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. 2020 Mar 4;15(3):e0229017.
doi: 10.1371/journal.pone.0229017. eCollection 2020.

Cell-free chromatin particles released from dying host cells are global instigators of endotoxin sepsis in mice

Indraneel Mittra  1 , Kavita Pal  1 , Namrata Pancholi  1 , Pritishkumar Tidke  1 , Sophiya Siddiqui  1 , Bhagyeshri Rane  1 , Jenevieve D'souza  1 , Alfina Shaikh  1 , Saili Parab  1 , Sushma Shinde  1 , Vishal Jadhav  1 , Soniya Shende  1 , Gorantla V Raghuram  1
Affiliations

Cell-free chromatin particles released from dying host cells are global instigators of endotoxin sepsis in mice

Indraneel Mittra et al. PLoS One. .

Abstract

We have earlier reported that cell-free chromatin (cfCh) particles that are released from dying cells, or those that circulate blood, can readily enter into healthy cells, illegitimately integrate into their genomes and induce dsDNA breaks, apoptosis and intense activation of inflammatory cytokines. We hypothesized that sepsis is caused by cfCh released from dying host cells following microbial infection leading to bystander host cell apoptosis and inflammation which are perpetuated in a vicious cycle with release of more cfCh from dying host cells. To test this hypothesis we used three cfCh inactivating agents namely 1) anti-histone antibody complexed nanoparticles which inactivate cfCh by binding to histones; 2) DNase I which inactivates cfCh by degrading its DNA component, and 3) a novel pro-oxidant combination of Resveratrol and Copper which, like DNase I, inactivates cfCh by degrading its DNA component. Female C57 BL/6 mice, 6-8 weeks old, were administered a single i.p. injection of LPS at a dose of 10 mg/Kg or 20 mg/Kg with or without concurrent treatment with the above cfCh inactivating agents. Administration of cfCh inactivating agents concurrently with LPS resulted in prevention of following pathological parameters: 1) release of cfCh in extra-cellular spaces of brain, lung and heart and in circulation; 2) release of inflammatory cytokines in circulation; 3) activation of DNA damage, apoptosis and inflammation in cells of thymus, spleen and in PBMCs; 4) DNA damage, apoptosis and inflammation in cells of lung, liver, heart, brain, kidney and small intestine; 5) liver and kidney dysfunction and elevation of serum lactate; 6) coagulopathy, fibrinolysis and thrombocytopenia; 7) lethality. We conclude that cfCh that are released from dying host cells in response to bacterial endotoxin represents a global instigator of sepsis. cfCh inactivation may provide a novel approach to management of sepsis in humans.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Release of cfCh into extra- nuclear spaces of vital organs and into the circulation following LPS treatment and its prevention by cfCh inactivating agents.
a. Fluorescence immuno-staining and confocal microscopy images of sections of mouse brain, lung and heart stained with fluorescent antibodies against DNA and histone H4 and examined by confocal microscopy. Co-localizing DNA (red) and histone H4 (green) fluorescent signals generate yellow / white coloured particles representing cfCh. Many yellow particles are seen outside the nucleus in the intra- / extracellular spaces in control animals with dramatic increase following LPS treatment. Treatment with CNPs, DNase 1 and R-Cu markedly reduced the number of yellow particles. b. Graphical representation of cfCh release into extracellular spaces of vital organs and its prevention by CNPs, DNase 1 and R-Cu. Six confocal fields were randomly captured (~150 cells) and their fluorescence intensities were recorded. Each group comprised of 2 animals and the histograms provide mean (± SEM) MFI values in each case. c. Release of cfCh into the circulation following LPS treatment and its prevention by cfCh inactivating agents. Serum cfCh was estimated using Cell Death Detection ELISA. Results (mean ±SE) are expressed in arbitrary units (a.u.) of absorbance values detected by spectrophotometry. Each group comprised of 5 animals and the histogram depicts mean (± SEM) values. Mean (± SEM) values of N = 5 between groups were compared using non parametric one-way ANOVA (Kurskal—Wallis test) with Dunn’s multiple comparison method at the significance and confidence level of p = 0.05.
Fig 2
Fig 2. Release of inflammatory cytokines into the circulation following LPS treatment and its prevention by cfCh inactivating agents.
LPS treatment resulted in marked increase in release of various inflammatory cytokines which were significantly reduced by concurrent treatment with CNPs, DNase 1 and R-Cu. Cytokines were estimated by ELISA at 18 h post LPS. Methodological details are given under Material and Methods section. Each group in all experiments comprised of 5 animals and the histograms provide mean (± SEM) values. Mean (± SEM) values of N = 5 between groups were compared using non parametric one-way ANOVA (Kurskal—Wallis test) with Dunn’s multiple comparison method at the significance and confidence level of p = 0.05.
Fig 3
Fig 3. DNA damage, apoptosis and inflammation in in cells of spleen and thymus following LPS treatment and their prevention by cfCh inactivating agents.
Treatment with LPS resulted in marked increase in DNA damage, apoptosis and inflammation in cells of spleen and thymus which were significantly reduced by concurrent treatment with CNPs, DNase 1 and R-Cu. The above parameters were estimated by indirect immuno-fluorescence performed at 72 h post LPS. Methodological details are given under Material and Methods section. Each group in all experiments comprised of 5 animals and the histograms provide mean (± SEM) values. Mean (± SEM) values of N = 5 between groups were compared using non parametric one-way ANOVA (Kurskal—Wallis test) with Dunn’s multiple comparison method at the significance and confidence level of p = 0.05.
Fig 4
Fig 4. DNA damage, apoptosis and inflammation in PBMCs following LPS treatment and their prevention by cfCh inactivating agents.
Treatment with LPS resulted in marked increase in DNA damage, apoptosis and inflammation in PBMCs which were significantly reduced by concurrent treatment with CNPs, DNase 1 and R-Cu. Reduction in WBCs count was also significantly ameliorated by concurrent treatment with above cfCh inactivating agents. DNA damage, apoptosis and inflammation were estimated by indirect immuno-fluorescence performed at 72 h post LPS. Methodological details are given under Material and Methods section. Each group in all experiments comprised of 5 animals and the histograms provide mean (± SEM) values. Mean (± SEM) values of N = 5 between groups were compared using non parametric one-way ANOVA (Kurskal—Wallis test) with Dunn’s multiple comparison method at the significance and confidence level of p = 0.05.
Fig 5
Fig 5. DNA damage, apoptosis and inflammation in vital organs following LPS treatment and their prevention by cfCh inactivating agents.
Treatment with LPS resulted in marked increase in DNA damage, apoptosis and inflammation in multiple organs which were dramatically reduced by concurrent treatment with CNPs, DNase 1 and R-Cu. The above parameters were estimated by indirect immuno-fluorescence performed at 72 h post LPS. Methodological details are given under Material and Methods section. Each group in all experiments comprised of 5 animals and the histograms provide mean (± SEM) values. Mean (± SEM) values of N = 5 between groups were compared using non parametric one-way ANOVA (Kurskal—Wallis test) with Dunn’s multiple comparison method at the significance and confidence level of p = 0.05.
Fig 6
Fig 6. Derangement of liver and kidney functions and elevation of serum lactate following LPS treatment and their prevention by cfCh inactivating agents.
Treatment with LPS resulted in marked increase in AST, ALT and LDH in the liver and creatinine and BUN in the kidney which were significantly reduced by concurrent treatment with CNPs, DNase 1 and R-Cu. Elevated serum lactate following LPS treatment was likewise significantly reduced following treatment with the cfCh inactivating agents. Liver and kidney functions were estimated by biochemical methods while serum lactate was estimated by colorimetric method, all performed at 72 h post LPS,. Methodological details are given under Material and Methods section. Each group in all experiments comprised of 5 animals and the histograms provide mean (± SEM) values. Mean (± SEM) values of N = 5 between groups were compared using non parametric one-way ANOVA (Kurskal—Wallis test) with Dunn’s multiple comparison method at the significance and confidence level of p = 0.05.
Fig 7
Fig 7. Hyper-coagulation, fibrinogen deposition in liver, fibrinolysis and low platelets count following LPS treatment and their prevention by cfCh inactivating agents.
Treatment with LPS resulted in increase in serum fibrinogen levels and reduction in those of anti-thrombin and protein C. Concurrent treatment with CNPs, DNase 1 and R-Cu significantly reversed these changes in case of serum fibrinogen and Protein C, but not in case of anti-thrombin. Fibrinogen deposition in liver increased following LPS treatment which was significantly reduced by cfCh inactivating agents. The reduction in TAT complex and platelet count following LPS treatment was significantly reversed by treatment with CNPs, DNase 1 and R-Cu. Serum fibrinogen, anti-thrombin and protein C and TAT complex were estimated by ELISA while fibrinogen deposition in the liver was measured by indirect immuno-fluorescence. Platelet count was performed by slandered procedure. All estimations were performed at 72 h post LPS. Methodological details are given under Material and Methods section. Each group in all experiments comprised of 5 animals and the histograms provide mean (± SEM) values. Mean (± SEM) values of N = 5 between groups were compared using non parametric one-way ANOVA (Kurskal—Wallis test) with Dunn’s multiple comparison method at the significance and confidence level of p = 0.05.
Fig 8
Fig 8. Kaplan Meier survival analysis of LPS induced lethality and its prevention by cfCh inactivating agents (10 mice in each group).
Only one of 10 animals survived following i.p. injection of LPS while three animals survived following treatment with CNPs and 5 animals each survived after treatment with DNase 1 and R-Cu. The survival curves were compared using log-rank test with the use of PRISM Version 6.0.
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This study was supported by the Department of Atomic Energy, Government of India, through its grant CTCTMC to Tata Memorial Centre awarded to I.M. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.