Sophorolipids block lethal effects of septic shock in rats...

Sophorolipids block lethal effects of septic shock in rats in a cecalligation and puncture model of experimental sepsis*

Martin H. Bluth, MD, PhD, FCAP†; Emad Kandil, MD†; Catherine M. Mueller, BS; Vishal Shah, PhD;Yin-Yao Lin, BS; Hong Zhang, PhD; Lisa Dresner, MD, FACS; Leonid Lempert; Maja Nowakowski, PhD;Richard Gross, PhD; Robert Schulze, MD, FACS; Michael E. Zenilman, MD, FACS

Septic shock is a common andfrequent cause of death in hos-pitals. Estimates of the inci-dence of septic shock in the

United States range from 300,000 to500,000 per annum, and mortality rates

ascribed to refractory hypotension fromseptic shock approach 90%. The overallestimated crude mortality rate from sep-tic shock is 35%, and the annual healthcare cost is estimated at $5 billion to $10billion (1). In patients with Gram-negative sepsis, bacterial components in-cluding DNA and endotoxin, specificallycell wall lipopolysaccharide (LPS), are be-lieved to be causative factors of septicshock via induction of cytokine cascades(2–6). Pro-inflammatory cytokines, spe-cifically monocyte-macrophage derivedtumor necrosis factor (TNF) and interleu-kin (IL)-1 (6), are now known to play animportant role in the inflammatory re-sponse. Septic shock can result in activa-tion of the coagulation cascade and apo-ptosis, causing further organ damage anddisseminated intravascular coagulation(DIC) (7).

Intra-abdominal sepsis is directly re-lated to delivery of endotoxin-producing

Gram-negative pathogens into the perito-neal cavity. This can occur directly fromspillage of fecal matter or by transloca-tion of endogenous bacteria from the gas-trointestinal tract. Despite significant im-provements in antibiotic therapy andaggressive surgical management, septicshock following peritonitis remains a dif-ficult clinical situation to manage (8, 9).Thus, identifying agents that could inter-fere with septic shock is of great clinicalimportance.

Sophorolipids are glycolipids having dis-accharide sophorose linked glycosidically tothe hydroxyl group at that penultimate car-bon of C16 to C19 chain fatty acids (Fig. 1).They are fermentatively produced by yeastssuch as Candida bombicola, Yarrowia lipoly-tica, Candida apicola, and Candida bogorien-sis when fed with carbohydrates, fatty acids,or their mixture thereof. First described in1961, sophorolipids occur as a mixture ofmacrolactone and free acid structures that

*See also p. 258.†These authors contributed equally to the content

of this paper.From the SUNY Downstate Medical Center, Depart-

ment of Surgery (MHB, EK, CMM, Y-YL, HZ, LD, RS,MEZ) and Department of Pathology (MHB, LL, MN); andPolytechnic University, National Science FoundationCenter for Biocatalysis and Bioprocessing of Macro-molecules (VS, RG), Brooklyn, NY.

The authors have no financial interests to disclose.Address requests for reprints to: Martin H. Bluth,

MD, PhD, FCAP, Director of Research, Assistant Pro-fessor of Surgery and Pathology, SUNY DownstateMedical Center, 450 Clarkson Avenue, Box 40, Brook-lyn, NY 11203. E-mail: [email protected]

Copyright © 2005 by the Society of Critical CareMedicine and Lippincott Williams & Wilkins

DOI: 10.1097/01.CCM.0000196212.56885.50

Objective: Sophorolipids, a family of natural and easily chemoen-zymatically modified microbial glycolipids, are promising modulatorsof the immune response. The potential of the therapeutic effect ofsophorolipids was investigated in vivo in a rat model of sepsis and invitro by analysis of nitric oxide and cytokine production.

Design: Prospective, randomized animal study.Setting: Experimental laboratory.Subjects: Male Sprague-Dawley rats, 200–240 g.Interventions: Intra-abdominal sepsis was induced in vivo in

166 rats via cecal ligation and puncture (CLP); 60 rats were usedto characterize the model. The remaining rats were treated withsophorolipids or vehicle (dimethylsulfoxide [DMSO]/physiologicsaline) by intravenous (iv) tail vein or intraperitoneal (IP) injectionimmediately post-CLP (25/group). Survival rates were comparedat 36 hrs after surgery. In vitro, macrophages were cultured inlipopolysaccharide (LPS) � sophorolipid and assayed for nitricoxide (NO) production and gene expression profiles of inflamma-tory cytokines. In addition, splenic lymphocytes isolated from CLPrats � sophorolipid treatment (three per group) were analyzed forcytokine production by RNase protection assay.

Measurements and Main Results: CLP with 16-gauge needlesoptimized sepsis induction and resultant mortality. Sophorolipidtreatment improved rat survival by 34% (iv) and 14% (IP) incomparison with vehicle controls (p < .05 for iv treatment).Sophorolipids decreased LPS-induced macrophage NO productionby 28% (p < .05). mRNA expression of interleukin (IL)-1� wasdownregulated by 42.5 � 4.7% (p < .05) and transforming growthfactor (TGF)-�1 was upregulated by 11.7 � 1.5% (p < .05) insplenocytes obtained 6 hrs postsophorolipid treatment. LPS-treated macrophages cultured 36 hrs with sophorolipids showedincreases in mRNA expression of IL-1� (51.7%), IL-1� (31.3%),and IL-6 (66.8%) (p < .05).

Conclusions: Administration of sophorolipids after induction ofintra-abdominal sepsis significantly decreases mortality in thismodel. This may be mediated in part by decreased macrophageNO production and modulation of inflammatory responses. (CritCare Med 2006; 34:E188)

KEY WORDS: sepsis; sophorolipid; mortality; glycolipid; nitricoxide; cytokines; lipopolysaccharide

E188 Crit Care Med 2006 Vol. 34, No. 1

are acetylated to various extents at the pri-mary hydroxyl position of the sophorose ring(10). Natural sophorolipid is a complex mix-ture of up to eight different compounds, withthe macrolactone and the sophorose glyco-side as the major constituents. The fatty acidportion forms a 1’,4” macrocyclic lactone ring(lactonic SLs) or has a free carboxylic end(nonlactonic SLs) (Fig. 1) (11).

Synthesis of glycolipids and their de-rivatives are of great interest because oftheir varied biological activities and po-tential use in other applications. Biolog-ical applications of glycolipids and theiranalogues have been reported in cancertreatment by cytokine upregulation/macrophage activation (12–16); treat-ment of autoimmune disorders (17);treatment of antiendotoxic (septic) shockby cytokine downregulation (18–20); reg-ulation of angiogenesis (21); and apopto-sis induction (22). Ongoing research withsophorolipids, which are unique mem-bers of the glycolipid family, has shownthat they have immense potential as ther-apeutic agents that can function as septicshock antagonists (23), anticancer agents(24), and antibacterial, antiviral, and an-tifungal agents (25). Furthermore, thesesophorolipid molecules can be chemoen-zymatically modified to provide a prom-ising new class of agents to combat septicshock (26, 27).

Preliminary data from our laboratory(23) show that sophorolipids can moder-ate macrophage production of NO. NO isan important mediator at the molecularlevel in refractory hypotension and shock(28). NO production has also been shownto be responsible for sepsis-related acutelung injury (29) and central neural apo-ptosis (30). Although agents that directlyinhibit NO production have been consid-

ered as therapeutic options in the treat-ment of sepsis, such therapy has not beenpromising and may prove detrimental(31–35).

We propose that natural sophorolipidsare a potential therapeutic tool in sepsisand act, as demonstrated here in both invivo and in vitro models, through modu-lation of inflammatory responses.

MATERIALS AND METHODS

Sophorolipid Synthesis

Sophorolipids were synthesized by fermen-tation of C. bombicola (11, 27). The fermen-tation media contained glucose (100 g), yeastextract (10 g), urea (1 g), and oleic acid (40 g)per 1000 mL of water. After 7 days of fermen-tation, sophorolipid was extracted three timeswith ethyl acetate. The extracts were pooledand the solvent was removed. The obtainedproduct was washed with hexane to removeresidual fatty acids. Liquid chromatography/mass spectrometry (LC/MS) and nuclear mag-netic resonance (NMR) analysis was carriedout to verify the purity of the compounds. Noresidual fatty acids or media components werefound in the sophorolipids.

In Vivo Sepsis Induction

To optimize a rat model of sepsis-relatedmortality and test the effect of sophorolipids inan in vivo CLP model of sepsis (36), 60 maleadult Sprague-Dawley rats (200–240 g; Taconic,Germantown, NY) were anesthetized with anintraperitoneal injection of Nembutal (40 mg/kg; Abbott Laboratories, North Chicago, IL). Theabdomen of each animal was shaved andscrubbed with Betadine. A midline laparotomywas performed and the cecum was ligated justbelow the ileocecal valve with a 3–0 silk ligature.To determine optimal sepsis induction, the an-

timesenteric cecal surface was punctured with a14, 16, 18, or 20-gauge needle proximal to theligature (15 rats/needle gauge). The abdominalincision was then closed in two layers with 2.0silk (36, 37). All subsequent experiments, in thepresence or absence of sophorolipid, employed16-gauge needle puncture.

Sophorolipid Treatment

The CLP-treated animals (n � 25/group)were treated with sophorolipid (5 mg/kg ratweight, in 4% DMSO-saline) following CLP, byeither intravenous (iv; tail vein) or intraperi-toneal (IP) injection. The two control groups(n � 25/group) received a similar volume ofplacebo (4% DMSO in saline) either iv (tailvein) or IP at the end of the operation. Allanimals were housed singly in standard cagesand had access to chow and water throughoutthe experiment. Animals were monitored over36 hrs, and the survival rate was comparedbetween the experimental and control groups.The study was approved by the Animal Careand Use Committee at SUNY-Downstate Med-ical Center.

Gene Profiles

The gene expression profiles of specific in-flammatory cytokines (tumor necrosis factor[TNF]-�, IL-1�, IL-1�, IL-6, IL-10, tumorgrowth factor [TGF]-�1, and macrophage in-hibitory factor [MIF]) were measured insplenic lymphocytes isolated from sophoro-lipid- or vehicle-treated rats 6 hrs after CLPand in a rat alveolar macrophage cell line,NR8383 (CRL-2192, ATCC, Manassas, VA),which were left untreated; treated (activated)with LPS (from Salmonella typhimurium;6511, Sigma-Aldrich, St. Louis, MO) to createa cellular model of sepsis (28, 38); treated withLPS and subsequently with sophorolipid; ortreated with sophorolipid only.

Splenic Lymphocytes. Spleens were har-vested from animals killed 6 hrs post-CLP withor without sophorolipid treatment (iv route;three per group) and placed in cold culturemedia (RPMI-1640, GIBCO, NY). Lymphocyteswere obtained by gently grinding spleens be-tween the frosted edges of two glass slides(Fisher Scientific, Suwannee, GA). Cell sus-pensions were poured through Nitex (nylon)mesh (Tetko, Elmsford, NY) to removestroma. Splenic lymphocytes were rinsedtwice with ice-cold phosphate-buffered saline(PBS) and quickly scraped into TRIzol (In-vitrogen, Carlsbad, CA) for isolation of totalRNA. Cytokines were assayed with the RNaseprotection assay system, as previously de-scribed (39), with the BD/Pharmingen Bio-sciences (San Diego, CA) RiboQuant Multi-Probe Template Sets rCK-1 and rCK-3,according to the manufacturer’s recommen-dations (rCK-1: IL-1�, IL-1�, TNF�, IL-3,IL-4, IL-5, IL-6, IL-10, TNF�, IL-2, interferon

Figure 1. Structure of various natural sophorolipids produced by Candida bombicola.


[IFN]-� cytokines; rCK-3: IFN�, TNF�, gran-ulocyte macrophage colony stimulating factor[GM-CSF], TGF�1, TGF�3, TGF�2, leukotri-ene [Lt]-�, TNF�, MIF, IFN� cytokines).Quantification of mRNA was performed (Phos-phorImager, Molecular Dynamics, Sunnyvale,CA) and analysis of radioactive bands was un-dertaken (ImageQuant software, MolecularDynamics) according to the manufacturer’srecommendations. Data are represented aspercent change in phosphoimager units.

Rat Macrophage Cell Line. NR8383 cellswere placed in 24-well plates (Corning, Corn-ing, NY) in Kaighn’s modification of F12[F12K] growth media (with 2 mM L-glu-tamine, 1.5g/L sodium bicarbonate, and 15%heat-inactivated fetal bovine serum, [GIBCO])at a concentration of 2.7 � 105/2 mL/well andcultured at 37°C in a 5% CO2 incubator (5500DH Autoflow, NuAire, Plymouth, MN). After24 hrs, the medium was changed and cellswere either left untreated or treated in a criss-cross pattern with increasing concentrationsof LPS (range, 0 [PBS vehicle] to 150 ng/mL)and after 90 mins by increasing concentra-tions of sophorolipid (range, 0 [DMSO/PBSvehicle] to 200 ng/mL). Optimal concentra-tion for LPS-stimulated macrophage activa-tion and subsequent response to sophorolipidtreatment, as determined by changes in cellgrowth, viability, morphology, and NO pro-duction, were found to be 100 ng/mL and 200ng/mL, respectively (data not shown).

Cells were then plated in 75 cm2 tissueculture flasks (BD Falcon, Bedford, MA) andrandomized into four groups: untreated cells,LPS-treated cells (100 ng/mL), LPS-treatedcells (100 ng/mL) treated 90 mins later withsophorolipid (200 ng/mL), and sophorolipid-only treated cells (200 ng/mL). At 36 hrs,TRIzol-isolated RNA from each group waspooled, quantitated (Smart-Spec 3000 spec-trophotometer, BioRad, Hercules, CA), andquality-checked with agarose/ethidium bro-mide gel electrophoresis. Cytokines were as-sayed (RNase Protection Assay) with the Ribo-Quant Multi-Probe Template Sets rCK-1 andrCK-3 described above. In brief, 15 �g of RNA

from each group was loaded, in duplicate, ontoa vertical polyacrylamide gel electrophoresis(PAGE) apparatus (IBI, Standard ThermoplateSequencer, New Haven, CT), along with theappropriate controls and probes, and run asper the manufacturer’s instructions. The gelwas then adsorbed to filter paper, dried undervacuum, placed in a cassette with X-AR film(Kodak, Rochester, NY) and an intensifyingscreen, developed at �80°C, and visualizedwith a medical film processor (SRX-101A,Konica, Taiwan). Radioactive bands were ana-lyzed and quantitated with the Gel Doc 2000System with specific software (The DiscoverySeries: Quantity One, BioRad). To adjust fordifferences in sample processing, hybridiza-tion signals in each sample were divided by thesignal for the housekeeping ribosomal proteinmRNA (L32). Data are represented as finaladjusted volume (area of band [mm2] � pixelintensity units [INT] � INT*mm2).

Effect of Sophorolipid onMacrophage Production of NO

To further test the in vitro effect of sopho-rolipids, mouse macrophages (RAW 264.7,ATCC TIB-71) were cultured with LPS (50ng/mL), with or without sophorolipids (25–100 ng/mL). Aliquots of culture supernatantswere collected at 5 days and NO content wasdetermined by measuring nitrite (modifiedGriess reaction), as previously described (28,40). In brief, triplicate 50-�L aliquots of cul-ture supernatant were mixed in wells of a96-well microtiter plate with 100 �L of Griessreagent containing a 1:1 (vol/vol) mixture of1% (wt/vol) sulfanilamide in 30% acetic acidand 0.5% (wt/vol) of N-(�1-Naphthyl) ethyl-enediamine dihydrochloride in 60% aceticacid. The chromophore generated by the reac-tion with nitrite was detected spectrophoto-metrically with a microtiter plate reader at550 nm (ELx 800, BioTek Instruments, Wi-nooski, VT). The concentration of nitrite wascalculated from a standard curve calibratedwith known concentrations of NaNO2.

Statistical Analysis

Survival data were compared by Kaplan-Meier analysis with log-rank test to comparesurvival function between conditions. Valuesother than survival data are expressed as mean� SEM. Significance was determined by eitherStudent’s t-test or analysis of variance(ANOVA) with Tukey posthoc analysis for pvalue adjustments, with use of SPSS (SPSS,Chicago, IL). In each case, significance was setat p � .05.

RESULTS

Sepsis Induction

Preliminary studies in our laboratoryrevealed average mortality rates of 39%,42%, 21%, and 27% when CLP was in-duced with 14-, 16-, 18-, and 20-gaugeneedles, respectively, for all cumulativetime points over the course of 36 hrs(Fig. 2). We chose 16 gauge for the re-mainder of our experiments because thisyielded the highest overall mortality.

Effect of Intravenous orIntraperitoneal SophorolipidTreatment on Survival after CLP

Intravenous Administration. Follow-ing CLP and iv administration of vehicle,the survival rate at 36 hrs in the controlgroup was 47.8%. The survival rate in-creased to 81.8% among animals given ivinjections of sophorolipid immediately af-ter CLP (p .05) (Fig. 3).

Intraperitoneal Administration. Fol-lowing CLP and IP administration of ve-hicle, the survival rate at 36 hrs in the

Figure 2. Cumulative mortality rates in experimental animal sepsis (cecal ligation and puncture) after12, 24, and 36 hrs with use of 14-, 16-, 18-, and 20-gauge needles. Data represent average of 15 animalsper group. Sixteen-gauge needle puncture yielded the highest mortality (42%) when averaged acrossall time points; there was an average of 19%, 48%, and 60% mortality at 12, 24, and 36 hrs,respectively.

Figure 3. Survival rate in intravenous naturalsophorolipid mixture–treated and control ani-mals (Kaplan-Meier); experiments ceased at 36hrs. Vehicle: cecal ligation and puncture (CLP) dimethylsulfoxide/saline, SL: CLP sophorolipid(SL) treatment. CLP was induced with a 16-gaugeneedle, as described in Materials and Methods.


control group was 53%. The survival rateincreased to 67% among animals giveninjections of sophorolipid immediately af-ter CLP (p � .08; data not shown).

Effect of Sophorolipid on NOProduction

When macrophage cells (RAW 264.7)were treated with 50 ng/mL LPS, in-creased levels of NO production were ob-served (34.8 � 3.9 �M, Fig. 4). In con-trast, when sophorolipid was added toLPS-treated cultures, NO production wassignificantly reduced by 25% to 26.3 �0.4 �M in comparison with LPS alone(Fig. 4). Cells cultured with or withoutsophorolipid in the absence of LPS gen-erated little to no NO production. (Fig. 4)

Effect of Sophorolipid onCytokine Production

mRNA derived from splenocytes, ob-tained from rats 6 hrs after CLP and so-phorolipid treatment, had a 42.5% �4.7% (p .05) reduction in IL-1� expres-sion in comparison with vehicle-treatedcontrols (Fig. 5A). In contrast, mRNA de-rived from splenocytes, obtained fromrats 6 hrs after CLP and sophorolipidtreatment, showed an 11.7 � 1.5% (p .05) increase in expression of TGF-�1(Fig. 5B). There was no difference in cy-tokine expression for IL-1�, TNF-�, IL-6,IL-10, and MIF from mRNA obtainedfrom rats 6 hrs after CLP/vehicle vs. CLP/sophorolipid treatment (data not shown).

Rat alveolar macrophage cell lines �LPS activation were subsequently cul-tured in the presence or absence of so-phorolipids and assessed for cytokinemRNA production by RNase ProtectionAssay (Fig. 6).

High levels of IL-1� and IL-1�, IL-6,IL-10, TNF-�, and MIF were observed inthe LPS-treated cultures in comparisonwith nontreated cells (Fig. 7). When so-phorolipid was added to LPS-treated cul-tures, expression of IL-1�, IL-1�, andIL-6 was significantly increased (by51.7%, 31.3%, and 66.8 %, respectively; p 0.05, ANOVA) (Fig. 7). There were nodifferences in IL-10, TNF�, or MIF whenLPS-treated cells were cultured in thepresence or absence of sophorolipids(Fig. 7). In addition, there were no differ-ences in TGF-�1 mRNA expression inLPS-treated, LPS-sophorolipid-treatedcells, or only-sophorolipid-treated cells incomparison with untreated cells (data notshown). In contrast, addition of sophoro-lipid to non-LPS-treated cells did notelicit changes in mRNA expression incomparison with cells alone (Fig. 7).

DISCUSSION

Despite advancements in the treat-ment of sepsis, mortality remains high(1). Gram-negative infections result inendotoxin-induced upregulation ofplasma cytokine levels (41–45), which in-duces septic shock. Current treatmentsfor septic shock caused by Gram-negativebacteria include antibiotic therapy andintensive care to support aberrations incardiovascular, endocrine, and other or-gan systems. Antibiotics may be harmfulwhen given in the setting of Gram-negative sepsis (9, 46, 47) because of fur-ther release of endotoxin and promotionof life-threatening complications. There-fore, there is a great interest in identify-ing novel strategies to treat not only in-fections but also the underlyinginflammatory responses. For this reason,agents that can modulate inflammatory

responses, in addition to having directantimicrobial activity, would be advanta-geous.

The multifactorial nature of septice-mia from an intra-abdominal source cre-ates a difficult environment for novel andeffective therapeutic intervention (48).Attempts to use immunomodulation tocontrol sepsis have met with mixed re-sults (49). Although experimental studiesin vitro and in animals with the anti-endotoxin antibody (50) showed promise,application in the clinical world met withfailure (51).

The only approved drug on the marketfor sepsis is recombinant protein C (Dro-trecogin alfa [activated], Xigris, Lilly, In-dianapolis, IN), an antithrombotic agentwhose mechanism is poorly understood.It has been shown to increase survival,through mechanisms which include re-duction of serum d-Dimer and IL-6 levels(52), and has a favorable benefit/risk ratiofor septic surgical patients (53, 54). De-spite a mild increase in risk of bleeding(3.5% vs. 2.0%; p � .06) (52), it is listedas contraindicated for patients with re-cent or active bleeding/coagulopathy,making it unsuitable for many septic,traumatic, or surgical patients. There-fore, development of additional pharma-ceutical agents that could be adminis-tered to treat sepsis would be valuable.

Sophorolipids are a unique class ofnatural microbial glycolipids that can bechemoenzymatically modified (11). Gly-colipids have been shown to modulateseveral disorders (12–16, 18–22). There-fore, the therapeutic potential of sopho-rolipids, a novel class of modified glyco-lipids, was investigated in sepsis. Alongwith conferring the immunomodulatoryeffect during septic shock, sophorolipidscould also be demonstrating an antibac-terial effect. Sophorolipids are biosurfac-tants that mediate antibacterial effectsthrough mechanisms involving mem-brane destabilization and increased per-meability (25). Although the concentra-tion of sophorolipids in the bloodstreamis far less than the minimal inhibitoryconcentrations for most of the organ-isms, the microbicidal property of sopho-rolipids may reduce the bacterial load inthe bloodstream through cell lysis (25).

A number of models for inducing in-tra-abdominal sepsis exist. A single inoc-ulum of one Gram-negative bacterial spe-cies has been the model of sepsis used forthe screening of antimicrobial drugs invitro (9). We prefer the cecal ligation andpuncture model of experimental sepsis,

Figure 4. Effect (mean � SEM) of sophorolipid on LPS-induced NO production in a mouse macrophagecell line. SL, sophorolipids; LPS, lipopolysaccharide; NO, nitric oxide. Statistical significance tested byanalysis of variance.


as it reproduces the clinical situation ofbowel perforation and subsequent mixedbacterial infection and polymicrobialperitonitis. This well described andwidely used animal model allows a con-sistent septic insult with predictable mor-tality, cytokine production, and alterationof physiologic parameters (36, 41). Highmortality in the cecal ligation and punc-ture model can be elicited, and in ourexperiments optimal mortality ap-proached 60% at 36 hrs with use of a16-gauge needle. Similar findings withuse of this gauge needle have been re-

ported by others (36). We felt that reduc-tion of mortality by administration of so-phorolipid could be effectively monitoredin this setting.

In the current study, polymicrobialsepsis was induced with the previouslyestablished cecal ligation and puncturemodel. We found that sophorolipid ad-ministered soon after the insult de-creased mortality at 36 hrs when admin-istered iv. It is possible that sophorolipidtreatment can be administered after thedevelopment of sepsis, making it an attrac-tive potential therapy. Because treatment ofsepsis generally requires iv administration,investigation of sophorolipid treatment us-ing this route was paramount, and IP in-stillation was used as a comparative pro-cess. Although it is unclear why only ivtreatment yielded a statistically significantdecrease in mortality, it may be due todifferences in the pharmacokinetics of so-phorolipid, which are route-dependent.These aspects of sophorolipid administra-tion are currently not known.

Sophorolipid dose and animal numberselection were predicated on preliminarydata from an investigation in which miceunderwent CLP � sophorolipid treat-ment (data not shown). In these experi-ments, maximum mortality decrease wasobtained with 5-mg/kg sophorolipidtreatment. Furthermore, this dose repre-sents the current maximum solubility forsophorolipids in DMSO for subsequent invivo administration.

The dose of sophorolipids was wellwithin the reported safety range. The le-thal dose (LD) of 50% of animals (LD50)of naturally occurring sophorolipid or itsderivatives in rodents is minimally 5.8g/kg when administered subcutaneouslyand 10g/kg when ingested orally (55), andthe oral no-adverse-effect level (NOAEL)

is reported to be approximately 200 mg/kg/day (56). Others have observed nomeasurable consequences with 12.5 g/kgsophorolipids in rats or 6 g/kg in mice(57). Although the therapeutic range andmetabolism of sophorolipid treatment insepsis are unknown, the relatively lowdose used in our studies was 5 mg/kg,which nonetheless demonstrated an ef-fect of decreasing sepsis-related mortal-ity. This dose approximated 1/1000 ofthat with reportable side effects.

NO production is a useful marker ofmacrophage response to bacterial LPS(28, 38). The ability of sophorolipid tomoderate macrophage NO productionwhen exposed to LPS was investigated ina tissue culture model. Addition of sopho-rolipid decreased NO production when itwas cultured with LPS-treated macro-phages. Although induction of NO has areported benefit in certain nonsepsis dis-ease states such as parasitic infections(55, 58, 59), such is not the case forsepsis. Furthermore, when NO was ad-ministered as therapy in acute lung in-jury, there was no observed decrease inmortality (60), and it may in fact worsenthe patient’s condition. Indeed, NO pro-duction is responsible for sepsis-relatedacute lung injury (29) and central neuralapoptosis (30). Furthermore, agents thatinhibit NO production have been consid-ered as therapeutic options for sepsis(31–33, 61, 62) with varying results (34,35). Our data show that addition of so-phorolipid did not cause an increase inNO production when it was cultured withhealthy cells. This is in critical contrastto other putative therapeutic candidates,which, although promising in the treat-ment of sepsis, appear to increase NOproduction (63). It is likely that sophoro-lipid-mediated mortality reduction actsthrough NO modulation in conjunctionwith additional mechanisms. However, itis also possible that NO production mayhave little if anything to do with ourobserved reduction in sophorolipid-mediated mortality and may represent abystander effect. Additional mechanismsresponsible for the sophorolipid effect,including inflammatory cytokine modu-lation, receptor-ligand and second mes-senger interactions, and other metabolicprocesses, warrant further investigation.

Septic shock results, in part, from al-teration of plasma cytokine levels (42–45), which can cause fever, hypotension,and prostration. Our data show that so-phorolipids modulate the inflammatoryresponse to sepsis. Splenocyte cytokine

Figure 6. RNAse protection assay: A, rCK-1, andB, rCK-3 template cytokine analysis as describedunder Materials and Methods. Lanes 1 and 14,unprotected CK probes; 2 and 3, untreated con-trols; 4 and 5, LPS (100 ng/mL), 6 and 7, LPS(100 ng/mL) plus sophorolipid (200 ng/mL), 8and 9, sophorolipid (200 ng/mL); 10 and 11, yeasttRNA background control; 12 and 13, CK seriesRNA control.

Figure 5. A, interleukin (IL)-1ß production, and B, tumor growth factor (TGF)-ß1 production insplenic lymphocytes obtained from rats 6 hrs after cecal ligation and puncture (CLP) � sophorolipid(SL) treatment. Data are expressed as percent control (CLP vehicle) � SEM; p .05 compared withcontrol, Student’s t-test.


profiles were determined at 6 hrs in orderto evaluate any sophorolipid-mediatedchanges during the early stages of sepsisand identify key mediators of the inflam-matory process that are required or por-tend disease progression. Similarly, thetranscription factor early growth re-sponse (EGR)-1 was identified as a keyregulator of another inflammatory dis-ease process, pancreatitis, and was foundto be upregulated in the early stages ofthe disease (64).

Splenocytes obtained from rats treatedwith sophorolipid immediately after CLPhad decreased production of IL-1� andincreased production of TGF-�1 in com-parison with rats treated with DMSO/saline vehicle. IL-1, including IL-1� andIL-1�, is a well established proinflamma-tory cytokine that is elevated in sepsis(65), and the reduction of IL-1 is an es-tablished marker for evaluating themechanisms of therapeutic candidates insepsis treatment (28). Increases in theplasma levels of the anti-inflammatorycytokine TGF�-1 are also considered ben-eficial in sepsis, but its role is less clearowing to widely reported ranges (66, 67)and differences in active vs. total forms(68). It is possible that the observed de-crease in sepsis mortality in the sophoro-lipid group is due to its direct effect oncytokine synthesis, making it similar toother therapies (46, 47, 19). It is morelikely that additional factors, includingNO and others as yet unidentified, are

part of the sophorolipid-mediated protec-tive mechanisms.

Using an in vitro model of sepsis, LPStreatment of rat macrophages, we ob-served significant increases in expressionof IL-1�, IL-1�, and IL-6 36 hrs aftersophorolipid treatment. The IL-1� obser-vation is the opposite of what we observedin splenocytes. Although these two sys-tems are not intended to linearly trans-late into mechanisms that explain ourobserved sophorolipid-mediated reduc-tion in sepsis-related mortality in vivo,there are several possible explanations forthe observed differences in cytokine ex-pression in these two in vitro systems.First, it may simply be due to the fact thatthe macrophage cell line is a single pop-ulation, whereas the splenocytes com-prise many different cell populations.Similar discrepancies in cytokine (69)and matrix metalloproteinase (70) ex-pression have been observed between or-gan-derived cell mixtures and malignantclonal cell lines. Differences in cytokine-activated mononuclear cell cytotoxic ac-tivity in cells obtained from cord bloodvs. peripheral blood have also been re-ported (71), demonstrating variability inorgan-derived cell responses. Finally,there may be a difference in the expres-sion of these cytokines at different timesfollowing sepsis induction. Similarchanges in IL-6, inducible NO synthase,and other genes have been observed inearly vs. late gene expression in two ex-

perimental models of acute pancreatitis(64). Either way, cytokines, including butnot limited to IL-1, are involved in theresponse to sophorolipid therapy.

CONCLUSIONS

Sophorolipid treatment after CLP po-tentially reduced mortality in experimen-tal sepsis and may exert its effectsthrough reduction of NO and the modu-lation of inflammatory responses. Fur-ther investigation is warranted to betterunderstand this novel candidate for usein treating sepsis and reducing sepsis-related mortality.

ACKNOWLEDGMENTS

We thank Victor Ocasio and NancyLoporcaro for contributions to this study.

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Figure 7. Cytokine gene expression profiles of rat alveolar macrophages 36 hrs post-sophorolipidtreatment, in comparison with controls (cells cultured in the absence of lipopolysaccharide [LPS],sophorolipid, or both). Data were obtained by RNAse protection assay and are presented as finaladjusted (Adj.) volume, mean of two experiments � SEM, as described in Materials and Methods (*p .05 for LPS-treated cells cultured with sophorolipid vs. LPS alone; analysis of variance with post-hoctests). Blue bars, untreated control; red bars, LPS (100 ng/mL); yellow bars, LPS (100 ng/mL) plussophorolipid (200 ng/mL); green bars, sophorolipid (200 ng/mL); INT, pixel intensity units; IL,interleukin; TNF, tumor necrosis factor; MIF, macrophage inhibitory factor.

S ophorolipid treat-

ment after cecal li-

gation and punc-

ture potentially reduced

mortality in experimental

sepsis and may exert its ef-

fects through reduction of

nitric oxide and the modula-

tion of inflammatory

responses.


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Sophorolipids block lethal effects of septic shock in rats...

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