HFD male mice with the nanocarriers for 4 hours before treating with PA01. We found that the COX-2 inhibited macrophages had improved bacterial phagocytosis (Figure 6A left), but no difference in killing (Figure 6A, right), consistent with results in Figure 4C. To test the therapeutic impact, the nanocarriers loaded with lumiracoxib were dosed at 1 mg/kg mouse weight and given with the bacterial infection in male mice. After 24 hours of infection, mice were euthanized and bacterial burden was quantified via CFU. We found that both ND and HFD male mice given the lumiracoxib-conjugated nanocarriers had improved bacterial clearance (Figure 6B) and PGE2 was significantly reduced in the lungs of the mice treated with the lumiracoxib-conjugated nanocarriers (Figure 6C). To investigate how lumiracoxib nanocarriers improved ability to phagocytize and clear bacteria, we evaluated expression of MARCO (Figure 6D) and FcγR1 (Figure 6E) in ND and HFD male macrophages treated with the lumiracoxib nanocarriers. We found a trend of increased MARCO expression in macrophages treated with the nanocarrier, and a significant increase in FcγR1 expression in HFD nanocarrier treated macrophages. These changes provide molecular insight into how lumiracoxib improves bacterial clearance.

Figure 6.

Figure 6.

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Blocking COX-2 improves bacterial phagocytosis and clearance. (A) Macrophages from ND and HFD male mice were treated with 10 uM control or coxib nanoparticles for 4 hours prior to phagocytosis and killing assay, combined from 2 experiments with n=5 mice per group. (B) Bacterial burden quantified 24 hours after infection. Control or lumiracoxib-conjugated nanoparticles dosed at 1 mg/kg and given at the same time as P. aeruginosa. Representative of 2 independent experiments with n=5 mice per group. (C) PGE2 measurement by ELISA from whole lung homogenate from experiments in (B). (D) MARCO and (E) FcγR expression in male ND and HFD alveolar macrophages treated with control or lumiracoxib nanoparticles as described in (A) combined from 2 experiments with n=4 per group. Statistical analysis by ANOVA with Tukey’s multiple comparisons. For all, error bars represent the mean ± SE. *p < 0.05, **p < 0.01, ****p < 0.0001.

Discussion

In this study, we aimed to understand how obesity impacts the alveolar macrophage response to P. aeruginosa pneumonia in both male and female mice. High fat diet fed mice of both sexes are more susceptible to bacterial pneumonia compared to normal diet mice. While P. aeruginosa can grow better in BALF from male and female HFD mice, alveolar macrophages from female HFD mice do not show impairments in bacterial phagocytosis and killing compared to alveolar macrophages from male HFD mice. Our results have identified that COX-2 and PGE2 production are elevated in male mice on HFD. We also show that PGE2 impacts macrophage function in male mice but macrophages from female mice are insensitive to this mediator, explaining some of the sexual dimorphism. Blocking COX-2, and subsequently PGE2 in male mice improves bacterial phagocytosis ex vivo and bacterial clearance in vivo, indicating a mechanism that could be explored further for therapeutics.

P. aeruginosa showed increased replication rates in the BALF of both male and female mice, indicating that the environment for bacterial replication is equally favorable in the lungs in both sexes. The increased growth noted in BALF from HFD mice may be due to increased glucose or lipid metabolites in the lung. It has been shown that increasing glucose concentrations in airway epithelial cell cultures increases growth of P. aeruginosa and decreasing glucose using metformin decreases the growth of P. aeruginosa6. Additionally, mice treated with metformin had decreased P. aeruginosa bacterial burden in the lung, indicating that blood glucose has an effect on bacterial growth and ability to establish an infection6. Our data and others28 identify that lipid mediators are changed in HFD fed lungs. The role of obesity-related lipid metabolites other than PGE2 in bacterial responses are poorly defined but may also be important regulators of pneumonia outcomes28.

While P. aeruginosa was able to grow well in BALF from HFD male and female mice, the alveolar macrophages from HFD female mice were better able to phagocytize and kill the bacteria compared to alveolar macrophages from HFD male mice. While both male and female HFD mice showed increased bacterial burden in the lung 24 hours after infection, female HFD mice had less bacteria than the male HFD mice. This may be due to the alveolar macrophages in the females having less impairment than the alveolar macrophages from the males and the fact that females overall had higher levels of neutrophils in the lung.

A sexual dimorphism in macrophages has been well defined. The prevalence and intensity of a variety of infections is lower in females compared to males, due to multiple mechanisms14. Both estrogen and testosterone are known to influence macrophage function29. Estrogen has been shown to increase the chemotaxis of neutrophils to the site of infection and enhances TLR expression on macrophages. In contrast, testosterone has been shown to have immunosuppressive effects by reducing TLR4 on macrophages and reducing cytokine production29,30. Additionally, antigen presenting cells from female mice have increased MHCII and co-stimulatory molecules compared to antigen presenting cells from male mice11. In the bone marrow, male mice on a HFD have increased myelopoiesis compared to female mice on a HFD, and the male HFD myeloid cells are more activated to pro-inflammatory stimuli, such as LPS and fatty acids12. These male mice also have increased circulating monocytes12. Therefore, our data that female mice, regardless of if they are on ND or HFD, are better able to phagocytize and kill bacteria is not surprising.

Previous work from our labs has shown that diabetic wound macrophages have an increase in COX-2/PGE2, which led to a failure to heal diabetic wounds. Additionally, we have shown that an increase in PGE2 post bone-marrow transplant leads to decreases in bacterial phagocytosis by alveolar macrophages; however, most of these studies have been done in male mice and thus, couldn’t address sexual dimorphism18,19,21,24. However, other animal models have shown a greater amount of COX-2 and PGE2 in peritoneal fluid in male mice and rats compared to females31. In human neutrophils stimulated with LPS, there is an increase in COX-2 and PGE2 in male neutrophils compared to female neutrophils32. Despite these studies which indicated higher production of PGE2 in males in other contexts, in our study PGE2 levels from the alveolar macrophages themselves were higher in females than males in our study (Fig. 4B) even though the male mice had higher COX-2 expression (Figure 4A). The reasons for this difference are not entirely clear, but our future studies will carry out a full lipidomics analysis to understand what other lipid metabolites may be altered. Interestingly, PGE2 has been shown to reduce translation and global protein synthesis in macrophages from male mice and to decrease TLR4 protein levels in rat alveolar macrophages, which may also contribute to decreased recognition for Gram-negative bacteria such as P. aeruginosa33,34. If protein levels of PGE2 synthetic machinery were impacted in males uniquely, this may explain why levels of PGE2 were not greater in male mice expressing more COX-2 in our study. This will be a focus for future studies to further understand mechanisms for greater PGE2 effect in males.

In evaluating signaling of PGE2 in our model we looked at receptors altered in HFD. A PGE2-dependent decrease in expression of the scavenger receptor MARCO was noted in the bone marrow transplant studies in male mice, and MARCO is a key receptor for recognition of non-opsonized P. aeruginosa21. Thus, as noted above, the increase in PGE2 in the HFD male lung would likely decrease the ability of these mice to use MARCO to clear the infection in vivo. Additionally, our ex vivo work used opsonized bacteria, and we also saw a trend to a decrease in FcγR in male HFD mice. This could limit recognition of bacteria opsonized by natural antibodies in vivo even in mice exposed for the first time. Previous work has also shown that PGE2 can lead to a decrease in downstream signaling of the FcγR in alveolar macrophages35. Taken together, our data adds to the literature explaining how PGE2 can lead to decreased phagocytosis of bacteria in the lung. We were surprised that PGE2 did not inhibit killing of P. aeruginosa in alveolar macrophages from ND male mice given that past studies have suggested that PGE2 can impair killing of Klebsiella pneumoniae in this context; thus, even within Gram-negative organisms, there may be unique host defense pathways that are differentially regulated by PGE224. One of the most interesting aspects of our study is the observation that macrophages from female HFD mice do not demonstrate an increase in cAMP in response to PGE2 stimulation. This likely explains why the female mice can maintain host defense function in the setting of high levels of PGE2 in the female mouse lungs. The reason for the dampened signaling by PGE2 in female macrophages is not completely clear but does not appear to be due to decreased EP2 or EP4 levels relative to males (Figure 5E and 5F). HFD male alveolar macrophages have increased EP2 and EP4, and prior research has shown that when macrophages were treated with EP2 or EP4 antagonists, they had improved bacterial killing24. Likewise, when macrophages were treated with EP2 or EP4 agonists, macrophages had decreased bacterial killing27.

Sex differences in COX-2 have been described, most notably in pain and inflammation. Female mice with genetic modifications to COX-2 show a decrease in contralateral allodynia and pain responses compared to male mice with these same modifications11. In contrast, male mice treated with NSAIDs to block COX-2 show decreased inflammation compared to female mice36. Possible explanations for these sex differences such as distribution of genes involved in drug transportation and metabolism have been suggested, but it is noteworthy that our observation that female mice do not generate cAMP in response to PGE2 stimulation (Fig. 4D) are consistent with these observations and may offer another explanation.

P. aeruginosa is one of the most common bacteria isolated from diabetic wounds and PGE2 is increased in diabetic wound macrophages, leading to a failure to clear bacteria and heal19. Given the myriad effects of PGE2 systemically, it is desirable to consider cell-specific targeting to reduce COX-2 or PGE2 levels in macrophages specifically. In past work from our labs, we used a nanoparticle approach to target phagocytic cells for macrophage-selective delivery of a COX-2 inhibitor in the skin where these therapies could be localized directly to the wound site by intradermal injection19. Additionally, similar nanocarriers have been used to target the histone demethylase JMJD3 and likewise improve diabetic wound healing24. However, this approach has not been tested in the lung where biodistribution to the cells of interest was less clear. Our work demonstrates the successful use of COX-2 inhibitory nanoparticles to target alveolar macrophage function in the inflamed and infected male lungs.

Our study was limited to a dietary obesity animal model and hence does have limitations. While both male and female mice on HFD do gain weight, only males develop significant metabolic impairment characteristic of human T2D. This is like human females, who even in obese states generally handle adipose tissue expansion better than human males, and females have a higher BMI threshold for T2D37. Therefore, this model is clinically relevant and can be used to answer experimental questions. Additionally, most patients who are obese that present with bacterial pneumonia, especially Pseudomonas species develop it as a secondary infection after a viral exposure or from ventilator induced pneumonia38. Regardless, it is important to understand why these patients have significant increases in susceptibility and mortality compared to patients of normal weight, as this can guide targeted treatment. Another important factor in interpreting our results is that PGE2 can also be produced by epithelial cells in the lung, but whether this is upregulated in diabetic epithelial cells is unknown39,40. How epithelial-derived secretions may affect the local PGE2 lung levels and the alveolar macrophages in this environment is not clear. While we do not believe the nanocarriers would effectively target this non-phagocytic cell type, that remains to be tested formally in future studies.

To conclude, our study provides a mechanism for the decreased ability for male HFD alveolar macrophages to phagocytize P. aeruginosa, an important pathogen in the diabetic patient population. Targeting COX-2 and PGE2 may be a beneficial therapeutic for male patients with obesity or T2D who are presenting with bacterial pneumonia.

Key Points.

Male mice fed high fat diet have increased P. aeruginosa in the lung post infection.

COX-2 and PGE2 are increased in alveolar macrophages of male mice on high fat diet.

Nanoparticles inhibiting COX-2 in alveolar macrophages improve bacterial clearance.

Acknowledgements

The authors would like to thank Dr. Emily Bowers for assisting with the bone marrow transplant studies. The authors acknowledge the University of Michigan Flow Cytometry Core for assistance with flow cytometry studies. Graphical abstract was created on Biorender.com.

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