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Cited by 102Adenosine and Purinergic SignalingInflammasome and immune disordersImmune Response and Inflammation

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Activation of P2X7 Receptor by ATP Plays an Important Role in Regulating Inflammatory Responses during Acute Viral Infection

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Lee, Benjamin H., Hwang, David M., Palaniyar, Nades, Grinstein, Sergio, Philpott, Dana J., Hu, Jim·2012·PLoS ONE
DOI 10.1371/journal.pone.0035812PMC PMC3338466
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Abstract

Acute viral infection causes damages to the host due to uncontrolled viral replication but even replication deficient viral vectors can induce systemic inflammatory responses. Indeed, overactive host innate immune responses to viral vectors have led to devastating consequences. Macrophages are important innate immune cells that recognize viruses and induce inflammatory responses at the early stage of infection. However, tissue resident macrophages are not easily activated by the mere presence of virus suggesting that their activation requires additional signals from other cells in the tissue in order to trigger inflammatory responses. Previously, we have shown that the cross-talk between epithelial cells and macrophages generates synergistic inflammatory responses during adenoviral vector infection. Here, we investigated whether ATP is involved in the activation of macrophages to induce inflammatory responses during an acute adenoviral infection. Using a macrophage-epithelial cell co-culture system we demonstrated that ATP signaling through P2X(7) receptor (P2X(7)R) is required for induction of inflammatory mediators. We also showed that ATP-P2X(7)R signaling regulates inflammasome activation as inhibition or deficiency of P2X(7)R as well as caspase-1 significantly reduced IL-1β secretion. Furthermore, we found that intranasal administration of replication deficient adenoviral vectors in mice caused a high mortality in wild-type mice with symptoms of acute respiratory distress syndrome but the mice deficient in P2X(7)R or caspase-1 showed increased survival. In addition, wild-type mice treated with apyrase or inhibitors of P2X(7)R or caspase-1 showed higher rates of survival. The improved survival in the P2X(7)R deficient mice correlated with diminished levels of IL-1β and IL-6 and reduced neutrophil infiltration in the early phase of infection. These results indicate that ATP, released during viral infection, is an important inflammatory regulator that activates the inflammasome pathway and regulates inflammatory responses.

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Figure 1 Treatment with oATP inhibited inflammatory responses in Ad infected MLE-Raw co-culture. The MLE-Raw co-culture was infected with Ad for 24 h in the presence of oATP at the designated concentrations. (A), The amount of NO produced in the culture was measured from the medium using Griess reagent. Data are expressed as mean ± SD. (B), The MLE-Raw co-culture was infected with Ad and ROS positive cells from were counted by flow cytometry after treating with 3-(p-aminophenyl) fluorescein (APF) without gating for specific cell types. Shown is a representative FACS histogram of three independent experiments with the percentage of ROS positive population. (C and D) Mouse IL-6 and KC, respectively, were measured from the medium using commercially available ELISA kits. Data are expressed as mean ± standard deviation (SD).
Figure 1 Treatment with oATP inhibited inflammatory responses in Ad infected MLE-Raw co-culture. The MLE-Raw co-culture was infected with Ad for 24 h in the presence of oATP at the designated concentrations. (A), The amount of NO produced in the culture was measured from the medium using Griess reagent. Data are expressed as mean ± SD. (B), The MLE-Raw co-culture was infected with Ad and ROS positive cells from were counted by flow cytometry after treating with 3-(p-aminophenyl) fluorescein (APF) without gating for specific cell types. Shown is a representative FACS histogram of three independent experiments with the percentage of ROS positive population. (C and D) Mouse IL-6 and KC, respectively, were measured from the medium using commercially available ELISA kits. Data are expressed as mean ± standard deviation (SD).CC BY · PubMed Central
Figure 2 Ad infection of the co-culture with P2X7R deficient Raw cells generated significantly less inflammatory mediators. The co-culture of MLE and a P2X7R deficient Raw cell (SF) was established as MLE-Raw co-culture. (A) MLE-Raw and MLE-SF co-cultures were infected with the designated MOI of Ad. The amount of NO produced in the culture medium was measured by Griess assay. Data are expressed as mean ± SD. (B) To verify that SF cells has no inherent defect in NO generation MLE-SF co-culture was treated with LPS (2.5 µg/ml) and NO generation was compared with Ad infected MLE-SF as well as MLE-Raw co-cultures. Data are expressed as mean ± SD. (C) The ROS positive cells in the Ad infected MLE-SF co-culture were counted by flow cytometry analysis after treating cells with APF. Shown is a representative FACS histogram of three independent experiments with the percentage of ROS positive population. (D) Mouse IL-6 was measured from the media of mono-cultures (MLE, Raw, and SF) and co-cultures (MLE-Raw and MLE-SF) 24 h after Ad infection using a commercially available ELISA kit. Data are expressed as mean ± SD.
Figure 2 Ad infection of the co-culture with P2X7R deficient Raw cells generated significantly less inflammatory mediators. The co-culture of MLE and a P2X7R deficient Raw cell (SF) was established as MLE-Raw co-culture. (A) MLE-Raw and MLE-SF co-cultures were infected with the designated MOI of Ad. The amount of NO produced in the culture medium was measured by Griess assay. Data are expressed as mean ± SD. (B) To verify that SF cells has no inherent defect in NO generation MLE-SF co-culture was treated with LPS (2.5 µg/ml) and NO generation was compared with Ad infected MLE-SF as well as MLE-Raw co-cultures. Data are expressed as mean ± SD. (C) The ROS positive cells in the Ad infected MLE-SF co-culture were counted by flow cytometry analysis after treating cells with APF. Shown is a representative FACS histogram of three independent experiments with the percentage of ROS positive population. (D) Mouse IL-6 was measured from the media of mono-cultures (MLE, Raw, and SF) and co-cultures (MLE-Raw and MLE-SF) 24 h after Ad infection using a commercially available ELISA kit. Data are expressed as mean ± SD.CC BY · PubMed Central
Figure 3 Ad infection of epithelial cell and macrophage co-culture induced secretion of IL-1β and IL-18. (A and B) MLE and J774 mono-culture as well as MLE-J774 co-culture were infected with Ad. IL-1β (A) and IL-18 (B) secretion was measured by ELISA assay using the media 24 h after infection. Data are expressed as mean ± SD. (C) MLE-J774 co-culture was infected with 0, 2, 20, or 100 MOI of Ad and IL-1β in the medium was measured 24 h after infection. Data are expressed as mean ± SD. (D) MLE-J774 co-culture was infected with Ad and medium was collected at designated time for Western blot analysis following immunoprecipitation. (E) Co-cultures of different ratio of MLE and J774 were established and IL-1β was measured in the medium 24 h after Ad infection. Data are expressed as mean ± SD. (F) A human lung epithelial cell line, A549, instead of MLE was used to establish the co-culture with J774 macrophages and infected with Ad. IL-1β was measured 24 h after infection. Data are expressed as mean ± SD.
Figure 3 Ad infection of epithelial cell and macrophage co-culture induced secretion of IL-1β and IL-18. (A and B) MLE and J774 mono-culture as well as MLE-J774 co-culture were infected with Ad. IL-1β (A) and IL-18 (B) secretion was measured by ELISA assay using the media 24 h after infection. Data are expressed as mean ± SD. (C) MLE-J774 co-culture was infected with 0, 2, 20, or 100 MOI of Ad and IL-1β in the medium was measured 24 h after infection. Data are expressed as mean ± SD. (D) MLE-J774 co-culture was infected with Ad and medium was collected at designated time for Western blot analysis following immunoprecipitation. (E) Co-cultures of different ratio of MLE and J774 were established and IL-1β was measured in the medium 24 h after Ad infection. Data are expressed as mean ± SD. (F) A human lung epithelial cell line, A549, instead of MLE was used to establish the co-culture with J774 macrophages and infected with Ad. IL-1β was measured 24 h after infection. Data are expressed as mean ± SD.CC BY · PubMed Central
Figure 4 Inhibition of P2X7R and caspase-1 reduced IL-1β secretion in the Ad infected co-culture. (A and B) MLE-J774 co-culture was infected with Ad in the presence of oATP (0∼400 µM) or z-YVAD-fmk (0∼50 µM) and IL-1β secretion was measured in the medium. Data are expressed as mean ± SD. (C) The co-culture of MLE and a P2X7R deficient J774 cell line (ATPR) was establish and IL-1β in the medium was measured 24 h after Ad infection. Data are expressed as mean ± SD. (D) MLE-J774 co-culture was treated with a P2X7R specific inhibitor, A438079 (0∼200 µM) at the designated concentration before Ad infection and IL-1β was measured from the medium 24 h after infection. Data are expressed as mean ± SD.
Figure 4 Inhibition of P2X7R and caspase-1 reduced IL-1β secretion in the Ad infected co-culture. (A and B) MLE-J774 co-culture was infected with Ad in the presence of oATP (0∼400 µM) or z-YVAD-fmk (0∼50 µM) and IL-1β secretion was measured in the medium. Data are expressed as mean ± SD. (C) The co-culture of MLE and a P2X7R deficient J774 cell line (ATPR) was establish and IL-1β in the medium was measured 24 h after Ad infection. Data are expressed as mean ± SD. (D) MLE-J774 co-culture was treated with a P2X7R specific inhibitor, A438079 (0∼200 µM) at the designated concentration before Ad infection and IL-1β was measured from the medium 24 h after infection. Data are expressed as mean ± SD.CC BY · PubMed Central
Figure 5 Co-cultures with primary macrophages from P2X7R and caspase-1 knockout mice secreted less IL-1β. (A) The co-culture was established with MLE and peritoneal macrophages obtained from wild-type (C56BL/6) mice and infected with Ad with or without oATP (200 µM) or A438079 (100 µM). IL-1β was measured from the medium 24 h after infection. Data are expressed as mean ± SD. (B–D), Peritoneal macrophages were collected from and P2X7R (B), caspase-1 (C), or NLRP3 (D) knockout mice and the co-cultures were established with MLE. IL-1β was measured from the medium 24 h (18 h and 24 h for NLRP3-KO) after Ad infection and compared to the wild-type counterpart. Data are expressed as mean ± SD.
Figure 5 Co-cultures with primary macrophages from P2X7R and caspase-1 knockout mice secreted less IL-1β. (A) The co-culture was established with MLE and peritoneal macrophages obtained from wild-type (C56BL/6) mice and infected with Ad with or without oATP (200 µM) or A438079 (100 µM). IL-1β was measured from the medium 24 h after infection. Data are expressed as mean ± SD. (B–D), Peritoneal macrophages were collected from and P2X7R (B), caspase-1 (C), or NLRP3 (D) knockout mice and the co-cultures were established with MLE. IL-1β was measured from the medium 24 h (18 h and 24 h for NLRP3-KO) after Ad infection and compared to the wild-type counterpart. Data are expressed as mean ± SD.CC BY · PubMed Central
Figure 6 Intranasal Ad infection in mice caused ARDS and fatality but inhibition of P2X7R and caspase-1 enhanced survival. Ad was intranasally administered to mice with matching age and gender. The mice were monitored and body weight was measured each day. The survival curves were generated based on the humane end point of 20% weight loss and the comparison was made by the log rank test. (A) The wild-type (C57/B6) mice were infected with Ad at two dosages, 5×1010 or 1×1011 vp/mouse. The mice that received 5×1010 vp (n = 5) showed no apparent symptoms and sustained only slight weight loss but recovered within a few days. The mice that received 1×1011 vp (n = 5) showed ARDS like symptoms and continuously lost their body weight to reach the experimental humane end point. In the subsequent in vivo experiments Ad dosage of 1×1011 vp/mouse was used. (B and C) The wild-type (n = 12) and the P2X7R-KO mice (n = 13) were intranasally infected with Ad. The individual body weight on day 3, when all the subject mice were still alive, was presented as the relative retained body weight. The bold line represents the average body weight. (D and E) Similar to B and C, but the wild-type (n = 6) and the caspase-1-KO mice (n = 6) were compared. (F) The wild-type mice (n = 15/group) were treated with A438079 (300 µmol/kg), z-YVAD-fmk (10 mg/kg), or apyrase (4 U/mice) on day 0 and day 1of Ad administration and their survival curve was generated.
Figure 6 Intranasal Ad infection in mice caused ARDS and fatality but inhibition of P2X7R and caspase-1 enhanced survival. Ad was intranasally administered to mice with matching age and gender. The mice were monitored and body weight was measured each day. The survival curves were generated based on the humane end point of 20% weight loss and the comparison was made by the log rank test. (A) The wild-type (C57/B6) mice were infected with Ad at two dosages, 5×1010 or 1×1011 vp/mouse. The mice that received 5×1010 vp (n = 5) showed no apparent symptoms and sustained only slight weight loss but recovered within a few days. The mice that received 1×1011 vp (n = 5) showed ARDS like symptoms and continuously lost their body weight to reach the experimental humane end point. In the subsequent in vivo experiments Ad dosage of 1×1011 vp/mouse was used. (B and C) The wild-type (n = 12) and the P2X7R-KO mice (n = 13) were intranasally infected with Ad. The individual body weight on day 3, when all the subject mice were still alive, was presented as the relative retained body weight. The bold line represents the average body weight. (D and E) Similar to B and C, but the wild-type (n = 6) and the caspase-1-KO mice (n = 6) were compared. (F) The wild-type mice (n = 15/group) were treated with A438079 (300 µmol/kg), z-YVAD-fmk (10 mg/kg), or apyrase (4 U/mice) on day 0 and day 1of Ad administration and their survival curve was generated.CC BY · PubMed Central
Figure 7 P2X7R-KO mice generated less inflammatory cytokines and neutrophil infiltration in the early stage of Ad infection. The wild-type (C57/B6) and the P2X7R-KO mice were infected with Ad (1×1011 vp/mice) by i.n. administration. (A) The mice were sacrificed on day 4 (n = 4) and the lungs were obtained for histological analysis. Representative microscopic sections are shown. See Figure S3 for sections from day 2 and 6. (B) The lung pathological scores were used to compare difference between the wild-type and the P2X7R-KO mice according to the criteria described in the Materials and Methods. Data are expressed as mean ± SD. (C and D) Twenty four hours after Ad administration BALF was collected from the wild-type (n = 10) and the P2X7R-KO mice (n = 8). IL-1β and IL-6 in the BALF were analyzed by ELISA (C). Differential cell counts were performed with the cell fraction of BALF (D). Data are expressed as mean ± SD.
Figure 7 P2X7R-KO mice generated less inflammatory cytokines and neutrophil infiltration in the early stage of Ad infection. The wild-type (C57/B6) and the P2X7R-KO mice were infected with Ad (1×1011 vp/mice) by i.n. administration. (A) The mice were sacrificed on day 4 (n = 4) and the lungs were obtained for histological analysis. Representative microscopic sections are shown. See Figure S3 for sections from day 2 and 6. (B) The lung pathological scores were used to compare difference between the wild-type and the P2X7R-KO mice according to the criteria described in the Materials and Methods. Data are expressed as mean ± SD. (C and D) Twenty four hours after Ad administration BALF was collected from the wild-type (n = 10) and the P2X7R-KO mice (n = 8). IL-1β and IL-6 in the BALF were analyzed by ELISA (C). Differential cell counts were performed with the cell fraction of BALF (D). Data are expressed as mean ± SD.CC BY · PubMed Central

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Metadata

Authors
Lee, B. H., Hwang, D. M., Palaniyar, N., Grinstein, S., Philpott, D. J., & Hu, J.
Affiliations
  • University of Toronto (CA)
  • University Health Network (CA)
Corresponding author
Hu, Jim
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2012-04-25
Volume
7
Issue
4
Pages
e35812
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0035812
PMC
PMC3338466
OpenAlex
W2072521717
Cited by
102
References
48
Citations by year
2026: 4 · 2025: 2 · 2024: 6 · 2023: 6 · 2022: 7 · 2021: 11 · 2020: 7 · 2019: 7 · 2018: 8 · 2017: 15 · 2016: 9 · 2015: 5 · 2014: 7 · 2013: 6 · 2012: 2
FWCI
4.49
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • Figshare (submittedVersion, cc-by)
Funders
  • Cystic Fibrosis Canada
  • Canadian Institutes of Health Research
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Physiology › Adenosine and Purinergic Signaling
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Inflammasome and immune disorders
  • Life Sciences › Immunology and Microbiology › Immunology › Immune Response and Inflammation
Keywords
P2X7 receptor, ATP signaling, acute viral infection, adenoviral vector, macrophage activation, inflammasome, caspase-1, IL-1β, interleukin-6, neutrophil infiltration, acute respiratory distress syndrome, innate immunity
MeSH terms
Acute Disease, Adenosine Triphosphate, Adenoviridae, Adenoviridae Infections, Animals, Cell Communication, Enzyme Inhibitors, Epithelial Cells, Immunity, Innate, Inflammation, Macrophages, Signal Transduction, Interleukin-6, Mice, Knockout, Coculture Techniques, Caspase 1, Neutrophil Infiltration, Mice, Interleukin-1beta, Receptors, Purinergic P2X7, Inflammasomes, Caspase Inhibitors

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