of February 17, 2018.This information is current as

New Class of Adjuvant?Immune Response against Influenza Virus: A17-HDHA Enhances the Antibody-Mediated The Specialized Proresolving Mediator

Martínez-Sobrido, David J. Topham and Richard P. PhippsKim, Eric A. Feldsott, Charles N. Serhan, Luis Sesquile Ramon, Steven F. Baker, Julie M. Sahler, Nina

ol.1302795http://www.jimmunol.org/content/early/2014/11/11/jimmun

published online 12 November 2014J Immunol 

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is published twice each month byThe Journal of Immunology

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The Journal of Immunology

The Specialized Proresolving Mediator 17-HDHA Enhancesthe Antibody-Mediated Immune Response against InfluenzaVirus: A New Class of Adjuvant?

Sesquile Ramon,*,† Steven F. Baker,* Julie M. Sahler,* Nina Kim,* Eric A. Feldsott,‡

Charles N. Serhan,† Luis Martınez-Sobrido,* David J. Topham,* and Richard P. Phipps*,‡

Influenza viruses remain a critical global health concern. More efficacious vaccines are needed to protect against influenza virus, yet

few adjuvants are approved for routine use. Specialized proresolving mediators (SPMs) are powerful endogenous bioactive reg-

ulators of inflammation, with great clinical translational properties. In this study, we investigated the ability of the SPM 17-HDHA to

enhance the adaptive immune response using an OVA immunization model and a preclinical influenza vaccination mouse model.

Our findings revealed that mice immunized with OVA plus 17-HDHA or with H1N1-derived HA protein plus 17-HDHA increased

Ag-specific Ab titers. 17-HDHA increased the number of Ab-secreting cells in vitro and the number of HA-specific Ab-secreting cells

present in the bone marrow. Importantly, the 17-HDHA–mediated increased Ab production was more protective against live

pH1N1 influenza infection in mice. To our knowledge, this is the first report on the biological effects of v-3-derived SPMs on the

humoral immune response. These findings illustrate a previously unknown biological link between proresolution signals and the

adaptive immune system. Furthermore, this work has important implications for the understanding of B cell biology, as well as

the development of new potential vaccine adjuvants. The Journal of Immunology, 2014, 193: 000–000.

Vaccines against infectious agents, such as influenza vi-ruses, rely on the ability of the adaptive immune systemto generate long-term memory and protection. An en-

hanced Ag-specific immune response increases the ability of theimmune system to eliminate pathogens and maintain homeostasis.Adjuvants increase a vaccine’s efficacy by enhancing the immuneresponse to the introduced Ag. Currently, alum is the only ap-proved adjuvant for routine use in vaccines in the United States(1). Influenza virus is responsible for seasonal flu outbreaks, aswell as deadly flu pandemics, which have recurred throughouthistory, such as the latest 2009 H1N1 pandemic (2, 3). Currentseasonal influenza vaccines include the inactivated influenzavaccine (IIV), live-attenuated influenza vaccine (LAIV) and the

recently approved recombinant influenza vaccine (RIV) (4, 5).These vaccines are designed to confer immune protection against

the most common seasonal influenza strains expected to circulate

each season. IIV, LAIV, and RIV do not use adjuvants in the

United States. Efficient vaccination is particularly important for

susceptible populations such as infants, older people, and immu-

nosuppressed patients (5). More efficacious vaccines are needed to

protect against seasonal influenza and possible pandemic strains.

The development of novel adjuvants could improve vaccines

against influenza and other pathogens.The acute inflammatory response is a self-limiting process that is

crucial to fight pathogens and for tissue repair and homeostasis (6, 7).

Specialized proresolving mediators (SPMs) are newly identified

lipid-derived molecules that are responsible for actively regulating

the resolution phase of inflammation (8–10). These endogenous

mediators are derived from either n-3 or n-6 polyunsaturated fatty

acids obtained from dietary sources, and they are found in the bone

marrow, spleen, and blood, among other tissues (11–13). SPMs are

classified into lipoxins, resolvins, protectins, and maresins (9, 10,

14). Docosahexaenoic acid (DHA) is a major n-3 polyunsaturated

fatty acid and is a precursor to the protectins, maresins, and D-series

resolvins families. 17-hydroxydocosahexaenoic acid (17-HDHA) is

an example of a DHA-derived SPM (10, 15).SPMs have many functions that can be cell and context de-

pendent. These functions include decrease of neutrophil cell

transmigration, enhancement of nonphlogistic monocyte recruit-

ment, and increase of macrophage engulfment of apoptotic neu-

trophils (16–18). In addition, SPMs decrease production of

proinflammatory mediators such as IL-12 and TNFa, and they

promote anti-inflammatory cytokine production, such as IL-10

(19–21). Little is known about the effects of SPMs on B cells

and the adaptive immune system. We recently reported the pres-

ence of DHA-derived resolvin D1 (RvD1), 17-HDHA, and pro-

tectin D1 in the spleen, and we have discovered that RvD1 and 17-

HDHA enhance human B cell Ab production (13). Furthermore,

*Department of Microbiology and Immunology, University of Rochester School ofMedicine and Dentistry, Rochester, NY 14642; †Center for Experimental Therapeu-tics and Reperfusion Injury, Department of Anesthesiology, Perioperative and PainMedicine, Brigham and Women’s Hospital and Harvard Medical School, Boston,MA 02115; and ‡Department of Environmental Medicine, University of RochesterSchool of Medicine and Dentistry, Rochester, NY 14642;

Received for publication October 16, 2013. Accepted for publication October 17,2014.

This work was supported by National Institutes of Health Grants AI103690 (toR.P.P.), ES01247 (to R.P.P.), GM038765 (to C.N.S.), RO1 AI077719 (to L.M.-S.),R21NS075611 (to L.M.-S.), and R03AI099681 (to L.M.-S.); University of RochesterCenter for Biodefense Immune Modeling Grant HHSN272201000055C; National In-stitute of Allergy and Infectious Diseases Center of Excellence for Influenza Researchand Surveillance Grant HHSN266200700008C; and Training Grants T90 DE021985,T32 AI007285, and T32 HL066988 (to the University of Rochester Medical Center).

Address correspondence and reprint requests to Dr. Richard P. Phipps, Box 850,MRBX 3-11001, Department of Environmental Medicine, University of RochesterSchool of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642.E-mail address: richard_phipps@urmc.rochester.edu

Abbreviations used in this article: DHA, docosahexaenoic acid; HA, hemagglutinin;17-HDHA, 17-hydroxydocosahexaenoic acid; IIV, inactivated influenza vaccine;LAIV, live-attenuated influenza vaccine; MDCK, Madin-Darby canine kidney;ODN, oligodeoxynucleotide; PFU, plaque forming unit; RIV, recombinant influenzavaccine; sciIAV, single-cycle infectious influenza A virus; SPM, specialized prore-solving mediator.

Copyright� 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302795

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our study showed that 17-HDHA promoted human B cell differ-

entiation toward an Ab-secreting phenotype, while not affecting

proliferation nor cytotoxicity (13). Abs produced solely by B cells

are pivotal for antiviral immunity because they mediate faster

pathogen clearance and promote long-term immune protection.

The biological roles of SPMs during the adaptive immune re-

sponse, specifically B cell–mediated immunity, are not known. In

this study, we used a preclinical influenza vaccination and infec-

tion in vivo mouse model to analyze the actions of the SPM 17-

HDHA on Ab production.

Materials and MethodsMouse immunization and viral challenge

OVA protein immunizations were done using 10 mg OVA emulsified inCFA (Sigma-Aldrich, St. Louis, MO; 1:1 ratio by volume). C57BL/6J malemice (8–10 wk old; The Jackson Laboratory, Bar Harbor, ME) were im-munized by i.p. injection (22). Mice were immediately given a secondinjection i.p. in the same site containing either 1 mg 17R-hydroxy-4Z, 7Z,10Z, 13Z, 15E, 19Z-docosahexaenoic acid (17-HDHA; Cayman ChemicalCompany, Ann Harbor, MI) or vehicle control (defined as PBS with 0.4%ethanol by volume). Sera were collected 2 and 6 wk after primary im-munization, and Ab levels were measured by ELISA. Ten weeks afterprimary immunization, mice were boosted by i.p. injection containing 10mg OVA suspended in PBS. Sera were collected 2 wk after boost and usedfor Ab ELISA.

Influenza hemagglutinin (HA) immunization was performed on C57BL/6J male mice (8–10 wk old; The Jackson Laboratory, Bar Harbor, ME).Mice were immunized with recombinant HA protein derived from influ-enza H1N1 A/Brisbane/59/2007 (5 mg) or A/California/04/2009 (2 mg;BEI Resources, Manassas, VA) plus 1 mg 17-HDHA or vehicle control.Mock immunization injection was defined as PBS with 0.4% ethanol byvolume without HA protein nor SPMs. HA immunizations were deliveredby i.m. injection in the left flank. Mice were given an initial immunizationat week 0 and boosted again at weeks 2 and 4 (23, 24). Sera were collected2 wk after each injection and used for analysis. Mice from experimentsshown in Fig. 2 were immunized with HA protein plus 10 mg CpG oli-godeoxynucleotides (ODN) 1826, sequence 39-TCCAATGAGCTTCCT-GAGTCT-59 (Integrated DNATechnologies, Coralville, IA) plus 1 mg 17-HDHA or vehicle control. Immunizations were delivered by i.m. injectionat weeks 0 and 3. Sera Ab titers were measured at weeks 3, 6, and 11.

For live influenza infections, C57BL/6J male mice were anesthetizedwith Avertin (2,2,2-tribromoethanol), injected i.p., and inoculated intra-nasally with H1N1 A/California/04/E3/2009 as described previously (25).Weight and survival were monitored for 14 d following infection. Allmouse experiments were approved by the University Committee on Ani-mal Resources at the University of Rochester Medical Center.

IgM and IgG ELISA and ELISPOT

Mouse sera Ag-specific Abs were measured by ELISA. Plates were pre-coated with HA protein (1 mg/ml), OVA (10 mg/ml), capture IgM, orcapture IgG Abs. Mouse-specific Ab ELISA kits were used to measureIgM and IgG (Bethyl Laboratories, Montgomery, TX) as suggested bythe manufacturer. For ELISPOT analysis, cells were incubated in HA-coated ELISPOT plates (Millipore, Billerica, MA). Alkaline phosphatase-conjugated goat anti-mouse IgM or IgG Abs (Southern Biotech, Bir-mingham, AL) were used as recommended by the manufacturer. ELISPOTplates were developed using Vector AP substrate kit III (Vector Labora-tories, Burlingame, CA) and quantified on a CTL plate reader andImmunoSpot software (Cellular Technologies, Shaker Heights, OH).

Cell staining

Bone marrow and spleen single-cell suspensions were stained for dead cellexclusion using Live/Dead fixable violet dead cell staining kit (InvitrogenLife Technologies, Grand Island, NY). Surface markers were stained witha mixture of fluorochrome-conjugated Abs, which included: CD19 (clone6D5; BioLegend, San Diego, CA), B220 (clone RA3-6B2; eBioscience,San Diego, CA), CD138 (clone 281-2; BD Biosciences, San Jose, CA),IgD (clone 11.26c.2a; BioLegend), IgM (clone II/41; eBioscience), IgG(eBioscience), MHC class II (clone 500A2), CD80 (clone I6-10A1; BDBiosciences) and CD86 (clone GL-1; BioLegend).

Fluorescence minus one controls were included in each staining protocoland were used to set specific gates (22, 26, 27). Six-peak validation beadswere used for calibration during the time course analysis. Samples were

run on a 12-color LSRII cytometer (BD Biosciences) and analyzed withFlowJo software (Tree Star, Ashland, OR).

B lymphocyte isolation and in vitro culture conditions

Mouse spleens were harvested, single-cell suspensions were prepared, andB cells were isolated using CD19 magnetic beads (Miltenyi, Auburn, CA).B cells (1 3 106 cells/ml, unless otherwise specified) were cultured inRPMI 1640 media (Invitrogen Life Technologies, Grand Island, NY)supplemented with 5% FBS (Thermo Fisher Scientific, Pittsburgh, PA), 50mM b-mercaptoethanol (Eastman Kodak, Rochester, NY), 10 mM HEPES(U.S. Biochemical, Cleveland, OH), 2 mM L-glutamine (Invitrogen LifeTechnologies, Carlsbad, CA), and 50 mg/ml gentamicin (Invitrogen LifeTechnologies, Grand Island, NY). For CD138+ B cell depletion, purifiedCD19+ B cells were stained with anti-CD138 (clone 281-2; BD Bio-sciences) Ab. Cells were then sorted using an 18-color FACS Aria II (BDBiosciences). Purified B cells were activated with CpG ODN 1826 (1 mg/ml) plus rabbit anti-mouse IgM Ab fragment (2 mg/ml; Jackson Immu-noResearch Laboratories, West Grove, PA) and cultured for up to 6 d (22).17-HDHA or vehicle control were added to cell culture 30 min beforemitogen stimulation. Because 17-HDHAwas suspended in ethanol, vehiclecontrol was defined as PBS with 0.03% ethanol by volume, equivalent to100 nM 17-HDHA. SPM treatments were added daily for the duration ofthe culture.

Proliferation analysis

Purified B cells (13 105 cells/ ml) were cultured in triplicate using 96-wellround-bottom plates for up to 6 d. B cell cultures were pulsed with [3H]-thymidine (1 mCi/well) 24 h prior to collection. [3H]-Thymidine incor-poration was measured by scintillation spectroscopy using a TopcountLuminometer (PerkinElmer, Boston, MA).

For CD138 depletion experiments, purified CD19+ CD1382 B cells (13106 cells/ml) were stained at day 0, using CellTrace CFSE Cell Prolifer-ation Kit (Invitrogen Life Technologies, Carlsbad, CA) as suggested by themanufacturer. CFSE staining was then monitored over time using a 12-color LSRII cytometer (BD Biosciences) and analyzed by FlowJo software(Tree Star, Ashland, OR).

Real-time PCR

Total RNAwas isolated using Qiagen RNeasy Mini Kit (Valencia, CA). RNAwas reversed transcribed using Superscript III and random primers (InvitrogenLife Technologies, Carlsbad, CA). Steady-state levels of Bcl-6, Blimp-1, and7S RNAwere measured by real-time PCR using iQ SYBR Green Supermix(Bio-Rad, Hercules, CA). Bcl-6 and Blimp-1 mRNA steady-state levels werenormalized to 7S. Primers used were: Bcl-6 sense 59-AGACGCACAGTG-ACAAACCATACAA-39, antisense 59-GCTCCACAAATGTTACAGCGAT-AGG-39; Blimp-1 sense 59-TTCTTGTGTGGTATTGTCGGGACTT-39, anti-sense 59-TTGGGGACACTCTTTGGGTAGAGTT-39, and 7S sense 59-AC-CACCAGGTTGCCTAAGGA-39, and antisense 59-CACGGGAGTTTTGA-CCTGCT-3 (22). Results were analyzed using Bio-Rad iCycler software(Hercules, CA).

Western blot

Protein was extracted from purified B cells using radioimmunoprecipita-tion assay buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% Na deoxycholate,50 mM Tris-base, 0.1% SDS [pH 8.0]). Protein was loaded onto gradientSDS-PAGE gels (Pierce/Thermo Fisher Scientific, Rockford, IL) and thentransferred onto PDVF membranes (Millipore). Membranes were probedwith Abs against Blimp-1 (clone NB600-235; Novus, Littleton, CO), Bcl-6(clone 4242; Cell Signaling Technologies, Danvers, MA), tubulin (Clone2146; Cell Signaling Technologies), and HRP-conjugated secondary Ab.ECL reagents (PerkinElmer Life Sciences, Boston, MA) was then used todevelop Western blots.

Virus neutralization assay

Madin-Darby canine kidney (MDCK) cells (ATCC CCL-34) were main-tained in DMEM (Mediatech) supplemented with 10% FBS (AtlantaBiologicals), and 1% P-S-G (penicillin, 100 U/ml; streptomycin, 100 mg/ml; L-glutamine, 2 mM; Mediatech). Cells were grown at 37˚C in a5% CO2 atmosphere. MDCK cells constitutively expressing HA frominfluenza H1N1 A/WSN/33 (WSN) or A/California_NYICE_E3/04/2009(pH1N1/E3) have been described previously (28). After viral infections,cells were maintained at 33˚C in a 5% CO2 atmosphere in DMEM con-taining 0.3% BSA, 1% P-S-G, and 0.3 or 1.0 mg/ml tosyl-sulfonyl phe-nylalanyl chloromethyl ketone–treated trypsin (Sigma-Aldrich) for WSNand MDCK cells, respectively.

2 SPM 17-HDHA ENHANCES THE HUMORAL RESPONSE IN VIVO

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The prototypic influenza pH1N1/E3 virus was prepared in eggs as de-scribed previously (25). Virus titers were determined by standard plaqueassay (plaque forming units [PFUs]) in MDCK cells as described (29).Single-cycle infectious influenza A virus (sciIAV) containing the pH1N1backbone (pH1N1/E3-sciIAV) was generated as previously published (30)and had the ORF of GFP instead of the fourth viral segment; it was amplifiedon MDCK cells constitutively expressing pH1N1/E3 HA. SciIAV titers weredetermined by plaque assay as above, but in MDCK-HA cells (28).

Virus-neutralizing Ab titers in mice sera were determined using a GFP-based microneutralization (28). Sera were heat-inactivated for 45 min at57˚C, and triplicates were serially diluted 2-fold in a 96-well plate. Twohundred PFUs of pH1N1/E3-sciIAV virus were then added to equivalentvolumes of sera dilutions and incubated for 1 h at room temperature.Confluent monolayers of MDCK WSN-HA cells (96-well plate format, 4 3104 cells) were then infected for 1 h at room temperature with virus–seramixture and incubated for 24 h at 33˚C. Cells were then washed twice withPBS and GFP was quantified for relative fluorescence units using a platereader (DTX-880; Beckman Coulter). Infection in the absence of sera wasused to normalize GFP expression to 100%. Neutralization titers representthe highest dilution at which the %GFP expression (6SD) was below 50%.

Viral replication assay

To assess the inhibition of influenza replication in vitro (31) monolayers ofMDCK cells (12-well plate format, 53 105 cells, triplicates) were infectedwith pH1N1/E3 at a multiplicity of infection of 0.001 for 48 h. Postin-fection media was uniformly supplemented with ,0.2% (v/v) ethanolalone (vehicle) or additionally with 5-fold serial dilutions of 17-HDHA oroseltamivir acid (Toronto Research Chemicals). Tissue culture super-natants were removed at 24 and 48 h postinfection, and viral titers cal-culated with immunofocus assay.

For antiviral analyses, viral titers were calculated by immunofocus assay(focus forming units per milliliter). Briefly, influenza NP positive cells fromserial supernatant dilutions were detected by indirect immunofluorescencewith 1 mg/ml of the primary Ab HT103 (a gift from Dr. Thomas M. Moranat Mount Sinai School of Medicine, New York, NY), and a secondary anti-mouse FITC (1:140; Dako) using a fluorescence microscope (DM IRB;Leica) as described previously (30).

Statistical analysis

Results are expressed as mean 6 SEM. Significance was determined bystatistical analysis using a two-tailed unpaired Student t test where ap-plicable, or one-way ANOVAwhen comparing three or more groups. Two-way ANOVA with a Bonferroni posttest was used where two or morevariables were analyzed. All tests were carried out using GraphPad Prism 5(GraphPad Software, La Jolla, CA). The p values # 0.05 were consideredstatistically significant.

ResultsSPM 17-HDHA enhances HA-specific Ab production

The in vivo effects of 17-HDHA on the humoral response are notknown. Therefore, an OVA immunization mouse model was initiallyused to study the effects of 17-HDHA under physiologic conditions.Mice were immunized with OVA protein plus 17-HDHA or vehiclecontrol. OVA-specific IgM and IgG titers in serum were measuredafter primary and secondary challenges (Fig. 1). OVA immunizationinduced a strong OVA-specific IgM and OVA-specific IgG levels.Mice that received 17-HDHA along with OVA had significantlyhigher OVA-specific IgM and IgG levels 6 wk after primary chal-lenge. OVA-specific IgM production was further increased aftersecondary OVA exposure, as measured at week 12. OVA-specificIgE was also measured in sera. Interestingly, 17-HDHA induceda decreasing trend in IgE levels (data not shown).Next, the effects of 17-HDHA were analyzed in a highly

translational preclinical influenza vaccination mouse model. Tothis end, recombinant hemagglutinin (HA) protein, derived fromH1N1 influenza virus, was used to elicit an Ag-specific humoral re-sponse. Mice were immunized and challenged with HA and CpGODN 1826 (a commonly used adjuvant) plus 17-HDHA or vehiclecontrol (Fig. 2A). HA-specific IgM and IgG serum levels weremeasured 3 wk after primary immunization (Fig. 2B, 2C) andagain after secondary challenge (Fig. 2D–G). In all cases, mice

that received immunizations containing 17-HDHA had signifi-cantly higher HA-specific IgG titers compared with the controlgroup. These results show that 17-HDHA enhances Ag-specificAb levels in both HA and OVA immunization mouse models.Therefore, the 17-HDHA-enhanced Ag-specific humoral responseis not limited to the HA protein.

17-HDHA enhances HA-specific Ab production and plasmacell differentiation

In the absence of adjuvants, recombinant HA induces a poor humoralresponse (24). Current seasonal influenza vaccines (IIV, LAIV, orRIV) do not use adjuvants (4, 5). Considering that 17-HDHA in-creased Ag-specific Ab levels (Figs. 1, 2), we asked whether 17-HDHA could increase the HA-specific humoral response without theuse of an adjuvant, such as CpG ODN 1826. Based on the dose-dependent HA-specific Ab titers from 17-HDHA immunized ani-mals (data not shown), mice were injected with PBS (mock) or givenimmunizations containing recombinant HA plus 1 mg 17-HDHA orHA without 17-HDHA (vehicle control; Fig. 3). After primary im-munization, vehicle-treated mice had no significant differences inthe HA-specific IgM and IgG titers compared with mock-immunizedmice. Interestingly, 17-HDHA-treated mice had a 2-fold increase inHA-specific IgM levels compared with the mock-treated group anda 9-fold increase in HA-specific IgG compared with mock- andvehicle-treated groups (Fig. 3B, 3C, respectively).Ag-experienced B cells undergo somatic hypermutation and

class switch recombination, which promotes Ag-specific Ab affinitymaturation and isotype switching and improves the immune responseupon Ag re-encounter (32, 33). Therefore, we measured the Abresponse following Ag challenge (Fig. 3D–G). HA-specific IgMlevels did not change between vehicle- and 17-HDHA–treatedgroups. However, HA-IgG titers were 100-fold higher comparedwith HA-IgM levels, reflective of Ab isotype switching. Remark-ably, mice immunized with HA plus 17-HDHA had a 3-fold in-

FIGURE 1. 17-HDHA increases OVA-specific Ab production. C57BL/6

mice were immunized i.p. at week 0 and at week 10 with OVA plus vehicle

control (vehicle) or with OVA plus 1 mg 17-HDHA (17-HDHA). Sera were

collected at weeks 2, 6, and 12. OVA-specific (A) IgM and (B) IgG titers

were measured by ELISA. Here is shown mean 6 SEM (n = 6/group).

Statistical analysis was done using two-way ANOVA. *p # 0.05.

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crease in HA-specific IgG titers by week 4, and a 9-fold increase byweek 6 compared with vehicle control group (Fig. 3E, 3G). Total

Ab levels were not different between vehicle- and 17-HDHA–

immunized groups (data not shown).Activated B cells can differentiate into plasma cells, which are

responsible for Ab production (34, 35). Long-lived plasma cells

reside mainly in the bone marrow, where they confer long-term

immune protection (36, 37). 17-HDHA promotes human B cell

differentiation toward an Ab-secreting cell in vitro (13). Thus, the

effects of 17-HDHA on B cell differentiation were analyzed using

the influenza immunization mouse model (Fig. 4). Two weeks

after completion of the HA-immunization protocol, B cell pop-

ulations were analyzed in the spleen and bone marrow by flow

cytometry, and CD138 expression was used to identify the plasma

cell population (Fig. 4A–C). Mice immunized with HA plus

FIGURE 2. 17-HDHA increases HA-specific IgG production. C57BL/6

mice were immunized i.m. with A/Brisbane/59/2007 recombinant HA plus CpG

plus vehicle control (CpG + Vehicle) or with A/Brisbane/59/2007 recombinant

HA plus CpG and 1 mg 17-HDHA (CpG + 17-HDHA), and sera was collected

as represented in (A) immunization schematic. HA-specific IgM and HA-spe-

cific IgG Ab levels were measured by ELISA at (B and C) week 3, (D and E)

week 6, and (F and G) week 11 (n = 6/group). Data are shown as mean6 SEM.

Statistical analysis was done using unpaired Student t test. *p # 0.05.

FIGURE 3. 17-HDHA enhances HA-specific Ab production without the

use of classic adjuvants. (A) C57BL/6 mice were immunized i.m. at weeks 0,

2, and 4 with PBS (mock), A/California/04/2009 recombinant HA plus ve-

hicle control (vehicle) or with A/California/04/2009 recombinant HA plus

1 mg 17-HDHA (17-HDHA). HA-specific IgM and HA-specific IgG Ab levels

were measured by ELISA at (B and C) week 2, (D and E) week 4, and (F and

G) week 6 (n = 10/group). Data shown as mean 6 SEM. Statistical analysis

was done using one-way ANOVA. *p # 0.05, **p # 0.01, ***p # 0.001.

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17-HDHA had a 2-fold increase in the percentage of CD138+

B cells present in the bone marrow compared with vehicle controlgroup (Fig. 4A, 4B). Furthermore, the distribution of CD138+

B cells in the spleen showed no differences (Fig. 4C).Next, the number of HA-specific Ab-secreting cells present in

the bone marrow was measured (Fig. 4D). ELISPOT analysisshowed that HA-specific IgG secreting cells were increased 2-foldin the 17-HDHA–treated group compared with mock and vehiclecontrols, consistent with flow cytometry results. The vehiclecontrol group had low numbers of HA-specific Ab-secreting cells,comparable to those of the mock-immunized control. Further-more, HA-specific IgM secreting cells were not detectable. To ourknowledge, these results are the first to show that 17-HDHAstrongly enhances the HA-specific Ab response, in part by in-creasing plasma cell differentiation in vivo. Thus, 17-HDHA hasadjuvant-like properties.

17-HDHA–mediated HA-specific Abs are protective againstlive influenza infection

Achieving an efficacious number of plasma cells and protectiveAbs are crucial elements for an effective antiviral immune responseand are important goals in vaccine development (38, 39). We testedwhether the 17-HDHA–mediated Ab increase also enhanced long-term immune protection against influenza viral infection (Fig. 5).

To this end, mice were injected with PBS (mock) or immunizedwith recombinant HA plus 17-HDHA or vehicle control at weeks0, 2, and 4 (see immunization schematic, Fig. 3A). Sera werecollected at week 6, and the neutralizing properties of the HA-specific Abs were tested in vitro (Fig. 5A). Results showed that44% of mice immunized with HA plus 17-HDHA had neutralizingAbs circulating in serum compared with 0% for both mock andvehicle controls, although one of the nine mock-immunized micedisplayed low levels of nonspecific neutralization (,1:20 seradilution).Next, the protective qualities of the 17-HDHA–mediated Ab

response were tested in vivo. Twenty-eight days after the lastimmunization, mice were infected with the mouse adapted liveinfluenza A/California/04/E3/2009 H1N1 virus as previously de-scribed (25). Weight and survival were monitored for 14 d fol-lowing influenza infection (Fig. 5B, 5C). The mock-immunizedgroup, which received no HA, had rapid weight loss and a 30%survival rate. By comparison, 17-HDHA–treated mice had mini-mal weight changes compared with the dramatic weight loss seenin both mock and vehicle-treated mice (Fig. 5B). Furthermore, 17-HDHA and vehicle control groups had significantly higher sur-vival rates of 100% and 80%, respectively (Fig. 5C).A recent report indicated that the SPM protectin D1 exhibited

direct antiviral activity against influenza Avirus (40). However, we

FIGURE 4. 17-HDHA enhances

plasma cell frequency in the bone mar-

row. C57BL/6 mice were immunized

i.m. with PBS (mock), A/California/

04/2009 HA plus vehicle control (ve-

hicle), or A/California/04/2009 HA

plus 17-HDHA (17-HDHA). Six weeks

later, bone marrow and spleen were

collected. Cells were isolated and

stained for flow cytometric analysis

(n = 10/group). (A) Gating strategy

(left panels) and representative histo-

gram of CD138 expression on CD19+

bone marrow B cells (right panel).

Quantification of (B) bone marrow

plasma cells (CD19+CD138+) and (C)

spleen plasma cells (CD19+CD138+).

(D) Quantification of HA-specific IgG

secreting bone marrow cells. Analysis

was done with ELISPOT (n = 6/groups).

Data are shown as mean 6 SEM. Sta-

tistical analysis was done using one-way

ANOVA. *p # 0.05, **p # 0.01.

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found that 17-HDHA, which is produced by the same biosyntheticpathway (10), did not inhibit the accumulation of live influenzavirus in tissue culture supernatants, unlike oseltamivir, a well-described antiviral medication (Fig. 5D). Overall, these resultsshow that the 17-HDHA–mediated Ab-increase is functional andprotects against live influenza virus infection, thus holding greatpotential as a vaccine adjuvant.

17-HDHA directly promotes B cell differentiation toward anAb-secreting phenotype

To interrogate the mechanisms by which 17-HDHA enhances thehumoral immune response in vivo, the effects of 17-HDHA weretested on purified B cells. Following B cell activation, the ex-pression of CD80, CD86, and MHC class II are upregulated on theB cell surface, because these activation markers are involved inAg presentation and the development of germinal center reac-tion within secondary lymphoid organs (41–44). Interestingly,17-HDHA strongly upregulated CD80 and CD86 expression onB cells 6 d after CpG plus anti-IgM stimulation (Fig. 6A, 6B).However, 17-HDHA did not affect MHC class II expression seenat day 6 after activation in this system (Fig. 6C). 17-HDHA wasnot cytotoxic, nor did it enhance viability of B cells (data notshown). Of interest, flow cytometric analysis of the draininglymph nodes of mice challenged with either HA plus 17-HDHA or

vehicle control showed no detectable changes above backgroundin the frequency of GC B cells (CD19+ B220+ GL7+ CD95+) andTfh cells (CD3+ CD4+ CXCR5+ PD-1+), at 6 or 10 d after chal-lenge, potentially because of a low response against HA (data notshown).Activated B cells can further differentiate into Ab-secreting cells

or plasma cells. Therefore, the effects of 17-HDHA onmouse B celldifferentiation were analyzed (Fig. 7). Activated B cells treatedwith 17-HDHA nearly doubled IgM and IgG production comparedwith vehicle controls (Fig. 7A, 7B). Blimp-1 mRNA and proteinlevels were also increased by a factor of three in 17-HDHA–treated B cells (Fig. 7B, 7C). Blimp-1 is the master regulator ofB cell differentiation toward a plasma cell phenotype (45, 46).Therefore, to determine whether the increased Ab levels were dueto enhanced Ab-secreting B cell differentiation, or due to a directeffect of 17-HDHA on existing plasma cells, CD138+ B cells weresorted out of purified CD19+ B cells. The remaining CD19+

CD1382 B cells were pretreated with 17-HDHA and then stim-ulated with CpG plus anti-IgM (Fig. 7D, 7H). 17-HDHA induceda 2-fold increase in the number of IgM- and IgG-secreting cells(Fig. 7D). No changes were seen in the spot size between thevehicle and 17-HDHA–treated samples, suggesting that 17-HDHAdoes not affect the amount of Ab produced per cell (data notshown). In addition, flow cytometric analysis showed that 17-HDHA

FIGURE 5. 17-HDHA–mediated HA-specific Abs are protective against live influenza infection. C57BL/6 mice were immunized i.m. with PBS (mock),

A/California/04/2009 HA plus vehicle control (vehicle), or A/California/04/2009 HA plus 17-HDHA (17-HDHA). (A) Neutralizing Ab titers in mice.

Serum from mice collected 14 d after final vaccination was used in a GFP microneutralization assay. The neutralization titer was determined as the highest

dilution providing .50% reduction in GFP expression from triplicate wells. Columns represent the geometric mean neutralization titer from nine mice. A

dotted line represents the limit of detection (,20) for Ag-specific Abs. Negative samples were arbitrarily assigned a value of 10. The number of mice with

a microneutralization titer equal to or greater than 40 is shown in parentheses. (B and C) Twenty-eight days after the last immunization, mice were infected

intranasally with 300 PFUs of H1N1 influenza virus (A/California/04/E3/2009; n = 10/group). (B) Weight and (C) survival were monitored daily for 14 d

following infection. Results are expressed as mean 6 SEM. For weight loss analysis, statistical analysis was performed by calculating the area under

the curve followed by a x2 test (***p # 0.001). Survival rates were analyzed by doing a log-rank (Mantel–Cox) test (*p # 0.05, **p # 0.01). (D)

Direct antiviral activity of 17-HDHA. MDCK cells were infected with pH1N1/E3 at a multiplicity of infection of 0.001 for 48 h in the presence of 5-

fold serial dilutions of 17-HDHA, oseltamivir, or vehicle control (,0.2% v/v; dotted line). Virus titers in triplicate wells were determined by

immunofocus assay. Results are expressed as mean 6 SEM. Significance was determined against vehicle using unpaired two-tailed Student t test.

***p # 0.001, **p # 0.01.

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increased the frequency of plasmablasts (IgD2IgM2IgG2; Fig.7E). These results show that 17-HDHA increases Ab productionby promoting B cell differentiation toward an Ab-secreting B cellphenotype.SPMs have been shown to affect cytokine production on different

immune cells, which can help to resolve inflammation whileavoiding immune suppression (19–21). Therefore, the effects of17-HDHA on CD19+CD1382 B cell cytokine production wereanalyzed. Interestingly, we discovered that 17-HDHA increasedproduction of IL-10. However, 17-HDHA did not affect IL-6 orTNF-a production (Fig. 7F). The increase in IL-10 is suggestiveof a proresolution cytokine profile, while maintaining IL-6 andTNF-a, which are important in B cell differentiation and survival(47, 48). Lastly, 17-HDHA did not affect B cell proliferation(Fig. 7G), nor was it cytotoxic (data not shown). Overall, thesenew findings show that 17-HDHA increased the humoral response,in part through promotion of B cell differentiation toward an Ab-secreting phenotype.

DiscussionWe report the novel finding that 17-HDHA strongly enhances theAg-specific Ab response in a preclinical influenza immunizationmouse model. Mice vaccinated with HA plus 17-HDHA had anenhanced Ab-mediated immune response, which was protectiveagainst live influenza virus infection. To our knowledge, this is thefirst report on the in vivo effects of DHA-derived lipid mediatorson the adaptive immune response, which provides a link betweenproresolution pathways and adaptive immunity.Long-lived plasma cells have a transient state in circulation

before they establish permanent residence in the bone marrow.Results showed an increase in the number of bone marrow CD138+

B cells and HA-specific Ab secreting cells in mice that received17-HDHA. Although the number of HA-specific Ab-secretingB cells and CD138+ cells could not be determined simulta-neously, 17-HDHA induced a 2-fold increase in the percentage ofCD138+ B cells, and a 2-fold increase in the number of HA-specific Ab secreting cells. These results suggest that the in-crease in HA-specific serum Ab levels is due to enhanced plasmacell frequency. Furthermore, no changes were observed in thenumber of splenic CD138+ or HA Ab-secreting cells at week 6, anexpected observation given the transient nature of plasma cells.Furthermore, in vitro analysis revealed that 17-HDHA directlyincreased Blimp-1 expression and B cell differentiation toward anAb-secreting cell phenotype. Considering the direct effects of 17-HDHA on B cells in vitro, it is possible that during the humoralresponse, 17-HDHA and other immunostimulatory SPMs promoteB cell activation and differentiation. These new results highlight

the potential use of 17-HDHA and other immunostimulatorySPMs as a vaccine adjuvant, as it strongly promotes Ag-specificAb production and plasma cell differentiation.The generation of long-lived plasma cells during an immune

response is fundamental for strong and long-term protection, whichis a highly desired outcome during vaccination. Furthermore, theduration of immune memory protection is also an importantmeasure of a vaccine’s efficacy. In this study, vaccinated micewere infected with influenza 28 d after the last immunization.Infections at later time points have not been tested. Future workshould analyze the actions of 17-HDHA using longer vaccinationprotocols to assess the long-term protective properties of the 17-HDHA-mediated humoral response.Taking advantage of the preclinical influenza vaccination mouse

model, the efficacy of the HA vaccination was tested by challengingmice with influenza virus. An important and exciting finding is thatmice vaccinated with HA plus 17-HDHA had minimal weight lossand complete survival as compared with vehicle and mock vac-cinated control groups. Vaccinating mice with recombinant HAalone generated a protective immune response (80% survival rate)that has been reported previously (24), although it did not producedetectable neutralizing Ab levels. Notably, 17-HDHA–vaccinatedmice had a 100% survival rate. No statistical significance was seenin the survival rate of vehicle control versus 17-HDHA–vacci-nated animals, both of which received recombinant HA. It ispossible that infection with a higher viral titer would further dif-ferentiate the level of immune protection between 17-HDHA andvehicle control group. Remarkably, 17-HDHA–vaccinated mice,unlike the vehicle control, had significantly decreased morbidity,as shown by minimal weight loss. This discovery is of greatimportance because it demonstrates that 17-HDHA–mediatedhumoral response is functional and provides the host with agreater level of protection against influenza infection. Therefore,17-HDHA possesses magnificent potential as a novel adjuvantcandidate.17-HDHA enhanced the humoral response in OVA and HA

immunization models. These results show that the 17-HDHA–mediated enhanced humoral response is not unique to the im-munization model or Ag used. An interesting finding is the abilityof 17-HDHA to increase Ag-specific IgG in both the OVA andHA models while also inducing a decreasing trend in IgE levels.Whether 17-HDHA specifically regulates B cell isotype switchingremains to be addressed.Others have reported on the direct antiviral properties of SPMs,

including protectin D1 (40, 49, 50). 17-HDHA, although highlybioactive, can also be biosynthesized into D-series resolvins andonly its 17-HpDHA precursor is converted to PD1 (51, 52). The

FIGURE 6. 17-HDHA increases B cell expression of CD80 and CD86, but not MHC class II. CD19+ B cells were isolated from the spleens of naive

C57BL/6 mice (n = 6). Purified B cells were cultured and stimulated with CpG 1826 ODN (1 mg/ml) plus anti-IgM (2 mg/ml). Cells were treated with 17-

HDHA or vehicle control for 30 min prior to B cell activation, and treatments were repeated daily for 6 d. Cells were collected and stained for (A) CD80,

(B) CD86, and (C) MHC class II and analyzed by flow cytometry at day 6. Results showed mean fluorescence intensity (MFI) 6 SEM. Statistical analysis

was performed using a one-way ANOVA with a Bonferroni posttest. *p # 0.05, **p # 0.01, ***p # 0.001.

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present results showed that 17-HDHA alone did not inhibit H1N1viral replication. It is possible that in vivo 17-HDHA is convertedinto other SPMs, which in turn could have antiviral properties;however, this does not detract from the increased Ab titers un-covered herein. More importantly, for the focus of our study, the17-HDHA–mediated increase in Ab levels was found to havevirus-neutralizing properties, and it remarkably conferred strongprotection against live influenza virus infection. In view of thedirect antiviral actions of PD1, our findings suggest that activationof the DHA metabolome pathways is critical to novel and previ-ously unappreciated viral host defense pathways.SPMs are endogenous mediators found in tissues such as the

bone marrow, spleen, tonsils, and blood (11–13, 53–55). Thesetissues provide an ideal environment under which SPMs can di-rectly influence immune cells, such as B cells. In this study, wediscovered that 17-HDHA increases B cell Ab production,increases Blimp-1 expression, and promotes B cell differentiation

toward an Ab-secreting cell. In addition, molecular analysisrevealed that 17-HDHA upregulates CD80 and CD86 expressionon B cells. These costimulatory molecules are upregulated fol-lowing B cell activation and play a crucial role during Ag pre-sentation and the germinal center reaction (41–43). The 17-HDHA–mediated increase in costimulatory molecules couldincrease the immunologic synapse signaling and with it the T cell–dependent immune response. Overall, the evidence presented inthis study shows that 17-HDHA enhances the humoral responsein part by upregulation of B cell activation and differentiationmarkers.Interestingly, 17-HDHA did not affect the levels of TNF-a or

IL-6. Although these two cytokines are generally consideredproinflammatory, they are involved in early B cell activation,proliferation, and Ab production (47, 48). In addition, 17-HDHAincreased IL-10 production, which has anti-inflammatory func-tions. The increase of IL-10 raises questions regarding the effects

FIGURE 7. 17-HDHA increases B cell Ab production and promotes B cell differentiation toward an Ab-secreting B cell phenotype. (A–C) CD19+ B cells

were isolated from the spleens of naive C57BL/6 mice (n = 6). Purified B cells were cultured in triplicate and stimulated with CpG 1826 ODN (1 mg/ml)

plus anti-IgM (2 mg/ml). Cells were treated with 17-HDHA at 10 nM and 100 nM or with vehicle control for 30 min prior to B cell activation. 17-HDHA or

vehicle control treatments were added daily for up to 6 d. Culture supernatants were collected, and (A) IgM and IgG were measured by ELISA. (B) Blimp-1

steady levels were measured by real-time quantitative PCR and normalized to 7S expression. (C) Blimp-1 protein levels were measured by Western blot,

representative blot images (left panel) and densitometry (right panel) are shown. (D–G) Purified CD19+ B cells isolated from naive mouse spleens were

depleted of plasma cells (CD19+CD138+) via FACS (n = 6). CD19+CD1382 B cells were then stimulated with CpG 1826 ODN plus anti-IgM. (D) IgM and

IgG-secreting cells were quantified after 5 d of activation using ELISPOT analysis. (E) Plasmablasts (IgD2IgM2IgG2) differentiation was measured by

flow cytometry after 6 d of activation. (F) Supernatants were collected from 6-d activated cultures and IL-10, IL-6, and TNF-a levels were measured with

ELISA. (G) The percentage of dividing cells was measured using CFSE tracing and flow cytometric analysis. Representative histogram (left panel) and

quantification of dividing cells (right panel) are shown. Results are shown as mean 6 SEM. Statistical analysis was performed using a one-way ANOVA

with a Bonferroni posttest. *p # 0.05, **p # 0.01.

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of 17-HDHA on IL-10–producing B cells, as well as the effects of17-HDHA on other B cell subsets (56). Whether, the increase inIL-10 and maintenance of IL-6 and TNF-a by 17-HDHA takesplace within the B cell microenvironment in vivo remains to beaddressed. Importantly, the B cell cytokine profile induced by 17-HDHA does not increase or decrease proinflammatory signals, butit does enhance anti-inflammatory signals while still promotingAb production.Alum is the only adjuvant approved by the U.S. Food and Drug

Administration and currently in routine use in the United States,with modest success. Current influenza vaccines do not even in-clude adjuvants. New adjuvants are needed to improve efficacy ofmany types of vaccines. This is particularly critical for susceptiblepopulations such as older adults, immunosuppressed patients, andinfants. We propose that SPM 17-HDHA is a new class of adjuvant.Our results show that 17-HDHA directly promotes B cell differ-entiation toward an Ab-secreting phenotype in vitro and in vivo.Whether the 17-HDHA-mediated effects are due to specific re-ceptor binding or further conversion of 17-HDHA into D-seriesresolvins is not known. It is also possible that 17-HDHA directlyaffects immune cell functions, such as APC Ag-processing capa-bilities. Determining the in vivo bioavailability of exogenous 17-HDHA in the local environment is critical yet challenging. Onedifficulty lays on the properties of 17-HDHA to undergo enzy-matic conversion rapidly into D-series resolvins (15, 57). In turn,resolvins can signal cells or can be inactivated enzymatically byoxidoreductases (reviewed in Ref. 52). Understanding how 17-HDHA and its derivatives enhance the adaptive immune responseis of great interest for the development of novel and more efficientadjuvants, and it clearly guarantees future studies.The present results provide new evidence of the immunosti-

mulatory properties of DHA-derived SPMs on the adaptive im-mune response, particularly on B cells. Overall, 17-HDHA hasgreat potential as an adjuvant considering that it is a naturallyoccurring small molecule, is not cytotoxic, and has a low pro-duction cost. Using SPMs to treat chronic inflammatory diseaseshas proved beneficial in models of periodontitis, asthma, arthritis,and cancer (58–63). Further study of the mechanisms regulatingthe resolution of inflammation and how they are linked to adaptiveimmunity has important implications for the understanding ofB cell biology, vaccine development, and the potential generationof a new class of adjuvant.

DisclosuresC.N.S. is an inventor on patents (resolvins) assigned to Brigham and

Women’s Hospital and licensed to Resolvyx Pharmaceuticals. C.N.S. is

a scientific founder of Resolvyx Pharmaceuticals and owns equity in the

company. C.N.S.’s interests were reviewed and are managed by Brigham

and Women’s Hospital and Partners HealthCare in accordance with their

conflict-of-interest policies. All other authors have no financial conflicts of

interest.

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