NLRP3 inflammasome activation in aged macrophages is diminished during Streptococcus pneumoniae infection

Abstract

Pneumococcal infections are the eigth leading cause of death in the United States, and it is estimated that older patients (≥65 yr of age) account for the most serious cases. The goal of our current study is to understand the impact of biological aging on innate immune responses to Streptococcus pneumoniae, a causative agent of bacterial pneumonia. With the use of in vitro and in vivo aged murine models, our findings demonstrate that age-enhanced unfolded protein responses (UPRs) contribute to diminished inflammasome assembly and activation during S. pneumoniae infection. Pretreatment of aged mice with endoplasmic reticulum chaperone and the stress-reducing agent tauroursodeoxycholic acid (TUDCA) decreased mortality in aged hosts that was associated with increased NLRP3 inflammasome activation, improved pathogen clearance, and decreased pneumonitis during infection. Taken together, our data provide new evidence as to why older persons are more susceptible to S. pneumoniae and provide a possible therapeutic target to decrease morbidity and mortality in this population.

INTRODUCTION

Pneumonia is the eighth leading cause of death in the United States with Streptococcus pneumoniae being the most causative organism in both immunocompromised and nonimmunocompromised populations (14, 34, 47). It is estimated that older patients, account for the most serious cases of pneumococcal infections with the majority of direct medical costs as well as the highest rate of hospitalizations, number of days hospitalized, emergency department visits, outpatient visits, and deaths (14, 18). It has been well established that aging affects various components of the immune response, which can lead to impaired host defense and defective vaccine responses, resulting in a significantly higher risk of elderly persons developing life-threatening bacterial infections (38, 42, 52). Due to increased prevalence of comorbidities in older persons, impaired adaptive immune responses to vaccination, and the pervasiveness of antibiotic-resistant bacterial strains, there is a pressing need to understand the molecular mechanisms that underlie these impairments and develop cutting-edge therapies that specifically target and amplify innate immune responses in older persons.

The NLRP3 inflammasome is a multiprotein complex consisting of the nucleotide-binding domain leucine-rich repeat containing (NLR) family member NLRP3, the adaptor protein ASC, and the cysteine protease caspase-1 (1). The NLRP3 inflammasome can activate caspase-1 in response to cellular danger resulting in the processing and secretion of the proinflammatory cytokines IL-1β and IL-18 (22, 31, 33). NLRP3 proteins are present within cells as preassembled inactive complexes and upon stimulation undergo a conformational change, assemble with additional components, and contribute to caspase-1 activation (8). A diverse array of stimuli can activate the NLRP3 inflammasome including both pathogen-associated molecular patterns (PAMPs) and endogenous host-derived molecules indicative of cellular damage (40, 44). Given the divergent qualities of NLRP3 inflammasome agonists, it is hypothesized that activators converge on a common pathway with a final endogenous ligand activating NLRP3. Activation of the NLRP3 inflammasome is reactive oxygen species (ROS) dependent, and interruption of ROS production with pharmacological inhibitors blocks inflammasome activation (6, 9, 11, 44, 56). Recent work has illustrated the importance of caspase-1 dependent responses, mediated by the NLRP3 inflammasome, in modulating innate immunity to S. pneumoniae (12, 23, 25, 31, 35, 36, 46, 53). When compared with wild-type, ASC-deficient mice as well as mice lacking IL-1β or IL-18 are highly susceptible to pneumococcal infection (21, 27, 29, 30).

Previous work has illustrated that under basal conditions NLRP3 localizes to the endoplasmic reticulum (ER) (56). In response to inflammasome activation, both NLRP3 and ASC redistribute to the perinuclear space where they colocalize with ER and mitochondria (56). NLRP3 inflammasome responses are tightly controlled and must remain inactive despite increased basal cellular stress or become inactivated once the inducing stimuli are no longer present, as to avoid unnecessary collateral damage to the host. In this context, negative feedback loops are an important aspect of the inflammatory response. As a host ages, due to enhanced exposure to increased basal levels of ER stress as well as augmented ROS production, negative feedback responses may be differentially regulated. Our previous findings demonstrate that the NLRP3 inflammasome is needed for protection and activation of the inflammasome is decreased and/or delayed in the aged lung in response to influenza infection (49). In addition, previous work has illustrated that the early production of IL-1β in response to a serotype 4 strain of S. pneumoniae declined with age and was associated with an ability of alveolar macrophages to respond to pneumococcal cell wall components (4). To expand upon these findings, we examined the impact of biological aging on inflammasome activation in macrophages in response to S. pneumoniae infection.

With the use of in vitro and in vivo aged murine models, our findings demonstrate that the NLRP3 inflammasome is essential for the survival of aged mice in response to a highly virulent serotype 3 strain of S. pneumoniae. Our results also illustrate that age-enhanced ER stress and increased unfolded protein response (UPR) signaling contribute to diminished inflammasome assembly and activation in the aged lung during S. pneumoniae infection. Pretreatment of aged mice with ER chaperone and stress-reducing agent tauroursodeoxycholic acid (TUDCA) resulted in significantly decreased UPR activation in control and S. pneumoniae-infected lung. Furthermore, when compared with saline-treated aged controls, there was decreased mortality in aged TUDCA-treated hosts that was associated with increased NLRP3 inflammasome activation, improved pathogen clearance, and decreased pneumonitis during infection. Taken together, our findings illustrate a potential mechanism by which an age-associated enhancement in ER stress and UPR activation during S. pneumoniae infection can contribute to diminished NLRP3 inflammasome activation.

MATERIALS AND METHODS

Mice

Young (2 mo) and aged (19 mo) male and female BALB/c mice were purchased from the National Institute on Aging rodent facility (Charles River). Young (2 mo) and aged (17 mo) NLRP3^−/− mice (C57Bl/6 background) were kindly obtained from Dr. Augustine M. Choi and evaluated by comparison with age-matched, wild-type C57BL/6 mice. Upon receipt, mice were handled under identical husbandry conditions and fed certified commercial feed. Body weights were measured daily, and mice were humanely euthanized if they lost >15% of their starting body weight. The Institutional Animal Care and Use Committee at Weill Cornell Medical College approved the use of animals in this study. No animals were used in the study if they had evidence of skin lesions, weight loss, or lymphadenopathy.

In Vivo Procedures

S. pneumoniae infection.

All mice were anesthetized with isoflurane (5% for induction and 2% for maintenance) before intranasal instillation with 1 × 10³ colony-forming units of S. pneumoniae (ATCC 6303) (50-μl vol in PBS).

TUDCA administration.

The mice received a 100-μl vol of a 500 mg/kg dose of TUDCA (Enzo Life Sciences; 1× PBS as vehicle) intraperitoneally for 21 days before infection. For all animal experiments, we used n = 10 per group and experiments were repeated at least two or more times.

Bacterial Culture

Streptococcus pneumoniae (ATCC 6303, ATCC, Manassas, VA) was grown on 10% sheep blood agar plates (BD Biosciences, San Jose, CA) overnight or for 4–24 h in brain heart infusion broth (BD Biosciences). Colony-forming units were quantified by dilution of samples in brain heart infusion, and titers were determined by colony counts × dilution.

Primary Bone Marrow Isolation and Cell Culture

Bone marrow cells (BMCs) were prepared from the femurs and tibias of mice as previously described (19, 55). Briefly, bone marrow cells were collected from the femur and tibia on day 0 and cultured with 30% L929 media containing DMEM, 10% FBS, and 1× antibiotic/antimycotic solution (Cat. No. 15240062; ThermoFisher Scientific, Carlsbad CA). On day 3, media were removed and cells were cultured in 25% L929 media containing DMEM, 10% FBS, and 1× antibiotic/antimyotic solution. On day 7, macrophages were harvested and replated with 20% L929 media. Macrophages were washed two times with PBS before culture in DMEM containing 10% FBS. Cells were cultured with media alone or media containing S. pneumoniae (50 colony-forming units). TUDCA was given 1–4 h before or at time of infection (100 μM). TUDCA was purchased from Enzo Life Sciences (Farmingdale, NY) and resuspended per manufacturer's recommendation. Toll-like receptor (TLR) stimulation was performed using the mouse TLR-1–9 agonist kit per manufacturer's recommended instructions and dilutions (InvivoGen, San Diego, CA).

Cytotoxicity Assay

Cytotoxicity was examined using CytoTox Glo assays (Promega, Madison WI) per manufacturer’s instructions.

Transfection with siRNA

Missense or gene specific siRNA (Flexitube Gene Solution siRN;, Qiagen, Germantown, MD; 50-nM final concentration) was complexed with GenMute siRNA transfection reagent (SignaGen Laboratories, Ijamsville, MD) for 15 min before addition to macrophage cultures (1 × 10⁶ cells per well of a 6-well plate, seeded 1 day before transfection). siRNA-transfected cells were cultured for 18–24 h before S. pneumoniae infection. Gene silencing was confirmed by real-time PCR and Western blot analysis.

Coimmunoprecipitation

Coimmunoprecipitation with NLRP3 was performed using the Pierce coimmunoprecipitation kit (ThermoFisher Scientific) according to the manufacturer’s instructions using rabbit anti-NLRP3 (Cell Signal Technology, Danvers, MA). Briefly, 250 μg of macrophage cell lysate or murine lung homogenates were incubated with a 1:200 dilution of anti-NLRP3 (48).

RNA Purification and Real-Time PCR

RNA samples were extracted and real-time PCR was performed using previously published methods (49). QuantiTect Primer Assays and RT² Profiler Assays (Unfolded Protein Response, PAMM-089ZA) were used to assess gene expression (Qiagen). All reactions were performed in triplicate. The relative levels of messenger RNA (mRNA) were calculated by the comparative cycle threshold method and either β-actin or β2M mRNA levels were used as the invariant control for each sample. X-box binding protein 1 (XBP1) splicing was quantified using previously described methods (20).

ELISA

Culture supernatants and lung homogenates were analyzed for IL-1β production using ELISA kits purchased from eBioscience (San Diego, CA). BIP/GRP78 protein expression was quantified using ELISA kits purchased from Enzo Life Sciences. NF-κB analysis was performed using the PathScan sandwich ELISA kit (No. 7276, Cell Signal Technologies). Protein levels were calculated using the Qubit protein assay (ThermoFisher Scientific) per manufacturer’s instructions.

Caspase-1 Assay

Cultured cells were lysed and caspase-1 activity was quantified using the caspase-1 colorimetric assay kit from Abcam (Cambridge, MA) according to the manufacturer’s instructions. Fold increase in caspase-1 activity was determined by comparing the results of treated samples with the level of the untreated control.

Western Blot Analysis

Equal amounts of protein (50–100 μg/lane) were loaded onto a 4–12% Bis-Tris Bolt gel (ThermoFisher Scientific) and run at 200 V for 35 min. Protein was transferred to a nitrocellulose membrane using the iBlot Western blotting system (ThermoFisher Scientific). Protein phosphorylation was assessed on samples using the Phosphoprotein purification kit (Qiagen). Immunodetection was performed using primary antibodies against NLRP3 (Cell Signal Technologies), ASC (Adipogen, San Diego, CA), inositol-requiring enzyme 1 (IRE1; Cell Signal Technologies), pro-caspase-1 (Biovision, Milpitas, CA), IL-1β (R&D Systems, Minneapolis, MN), β-actin (Cell Signaling Technologies), and the ECL Western Blotting Analysis System (ThermoFisher Scientific). All antibodies were diluted 1:1,000 in membrane blocking solution (ThermoFisher Scientific). Images were acquired on film or by using Multi-Gauge software (Fujifilm, Greenwood, SC).

Statistical Analysis

Survival analysis between groups was calculated using the Mantel Cox test. Comparison of groups was performed using a two-tailed t-test or one-way or two-way ANOVA, when appropriate. Samples obtained were normally or approximately normally distributed. All samples were independent and contained the same sample size for analysis. Variances of the populations were equal. All data were analyzed using GraphPad prism software (San Diego, CA). P < 0.05 was considered statistically significant.

RESULTS

NLRP3 Expression in Aged Lung Is Necessary for Survival During S. Pneumoniae

Previous work has illustrated that the NLRP3 inflammasome contributes to host defense in response to S. pneumoniae (serotypes 1, 8, and 7F)-induced pneumococcal pneumonia (53). The goal of current work is to expand on these findings and examine if the NLRP3 inflammasome is necessary for survival of aged mice in response to serotype 3 S. pneumoniae. We intranasally instilled young (2 mo of age) and aged (18–20 mo of age) male and female mice with ATCC 6303, a highly virulent type 3 strain of S. pneumoniae commonly associated with increased relative risk of death in older persons (32). In agreement with previous studies, in response to S. pneumoniae, aged mice have increased inflammation and alveolitis (Fig. 1A), resulting in increased morbidity during S. pneumoniae infection (26, 50). When compared with wild-type controls, mortality in aged mice in response to S. pneumoniae was significantly increased in the absence of NLRP3 (Fig. 1B, Mantel-Cox test, P = 0.0026). In addition, when compared with age-matched wild-type mice, bacterial clearance was significantly decreased in aged NLRP3-deficient mice (NLRP3^−/−) (Fig. 1C, t-test, P < 0.05).

Fig. 1.

NLRP3 expression in the aged lung is necessary for survival during Streptococcus pneumoniae. A, D–H: young (2 mo) and aged (18 mo) male and female BALB/c mice received 1 × 10³ colony-forming untis (CFU) of S. pneumoniae (S. pne) (ATCC 6303) or saline via intranasal instillation. A: hematoxylin and eosin (H&E) staining of lung tissue from young and aged control and S. pneumoniae-infected mice collected at 72 h postinfection. B and C: young (2 mo) and aged (17 mo) male and female wild-type C57BL/6 or C57Bl/6-NLRP3^−/− mice were instilled with 1 × 10³ CFU of S. pneumoniae. Survival (B) (Mantel-Cox: P = 0.0026) and bacterial titer (C) in lung was examined in lung homogenates at 24 h postinfection (t-test, **P < 0.05). D: pro-caspase-1, NLRP3, and ASC mRNA expression was calculated at 24 h postinfection (NLRP: t-test, *P = 0.0312; ASC: t-test, ***P = 0.0007). E: protein was collected from control and S. pneumoniae-infected lung tissue and expression of NLRP3, ASC, and processed IL-1β was determined at 24 and 48 h postinfection. F: lung homogenate samples were isolated at 24 h postinfection and coimmunoprecipitation (IP) was performed against anti-ASC. Eluted proteins were separated by electrophoresis and immunoblotted (IB) with anti-mouse NLRP3 and pro-caspase-1. G and H: β-actin expression was used to confirm equivalent protein input. Caspase-1 activity (G) (aged saline vs. S. pneumoniae: t-test, P < 0.0001; young vs aged S. pneumoniae: t-test, **P = 0.0016) and IL-1β production (H) (t-test, ***P < 0.0001) was determined in young and aged lungs homogenates isolated 24 h post-S. pneumoniae instillation. Similar results were obtained from at least 2 or more independent experiments with n = 10 per experiment. Data are expressed as the means ± SE.

Given the importance of NLRP3 for survival, we next examined the impact of biological aging on the inflammasome expression and activation in lung in response to S. pneumoniae. In response to S. pneumoniae, there was increased mRNA and protein expression of NLRP3 and ASC (Fig. 1D, t-test, P < 0.05, and Fig. 1E), inflammasome complex formation (Fig. 1F), caspase-1 cleavage (Fig. 1G), and IL-1β production in young lung (Fig. 1H, t-test, P < 0.0001). In contrast, ASC-NLRP3 and ASC-caspase-1 association, caspase-1 activation, and production of mature IL-1β in the aged lung during S. pneumoniae infection were significantly decreased (Fig. 1, D–H).

TLR Signaling in Aged Lung and Macrophages in Response to S. Pneumoniae Infection

To gain a better understanding of how the process of biological aging might contribute to decreased NLRP3 inflammasome activation in response to S. pneumoniae, we first examined the impact of aging on the activation of pathogenic recognition receptors. Previous work has illustrated an important role for TLRs in the innate immune response to S. pneumoniae and dysregulated TLR signaling in the aged lung contributes to increased susceptible to infection (2, 4, 5, 16, 24, 41). In agreement with previous work, we detected a significant decrease in TLR-1 and TLR-9 mRNA expression in response to S. pneumoniae infection (Fig. 2A, t-test, P < 0.05) (16). Similarly, when compared with young, in response to a serotype 3 strain of S. pneumoniae, there was a significant increase in TLR-2 and TLR-4 mRNA expression in the aged lung (Fig. 2A) (4).

Fig. 2.

Macrophage and lung Toll-like receptor (TLR) expression in response to S. pneumoniae. A–C: young (2 mo) and aged (18 mo) male and female BALB/c mice received 1 × 10³ CFU of S. pneumoniae (S. pne) (ATCC 6303) or saline via intranasal instillation. A: changes in mRNA expression of TLR-1 (t-test, *P = 0.01354), 2 (t-test, **P = 0.0082), 4 (t-test, *P = 0.0487), 6, and 9 (t-test, *P = 0.0144) in young and aged lungs was evaluated by real-time PCR. B: changes in mRNA expression of NOD1 (t-test, *P = 0.0054), NOD2 (t-test, **P = 0.015), RIP2, and NF-κB at 24 h postinfection were quantified by real-time PCR. C: inflammatory cytokine expression was investigated in young and aged lungs, and changes in pro-IL-1β, pro-IL-18, IL-6 (t-test, ****P < 0.0001), and TNF (t-test, **P = 0.002) were determined by real-time PCR. D–F: young and aged macrophages were cultured with S. pneumoniae (50 CFU) and mRNA expression was quantified at 4 and 24 h postinfection. D: changes in TLR-1, -2 (24 h: t-test, P = 0.0352), -4, -6, -9, and MYD88 were examined by real-time PCR. E: mRNA expression of NOD1, NOD2, RIP2, and NF-κB (24 h: t-test, *P = 0.0137) by real-time PCR were also assessed. F and G: pro-IL-1β (4 h: t-test, **P = 0.0093; 24 h: t-test, **P = 0.0083), pro-IL-18, IL-6, and TNF (t-test, P = 0.0024) mRNA expression was also quantified. Similar results were obtained from at least 3 or more independent experiments with n = 5 per experiment. Data are expressed as the means ± SE.

Previous work has illustrated that in response to streptococcal peptidoglycan NOD-like receptor (NLR), NOD2, plays an important role in the production of inflammatory mediators, such as TNF-α, IL-1β, and IL-6 (7, 10). Given the importance of the NOD1/NOD2 pathway on innate immune responsiveness to multiple pulmonary pathogens, we next examined if changes in NOD1, NOD2, or RIP2 mRNA expression might contribute to diminished NLRP3 inflammasome activation in the aged lung. In response to S. pneumoniae, there was decreased NOD1 and NOD2 mRNA expression in the aged lung (Fig. 2B, t-test, P < 0.05). We next evaluated the impact of biological aging on inflammatory cytokine expression in response to S. pneumoniae. While there was similar pro-IL-1β and pro-IL-18 expression, IL-6 and TNF mRNA expression was significantly elevated in aged S. pneumoniae-infected lung (Fig. 2C, t-test, P < 0.05).

As macrophages play an important role in the innate immune response to S. pneumoniae, we next examined the impact of biological aging on macrophage-specific responses to S. pneumoniae. To this extent, we examined baseline and S. pneumoniae stimulated mRNA expression of TLR-1, -2, -4, -6, and -9 as well as adaptor protein MYD88 in young and aged macrophages. Despite slightly higher baseline expression of TLR-1 in aged macrophages, there was similar expression of TLRs 2, 4, 6, 9, and MYD88 in both young and aged macrophages (data not shown). In response to S. pneumoniae infection, there was a similar increase in TLR-1, -2, and -6 mRNA expression in both young and aged macrophages (Fig. 2D). We next evaluated baseline and S. pneumoniae stimulated mRNA expression of NOD1, NOD2, and RIP2, as well as downstream effector, NF-κB, in young and aged macrophages. Despite slightly higher baseline expression of RIP2 in aged macrophages, there was similar mRNA expression of NOD1, NOD2, and NF-κB in both young and aged macrophages (data not shown). In response to S. pneumoniae infection, there was similar NOD1, NOD2, and NF-κB mRNA expression in both young and aged macrophages, with levels remaining significantly elevated in aged macrophages at 24 h postinfection (Fig. 2E). Based on these findings, we next examined the impact of biological aging on pro-IL-1β, pro-IL-18, IL-6, and TNF-α mRNA expression in macrophages in response to S. pneumoniae infection. When compared with young, there was a significant increase in pro-IL-1β and IL-6 mRNA expression in aged macrophages at baseline (Fig. 2, F and G). In response to S. pneumoniae, pro-IL-1β and TNF-α mRNA expression was significantly increased in aged macrophages, with levels remaining elevated at 24 h postinfection (Fig. 2, F and G).

Given the importance of NF-κB on inflammatory cytokine transcription, we next evaluated if increased NF-κB expression might contribute to elevated inflammatory cytokine mRNA expression in response to S. pneumoniae infection. In response to treatment with TLR-1/2, -2, -3, -4, -5, -2/6, -7, and -9 stimulatory compounds, there was similar or significantly elevated TNF-α expression by aged macrophages (Fig. 3A). We next examined if inflammatory cytokines, such as IL-1β, might also be altered in response to direct TLR stimulation. When compared with young, in response to TLR-1/2, -2, -4, -2/6, and -9 stimulatory compounds, there was a significant increase in IL-1β production by aged macrophages (Fig. 3B).

Fig. 3.

Similar NF-κB expression in young and aged macrophages in response to S. pneumoniae. A–D: young and aged macrophages were stimulated with TLR-1/2 (Pam3CSK4, 100 ng/ml), TLR-2 (HKLM, 10⁷ cells/ml), TLR-3 (HMW poly I:C, 10 ng/ml), TLR-3 (LMW poly I:C, 30 ng/ml), TLR-4 (LPS-EK, 10 ng/ml), TLR-5 (FLA-st, 100 ng/ml), TLR-6 (FSL-1 10 ng/ml), TLR-7 (ssRNA40, 1 μg/ml), or TLR-9 (ODN1826, 1 μM) agonists. TNF-α (TLR-1/2: t-test, P = 0.0446; TLR-3 (HMW): t-test, *P = 0.0114; TLR-3 (LMW): t-test, ***P = 0.0006; TLR-9: t-test, *P = 0.0462) (A) and IL-1β (TLR-1/2: t-test, **P = 0.0037; TLR-2: t-test, **P = 0.0016; TLR-4: t-test, *P = 0.0155; TLR-6: t-test, **P = 0.0097; TLR-9: t-test, ***P = 0.0002) (B) production was quantified by ELISA. C and D: cytosolic and nuclear protein extracts were isolated from young and aged macrophages at 2 h post-S. pneumoniae (50 CFU) infection. NF-κB (t-test, ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05) (C) and phosphorylated NF-κB (t-test, ***P < 0.001, **P < 0.01, *P < 0.05) (D) expression was evaluated by PathScan sandwich ELISA and normalized to protein concentration for each sample. Similar results were obtained from at least 3 or more independent experiments with n = 5 per experiment. Data are expressed as the means ± SE.

As nuclear translocation of NF-κB is essential for cytokine transcription, we next examined NF-κB expression in cytosolic and nuclear protein fractions from young and aged macrophages at baseline and during S. pneumoniae infection. In response to S. pneumoniae, there was similar NF-κB (Fig. 3C) and phosphorylated NF-κB (Fig. 3D) nuclear expression in both young and aged macrophages. Taken together, our results illustrate that in response to S. pneumoniae there are similar levels of TLR expression and NF-κB expression in both young and aged macrophages.

NLRP3 Inflammasome Activation in Response to S. Pneumoniae Is Diminished in Aged Macrophages

Based on these findings, we next evaluated the impact of biological aging on NLRP3 inflammasome activation during S. pneumoniae infection. Despite similar NLRP3 mRNA upregulation, when compared with young, there is a significant decrease in pro-caspase-1 and ASC mRNA expression in aged macrophages in response to S. pneumoniae (Fig. 4A). In response to S. pneumoniae, there was an increase in cytosolic and perinuclear ASC expression in young macrophages that was associated with increased NLRP3 expression and caspase-1 cleavage (Fig. 4, B and C). In contrast, while ASC, NLRP3, and cleaved caspase-1 expression was increased in aged macrophages in response to S. pneumoniae, these levels were decreased in magnitude when compared with young macrophages (Fig. 4, B and C). As association of the NLRP3 and ASC components is essential for inflammasome activation, we next evaluated if assembly of these proteins in response to S. pneumoniae might be altered in aged macrophages. In response to S. pneumoniae infection, there is a significant increase in NLRP3 inflammasome assembly in young macrophages that corresponds with an increase in caspase-1 activity and IL-1β production (Fig. 4, D–F). When compared with young, in response to S. pneumoniae, NLRP3 inflammasome assembly is significantly reduced in aged macrophages (Fig. 4D). Interestingly, despite decreased caspase-1 activity, there are similar levels of IL-1β production by both young and aged macrophages in the early response at 2–4 h post-S. pneumoniae infection (Fig. 4, E and F). However, when compared with young, by 24 h postinfection, IL-1β production by aged macrophages is significantly decreased (Fig. 4F). We next evaluated if the production of additional proinflammatory cytokines was altered in aged macrophages in response to S. pneumoniae. There were similar levels of TNF-α and IL-6 production by both young and aged macrophages at 2 and 4 h post-S. pneumoniae infection, with significantly increased TNF-α being detectable in aged macrophages at 24 h postinfection (Fig. 4, G and H, t-test, P = 0.0006).

Fig. 4.

Diminished NLRP3 inflammasome activation in aged macrophages in response to S. pneumoniae. Young (2 mo) and aged (18 mo) male and female BALB/c macrophages were treated with media alone or media containing 50 CFU of S. pneumoniae (S. pne). A: baseline and S. pneumoniae upregulated pro-caspase-1, NLRP3, and ASC mRNA expression was evaluated by real-time PCR (t-test, ***P < 0.001, **P < 0.01). B: cytosolic and nuclear protein expression of ASC at 2 h postinfection was quantified by Western blot. C: caspase-1 cleavage and NLRP3 expression in young and aged macrophages in response to S. pneumoniae was examined in whole cell extracts collected at 2 h postinfection. D: NLRP3 inflammasome assembly was evaluated by coimmunoprecipitation of young and aged whole cell protein lysates collected at 2, 4, and 24 h postinfection. E: caspase-1 activity in young and aged macrophages was quantified at 4 h postinfection (t-test, **P < 0.01). F–H: IL-1β (F), TNF-α (G), and IL-6 (H) production by young and aged macrophages in response to S. pneumoniae was analyzed at 2, 4, and 24 (IL-1β: t-test, ***P = 0.001; TNF-α: t-test, ***P = 0.0006) h. Histone H3 and β-Actin were used as protein loading controls. Similar results were obtained from at least 5 or more independent experiments with n = 5 per experiment. Data are expressed as the means ± SE.

The AIM2 Inflammasome Is not Essential for Caspase-1 Activation and IL-1β Production in Response to Serotype 3 S. Pneumoniae

Previous work has illustrated that in addition to NLRP3, the AIM2 inflammasome plays an important role in caspase-1 activation in response to a serotype 2 strain of S. pneumoniae (13). Based on these findings, we next examined the role of the AIM2 inflammasome on IL-1β production in response to a serotype 3 strain of S. pneumoniae. To this extent, we first examined TLR, NOD1/NOD2, and inflammatory cytokine mRNA expression in young wild-type and AIM2 knockout (Aim2^−/−) macrophages in response to S. pneumoniae infection. Examination of mRNA expression at both 4 and 24 h postinfection illustrated no significant difference in TLR- 1, 2, 4, 6, 9, or MYD88 mRNA expression (Fig. 5A). Similarly, there was no significant difference in NOD1, NOD2, RIP2, or NF-κB mRNA expression between wild-type and Aim2^−/− macrophages (Fig. 5B). In addition, pro-IL-1β, pro-IL-18, IL-6, and TNF-α mRNA expression in response to S. pneumoniae was similar between wild-type and Aim2^−/− macrophages (Fig. 5C). Despite a trend toward higher NLRP3 mRNA expression in Aim2^−/− macrophages, there was similar expression of pro-caspase-1 and ASC in both wild-type and Aim2^−/− macrophages in response to S. pneumoniae (Fig. 5D). We next evaluated cytosolic (CYT) and perinuclear (P) protein localization of ASC and NLRP3 in wild-type and Aim2^−/− macrophages in response to S. pneumoniae infection. When compared with wild-type, there was a similar level of ASC and NLRP3 protein upregulation in the perinuclear fraction in response to S. pneumoniae infection (Fig. 5E). Despite elevated cytosolic NLRP3 protein expression in Aim2^−/− macrophages at baseline, ASC-dependent NLRP3 and cleaved caspase-1 expression in response to S. pneumoniae was similar between wild-type and Aim2^−/− macrophages (Fig. 5, E and F). We next evaluated the importance of AIM2 for IL-1β production and S. pneumoniae clearance. There was similar IL-1β production and bacterial clearance between wild-type and Aim2^−/− macrophages (Fig. 5, G and H).

Fig. 5.

AIM2 inflammasome responses are not essential for caspase-1 activation or IL-1β production in response to a serotype 3 strain of S. pneumoniae. Young wild-type (WT) and Aim2^−/− macrophages were cultured with S. pneumoniae (S. pne) (50 CFU) and mRNA and protein expression was examined at 4 and 24 h postinfection. A: changes in TLR- 1, 2, 4, 6, and 9, and MYD88 were quantified by real-time PCR. B: mRNA expression of NOD1, NOD2 (4 h: t-test, P < 0.05), RIP2, and NF-κB by real-time PCR were also evaluated. C: pro-IL-1β, pro-IL-18, IL-6, and TNF mRNA expression was also determined. D: pro-caspase-1, NLRP3, and ASC mRNA expression was quantified by real-time PCR. E: ASC and NLRP3 protein expression in cytosolic (CYT) and perinuclear (P) protein extracts isolated from wild-type and Aim2^−/− macrophages at 2–4 h post-S. pneumoniae infection. F: NLRP3 and cleaved caspase-1 protein expression in whole cell protein extracts isolated at 4 h post-S. pneumoniae infection. G and H: IL-1β production (G) and bacterial titer (H) in wild-type and Aim2^−/− macrophages at 24 h postinfection. I: IL-1β production by young and aged macrophages pretreated with missense of NLRP3 specific siRNA before infection with S. pneumoniae (50 CFU) (t-test, **P < 0.01). Similar results were obtained from at least 3 or more independent experiments with n = 5 per experiment. Data are expressed as the means ± SE.

Given these findings, we next evaluated the contribution of AIM2 to IL-1β production by young and aged macrophages in response to S. pneumoniae. In response to NLRP3 siRNA treatment, IL-1β production by both young and aged macrophages was significantly diminished (Fig. 5I). Taken together, our findings illustrate that the AIM2 inflammasome does not play an essential role in caspase-1 activation or IL-1β production in response to a serotype 3 strain of S. pneumoniae.

ER Stress in the aged lung Is Enhanced during S. Pneumoniae Infection

In response to prolonged ER stress, three main branches of the UPR contribute to restoring ER homeostasis: inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6), and PKR-like ER resident kinase (PERK). In agreement with our previous results, enhanced BIP/GRP78 expression (Fig. 6A, t-test, P < 0.05) in the aged lung is associated with increased IRE1 and PERK gene expression (Fig. 6, B and C) (37). As age enhanced ER stress may contribute to altered inflammasome function, we next examined the impact of aging on IRE1 expression and phosphorylation as well as subsequent XBP1 splicing in lung during S. pneumoniae infection. When compared with young, there was increased IRE1 expression and phosphorylation present in the aged lung (Fig. 6D). To examine if IRE1 endonuclease activity was similarly increased, we next examined the impact of biological aging on XBP1 expression and splicing in lung during S. pneumoniae infection. When compared with young, there is enhanced XBP1 expression and splicing present in the aged lung in response to S. pneumoniae (Fig. 6E: t-test, P < 0.05).

Fig. 6.

Endoplasmic reticulum (ER) stress in the aged lung is enhanced during S. pneumoniae infection. Young (2 mo) and aged (18 mo) male and female BALB/c mice received 1 × 10³ CFU of S. pneumoniae (S. pne) (ATCC 6303) or saline via intranasal instillation. A: changes in BIP/GRP78 expression in lung homogenates was calculated at baseline and at 24, 48, and 72 h postinfection (24 h: t-test, *P = 0.0189; 48 h: t-test, **P = 0.0028; 72 h, t-test: **P = 0.0020). B and C: inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6) (B) and PKR-like ER resident kinase (PERK) (C) mRNA expression was quantified by real-time PCR at 24 h postinfection (IRE1: t-test, *P = 0.0119; PERK: t-test, **P < 0.01). D: protein expression of IRE1 was determined by Western blot analysis of young and aged lungs tissue homogenates at 24 and 48 h postinfection. IRE1 phosphorylation was examined in phosphoprotein enriched lung tissue homogenates and detected by Western blot. E: X-box binding protein 1 (XBP1) splicing was evaluated in young and aged lungs tissue at 24 h postinfection and the ratio of XBP1 spliced to total XBP1 was quantified [t-test: control (young vs. aged), *P = 0.0285; infected (young vs. aged), P = 0.0298; aged (control vs. infected), P = 0.0056; young (control vs. infected). P = 0.0179). Similar results were obtained from at least 2 or more independent experiments with n = 10 per experiment. Data are expressed as the means ± SE.

Treatment with TUDCA Rescues NLRP3 Inflammasome Activation in Aged Lung During S. Pneumoniae Infection

We next investigated the impact of TUDCA, an ER stress-reducing compound, on IL-1β production by aged macrophages. When compared with untreated aged macrophages, TUDCA treatment significantly decreased IRE1 mRNA expression in response to S. pneumoniae (Fig. 7A, t-test, P = 0.0008). Furthermore, in response to TUDCA, there was decreased IRE1 phosphorylation and XBP1 splicing in aged macrophages (Fig. 7, B and C, t-test, P = 0.0032). When compared with control, a reduction of ER stress in response to TUDCA treatment resulted in a significant decrease in cytotoxicity during S. pneumoniae infection (Fig. 7D, t-test, P = 0.0011). Given these findings, we next evaluated if a reduction in ER stress might impact NLRP3 inflammasome responses in aged macrophages during S. pneumoniae infection. In response to TUDCA, there was an increase in ASC, pro-caspase-1, and NLRP3 mRNA expression (Fig. 7E, t-test, P < 0.05) in S. pneumoniae-infected aged macrophages. Furthermore, in response to TUDCA, caspase-1 activity (Fig. 7F, P = 0.0002) and IL-1β cleavage (Fig. 7G, t-test, P < 0.0001) were increased in aged macrophages during S. pneumoniae infection.

Fig. 7.

Treatment with tauroursodeoxycholic acid improves NLRP3 inflammasome activation in aged macrophages during S. pneumoniae infection. Young (2 mo) and aged (18 mo) male or female BALB/c macrophages were pretreated with tauroursodeoxycholic acid (TUDCA) (100 μM) for 24 h before culture with media alone or media containing 50 CFU of S. pneumoniae (S. pne) (ATCC 6303). A: mRNA expression of ATF6, IRE1, and PERK in untreated and TUDCA-treated aged macrophages at 24 h postinfection was quantified by real-time PCR (IRE1: t-test, ***P = 0.0008). B: IRE1 phosphorylation in untreated and TUDCA pretreated aged macrophages at 24 h postinfection was examined by Western blot. C: XBP1 splicing was evaluated by PCR and % of XBP1 splicing was quantified (t-test, **P = 0.0032). D: cytotoxicity in young and aged macrophages in response to TUDCA was examined at 24 h post-S. pneumoniae infection (t-test, **P < 0.05). E: mRNA expression of pro-IL-1β, pro-IL-18, ASC, caspase-1, and NLRP3 at 24 h postinfection was evaluated in aged macrophages treated with media alone or media containing TUDCA (ASC: t-test, *P < 0.01; caspase-1, t-test: ***P = 0.0005; NLRP3: t-test, **P = 0.0042). F and G: caspase-1 activity (F) and production of IL-1β (G) was quantified in aged macrophages in response to TUDCA pretreatment (caspase-1: t-test, ***P = 0.0002; IL-1β: t-test, ****P < 0.0001). Similar results were obtained from at least 5 or more independent experiments with n = 5 per experiment. Data are expressed as the means ± SE.

To expand on these results, we next examined the in vivo effects of TUDCA on UPR activation in the aged lung during S. pneumoniae infection. Briefly, we pretreated aged mice with TUDCA (500 mg/kg ip, daily) for 21 days before infection with S. pneumoniae. In contrast to saline treatment, pretreatment with TUDCA reduced cellular apoptosis and injury in the aged lung in response to S. pneumoniae infection (Fig. 8A). Pretreatment with TUDCA was also associated with improved bacterial clearance in the aged lung (Fig. 8B, t-test, P < 0.05). Next, we examined the impact of TUDCA pretreatment on the expression of genes associated with the UPR. Our results illustrate a global decrease in lung expression of genes associated with unfolded protein binding, ER protein folding control, and apoptosis at baseline and during S. pneumoniae infection in TUDCA-treated mice (Fig. 8C and Table 1). Based on these findings, we investigated the impact of TUDCA pretreatment on IRE1 expression and activation in response to S. pneumoniae. When compared with saline-treated controls, pretreatment with TUDCA resulted in a significant decrease in IRE1 mRNA (Fig. 8D: t-test, P < 0.0001) and protein expression (Fig. 8E). In addition, pretreatment with TUDCA also resulted in a decrease in IRE1 endonuclease activity, as illustrated by a decrease in XBP1 splicing (Fig. 8F: t-test, P = 0.0032) in the aged lung during S. pneumoniae infection.

Fig. 8.

Treatment with TUDCA decreases UPR gene expression in the aged lung during S. pneumoniae infection. A–F: young (2 mo) and aged (18 mo) male and female BALB/c mice received saline or TUDCA (500 mg·kg⁻¹·day⁻¹) via intraperitoneal injection before intranasal instillation with saline or 1 × 10³ CFU of S. pneumoniae (S. pne) (ATCC 6303). A: lung tissue was collected at 72 h postinfection and evaluated for histological changes. B: bacterial titer in lung was quantified in lung homogenates at 24 h postinfection (***P = 0.0001). C: lung gene expression in control and TUDCA pretreated aged mice at 24 h post-S. pneumoniae infection was determined by real-time PCR using UPR specific primer assays (SABiosciences). D: mRNA expression of IRE1 (t-test, ****P < 0.0001) was quantified by real-time PCR at 24 h postinfection. E: IRE1 and phosphorylated IRE1 protein expression was evaluated at 24 and 48 h postinfection by Western blot analysis. F: XBP1 splicing was examined in young and aged lungs tissue at 24 h postinfection and the ratio of XBP1 spliced to total XBP1 was calculated (t-test: ***P = 0.0001). Similar results were obtained from at least 2 or more independent experiments with n = 5 per experiment. Data are expressed as the means ± SE.

Table 1.

Expression level of genes associated with the unfolded protein responses

		Aged + S. Pneumoniae (Compared with Aged Control)		Aged, TUDCA (Compared with Aged Control)		Aged, TUDCA + S. Pneumoniae (Compared with Aged + S. Pneumoniae)
Refseq	Symbol	Fold Regulation	SD	Fold Regulation	SD	Fold Regulation	SD
NM_011787	Amfr	4.1989	0.153	4.1068	0.28	1.6569	0.3205
NM_009716	Atf4	5.1569	0.2255	1.629	0.1015	1.4211	0.53
NM_001081304	Atf6	5.6149	0.44675	1.6777	0.40525	1.1909	0.292
NM_017406	Atf6b	2.4112	0.34025	1.7053	0.3255	−1.3972	0.0595
NM_029705	Atxn3	2.7146	0.19425	1.7654	0.0105	−1.2184	0.473
NM_007527	Bax	6.0997	0.83125	1.3013	0.056	−1.7441	0.3165
NM_007591	Calr	4.7585	0.5405	1.3195	0.11925	−1.8075	0.365
NM_007597	Canx	5.9639	0.34475	2.9261	0.1975	1.2201	0.273
NM_009837	Cct4	5.7657	0.2305	2.1332	0.153	1.805	0.375
NM_007638	Cct7	3.4804	0.41075	1.3841	0.03	−1.5358	0.474
NM_009883	Cebpb	4.1025	0.7745	1.2083	0.19525	−1.7752	0.017
NM_013497	Creb3	4.3726	0.5245	1.8138	0.1925	−1.3486	0.2315
NM_145365	Creb3l3	6.6059	0.43725	2.7664	0	−4.212	1.5145
NM_007837	Ddit3	8.1753	0.4175	1.8789	0.06975	1.1167	0.2195
NM_024207	Derl1	3.3346	0.7765	−1.5944	0.08225	−8.4855	0.462
NM_033562	Derl2	6.5887	0.058	4.8635	0.5135	6.8258	0.55
NM_178055	Dnajb2	3.3456	0.52275	2.8069	0.0895	1.8745	0.1065
NM_013760	Dnajb9	2.8749	0.0295	3.7869	0.531	1.7381	0.2375
NM_024181	Dnajc10	3.9007	0.26525	2.4368	0.34425	1.1239	0.2115
NM_008929	Dnajc3	4.9365	0.4825	1.654	0.1065	−1.3727	0.446
NM_020566	Dnajc4	2.7506	0.45425	1.669	0.24325	−1.0886	0.1575
NM_138677	Edem1	5.8848	0.267	4.1583	0.2605	1.9986	0.42
NM_001039644	Edem3	4.2317	0.52775	1.5605	0.19425	−1.2839	0.3145
NM_001005509	Eif2a	2.6689	0.01375	2.9404	0.51725	1.4545	0.0575
NM_010121	Eif2ak3	3.3905	0.4135	1.7411	0.216	−1.6592	0.3035
NM_023913	Ern1	14.3334	0.6145	−1.6178	0.09575	−2.3561	0.3015
NM_012016	Ern2	6.6059	0.43725	2.7664	0	−4.212	1.5145
NM_015774	Ero1l	4.3439	0.354	1.9212	0.2155	−1.2772	0.39
NM_026184	Ero1lb	6.3017	0.54025	1.7053	0.04725	−1.5379	0.328
NM_029572	Erp44	4.7437	0.242	2.7019	0.24125	1.2436	0.2855
NM_015797	Fbxo6	4.5972	0.36925	2.7245	0.15225	1.2121	0.5125
NM_008060	Ganab	3.0262	0.4715	1.0966	0.153	−2.1332	0.265
NM_172672	Ganc	4.6889	0.01625	4.1353	0.269	2.8031	0.137
NM_024439	Vimp	5.712	0.252	1.9697	0.01475	1.0996	0.327
NM_022331	Herpud1	4.6589	0.468	1.4369	0.1095	2.7818	0.507
NM_013558	Hspa1l	4.5512	0.71075	1.4006	0.1585	1.0581	0.1995
NM_008301	Hspa2	5.0403	0.2575	1.6279	0.0595	1.2316	0.2725
NM_008300	Hspa4	4.5718	0.62925	1.5823	0.02625	−1.5867	0.577
NM_011020	Hspa4l	4.5198	0.53275	1.1431	0.02575	−1.676	0.164
NM_022310	Hspa5	4.2938	0.16775	3.4534	0.552	1.4616	0.2045
NM_029307	Hspb9	5.056	0.599	1.9986	0.417	−1.3496	0.3855
NM_013559	Hsph1	5.4002	0.657	1.3538	0.0985	−1.1274	0.168
NM_019752	Htra2	4.53	0.1695	4.4015	0.45525	2.4284	0.222
NM_001081187	Htra4	2.3469	0.43725	5.9134	0.41425	4.7916	0.2085
NM_153526	Insig1	3.4468	0.20775	2.0835	0.2	1.0059	0.0645
NM_178082	Insig2	3.7115	0.233	3.3035	0.32325	1.8751	0.22
NM_029103	Manf	5.312	0.44575	3.1821	0.11325	1.3255	0.2705
NM_009158	Mapk10	2.052	0.121	−2.0936	0.01775	−3.7425	0.841
NM_016700	Mapk8	3.9055	0.4325	1.244	0.1225	−1.6268	0.386
NM_016961	Mapk9	5.609	0.18325	3.3543	0.27275	1.8954	0.1855
NM_019709	Mbtps1	3.5844	0.43625	3.8264	0.27675	1.629	0.382
NM_172307	Mbtps2	3.1064	0.27375	1.1615	0.21225	−1.8875	0.3405
NM_199469	Nploc4	3.1096	0.15925	1.2501	0.263	−1.0042	0.324
NM_008749	Nucb1	5.8361	0.41	2.291	0.22425	1.1254	0.2495
NM_177614	Os9	4.172	0.36525	1.7339	0.24875	−1.4088	0.2965
NM_007952	Pdia3	4.6316	0.3485	1.9917	0.20775	1.0729	0.2525
NM_011070	Pfdn2	3.7503	0.527	−1.0049	0.02425	−2.1287	0.49
NM_027044	Pfdn5	4.6954	0.49875	2.8069	0.181	1.0733	0.332
NM_008907	Ppia	3.8397	0.304	1.7065	0.04525	−1.4384	0.5195
NM_133819	Ppp1r15b	5.3129	0.3505	1.6166	0.204	−1.0472	0.2955
NM_008925	Prkcsh	3.2132	0.489	1.1127	0.16975	−1.6783	0.19
NM_175226	Rnf139	4.0869	0.137	3.0209	0.5615	1.6071	0.1465
NM_019403	Rnf5	3.5944	0.65325	−1.0007	0.01675	−2.2934	0.1955
NM_133933	Rpn1	3.9299	0.1595	2.8999	0.35125	1.5079	0.3835
NM_001001144	Scap	3.6865	0.41875	2.2454	0.119	1.0475	0.59
NM_027016	Sec62	4.7478	0.05575	4.353	0.43375	3.4414	0.166
NM_153055	Sec63	3.5671	0.46925	−1.1142	0.02525	−2.2966	0.4055
NM_001039089	Sel1l	3.0001	0.255	1.3287	0.31475	−1.2596	0.214
NM_030685	Serp1	8.0654	0.42475	4.7371	0.33125	10.9473	0.0485
NM_030749	Sil1	4.6938	0.57025	2.0335	0.074	1.0122	0.2565
NM_011480	Srebf1	3.1624	0.514	1.2176	0.01825	−1.6392	0.22
NM_033218	Srebf2	3.5265	0.54875	−1.1088	0.09775	−1.9616	0.317
NM_028769	Syvn1	4.53	0.6005	2.178	0.13225	−1.1495	0.198
NM_013686	Tcp1	3.5223	0.1625	2.8739	0.18425	1.5385	0.2655
NM_144884	Tor1a	3.8987	0.338	1.8013	0.119	−1.2509	0.465
NM_019803	Ube2g2	3.9279	0.70925	−1.3519	0.03725	−3.3346	0.3755
NM_001039157	Ube2j2	4.1554	0.525	1.8302	0.12	−1.1258	0.445
NM_026390	Ubxn4	4.4514	0.03375	4.8366	0.27025	2.2046	0.1555
NM_011672	Ufd1l	3.8798	0.477	1.6748	0.0545	−1.5637	0.382
NM_198899	Uggt1	3.9163	0.3535	1.8075	0.229	−1.429	0.197
NM_001081252	Uggt2	4.3969	0.4625	−1.3241	0.0235	−1.5449	0.4595
NM_021522	Usp14	4.5182	0.15125	2.6629	0.207	1.6016	0.2235
NM_009503	Vcp	3.9068	0.495	1.8468	0.0635	−1.2531	0.4615
NM_013842	Xbp1	3.4888	0.22425	3.934	0.3955	1.9159	0.129

Lung tissue was isolated at 24 h postinfection. mRNA expression was assessed by real-time PCR using the RT Profiler primer specific array (Mouse Unfolded Protein Response, Cat. No. 330231, PAMM-089ZA, Qiagen). TUDCA tauroursodeoxycholic acid. S. pneumoniae, Streptococcus pneumoniae.

On the basis of these findings, we examined if heightened ER stress in the aged lung might underlie diminished NLRP3 inflammasome activation in response to S. pneumoniae infection. As shown in Fig. 7, a decrease in ER stress post-TUDCA treatment improved ASC, NLRP3, and pro-caspase-1 gene expression (Fig. 9, A and B: t-test, P < 0.05). We next evaluated the impact of TUDCA pretreatment on NLRP3 and ASC protein expression in response to S. pneumoniae infection. When compared with saline-treated controls, there was enhanced ASC expression that was associated with increased NLRP3-ASC complex formation (Fig. 9C). In addition, pretreatment of aged mice with TUDCA also resulted in a significant increase in caspase-1 activity (Fig. 9D: t-test, P < 0.0001) and IL-1β production (Fig. 9E; t-test, P < 0.0001) in lung during S. pneumoniae infection. Taken together, our data illustrate that age-enhanced levels of ER stress and initiation of the UPR contribute to impaired inflammasome function in aged macrophages and lung in response to S. pneumoniae. A decrease in ER stress improved NLRP3 inflammasome-mediated production of IL-1β in response to S. pneumoniae infection.

Fig. 9.

Treatment with TUDCA rescues NLRP3 inflammasome activation in the aged lung during S. pneumoniae infection. A–E; young (2 mo) and aged (18 mo) male and female BALB/c mice received saline or TUDCA (500 mg·kg⁻¹·day⁻¹) via intraperitoneal injection before intranasal instillation with saline or 1 × 10³ CFU of S. pneumoniae (S. pne) (ATCC 6303). A: gene expression in lung tissue collected from young, aged, and TUDCA-treated aged mice at 24 h postinfection was quantified by real-time PCR using inflammasome specific primer assays (SABiosciences). ASC, NLRP3, and pro-caspase-1 mRNA expression was evaluated by real-time PCR (ASC: t-test, ****P < 0.0001; NLRP3: t-test, ****P < 0.0001). Results were normalized to β-actin and are relative to uninfected control. B: Western blot analysis of NLRP3, ASC, and cleaved IL-1β protein expression in lung tissue collected from saline or TUDCA-pretreated aged mice at 24 h post-S. pneumoniae infection. C: lung homogenate samples were isolated at 24 h postinfection and coimmunoprecipitation was performed against anti-NLRP3. Eluted proteins were separated by electrophoresis and immunoblotted with anti-ASC. β-Actin was used as loading control. D: caspase-1 activity was quantified in saline or TUDCA pretreated lung at 24 h post-S. pneumoniae infection (saline vs. TUDCA- uninfected, t-test, **P = 0.0067; S. pneumoniae-infected, t-test, ****P < 0.0001). E: IL-1β production was evaluated by ELISA in saline and TUDCA-pretreated lung at 24 h postinfection (t-test, ****P < 0.0001). Similar results were obtained from at least 2 or more independent experiments with n = 10 per experiment. Data are expressed as the means ± SE.

DISCUSSION

The results of our current study demonstrate that age-enhanced ER stress and UPR activation contributes to diminished inflammasome assembly and activation during S. pneumoniae infection. Pretreatment of aged mice with the ER chaperone and stress-reducing agent TUDCA resulted in significantly decreased IRE1 phosphorylation and RNase activity in control and S. pneumoniae-infected lungs. A decrease in age-enhanced ER stress improved NLRP3 inflammasome activation and production of IL-1β in the aged lung in response to S. pneumoniae infection. Furthermore, increased NLRP3 inflammasome activation in TUDCA-pretreated aged lungs resulted in decreased bacterial load and improved lung pathology during S. pneumoniae infection.

In agreement with our previous work, there is significant enhancement in multiple components of the ER stress response in the aged lungs during S. pneumoniae infection (37). Our current results illustrate that modulation of IRE1 activity can restore NLRP3 inflammasome activation in aged macrophages in response to S. pneumoniae. IRE1 senses ER unfolded proteins through the ER luminal domain and becomes oligomerized, thereby activating its ribonuclease domain through autophosphorylation. Activation of IRE1 results in dissociation from GRP78/BIP, followed by cleavage and splicing of XBP1, a transcription factor that controls the transcription of chaperones that aid in restoring ER function, into its activated form (3, 28, 43, 54). It is plausible that enhanced IRE1 expression and function in aged macrophages and lung may play an important role in maintaining cellular homeostasis; thereby, reducing the potential for excessive inflammasome activation in the absence of pathogenic stimuli. Interestingly, direct TLR-1/2, -2, -4, -6, and -9 stimulation results in a significant increase in IL-1β production by aged macrophages; thereby illustrating the potential for heightened IL-1β production to occur by aged macrophages in response to treatment with specific TLR-stimulatory agents (Fig. 3F). Similar to other innate immune pathways, excessive inflammasome activation can be detrimental to the host, resulting in extensive tissue damage. Recent work, using a model of pneumococcal meningitis, has illustrated that the NLRP3 inflammasome can contribute to increased host pathology instead of pathogen protection and clearance (17). Our recent work has illustrated that in response to heightened ROS and DNA damage in response to bleomycin sulfate instillation, aged macrophages can contribute to harmful NLRP3-mediated inflammation and development of pulmonary fibrosis (48). Similarly, in the presence of high levels of free fatty acids, excessive NLRP3 activation can contribute to increased morbidity in response to S. pneumoniae (39). NLRP3 inflammasome responses are tightly regulated and the balance between harmful and beneficial responses to pathogenic stimuli has not been fully elucidated. As inflammasomes play an important role in the response to S. pneumoniae, future work will examine the impact of location, magnitude, the context of inflammasome activation, and the impact of biological aging on these responses.

Results from our current study demonstrate that treatment with TUDCA can rescue NLRP3 inflammasome activation in the aged lung during S. pneumoniae infection. Previous work has illustrated that TUDCA can efficiently inhibit the expression of UPR-dependent genes in response to the ER stressor tunicamycin (51). Of note, TUDCA can inhibit upstream signaling in the PERK, ATF6, and IRE1 branches of the ER stress response cascade as well as diminish binding of ER stress-inducible transcription factors to their respective promoters. It is possible that mechanisms that underlie host responsiveness to increased basal ER stress and mitochondrial ROS may also contribute to enhanced pneumococcal pathogenesis in the aged lung. Future work will need to be performed to fully elucidate this relationship.

Previous work has illustrated an essential role for the AIM2 inflammasome in caspase-1 activation in response to a serotype 2 strain of S. pneumoniae (13). Interestingly, while our results do not illustrate an essential role of AIM2 for caspase-1 activation, we did note that in the absence of AIM2, there is heightened NLRP3 protein expression in response to a serotype 3 strain of S. pneumoniae (Fig. 5). Previous work has illustrated a protective role for AIM2 in response to dsDNA release during influenza infection (45). Increased susceptibility of AIM2 knockout mice was attributed to a protective role of AIM2 in dampening inflammatory responses that contribute to excessive immunopathology in response to influenza (45). It is therefore plausible that AIM2 might also have a regulatory role in mediating the inflammatory response to S. pneumoniae. Specifically, as our results illustrate, while there was not excessive proinflammatory cytokine production in Aim2^−/− macrophages at 24 h post-S. pneumoniae infection, there was heightened NLRP3 expression and a trend toward increased IL-1β production (Fig. 5). As AIM2 recognition of both bacterial DNA and host-derived DNA plays an important role in shaping inflammatory signaling, it will be essential for future studies to investigate the impact of biological aging on AIM2 expression and the contribution of AIM2 regulation on excessive inflammation in lung during S. pneumoniae.

In agreement with previously published studies, we noted a decrease in NF-κB upregulation in response to TLR-1/2 and -6 stimulation (Fig. 3) (15, 16). Similarly, our results illustrate a decrease in TLR-1 mRNA expression in the aged lung in response to S. pneumoniae infection. Interestingly, in agreement with previously work, we did also note a significant increase in TLR-2 mRNA expression (4). It is possible that in agreement with previously published work, a chronic inflammatory environment contributes to increased TLR expression and dysfunction (16). It is important to note that our study investigated a highly virulent serotype 3 strain of S. pneumoniae with a higher incidence of occurrence in older adults. Given the divergent nature and secretion of pneumolysin by different S. pneumoniae strains, it will be of great interest to investigate if specific changes in TLR mRNA expression are conserved in response to multiple S. pneumoniae serotypes. These avenues of discovery may aid in the development of targeted therapeutics that can improve innate antibacterial responses against multiple pathogens.

In summary, the findings of our current study demonstrate a potential mechanism by which an age-associated enhancement in ER stress during S. pneumoniae infection results in decreased NLRP3 inflammasome activation. Treatment with TUDCA, an ER stress reducing agent, results in improved inflammasome activation in the aged lung in response to S. pneumoniae infection.

GRANTS

The work was supported by National Institute on Aging Grants R21-AG-044755, K01-AG-034999, and R01-AG-052530 (to H. W. Stout-Delgado) and a Department of Medicine Seed Grant for Innovative Research (to H. W. Stout-Delgado).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

S.J.C., A.M.C., and H.W.S.-D. conceived and designed research. S.J.C., K.R., and H.W.S.-D. performed experiments; S.J.C., K.R., and H.W.S.-D. analyzed data. S.J.C. and H.W.S.-D. interpreted results of experiments; S.J.C. and H.W.S.-D. prepared figures. S.J.C., A.M.C., and H.W.S.-D. edited and revised manuscript.