Microbe capture by splenic macrophages triggers sepsis via T cell-death-dependent neutrophil lifespan shortening

The mechanisms linking systemic infection to hyperinflammation and immune dysfunction in sepsis are poorly understood. Extracellular histones promote sepsis pathology, but their source and mechanism of action remain unclear. Here, we show that by controlling fungi and bacteria captured by splenic macrophages, neutrophil-derived myeloperoxidase attenuates sepsis by suppressing histone release. In systemic candidiasis, microbial capture via the phagocytic receptor SIGNR1 neutralizes myeloperoxidase by facilitating marginal zone infiltration and T cell death-dependent histone release. Histones and hyphae induce cytokines in adjacent CD169 macrophages including G-CSF that selectively depletes mature Ly6Ghigh neutrophils by shortening their lifespan in favour of immature Ly6Glow neutrophils with a defective oxidative burst. In sepsis patient plasma, these mediators shorten mature neutrophil lifespan and correlate with neutrophil mortality markers. Consequently, high G-CSF levels and neutrophil lifespan shortening activity are associated with sepsis patient mortality. Hence, by exploiting phagocytic receptors, pathogens degrade innate and adaptive immunity through the detrimental impact of downstream effectors on neutrophil lifespan.

D 0 The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement D 0 A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly D 0 The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section. 0 D A description of all covariates tested D 0 A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons D 0 A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) □ l'xl For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted L.'.:.J Give P values as exact values whenever suitable. 0 D For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings 0 D For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes 0 D Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
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Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our QQ_1ig,_ The high throughput RNA sequencing expression profile data is publically made available and can be accessed at NCBI Gene Expression Omnibus (GSE160301) and the token (ydyjocqyjpczjgt). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD034391. All other source data are provided with this paper. No data was excluded in this study.
Most experiments were validated with repeat experiments. Few exceptions include costly experiments, such as Cytokine arrays, proteomics and RNAsequencing were biological replicates were employed. Sufficient sample size was included and when possible, samples were processed simultaneously to prevent batch variations Treatment groups in animal experiments were mixed within the same cage, to prevent variation among different cages, and groups were assigned randomly. Proteomic analysis was also randomized to prevent run bias. The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).

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The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
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This study did not involve wild aninals
This study did not involve samples collected in the field.
Peripheral blood was isolated from consenting healthy adult volunteers, according to approved protocols of the ethics board of the Francis Crick Institute and the Human Tissue act. Sepsis patient samples were provided by the Hannover Medical School approved the ethics committee under the study protocol (No. 2786(No. -2015. Written informed consent was obtained from participants or authorized representatives. The study was performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.

Healthy adult volunteers donated peripheral blood for in vitro experiments, and septic patients were included for plasma analysis
All donors were recruited voluntarily and consented to the study objectives.
Ethics board of the Francis Crick Institute, the Human Tissue act and the Hannover Medical School ethics committee.

No. 2786-2015
Describe the settings and locales of data collection, noting the time periods of recruitment and data collection.
Describe how you pre-defined primary and secondary outcome measures and how you assessed these measures.

Methodology
Sample preparation Peripheral venous blood was collected into S-Monovette 7.5 ml sodium heparin tubes (Laborimpex; cat. 01.1613.100) and kept at room temperature (RT) until start of neutrophil isolation. 5ml whole blood was carefully layered on top of 5ml prewarmed Histopaque 1119 (Sigma-Aldrich; cat. 11191-100ml) at 37oC in a 15ml Falcon tube. Tubes were then centrifuged for 20 min at 800xg at RT, with acceleration speed 7 and deceleration speed 3. The transparent top layer containing plasma was placed in a new Falcon tube and spun for 10 min at 300xg at RT to ensure acellularity. The subsequent layers containing peripheral blood mononuclear cells (PBMCs) and neutrophils were collected separately in new 15ml Falcon tubes and washed in Cytiva HyClone Hank's balanced salt solutions (HBSS) without calcium, magnesium or henol red (Fisher Scientific; cat. SH3058801) supplemented with 10mM HEPES (Invitrogen; H0887-100ml) 0.1% plasma and spun for 10min at 300xg at RT. PBMCs and neutrophils were resuspended in 2ml washing buffer before being further fractionated on a discontinuous Percoll (GE Healthcare; cat. 17-5445-02) gradient. Neutrophils were overlayed on a density gradient in a 15ml Falcon tube containing 2ml of each of the following densities: 1105 g/ml (85%), 1100 g/ml (80%), 1093 g/ml (75%), 1087 g/ml (70%), and 1081 g/ml (65%), containing the highest density at the bottom of the tube. Neutrophils were then centrifuged for 20 min at 800xg, with acceleration speed 7 and deceleration speed 3. Neutrophil enriched layers (75-65% layer) were collected and washed in washing buffer, and spun for 10min at 300xg at RT. Neutrophils were resuspended in 1ml washing buffer and manually counted with a cell-counting chamber.
For murine blood collection, mice were euthanised with pentobarbital (600mg/kg) with mepivacaine hydrochloride (20mg/ ml). When animals became irresponsive, blood was collected via cardiac puncture using a 25Gx5/8" needle (BD; cat. 300600) and an 1ml syringe (BD; 303172). After collection, the needle was disposed and blood sample put in a blood collection 1.3ml microvette tube with a lithium-heparin ring (Sarstedt; cat. 41/09). The heparin tube was gently inverted 10 times and left at RT until proceeding. To separate the leukocytes from plasma and remove a large proportion of red blood cells, a maximum of 300%l whole blood was overlayed on 1ml Histopaque-1119 (Sigma; cat. 11191-100ML) at RT in an Eppendorf and spun for 20min at 800xg RT, with acceleration speed 7 and deceleration speed 3. The top transparent plasma layer was carefully collected without collecting cells. The remaining supernatant containing the leukocytes and was put in a 15ml Falcon tube, topped up with PBS 3% FCS and spun for 10min at 300xg at 4oC. Pellet was resuspended in 1ml PBS 3% FCS and left on ice until proceeding.
For leukocytes from murine spleens, kidney and bone marrow (tibia and femur), the organs were collected organs were kept in 1ml sterile PBS 3% FCS on ice. For splenocytes, spleens were gently meshed in PBS 3% FCS (Sigma) using a 40%M nylon mesh cell strainer (Fisher Scientific; cat. 22363547) and a plunger from a 2ml syringe (BD; cat. 307727), to prepare single cell suspensions. Cell suspension was collected in 15ml Falcon tubes, topped up with PBS 3% FCS and left on ice until proceeding. For kidneys cells, organs were chopped up with a scalpel and incubated in 10ml digestion medium containing 0.2mg/ml Liberase TL (Roche Diagnostics; cat. 05401020001) and 0.1mg/ml DNase I (Roche; cat. 11284932001) in a 50ml Falcon for 20 minutes while shaking at 37oC. The digestion medium and tissues were filtered and meshed through an 100%M nylon mesh cell strainer (Fisher Scientific; cat. 352360) and a plunger from a 2ml syringe, to prepare single cell suspensions in a new 50ml Falcon tube. Cell suspension was spun for 10 min at 300xg at RT and the pellet was resuspended in 50ml PBS 3% FCS to wash away the digestion buffer. The samples were spun again for 10 min at 300xg at RT and the pellet was resuspended in 8ml or 40% Percoll (GE Healthcare; cat. 17-5445-02), which was carefully overlayed on top of 3ml 70% Percoll in a 15ml Falcon tube. This step was performed to eliminate debris and non-leukocytes. The Percoll gradient was spun for 20min at 800xg RT, with acceleration speed 7 and deceleration speed 3. The top layer represents fat and debris, which is carefully collected and discarded without interrupting inferior layers. Leukocytes were present mostly in the interphase between 40% and 70%, which were then collected and put in a 15ml Falcon tube, topped up with PBS 3% FCS and spun down for 10min at 300xg at 4oC. The cells were then washed and spun twice more in PBS 3% FCS and kept on ice until proceeding.
For bone marrow cells femur and tibia were cut with surgical scissors on one end of the bone and placed in a perforated 0.5ml Eppendorf inside a 1.5ml Eppendorf tube. Eppendorf's were spun for 15 seconds at 10.000xg at RT. The pellet was resuspended in PBS 3% FCS and kept on ice until proceeding. To deplete erythrocytes, all cell suspensions were spun for 10min at 300xg in 4oC and the pellet was resuspended in 2ml ammonium-chloride-potassium (ACK; Gibco; cat. A10492-01) lysing buffer and incubated for 3min at RT. The tubes were topped up with PBS 3% FCS and kept on ice until proceeding. All samples were then filtered again with an 40%M nylon mesh cell strainer (Fisher Scientific; cat. 22363547) and were then spun for 10min at 300xg at 4oC. The pellets were resuspended in 300-1000%l PBS 3% FCS and counted.
For flow cytometry, single cell suspensions were obtained as indicated previously. Maximum 5x106 cells were plated in a Ubottom 96-well plate and spun for 10min at 300xg at 4oC. Cells were washed by resuspending the pellets in 200%l PBS and centrifuging for 10min at 300xg at 4oC. Cells were then stained for 25 mins at 4oC in the dark in 100%l PBS with fixable livedead stain and fluorescently-labelled antibodies. After staining, 100%l PBS 3% FCS was added to each well and the plate was spun for 10min at 300xg at RT. Cells were then washed once more in 200%l PBS 3% FCS and spun. The pellet was resuspended in 100%l 4% PFA (Thermo Scientific; cat. 28908) in PBS for fixation 25min at 4oC in the dark. Then, 100%l PBS 3% FCS was added to each well and the plate was spun for 10min at 300xg at RT. Cells were then resuspended in 200ml PBS, stored at 4oC and analysed within 2 days. Cells were filtered with 40%M cell strainers or 50%M filter mesh before analysis.
When intracellular staining was required, Foxp3 / transcription factor staining buffer set (ThermoFisher Scientific eBiosciences; cat. 00-5523-00) was used. Cells were fixed in fix-perm buffer instead of 4% PFA. Washed fixed cells were then first left in 50%l 1x permeabilization (perm.) buffer for 10minutes at 4oC in the dark. Then 50ul of 2x MPO (clone 8F4; Hycult Biotech; dilution 1/50) in 1x permeabilization buffer was added and incubated for 25min at 4oC in the dark. After secondary staining, 100%l perm. buffer was added to each well and the plate was spun for 10min at 300xg at RT. Cells were then washed twice in 200%l perm buffer and spun. Cells were then resuspended in 200ml PBS, stored at 4oC and analysed within 2 days.
For the V# T cell analysis, single cell suspensions were obtained as indicated above and the mouse V# TCR Screening Panel