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. Author manuscript; available in PMC: 2013 Mar 15.
Published in final edited form as: J Immunol. 2012 Feb 10;188(6):2815–2824. doi: 10.4049/jimmunol.1103051

Impaired NLRP3 Inflammasome Function in Elderly Mice during Influenza Infection is Rescued by Treatment with Nigericin1

Heather W Stout-Delgado 2, Sarah E Vaughan 2, Anushree C Shirali 3, Richard J Jaramillo 2, Kevin S Harrod 2
PMCID: PMC3294083  NIHMSID: NIHMS350370  PMID: 22327078

Abstract

The NLRP3 inflammasome is activated in the lung during influenza viral infection; however the impact of aging on inflammasome function during influenza infection has not been examined. Here, we show that elderly mice infected with a mouse adapted strain of influenza produced lower levels of IL-1β during in vitro and in vivo infection. Dendritic cells from elderly mice exhibited decreased expression of ASC, NLRP3, and capase-1, but increased expression of pro-IL-1β, pro-IL-18, and pro-IL-33 when compared to young infected mice. Treatment with nigericin during influenza infection augmented IL-1β production, increased caspase-1 activity, and decreased morbidity and mortality in elderly mice. Our study demonstrates for the first time that during influenza viral infection, elderly mice have impaired NLRP3 inflammasome activity and that treatment with nigericin rescues NLRP3 activation in elderly hosts.

Introduction

Influenza viral infections are responsible for significant morbidity and mortality involving approximately five million people worldwide, with the highest infections rates found in the elderly (>65 years) population (1). It has been well established that innate and adaptive immune responses to influenza are impaired with aging, however the mechanisms that underlie this impairment and thereby contribute to higher levels of mortality and morbidity in elderly hosts have not been well elucidated (2-8). The innate immune system expresses several germline-encoded pathogen receptors, known as pattern recognition receptors (PRRs), that recognize general motifs that are essential to the function of microbes (9). Toll like receptors (TLRs), nucleotide-binding domain and leucine-rich-repeat receptors (NLR), and retinoic acid-inducible gene-I like receptors (RLR) are several classes of PRRs that have been shown to be involved in the recognition of influenza virus (10-13). Previous work has highlighted age-associated decreased TLR function in dendritic cells, dysregulated signaling cascades, and decreased cytokine production, which contribute to impaired innate immune responses (14-17). Similarly, age associated defects in RIG-I signaling, specifically impaired interferon signaling post RIG-I stimulation with West Nile Virus, have been observed (18). Recent work has illustrated a critical role for NLRs in immunity against influenza however age-associated changes in this pathway have not been reported (11).

Influenza virus activates inflammasome-dependent innate and adaptive immune responses (11). Influenza infection stimulates dendritic cells and macrophages via TLR7, leading to the synthesis of pro-IL-1β, pro-IL-18, and pro-IL-33. Recent work has shown that the proton-specific ion channel, M2, which plays a key role in both fusion and during viral entry and synthesis of new virions, can provide a secondary signal that is sufficient to trigger inflammasome assembly and activation (19). Activation of the NLRP3 inflammasome can be attributed to changes in potassium efflux and is ROS dependent (20, 21). Stimulation of the P2X7 receptor by ATP or nigericin treatment can stimulate NLRP3 inflammasome function in LPS primed dendritic cells (11, 21, 22). Once activated, NLRP3-dependent production of IL-1β by alveolar macrophages and dendritic cells leads to the production of chemokines and subsequent recruitment of inflammatory cells to the site of infection (11). Activation of the NLRP3 inflammasome during influenza viral infection leads to the migration of dendritic cells from the lung to the draining lymph node where subsequent T cell priming contributes to adaptive immune responses, specifically the establishment of Th1, CTL, and IgA responses to influenza viral infection (11, 23). Deficiency of NLRP3 inflammasome function results in decreased production of IL-1β and increased mortality upon challenge with a sublethal dose of influenza virus (11, 23, 24). Thus, NLRP3 inflammasome function is required for innate and adaptive immune responses that limit disease during influenza infection.

In the present study, we investigated the effect of aging on NLRP3 inflammasome activation in a murine model of influenza. Our results demonstrate that elderly hosts have impaired inflammasome activation, decreased gene expression of several key components of the NLRP3 inflammasome signaling pathway, decreased caspase-1 activity, and decreased production of IL-1β and IL-18 in response to in vitro and in vivo infection with HKx31, a mouse adapted strain of influenza. Adoptive transfer of young dendritic cells into elderly influenza infected hosts decreased morbidity, increased leukocyte infiltration, and increased IL-1β secretion when compared to elderly influenza infected mice that did not receive an adoptive transfer or were adoptively transferred with elderly dendritic cells. Interestingly, while pro-IL-1β, pro-IL-18, and pro-IL-33 mRNA expression was preserved in elderly dendritic cells, expression of NLRP3, ASC, and caspase-1 mRNA was decreased during HKx31 infection. Stimulation with ATP or nigercin, which have been previously shown to promote K+ efflux, augmented IL-1β and IL-18 release by elderly dendritic cells as well as increased expression of NLRP3 inflammasome components and augmented caspase-1 activity. Treatment of HKx31 infected elderly mice with nigericin resulted in increased IL-1β secretion as well as improved morbidity and mortality during the course of infection. Taken together, our data provide the first evidence for an age-associated decline in NLRP3 inflammasome function, which confers increased susceptibility of elderly hosts to influenza viral infection, and an early intervention, such as treatment with nigericin, can rescue function and improve clinical outcomes.

Materials and Methods

Mice

Young (2-4 months) and middle-aged (8-9 months) male and female BALB/c mice were either purchased from Taconic (Hudson, NY) or the National Institute of Aging (NIA) rodent facility (Charles River Laboratories, New York, NY). Elderly (15-16 months) male and female BALB/c mice were purchased from the NIA rodent facility (Charles River Laboratories). Upon receipt, mice were handled under identical husbandry conditions and fed certified commercial feed. Examination of NLRP3 inflammasome activation in young and middle-aged mice purchased from either Taconic or the NIA rodent facility showed that activation of the NLRP3 inflammasome and production of IL-1β was consistent despite the source of the animals. Body weights were measured daily and mice were humanely euthanized if they lost more than 20% of their starting body weight. The IACUC at Lovelace Respiratory Research Institute 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.

Viral propagation

HKx31 virus was generously provided by Dr. Ralph Tripp (University of Georgia, Department of Infectious Diseases) and grown in MDCK cells (ATCC, Manassas, VA) as previously described (25).

Primary bone marrow cell isolation and culture

Bone marrow cells were prepared from the femurs and tibias of mice as previously described (26). Bone marrow cells were cultured in complete RPMI (Invitrogen, Carlsbad, CA) containing 10% FBS (Invitrogen), and 1X penicillin/streptomycin (Invitrogen), and 25 ng/mL of GMCSF (Cell Signal, Danvers, MA) for 5 days at 37°C with 5% CO2. For in vitro stimulation experiments the following treatments were given in complete RPMI for specified times: HKx31 (MOI=10; 1, 2, or 24 hours), ATP (5mM; 0.5 - 1 hour) (Invivogen, San Diego, CA), nigericin (1μM; 0.5 – 1 hour) (Invivogen), and /or LPS (100-500 ng/mL; 3 hours) (Invivogen). Treatment with rotenone: cells were subsequently treated with rotenone (5μM, Enzo Life Sciences, Plymouth Meeting, PA) for 1 hour prior to ATP (5mM, Invivogen) stimulation (30 minutes). Treatment with mitoTEMPO: cells were treated with mitoTEMPO (100μM, Enzo Life Sciences) for 1 hour prior to culture with LPS (100ng/mL) or HKx31 (MOI=10) for 4 hours. Cells were then stimulated with ATP (5mM, Invivogen) for 30 minutes. Alum stimulation: cells were stimulated with LPS (100ng/mL) for 4 hours prior to alum stimulation (250μg/mL, 24 hours; Invivogen).

Pulmonary dendritic cell isolation

Pulmonary lymphocytes were isolated as previously described (27). Briefly, lung samples were digested at 37°C for 1 hour in freshly prepared collagenase digestion solution [300 U/ml collagenase type II (Roche), 10 ml PBS (Invitrogen), and 150μl DNase I of a 10 mg/ml stock solution (Roche)] prior to passage through a 40μm sieve. Cells were negatively selected using the EasySep dendritic cell selection kit (StemCell Technologies, Vancouver, Canada). Cell purity was assessed by flow cytometry (>95% purity).

RNA purification and real time PCR

RNA samples were extracted using the Qiagen RNAeasy mini kit (Qiagen, Valencia, CA) and quantified by A260/A280 absorbance readings. Superscript III Platinum SYBR Green One-Step qRT-PCR kit (Invitrogen) was used according to manufacturer’s instructions to assess primer specific gene expression. QuantiTect Primer Assays were purchased from Qiagen and used at a 1X concentration per reaction. PCR was conducted in 96-well plates using the Applied Biosystems 7300 detection system (Applied Biosystems, Carlsbad, CA). All reactions were performed in triplicate. Relative levels of messenger RNA (mRNA) were calculated by the comparative cycle threshold method (User Bulletin No. 2, Applied Biosystems) and 18sRNA mRNA levels were used as the invariant control for each sample.

ELISA

Culture supernatants or serum were analyzed for IL-1β or IL-18 production using ELISA kits purchased from eBiosciences (San Diego, CA) according to the manufacturer’s instructions.

Caspase-1 assay

Cultured cells were lysed and caspase-1 activity was assessed 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 blotting

Cells were washed and lysed in buffer containing 50mM Tris, pH 7.5, 1% (vol/vol) Triton X-100, 150mM NaCl, 10% (vol/vol) glycerol, 1mM EDTA and a protease inhibitor cocktail. Protein concentrations were assayed using the Bio-Rad protein assay reagents (Bio-Rad Laboratories, Hercules, CA) and using BSA as a standard. Equal amounts of protein (15μg/lane) were separated on 4-15% bis-acrylamide gels (Invitrogen) by SDS-PAGE and electrophoretically transferred to nitrocellulose membrane (Bio-Rad). Immunodetection was performed using primary rabbit anti-ASC (Genway Biotech, San Diego, CA), rabbit anti-NLRP3 (Abcam, Cambridge, MA), rabbit anti-capase-1 (Abcam), and secondary HRP-conjugated goat anti-rabbit IgG (Cell Signaling Technologies, Cambridge, MA) and the ECL Western Blotting Analysis System (Santa Cruz Biologicals, Santa Cruz, CA). Images were acquired using Multi-Guage software (Fujifilm, Greenwood, SC).

In vivo procedures

Influenza viral infection: All mice were anesthetized with isoflurane (5% for induction and 2% for maintenance) prior to intranasal instillation with 1×106 pfu of HKx31 (50-μL volume in PBS). Mice were observed twice daily for 16 days post HKx31 administration. As previously described, the following clinical scores were assigned: 0=normal, 1=slightly ruffled, 2=ruffled fur, 3= ruffled fur and inactive, 4=hunched/moribund, and 5=dead (28). Adoptive transfer: Mice were given 1 × 105 young or elderly bone marrow derived dendritic cells (200-μL volume in PBS) via intravenous injection of the tail vein one day prior to influenza instillation. Nigericin administration: Mice received a 250-μL volume of 0.005 mg/gram body weight dose of nigericin (Invivogen) (1X PBS as vehicle) intraperitoneally at 24 hours post influenza viral infection. On completion of each of these procedures, mice were monitored until normal breathing and postural reflexes returned.

Statistical Analysis

Survival analysis between groups was calculated using the Log rank method and the Mantel Cox test. Comparison of means was performed using a two-tailed T test, one-way or two-way ANOVA, when appropriate, and repeated measures using analysis of variance. All data were analyzed using GraphPad prism software (San Diego, CA). Statistical significance was considered by a p value < 0.05.

Results

Increased morbidity in elderly mice during influenza viral infection with HKx31

It is well established that the elderly have increased morbidity and mortality to influenza infections and it is believed that decreased immune function is the reason. Previous work has illustrated age associated impairments in immune responses during influenza viral infection with A/Puerto Rico/8/1934 (H1N1) (2, 29, 30). To further examine this phenotype, we infected young (2-4 months of age), middle-aged (8-9 months of age) and elderly (15 months of age) BALB/c mice with HKx31 (H3N2), a mouse-adapted strain of influenza (29). During influenza viral infection, elderly mice had higher clinical scores (Figure 1A; one-way ANOVA, p=0.0108) and increased mortality (Figure 1B; Mantel Cox test, p=0.0015) when compared to young mice. Further, when compared to young mice, elderly mice had decreased cellular infiltration of leukocytes to the site of infection (Figure 1C; t-test, p<0.01). At specific time points during influenza viral infection, lung tissue was harvested from young and elderly mice and lung viral titers were assessed by plaque assay. As illustrated in Figure 1D, elderly mice also had increased viral load in the lung (two-way ANOVA, p<0.0001) when compared to young virally infected mice. Taken together, these results illustrate that compared to young mice, elderly mice infected with HKx31 display increased morbidity with decreased leukocyte infiltration and increased viral load in the lung.

Figure 1. Increased morbidity and mortality in elderly mice during viral infection with HKx31.

Figure 1

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Young (2-4 months), middle-aged (8-9 months), and elderly (15 months) BALB/c mice were infected with 1×106 pfu of HKx31 via intranasal instillation. Throughout the duration of HKx31 infection, weights and clinical scores were recorded. (A) Clinical scores were recorded for young and elderly BALB/c mice post infection with HKx31, (one-way ANOVA, p=0.0108). Young and elderly PBS controls did not exhibit a significant change in clinical scores. (B) Survival of young and elderly BALB/c mice during HKx31 infection, (Mantel Cox test, p=0.0015). No lethality occurred in young and elderly PBS treated mice. (C) Lungs were harvested from HKx31 infected young and elderly BALB/c on day 5 of infection (t-test, p <0.01). Lung tissue was digested with collagenase A and the number of leukocytes is shown. (D) Viral titer was evaluated in lung homogenates from young and elderly mice by plaque assay (two-way ANOVA (p <0.0001). Similar results were obtained from at least three independent experiments with greater than N=5 per group and results are shown as the mean ± SEM.

Decreased activation of IL-1β in elderly dendritic cells during influenza viral infection

The NLRP3 inflammasome activates caspase-1 in response to influenza viral infection resulting in the processing and secretion of IL-1β and IL-18 (11, 31-33). As production of IL-1β and IL-18 is an important component of the acute phase responses to influenza, we examined if aging alters NLRP3 inflammasome mediated activation of IL-1β and IL-18. Young and elderly bone marrow derived dendritic cells (BMDCs) were infected with active or heat-inactivated HKx31 (MOI=10) for 24 hours and the production of IL-1β and IL-18 was assessed by ELISA. As shown in Figure 2, elderly BMDCs had significantly decreased secretion of IL-1β (Figure 2A; two-way ANOVA, p<0.0001) and IL-18 (Figure 2B; two-way ANOVA, p=0.009) in response to HKx31 when compared to young BMDCs. Production of mature IL-1β and mature IL-18 by young and elderly BMDCs in response to HKx31 was caspase-1 dependent, as treatment with caspase-1 inhibitor (V-ZAD-FMK) decreased IL-1β and IL-18 secretion in both young and elderly BMDCs (data not shown). We next investigated IL-1β and IL-18 production in elderly mice during HKx31 viral infection. Young, middle-aged, and elderly BALB/c mice were infected with HKx31 and lung, serum, and BAL were collected at specific time points during viral infection. When compared to young and middle-aged HKx31 infected groups, IL-1β and IL-18 production was significantly decreased in lung homogenates (Figure 2C; two-way ANOVA, p=0.0001, Figure 2D; two-way ANOVA, p=0.0003), serum (Figure 2E; two-way ANOVA, p<0.0001), and BAL (Figure 2F; t-test, p<0.0001, Figure 2G; t-test, p=0.0041) isolated from elderly mice. Taken together, these data indicate that aging decreases both in vitro and in vivo production of IL-1β and IL-18 in response to influenza viral challenge.

Figure 2. IL-1β expression is decreased in elderly mice during HKx31 viral infection.

Figure 2

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Young and elderly bone marrow cells were cultured with GM-CSF (25ng/mL) for 5 days in 37°C, 5% CO2. (A-B) Cells were cultured with media alone, heat inactivated (65°C 1 hour) HKx31 (MOI=10), or with HKx31 (MOI=10) for 24 hours. Cell culture supernatants were collected and IL-1β production (Figure 2A; two-way ANOVA, p<0.0001) and IL-18 expression (Figure 2B; two-way ANOVA, p=0.009) was assessed by ELISA. Young (2-4 months) and elderly (15 months) BALB/c mice were infected with 1×106 pfu of HKx31 via intranasal instillation. (C-E) Lung homogenates and serum were collected at specific time points during HKx31 infection and IL-1β and IL-18 production was assessed by ELISA [(C; two-way ANOVA, p=0.0001), (D; two-way ANOVA, p=0.0003), (E; two-way ANOVA, p<0.0001)]. (F-G) BAL was collected on day 5 of HKx31 infection and (F) IL-1β (t-test, p<0.0001) and (G) IL-18 (t-test, p=0.0041) production was assessed by ELISA. Similar results were obtained from three or more independent experiments. For in vitro experiments, the values represent N=3 or greater per experiment and are expressed as the mean ± SEM. For in vivo experiments, the values are representative of three or more mice per group and are expressed as the mean ± SEM.

Previous work has illustrated that activation of the NLRP3 inflammasome and subsequent processing of IL-1β by caspase-1 requires two distinct signals. The first signal involves Toll-like receptor (TLR) activation which induces the synthesis of pro-IL-1β, pro-IL-18, and pro-IL-33 (11). A second signal leads to the activation of the NLRP3 inflammasome and subsequent processing of IL-1β, IL-18, and IL-33 by caspase-1 (11). To examine if decreased IL-1β and IL-18 production by elderly dendritic cells was due to impaired TLR activation, we examined the expression of pro-IL-1β and pro-IL-18 during HKx31 infection. As shown in Figure 3A, synthesis of pro-IL-1β mRNA in elderly BMDCs was significantly higher during HKx31 infection when compared to young BMDCs (t-test, p=0.0053). Synthesis of pro-IL-18 and pro-IL-33 mRNA during HKx31 infection was similar between young and elderly BMDCs (Figure 3B and 3C, respectively). Nuclear translocation of the p65 subunit of NF-κB (data not shown) as well as the synthesis of IL-6 and TNF-α mRNA in response to HKx31 infection was similar between young and elderly BMDCs (data not shown). Next, we examined the expression of pro-IL-1β and pro-IL-18 in pulmonary dendritic cells isolated from young, middle-aged, and elderly control and HKx31 infected mice. As shown in Figure 3D, pro-IL-1β mRNA expression levels in pulmonary dendritic cells isolated from HKx31 infected mice were similar between all age groups. When compared to young, expression of pro-IL-18 and pro-IL-33 in pulmonary dendritic cells during HKx31 infection was increased in middle-aged and elderly mice (Figure 3E and F, respectively). Similarly, elderly pulmonary dendritic cells isolated from HKx31 infected mice had higher expression of IL-6 and TNF-α mRNA when compared to young (data not shown). Our results indicate that, despite increased mRNA transcript levels, IL-1β and IL-18 production by elderly dendritic cells is decreased during influenza viral infection.

Figure 3. Expression of pro-IL-1β, pro-IL-18, and pro-IL-33 is preserved in elderly dendritic cells during HKx31 infection.

Figure 3

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Young and elderly bone marrow cells were cultured with GM-CSF (25ng/mL) for 5 days in 37°C, 5% CO2. On day 5, cells were cultured in media alone or media containing HKx31 (MOI=10) for 24 hours. RNA was isolated and (A) pro-IL-1β (t-test, p=0.0053), (B) pro-IL-18, and (C) pro-IL-33 mRNA expression was assessed by real time PCR. Pulmonary dendritic cells were isolated from lung tissue collected from control and HKx31 infected young and elderly BALB/c mice on day 5 of infection. RNA was isolated and (D) pro-IL-1β, (E) pro-IL-18, and (F) pro-IL-33 (t-test, p=0.0306) mRNA expression was assessed by real time PCR. Similar results were obtained from three or more independent experiments with an N=3 or greater per experiment and are expressed as the mean ± SEM.

We next examined if activation of the NLRP3 inflammasome and subsequent activation of caspase-1 was altered in elderly dendritic cells during influenza infection. To this extent, young and elderly BMDCs were infected with HKx31 and gene expression of ASC, NLRP3, and caspase-1 was examined. As shown in Figure 4, when compared to young BMDC, elderly BMDC have decreased synthesis of ASC (Figure 4A; t-test, p=0.0025), NLRP3 (Figure 4B; t-test, p=0.0085), and caspase-1 (Figure 4C; t-test, p=0.0258) mRNA during HKx31 infection. We next investigated if caspase-1 activity in response to HKx31 infection was altered in elderly dendritic cells. As shown in Figure 4D, capase-1 activity in elderly BMDCs during HKx31 infection was decreased when compared to young HKx31 infected BMDCs (t-test, p=0.0015). To expand upon these results, we examined ASC, NLRP3, and caspase-1 mRNA synthesis in young, middle-aged, and elderly pulmonary dendritic cells during HKx31 infection. Dendritic cells isolated from lungs of elderly HKx31 infected mice had decreased expression of ASC, NLRP3, and caspase-1 when compared to dendritic cells isolated from lungs of young HKx31 infected mice (data not shown).

Figure 4. NLRP3 inflammasome mediated activation of IL-1β in elderly dendritic cells during influenza viral infection is decreased.

Figure 4

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Young and elderly bone marrow cells were cultured with GM-CSF (25ng/mL) for 5 days in 37°C, 5% CO2. On day 5, cells were cultured in media alone or media containing HKx31 (MOI=10) for 24 hours. RNA was isolated and (A) ASC [t-test (p=0.0025)], (B) NLRP3 [t-test (p=0.0085)], and (C) caspase-1 [t-test (p=0.0258)] mRNA expression was assessed by real time PCR. (D) Caspase-1 activity was measured in young and elderly BMDCs culture with HKx31 for 4 hours (37°C, 5% CO2), [t-test (p=0.0015)]. Similar results were obtained from three or more independent experiments with an N=3 or greater per experiment and are expressed as the mean ± SEM.

Next we examined if adoptive transfer of young dendritic cells to elderly hosts could rescue impaired NLRP3 function in elderly mice during influenza infection. To test this, one day prior to influenza viral infection, young and elderly BALB/c mice were adoptively transferred with dendritic cells isolated from either young or elderly BALB/c mice. As shown in Figure 5A, adoptive transfer of young dendritic cells to elderly mice attenuated the decreased weight loss when compared to HKx31 infected elderly controls (two-way ANOVA, p<0.0001). Further, elderly mice receiving an adoptive transfer of young dendritic cells prior to influenza viral infection had increased cellular infiltration, as illustrated by increased lung weight (Figure 5B; two-way ANOVA, p<0.0001) and total leukocyte numbers (Figure 5C; two-way ANOVA, p=0.0002), when compared to elderly HKx31 infected controls. We next examined if adoptive transfer of young dendritic cells increased production of IL-1β in elderly hosts during HKx31 infection. As shown in Figure 5D-E, elderly mice receiving an adoptive transfer of young dendritic cells had increased IL-1β release in serum (Figure 5D; two-way ANOVA, p<0.0001) and lung homogenates (Figure 5E; two-way ANOVA, p<0.0001) during influenza infection when compared to elderly HKx31 infected controls.

Figure 5. Adoptive transfer of young dendritic cells to aged hosts improves morbidity during HKx31 infection.

Figure 5

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Young (2-4 months) or elderly (15 months) BALB/c mice received an adoptive transfer of 1×105 young or elderly dendritic cells one day prior to infection with 1×105 pfu of HKx31 via intranasal instillation. Throughout the duration of HKx31 infection, weights and clinical scores were recorded. (A) Percentage weight change for each time point was defined as percentage of weight loss from baseline (day 0) for each mouse (two-way ANOVA, p<0.0001). (B) Lung tissue was isolated on specific time points during HKx31 infection and percentage weight loss was defined as change in lung weight at time of necropsy from PBS treated controls (two-way ANOVA, p <0.0001). (C) Lung tissue was digested and leukocytes were harvested. Trypan blue staining was performed and total leukocyte numbers were quantified (two-way ANOVA, p=0.0002). Dead cells, as defined as trypan blue positive, were excluded. (D) Serum (two-way ANOVA, p<0.0001) and (E) lung homogenates (two-way ANOVA, p<0.0001) were collected at specific time points during HKx31 infection and IL-1β production was assessed by ELISA. Similar results were obtained from three or more independent experiments with four or more mice per group and are expressed as the mean ± SD.

Decreased NLRP3 inflammasome mediated production of IL-1β by elderly dendritic cells is not unique to influenza viral infection

Previous work has illustrated that in the presence of pathogen associated molecular patterns (PAMPs), alum crystals can activate the NLRP3 inflammasome (11, 33-37). Activation of the NLRP3 inflammasome by alum requires phagocytosis, which causes lysosomal swelling and damage and involves cathepsin B, a lysosomal cysteine protease (35-37). To this extent, we examined production of IL-1β by in response to alum in LPS stimulated young and elderly BMDCs. As shown in Supplemental Figure 1A, despite similar lysosomal trafficking and cathepsin B mRNA expression (data not shown), treatment of LPS stimulated elderly BMDCs with alum resulted in decreased production of IL-1β when compared to similarly stimulated young BMDCs (t-test, p=0.0015). Further, when compared to young BMDC, NLRP3 mRNA expression (Supplemental Figure 1B; t-test, p=0.0066) and caspase-1 activity (Supplemental Figure 1C; t-test, p=0.0328) were also decreased in elderly BMDC post alum stimulation.

An alteration in mitochondrial ROS does not enhance the production of IL-1β by elderly dendritic cells

Mitochondrial ROS is required for the formation of the NLRP3 inflammasome (20, 21). As aging is associated with increased levels of ROS, we next examined the impact of aging on mitochondrial integrity and production of ROS on NLRP3 activation (38). Young and elderly BMDCs cultured with LPS or HKx31 were treated with rotenone, a mitochondrial complex I inhibitor, followed by stimulation with ATP. As shown in Supplemental Figure 2A, both young and elderly LPS primed BMDCs that received rotenone treatment had increased release of IL-1β following ATP stimulation. Despite an overall increase in IL-1β production, when compared to young, elderly LPS primed BMDCs had significantly decreased levels of IL-1β regardless of treatment with rotenone (Supplemental Figure 2A; t-test, p=0.0030), stimulation with ATP (Supplemental Figure 2A; t-test, p=0.0095), or treatment with both rotenone and ATP (Supplemental Figure 2A; t-test, p=0.0058). While rotenone treatment during stimulation with LPS and ATP increased NLRP3 mRNA expression in both young and elderly BMDCs, NLRP3 expression in elderly BMDCs was still significantly decreased when compared to young BMDCs after rotenone treatment (Supplemental Figure 2B; two-way ANOVA, p=0.001). To investigate if alterations in mitochondrial integrity could enhance NLRP3 inflammasome activity in elderly BMDCs during influenza viral infection, we next examined IL-1β production post treatment with rotenone. As illustrated in Supplemental Figure 2C, treatment with rotenone stimulated IL-1β production by elderly BMDCs. In the presence of ATP, IL-1β production by both young and elderly BMDCs during HKx31 infection was increased and levels were similar between the groups (Supplemental Figure 2C). Rotenone treatment during HKx31 infection increased NLRP3 mRNA expression similarly in both young and elderly BMDCs (Supplemental Figure 2D). Interestingly, ATP stimulation of HKx31 infected elderly BMDCs resulted in a significant up-regulation of NLRP3 mRNA when compared to ATP stimulated young HKx31 infected BMDCs (Supplemental Figure 2D; t-test, p=0.0381). Given these findings, we next examined NLRP3 mediated production of IL-1β in the absence of mitochondrial ROS. Thus, prior to LPS stimulation or HKx31 infection, we treated both young and elderly BMDCs with mitoTEMPO, a mitochondrial targeted antioxidant. As shown in Supplemental Figure 2E, treatment with mitoTEMPO decreased IL-1β production in both young and elderly BMDCs during both LPS stimulation (Supplemental Figure 2E; t-test, p=0.0250) and HKx31 infection (Supplemental Figure 2F). Taken together, these results illustrate that IL-1β production by young and elderly dendritic cells is ROS dependent and decreased mitochondrial-specific generation of ROS equally impaired IL-1β release by young and elderly BMDCs.

Caspase-1 activity in elderly dendritic cells during influenza viral infection is recovered by depletion of intracellular potassium (K+)

Previous work has illustrated that stimulation of the P2X7 receptor can provide a signal that leads to the maturation and release of active IL-1β (21, 22, 39, 40). Upon activation, the P2X7 receptor induces a rapid potassium efflux from the cytosol (39, 40). While potassium efflux alone is not sufficient for activation of the NLRP3 inflammasome, association with pannexin-1 and subsequent opening of a larger pore that mediates the delivery of PAMPs to the cytosol can lead to the activation of caspase-1 and release of IL-1β (40, 41). As stimulation of the P2X7 receptor can serve as a strong second signal for NLRP3 inflammasome activation, we examined if stimulation of the P2X7 receptor with ATP could augment caspase-1 activation and release of IL-1β by elderly dendritic cells during influenza infection. Young and elderly BMDCs were infected with HKx31 followed by stimulation with ATP and NLRP3 mRNA expression was evaluated. When compared to young HKx31 infected BMDCs, ATP stimulation significantly increased NLRP3 mRNA expression in elderly HKx31 infected BMDCs (Figure 6A; t-test, [*] p=0.0175 and [*2] p=0.0381). We next examined if ATP stimulation could increase caspase-1 activity in elderly HKx31 infected BMDCs. As shown in Figure 6, when compared to HKx31 infection alone, stimulation of elderly HKx31 infected BMDCs with ATP significantly increased caspase-1 activity (Figure 6B; t-test, p=0.0015), IL-1β release (Figure 6C; t-test, p=0.0278), and IL-18 production (Figure 6D, two-way ANOVA, p<0.0001). Interestingly, despite increased P2X7 receptor expression in elderly BMDCs (data not shown), when compared to young, ATP stimulation of elderly HKx31 infected BMDCs resulted in similar caspase-1 activity, IL-1β release, and IL-18 production (Figure 6B-D, respectively).

Figure 6. Capase-1 activity in elderly dendritic cells is recovered by treatment with ATP or nigericin.

Figure 6

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Young and elderly bone marrow cells were cultured with GM-CSF (25ng/mL) for 5 days in 37°C, 5% CO2. On day 5, cells were cultured for 24 hours with HKx31 (MOI=10) prior to stimulation with ATP (5mM) (A-D, H) or nigericin (10μM) (E-G, H) for 30 minutes. (A) HKx31 infected cells were collected 30 minutes post ATP stimulation and NLRP3 mRNA expression was assessed by real time PCR [t-test, (*, p=0.0175; *2, p=0.0381)]. (B) Caspase-1 activity was assessed 30 minutes post ATP stimulation (t-test, p=0.0015). (C-D) Cell culture supernatants were collected post ATP stimulation and IL-1β (t-test, p=0.0278) and IL-18 (two-way ANOVA, p<0.0001) production was assessed by ELISA. (E) Caspase-1 activity was assessed 30 minutes post nigericin stimulation (t-test, p=0.0117). (F-G) Cell culture supernatants were collected post nigericin stimulation and IL-1β (two-way ANOVA, p<0.0001) and IL-18 (two-way ANOVA, p<0.0001) production was assessed by ELISA. (H) Dendritic cells were collected post ATP or nigericin stimulation and NLRP3, ASC, and caspase-1 protein expression was examined by western blotting. GAPDH was used as a loading control. Similar results were obtained from two or more independent experiments with an N=3 or greater and are expressed as the mean ± SEM.

We next investigated if nigericin, a pore forming toxin that has been shown to promote P2X7 receptor independent potassium efflux, could increase NLRP3 activation in elderly BMDCs during influenza viral infection (21). Young and elderly HKx31 infected BMDCs were stimulated with nigericin and caspase-1 activity was assessed. Treatment with nigericin resulted in a significant increase in caspase-1 activity in elderly HKx31 infected BMDCs (Figure 6E; t-test, p=0.0117) when compared to untreated, elderly HKx31 infected BMDCs. When compared to young, nigericin stimulation of elderly HKx31 infected BMDCs resulted in significantly increased IL-1β release (Figure 6F; two-way ANOVA, p<0.0001) and IL-18 production (Figure 6G; two-way ANOVA, p<0.0001).

Based upon these findings, we next investigated if treatment of elderly BMDCs with ATP or nigericin could rescue NLRP3, ASC, or caspase-1 protein expression during influenza infection. To this extent, young and elderly BMDCs were infected with HKx31 followed by stimulation with either ATP or nigericin and protein expression of ASC, NLRP3, and caspase-1 was examined by western blot. As shown in Figure 6H, protein expression of NLRP3, ASC, and caspase-1 was increased in young BMDCs during HKx31 infection as well as in response to subsequent ATP or nigericin stimulation. In contrast, NLRP3, ASC, and caspase-1 levels are only detectable in elderly HKx31 infected BMDCs post ATP or nigercin stimulation (Figure 6H).

As nigericin treatment can increase caspase-1 activity and IL-1β release during in vitro influenza viral infection, we examined if administration of nigericin during influenza viral infection would stimulate NLRP3 inflammasome mediated production of IL-1β in elderly hosts. At 24 hours post influenza administration elderly mice were treated with PBS or nigericin (0.005 mg/kg). As shown in Figure 7, elderly mice that received nigericin had decreased morbidity and mortality during influenza infection, as illustrated by decreased weight loss (data not shown), decreased clinical scores (Figure 7A, two-way ANOVA, p<0.0001), and decreased lethality (Figure 7B; Mantel Cox test, p=0.0004) when compared to untreated, HKx31 infected elderly controls. Further, when compared to PBS treated controls, elderly mice that received nigericin treatment had increased infiltration of leukocytes (Figure 7C; t-test, p=0.009) as well as increased IL-1β release (Figure 7D; t-test, p=0.007). Taken together, these results illustrate that nigericin treatment during early influenza infection augments IL-1β production and improves clinical outcomes in elderly mice.

Figure 7. Nigericin treatment during influenza infection results in decreased mortality and increased IL-1β expression.

Figure 7

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Elderly (15 months) BALB/c mice were infected with 1×106 pfu of HKx31 via intranasal instillation. At 24 hours post infection, mice received a weight based dose of nigericin (0.005mg/kg body weight). (A) Clinical scores were recorded for untreated and nigericin treated elderly BALB/c mice during the course of infection with HKx31 (two-way ANOVA, p<0.0001). (B) Survival of untreated and nigericin treated elderly BALB/c mice during HKx31 infection (Mantel Cox test, p=0.0004). (C) On day 5 of HKx31 infection, lung tissue was digested and leukocytes were collected. Trypan blue staining was performed and total leukocyte numbers on day 5 were quantified (t-test, p=0.009). Dead cells, as defined as trypan blue positive, were excluded. (D) Day 5 lung homogenates (t-test, p=0.007) were collected post nigericin treatment and IL-1β production was assessed by ELISA. Similar results were obtained for two or more independent experiments and the values are representative of N=4 or more mice per group. Results are expressed as the mean ± SEM.

Discussion

While the increased burden of influenza infections in the elderly (>65 years) population is well know, the molecular mechanisms that influence this increased susceptibility have not been well elucidated. In the current study, we examined the impact of aging on inflammasome activation during influenza viral infection. We found that NLRP3 inflammasome mediated activation of IL-1β by elderly dendritic cells is decreased during influenza infection (Figure 2). This was not due to impaired NF-κB mediated gene transcription as expression of the pro-forms of IL-1β, IL-18, IL-33, as well as IL-6 and TNF-α mRNAs was elevated in both young and elderly dendritic cells during influenza infection (Figure 3). In contrast, synthesis of NLRP3, ASC, and caspase-1 was impaired in elderly dendritic cells during influenza infection (Figure 4, Figure 6). A similar decrease in NLRP3 and ASC mRNA expression was detected post stimulation with alum, despite increased cathepsin B expression and similar lysosomal trafficking (Supplemental Figure 1). Collectively, these results illustrate that decreased caspase-1 activity and decreased expression of mature IL-1β in elderly dendritic cells occurs at a proximal step of NLRP3 inflammasome activation, such as assembly of the NLRP3 complex or activation of the NLRP3 subunit, and is decreased despite treatment with alum or during influenza infection. Interestingly, IL-1β is known to play an important role in the migration of dendritic cells from the lung to the draining lymph nodes (42). Impaired IL-1β expression in elderly hosts early during influenza infection may contribute to decreased mobilization of dendritic cells, thereby resulting in an impaired immune response and decreased viral clearance.

Our data demonstrates that stimulation with ATP or nigericin can amplify NLRP3 mediated activation of IL-1β by increasing potassium efflux in elderly dendritic cells (Figure 6). Our results illustrate that ionic changes, such as potassium efflux, in the cytosol are critical for activating the NLRP3 inflammasome in elderly dendritic cells. Further, despite impaired inflammasome activation during influenza infection, treatment with ATP or nigericin can enhance caspase-1 activity and expression of mature IL-1β and IL-18 (Figure 6). Following ligation, the channels formed by the P2X7 receptor rapidly transform to pores that allow passage of PAMPs and DAMPs into the cytosol. Therefore, treatment of elderly dendritic cells with ATP may also further amplify TLR stimulation and increase inflammasome activity. Of note, mRNA and protein expression of NLRP3, ASC, and caspase-1 in elderly dendritic cells was enhanced with ATP or nigericin stimulation, indicating that transcription and translation of these inflammasome components is not impaired in aging (Figure 6). Further, activation of caspase-1 and expression of IL-1β and IL-18 was also enhanced by ATP or nigericin stimulation, illustrating that expression and activity of these components is also conserved in elderly dendritic cells (Figure 6).

The NLRP3 inflammasome signaling pathway is a multi-faceted signaling pathway that requires two signals to induce inflammasome complex formation and secretion of IL-1β and IL-18. Based on our current results, LPS priming and secondary stimulation with ATP does not fully rescue impaired inflammasome activation in elderly dendritic cells and this may be due to a residual age-induced impairment in NLRP3 activation. ATP stimulation leads to changes in ionic concentrations within the cytoplasm and NLRP3 senses these disturbances, thus leading to inflammasome complex formation. We have examined NLRP3 mRNA expression in response to LPS priming and subsequent ATP stimulation and have found that NLRP3 expression in elderly dendritic cells does increase (approximately 15-fold), but these levels are lower than those detected in similarly treated, young dendritic cells (Supplemental Figure 2). Previous work has shown that inflammasome activation during influenza viral infection is mediated by the M2 ion channel, which via pH neutralization of the trans-golgi network results in potassium efflux and increased ROS production (27). Hence, influenza can stimulate the NLRP3 inflammasome via TLR7 stimulation and M2 ion channel mediated activation of NLRP3. Treatment of young and elderly dendritic cells with heat inactivated HKx31 resulted in a significant decrease in IL-1β and IL-18 expression (Figure 2) indicating that actively replicating HKx31 was necessary for IL-1β and IL-18 expression. Based on our current findings, there may be a defect in viral mediated production of M2, translocation of M2 through the trans-Golgi network, or M2 mediated activation of the NLRP3 inflammasome in elderly dendritic cells. Subsequent treatment with ATP or nigericin possibly perturbs the ionic concentration within the cytoplasm and thereby, coupled with influenza infection, serves as a potent stimulus that rescues this impairment and increases NLRP3 mRNA and protein expression (Supplemental Figure 2 and Figure 6, respectively). Based upon our current findings, it is possible that when influenza infected elderly dendritic cells are treated with ATP or nigericin, this dual stimulation overcomes defective NLPR3 activation whereas LPS priming and subsequent ATP stimulation, while it does increase NLRP3 activation and IL-1β production, does not provide a robust enough signal that fully restores this aged induced impairment.

Aging is associated with an accumulation of ROS within cells, which augments oxidative stress and subsequently impairs cellular functions (43). Similar to previously published studies, our data demonstrate that mitochondrial generation of ROS is important for NLRP3 inflammasome mediated activation of IL-1β (20). Interestingly, enhancement of ROS by treatment of young and elderly dendritic cells with rotenone resulted in similar levels of IL-1β expression during influenza infection (Supplemental Figure 2). Treatment with mitoTEMPO equally decreased IL-1β expression by young and elderly dendritic cells during influenza infection (Supplemental Figure 2). Taken together, these results illustrate that while ROS synthesis is necessary for inflammasome formation and function, aged induced impairments in caspase-1 activity and IL-1β production during influenza infection was not solely due to an increased accumulation of ROS within elderly dendritic cells.

Pneumonia has become an increasingly significant cause of morbidity and mortality in the aging population (44-46). Although primary influenza infection alone can lead to adverse outcomes, secondary bacterial infections during and shortly after recovery from influenza infections are more common reasons for influenza-associated illness (47). Previous work has illustrated multiple bacterial pathogens can activate the NLRP3 inflammasome. Secretion of pneumolysin by Streptococcus pneumoniae can induce NLRP3 inflammasome activation through potassium efflux and lysosomal leakage induction and absence of NLRP3 expression resulted in impaired bacterial clearance (48). As NLRP3 inflammasome activity during influenza infection is impaired, it will be important to examine whether elderly hosts display altered responses to secondary bacterial infections following primary influenza infection.

In summary, our findings show that NLRP3 inflammasome activation during influenza viral infection is impaired in elderly dendritic cells. An increase in potassium efflux by ATP or nigericin stimulation rescued this defect. The data presented in this study provide new evidence as to why older persons are more susceptible to influenza viral infection and provide a possible mechanism to enhance these responses, thereby decreasing morbidity and mortality in this population.

Supplementary Material

1
2

Acknowledgments

We graciously thank Ralph Tripp for the HKx31 virus; Jennifer Tipper for propagation of the HKx31 virus; Steven Belinsky, Yohannes Tesfaigzi, Julie Wilder, and Mohan Sopori for valuable discussions; Zemmie Pollock, Dana Mitzel, and Jessie vanWestrienen for their assistance with experimental techniques.

Abbreviations

DC

dendritic cell

NLRP3

NLR family, pyrin domain containing 3

ASC

Apoptosis-associated speck-like protein containing a CARD

IL

interleukin

IAV

influenza A virus

P2X7R

P2X purinoceptor 7

Footnotes

1

This work was supported by NIH K01AG034999-01A1 (H.W.S).

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2
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