Amphipathic Liponecrosis Impairs Bacterial Clearance and Causes Infection During Sterile Inflammation

BACKGROUND & AIMS: Although transient bacteremia is common during dental and endoscopic procedures, infections developing during sterile diseases like acute pancreatitis (AP) can have grave consequences. We examined how impaired bacterial clearance may cause this transition. METHODS: Blood samples from patients with AP, normal controls, and rodents with pancreatitis or those administered different nonesterified fatty acids (NEFAs) were analyzed for albumin-unbound NEFAs, microbiome, and inflammatory cell injury. Macrophage uptake of unbound NEFAs using a novel coumarin tracer were done and the downstream effects—NEFA–membrane phospholipid (phosphatidylcholine) interactions—were studied on isothermal titration calorimetry. RESULTS: Patients with infected AP had higher circulating unsaturated NEFAs; unbound NEFAs, including linoleic acid (LA) and oleic acid (OA); higher bacterial 16S DNA; mitochondrial DNA; altered β-diversity; enrichment in Pseudomonadales; and increased annexin V–positive myeloid (CD14) and CD3-positive T cells on admission. These, and increased circulating dead inflammatory cells, were also noted in rodents with unbound, unsaturated NEFAs. Isothermal titration calorimetry showed progressively stronger unbound LA interactions with aqueous media, phosphatidylcholine, cardiolipin, and albumin. Unbound NEFAs were taken into protein-free membranes, cells, and mitochondria, inducing voltage-dependent anion channel oligomerization, reducing ATP, and impairing phagocytosis. These were reversed by albumin. In vivo, unbound LA and OA increased bacterial loads and impaired phagocytosis, causing infection. LA and OA were more potent for these amphipathic interactions than the hydrophobic palmitic acid. CONCLUSIONS: Release of stored LA and OA can increase their circulating unbound levels and cause amphipathic liponecrosis of immune cells via uptake by membrane phospholipids. This impairs bacterial clearance and causes infection during sterile inflammation.

serum or PBS (~100µl) was mixed with MagMAX Cell-free DNA Magnetic beads (10µl), and binding solution (400µl).The mixture was vortexed thoroughly (10min, 25⁰C), placed the tubes on the DynaMag Magnet (5 min, 25⁰C) to separate beads from the supernatant, and carefully discarded the supernatant.The beads were washed with wash solution thrice, and with ethanol (80%) twice.The beads were dried up, and cfDNA was eluted out from the bead with ultrapure water (10µl).The concentration of the DNA in the eluent was measured by NanoDrop Microvolume Spectrophotometers (Thermo Fisher Scientific, MA, USA), and processed for qPCR assay.Sterile PBS (2 samples/ batch of 10-14 samples) served as extraction controls in place of serum to assess background noise for the cell free DNA extraction.Each batch had samples from all patient groups.The extraction controls determined the threshold cycles to compare and quantify the 16S bacterial DNA amounts using primers corresponding to V3-V4 hypervariable regions (please see below) for each batch.The cell free DNA was isolated from human samples in 7 batches.Amplification: PCR reaction was performed with the universal 16S bacterial primer pair 5'-CCTACGGGNGGCWGCAG-3', and 5'-GACTACHVGGGTATCTAATCC-3' as recommended 1 .In a qPCR plate, the reaction volume (20µl) was prepared with SYBR Green Master Mix (10µl), F-primer (1µl), R-primer (1µl), cfDNA (1µl), and ultrapure water (7µl).The following PCR conditions were used, and run on the LightCycler 480 II instrument (Roche, Indianapolis, IN, USA): Initial denaturation at 95⁰C for 5 min, followed by 40 cycles consisting of denaturation (95⁰C for 40 s), annealing (55⁰C for 2 min) and extension (72⁰C for 1 min) and a final extension step at 72⁰C for 7 min.The 16S copy number was calculated from the qPCR cycle number after subtracting extraction control's cycle number.

Microbiome library preparation methodology:
This was done in the genomics core facility at Arizona State University.Bacterial community analysis was performed via next generation sequencing in MiSeq Illumina platform.Amplicon sequencing of the V4 region of the 16S rRNA gene was performed with the barcoded primer set 515f/806r designed by Caporaso et al 2 and following the protocol by the Earth Microbiome Project (EMP) (http://www.earthmicrobiome.org/emp-standard-protocols/)for the library preparation.PCR amplifications for each sample were done in triplicate, then pooled and quantified using Quant-iT™ PicoGreen® dsDNA Assay Kit (Invitrogen, Waltham, MA).A no template control sample was included during the library preparation as a control for extraneous nucleic acid contamination.240 ng of DNA per sample were pooled and then cleaned using QIA quick PCR purification kit (QIAGEN, Germantown, MD).The pool was quantified by Illumina library Quantification Kit ABI Prism ® (Kapa Biosystems, Wilmington, MA).Then, the DNA pool was diluted to a final concentration of 4 nM then denatured and diluted to a final concentration of 4 pM with a 15% of PhiX.Finally, the DNA library was loaded in the MiSeq Illumina and run using the version 2 module, 2x250 paired-end, following the directions of the manufacturer.

Quality Control of microbiome studies:
1) Steps to minimize bacterial contamination A) Samples were collected, transported (4 0 C) and stored (-80 0 C) in sterile containers.B) All plasticware used for handling samples was sterile, DNA, RNA, DNAse free, with barrier tips.C) Samples were always opened using mask, gloves, and behind a shield using standard microbiology, and molecular biology precautions.2) Using extraction controls as negative controls for each serum batch, and their post-hoc analysis; These controls were present at all steps from cell free DNA extraction till microbiome analysis: Therefore, these organisms are present early in AP, but are cleared by the competent immune cells in the group that does not develop infections, while the group with immune cell injury and impaired clearance develops infections.

ANIMAL STUDIES:
All experiments were approved by the Institutional Animal Care and Use Committee of the Mayo Clinic; care and handling of animals were in accord with the Guide for the Care and Use of Laboratory animals by the Institute for Laboratory Animal Research.10-12weeks old male CD1 mice (Charles River Laboratories, Wilmington, MA) or C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME) were acclimatized for at least 2 days before experimentation.Mice were housed at temperatures ranging from 21-25 °C with a 12-hour light/dark cycle, fed standard laboratory chow and allowed to drink ad libitum.200-300 gm male Wistar rats were purchased from Charles River laboratories (Wilmington, MA) were acclimatized and fed in a similar fashion.
Acute Pancreatitis models: To induce pancreatitis, the murine recombinant IL-12 (Peprotech, Cranbury, NJ, 150 ng/30g) and IL-18 (R&D Systems, Minneapolis, MN, 750 ng/30g) were injected into peritoneal cavity, two consecutive doses at 24h intervals as described previously 11 .A single dose of 150µl (0.5% body weight) glyceryl trilinoleate (GTL, TCI Inc, Philadelphia, PA), GTP (Sigma Aldrich; surgically placed 150 mg powder) or GTL+orlistat (Cayman Chemical, Ann Arbor, MI, 50 mg/kg dissolved in GTL) was given intraperitoneally 2 hours after the second dose of IL12&18 as described previously 11 .Animals were followed for 3 days after the second injection of IL12&18 or till they were moribund, which ever came first, at which time they were euthanized in a carbon dioxide chamber before harvesting blood and tissues.90% mice in the IL12&18+ GTL group became moribund between 48-72 hours after the second IL12&18 injection as shown previously 11 .
Cerulein AP was induced in rats as previously 12 by intraperitoneal caerulein injection (20 mcg/kg 2 times a day) for 3 days.For rats co-administered the triglyceride of oleic acid (glyceryl trioleate; GTO or triolein; Sigma-Aldrich), GTO was given at 3% of body weight based on a previously optimized protocol 12 that simulates human obesity.GTO alone, as shown previously 12 (see supplementary figure 5 of 12 ) does not cause any deleterious effects or pancreatitis.In some rats with caerulein pancreatitis, the lipase inhibitor orlistat (Cayman Chemical, Ann Arbor, MI) was co-administered (at 50 mg/kg) dissolved in GTO (i.e.CER+GTOO group), with a goal to prevent lipolysis of GTO to oleic acid.Rats were followed for 3 days and sacrificed 3-4 hours after the last injection or when the CER+GTO group was noted to be moribund, which was typically on the third day (Fig 4Q ).
Carotid pulse distension (MouseOx Oximeter, STARR Life Sciences, Pittsburgh, PA) was used as a measure for noninvasive blood pressure monitoring.Shock was defined as reduction tp <40% of baseline.Mice and rats were observed four times daily.Terminal blood and tissue harvest was done and used in the analyses.Tissues were collected for histology and TUNEL staining (formalin).Blood was collected for flow cytometry and processed as below.Serum for lipidomics, Luminex and biochemical assays (serum) was processed as described below and detailed previously 13,14 .

Intraperitoneal GFP-E coli and NEFAs injection and mimicking sepsis like model in vivo:
CD-1 mice were used for this experiment.These were divided into different groups, i.e. control, GFP-E.coli (ATCC® 25922GFP™) alone, and GFP-E.coli given along with Oleic acid (OA, 0.3% body weight), and Palmitic acid (PA, 0.3% body weight).Additionally, Linoleic acid (LA, 0.2% body weight) +fatty acid free bovine serum albumin (BSA) or GFP-E.coli with LA ± Alb were injected into CD1 mice in different groups.For the LA+ albumin group LA was pre-conjugated with BSA and Calcium (60µl LA with 1.2 ml of 25% BSA and 20mM CaCl2) at 37⁰C for 2-4h.There were 8-12 mice per group.10 6 GFP-E coli/mouse in 0.1ml saline were injected IP in the respective group of mice.After 30 min of GFP-E coli injection, pulse distension, and rectal temperatures were measured at baseline and daily thereafter.14 hours later, pHrodo E. coli (10 6 /mouse) were given intraperitoneally, and mice were sacrificed using carbon dioxide and the cells in the peritoneal fluid were stained with live dead marker.To quantitate bacterial load, ascitic fluid/peritoneal lavage was collected.
Postmortem blood was collected for total WBC count, processed for flow cytometry from heparinized samples (as below) and plasma was collected for measuring blood urea nitrogen, albumin, Ca 2+ , uFA.Kidney, lung, and spleen samples were formalin fixed; paraffin embedded.These were stained for TUNEL, using the method described below under immunofluorescence staining.

Peritoneal bacterial loads:
To quantify bacterial loads the peritoneal cavity was lavaged with 1-2 ml saline at the time of sacrifice and the sediment was pelleted at 10,000g for 30 minutes.The pellet was resuspended in 1 ml saline, sonicated with a probe sonicator (15 Seconds), and centrifuged again at 10,000 g/10 min.Fluorescence was measured in the supernatant with excitation at 488nm and emission at 510 nm Flex Station 3 microplate reader (Molecular Devices, Sunnyvale, CA), and normalized to peritoneal washings from rodents to which no bacteria were administered.This was compared to a standard curve prepared with known amounts of E. coli GFP bacteria to quantify the bacterial load in the peritoneal cavity.The bacterial counts were confirmed by using a Neubauer chamber.
Gram's staining: This was done using the commercially available Kit from Sigma-Aldrich (St. Louis, MO) on deparaffinized formalin fixed paraffin embedded sections of the pancreas and surrounding fat of rodents used in the study.Imaging was done using an Olympus microscope with a 100x/1.3NAoil objective with a DP74 Digital Color Camera, and cellSens entry imaging software.Slides were scanned for bacteria using a 10x-40x objective.2-4 representative images of bacteria or their absence were taken at 100x for each pancreas.Bacteria when present were usually in large numbers and multiple fields.Number of pancreas from each group that were positive for bacteria, along with type of bacteria were recorded.
Luminex studies for serum cytokines: These were done on human 15 and rodent serum samples 12,14 as described previously.

Serum Biochemical and blood hematological assays and lipidomics assays:
Pancreatic lipase was assayed using the Kit (L7503) from Pointe Scientific (Canton, MI) as per protocol for the clinically established method.This uses a 1,2-diglyceride substrate, with colipase as the pancreatic lipase cofactor, and deoxycholate (1.0 mM) which acts both as an activator for pancreatic lipases and inhibitor of lipoprotein lipase.Serum amylase and blood urea nitrogen (BUN) were also assayed per clinically established methods using kits from Pointe Scientific using A7564 and B7552 respectively.Total peripheral WBC counts was done manually using a hemocytometer as per established protocols after lysing EDTA blood or heparinized blood in 1% acetic acid.For serum fatty acids, the samples were aliquoted and stored at -80'C.Batch analyses for total serum NEFA were done using the Wako HR series NEFA-HR(2) enzymatic colorimetric method (Fujifilm, Valhalla, NY) as previously 11,15 .Unbound fatty acid were measured using the Kit from FFA sciences (San Diego, CA) as described previously 11,15 .
TUNEL staining in Spleen sections: Spleen tissue from FFPE sections (5 μm) was used for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL).In brief, after deparaffinization, the TUNEL Andy Fluor™ 647 Apoptosis Detection Kit (GeneCopoeia, Rockville, MD) was used to detect DNA cleavage in apoptotic cells in tissue sections as per the mouseocmanufacturer's instructions.The staining was completed with fluorescent nuclear counterstain with Hoechst 33342 solution (1:1000, Thermo Scientific).Digital images of section were captured with a digital microscope Axio Imager.M2 or Axio Observer.Z1 (Zeiss, Oberkochen, Germany) and quantified as percentage of TUNEL positive nuclei in the spleen tissue.
Flow cytometry: Human samples collected in citrated and EDTA blood vials (after performing routine laboratory test) were used.RBCs were lysed in FACS lysing solution (BD Biosciences, CA, USA) for 15 min at room temperature.WBCs were washed with FACS staining solution (BD Bioscience, CA, USA) three times at 1,600 rpm for 5 min at 4⁰C.Approximately, 1 x 10 6 cells were suspended in FACS staining solution (100 µl/tube) having 1mM CaCl2 since annexin-V and phosphatidylserine binding is calcium dependent.The cells were incubated with a mixture of Annexin V-Alexa Fluor 488 (Thermo Fisher Scientific, MA, USA) and different cell lineage markers (Brilliant Violet 711 anti-human-CD3 and APC/Cy7 anti-human-CD14) from Bio-legend, CA, USA for 45 min at 4⁰C in dark.Then excess antibodies were washed off two times with FACS staining solution and finally cells were suspended in same buffer (500 µl) for acquisition.Annexin V positivity of total, CD3 sub-population, and CD14 sub-population were analyzed.Similarly, Brilliant Violet 711™ Mouse IgG2a kappa and APC/Cy7 Mouse IgG1 kappa also from Biolegend, CA, USA were used for respective isotype control of CD3 and CD14.
Absolute CD3 number was calculated from the total WBC count multiplied by percentage of CD3 population in flow analysis for each mouse.

CELLULAR STUDIES:
Cell lines: Peritoneal macrophages were collected from CD1 wild type mouse.Briefly, 10-ml syringe was filled with 10 mL of cold harvest medium (PBS) and with the beveled end of a 20-G needle facing inward, the needle was inserted through peritoneal wall and all the 10 ml of the cold harvest medium was injected into each mouse.The mice were euthanized using CO2 and the peritoneal fluid was aspirated from the peritoneum using a 10mLsyringe and needle (24-G).The fluid recovery was ~8 ml per mouse.Further, the collected fluids were spun in at 1600g for 2 min to pellet down the peritoneal macrophages.The supernatant was removed, and cells were resuspended in PBS containing 0.1% albumin.These were then washed in pure PBS x3 to remove albumin and kept on ice before use.J774A.1 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Corning, Manassas, VA) supplemented with 10% fetal bovine serum (Corning, Woodland, CA) and 1% penicillin/streptomycin (Life Technologies, Grand Island, NY).The cells were maintained in a humidified 5% CO2 atmosphere at 37°C.The day before live imaging studies, the J774A.1 cells were seeded on glass bottom dish (MatTek, Ashland, MA) and incubated overnight at 37°C under 5% CO2.

Mitochondrial depolarization in suspension:
For in vitro mitochondrial depolarization studies, cells were loaded with (JC-1, 5 µg/ml: Enzo Life Sciences, Farmingdale, NY) at 37°C for 15 min in HEPES buffer (20 Mm HEPES, 120 mM NaCl, 5 mM KCl, 10 mM glucose, 10 mM sodium pyruvate) containing 0.1% albumin media.The cells were washed twice in HEPES buffer and after this stored on ice till just before use.Just before the experiment, the cells (e.g.peritoneal macrophage cell suspension (40 µl) was added to 1950 µL of PBS in a plastic fluorimeter cuvette equipped with a stir bar.Mitochondrial inner membrane potential values (Ψm) were determined by recording time-based fluorescence for excitation at 490 nm and alternate measuring emission at 530 and 590 nm using the temperature controlled F2710 Hitachi Fluorescence Spectrophotometer (excitation and emission slits 10.0 nm, Hz, stirring ~800 rpm).For e.g. for Figure 1K, after measurement for at least 100 s to ensure a stable signal, fatty acids (Oleic acid or Myristic acid or Lauric acid or Palmitoleic acid) were added at 2µM concentration to the above stirred cell suspensions through the injection port in the lid of the fluorimeter chamber, and fluorescence was further monitored for at least 500s.The data were collected every 10 s.The first 100s of data were averaged and subtracted, and the net increase was plotted as a function of time or FA concentrations.The 530/590 emission ratio for mito-depolarization were averaged and presented from at least five independent experiments.
Mitochondrial reactive oxygen species (MROS): cells were loaded with MitoSOX red using a protocol similar to JC1 mentioned above following the manufacturer's instructions (ThermoFisher).MROS were then measured in a stirred cuvette using excitation of 396nm and emission of 610nm in a manner similar to JC1.Mouse red blood cell lysate (final hemoglobin concentration of 0.5-0.7 mg/ml) with Hydrogen peroxide (1mM final concentration) were used as a positive control.
MitoTracker staining in adherent cells: MitoTracker Red CMXRos probes (Thermo Fisher Scientific, Eugene, OR) were dissolved in DMSO to make a 1mM stock.The stain was further diluted in a complete DMEM to a working concentration of 50nM.The adhering cells were stained for 20-30 minutes at 37°C, then washed twice with pre-warmed fresh HEPES buffer and studied with the ZEISS LSM 800 confocal microscope equipped with a 63x oil immersion lens and airyscan detector.Images were collected every 5 seconds before and after LA-Coumarin treatment.

FA-coumarin uptake into cells:
Coumarin-fatty acids of Linoleic acid (LA; C18:2), oleic acid (OA; C18:1) and palmitic acid (PA; C16:2) with the coumarin group attached to the methyl terminus were custom-sythesized by Nanosyn Inc (Santa Clara, CA).Purity on LC-MS was confirmed to be >95%.These were dissolved in a chloroform to obtain a concentration of 2 mg/ml, aliquoted and stored at -87 °C until use.On the day of the assay, 7.5μl of chloroform solution of FA-Coumarin was placed into a glass vial and dried under nitrogen.The dry FA-Coumarin was then resuspended in 1μl 3.2M corresponded FA and 320μl warm PBS and sonicated to make a mixture of 10mM FA + 167μM FA-Coumarin.A reconstituted FA-Coumarin suspension was diluted in a warm HEPES buffer to get a proper concentration, sonicated after each dilution, and used for cell treatment at 37°C.In adherent live cell monolayers LA-coumarin was reproducibly visualized at ≈1M (total LA > 30 M; Supplementary figure 6).This was necessary to prevent photobleaching, allow reliable visualization despite uptake into cells via only one cell surface exposed to the NEFA.Additionally, avoidance of stirring was necessary for phagocytosis studies described below.Moreover, imaging of single cell monolayers can inherently reduce sensitivity compared to the several cells (100s or more) in the light path of a stirred cell suspension in a cuvette.
Phagocytosis assays: pHrodo™ Red E. coli BioParticles™ Conjugate (pH dependent dye conjugated with E. coli, (Thermo Fisher Scientific, Eugene, OR) were resuspended and sonicated in HEPES buffer to make 1 mg/ml stock.On the day of the assay, adhering J774A.1 cells were stained with 50nM LysoTracker Green DND-26 (Thermo Fisher Scientific, Eugene, OR) for 20-30 minutes at 37°C, then washed twice with prewarmed fresh HEPES.BioParticles were added to cells at 1:15 for a final concentration of 70μg/ml after LA-Coumarin treatment.Live imaging was performed for 90 min, with 2-minutes intervals between shots.
Phagocytosis was quantified by measuring the fluorescence intensity of colocalization between LysoTracker and pHrodo™ Red E. coli using Fiji (ImageJ) software.

ATP levels on live imaging:
To visualize ATP levels inside individual living cells we used FRET-based ATeam sensor consisting of the ε-subunit of the bacterial F0F1-ATP synthase sandwiched by CFP and YFP (Addgene, cat#: 51958).J774A.1 cells were transiently transfected with ATeam1.03-nD/nA/pcDNA3construct using Xfect Transfection Reagent (TaKaRa, cat#: 631317).Live cell images were taken using confocal microscope equipped with a 405-nm diode laser every 30 seconds before and after LA/OA treatment.The band-pass emission filters were used as follows: 455-550 nm (CFP) and 507-586 nm (YFP).Quantification and processing including background subtraction, were done in Fiji (ImageJ) software.Ratios of CFP/YFP were used to measure ATP levels.

Bioluminescent Measurement of ATP:
A bioluminescent-based assay kit was used to measure intracellular ATP levels (CellTiter-Glo® 2.0 Assay, Promega, Madison, USA) following manufacturer's instructions in cells (Fig 3D).Briefly, J774A.1 and peritoneal macrophages were suspended in HEPES buffer (with 0.01% albumin for e.g. in Fig 3D ) in appropriate dilution and added to white 96 well plate (Greiner bio-one, Germany) and exposed to respective fatty acids at unbound concentrations calculated based on the calibration curve in supplemental figure 7. and incubated at 37 o C with shaking at 120rpm for designated time points.At the end point, equal volume of assay buffer containing luminescent substrate was added to each well and measured on a Promega Glomax 20/20 Luminometer.Percentage of ATP coming from the viable cells for a particular dose and time was calculated considering the control set ATP level as 100%.
Biochemical assays: Lactate dehydrogenase (LDH) was measured in the medium of cells before and after treatment.The increase in leakage was normalized to total LDH content in the medium after cell lysis using 1% tritone for 1 hour or more.
Voltage dependent anion channel (VDAC) oligomerization studies: The J774A.1 cells were cultured in 10cm plates in DMEM supplemented with 10% FBS in a humidified atmosphere at 37 °C with 5% CO2.When cells reached 80% confluency, the medium was replaced with HEPES buffer (3 washes) and cells were treated with different concentrations of fatty acids within different time frames.To reverse fatty acid induced VDAC oligomerization, cells were treated with 1% albumin for 5 min at 37 °C.The cells were then incubated with cross-linking reagent (Ethylene glycol-bis(succinic acid N-hydroxysuccinimide ester), EGS) (Sigma-Aldrich, St. Louis, MO) for 15 min at 37 °C.After that, the EGS reagent was discarded, Following that, cells were harvested with a lysis buffer composed of Native PAGE Sample Buffer (Life Technologies, Carlsbad, CA), Digitonin (1.25% final concentration) (Life Technologies, Carlsbad, CA) and Benzonase Nuclease (2 units/μl final) (Sigma-Aldrich, St. Louis, MO), incubated at room temperature for 40 min and centrifuged at 20,000 x g for 30 min at 4 °C.The supernatant was aliquoted into sterile microcentrifuge tubes and stored at -87 °C until use.Protein concentration was determined using Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL).Then, G-250 Sample Additive (0.32% final concentration) (Life Technologies, Carlsbad, CA) was added to the samples.For Western blotting, sample (10 to 20 µg) were separated by electrophoresis using SDS-PAGE gels.Gels were electrotransferred onto Immobilon-P membranes (Millipore, Carrigtwohill, Ireland).The blots were blocked in a blocking solution containing 5% non-fat dry milk and 0.1% Tween-20 in Tris-buffered saline, followed by incubation with anti-VDAC1/2 (1:1000) antibodies (Abcam, ab154856).

LIPIDOMICS STUDIES:
Unbound fatty acid characterization by gas chromatography and mass spectrometry (GC-MS) : These were previously detailed by Cartin-Ceba et al 15 .Individual steps in sequence were albumin depletion using the Pierce Albumin Depletion Kit (Rockford, lL), followed by lipid extraction, derivatization using Deoxo-Fluor method by Kangani et al 16 .Derivatized fatty acids were quantified using GC-MS on the Agilent GC 7890B system attached to an Agilent 5977A MSD, with a HP-5MS (UI 30 m x .25mmI.D. with a .25µmfil thickness) capillary column.The internal standard added to every sample at the time of extraction to account for extraction efficiency comprised of 4.5µM C16:2, 9.1µM C17:0, and 9.1µM C19:2.A 5ppm caffeine in hexane mixture was used for injection to determine the GC consistency throughout a run.A standard curve was made in heptane and serial diluted from 20µM down to 0.1µM comprised of C12:0, C14:0, C16:0, C16:1, C18:0, C18:1, C18:2, C18:3 and C20:4 which were all quantified for each sample.Blanks of hexane were taken through the extraction process and subtracted out of the GC response for each sample and standard.The individual fatty acid values for each sample are then calculated based on the linear equation based on the standard curve.Values were reported a micromolars after being adjusted based on the internal standards, and accounting for extraction efficiency.

Serum non esterified fatty acid assay by GC:
The serum samples frozen at -80C were transported on dry ice to the Vanderbilt university medical center lipidomics core, and analyzed for individual NEFA composition using GC as described previously 11,14,15 .The result were reported as micromolars.
Thin layer chromatography (TLC) of J774A.1 lipids for LA uptake, reversal by albumin: J774A.1 cells were grown in 10cm plate and once they reach 80-90 % confluency cells were recovered by trypsin digestion and collected in 1.5ml microfuge tube.Linoleic acid stock (3.2M) was initially sonicated (three pulses -10 sec ON, 5 sec OFF) into the HEPES buffer (pH 7.4) (20 Mm HEPES, 120 mM NaCl, 5 mM KCl, 10 mM glucose, 10 mM sodium pyruvate).This was further diluted to working stock of 10mM linoleic acid concentration by immediately diluting in HEPES buffer followed by sonication as previously described.The working stock prepared was either promptly used or else was incubated at 37°C till the time of use.This stock was used to get the final concentration of 50µM linoleic acid in the cell suspension.
Cells from each 10cm plate were either treated with 50µM Linoleic acid (LA) alone for 2 min or with LA (50µM) followed by albumin (0.5%) after 2 min of LA addition or untreated (control).The cells were washed once with PBS and further proceeded with lipid extraction.Total lipids were extracted by the Folch method and the lipids were recovered in the chloroform phase and further separated by thin-layer chromatography using Silica Gel G plates (Analtech, Newark, DE).Standards were loaded next to the samples to identify the lipids.Lipids were visualized by spraying with primulin (25mg of primulin in 10mL of water and 40mL of acetone) followed by viewing on UV transilluminator.
At the time of TLC, the TLC tank was filled with 100mL of a 1:1 methanol chloroform mixture.A filter paper was placed along the wall of the tank and allowed it to saturate.Once the filter paper had saturated, the Silica Gel G plate was placed on the opposite wall facing towards the filter paper.Once the solvent front migrated to the top of the plate, the plate was taken out and allowed to air dry in a fume hood for 5-10 minutes before moving it to a 60°C dry incubator for a minimum of 4 hours.Prepare a running solvent of 60mL chloroform, 70mL of ethanol, 14 mL of D.I. water and 70mL of triethylamine and add that to the TLC tank.Place the filter paper on the wall and allow to saturate again.Reactivate the silica on the dry TLC plate by placing it onto a hot plate set to 130°C for 3 minutes.Spot the standards and samples on the plate and then allow the plate to dry for 5-10 minutes after spotting.The plate was added to the opposite wall of the filter paper in the tank like before and allow the solvent to migrate until the solvent front is about 1 inch from the top of the plate.Then, the plate was removed from the tank and allowed to air dry for 5 minutes before spraying the plate with the primulin solution.After the spray dried up, the plate was viewed under UV light.

Liposome Fatty acid interaction/binding:
We used a modified method, adapted from Manley and Gordon (2008) using dehydration and rehydration of large unilamellar vesicles (LUVs) to produce giant unilamellar vesicles (GUVs) 17 .The protocol for LUV preparation is a variation of a previously established method 18 .
Briefly, 4.2 μmol of total lipids containing a mixture of POPC, and Cy5-DOPC (0.5 mol %) was dried in a glass tube under gentle nitrogen stream.This was further incubated in a vacuum desiccator for a minimum of 3 hours to overnight.After incubation, the lipid film in the vial was hydrated with 1 mL of 100 mM NaCl containing 1 vol% of glycerol.This was subjected to sonication in a bath sonicator for 2 minutes to dissociate the lipid film from the glass bottom and make a uniform lipid suspension.Further, this suspension was given three cycles of freezethaw (freezing in liquid nitrogen and thawing in a 37 °C water bath) and was further extruded in an Avanti Mini Lipid Extruder by giving 21 passes through 400nm trac-ketch membrane followed by 15 passes through 200-nm track-etch membrane to generate the 200nm LUVs.These LUVs were kept at 4 °C and were used within 2 days.From the above LUV suspension, 10 μL was spread onto a glass cover slip.This was allowed to dry for 30 min and further rehydrated with 50 μl of buffer (50 mM HEPES-NaOH, pH 7.4, 100 mM NaCl).After around 5 minutes, giant unilamellar vesicles (GUVs) start to form which are then collected and visualized.Since the lipid mixture contained light-sensitive Cy5-DOPC, liposomes (LUVs or GUVs) were protected from light throughout the experiment.For fatty acid (Oleic acid) binding experiment with GUVs, sonicated oleic acid stocks solutions containing Top Fluor OA as tracer were freshly prepared and kept at 37 ºC during the experiment.Liposomes were mixed with Oleic acid stock solution on slides just before imaging to get a final concentration of 150µM of OA and 2.0µM of Top Fluor OA.These were imaged at 37ºC under a 63X oil immersion objective in a Zeiss LSM 800 confocal microscope (Zeiss, Oberkochen, Germany).
A) Sterile PBS (2 samples/ batch of 10-14 samples [now mentioned in the suppletory section]) served as extraction controls in place of serum to assess background noise for the cell free DNA extraction, and amplification.Each batch had samples from normal controls, non-infected and infected patients.B) Extraction control noise is a reference for all microbiome data.For e.g. in Fig 1D, the "threshold" for copy number is determined by extraction controls.This was typically at 26 cycles.Comparing identified organisms to published literature: The main types of organisms in our infected patients (ie.Pseudomonadales including Acinetobacter and Pseudomonas; shown in figure 1H and the Linear discriminant analysis in Supplementary Fig 2) are similar to what others have identified by cultures of infected AP patients.For e.g., we identify Pseudomonadales to average at ≈30% of our microbiome (fig 1H; B in image below).similarly, Li et al show Acinetobacter being the highest prevalence at 26% and Pseudomonas at 8%.Additionally, Lu et al 10 identify Pseudomonas and Acinetobacter as the most common organisms isolated.Please see comparison to their table below: