Environmental enrichment alters splenic immune cell composition and enhances secondary influenza...

INTRODUCTIONStress can be defined as a relationship

between a person and his or her environ-

ment that is appraised as being taxing orexceeding the ability to cope (1). The per-ception of a stressor activates a cascade

of physiological responses in the bodyincluding the sympathetic-adrenal- medullary and hypothalamic–pituitary–adrenal (HPA) axes, culminating in sys-temic release of catecholamines and glu-cocorticoids, respectively. Cells of the im-mune system express receptors for thesestress mediators and are susceptible toparacrine immunomodulation as well assignaling via direct innervation of lym-phoid organs (2).

Accumulating evidence suggests thatacute stressors (those lasting minutes tohours) can augment immune responses.

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Environmental Enrichment Alters Splenic Immune CellComposition and Enhances Secondary Influenza VaccineResponses in Mice

Blake T Gurfein,1,2 Olga Davidenko,3,4 Mary Premenko-Lanier,2 Jeffrey M Milush,2 Michael Acree,1

Mary F Dallman,5 Chadi Touma,6 Rupert Palme,7 Vanessa A York,2 Gilles Fromentin,4 Nicolas Darcel,3,4*

Douglas F Nixon,2* and Frederick M Hecht1*

1Osher Center for Integrative Medicine, University of California, San Francisco, California, United States of America; 2Division ofExperimental Medicine, University of California, San Francisco, California, United States of America; 3Chaire ANCA, Food Science,Nutrition and Eating Behavior, AgroParisTech, Paris, France; 4INRA, Unit 914 Nutrition Physiology and Ingestive Behavior,AgroParisTech, Paris, France; 5Department of Physiology, University of California, San Francisco, California, United States ofAmerica; 6Research Group of Psychoneuroendocrinology, Max Planck Institute of Psychiatry, Munich, Germany; and 7Department ofBiomedical Sciences/Biochemistry, University of Veterinary Medicine, Vienna, Austria

Chronic stress has deleterious effects on immune function, which can lead to adverse health outcomes. However, studies in-vestigating the impact of stress reduction interventions on immunity in clinical research have yielded divergent results, potentiallystemming from differences in study design and genetic heterogeneity, among other clinical research challenges. To test the hy-pothesis that reducing glucocorticoid levels enhances certain immune functions, we administered influenza vaccine once(prime) or twice (boost) to mice housed in either standard control caging or environmental enrichment (EE) caging. We haveshown that this approach reduces mouse corticosterone production. Compared with controls, EE mice had significantly lower lev-els of fecal corticosterone metabolites (FCMs) and increased splenic B and T lymphocyte numbers. Corticosterone levels werenegatively associated with the numbers of CD19+ (r2 = 0.43, p = 0.0017), CD4+ (r2 = 0.28, p = 0.0154) and CD8+ cells (r2 = 0.20, p =0.0503). Vaccinated mice showed nonsignificant differences in immunoglobulin G (IgG) titer between caging groups, although EEmice tended to exhibit larger increases in titer from prime to boost than controls; the interaction between the caging group (con-trol versus EE) and vaccine group (prime versus boost) showed a strong statistical trend (cage-group*vaccine-group, F = 4.27, p = 0.0555), suggesting that there may be distinct effects of EE caging on primary versus secondary IgG vaccine responses. Vaccine-stimulated splenocytes from boosted EE mice had a significantly greater frequency of interleukin 5 (IL-5)-secreting cellsthan boosted controls (mean difference 7.7, IL-5 spot-forming units/106 splenocytes, 95% confidence interval 0.24–135.1, p =0.0493) and showed a greater increase in the frequency of IL-5–secreting cells from prime to boost. Our results suggest that cor-ticosterone reduction via EE caging was associated with enhanced secondary vaccine responses, but had little effect on primaryresponses in mice. These findings help identify differences in primary and secondary vaccine responses in relationship to stress me-diators that may be relevant in clinical studies.Online address: http://www.molmed.orgdoi: 10.2119/molmed.2013.00158

*ND, DFN, and FMH are co–senior authors.

Address correspondence to Blake T Gurfein, 1001 Potrero Avenue, Building 3, Room 609,

UCSF Division of Experimental Medicine, San Francisco, CA 94110. Phone: 415-206-4981;

Fax: 415-206-5581; E-mail: [email protected].

Submitted December 4, 2013; Accepted for publication March 27, 2014; Epub

(www.molmed.org) ahead of print March 27, 2014.

Studies in rodents and humans havedemonstrated that acute stress near thetime of antigen exposure can amplify de-layed-type hypersensitivity (DTH) reac-tions, increase natural killer cell lytic ac-tivity and enhance antibody production inresponse to vaccination (3–5). In contrast,chronic stressors (those lasting weeks toyears) are associated with dysregulationof the HPA axis, impaired innate andadaptive immune responses, and adversehealth outcomes. For example, chronicallystressed rodents exhibit increased lym-phocyte apoptosis, suppressed DTH re-sponses and accelerated tumor growth(5–7). Human studies have demonstratedthat chronically stressed populations ex-hibit a variety of immune impairments,such as diminished wound healing andincreased susceptibility to upper respira-tory infection (8–10). Vaccination has be-come an important model for studyingthe impact of stress on immune function.Studies have shown that chronicallystressed individuals mount weaker re-sponses to influenza, hepatitis B andpneumococcal vaccines, compared withmatched control subjects, and repeatedactivation of the stress response and expo-sure to stress mediators are thought to berelated to the observed immune dysfunc-tion (11–13). Interestingly, these studies, aswell as others, have reported a negativeassociation between psychological stresslevel and performance in a variety of testsof immune function (14,15).

Although prior data reporting an associ-ation between stress and immune re-sponses to vaccination in humans havecome from observational studies, severalstudies have reported the effects of stressreduction interventions on vaccine re-sponses. A key intervention that has beentested is mindfulness-based stress reduc-tion (MBSR), a standardized meditation-based intervention that has becomewidely used in hospitals, schools andcommunity settings internationally. MBSRhas been shown to reduce anxiety and de-pression, improve mood and may influ-ence HPA axis activity (16–21). Given thenegative relationship between chronicpsychological stress and indices of im-

mune function, several groups have hy-pothesized that stress reduction interven-tions may enhance responses to an im-mune challenge, in part, due to reducedexposure to neuroendocrine stress media-tors. To date, reports from three random-ized controlled trials have been publishedin which vaccination has been used tocompare antigen-specific immune re-sponses in MBSR-trained subjects andcontrols (22–24). Of the three studies, thefirst reported that MBSR-trained subjectshad greater antigen-specific antibodytiters, the second reported no differencebetween MBSR-trained subjects and con-trols and the third reported that MBSR-trained subjects had significantly lowerantibody titers than controls. These contra-dictory findings may stem from differencein study design such as choice of immuno-gen, sampling time points, subject agerange, how the intervention was deliveredor statistical power. Additionally, inherentchallenges of human research, such as ge-netic heterogeneity, unforeseen life eventsand variability in previous exposure to theimmunogen, may add to the complexityof identifying relationships between stressreduction and vaccine responses.

In previous work, we described a stan-dardized approach to environmental en-richment (EE) in which we housedBALB/c mice, a mouse strain known to berelatively anxious, in caging containing atargeted collection of enrichments, to re-duce corticosterone production (25). EEcages are larger and contain shreddednesting material, a cardboard nest box anda polycarbonate tube, which allow themice to create a customized habitat includ-ing the construction of a three- dimensional nest. Mice housed in stan-dard cages did not have access to anyenrichment materials. We showed that EE-caged mice exhibited significant reduc-tions in corticosterone excretion and in-creased body mass when compared withmice housed in standard control caging,although no differences in elevated-plus-maze behavior were observed. Addition-ally, we found that EE-caged mice hadgreater spleen mass than controls, whichled us to hypothesize that EE-caged mice

may have altered spleen composition andenhanced response to an immune chal-lenge and that these results may be relatedto reduced glucocorticoid signaling.

Given the incongruent findings inhuman studies of MBSR and vaccine re-sponses, we reasoned that conducting arodent vaccination study, focused onmodeling reduced HPA axis activity,would provide insights into the relation-ship between glucocorticoid signaling andimmune function. Such a study designwould also be useful for shaping futureimmunologically driven studies of mind-body interventions. In the current study,we investigated whether control- and EE-caged mice exhibited differences in spleencomposition and/or humoral and cellularimmune responses to influenza vaccina-tion. Through studying genetically identi-cal inbred mice, with a known immunogenexposure history, and housing the animalsin a controlled environment, we were ableto circumvent some of the human researchchallenges described above.

MATERIALS AND METHODS

AnimalsSix-week-old male BALB/c mice were

purchased from The Jackson Laboratoryand housed in the Laboratory Animal Re-source Center at San Francisco GeneralHospital. Cages were kept in a temperature-controlled room (22°C) witha light–dark 12:12 cycle (lights on0600–1800 h). All cages were providedwith water and PicoLab Rodent Diet 20(LabDiet, PMI Nutrition International [seehttp://www.labdiet.com/ [LabDiet, St.Louis, MO, USA]) ad libitum. All studieswere approved by the University of Cali-fornia San Francisco Institutional AnimalCare and Use Committee and were con-ducted in accordance with national guide-lines of humane laboratory animal care.

Caging and EnrichmentMice were randomly assigned to control

or EE caging (n = 20 per group). For bothgroups, each cage housed five animals(four cages per group). All caging, beddingand enrichment items were autoclaved

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before use or sterilized with Coverage dis-infectant spray (STERIS Corporation,Mentor, OH). Standard wire-bar lids forfood and water and filter-top bonnets wereused for all cages. Cages for the controlgroup (189 × 297 × 128 mm; 484 cm2 sur-face area; Allentown Inc., Allentown, NJ,USA) contained standard Paperchip bed-ding (Shepherd Specialty Papers,http://www.ssponline. com/ index. htm)and lacked enhancements. Cages for theEE group were larger (257 × 483 × 152 mm; 980 cm2 surface area) and con-tained standard bedding, one paper nestbox (Bio-Serv, Frenchtown, NJ, USA), onered polycarbonate mouse tunnel (Bio-Serv)and 236 cm3 (1 cup) of compressed Enviro-dri Eco-bedding shredded paper strips(FiberCore, Cleveland, OH, USA). Onceweekly, cages and washable enrichmentitems were cleaned, and bedding, Enviro-dri strips and nest boxes were replaced.

Influenza Vaccination TimelineFigure 1 summarizes the cage enrich-

ment and vaccination timeline carriedout in these studies. After arrival fromthe vendor, all mice were group-housed(five animals per cage) in standard con-trol caging for a period of 14 d (d –14 to 0) to allow them to acclimatize to theLaboratory Animal Resource Centerhusbandry environment. Body mass andfecal corticosterone measurements

began 1 wk before the onset of experi-mental caging (d –7 onward). After ac-climatization, animals were placed intheir respective caging conditions andremained there for an additional periodof either 56 or 63 d. Animals wereweighed during weekly cage cleaning.Additionally, fecal samples were takenfrom each animal at regular intervalsand kept at –20°C for batched fecal cor-ticosterone metabolite analysis (seebelow). Inactivated Fluarix trivalent in-fluenza vaccine (GlaxoSmithKline,Brentford, Middlesex, UK) was dilutedin sterile phosphate-buffered saline(PBS). The vaccine contained equalquantities of hemagglutinin (HA) fromthe following strains: A/California/7/2009 (H1N1), A/Perth/16/2009 (H3N2),B/Brisbane/ 60/2008. Mice were placedunder light isoflurane anesthesia and in-jected intramuscularly in the righthindlimb with dilute vaccine (1 μg HAin 50 μL) or vehicle (50 μL PBS). Micewere injected with vaccine once (prime,n = 5 per caging group) or twice (boost,n = 5 per caging group) or were injectedwith PBS alone (naive, n = 10 per caginggroup). Primed mice were vaccinatedafter 4 wks of experimental caging andeuthanized 4 wks after injection.Boosted mice received the first injection4 wks after the onset of experimentalcaging and the second injection 4 wks

after the first injection, and were eutha-nized 1 wk after boost.

Fecal Corticosterone MetabolitesDuring cage changing, animals were

temporarily placed in individual con-tainers, and fecal pellets produced atthis time were collected and stored at–20°C. Weekly samples were taken fromeach animal at the same time of day for7 wks. Glucocorticoid metabolite quan-tification in fecal samples has been ex-tensively validated for laboratory mice(26–29). Collected samples were ana-lyzed for immunoreactive fecal corticos-terone metabolite (FCMs) by using a 5α- pregnane-3β,11β,21-triol-20-one en-zyme immunoassay (EIA) as describedpreviously (27). Samples were run inone batch to enhance data measure con-sistency. The intraassay coefficient ofvariation was 8.8%. Area under thecurve was determined by using thetrapezoidal rule. The 7-wk mean FCMwas calculated by averaging the weeklyFCM data for each animal beginning atthe first measurement after the onset ofexperimental caging (7 d) and ending atthe last time point in which FCM datawere collected for all animals in allcaging conditions (49 d). Group means(Figure 2B) were determined by averag-ing the 7-wk mean FCM values for allanimals in each caging condition,

R E S E A R C H A R T I C L E


Figure 1. Caging enrichment and influenza vaccination timeline. After arriving at the husbandry facility, 6-wk-old male BALB/c mice werehoused in standard control caging for 14 d (day –14 to 0) for acclimatization. At d 0, mice were placed, five per cage, into either stan-dard control caging (n = 20) or enriched EE caging (n = 20) for either 56 or 63 d. At d 28, all mice were injected intramuscularly with triva-lent influenza vaccine (1 μg HA in 50 μL PBS) or vehicle (50 μL PBS). At 28 d after primary vaccination, half of the animals were euthanizedfor blood and tissue collection and half were given a second intramuscular vaccination (boost). Remaining animals were euthanized forblood and tissue collection 7 d after boost.

whereas regression analyses used 7-wkmean FCM data from each individualanimal.

EuthanasiaAt the experimental endpoint, d 56 or

63, cardiac blood was collected while theanimals were under deep isoflurane anes-thesia, and animals were then euthanized

via cardiac puncture one at a time. Depthand duration of anesthesia were similarfor all animals, and sample collection wasdone within 2 h of lights on in the hus-bandry facility.

Flow CytometrySpleens were taken and cells were

mechanically dissociated. Cell number

was counted using standard methodsafter red blood cell lysis (eBioscience, SanDiego, CA, USA). Splenocytes wereplated at 1.0 × 106 cells per well in a V-bottom 96-well plate. Cells werestained with phycoerythrin (PE)- conjugated anti-Thy1.2, Alexa 700– conjugated anti-CD4, phycoerythrin-Texas Red (PE-Tx)-conjugated anti-CD8,



Figure 2. EE-caged mice have greater body mass and reduced corticosterone concentration. (A) EE-caged mice exhibited greater bodymass than control-caged mice over the course of the study (caging, p = 0.0037; time*caging, p < 0.0001). (B) Longitudinal FCM levels, anoninvasive measure highly correlated with serum corticosterone concentration, were consistently lower in EE-caged mice compared withcontrols (caging, p < 0.0001; area under curve, p < 0.0001). (C) Mean FCM concentration, averaged over the course of the experiment, wasalso significantly lower in EE mice compared with control-caged mice (p < 0.0001). (D) Serum corticosterone concentration measured atthe time of euthanasia was 43% lower in EE mice than control mice (p = 0.0029). (E) A negative relationship was observed between bodymass and mean FCM concentration across caging groups (r2 = 0.30, p = 0.0002). Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.



Figure 3. Splenic B and T lymphocyte numbers are greater in naive EE-caged mice than naive controls and negatively associated withcorticosterone concentration. (A) Naive (PBS-injected) EE mice exhibited a nonsignificant trend toward a greater number of splenocytesthan naive controls (p = 0.0588). (B–D) The absolute number of splenic CD19+, CD4+ and CD8+ cells was greater in naive EE mice thannaive controls (CD19+, p = 0.0003; CD4+, p = 0.0321; CD8+, p = 0.0605). (E–G) Negative relationships were observed between the absolutenumber of splenic CD19+, CD4+ and CD8+ cells and serum corticosterone concentration (CD19+, r2 = 0.43, p = 0.0017; CD4+, r2 = 0.28, p =0.0154; CD8+, r2 = 0.20, p = 0.0503). (H–J) Similarly, the proportion (% of total splenocytes) of CD19+, CD4+, and CD8+ cells exhibited a signifi-cant negative relationship with serum corticosterone concentration (CD19+, r2 = 0.61, p < 0.0001; CD4+, r2 = 0.39, p = 0.0034; CD8+, r2 =0.20, p = 0.0492). Data are mean ± SEM. *p < 0.05, ***p < 0.001.

and allophycocyanin-Cy7 (APC-Cy7)-conjugated anti-CD19. Dead cells werelabeled and excluded from analyses byusing Alexa 430 amine-reactive live/ deaddye (BD Biosciences [San Jose, CA, USA]and Molecular Probes/Life Technologies[Carlsbad, CA, USA]). After staining,cells were washed with fluorescence- activated cell sorter buffer (PBS + 0.5%

bovine serum albumin + 2 mmol/L eth-ylenediaminetetraacetic acid [EDTA]),fixed with 2% paraformaldehyde in PBS,and collected on an LSR II (BD Bio-sciences). Anti-rat immunoglobulin G(IgG)-coated beads were stained witheach fluorochrome-conjugated rat anti-body separately and used for software-based compensation. All samples were

analyzed on a custom four-laser LSR II(BD Biosciences) equipped with a 50 mWblue (488 nmol/L), 50 mW violet(406 nmol/L), 40 mW red (640 nmol/L)and 150 mW green (532 nmol/L) laser.

Gating StrategyFlow cytometry analyses were carried

out using FlowJo software, version 9.5



Figure 4. Effects of EE caging on primary and secondary vaccine responses and spleen composition. (A) Influenza vaccine–specific IgGtiters were measured in serum from mice vaccinated once (prime) or twice (boost) using an ELISA. In primed groups, EE mice had slightlylower IgG titers than control-caged mice (p = 0.0943), whereas in boosted groups, EE mice had slightly higher IgG titers than controls (p =0.1950), although neither difference was statistically significant. Control mice exhibited an increase in IgG titer, from prime to boost, of11.4%, whereas EE mice exhibited an increase of 23.8% (control, p = 0.0138; EE, p = 0.0006). (B) 1 × 106 splenocytes were stimulated witheither PBS or vaccine for 24 h in an IL-5 ELISPOT assay. Primed animals showed no difference between caging groups, whereas spleno-cytes from boosted EE mice that were stimulated with vaccine had a significantly greater frequency of IL-5–secreting cells than boostedcontrols (p = 0.0493). The increase in frequency of IL-5–secreting cells from prime to boost was larger in EE mice (334.2%) than controls(177.9%) (control, p = 0.0230; EE, p = 0.0005). (C) A nonsignificant negative trend was observed between IL-5 production and mean FCMconcentration (r2 = 0. 38, p = 0.0781). (D) Spleen mass per unit body mass was found to be significantly lower in boosted control-cagedmice than boosted EE-caged mice (p = 0.0001). (E) A negative relationship was observed between mean FCM concentration andspleen mass per unit body mass in vaccine-boosted animals (r2 = 0.90, p < 0.0001). (F, G) Negative relationships were also observed be-tween mean FCM concentration and CD19+ B cell number and CD4–CD8– lymphocyte number (CD19+, r2 = 0.39, p = 0.0533; CD4–CD8–,r2 = 0.52, p = 0.0190). Data are mean ± SEM. *p < 0.05, ***p < 0.001.

(TreeStar, Ashland, OR, USA). Cell dou-blets were excluded by plotting forwardscatter height and area parameters. Alymphocyte gate was created using sidescatter area and forward scatter heightarea parameters. Live cells were selectedas those not stained positively by Alexa430 amine-reactive live/dead dye. B lym-phocytes were identified as Thy1.2–CD19+

cells. CD4+ T lymphocytes were identifiedas CD19-Thy1.2+CD4+CD8– cells. CD8+

T lymphocytes were identified asCD19–Thy1.2+CD4–CD8+ cells. CD4–CD8–

T lymphocytes were identified asCD19–Thy1.2+CD4–CD8– cells.

IgG TiterVaccine-specific IgG levels were as-

sessed by using an enzyme-linked im-munosorbent assay (ELISA). Briefly, 96-well Immulon-2 High Binding flat-bottommicrotiter plates (Thermo Scientific[Thermo Fisher Scientific Inc., Waltham,MA, USA]) were coated with 100 μL di-luted Fluarix vaccine (1 μg/mL HA pro-tein) and incubated overnight at 4°C. Ani-mal sera were diluted with blockingbuffer (0.5% gelatin, 0.15% Tween-20, 1%bovine serum albumin in PBS) and incu-bated for 1 h at 37°C. Plates were washedand blocked. Serum samples weretwofold serially diluted starting from1:200, plated in duplicate and incubatedfor 1.5 h at 37°C. Plates were washed andthen incubated with horseradish peroxidase–conjugated anti-mouse IgG(Abcam, Cambridge, UK) for 45 min at37°C. Plates were washed and tetram-ethylbenzidine (TMB) substrate (Invitrogen/ Life Technologies) wasadded, followed by 2 mol/L H2SO4 stopsolution. A SPECTRAmax absorbance mi-croplate reader (Molecular Devices/LifeTechnologies) was used to read each plateat optical densities of 450 and 650 nm.The 650-nm values were subtracted fromthe 450-nm values to account for platebackground. Serum from one vaccinatedmouse was added to each plate in tripli-cate and used as an interplate normaliza-tion control (coefficient of variation: in-terassay 7.2%, intraassay 6.7%). Negativecontrol wells were not coated with vac-

cine and incubated with serum samplesor coated with vaccine and incubatedwith blocking buffer only. Optical density(OD) values from negative control wellswere subtracted from serum sample ODvalues as a background correction. IgGtiter was set as the greatest dilution ofserum generating an OD value greaterthan twice the vaccine-uncoated negativecontrol OD value.

ELISPOT AssayInterleukin (IL)-5 production was mea-

sured with a mouse IL-5 ELISPOTplus kit(Mabtech, Cincinnati, OH, USA) accord-ing to the manufacturer’s instructions.Briefly, splenocytes were plated in 96-well ELISPOT polyvinylidene fluorideplates (EMD Millipore, Billerica, MA,USA) at 1 × 106 cells per well. Wells weretreated with Fluarix vaccine (3 μg/mL),concanavalin A (2 μg/mL) or PBS in du-plicate. Plates were then incubated for24 h at 37°C, washed with PBS and de-veloped per the manufacturer’s instruc-tions. Spots were counted by using AIDEliSpot Reader and Software (Autoim-mun Diagnostika, Straßberg, Germany).To correct for background signal, twicethe number of spots counted in PBS-treated negative control wells was sub-tracted from each sample spot number.

Serum CorticosteroneWhole blood was collected via cardiac

puncture at the study endpoint within 2 h of lights on, kept at 4°C overnightuntil coagulated and centrifuged for 10 min at 17,970 × g. Supernatants werefrozen at –20°C. ELISA kits were used asinstructed by the manufacturer to mea-sure serum concentrations of corticos-terone (IBL America, Minneapolis, MN,USA). Samples were assayed in onebatch (intraassay coefficient of variationwas ≤8.3%).

Statistical AnalysisData are expressed as mean ± standard

error of the mean (SEM). Analyses wereperformed by using Prism software v5.0a(GraphPad Software Inc., La Jolla, CA,USA) or SAS PROC GLM 9.2. Unpaired

t tests were used to compare control-caged animals with EE-caged animals orvaccine-primed animals with vaccine-boosted animals (Figures 2A–D, 3A–D,and 4A, B, D). Two-way repeated- measures analysis of variance (ANOVA)was used to compare increases in bodymass between caging groups over time(Figure 2A) and FCM level over time(Figure 2B). Relationships between corti-costerone concentration and body mass(Figure 2E), lymphocyte number (Fig-ures 3E–J and 4F, G), IL-5 production(Figure 4C) and spleen mass (Figure 4E)were measured by using a linear regres-sion followed by two-tailed statisticalcomparison of regression coefficients. Interaction between cage group and vac-cine group (Figure 4A, B) was determinedby using a 2 × 2 ANOVA. Partial η2, theproportion of residual variation ex-plained by a given effect, was used for effect size in ANOVA (Figure 4A, B). Inall cases, p < 0.05 was considered statisti-cally significant.

RESULTS

Body Mass and CorticosteroneConcentration

Mice housed in EE caging gained sig-nificantly greater body mass than control-housed animals within 1 wk ofthe onset of experimental caging (7 d,difference in means [Md] 1.15 g, 95% con-fidence interval [CI] 0.32–1.99, p =0.0082) and significantly greater bodymass over the course of the study(caging, p = 0.0037; time*caging, p <0.0001; Figure 2A). These results replicateour previous findings (25). To monitorstress hormone production noninva-sively and longitudinally, we measuredFCM concentrations from each animalweekly (a measure highly correlatedwith plasma corticosterone concentra-tion) (29). EE-caged animals had signifi-cantly lower FCM concentrations thancontrol-caged animals over the course ofthe experiment (caging, p < 0.0001; areaunder curve [trapezoidal rule], Md

–425.3 ng/ 0.05 g feces, CI –247.2 to–603.4, p < 0.0001; Figure 2B) and a sig-



nificantly lower 7-wk mean FCM concen-tration (Md –11.1 ng/0.05 g feces, CI –6.7to –15.5, p < 0.0001; Figure 2C). Interest-ingly, in the first week of experimentalcaging (d 0–7) both caging groups exhib-ited a reduction in FCM level, whichmay have been related to latent acclima-tization or habituation to regular han-dling. Serum corticosterone concentra-tions were also measured at the time ofeuthanasia and, similarly, were 43%lower in EE-caged animals comparedwith controls (Md –43.1 ng/mL, CI –15.7to –70.5, p = 0.0029; Figure 2D). In regres-sion analyses, we found a negative rela-tionship between body mass and meanFCM concentration (r2 = 0.30, p = 0.0002;Figure 2E), a finding in line with previ-ous reports of the relationship betweendominance hierarchy and corticosteroneproduction (30).

Spleen Immunophenotype of NaiveMice

Consistent with our previous findingsof increased spleen mass in EE mice, wefound that PBS-injected (naive), EE-caged animals exhibited a trend towardgreater numbers of splenocytes after ery-throlysis compared with PBS-injectedcontrols (Md 11.7 × 106 cells, CI –0.49 ×106 to 24.0 × 106, p = 0.0588, Cohen’s d =0.90; Figure 3A). By using multicolorflow cytometry, we enumerated splenicThy1.2+, CD4+, CD8+ and CD19+ cells toidentify potential shifts in the absolutenumber and proportion of these cell pop-ulations between caging groups (see Ma-terials and Methods). We found that EE-caged mice had a markedly greaternumber of CD19+ B lymphocytes perspleen compared with control-cagedmice (Md 9.0 × 106 CD19 cells per spleen,CI 4.8 × 106 to 13.3 × 106, p = 0.0003; Fig-ure 3B). We also found a trend towardgreater numbers of Thy1.2+ T lympho-cytes in spleens from EE-caged mice (Md 20.0 × 105 CD3+ cells per spleen, CI–0.06 × 105 to 40.0 × 105, p = 0.0506; datanot shown). Among the T cells, we foundthat EE-caged mice also had greater num-bers of CD4+ and CD8+ T-cell subsetsthan controls, although the differences in

CD8+ T cell numbers were not statisti-cally significant (Md 11.2 × 105 CD4+ cellsper spleen, CI 1.1 × 105 to 21.4 × 105, p =0.0321; Figure 3C; Md 8.4 × 105 CD8+ cellsper spleen, CI –0.4 × 105 to 17.25 × 105,p = 0.0605; Figure 3D).

To determine whether serum corticos-terone concentration was related to thedifferences observed in the number ofsplenic immune cell subpopulations, we applied regression analyses focusedon absolute cell number per spleen (Fig-ure 3E–G) and the proportion of eachsubpopulation per spleen (Figure 3H–J).We found a statistically significant nega-tive relationship between the absolutenumber of CD19+ B lymphocytes andserum corticosterone concentration (r2 =0.43, p = 0.0017; Figure 3E). CD4+ andCD8+ T lymphocyte counts also dis-played a negative relationship with corti-costerone concentration (CD4+, r2 = 0.28,p = 0.0154, Figure 3F; CD8+, r2 = 0.20, p =0.0503, Figure 3G). Similarly, the propor-tion of CD19+, CD4+ and CD8+ cells, as apercentage of total splenocytes, exhibiteda statistically significant negative rela-tionship with corticosterone concentra-tion (CD19+, r2 = 0.61, p < 0.0001, Figure3H; CD4+, r2 = 0.39, p = 0.0034, Figure 3I;CD8+, r2 = 0.20, p = 0.0492, Figure 3J).These results demonstrate that in naivemice, decreased glucocorticoid levelswere associated with greater spleen cel-lularity, a pronounced increase in thenumber of CD19+ B lymphocytes and amodest increase in CD4+ and CD8+

T lymphocyte numbers per spleen.

Vaccine-Specific IgG TiterTo test the hypothesis that EE caging

and related corticosterone reduction mayenhance vaccine responses, we assessedhumoral and cellular antigen-specific im-munity after vaccination. We measuredinfluenza vaccine–specific IgG titers inserum from control- and EE-caged micethat were injected with influenza vaccineintramuscularly either once (prime) ortwice (boost). Naive mice injected withPBS served as negative controls and exhib-ited uniform influenza vaccine–specificIgG titers near or below the detection

threshold (data not shown). For vaccine-primed groups, EE-caged mice had lowerIgG titers than control-caged mice,whereas for boosted groups, EE-cagedmice had higher IgG titers than control-caged mice, although neither differencewas found to be statistically significant(prime, Md –0.18 log IgG titer, CI 0.04 to–0.40, p = 0.0943, Cohen’s d = 1.20; boost,Md 0.30 log IgG titer, CI –0.20 to 0.79, p =0.1950, Cohen’s d = 0.89; Figure 4A).Within caging groups, we found that con-trol-caged animals exhibited an increasein IgG titer, from prime to boost, of11.4%, whereas EE-caged mice exhibitedan increase of 23.8% (control, Md 0.4816log IgG titer, CI 0.13–0.84, p = 0.0138; EE,Md 0.96 log IgG titer, CI 0.56–1.37, p =0.0006; Figure 4A). There was a strongstatistical trend toward an interaction be-tween caging group (control versus EE)and vaccine group (prime versus boost),suggesting there may be distinct effects ofenriched caging on primary versus sec-ondary IgG response to vaccination(cage-group*vaccine-group, F = 4.27, p = 0.0555, partial η2 = 0.21).

Vaccine-Specific IL-5 ResponsesBALB/c mice display a genetic bias to-

ward T helper type 2 (TH2)-mediated im-mune responses and have been reportedto produce greater quantities of IgG anti-bodies in response to vaccination whencompared with other strains such asC57BL/6 (31,32). We investigated differ-ences between caging groups in spleno-cyte production of IL-5, a TH2 cytokineknown to promote B lymphocytegrowth, differentiation and increased IgGsecretion (33). Splenocytes were stimu-lated with either PBS or vaccine for 24 hin an IL-5 ELISPOT assay. We found thatin primed animals, IL-5 responses to vac-cine stimulation were similar in control-and EE-caged groups (Figure 4B). How-ever, for vaccine-boosted animals, a sig-nificantly greater frequency of spleno-cytes from EE-caged mice produced IL-5after vaccine stimulation when comparedwith control-caged mice (Md 67.7 IL-5spot-forming units (SFU)/106 splenocytes,CI 0.24–135.1, p = 0.0493; Figure 4B). The



increased frequency in IL-5–secretingcells from prime to boost was larger inmagnitude in EE-caged mice (334.2%)compared with control-caged mice(177.9%) (control, Md 60.3 IL-5 SFU/106

splenocytes, CI 10.7–109.9, p = 0.0230; EE,Md 124.7 IL-5 SFU/106 splenocytes, CI75.8–173.6, p = 0.0005; Figure 4B). We ob-served a statistically significant interac-tion between caging group and vaccinegroup, suggesting there may be distincteffects of corticosterone reduction cagingon primary versus secondary cellular immune responses to vaccination (cage-group*vaccine-group, F = 4.60, p =0.0487, partial η2 = 0.23). Given these re-sults, we applied regression analyses toinvestigate the relationship between thefrequency of IL-5–secreting splenocytesin boosted mice and the 7-wk mean FCMconcentration. We observed a trend sug-gesting a negative relationship betweenmean FCM concentration and the fre-quency of IL-5–secreting cells (r2 = 0.38,p = 0.0781; Figure 4C). Together, these re-sults suggest that the caging environ-ment influenced secondary, but not pri-mary, cell-mediated immune responsesupon vaccine stimulation.

Spleen Composition of Vaccine-Boosted Mice

Our observations of caging-group ef-fects on functional immunity led us tofurther investigate splenic differences invaccine-boosted animals. To account forvariance in body mass within cages andacross groups, we quantified spleen massas a function of body mass (g/kg). Wefound that boosted EE-caged mice had asignificantly greater spleen mass per unitbody mass than boosted control-cagedmice (Md 1.55 g/kg, CI 1.05–2.06, p =0.0001; Figure 4D). Notably, regressionanalysis demonstrated a strong negativerelationship between the 7-wk meanFCM concentration and spleen mass perunit body mass in vaccine-boosted ani-mals (r2 = 0.90, p < 0.0001; Figure 4E). Onthe basis of the large increase we ob-served in CD19+ B lymphocyte numbersin naive EE-caged mice, we anticipatedthat the FCM-associated differences in

spleen mass in boosted mice might bepartially attributed to differences in B-cell number. To investigate this rela-tionship further, we looked at regressionsof mean FCM concentration and absolutenumbers of splenocyte subpopulationsgenerated from our flow cytometry–basedphenotype analysis of vaccine-boostedanimals. We observed negative relation-ships between mean FCM concentrationand CD19+ B-cell number and, unexpect-edly, CD4–CD8– T lymphocyte number(CD19+, r2 = 0.39, p = 0.0533; CD4–CD8–,r2 = 0.52, p = 0.0190; Figure 4F–G). Theseresults suggest that, among vaccine-boosted animals, spleen mass is influ-enced by caging condition and mean glu-cocorticoid exposure and that distinctsubpopulations of splenocytes, includingCD19+ B cells and CD4–CD8– T cells, ap-pear to be more influenced by glucocorti-coid exposure.

DISCUSSIONIn the current work, we demonstrate

that our standardized approach of EEhousing reduces corticosterone levels inmice and modulates splenic immune cellcomposition and influenza vaccine re-sponses. Naive EE-caged mice had a sig-nificantly greater number and proportionof splenic B and T lymphocytes than con-trols, and we observed a negative rela-tionship between corticosterone concen-tration and B and T lymphocyte numberand proportion. The difference in pri-mary influenza vaccine–specific IgGtiters between caging groups was notstatistically significant; however, EE-caged mice tended to exhibit a greater in-crease in titer from prime to boost thancontrols, although this finding did notquite reach the level of statistical signifi-cance (p = 0.0555). Vaccine-stimulatedsplenocytes from boosted EE-caged micehad a significantly greater frequency ofIL-5–secreting cells than boosted con-trols, whereas there was little differencebetween groups in the frequency of IL-5–secreting cells in primed mice.Lastly, we found that, in boosted mice,spleen mass per unit body mass was sig-nificantly lower in control-caged mice

compared with EE-caged mice and thenumber of splenic B lymphocytes in bothcaging groups was negatively correlatedwith the average corticosterone level.

In previous studies, by using the sameenriched environment parameters andmouse strain used here, we found thatEE-caged mice displayed no significantdifferences in elevated-plus-maze behav-ior (25). We have since repeated this ex-periment by using an elevated-zero-mazeand again found no significant behav-ioral differences between EE- and control-caged animals (data not shown),although a marked reduction of corticos-terone production in EE-caged mice re-mained reproducible. We speculate thatthe lack of behavioral differences may berelated to strain, such as BALB/c micebeing relatively less exploratory thanother strains, and the sensitivity of thebehavioral tests we have used to detectsubtle behavioral changes that may re-sult from caging enrichment. As a prod-uct of these findings, we have deter-mined that further behavioral testingneeds to be done to validate this ap-proach to caging enrichment as a stressreduction model. In this article, we havereferred to the enhanced caging as “en-riched environment” instead of the for-mer label “Calm” caging.

Other studies have demonstrated thatenriched environment has effects on im-mune function such as increased naturalkiller activity, elevated tumor resistanceand increased antibody production aftervaccination (34–36). A key detail of theenrichment protocols in these studies isthat they included access to a runningwheel. In previous work, we have shownthat, compared with EE caging alone, EEcaging with the addition of a runningwheel enhances the observed effects onspleen mass and corticosterone reduc-tion. A limitation of the current study isthat we did not have resources to includeseparate EE and control caging condi-tions with and without exercise wheelsand hence cannot distinguish the effectof exercise from that of other elements ofthe EE caging. In contrast to our data, theabove studies, and others, also reported



that mice housed in enriched conditionshad similar or increased concentration ofplasma corticosterone compared withcontrol-caged mice (37,38). These dis-parate findings may be related to differ-ences in the approach to EE. Some en-richment protocols call for frequentrotation of enrichment items into and outof the cage, which increases the level ofenrichment novelty but may also be asource of mild stress. In our studies, theenrichment items and locations withinthe cage were kept consistent throughoutthe experiment so as to minimize cagedisruption and its potential effect as amild stressor. Additionally, genetic differ-ences among the strains of mice used inEE studies may, in part, contribute to thedivergent results. The BALB/c strainused in our studies is relatively anxious,with higher concentrations of basalserum corticosterone and lower rates ofcorticosterone catabolism, which mayamplify the effects of cage enrichment onreduction of corticosterone production(39,40). In contrast, the above studiesthat found increased corticosterone con-centrations in EE-caged mice usedC57BL/6 and C3H/eB strains. It will beinteresting to use other strains of labora-tory mice in our standardized approachto EE, both to determine whether it is aneffective tool for corticosterone reductionacross genetic strains and to address thehigh degree of variability that exists inimmune phenotype across strains (41).

Previous studies in rodents haveshown that restraint stress or treatmentwith exogenous corticosterone is nega-tively associated with spleen mass andsplenic lymphocyte number (42). Simi-larly, adrenalectomized mice havegreater numbers of splenic IgG-secretingcells compared with sham surgery con-trols, although a statistically significantcorrelation between corticosterone andIgG-secreting cell number was not ob-served as it was in our data (43). In astudy investigating how glucocorticoidsaffect B lymphocyte populations, it wasfound that chronic corticosterone expo-sure causes depletion of developing B-lineage cells in the bone marrow by re-

ducing the number of cycling precursorcells and inducing apoptosis (44). Datafrom our enriched environment ap-proach (a mild intervention relative to re-straint, corticosterone treatment oradrenalectomy) are consistent with theabove findings. Our results also suggesta relationship between corticosteroneand the immune system in which small,physiologically relevant decrements incorticosterone production are associatedwith substantial increases in splenic Band, to a lesser extent, T lymphocytenumbers. Though further work is neededto establish the immunologic and clinicalsignificance of having a greater numberof naive B lymphocytes in the spleen, wespeculate that, under conditions of infec-tion or vaccination, there may be an ad-vantage to having a larger pool of B lym-phocytes available such that a greaterdiversity of antigen-specific antibodiescan be generated. This might be of par-ticular importance for infectious diseasesin which the pathogen undergoes rapidmutations such as influenza. Further, B cells are also capable of functioning asantigen-presenting cells, and an in-creased number of B cells in the spleen,and possibly other lymphoid tissues,could result in enhanced antigen presen-tation (45,46).

Both human and animal studies havedemonstrated the suppressive effects ofchronic stress on humoral immunity. Forexample, a study of caregivers of spouseswith dementia demonstrated that thepercentage of caregivers who mounted aclinically protective IgG antibody re-sponse after influenza vaccination wassignificantly lower than in vaccinatednonstressed controls (47). In relatedwork, restraint stress altered the kineticsof the antibody response and delayedclass switching from IgM to IgG and IgAisotypes in mice exposed to influenzavirus (48). Repeated activation of thestress response and exposure to stressmediators are believed to be related tothe immune modulation observed inthese studies. Given these data, we hy-pothesized that mice with reduced corti-costerone, a principal stress hormone,

would exhibit enhanced humoral re-sponses after vaccination. However, in-fluenza vaccine–specific antibody titersin EE-caged mice were not significantlydifferent from controls. Inspection of thedata revealed a notable pattern, however,in that EE mice had lower IgG titers thancontrol-caged mice after primary vacci-nation, but tended to have higher titersthan controls after vaccine boost. Further,EE animals tended to have a greater in-crease in titer from prime to boost thandid control mice. This interaction be-tween caging group and number of vac-cine administrations was nearly statisti-cally significant and is consistent withanother study in which keyhole limpethemocyanin was administered once ortwice to male mice housed from 1 percage up to 12 per cage (49). It was ob-served that increased housing densitywas positively associated with corticos-terone production. Whereas singlyhoused mice receiving a booster immu-nization had significantly greater IgMand IgG titers than group-housed mice,there was no effect of caging density onantibody production in mice that only re-ceived a primary immunization withkeyhole limpet hemocyanin. Notably, theauthors report administering 5–150 μg ofkeyhole limpet hemocyanin antigen pervaccination, whereas we administered 1μg HA antigen in our studies. In follow-up work, it will be important to deter-mine the degree to which the quantity ofimmunogen has effects on the interactionbetween stress level and humoral im-mune responses.

Cellular immune function is modifiedby stress and stress hormone signaling.For example, restraint stress has beenshown to suppress IL-2 production 14 dafter influenza viral infection and also toattenuate delayed-type hypersensitivityreactions (5,48). However, no investiga-tions have been made into the effects ofenriched environment and reduced glu-cocorticoid production on cell-mediatedresponses after vaccination. BecauseBALB/c mice display a genetic bias to-ward TH2-predominant immune re-sponses, we investigated differences be-



tween caging groups in splenocyte pro-duction of IL-5, a TH2 cytokine known topromote B lymphocyte growth, differen-tiation and increased IgG secretion (33).We anticipated that splenocytes fromboth primed and boosted EE-caged micewould exhibit enhanced cellular immuneresponses when exposed to vaccine invitro. While we observed that vaccine-stimulated splenocytes from boosted EEmice had a significantly greater fre-quency of IL-5–secreting cells thanboosted control-caged mice, we found nodifferences in the frequency of IL-5–se-creting cells between vaccine-primed EEand control animals. Similar to the IgGtiter data, we found that EE mice dis-played a greater increase in IL-5–secreting cells from prime to boostthan did controls, and this result wassupported by a statistically significant in-teraction between the caging group andnumber of vaccine administrations. To-gether with the humoral immune data,these findings support the argument thatenriched environment and associatedchanges in corticosterone may preferen-tially affect secondary humoral and cel-lular immune responses and have less ofan impact on primary responses. Addi-tionally, while these preliminary studieswere mainly focused on adaptive immu-nity, glucocorticoid signaling is alsoknown to influence important innate celltypes such as macrophages and dendriticcells, which are important for efficaciousvaccine responses. In future studies, weintend to assess cell type–specific func-tional effects across innate and adaptivearms of the immune system.

Analysis of the spleen data revealedthat spleen mass per unit body mass wassignificantly lower in boosted controlmice than boosted EE mice. We alsofound that in boosted animals, glucocor-ticoid concentration was negatively asso-ciated with spleen mass and the absolutenumber of splenic CD19+ B cells andCD4–CD8– T cells. These findings areconsistent with our observations relatedto glucocorticoid levels and spleen com-position in naive animals as well as workby others reporting the effects of corti-

costerone signaling on B lymphocytepopulations (42–44).

It is well established that both gluco-corticoids and autonomic nervous systemmediators, such as epinephrine and nor-epinephrine, are important for regulatingimmune cell distribution and function.Our approach in the current work was tominimize animal handling and stressfulprocedures such as blood collection toprevent regular activation of the acutestress response. We were able to collectdetailed longitudinal data on glucocorti-coid production by measuring fecal corti-costerone metabolites noninvasively.However, one disadvantage of this ap-proach was that we were not able to mea-sure plasma or urinary catecholamines.

Lastly, it is important to note that therelationship between stress and HPA axisactivity is complex and remains an areaof ongoing investigation. For example, insome scenarios of chronic stress, such asposttraumatic stress disorder, humansand rodent models can display increasedor decreased glucocorticoid production,findings that are currently poorly under-stood (50,51). In the present work, al-though we found consistent relationshipsbetween corticosterone and a number ofimmunologic outcomes, the complexityof the stress-HPA axis relationshipshould be carefully considered when re-lating our results to stress physiology.

CONCLUSIONOverall, the current data highlight key

issues that should be carefully addressedin future human studies investigating therelationship between psychologicalstress, glucocorticoid signaling and vac-cine responses. In particular, it may beimportant to use an experimental designcapable of testing the differential effectsof stress mediators (that is, glucocorti-coids and catecholamines) on primaryversus secondary immune responses.Furthermore, assessment of both hu-moral and cell-mediated immune re-sponses in this context will provide amore thorough characterization of theimpact of behavioral interventions onimmune function and health.

DISCLOSUREThe authors declare that they have no

competing interests as defined by Molecu-lar Medicine, or other interests that mightbe perceived to influence the results anddiscussion reported in this paper.

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