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

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

doi:10.1371/journal.pone.0229017

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

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

Editor: Partha Mukhopadhyay²

PMCID: PMC7055819 PMID: 32130239

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.

Introduction

Sepsis is a common and lethal syndrome with a hospital death rate of 40–50% [1]. It has been estimated that 30 million people are affected by sepsis and 6 million succumb to it globally every year [2]. Sepsis is a complex disorder characterized by: a hyper-inflammatory state with marked release of inflammatory cytokines in circulation [3]; immune paralysis due to apoptosis of lymphoid cells, especially of thymus and spleen [4]; inflammation and apoptosis of parenchymal cells leading to multi-organ dysfunction, especially of kidney and liver [5]; disseminated intravascular coagulation [6] and deposition of fibrin causing micro-vascular thrombi which exacerbate organ dysfunction [7]; consumption of clotting factors leading to fibrinolysis and life-threatening haemorrhage [8].

In spite of intensive research, patho-physiology of sepsis remains poorly understood hindering development of effective therapies [9]. Numerous trials of anti-inflammatory drugs having failed [10], a global clarion call has been sounded for new approaches to treatment of sepsis [11]. Herein, we provide one such new approach that involves inactivating cell-free chromatin (cfCh) particles that are released from dying host cells following severe microbial infection. We have earlier reported that 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 [12–17]. The uptake of cfCh particles by cells was found to be rapid and spontaneous. In vitro experiments in which mouse fibroblast cells were co-cultured with dying cells, maximum uptake of cfCh released from the dying cells was reached at 6 h, and microarray analysis at this time point showed up-regulation of pathways related to phagocytosis, suggesting a possible mechanism by which cfCh are ingested by cells [12]. The intracellular cfCh associated themselves with host cell chromosomes followed by their genomic integration [13, 12]. The latter involved dsDNA breaks as indicated by activation of H2AX and repair of the integrated cfCh particles by non-homologous end joining [13, 12, 14]. The extensive DNA damage also evoked activation of apoptotic pathways leading to death of a proportion of cells [13, 12, 14]. Surprisingly, genomic integration of cfCh and the resulting dsDNA breaks triggered marked activation of inflammatory cytokines to include NFκB, IL-6, IFNγ and TNFα [13, 15, 16]. Fluorescent NFκB signals were found to co-localise with those of γH2AX suggesting that inflammation is a direct response to dsDNA breaks [13, 15, 16]. In summary, cfCh from dying cells, or those that circulate in blood, can lead to extensive DNA damage, apoptosis and inflammation in healthy cells [17].

Based on the above findings, we hypothesized that sepsis may be caused by release of cfCh from dying host cells that follow microbial infection to trigger DNA damage, apoptotic and inflammatory responses in healthy cells of the host. The released cfCh from dying cells trigger a vicious cycle with release of more cfCh from dying host cells thereby perpetuating and amplifying the pathological complications of sepsis. cfCh released from dying host cells as a cause of sepsis would be consistent with the consensus definition of the International Sepsis Forum as “a life-threatening condition that arises when the body's response to an infection injures its own tissues and organs” [18]. Herein we show, in the mouse endotoxin model, that three different agents that have the ability to inactivate cell-free chromatin (cfCh), namely: 1) anti-histone antibody complexed nanoparticles (CNPs) which inactivates cfCh by binding to histones; 2) DNase I which inactivates cfCh by degrading its DNA components and 3) a novel pro-oxidant combination of Resveratrol and Copper (R-Cu) which, like DNase I, can degrade the DNA components of cfCh prevent multiple pathological parameters of sepsis following intra-peritoneal administration of LPS leading to improved survival of mice.

Materials and methods

Aim, design and setting of the study

The aim of the study is to investigate whether cfCh inactivating agents would prevent LPS induced sepsis. In a pre-clinical setting, mice were given i.p. injections of LPS with and without concurrent administrations of cfCh inactivating agents such as 1) anti-histone antibody complexed nanoparticles (CNPs); 2) DNase I, and 3) a combination of Resveratrol and Copper (R-Cu), and various pathological parameters of sepsis were estimated at appropriate time points.

Animal ethics approval

The experimental protocol was approved by the Animal Ethics Committee of Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Navi Mumbai, India under two projects: the 1st project was entitled ‘To evaluate the ability of 1) Resveratrol-Cu²⁺, 2) anti-histone antibody complexed nanoparticles (CNPS) and 3) DNase I preventing lethality induced by lipopolysaccharide (LPS)’ (Project no. 20/2016); the 2nd project was entitled ‘To evaluate the ability of 1) Resveratrol-Cu²⁺, 2) anti-histone antibody complexed nanoparticles (CNPS) and 3) DNase I in preventing tissue toxicity induced by Lipopolysaccharide (LPS)’ (Project no. 2/2017). The experiments were carried out according to the Committee’s animal safety guidelines and ARRIVE guidelines.

ACTREC IAEC maintains that the respectful treatment, care and use of animals involved in research is an ethical and scientific necessity and that the use of animals in research and teaching contributes to the advancement of knowledge and the acquisition of understanding. All medical and biological scientists involved in this study have undergone training in ethical treatment and management of animals under supervision of attending veterinarian. They affirm that respect for all forms of life is an inherent characteristic of biological and medical scientists who conduct research involving animals.

Animals

We used inbred female C57Bl/6 mice obtained from the Institutional Animal Facility for our study. All mice were maintained in agreement with Institutional Animal Ethics Committee (IAEC) standards. Mice of age 6–8 weeks were randomly assigned to control and experimental groups. All animals had free access to water and food. They were housed in pathogen-free cages containing husk bedding under 12-h light/dark cycle. HVAC system was used to provide a controlled room temperature, humidity and air pressure. The study involved three experiments 1) Effect of cfCh inactivating agents in reducing the surge in chromatin levels and inflammatory cytokines in serum after 18h of LPS administration, 2) Effect of cfCh inactivating agents in reducing tissue DNA damage, apoptosis and inflammation after 72h administration of LPS and 3) Effect of cfCh inactivating agents in reducing the mortality induced by LPS. All experiments were carried out at a sub lethal LPS dose of 10 mg/Kg except for the survival study in which a dose of 20 mg/Kg was used. The various experiments were either of 18 h or of 72 h duration, with one experiment on lethality lasting for 10 days. Humane endpoints were defined as reduced physical activity and weight loss. These parameters were not assessed in experiments lasting 18 hours; however, no visible loss of activity was observed. For experiments lasting 72 h, both weight and activity of animals were recorded (S1 Table). The experiment that lasted for 10 days to evaluate the effects of cfCh inactivating agents in preventing lethality following LPS treatment, detailed record of weight and activity were kept and are given in S2A and S2B Table. At the end of each study, the animals were anaesthetized under isoflurane in a fume hood and blood was collected. In certain studies, organs were collected after CO2 euthanization followed by cervical dislocation under the supervision of FELASA certified attending veterinarian. Any animal reaching humane endpoints were euthanized using CO2 inhalation followed by cervical dislocation under the supervision of attending veterinarian, irrespective of the control or test groups.

Ethical approval and consent to participate

Our study does not involve human subjects; hence ethical approval and patient consent is not applicable to our manuscript.

Materials

Details of analytical kits and antibodies used in this study are given in S3 Table. LPS of Salmonella enteric serotype typhimurium was obtained from Sigma, USA (catalogue no. L6511). LPS was dissolved in PBS and administered as a single i.p. injection.

Preparation of cfCh inactivating agents, their sources, dosage and routes of administration

We used three cfCh inactivating agents in our study, namely: 1) anti-histone antibody complexed nanoparticles (CNPs); 2) DNase I, and 3) a combination of Resveratrol and Copper (R-Cu). 1) CNPs were prepared according to our earlier report [19,20] except that histone H4 IgG was exclusively used to prepare CNPs (S3 Table). CNPs, 50 μg in 100 μl of buffer, was administered once a day i.p; the 1st CNPs dose being administered four hours prior to LPS administration. 2) DNase I (Sigma-Aldrich; Catalogue No- DN25-1G) dissolved in saline was administered at a twice daily dose of 15 mg/kg i.p. The 1st dose of DNase I was administered 4 h prior to LPS treatment. 3) The plant polyphenol Resveratrol (R) is an antioxidant which has been extensively researched for its health promoting properties [21]. R can paradoxically act as a pro-oxidant in presence of copper (Cu) because its ability to reduce Cu (II) to Cu (I) resulting in the generation of free radicals which can cleave plasmid DNA [22, 23]. We have shown that R-Cu can not only cleave, but also degrade, genomic DNA in vitro.

Light metals, such as Zn, are not active when combined with resveratrol (unpublished data). The DNA degrading activity of R-Cu is maintained even when the molar concentration of Cu is reduced more than 10,000 fold with respect to that of R [24]. We have also shown that R-Cu is active in vivo, and can degrade the DNA component of cfCh thereby inactivating it even when the molar ratio of R:Cu is kept at 1: ⁻10 000 [20, 25]. The final concentration of R in the current study was 1 mg/kg and that of Cu was 0.1 μg/kg, i.e. at a final ratio of 1:10⁻⁴. R and Cu were administered orally separately one after the other. The 1st dose of R-Cu was administered four hours prior to LPS challenge. The sources of R and Cu were (Resveratrol, Trade name—TransMaxTR, Biotivia LLC, USA; Copper, Trade name—Chelated Copper, J.R. Carlson Laboratories Inc. USA).

Detection of cfCh generated in vital organs by fluorescence Immune-staining and confocal microscopy

Animals were injected with LPS with or without cfCh inactivating agents. Animals (2 in each group) were sacrificed after 72 h and brain, lung and heart were harvested and fixed in formalin. Unstained FFPE slides were processed for dual immune-staining using antibodies against DNA (1:100 dilution) (Novus Biologicals LLC, Littleton, USA, Catalogue No. NB110-89473) and histone H4 IgG (1: 100 dilution) (custom synthesised by Bioklone Biotech Pvt Ltd, Chennai, India). The secondary antibody used was Rhodamine labelled anti-mouse antibody (red, in case of DNA) (Merck Millipore, USA, Catalogue No. AP160R) and FITC labelled anti-rabbit antibody (green, in case of histone H4) (Abcam®, UK, Catalogue No. ab6717). It should be pointed out that while the primary antibody against DNA was mouse IgM (Novus Biologicals LLC, Littleton, USA, Catalogue No. NB110-89473), the secondary antibody was IgG, but which also reactes with the light chains from all Mouse immunoglobulin classes (including mouse IgM) (Sigma-Aldrich, AP160R). Slides were mounted with vectashield DAPI mounting medium (Vector Laboratories, Catalog#H-1200). Immune-stained slides were examined by confocal microscopy and images were captured in LSCM780 (Carl-Zeiss AG, Oberkochen, Germany) microscope and analysed in ZEN 2012 software version 8.1. cfCh in vital organs of mice were identifiable by co-localizing fluorescence signals of DNA (red) and histone H4 (green) generating yellow / white coloured particles. Fluorescence intensity of six randomly chosen confocal fields (~ 150 cells) from each slide was recorded and the mean (± S.E.M) fluorescence intensity (MFI) was calculated.

Estimation of cfCh in serum

This estimation were performed at 18 h post LPS. Blood was obtained by orbital puncture followed by separation of serum by standing at room temperature for 3 h without centrifugation. cfCh estimation was performed as described by us earlier using the Cell Death Detection ELISAPLUS kit [20, 25]. Results were expressed in arbitrary units in the form of absorbance value in spectrophotometer.

Estimation of serum inflammatory cytokines in serum

These estimations were performed at 18 h post LPS. Blood was obtained by orbital puncture followed by separation of serum by standing at room temperature for 3 h. followed by centrifugation at 1800 x g for 10 min. Estimation of CRP, IL-6, IL-1β, TNF-α and IFN-γ were performed by ELISA according to vendors’ instructions as described by us earlier [20].

WBC and platelets count

These estimations were performed at 18 h post LPS. Total WBC and Platelet counts were estimated in the blood collected in EDTA chelated tubes and processed immediately for the cell count using the ADVIA^® 2120i (Siemens) automated machine.

Detection of cellular DNA-damage, apoptosis and inflammation in organs, tissues and PBMCs by immuno-fluorescence

These estimations were performed at 72 h post LPS. Organs were snap frozen and immune-staining was done on cryosections. For PBMC, blood was collected by orbital puncture in EDTA chelated tubes. PBMCs were separated using Ficoll gradient centrifugation technique and smears were prepared on slides. Analysis of γH2AX, active caspase-3, Bcl-2, NF-κB, IL-6, TNF-α and IFN-γ was done by indirect immuno-fluorescence as described by us earlier [20]. In brief, PBMC or cryo-sectioned slides were washed with PBS and fixed in 4% paraformaldehyde for 20 minutes followed by permeabilization with 0.2% Triton X-100 at room temperature for 30 minutes. Slides were washed thrice with 1X PBS for 5 minutes each and blocked using Bovine Serum Albumin (3% (BSA) at room temperature for 1 h. The sections were immuno-stained with primary antibody specific for γH2AX, active caspase-3, Bcl-2, NF-κB, IL-6, TNF-α and IFN-γ (in 3% BSA) at 4°C overnight followed by secondary antibody (in 3% BSA) incubation in dark at room temperature for 1 h. After three successive 1x PBS washes, tissue sections were stained with DAPI containing mounting medium (VECTASHIELD, Vector laboratories). Images were acquired and analysed under Applied Spectral Bio-imaging system (Applied Spectral Imaging, Israel). Mean fluorescence intensity (MFI) was determined from 1000 cells for each animal and final results were expressed as mean (± S.E.M.).

Serum biochemical analysis for measurement of organ dysfunction in serum

These estimations were performed at 72 h post LPS. Blood was obtained by orbital puncture under anaesthesia, followed by separation of serum by standing at room temperature for 3 h followed by centrifugation at 1800 x g for 10 min. Alanine aminotransaminase (ALT), aspartate transaminase (AST), lactate dehydrogenase (LDH), creatinine and blood urea nitrogen (BUN) levels were estimated using the automated Dimension EXL with LM machine (Siemens). Serum lactate was estimated by colorimetric assay kit (S3 Table).

Blood plasma analysis for coagulopathy

These estimations were performed at 72 h post LPS. Blood was collected by orbital puncture in EDTA tubes followed by centrifugation at 1800 x g for 10 min. Fibrinogen, antithrombin, protein C and TAT complex were estimated in plasma using ELISA kits (S3 Table) and per vendors’ instructions. Fibrinogen deposition in cryo-sectioned liver sections was performed by indirect Immuno-fluorescence using anti-fibrinogen antibody at 1:200 dilution. Goat anti-mouse FITC conjugated secondary antibody was used at concentration of 1:500. Slides were mounted with vectashield DAPI mounting medium. The immuno stained slides were examined by fluorescence microscope under 40X magnification and images were captured. Thousand cells per slide were counted for percent positive cells and mean ± SEM was calculated.

Survival analysis

Forty animals were divided into 4 groups of 10 mice each and all groups received a lethal dose of LPS (20 mg/kg). CNPs, DNase I and R-Cu were administered in doses and frequencies as described above. The cfCh inactivating agents were commenced 4 h prior to LPS injection. Survival between groups was compared by Kaplan–Meier survival analysis using log-rank test.

Statistical analysis

Statistical analyses were done using GraphPad Prism 6 (GraphPad Software, Inc., USA. Version 6.0). 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. Comparisons of control and treatment groups were made with LPS treated groups separately for each organ/tissue. Kaplan-Meier curves were used to express survival rates and survival curves were compared using log-rank test with the use of PRISM Ver.6.0.

Results

Release of cfCh into extracellular spaces of vital organs and in circulations and its prevention

LPS administration is known to cause tissue injury and cellular apoptosis via the production of free radicals and inflammatory cytokines [26, 27]. We investigated by fluorescence immune-staining and confocal microscopy using antibodies against DNA and histone H4, if cellular apoptosis following LPS challenge would lead to release of cfCh into extracellular spaces of vital organs, namely brain, lung and heart. cfCh could be identified as yellow / white particles resulting from co-localizing fluorescent signals of DNA (red) and histone H4 (green) (Fig 1A).

Fig 1 — 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.

It should be noted that in some DAPI positive areas of the nuclei are not stained with DNA and / or histone H4 antibodies. This is likely to be due to unevenness of cut surfaces of paraffin sections resulting in the antibodies not being able to access these DNA / histone epitopes in the nuclei. Multiple dual labelled fluorescence particles were detectable in areas outside the nuclei of vital organs of untreated control mice which are strongly suggested of being cfCh particles (Fig 1A). It cannot be excluded, however, that some of these may represent non-specific binding of antibodies to dead tissues. However, following LPS treatment, copious effusion of cfCh particles into extra-nuclear areas was evident (Fig 1A). The efflux of cfCh could be virtually abolished by concurrent treatment with cfCh inactivating agents, viz anti-histone antibody complexed nanoparticles (CNPs), DNase I and R-Cu (Fig 1A and Fig 1B). LPS treatment also led to marked release of cfCh into the circulation which could also be prevented by the cfCh inactivating agents (Fig 1C).

Release of inflammatory cytokines in circulation and its prevention

Inflammation is a hallmark of sepsis with release of inflammatory cytokines in circulation [27, 28]. A surge of CRP, IL-6, IL-1β, TNFα, IFNγ in blood occurred following LPS challenge which was significantly reduced by all three cfCh inactivating agents (Fig 2).

Fig 2 — 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.

DNA damage, apoptosis and inflammation in cells of thymus and spleen and their prevention

Apoptotic cell death of lymphocytes leading to immune paralysis is a major cause of death from sepsis [29] which is known to be associated with apoptosis of thymocytes and splenocytes [29]. A marked activation of H2AX following LPS treatment was seen in cells of thymus and spleen as evidence of DNA damage, and of apoptosis by up-regulation of active Caspase 3 with simultaneous down-regulation of BCL-2. Activation of all three parameters could be reversed by CNPs, DNase I and R-Cu (Fig 3 and S1A–S1G Fig).

Fig 3 — 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.

LPS treatment also led to pronounced activation of inflammatory cytokines which are important triggers for lymphocyte apoptosis in thymus and spleen [30]. The up-regulated levels of NFκB, IL-6, TNF-α and INF-γ in cells of spleen and thymus were significantly reduced following concurrent treatment with cfCh inactivating agents (Fig 3). Peripheral blood mononuclear cells (PBMCs) also showed evidence of DNA damage, apoptosis and inflammation [31] following LPS administration which were abrogated by CNPs, DNase I and R-Cu (Fig 4, upper three panels and S2A and S2B Fig).

The depletion of total leukocyte count following LPS was also reversed by the three cfCh agents (Fig 4, lowest panel).

DNA damage, apoptosis and Inflammation of parenchymal cells of vital organs and their prevention

Inflammation and apoptosis of parenchymal cells of vital organs leading to multi-organ dysfunction is another cardinal feature of sepsis [32]. We observed extensive DNA damage, apoptosis and inflammation in cells of lung, liver, heart, brain, kidney and small intestine following LPS challenge (Fig 5 and S3A–S3F Fig).

Fig 5 — 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.

This was evidenced by marked activation of H2AX, active Caspase 3, NFκB, IL-6, TNFα and INFγ. The effects of CNPs, DNase I and R-Cu on these parameters was dramatic, and levels of all parameters were reduced to near normal levels. (Fig 5 and S3A–S3F Fig)

Liver and kidney dysfunction and elevation of serum lactate and their prevention

Parenchymal DNA damage, apoptosis and inflammation is accompanied by liver and kidney dysfunction which are prominent features of sepsis [33]. Liver dysfunction following LPS treatment resulted in elevation of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH). Levels of these enzymes were significantly reduced by concurrent treatment with CNPs, DNase I and R-Cu (Fig 6, upper panel).

Fig 6 — 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.

Kidney dysfunction following LPS evidenced by elevation of creatinine and blood urea nitrogen (BUN) levels were also significantly attenuated by concurrent treatment with the three cfCh inactivating agents (Fig 6, middle panel). The elevated levels of serum lactate in LPS treated animals, indicative of tissue hypo-perfusion resulting from micro-vascular damage and organ dysfunction [34], was also significantly reduced by the three cfCh inactivating agents (Fig 6, lowest panel).

Coagulopathy, fibrinolysis and low platelet count and their prevention

LPS treatment led to marked dysregulation of parameters of blood coagulation akin to that seen in disseminated intravascular coagulation [35]. A pro-thrombotic state was evident at 18 h post LPS with elevation of fibrinogen levels in serum [36], and decrease in levels of anti-thrombin [37] and protein C [38] (Fig 7, upper panel).

Abnormal levels of all three parameters could be reversed, except in case of anti-thrombin, by concurrent treatment of CNPs, DNase I and R-Cu (Fig 7, upper panel). We also observed increase in fibrinogen deposition in liver following LPS treatment apparently as a result of micro-vascular coagulopathy [39], which was significantly reduced by treatment with cfCh inactivating agents (Fig 7, middle panel and S4 Fig). Consumption of clotting factors apparently led to a fibrinolytic state with decrease in levels of thrombin-anti-thrombin complex (TAT complex)—a surrogate marker of plasma thrombin [40]—which was restored by the administration of cfCh inactivating agents (Fig 7, lowest panel, left hand image). Also evident was a drastic reduction in platelets count in LPS treated mice [41] suggesting a decrease in clotting capacity (Fig 7, lowest panel, right hand image). The low platelets count was significantly restored by treatment with CNPs and R-Cu, but not by DNase I.

Fatality and its prevention

Finally, we show that treatment with cfCh inactivating agents can reduce fatality from LPS treatment (Fig 8).

Ninety percent of mice given a lethal dose of LPS died by day 10, while treatment with CNPs and R-Cu reduced the fatality rate to 50% whereas DNase I reduced it to 40%.

Discussion

Patho-physiology of sepsis is complex and poorly understood [10]. Of date, no specific treatment for sepsis exists [10]. For long it was assumed that sepsis is caused by an intense inflammatory reaction to microbial infection [3]. However, failure of numerous clinical trials of anti-inflammatory agents to improve survival [10] has led to the conclusion that other unknown host factors might be responsible for the high mortality rates [18]. Recent research by our group suggests that cfCh from dying cells of the host might be one such factor [12–17]. cfCh released from dying cells, or those that circulate in blood, have the capacity to freely enter into healthy bystander cells to illegitimately integrate into their genomes and inflict dsDNA breaks [12–14,17]. The latter not only leads to apoptosis of cells [12, 13], but also to intense activation of inflammatory cytokines in the affected cells [15–17]. We have made the striking observation that fluorescent NFκB signals co-localize with those of γH2AX, which are activated at the sites of cfCh integration [12]. This has led to our proposal that inflammation is a direct consequence of dsDNA breaks induced by illegitimate integration of cfCh into the genome [15–17]. A close association of cfCh induced dsDNA breaks and inflammation was seen in multiple cell types in vitro [12] as well as in all organs and tissues examined in vivo [12]. cfCh inactivating agents not only prevented dsDNA breaks but also prevented inflammation [12]. In the present study, LPS injection led to copious release of cfCh particles from dying cells into extracellular spaces of vital organs (Fig 1A and 1B) and into the circulation (Fig 1C). We propose that a global activation of apoptosis and inflammation in all organs and tissues in response to LPS, including in cells of spleen, thymus and PBMCs, are a direct consequence of dsDNA breaks and inflammation induced by cfCh integration in their genomes leading to organ dysfunction and immune suppression. We hypothesise that trials of anti-inflammatory agents might have failed because they did not address the root cause of inflammation, i.e. genomic cfCh integration and dsDNA breaks, but rather were targeting the symptoms or consequences of it.

We undertook a separate experiment to demonstrate that cfCh inactivating agents themselves have no bystander damaging effects on host cells. Animals which were administered CNPs, DNAse I and R-Cu in the absence of LPS showed no DNA damaging effects (activation of H2AX) in brain cells of mice (S5 Fig).

Taken together, these findings support the theory that sepsis is caused by cfCh released from dying host cells following pathogen invasion leading to bystander host cell apoptosis and inflammation which are perpetuated in a vicious cycle with release of more cfCh from dying bystander cells. Clinical relevance of our finding is suggested by reports that blood cfCh levels are grossly elevated in patients with sepsis [42] and act as predictors of organ dysfunction and death [43]. It is pertinent to note here that we have proposed a similar vicious cycle to explain chemotherapy toxicity [20]. We have shown that toxicity of cancer chemotherapy is not so much due the damaging effects of the drugs themselves, but rather, like in case of sepsis, is due to release of cfCh from initial round of drug-induced cell death leading to a vicious cycle of DNA damage, apoptosis and inflammation perpetuated by more cfCh being released from dying cells of the host in a cascading manner [20]. We have also reported that bystander DNA damage and inflammation in healthy cells following radiotherapy is due to cfCh released from irradiated dying cells and that the bystander effect can occur both locally and in distant organs [25].

It has been reported that highly toxic extracellular histones are released in circulations in response to hyper-inflammatory challenge and can lead to endothelial dysfunction, organ failure and death from sepsis [44, 45]. The damaging effects of histones are mediated through Toll-like receptors 2 and 4 [46], and can be reduced by antibody to histones or activated protein C [44]. Neutrophil extracellular traps (NETs) are known to release cell-free DNA, histones as well as chromatin which have been implicated in sepsis [47, 48]. In this context, it should be noted from our results that inactivation of cfCh was more effective in preventing tissue damage and inflammation (Figs 3–5) and organ dysfunction (Fig 6), but was relatively less effective in reducing inflammatory cytokines in circulation (Fig 2) and in preventing fatality (Fig 8). Thus, it is possible that alarmin effects of histone and DNA and other nuclear proteins also contribute to endotoxin sepsis. However, the fact that sepsis inducing effects of cfCh were nullified to an equal extent by both anti-histone antibody complexed nanoparticles as well as anti-DNA agents such as DNase I and R-Cu suggest that these agents were inactivating a common target, viz cfCh.

The three cfCh inactivating agents not only had a global ameliorating effect on various parameters of LPS induced sepsis, but also improved survival rates of mice following a lethal dose of LPS (Fig 8). R-Cu and DNase I treatment achieved a survival figure of 50%, while in case of CNPs it was 30%, compared to 10% in LPS controls. CNPs, DNase I and R-Cu on their own had little bystander toxic side effects on healthy cells suggesting the possibility of their use in therapy of sepsis. It should be noted, however, that treatment with cfCh inactivating agents, in our experiments was started 4 h prior to LPS injection, unlike in patients with sepsis, wherein treatment is commenced only after signs and symptoms of sepsis have set in. Our study also does not exclude the possibility that host factors other than cfCh may be involved in LPS induced sepsis which remain to be identified [49]. Nonetheless, our results provide evidence for an association between cfCh released from dying host cells and the aetio-pathology of sepsis, suggesting a possible novel approach to treatment of sepsis with the use of cfCh inactivating agents.

Conclusion

cfCh that are released from dying host cells in response to bacterial endotoxin may represent a global instigator of sepsis. cfCh inactivation may provide a novel approach to management of sepsis in humans.

Supporting information

S1 Fig. Representative immuno-fluorescence images of spleen and thymus showing activation of DNA damage, apoptosis and inflammation in splenocytes and thymocytes following LPS challenge and their prevention by cfCh inactivating agents.

The analyses were performed at 72 h post LPS. Methodological details are given under Material and Methods section.

(TIF)

Click here for additional data file.^{(24.9MB, tif)}

S2 Fig. Representative immuno-fluorescence images of PBMCs showing DNA damage, apoptosis and inflammation following LPS treatment and their prevention by cfCh inactivating agents.

The above parameters were estimated by indirect immuno-fluorescence performed at 72 h post LPS. Methodological details are given under Material and Methods section.

(TIF)

Click here for additional data file.^{(8.8MB, tif)}

S3 Fig. Representative immuno-fluorescence images of vital organs showing activation of DNA damage, apoptosis and inflammation following LPS challenge and their prevention by cfCh inactivating agents.

The analyses were performed at 72 h post LPS. Methodological details are given under Material and Methods section.

(TIF)

Click here for additional data file.^{(15.7MB, tif)}

S4 Fig. Representative immuno-fluorescence images of liver showing fibrinogen deposition following LPS challenge and its prevention by cfCh inactivating agents.

The analyses were performed at 72 h post LPS. Methodological details are given under Material and Methods section.

(TIF)

Click here for additional data file.^{(554.5KB, tif)}

S5 Fig. Histograms to demonstrate that the three cfCh inactivating agents were themselves not toxic to mice.

Animals were divided into four groups: 1) control (n = 10) and those receiving 2) CNPs (n = 5), 3) DNase I (n = 5) and 4) R-Cu (n = 5) in doses as described in material and methods section. Animals were sacrificed on day 7 and their brain tissues were removed and cryo-sections were prepared for estimation of γ - H2AX by immunofluorescence as described in materials and methods section. The results show that the three cfCh inactivating agents did not lead to any increase in DNA damage in terms of H2AX activation.

(TIF)

Click here for additional data file.^{(161.9KB, tif)}

S1 Table. Cage side parameters for assessment of body weight to evaluate side effects in experiments lasting 72 h (10 mg/kg LPS).

Results show no change in body weight during the 72h period. Changes in physical activity were not monitored in these experiments.

(DOCX)

Click here for additional data file.^{(16.8KB, docx)}

S2 Table. A: Cage side parameters for assessment of body weight to evaluate side effects in the lethality experiment (20 mg/kg LPS).

Results show loss of body weight in LPS alone group but not in groups receiving LPS plus DNase I, R-Cu, and CNPs. B: Cage side parameters for assessment of physical activity to evaluate side effects in experiments in the lethality experiments (20 mg/kg LPS). Results show no loss of physical activity in control group. In LPs alone group loss of activity was observed. In LPS plus DNase, CNPs and R-Cu groups, a variable degree of recovery was observed.

(DOCX)

Click here for additional data file.^{(44.2KB, docx)}

S3 Table

Analytical kits used and their procurement sources (Upper Table). Antibodies used and their procurement sources (Lower table).

(DOCX)

Click here for additional data file.^{(27.7KB, docx)}

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

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.

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PLoS One. doi: 10.1371/journal.pone.0229017.r001

Decision Letter 0

Partha Mukhopadhyay

18 Nov 2019

PONE-D-19-28396

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

PLOS ONE

Dear Prof. Mittra,

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Reviewer #1: In the present study “Cell-free chromatin particles released from dying host cells are global instigators of endotoxin sepsis in mice” the authors studied the possible role of cell-free chromatin in sepsis. This is a consecutive study after the discussion of cell-free chromatin particles in other disease models from the same authors. Here are some major concerns:

1. The authors claimed Figure 1A is the proof of the existence of cell free chromatin particles in LPS induced sepsis model. However, there are some major defects in these images. First, the images cannot reflect the typical structures of brain, lung and heart tissues. Second, the DNA antibody listed by the authors is mouse IgM, but the secondary antibody is against mouse IgG. Theoretically this immunostaining cannot work. Third, a lot of areas have positive DAPI staining but not DNA staining. Forth, the “positive” staining of cell free chromatin particles cannot be simply described as chromatin in the extracellular area. According to the data, they can only be described as somewhere not in the nucleus. Fifth, the specificity of these staining, especially the possibility that they are the result of unspecific binding of antibodies to dead tissues, should be better defined. The authors need to revise all these defects.

2. Although the authors used three reagents to neutralize or degrade cell free chromatin, necessary controls were not used. For neutralizing antibody, an isotype control Ig antibody should be used. For an enzyme, a deactivated enzyme should be used. For a compound containing heavy metal, the same organic compound with a light metal should be used.

3. In Figure 6, LDH is not a tissue specific parameter so the authors should not use it to define liver damage. Creatinine and BUN are kidney parameters but they are not produced by kidneys. So the authors should not put “renal” before them. Also, AST is not liver specific, so the authors should remove “liver” before AST.

4. The alarmin effects of DNA, histone and other nuclear proteins have been well defined. Do the authors claim a unique function of the cell-free chromatin particles as a whole or a combined phenomenon of the already defined alarmins?

Reviewer #2: In the study 'Cell-free chromatin particles released from dying host cells are global instigators of endotoxin sepsis in mice', the authors have shown convincing proof that chCh particles are involved in sepsis, and neutralizing them helps alleviate the adverse pathophysiological effects associated with it. This is an important study, and hopefully can help develop effective strategies to deal with sepsis. Some minor revisions are recommended below as follows:

1) Abstract (Line 29) and Introduction (Line 69): Please check sentence construction for '..those that circulated blood..'.

2) Introduction: It would be nice if the authors can elaborate more on the mechanisms by which cfCh particles induces DNA damage, apoptosis and activation of the inflammatory cytokine cascades. How are cfCh particles even transported from the extracellular domain into the cell? Elaborate more on findings from previous work (Refs 12-17).

3) Materials and Methods: Animals: Please include data about any side effects experienced by the animals in the study plus their weight in the manuscript. This is important in order to rule out any harmful effects on 'bystander' healthy cells which the authors mention, but don't discuss in detail later in the discussion section.

4) Materials and Methods: Detection of cellular DNA damage, apoptosis and inflammation in organs, tissues and PBMCs by IF: Please elaborate on the indirect immuno-fluorescence method used to estimate levels of gH2AX, caspase-3, Bcl-2, NF-kB, IL-6, TNFa and IFNg.

5) Materials and Methods: Statistical analysis: As multiple groups are being compared simultaneously, a simple t-test won't suffice. Please use ANOVA or a non parametric test such as Mann Whitney.

6) Results: Please eliminate all figure captions. This is unecessary, or can be used as Figure legend captions.

7) Results: Result titles/caption should reflect actual findings and not just what was done.

8)Results: Figure 3: How were thymocytes and splenocytes isolated?

9) Figures: Image quality is poor. Please upload high quality tiff images.

10) Discussion: Please discuss the potential of side effects by using all of these cfCh particle-inactivating agents on healthy cells. Elaborate on bystander effect.

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Reviewer #2: No

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PLoS One. 2020 Mar 4;15(3):e0229017. doi: 10.1371/journal.pone.0229017.r002

Author response to Decision Letter 0

6 Jan 2020

Reviewers’ Comments to the Author

Reviewer #1:

1. First, the images cannot reflect the typical structures of brain, lung and heart tissues.

Our Response : We agree. For typical structures of brain, lung and heart to be reflected we need H&E stained slides. However, in that case immunohistochemical analysis will not be possible.

Second, the DNA antibody listed by the authors is mouse IgM, but the secondary antibody is against mouse IgG. Theoretically this immunostaining cannot work.

Our Response : We were aware of this. We ensured that the secondary antibody also reacted with mouse IgM. We quote from the vendor’s-Datasheet: “the secondary antibody reacts with Mouse IgG gamma chain, as well as the light chains from all Mouse immunoglobulin classes [including mouse IgM (sic)]” (Sigma-Aldrich, AP160R).

This has now been clarified in the text (lines 202-206, page 9).

Third, a lot of areas have positive DAPI staining but not DNA staining.

Our Response : It needs to be pointed out that these are paraffin sections wherein the cut surfaces are not perfectly even. The unevenness is clearly seen in the magnified confocal images. Unevenness of paraffin sections is likely to cause the antibodies not being able to uniformly access the DNA / histone epitopes in the nuclei.

This issue has been discussed in the text (lines 307-311, page 13).

Fourth, the “positive” staining of cell free chromatin particles cannot be simply described as chromatin in the extracellular area. According to the data, they can only be described as somewhere not in the nucleus.

Our Response : We agree, and we have made the necessary amendments in the text (lines 311-313 and line 316, page 13).

Fifth, the specificity of these staining, especially the possibility that they are the result of unspecific binding of antibodies to dead tissues, should be better defined.

Our Response : This may be a remote possibility. We have made the necessary amendments in the text (lines 313-315, page 13).

2. Although the authors used three reagents to neutralize or degrade cell free chromatin, necessary controls were not used.

For neutralizing antibody, an isotype control Ig antibody should be used.

Our Response : The use of an isotype control Ig antibody would have been important had we been characterizing the specificity of anti-histone antibody complexed nanoparticles (CNPs) for the first time. We are past that stage. We have already published in detail characteristics of CNPs (Ref. 19) and even obtained a US Patent (U.S. Patent No. 9,096,655, Issued: August 4, 2015). The high specificity of CNPs was fully addressed in the publication / patent application. We do not think we need to re-invent the wheel for this paper. We have also published several papers using CNPs to neutralize cfCh in mice, without Ig antibody controls, which did not evoke any queries from reviewers (Ref. 12, 13, 20, 25). We do not think it is necessary to use negative controls in a study which uses a well characterized and well established antibody complex.

For an enzyme, a deactivated enzyme should be used.

Our Response : As the name suggests, DNase I degrades DNA. We do not think that it is necessary to use a deactivated enzyme as a negative control for such a specific enzyme which is universally accepted. We have also published several articles using DNase I to inactivate cfCh without any deactivated enzymes as a negative control and without any objection from reviewers (Ref. 12, 13, 20, 25).

For a compound containing heavy metal, the same organic compound with a light metal should be used.

Our Response : Again, we are not addressing as to which is the best metal to use along with Resveratrol in this paper. This was done in the past when we had undertaken extensive research to come to the conclusion that Resveratrol and Copper is the best combination to degrade genomic DNA (Ref. 24), and subsequently to show that it can degrade the DNA component of cfCh to inactivate it in mice without any comments from reviewers (Ref. 12, 20, 25). During our initial extensive research we had tried out various combinations of plant poly-phenols and heavy and light metals to ultimately conclude that R-Cu was the most active combination. Light metals such as Zinc were found not to be active in combination with Resveratrol.

Nonetheless, we have now added a line to clarify that light metals are not active in combination with Resveratrol (lines 180-181, page 8).

Our Response : We completely agree. The necessary corrections have been made in figure no. 6.

Our Response : Yes, we believe that the role of cell-free chromatin (cfCh) in sepsis is unique and independent of any alarmin effects that DNA or histones might have. The sepsis inducing effects of cfCh are nullified to an equal extent by anti-histone antibody complexed nanoparticles as well as anti-DNA agents such as DNase I and R-Cu indicating that they are inactivating a common target, viz cfCh. We do not deny that free DNA and / or histones may also have independent alarmin effects; but in many of our experiments the three cfCh inactivating agents reduced the sepsis biomarkers to near control levels suggesting that cfCh is the key instigator of sepsis (Figures 3,5 and 6 for example). We have addressed this issue in the penultimate paragraph of the discussion section of the paper.

References: The references above pertain to those in the manuscript.

Reviewer #2:

1. Abstract (Line 29) and Introduction (Line 69): Please check sentence construction for '…those that circulated blood..'.

Our Response : We thank the reviewer for pointing out these minor errors. These have now been rectified.

2. Introduction: It would be nice if the authors can elaborate more on the mechanisms by which cfCh particles induces DNA damage, apoptosis and activation of the inflammatory cytokine cascades. How are cfCh particles even transported from the extracellular domain into the cell? Elaborate more on findings from previous work (Refs 12-17).

Our Response : Thanks are due again. This issue has now been elaborated in the introduction section (lines 73-88, Pages 3 & 4).

3. Materials and Methods: Animals: Please include data about any side effects experienced by the animals in the study plus their weight in the manuscript. This is important in order to rule out any harmful effects on 'bystander' healthy cells which the authors mention, but don't discuss in detail later in the discussion section.

Our Response : Thanks. The issue of side effects in terms of physical activity and weight loss have now been addressed in the text (lines 131-138 page 6 and supplementary tables 1 and 2a & 2b.

With respect to harmful side effects (bystander effects), we especially undertook an experiment to assess the independent toxicity, if any, of the three cfCh inactivating agents and observed that CNPs, DNase I, and R-Cu have little toxicity of their own as assessed by activation of H2AX (DNA damage) in brain cells. The results are given in supplementary figure 5 and discussed in the text (lines 442-445, pages 18 & 19) and lines 482-483, page 20.

4. Materials and Methods: Detection of cellular DNA damage, apoptosis and inflammation in organs, tissues and PBMCs by IF: Please elaborate on the indirect immuno-fluorescence method used to estimate levels of gH2AX, caspase-3, Bcl-2, NF-kB, IL-6, TNFa and IFNg.

Our Response : Details of indirect immuno-fluorescence method has now been given in the methods section (lines 243 – 254, pages 10 and 11).

5. Materials and Methods: Statistical analysis: As multiple groups are being compared simultaneously, a simple t-test won't suffice. Please use ANOVA or a non- parametric test such as Mann Whitney.

Our Response : The reviewer is absolutely right. We have reanalyzed the data using ANOVA in all our experiments. The ANOVA analytical details are given in lines 287–294, page 12.

6. Results: Please eliminate all figure captions. This is unnecessary, or can be used as Figure legend captions.

Our Response : Figure captions have now been removed.

7. Results: Result titles/caption should reflect actual findings and not just what was done.

Our Response : The actual findings have now been included in all figure legends.

8. Results: Figure 3: How were thymocytes and splenocytes isolated?

Our Response : Perhaps we have created this confusion. Thymocytes and splenocytes were not isolated, rather, the various parameters were assayed on histological sections of thymus and spleen. This issue has been clarified in the text (lines 330, 335, 340 - 341, page 14).

9. Figures: Image quality is poor. Please upload high quality tiff images.

Our Response : We have now improved the quality of the figures.

10. Discussion: Please discuss the potential of side effects by using all of these cfCh particle-inactivating agents on healthy cells. Elaborate on bystander effect.

Our Response : In response to the reviewer’s query, we especially undertook an experiment to assess the independent toxicity, if any, of the three cfCh inactivating agents and observed that CNPs, DNase I, and R-Cu have little toxicity of their own as assessed by activation of H2AX (DNA damage) in brain cells. The results are given in supplementary figure 5 and discussed in the text (lines 442-445, pages 18 & 19) and lines 482 – 483, page 20.

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PLoS One. doi: 10.1371/journal.pone.0229017.r003

Decision Letter 1

Partha Mukhopadhyay

23 Jan 2020

PONE-D-19-28396R1

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

PLOS ONE

Dear Prof. Mittra,

Your manuscript was reviewed by two experts and one reviewer raised minor comments. A quick editorial decision will be taken after satisfactory revision and the manuscript will not send to reviewers.

We would appreciate receiving your revised manuscript by Mar 08 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Partha Mukhopadhyay, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: The current version of “Cell-free chromatin particles released from dying host cells are global instigators of endotoxin sepsis in mice.” made a lot of improvement compared to the last version. Here is a minor concern the authors still have to address:

The authors claimed that the effect of cell free chromatin is unique from already known alarmin effect of histone and DNA because their neutralization can bring inflammation to baseline level. However, this is not entirely true according to the result of circulating inflammatory factors (Figure 2) and sepsis survival (Figure 8). The authors need to provide a better discussion of the contribution from each inflammatory element. Also the authors need to provide some clues why tissue inflammation is resolved while circulating inflammatory factors are still high.

Reviewer #2: (No Response)

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Reviewer #1: No

Reviewer #2: No

PLoS One. 2020 Mar 4;15(3):e0229017. doi: 10.1371/journal.pone.0229017.r004

Author response to Decision Letter 1

27 Jan 2020

6. Review Comments to the Author

Our response: The reviewer’s concerns have now been addressed on page 19, lines 451-460.

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PLoS One. doi: 10.1371/journal.pone.0229017.r005

Decision Letter 2

Partha Mukhopadhyay

29 Jan 2020

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

PONE-D-19-28396R2

Dear Dr. Mittra,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Partha Mukhopadhyay, Ph.D.

Section Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0229017.r006

Acceptance letter

Partha Mukhopadhyay

4 Feb 2020

PONE-D-19-28396R2

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

Dear Dr. Mittra:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

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Section Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

The analyses were performed at 72 h post LPS. Methodological details are given under Material and Methods section.

(TIF)

Click here for additional data file.^{(24.9MB, tif)}

S2 Fig. Representative immuno-fluorescence images of PBMCs showing DNA damage, apoptosis and inflammation following LPS treatment and their prevention by cfCh inactivating agents.

The above parameters were estimated by indirect immuno-fluorescence performed at 72 h post LPS. Methodological details are given under Material and Methods section.

(TIF)

Click here for additional data file.^{(8.8MB, tif)}

The analyses were performed at 72 h post LPS. Methodological details are given under Material and Methods section.

(TIF)

Click here for additional data file.^{(15.7MB, tif)}

S4 Fig. Representative immuno-fluorescence images of liver showing fibrinogen deposition following LPS challenge and its prevention by cfCh inactivating agents.

The analyses were performed at 72 h post LPS. Methodological details are given under Material and Methods section.

(TIF)

Click here for additional data file.^{(554.5KB, tif)}

S5 Fig. Histograms to demonstrate that the three cfCh inactivating agents were themselves not toxic to mice.

(TIF)

Click here for additional data file.^{(161.9KB, tif)}

S1 Table. Cage side parameters for assessment of body weight to evaluate side effects in experiments lasting 72 h (10 mg/kg LPS).

Results show no change in body weight during the 72h period. Changes in physical activity were not monitored in these experiments.

(DOCX)

Click here for additional data file.^{(16.8KB, docx)}

S2 Table. A: Cage side parameters for assessment of body weight to evaluate side effects in the lethality experiment (20 mg/kg LPS).

(DOCX)

Click here for additional data file.^{(44.2KB, docx)}

S3 Table

Analytical kits used and their procurement sources (Upper Table). Antibodies used and their procurement sources (Lower table).

(DOCX)

Click here for additional data file.^{(27.7KB, docx)}

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Submitted filename: Response to Reviewers 1 Jan 2020.doc

Click here for additional data file.^{(52KB, doc)}

Attachment

Submitted filename: Response to Reviewers.docx

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Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

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PERMALINK

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

Indraneel Mittra

Kavita Pal

Namrata Pancholi

Pritishkumar Tidke

Sophiya Siddiqui

Bhagyeshri Rane

Jenevieve D’souza

Alfina Shaikh

Saili Parab

Sushma Shinde

Vishal Jadhav

Soniya Shende

Gorantla V Raghuram

Roles

Abstract

Introduction

Materials and methods

Aim, design and setting of the study

Animal ethics approval

Animals

Ethical approval and consent to participate

Materials

Preparation of cfCh inactivating agents, their sources, dosage and routes of administration

Detection of cfCh generated in vital organs by fluorescence Immune-staining and confocal microscopy

Estimation of cfCh in serum

Estimation of serum inflammatory cytokines in serum

WBC and platelets count

Detection of cellular DNA-damage, apoptosis and inflammation in organs, tissues and PBMCs by immuno-fluorescence

Serum biochemical analysis for measurement of organ dysfunction in serum

Blood plasma analysis for coagulopathy

Survival analysis

Statistical analysis

Results

Release of cfCh into extracellular spaces of vital organs and in circulations and its prevention

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.

Release of inflammatory cytokines in circulation and its prevention

Fig 2. Release of inflammatory cytokines into the circulation following LPS treatment and its prevention by cfCh inactivating agents.

DNA damage, apoptosis and inflammation in cells of thymus and spleen and their prevention

Fig 3. DNA damage, apoptosis and inflammation in in cells of spleen and thymus following LPS treatment and their prevention by cfCh inactivating agents.

Fig 4. DNA damage, apoptosis and inflammation in PBMCs following LPS treatment and their prevention by cfCh inactivating agents.

DNA damage, apoptosis and Inflammation of parenchymal cells of vital organs and their prevention

Fig 5. DNA damage, apoptosis and inflammation in vital organs following LPS treatment and their prevention by cfCh inactivating agents.

Liver and kidney dysfunction and elevation of serum lactate and their prevention

Fig 6. Derangement of liver and kidney functions and elevation of serum lactate following LPS treatment and their prevention by cfCh inactivating agents.

Coagulopathy, fibrinolysis and low platelet count and their prevention

Fig 7. Hyper-coagulation, fibrinogen deposition in liver, fibrinolysis and low platelets count following LPS treatment and their prevention by cfCh inactivating agents.

Fatality and its prevention

Fig 8. Kaplan Meier survival analysis of LPS induced lethality and its prevention by cfCh inactivating agents (10 mice in each group).

Discussion

Conclusion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Partha Mukhopadhyay

Roles

Author response to Decision Letter 0

Decision Letter 1

Partha Mukhopadhyay

Roles

Author response to Decision Letter 1

Decision Letter 2

Partha Mukhopadhyay

Roles

Acceptance letter

Partha Mukhopadhyay

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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