Spleen supports a pool of innate-like B cells in whiteadipose tissue that protects against obesity-associatedinsulin resistanceLan Wua,1, Vrajesh V. Parekha, Joseph Hsiaoa, Daisuke Kitamurab, and Luc Van Kaera,1

aDepartment of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232; and bResearch Institute forBiomedical Sciences, Tokyo University of Science, Noda, Chiba 278-0022, Japan

Edited by Ajay Chawla, University of California, San Francisco, CA, and accepted by the Editorial Board September 15, 2014 (received for review December24, 2013)

Lipid accumulation in obesity triggers a low-grade inflammationthat results from an imbalance between pro- and anti-inflammatorycomponents of the immune system and acts as the major un-derlying mechanism for the development of obesity-associateddiseases, notably insulin resistance and type 2 diabetes. Innate-likeB cells are a subgroup of B cells that respond to innate signals andmodulate inflammatory responses through production of immu-nomodulatory mediators such as the anti-inflammatory cytokineIL-10. In this study, we examined innate-like B cells in visceralwhite adipose tissue (VAT) and the relationship of these cells withtheir counterparts in the peritoneal cavity and spleen during diet-induced obesity (DIO) in mice. We show that a considerablenumber of innate-like B cells bearing a surface phenotype distinctfrom the recently identified “adipose natural regulatory B cells”populate VAT of lean animals, and that spleen represents a sourcefor the recruitment of these cells in VAT during DIO. However,demand for these cells in the expanding VAT outpaces their re-cruitment during DIO, and the obese environment in VAT furtherimpairs their function. We further show that removal of splenicprecursors of innate-like B cells through splenectomy exacerbates,whereas supplementation of these cells via adoptive transferameliorates, DIO-associated insulin resistance. Additional adoptivetransfer experiments pointed toward a dominant role of IL-10 inmediating the protective effects of innate-like B cells against DIO-induced insulin resistance. These findings identify spleen-suppliedinnate-like B cells in VAT as previously unrecognized players andtherapeutic targets for obesity-associated diseases.

innate-like B cells | spleen | diet-induced obesity | inflammation |insulin resistance

The current obesity epidemic has led to an increase in theincidence of a variety of disorders that are collectively re-

ferred to as obesity-associated diseases. Changes in diet andlifestyle, especially the abundance of energy-dense high-fat foods,have played a central role in the emergence of this epidemic. Lipidaccumulation in obesity triggers a low-grade inflammation thatresults from an imbalance between pro- and anti-inflammatorycomponents of the immune system. This chronic inflammationacts as the major underlying mechanism for the development ofobesity-associated diseases, notably insulin resistance and type 2diabetes (T2D) (1–5). Both the innate and adaptive branches ofthe immune system are activated during obesity and participatein the induction and maintenance of obesity-triggered inflamma-tion (1–7). In metabolic organs, most notably visceral white adiposetissue (VAT), increases in proinflammatory cells and decreases inanti-inflammatory cells create an insulin-antagonizing environmentthat interferes with normal metabolic pathways (1–5). Whereasit is now clear that multiple immune cells play a role in thisprocess, the full spectrum of cellular and molecular signals thatinitiate and sustain the chronic inflammation in obesity remainsto be delineated.

Lymphocytes of the T- and B-cell lineages are traditionallycategorized as components of the adaptive immune system, asthey recognize specific pathogen-derived antigens and are ca-pable of developing long-lasting immune memory. Among theseconventional adaptive lymphocytes, CD8+ T cells and follicular(B-2) B cells have been shown to exacerbate, whereas CD4+Foxp3+

regulatory T (Treg) cells have been shown to protect against, obesity-triggered inflammation and insulin resistance (8–11). Over the pastfew decades, a growing family of lymphocyte subsets with innate-like properties and functions has been identified (12–17).These cells, through recognition of nonspecific innate immunesignals and production of immunomodulatory cytokines, interactwith and influence the function of multiple cell types of the in-nate and adaptive branches of the immune system and thusshape subsequent immune and inflammatory responses and im-pact disease outcomes. In the T-cell lineage, several researchgroups have investigated the role of natural killer T (NKT) cellsin obesity and insulin resistance (18). A recent report identifieda subset of B cells in white adipose tissue that expressed a uniquesurface phenotype and protected mice against obesity-inducedinflammation (19). However, whether other subsets of innate-like Bcells capable of influencing insulin sensitivity also populate VAT iscurrently unknown. Additionally, the source(s) for the recruitmentof these cells in VAT during obesity remains unclear.Several subsets of innate-like B cells, including B-1a and B-1b

B cells, marginal zone (MZ) B cells, regulatory B cells, and in-

Significance

The rise in obesity-associated diseases has led to increased effortsto identify new therapeutic targets. Lipid accumulation inobesity triggers a low-grade inflammation that links obesity toits associated diseases, notably insulin resistance and type 2diabetes. In this report, we have studied a group of innate-likeB lymphocytes in visceral white adipose tissue (VAT) of micethat are (i) capable of producing the anti-inflammatory medi-ator IL-10; (ii) replenished from precursor cells in spleen duringdiet-induced obesity (DIO); and (iii) impaired in VAT of DIO mice,resulting in diminished protection against obesity-associatedinsulin resistance that can be ameliorated by supplementation ofthese cells. These findings identify spleen-supplied innate-like Bcells in VAT as previously unidentified therapeutic targets forobesity-associated diseases.

Author contributions: L.W. and L.V.K. designed research; L.W., V.V.P., and J.H. performedresearch; D.K. contributed new reagents/analytic tools; L.W. analyzed data; D.K. assistedin experimental design and data analysis; and L.W. and L.V.K. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. A.C. is a guest editor invited by the EditorialBoard.1To whom correspondence may be addressed. Email: luc.van.kaer@vanderbilt.edu or lan.wu@vanderbilt.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1324052111/-/DCSupplemental.

E4638–E4647 | PNAS | Published online October 13, 2014 www.pnas.org/cgi/doi/10.1073/pnas.1324052111

Dow

nloa

ded

by g

uest

on

Dec

embe

r 24

, 202

0

nate response activator (IRA) B cells have been identified inlymphoid organs and in peritoneal cavity (PerC) of mice (12, 14–16, 19–21). These innate-like B-cell subsets share several phe-notypic and functional characteristics but also display importantdifferences. Compared with conventional adaptive B-2 B cells,innate-like B cells exhibit increased responsiveness to innatesignals through a variety of innate receptors such as toll-likereceptors (TLRs) (12, 14–16, 20, 21). Although these cells onlyconstitute a small subpopulation of B cells in spleen and mainlyreside in the PerC under steady-state conditions (12), adult mousespleen houses progenitors and precursors of these cells and thisorgan therefore plays a critical role in maintaining a functionalpool of innate-like B cells (22–24). Among innate-like B cells,IL-10–producing B cells, often referred to as regulatory B cellsor B10 cells, modulate inflammatory responses primarily throughproduction of the anti-inflammatory cytokine IL-10 (14–16).Furthermore, B-1a B cells are an important source of naturalIgM antibodies that protect against atherosclerosis, a chronicinflammatory disease that shares several mechanistic featureswith obesity-associated diseases (25, 26).Several recent studies are consistent with a protective role of

spleen-derived IL-10–producing B cells in obesity-induced in-flammation and insulin resistance. Removal of spleen in diet-induced obesity (DIO) mice exacerbated VAT inflammation,which could be ameliorated by supplementation of IL-10 (27).Unfractionated B cells of DIO mice and of patients with T2Dproduced reduced levels of IL-10 in response to in vitro stimu-lation (10, 28). Whereas these findings suggest a critical role ofspleen for provision of IL-10, possibly through IL-10–producingB cells, in protecting against obesity-associated insulin resistance,the cellular mechanisms of action remain unclear.We show here that a substantial number of B cells with

a surface phenotype resembling innate-like B-1a and regulatoryB10 B cells (12, 16, 29) but distinct from recently identified“adipose natural regulatory B cells” (19) populate VAT understeady-state conditions. Similar to their counterparts in spleenand PerC, these cells in VAT spontaneously produce IgM anti-bodies and constitute the majority of IL-10–competent B cells atthis anatomic location. Whereas the spleen supports a pool ofinnate-like B cells in VAT, the demand for these cells in theexpanding VAT during DIO outpaces their recruitment and theobese environment in VAT further impairs their IL-10 compe-tence. Consequently, splenectomy exacerbates, whereas supple-mentation with these innate-like B cells via adoptive transferameliorates, DIO-induced systemic insulin resistance. Overall,our findings have identified a previously unrecognized subset ofIL-10–competent innate-like B cells in VAT and provided evi-dence for a critical role of spleen in supplying these cells to VATfor protection against obesity-associated insulin resistance.

ResultsA Substantial Number of B Cells with a Surface Phenotype ResemblingInnate-Like B-1a and B10 B Cells Populate VAT and Are Competent inProducing IL-10. To investigate whether a subset(s) of innate-likeB cells other than adipose natural regulatory B cells populatesVAT, we examined B-cell subpopulations in VAT and comparedthem with B cells in several other anatomic locations that areknown to house innate-like B cells. We purified stromal vascularfractions (SVFs) from perigonadal fat, PerC cells from PerCwash, splenocytes (SPLs) from spleen, intrahepatic leukocytes(IHLs) from liver, and single lung cells (SLCs) from lung ofC57BL/6J (B6) mice fed a regular chow diet (RCD) (Table S1).We stained the cells with antibodies recognizing surface markerspreviously described for innate-like B cells (12, 16, 29) and an-alyzed them by flow cytometry. Among TCRβ−CD19+ B cells inSVFs, we detected a subpopulation expressing surface CD5(Fig. 1A), a marker that distinguishes B-1a from B-1b B cells (12)and is lacking in adipose natural regulatory B cells (19). These

TCRβ−CD19+CD5+ B cells, hereafter referred to as CD5+ Bcells, could also be detected in other anatomic locations (Fig. 1A).We then compared their prevalence in different tissues. Re-markably, the prevalence of these cells among the B-cell popu-lation in VAT was nearly as high as that in PerC (Fig. 1B, Left).We further determined their absolute numbers, using tissueweight as the denominator to standardize (we used perigonadalfat weight as the denominator for cells in PerC due to the lack oftissue weight in this location and considering the intimate physicalrelationship between perigonadal fat and PerC). The resultsshowed that a significant number of CD5+ B cells populatedVAT. Whereas the spleen and lung housed higher numbers ofthese cells due to a larger pool of leukocytes per gram of tissue,these cells were more abundant in VAT than in liver (Fig. 1B,Center). Among the B-cell populations, VAT contained signifi-cantly more CD5+ B cells per gram of tissue than liver, spleen,and lung (Fig. 1B, Right).We next examined the phenotypic and functional features of

these VAT-resident CD5+ B cells and compared them with thewell-characterized subsets of innate-like B cells in PerC andspleen (12, 16, 29). Similar to the cells in the latter two anatomiclocations, the CD5+ B cells in VAT were primarily containedwithin the B-1 B-cell subpopulation (TCRβ−CD19hiB220int)rather than the conventional B-2 B-cell subpopulation (TCRβ−CD19intB220hi) (Fig. 1C). Notably, approximately half of the B cellsin VAT expressed the surface phenotype of B-1 B cells, with aboutequal numbers of B-1a and B-1b B cells (Fig. 1C). The CD5+

B cells in VAT were mostly within the subpopulation of B cellsexpressing surface IgM (TCRβ−CD19+IgM+IgD− cells, hereafterreferred to as IgM+IgD− B cells), again resembling their coun-terparts in PerC and spleen (Fig. 1D). Roughly one-third of theCD5+ B cells in VAT were CD1dhi (TCRβ−CD19+CD5+CD1dhi

cells in Fig. 1E and Fig. S1A, hereafter referred to as CD5+CD1dhi

B cells), a surface phenotype previously described for regulatoryB10 cells in lymphoid organs (29). Finally, the vast majority ofCD5+ B cells in VAT were CD43+ (Fig. S1B) and CD11b+ (Fig.S1C). Therefore, the VAT-resident CD5+ B cells investigatedhere express a surface phenotype resembling innate-like B-1a Bcells and regulatory B10 B cells but are distinct from adiposenatural regulatory B cells that are CD5−/lowCD1dlow (19).One of the key functional features of innate-like B cells in

lymphoid tissues is their anti-inflammatory properties mediatedby production of anti-inflammatory mediators such as IL-10 ornatural IgM antibodies (14–16, 25, 26). We therefore examinedwhether the CD5+ B cells detected in VAT could function ina similar manner. Unlike adipose natural regulatory B cells (19),but resembling IL-10–producing B cells in lymphoid organs (12,16, 29), we were unable to detect intracellular IL-10 in freshlyprepared VAT CD5+ B cells (Fig. 1F, Left), and these cells failedto spontaneously produce (solid lines in Fig. 1F) or release IL-10(Fig. 1G, Left and Fig. S1D) in the absence of exogenous stimuliduring in vitro culture. Next, we stimulated these cells with eithera combination of phorbol 12-myristate 13-acetate (PMA), ion-omycin and Escherichia coli lipopolysaccharide (LPS) (PIL) (Fig.1F, dashed lines) or with LPS alone (Fig. 1G, Right and Fig.S1D), which represent methods that are frequently used toevaluate IL-10–producing B cells (12, 16, 29). Similar to theircounterparts in PerC and spleen, CD5+ B cells in VAT consti-tuted the majority of the IL-10–competent B cells as these cellsproduced IL-10 upon stimulation with PIL (Fig. 1F) or with LPSalone (Fig. 1G and Fig. S1D). Such a response was not detectedin B cells isolated from VAT of IL-10–deficient mice (Fig. S1E).Stimulation with LPS is of potential relevance to obesity, as itrepresents the effects of ligands for TLR4 that include saturatedfree fatty acid (30–32). Similar to their counterparts in PerC andspleen, freshly prepared VAT CD5+ B cells contained significantamounts of intracellular IgM (Fig. 1H) and spontaneously re-leased IgM during the in vitro cultures in the absence of exogenous

Wu et al. PNAS | Published online October 13, 2014 | E4639

IMMUNOLO

GYAND

INFLAMMATION

PNASPL

US

Dow

nloa

ded

by g

uest

on

Dec

embe

r 24

, 202

0

stimuli (Fig. 1I). Upon in vivo stimulation with LPS (21), innate-like B cells in VAT also produced granulocyte macrophage colony-stimulating factor (GM-CSF) (Fig. S1F).Together, the above results indicate that VAT contains a signifi-

cant proportion of innate-like B cells that spontaneously produceIgM. Many of these cells are also capable of producing IL-10 uponstimulation.

DIO Progressively Impairs the Number and IL-10 Competence ofInnate-Like B Cells in VAT and PerC. We next sought to investigatethe behavior of innate-like B cells during the development ofobesity. To examine whether expansion of VAT during obesityinfluenced these cells, we used the DIO mouse model, usinga high-fat diet (HFD) and a low-fat diet (LFD) (the compositionof the latter diet was comparable to the RCD, Table S1). We first

Fig. 1. Characterization of innate-like B cells in VAT. B6 mice at 10–15 wk of age on the RCD were used. (A–E) Cells were analyzed for the prevalence andnumber of innate-like B cells by flow cytometry. Representative plots or summary of the results from three independent experiments (n = 10–15 per group)are shown. (F) Cells were either freshly analyzed or cultured in the absence or presence of PMA, ionomycin, and LPS (PIL) for 5 h, and analyzed for intracellularIL-10 by flow cytometry. Representative plots from two independent experiments (n = 8–10 per group) are shown. (G) FACS-sorted B-cell subsets werecultured in the absence or presence of LPS for 40 h. IL-10 secreted during culture was examined by ELISPOT assay. Representative photographs from twoindependent experiments (n = 5 per group) are shown. (H) Freshly isolated cells were characterized for intracellular IgM level by flow cytometry. Repre-sentative plots from three independent experiments (n = 10–12 per group) are shown. (I) FACS-sorted B-cell subsets were cultured for 16 h. IgM secreted duringculture was examined by ELISPOT assay. Representative photographs from two independent experiments (n = 5 per group) are shown. ISO, isotype control.

E4640 | www.pnas.org/cgi/doi/10.1073/pnas.1324052111 Wu et al.

Dow

nloa

ded

by g

uest

on

Dec

embe

r 24

, 202

0

fed male B6 mice with the two diets for 12 wk. Mice on the HFDdeveloped overt obesity with markedly higher body weight ac-companied by significant enlargements in VAT, liver, and lung(Fig. S2A). As the organ weights increased, so did the overallnumbers of SVFs in the VAT, of leukocytes in the liver, and ofSLCs in the lung (Fig. S2B). In VAT, this caused an increase inthe number of SVFs per gram of fat tissue (Fig. S2C). However,rises in the total numbers of leukocytes were not reflected in thenumbers of innate-like B cells in VAT. Instead, the CD5+ B cellswere significantly reduced in both the prevalence and numbersper gram of tissue in VAT and PerC of obese mice (Fig. 2 A andB). Reductions in CD5+ B cells in VAT and PerC of obese micewere reflected in the overall numbers of B-1 B cells and in thenumbers of IgM+IgD− B cells (Fig. S2 D and E). The VAT ofobese mice also contained significantly reduced numbers ofCD5+CD1dhi B cells compared with lean mice (Fig. S2F). Suchnumerical impairments were specific to VAT and PerC becausethe numbers of these cells in the liver and lung remained com-parable between the two feeding groups (Fig. 2 A and B). TheCD5+ B cells in VAT and PerC of obese animals contained

a higher prevalence of proliferating cells than their counterpartsin lean mice (Fig. 2C). No significant differences in the preva-lence of overall dead cells among the CD5+ B cells in eitherVAT or PerC were observed between the two feeding groups (Fig.2D, Sum). However, the CD5+ B cells in VAT of obese mice hadsignificantly fewer early apoptotic cells (Fig. 2D, AV+PI− cells) butincreased numbers of dead cells that had lost their membraneintegrity (Fig. 2D, AV−PI+ cells). Because SVF preparation fromVAT involves enzyme digestion that is not used for collection ofPerC cells, these results might indicate that the CD5+ B cells inobese mice are susceptible to stress-induced cell death. Func-tionally, the CD5+ B cells from VAT and PerC of obese miceexhibited reduced capacity to produce IL-10 during the in vitrostimulation assays, as evidenced by the levels of intracellular (Fig.2E) and released (Fig. 2F) IL-10. Nevertheless, the CD5+ B cellsof obese mice maintained their capacity to produce IgM anti-bodies (Fig. S2G). These results indicate that expansion of VAT inobese mice is accompanied by decreases in the numbers and IL-10competence of innate-like B cells in VAT and PerC.

Fig. 2. DIO impairs the number and IL-10 competence of innate-like B cells in VAT. Mice were placed on the test diets for 12 wk (A–F) or 4 wk (G–I). (A, B, G,and H) Cells were analyzed for percentage and number of CD5+ B cells by flow cytometry. Summaries of three independent experiments (n = 10–15 pergroup) are shown. (C) Cells were examined for the proliferation marker Ki-67 by flow cytometry. Isotype control staining was performed but is omitted in thefigure for clarity. Summaries of two independent experiments (n = 8 per group) are shown. (D) Cells were examined for cell death by flow cytometry.Representative plots are shown for characterizing subsets of dead cells, and summaries are shown in the panels below the flow cytometry plots (n = 8 pergroup). (E and I) Cells were cultured in the absence or presence of PIL for 5 h and analyzed for intracellular IL-10 by flow cytometry. A summary of the resultsfor three independent experiments (n = 10–15 per group) is shown. (F) FACS-sorted CD5+ B cells were cultured in the presence of LPS for 40 h. IL-10 secretedduring culture was examined by ELISPOT assay. Statistical analyses are shown (n = 5 per group). *P < 0.01 and #P < 0.05.

Wu et al. PNAS | Published online October 13, 2014 | E4641

IMMUNOLO

GYAND

INFLAMMATION

PNASPL

US

Dow

nloa

ded

by g

uest

on

Dec

embe

r 24

, 202

0

To gain insight into the progression of innate-like B-cell im-pairment as obesity develops, we fed mice with the two diets for4 wk. Such a shortened HFD feeding caused detectable weightgain and enlargements of VAT (Fig. S2H). Leukocyte recruitmentto VAT appeared to lag behind the rise in organ weight. As such,the numbers of SVFs per gram of fat were significantly lower inVAT of obese mice after 4 wk of HFD feeding (Fig. S2 I and J). Atthis time, a reduction in the prevalence of CD5+ B cells was onlyevident in VAT, leading to reduced numbers of these cells pergram of VAT (Fig. 2 G and H). Although the prevalence of CD5+

B cells in PerC was not different between the HFD- and LFD-fedgroups, when normalized by the weight of VAT, the numbers ofthese cells were decreased at this location (Fig. 2 G and H).Similar to our observations after 12 wk of HFD feeding, thechanges in CD5+ B cells in VAT and PerC were reflected in thetotal population of B-1 B cells as well as in IgM+IgD− B cells(Fig. S2 K and L). At this stage of DIO, the IL-10 competenceof CD5+ B cells was only impaired in VAT of the HFD-fed mice(Fig. 2I).Together, the above results indicate that DIO progressively

impairs the number and IL-10 competence of innate-like B cellsin VAT and PerC.

Spleen Supports a Pool of Innate-Like B Cells in VAT and PerC DuringDIO. Considering the role of spleen in maintaining innate-like Bcells in PerC (22–24), we analyzed splenic CD5+ B cells duringDIO. In mice with overt obesity after 12 wk of HFD feeding,a significantly enlarged spleen housed increased numbers ofSPLs per gram of tissue (Fig. S3A). Different from our obser-vations in VAT and PerC described above, the prevalence ofsplenic CD5+ B cells (Fig. 3A, Left), B-1 B cells, IgM+IgD− Bcells, and CD5+CD1dhi B cells (Fig. S3B) was comparable be-tween the HFD- and LFD-fed groups. Taking into account theexpanded SPL cell pool, the obese mice had significantly moreCD5+ B cells per gram of spleen (Fig. 3A, Center). The observedchanges in spleen were somewhat reflected in peripheral blood,as the obese mice had more leukocytes (Fig. S3C) and a some-what higher number of CD5+ B cells per unit of blood (Fig. 3B).The HFD-induced changes in splenic CD5+B cells also appeared tobe progressive, as a 4-wk HFD feeding period induced less signifi-cant rises in the number of these cells (Fig. 3C, Left and Center andFig. S3D). We further examined the capacity of splenic CD5+ Bcells to produce IL-10. The results showed that these cells frommice with overt obesity after 12 wk of HFD feeding were at least ascompetent as their counterparts from lean mice in producing IL-10(Fig. 3A, Right). In mice fed the HFD for 4 wk, the splenic CD5+ Bcells appeared to be more competent in producing IL-10 comparedwith their counterparts from lean mice (Fig. 3C, Right). Together,these results suggest that CD5+ B cells expand in spleen duringDIO, and that these cells maintain their IL-10 competence.Our observation that CD5+ B cells tend to be overrepresented

in peripheral blood of obese mice, in conjunction with ourfinding that these cells are expanded in spleen, suggests thatspleen responds to dietary lipid excess to provide a supply ofthese cells to meet the demand in the expanding VAT. To ad-dress this possibility, we splenectomized male B6 mice, placedthem on the HFD, and subsequently analyzed immune cells inVAT. The surgical procedures did not influence the progressionof obesity. The weight gains of whole body and VAT were com-parable between splenectomized and sham-operated mice on theHFD (Fig. S3 E and H). Recruitment of leukocytes into VAT wasalso comparable between splenectomized and sham-operatedanimals (Fig. S3 F, G, I, and J). When the lymphocytes in VATwere analyzed, it became evident that absence of a spleen in thecontext of dietary lipid excess affected CD5+ B cells. Decreasesin these cells during DIO were accelerated by removal of spleen.After 4 wk of HFD feeding, when decreases in CD5+ B cellswere detectable only in VAT of spleen-sufficient mice (Fig. 2G),

these cells were also significantly reduced in PerC of splenec-tomized mice (Fig. 3D). These changes worsened with prolongedHFD feeding (Fig. 3E). Removal of spleen in HFD-fed mice didnot significantly affect the IL-10–producing capacity of remain-ing CD5+ B cells in VAT and PerC (Fig. 3F) nor did it signifi-cantly affect the prevalence of CD5+CD1dhi B cells in VAT (Fig.S3K), when the animals were examined after 10 wk of feeding.The impact of splenectomy on lymphocytes in VAT was specificto innate-like B cells, as the prevalence of other lymphocytepopulations, including total B and TCRαβ+ T cells, CD8+, andCD4+ T cells, and Treg cells, was not significantly different be-tween the splenectomized and sham-operated animals fed theHFD for 10 wk (Fig. 3G).These findings show that spleen responds to dietary lipid ex-

cess and expands innate-like B cells and thus serves as a sourcefor the recruitment of these cells to VAT. Our observation thatDIO-induced decreases in CD5+ B cells occurs earlier in VATthan PerC, together with our finding that removal of spleenaccelerates DIO-induced decreases in CD5+ B cells in PerC, isconsistent with the proposed role of spleen in supporting a poolof innate-like B cells in PerC that can be recruited to the

Fig. 3. CD5+ B cells in spleen and peripheral blood of DIO mice and theeffect of splenectomy. (A–C) B6 mice were placed on the test diets for 12 wk(A and B) or 4 wk (C). (A and C) Cells were analyzed for percentage andnumber of CD5+ B cells (Left and Center) or cultured in the absence orpresence of PIL for 5 h and analyzed for intracellular IL-10 by flow cytometry(Right). Summaries of three independent experiments (n = 10–15 per group)are shown. (B) PBLs were analyzed for percentage and number of CD5+ Bcells. A summary of the results for three independent experiments (n = 10–15 per group) is shown. (D–G) B6 mice underwent splenectomy or shamoperation and were placed on the HFD. (D and E) Mice were killed after 4 wkor 10 wk of feeding for analyses of CD5+ B cells. (F) Mice were killed after10 wk of feeding. Cells were cultured in the absence or presence of PIL for5 h and analyzed for intracellular IL-10 by flow cytometry. (G) Mice werekilled after 10 wk of feeding. SVFs were analyzed for lymphocytes by flowcytometry. Summaries of the results for three independent experiments(n = 9–12 per group) are shown. *P < 0.01 and #P < 0.05.

E4642 | www.pnas.org/cgi/doi/10.1073/pnas.1324052111 Wu et al.

Dow

nloa

ded

by g

uest

on

Dec

embe

r 24

, 202

0

expanding VAT. Together with our earlier observations thatDIO gradually results in a decrease in innate-like B cells in VATover time, these data suggest that demand for innate-like B cellsin the expanding VAT during obesity outpaces their recruitmentfrom spleen and PerC. The obese environment further impairsthe IL-10 competence of these cells and may compromise thesurvival of innate-like B cells in VAT.

Splenectomy Exacerbates DIO-Associated Insulin Resistance. To addressthe role of spleen-supplied innate-like B cells in obesity-inducedinsulin resistance, we placed splenectomized or sham-operatedmale B6 mice on the HFD and evaluated insulin sensitivity. Sple-nectomized and sham-operated mice gained similar body weightduring the entire 10-wk feeding period (Fig. 4A, Left), and theweights of VAT were comparable between the two groups whenkilled (Fig. 4A, Right). However, asplenic mice exhibited reducedinsulin sensitivity during an i.p. insulin tolerance test (IPITT)after 10 wk on the HFD (Fig. 4B). When killed after 10 wk ofHFD feeding, VAT of the asplenic mice accumulated increasednumbers of macrophages (CD11b+F4/80+ cells) (Fig. 4C, Left).Proinflammatory M1 macrophages, identified as CD11b+F4/80+

CD11c+ MHC-II+ cells (Fig. S4) were also increased (Fig. 4C,Right). VAT of the asplenic mice expressed reduced levels ofmRNA transcripts for the insulin-sensitizing adipokine adiponectin(Fig. 4D). However, we did not detect differences in the levels ofmRNA transcripts for TNF-α in VAT of HFD-fed mice betweenthe splenectomized and sham-operated groups (Fig. 4D). In theentire VAT, IL-10 mRNA transcripts were similar between the twotreatment groups, and mRNA transcripts for the proinflammatorycytokine IL-17A were not detectable in our assay (Fig. 4D). Thesestudies provide evidence that spleen-supplied innate-like B cellsafford protection against DIO-associated systemic insulin resistance.

Supplementation with Innate-Like B Cells Ameliorates DIO-AssociatedInsulin Resistance.We next investigated whether supplementationof innate-like B cells during the development of DIO impactsobesity-associated insulin resistance. For this purpose, we firstevaluated primary CD5+ B cells. We purified these cells frommale B6 mice fed the RCD and adoptively transferred them intoPerC of male B6 mice fed the HFD. In pilot experiments,transfer of 2 × 106 PerC B cells (collected from three donor

mice) into B-cell–deficient μMT mice (33) did not lead to sig-nificant numbers of B cells in PerC, spleen, or VAT when therecipients were examined 3 wk after the adoptive transfer (Fig.S5A). In wild-type (WT) B6 mice, significant numbers of thetransferred B cells, including CD5+ B cells, migrated to VATand this population could be further enlarged by increasing thenumber of donor cells (Fig. S5B). Under the setting of DIO,CD5+ B cells migrated more efficiently to VAT than spleen (Fig.S5B), which is consistent with the documented migration of in-nate-like B cells toward sites of inflammation (12, 21). In oursubsequent studies, we purified CD5+ B cells by fluorescence-activated cell sorting (FACS) (Fig. S6A) and performed twoadoptive transfers in each recipient animal fed the HFD (Fig.S6B). In our initial experiments, we transferred 5 × 105 CD5+ Bcells harvested from the PerC of three donor mice into eachrecipient mouse. This experimental manipulation did not in-fluence the progression of obesity (Fig. S6C). The transferredcells appeared to migrate from PerC into VAT, as the mice thatreceived transferred cells had more CD5+ B cells in VAT ratherthan in PerC (Fig. S6D). During the IPITT assay, we detecteda modest but statistically significant improvement in insulinsensitivity at the 15-min time point after insulin challenge in micethat received CD5+ B cells (Fig. S6E). Because increasing thenumber of donor cells could further enlarge the CD5+ B-cellpool in VAT, and because our findings suggested that spleenrepresents a source for the recruitment of CD5+ B cells intoVAT of DIO mice, we next combined CD5+ B cells purifiedfrom spleen and PerC and transferred 1.5 × 106 cells into eachrecipient animal and performed two adoptive transfers per re-cipient. To evaluate the role of IL-10, we included in this set ofexperiments donor cells purified from IL-10–deficient mice. Theresults showed that mice receiving CD5+ B cells from WT B6,but not IL-10–deficient, mice exhibited significantly improvedinsulin sensitivity during IPITT (Fig. 5B) and better glucosetolerance during an i.p. glucose tolerance test (IPGTT) (Fig.5C). Again, this experimental manipulation did not affect theprogression of obesity (Fig. 5A). The improved insulin sensitivitywas also reflected in blood insulin levels, as the mice receivingWT CD5+ B cells had lower fasting blood insulin than mice re-ceiving the vehicle control or IL-10–deficient CD5+ B cells (Fig.5D). When killed, the VAT of DIO mice that received WT CD5+

B cells expressed higher levels of adiponectin but low levels ofTNF-α (Fig. 5E). Supplementation of innate-like B cells in DIOmice did not alter the IL-10 mRNA transcript levels and IL-17AmRNA transcripts were not detectable in the entire VAT (Fig.5E), possibly due to dilution of the effects of CD5+ B cells onthese cytokines when the entire VAT is examined. As such, wenext performed coculture experiments. Consistent with theresults presented in Fig. 1 F andG, and Fig. S1D, enriched CD5+

B cells only released IL-10 in the presence of LPS (Fig. S6F).Addition of these cells to the in vitro cultured SVFs of obesemice influenced production of both IL-10 and IL-17A. Levels ofIL-10 in the culture supernatant were significantly increased withthe addition of innate-like B cells under both unstimulated andLPS-stimulated conditions (Fig. 5F). Whereas levels of IL-17A inthe culture supernatant were nearly undetectable in the absenceof stimulation, addition of innate-like B cells significantly de-creased LPS-stimulated IL-17A production (Fig. 5F).Because our earlier studies showed that VAT-resident CD5+

B cells spontaneously produce IgM and because both IgM-producing B-1a B cells and natural IgM antibodies were reportedto protect mice against atherosclerosis (25, 26), we also exam-ined whether IgM may contribute to the beneficial effects ofCD5+ B cells. We i.p. injected DIO mice with either vehicle ornormal mouse IgM at a dose of 0.4 mg per mouse per week fora total of 8 wk, using a protocol adapted from an atherosclerosisstudy (26), and evaluated insulin sensitivity (Fig. S6G). As shownin Fig. S6 H and I, this treatment regimen did not affect the

Fig. 4. Splenectomy exacerbates DIO-induced insulin resistance. B6 miceunderwent operation and were placed on the HFD. Body weight was mon-itored (A, Left). Perigonadal fat weight was examined at time of killing (A,Right). IPITT was examined after 10–12 wk of feeding (B). VAT was examinedfor macrophages by flow cytometry (C) and expression of cytokines/adipo-kines by real-time PCR (D). Summaries of the results for three independentexperiments (n = 12–15 per group) are shown. #P < 0.05.

Wu et al. PNAS | Published online October 13, 2014 | E4643

IMMUNOLO

GYAND

INFLAMMATION

PNASPL

US

Dow

nloa

ded

by g

uest

on

Dec

embe

r 24

, 202

0

progression of obesity nor did it influence insulin sensitivity inHFD-fed mice. Together with our earlier observations thatCD5+ B cells in VAT of DIO mice maintained their capacity toproduce IgM and that CD5+ B cells of IL-10–deficient micefailed to protect against insulin resistance during DIO, ourstudies point toward a dominant role of IL-10 in mediating thebeneficial effects of CD5+ B cells on insulin sensitivity.IL-10–competent CD5+ B cells can be ex vivo expanded from

naïve splenic B cells under conditions favoring B10 cell differenti-ation (34, 35). These ex vivo expanded CD5+ B cells are protectivein a mouse model of multiple sclerosis, a chronic inflammatorydisease of the central nervous system (35). Because our earlierstudies indicated that the decreases in CD5+B cells in VAT of DIOmice were primarily due to insufficient supply, our final experimentsevaluated the therapeutic potential of this ex vivo approach. In ourexperimental conditions, ex vivo expanded CD5+ B cells but not

CD5− B cells increased intracellular IL-10 after stimulation withPIL (Fig. S7). We then FACS sorted CD5+ B cells and CD5− Bcells and transferred them into PerC of DIO mice following ourprotocol used for primary CD5+ B cells (Fig. S6B). Similar to theresults for primary B cells, transfer of ex vivo expanded B cells didnot affect body weight gain or VAT expansion (Fig. 5G), but im-proved insulin sensitivity in DIO mice (Fig. 5 H and I).Collectively, these findings indicate a critical role of IL-10

production for the protective effects of innate-like B cells inDIO-associated insulin resistance.

DiscussionIn this report, we describe a previously unrecognized group ofinnate-like B cells in VAT. These cells express phenotypicmarkers resembling B-1a and regulatory B10 B cells found inother anatomic locations but are distinct from the recently

Fig. 5. Supplementation of innate-like B cells ameliorates DIO-induced insulin resistance. (A–E) Mice on the HFD were adoptively transferred with vehicle orFACS-sorted cells. Body weight was monitored (A, Left), and perigonadal fat weight was examined at time of killing (A, Right). IPITT, IPGTT, and fasting levelof blood insulin were examined after 10–12 wk of feeding (B–D). VAT was analyzed for expression of cytokines/adipokines by real-time PCR (E). Summaries ofthe results for two independent experiments (n = 7–8 per group) are shown. (F) SVFs of mice on the HFD for 12 wk were cocultured with or without PerC Bcells purified from mice on the RCD in the absence (left scales) or presence (right scales) of LPS. Cytokines in the culture supernatant were examined.Summaries of the results for two independent experiments (n = 8 per group) are shown. (G–I) Mice on the HFD were transferred with vehicle or FACS-sortedcells. Body weight was monitored (G, Left) and perigonadal fat weight was examined at time of killing (G, Right). IPITT (H) and fasting blood insulin (I) wereexamined after 10–12 wk of feeding. Summaries of the results for two independent experiments (n = 9–10 per group) are shown. *P < 0.01 and #P < 0.05.

E4644 | www.pnas.org/cgi/doi/10.1073/pnas.1324052111 Wu et al.

Dow

nloa

ded

by g

uest

on

Dec

embe

r 24

, 202

0

identified adipose natural regulatory B cells (19) and are com-petent in producing the anti-inflammatory cytokine IL-10. UsingHFD feeding of mice to model obesity in humans, we haveshown that dietary lipid excess progressively decreases thenumber and IL-10–producing capacity of these cells in VAT. Onthe other hand, dietary lipid excess promotes expansion of thesecells in the spleen. Our findings indicate that spleen serves, atleast in part, as a source for the recruitment of innate-like B cellsto VAT of obese mice. In the expanding VAT, the demand forinnate-like B cells appears to outpace their recruitment. Fur-thermore, the obese environment in VAT impairs their IL-10competence and may also compromise their survival. The netresult of this process is a diminished protection of innate-like Bcells against insulin resistance in DIO mice. However, supple-mentation of such B cells during the development of obesity canpartially ameliorate insulin resistance. The present study pri-marily examined innate-like B cells in VAT, and additional workis needed to address whether these cells also function in otheranatomical locations that collectively achieve their beneficialeffects during DIO.Multiple cell types of the immune system participate in obe-

sity-triggered inflammation, contributing either positively ornegatively to obesity-associated insulin resistance (1–7). Our find-ings have identified a group of innate-like B cells as previouslyunrecognized players in curbing the insulin-antagonizing envi-ronment in VAT of obese mice. Our studies therefore placethese cells within the complex interplay between immune andparenchymal cells during the development of obesity. Ourstudies have further demonstrated an active involvement of thespleen in maintaining such innate-like B cells in VAT of obesemice, thus highlighting the role of this secondary lymphoid organin obesity-associated abnormalities. Previous studies by otherinvestigators have implicated a beneficial role of spleen-derivedIL-10 in protecting mice against DIO-mediated inflammation(27), and additional studies have demonstrated a reduced ca-pacity of unfractionated B cells from obese mice or patients withT2D to produce IL-10 (10, 28). Our results advance theseobservations by identifying a set of spleen-supplied innate-like Bcells in VAT as an important source of IL-10 to ameliorate theinsulin-antagonizing environment in obese mice. In the previousstudies investigating the role of B cells in obesity and insulinresistance (9, 10), the contribution of innate-like B cells waslikely underestimated, due to experimental procedures that en-rich conventional B2 B cells but deplete innate-like B cells basedon cell surface expression of CD43 (29). Our findings also raisesome caution for developing immunotherapies of obesity-asso-ciated diseases based on B-cell depletion (9), as they may bringalong unintended consequences.Accumulating evidence demonstrates an active yet complex

role of the spleen in immune responses combating infections andin inflammatory reactions occurring in sterile inflammatory dis-eases (21, 22, 36–38). The lymphoid and myeloid cell progenitorsthat are housed in and/or are mobilized to adult mouse spleenrapidly proliferate upon sensing stimuli to produce effector cellsthat subsequently migrate to sites of inflammation (21, 22, 36–38). In addition to supporting a pool of innate-like B cells, extra-medullary hematopoiesis in spleen supplies monocytes that canfurther differentiate into tissue macrophages (36–38). In ourstudies using splenectomy, we observed a progressive increase inM1 macrophages but a gradual decrease in Ly6Chi monocytes inVAT of asplenic DIO mice (Figs. S4 and S8A). The latter cellshave been previously detected in VAT (39). The influence ofsplenectomy on monocytes and macrophages appeared to trailits effects on innate-like B cells in VAT (Fig. 3 D and E and Fig.S8A). Further studies are needed to examine whether such acomplex role of spleen in supporting different lineages of immunecells in VAT could have contributed to the lack of differences inTNF-α expression observed between splenectomized and sham-

operated DIO mice. In our adoptive transfer experiments, althoughwe failed to detect a reduction in the numbers of CD11b+F4/80+

macrophages in VAT of DIO mice after receiving WT CD5+

B cells, we observed a modest increase in Ly6Chi monocytes ac-companied by a decrease in M1 macrophages in VAT (Fig. S8B).Putting together our observations from the splenectomy andadoptive transfer studies, one possible scenario is that spleen-supplied innate-like B cells inhibit the transition of monocytesto M1 macrophages, thus reducing the inflammatory environ-ment in VAT during obesity.The specific stimuli and signaling pathways that expand

splenic innate-like B cells and mobilize them to VAT duringdietary lipid excess remain unknown. Innate-like B cells respondto TLR agonists, including ligands for TLR2 and TLR4 (14–16).It is therefore tempting to speculate that dietary lipid excesspromotes expansion of these cells through binding of HFD-supplied saturated fatty acids with these pattern recognitionreceptors (30, 40). B-cell–activating factor (BAFF), which can beproduced by VAT and is elevated in obesity (41, 42), inducesexpansion of innate-like B cells in spleen (34, 43) and thus couldalso play a role in controlling the activation and function of thesecells in VAT during obesity. Understanding the mechanisms bywhich an obese environment in VAT impairs the IL-10 compe-tence and survival of innate-like B cells will aid the developmentof novel therapeutic interventions in obesity. The interactionsbetween innate-like B cells and Treg cells during inflammatoryconditions (14, 16, 44, 45), in conjunction with their impairmentin VAT of DIO mice (the present work and refs. 11, 19), raisethe possibility that these two populations are mechanistically linkedduring the development of obesity-associated inflammation andmetabolic diseases. Further studies are needed to decipher thecomplex interactions among different lineages of immune cells andbetween immune cells and parenchymal cells in VAT, during thedevelopment of obesity. The results from our in vitro cocultureexperiments indicate a regulatory role of innate-like B cells in theproduction of cytokines by SVF cells that are relevant to obesity-triggered inflammation. Our future investigations will explore therelative contribution of different cell types in VAT that are in-volved in this process. In this context, we have previously identifieda pathogenic role of innate-like invariant natural killer T (iNKT)cells in obesity-triggered inflammation and obesity-associated in-sulin resistance (46). Our preliminary studies have suggested thatthe presence of iNKT cells in DIO mice exacerbates the abnor-malities observed for innate-like B cells in VAT.In conclusion, our studies have identified a group of spleen-

supplied innate-like B cells in VAT as previously unrecognizedplayers in curbing the insulin-antagonizing environment duringobesity. Therefore, our findings point to these cells as new targetsfor therapeutic intervention in obesity-associated inflammation andinsulin resistance.

Materials and MethodsMice and Diets. We used male B6 mice (CD45.2+) that were either purchasedfrom The Jackson Laboratory or bred in the animal facility at VanderbiltUniversity Medical Center from breeding pairs originally purchased from TheJackson Laboratory (stock no. 000664). For distinguishing donor cells fromrecipient cells in the adoptive transfer experiments, we used CD45.1+ micefrom The Jackson Laboratory (stock no. 002014) to prepare donor cells andtransferred them into CD45.2+ WT B6 or μMT mice on the B6 background.IL-10 mutant mice (stock no. 002251) and B-cell–deficient μMT mice (stockno. 002288) were originally purchased from The Jackson Laboratory. For dietarymanipulation, mice at 5–6 wk of age were placed on the test diets for thedurations indicated in Results and in the figure legends. The compositionsand manufacturers of the test diets are listed in Table S1. For surgicaltreatment followed by dietary manipulation, mice at 4–5 wk of age un-derwent splenectomy or sham operation and were placed on the test diets1 wk later for the durations described in Results and in the figure legends. Inthe adoptive transfer experiments, mice at 5–6 wk of age were placed on theHFD for 2 wk and then received transfers of cells while maintained on the

Wu et al. PNAS | Published online October 13, 2014 | E4645

IMMUNOLO

GYAND

INFLAMMATION

PNASPL

US

Dow

nloa

ded

by g

uest

on

Dec

embe

r 24

, 202

0

HFD. Animals age matched between the experimental and control groupswere used in each experiment and were housed under specific pathogen-freeconditions. The experimental procedures were approved by the InstitutionalAnimal Care and Use Committee at Vanderbilt University Medical Center.

Reagents. We used fluorescently labeled antibodies purchased from BDBiosciences or eBioscience. These included antibodies against mouse TCR-β,CD90, CD19, B220, CD5, CD43, CD21, CD23, CD1d, CD11b, CD11c, CD45.1,CD45.2, F4/80, IgM, IgD, Ly-6C, I-Ab, Ly-6G, PanNK, NK1.1, IL-10, CD45, GM-CSF, Ki-67, and appropriate isotype controls for surface and intracellularlabeling. Viability staining solutions 7-amino-actinomycin D (7AAD) andpropidium iodide (PI) were from eBioscience. PMA, ionomycin, LPS, DNase I,and collagenase type IV were obtained from Sigma-Aldrich. Collagenasetype I was from Worthington Biochemical Corp. Reagents for cell culturewere from either Invitrogen Life Technologies or Mediatech. Other reagentsare described below in each specific assay.

Cell Preparation. We analyzed cells purified from perigonadal fat pads, PerCwash, spleen, liver, lung, and peripheral blood simultaneously in each mouse.Purification of SVFs from VAT, IHLs from liver, and SPLs from spleen wasperformed as previously described (46). SLC suspensions were prepared bydigesting minced lung tissue with 1 mg/mL collagenase IV and 10 units/mLDNase I dissolved in RPMI medium 1640 at 37 °C for 1 h, followed by washingto remove enzymes. PerC was washed with PBS containing 5% (vol/vol) FBS(Life Technologies) to collect PerC cells. Peripheral blood was collected by heartpuncture with EDTA as anticoagulant. Peripheral blood leukocytes (PBLs) wereprepared by gradient centrifugation using Ficoll-Paque (GE Healthcare LifeSciences). Red blood cells were lysed using ACK buffer (Lonza).

Surface and Intracellular Labeling and Flow Cytometry Analyses. We per-formed surface and intracellular labeling as previously described (46). Weexamined innate-like B cells by labeling their surface markers. CD5+ B cellswere characterized as TCRβ−CD19+CD5+ cells. We quantified B-1 B cells asTCRβ−CD19hiB220int cells and B-2 B cells as TCRβ−CD19intB220hi cells (12). B-1B cells were further divided as B-1a B cells (TCRβ−CD19hiB220int CD5+) and B-1b B cells (TCRβ−CD19hiB220int CD5−). Total αβ T cells, total B cells, CD8+

T cells, CD4+ T cells, and CD4+CD25+Foxp3+ Treg cells in VAT were analyzedand quantified as previously described (46). Macrophages in VAT were iden-tified as CD11b+F4/80+ cells as previously described (46). M1 macrophageswere identified as Lin−CD11b+F4/80+CD11c+I-Ab+Ly-6C−/lo cells, and Ly-6Chi

monocytes were identified as Lin−CD11b+F4/80−CD11c−I-Ab−Ly-6Chi cells, whereLin represents a mixture of antibodies against CD90, B220, PanNK, NK1.1,and Ly-6G (36). Intracellular staining of IL-10 was performed as described (29,46) on either freshly prepared cells or after in vitro stimulation (see below). Forintracellular staining of IgM, freshly prepared cells were first surface stainedwith anti-CD19, -IgD, and -IgM antibodies to gate out IgM+IgD− B cells, fixed,and permeabilized with Cytofix/Cytoperm (BD Biosciences), and stained forintracellular IgM using the anti-IgM antibody conjugated to a different fluo-rochrome. For intracellular staining of Ki-67 on freshly prepared cells, we usedthe anti-mouse Ki-67 antibody from eBioscience following the manufacturer’sprotocol. Annexin V Apoptosis Detection kit (eBioscience) was used to quantifycell death on freshly prepared cells following the manufacturer’s protocol.Flow cytometry was performed on either a FACSCalibur or LSR II (BD Bio-sciences; Immunocytometry Systems). The acquired data were analyzed usingFlowJo software (Tree Star).

Isolation of B-Cell Subsets and Stimulation Assays. Total B cells were purifiedfrom splenocytes or PerC cells by positive selection using mouse CD19microbeads and ancillary columns (Miltenyi Biotec) following the manu-facturer’s protocol. We isolated subsets of B cells from splenic CD19+ B cells,PerC cells, or SVFs by FACS using BD FACSAria cell sorter (BD Biosciences). Weperformed the in vitro stimulation assays as described (10, 29) to examinethe capacity of innate-like B cells to produce IL-10. For intracellular stainingof IL-10, we analyzed SVFs, PerC cells, and splenocytes simultaneously in eachmouse. Briefly, purified cells (1–1.5 × 106 cells) were added into each well ofa 96-well round-bottomed tissue culture plate. Cells were cultured for 5 h inthe presence of GolgiStop (BD Biosciences) at 37 °C in a humidified cellculture incubator with 5% CO2. The unstimulated samples were cultured withGolgiStop alone, and the stimulated samples were cultured with GolgiStopplus PMA (50 ng/mL), ionomycin (500 ng/mL), and LPS (10 μg/mL). At the end ofculture, cells were harvested for surface and intracellular labeling as describedabove. To measure secreted IL-10 by ELISPOT, purified subsets of B cells fromVAT or PerC (25,000 cells) were added into each well of an ELISPOT plate(Millipore) and cultured in the absence or presence of LPS (1 μg/mL) for 40 h at37 °C in a humidified cell culture incubator with 5% CO2. IL-10 released during

the culture was subsequently detected by ELISPOT using the Mouse IL-10ELISPOT Pair and its ancillary reagents from BD Biosciences following themanufacturer’s protocol. To measure spontaneous secretion of IgM by ELISPOT,purified subsets of B cells (7,500 cells per well) were cultured for 16 h as de-scribed above in the absence of LPS. Released IgM was detected using theMouse IgM ELISpotplus kit (Mabtech) following the manufacturer’s pro-tocol. The stained spots were examined using an ImmunoSpot Analyzer (CTL).We followed a published protocol (21) to examine GM-CSF production insubsets of B cells. We injected vehicle (PBS) or LPS dissolved in vehicle (10 μgper mouse) into PerC and killed the mice 4 d after the treatment. Freshlyprepared SVF cells were analyzed for GM-CSF in subsets of B cells by surfaceand intracellular labeling as described above, followed by flow cytometry.

Splenectomy and Sham Operation. We performed splenectomy and shamoperations as described (36). Briefly, mice at 4–5 wk of age underwent the op-eration under anesthesia. The abdomen of the mouse was opened and thespleen was exposed. The blood vessels were cauterized, and the spleen wascarefully removed. For sham operation, the abdomen was opened, but thespleen was not removed. The abdomen was closed by suturing first the musclelayer and then the skin layer. Mice were allowed to recover for 1 wk in auto-claved cages with autoclaved food and water before any further manipulation.

Ex Vivo Expansion of CD5+ B Cells.We ex vivo expanded CD5+ and CD5− B cellsas described (34, 35). Male B6 mice on the RCD were used to purify splenicCD19+ B cells by positive selection as described above. Total splenic B cellswere cultured on a confluent layer of γ-ray irradiated feeder cells expressingCD40-ligand (CD40L) and BAFF (40LB cells) in the presence of mouserecombinant IL-4 (eBioscience) for 4 d and recultured on new feeder cells foranother 4 d in the presence of mouse recombinant IL-21 (eBioscience). Cellswere then analyzed by surface and intracellular labeling followed by flowcytometry. Alternatively, cells were surface labeled and FACS sorted intoCD5+ and CD5− B cells using BD FACSAria cell sorter.

Adoptive Transfer of B Cells. To track transferred cells, we used donor cellsfrom CD45.1+ mice fed the RCD. We prepared positively selected CD19+ Bcells from PerC wash and injected them i.p. into CD45.2+ mice fed the HFD,and analyzed the recipients 3 wk later. For transfer of primary CD5+ B cells,we harvested donor cells from PerC and spleen of WT B6 or IL-10–deficientmice. Positively selected CD19+ splenic B cells or PerC cells were surface la-beled and FACS sorted, washed with PBS, and injected i.p. into recipientmice fed the HFD. FACS-sorted CD5+ and CD5− B cells expanded ex vivo weretransferred similarly.

IgM Treatment. We purchased normal mouse IgM whole molecule fromRockland Immunochemicals. The antibody was dialyzed against PBS, filteredto sterilize, and i.p. injected into mice on the HFD at a dose of 0.4 mg permouse per week for 8 wk.

Cell Cocultures and Cytokine Measurements. We cocultured SVFs with PerC-derived CD19+ B cells and examined cytokine secretion. Briefly, we preparedSVFs as described above from DIO mice fed the HFD for 12 wk. SVF cells werefurther purified by gradient centrifugation using Percoll from GE Healthcare(46). CD19+ B cells were positively selected from PerC wash of B6 mice fedthe RCD as described above. SVF and B cells were cocultured at differentratios in U-bottomed 96-well plates at 37 °C in a humidified cell culture in-cubator with 5% CO2 in the absence or presence of LPS (1 μg/mL) for dif-ferent durations. Supernatant was collected at the end of culture and storedat −80 °C. Cytokines in the supernatant were assayed using the BD Cyto-metric Bead Array Mouse Th1/Th2/Th17 Cytokine kit (BD Biosciences) fol-lowing the manufacturer’s protocol.

Analysis of Metabolic Parameters. Metabolic parameters were examined asdescribed previously (46). Body weight was measured between 9:00 and 10:00AM. We performed IPITT and IPGTT on mice that had been fed the HFD for 10–12 wk. For IPITT, mice were fasted for 6 h, examined for blood glucose throughsaphenous vein sampling, received an i.p. injection of Novolin R (Novo Nordisk)at 2 units per kilogram of body weight, followed by further glucose sampling at15, 30, 60, and 120 min. For IPGTT, mice were fasted for 16 h and examined forblood glucose as described for IPITT, using an i.p. injection of glucose at 1 g perkilogram of body weight. We fasted mice for 16 h and examined the level ofblood insulin as previously described (46). Mice were killed at nonfasting statewith body and organ weights measured.

E4646 | www.pnas.org/cgi/doi/10.1073/pnas.1324052111 Wu et al.

Dow

nloa

ded

by g

uest

on

Dec

embe

r 24

, 202

0

Quantitative Real-Time PCR. At the time of killing, VAT was snap frozen inliquid nitrogen and stored at −80 °C. We measured mRNA levels of TNF-α,adiponectin, and IL-10 as described (46). We used the previously publishedprimer sequences for measurement of IL-17A mRNA transcripts (47). Real-time PCR was performed using SYBR green in a MyiQ2 instrument (Bio-Rad)as described (46).

Statistical Analyses. Data are presented as means ± SEM. Statistical anal-yses were performed using unpaired two-tailed t test after examiningdistribution normality of the data. A P value of <0.05 was consideredstatistically significant.

ACKNOWLEDGMENTS. We thank Dr. Daniel Moore for μMT mice andDr. Feng Yang for IL-10 mutant mice. This work was supported by a JuniorFaculty Award from the American Diabetes Association (to L.W.), a Pilot andFeasibility grant from the Diabetes Research and Training Center at Vander-bilt (to L.W.), National Institutes of Health (NIH) Grants DK081536 (to L.W.and L.V.K.), AI070305 (to L.V.K.), and HL089667 (to L.V.K.). J.H. was a partic-ipant in the NIH-sponsored Vanderbilt Student Research Training Program(T35 DK07383-27). FACS sorting was performed in the Flow CytometryShared Resource at Vanderbilt University Medical Center, supported by theVanderbilt Ingram Cancer Center (P30 CA68485) and the Vanderbilt Diges-tive Disease Research Center (DK058404).

1. Osborn O, Olefsky JM (2012) The cellular and signaling networks linking the immune

system and metabolism in disease. Nat Med 18(3):363–374.2. Lumeng CN, Saltiel AR (2011) Inflammatory links between obesity and metabolic

disease. J Clin Invest 121(6):2111–2117.3. Gregor MF, Hotamisligil GS (2011) Inflammatory mechanisms in obesity. Annu Rev

Immunol 29:415–445.4. Mathis D, Shoelson SE (2011) Immunometabolism: An emerging frontier. Nat Rev

Immunol 11(2):81.5. Odegaard JI, Chawla A (2013) Pleiotropic actions of insulin resistance and in-

flammation in metabolic homeostasis. Science 339(6116):172–177.6. Odegaard JI, Chawla A (2013) The immune system as a sensor of the metabolic state.

Immunity 38(4):644–654.7. Jin C, Henao-Mejia J, Flavell RA (2013) Innate immune receptors: Key regulators of

metabolic disease progression. Cell Metab 17(6):873–882.8. Nishimura S, et al. (2009) CD8+ effector T cells contribute to macrophage recruitment

and adipose tissue inflammation in obesity. Nat Med 15(8):914–920.9. Winer DA, et al. (2011) B cells promote insulin resistance through modulation of T

cells and production of pathogenic IgG antibodies. Nat Med 17(5):610–617.10. DeFuria J, et al. (2013) B cells promote inflammation in obesity and type 2 diabetes

through regulation of T-cell function and an inflammatory cytokine profile. Proc Natl

Acad Sci USA 110(13):5133–5138.11. Feuerer M, et al. (2009) Lean, but not obese, fat is enriched for a unique population

of regulatory T cells that affect metabolic parameters. Nat Med 15(8):930–939.12. Baumgarth N (2011) The double life of a B-1 cell: Self-reactivity selects for protective

effector functions. Nat Rev Immunol 11(1):34–46.13. Brennan PJ, Brigl M, Brenner MB (2013) Invariant natural killer T cells: An innate

activation scheme linked to diverse effector functions. Nat Rev Immunol 13(2):101–117.

14. Vadasz Z, Haj T, Kessel A, Toubi E (2013) B-regulatory cells in autoimmunity andimmune mediated inflammation. FEBS Lett 587(13):2074–2078.

15. Kalampokis I, Yoshizaki A, Tedder TF (2013) IL-10-producing regulatory B cells (B10

cells) in autoimmune disease. Arthritis Res Ther 15(Suppl 1):S1.16. Mauri C, Bosma A (2012) Immune regulatory function of B cells. Annu Rev Immunol

30:221–241.17. Vantourout P, Hayday A (2013) Six-of-the-best: Unique contributions of γδ T cells to

immunology. Nat Rev Immunol 13(2):88–100.18. Wu L, Van Kaer L (2013) Contribution of lipid-reactive natural killer T cells to obesity-

associated inflammation and insulin resistance. Adipocyte 2(1):12–16.19. Nishimura S, et al. (2013) Adipose natural regulatory B cells negatively control adi-

pose tissue inflammation. Cell Metab. pii: S1550-4131(13)00386-0.20. Cerutti A, Cols M, Puga I (2013) Marginal zone B cells: Virtues of innate-like antibody-

producing lymphocytes. Nat Rev Immunol 13(2):118–132.21. Rauch PJ, et al. (2012) Innate response activator B cells protect against microbial

sepsis. Science 335(6068):597–601.22. Ghosn EE, Sadate-Ngatchou P, Yang Y, Herzenberg LA, Herzenberg LA (2011) Distinct

progenitors for B-1 and B-2 cells are present in adult mouse spleen. Proc Natl Acad Sci

USA 108(7):2879–2884.23. Wardemann H, Boehm T, Dear N, Carsetti R (2002) B-1a B cells that link the innate and

adaptive immune responses are lacking in the absence of the spleen. J Exp Med

195(6):771–780.24. Kretschmer K, Stopkowicz J, Scheffer S, Greten TF, Weiss S (2004) Maintenance of

peritoneal B-1a lymphocytes in the absence of the spleen. J Immunol 173(1):197–204.

25. Kyaw T, et al. (2011) B1a B lymphocytes are atheroprotective by secreting natural IgMthat increases IgM deposits and reduces necrotic cores in atherosclerotic lesions. CircRes 109(8):830–840.

26. Cesena FH, et al. (2012) Immune-modulation by polyclonal IgM treatment reducesatherosclerosis in hypercholesterolemic apoE-/- mice. Atherosclerosis 220(1):59–65.

27. Gotoh K, et al. (2012) A novel anti-inflammatory role for spleen-derived interleukin-10in obesity-induced inflammation in white adipose tissue and liver. Diabetes 61(8):1994–2003.

28. Jagannathan M, et al. (2010) Toll-like receptors regulate B cell cytokine production inpatients with diabetes. Diabetologia 53(7):1461–1471.

29. Matsushita T, Tedder TF (2011) Identifying regulatory B cells (B10 cells) that produceIL-10 in mice. Methods Mol Biol 677:99–111.

30. Shi H, et al. (2006) TLR4 links innate immunity and fatty acid-induced insulin re-sistance. J Clin Invest 116(11):3015–3025.

31. Lee JY, Sohn KH, Rhee SH, Hwang D (2001) Saturated fatty acids, but not unsaturatedfatty acids, induce the expression of cyclooxygenase-2 mediated through Toll-likereceptor 4. J Biol Chem 276(20):16683–16689.

32. Kim F, et al. (2007) Toll-like receptor-4 mediates vascular inflammation and insulinresistance in diet-induced obesity. Circ Res 100(11):1589–1596.

33. Kitamura D, Roes J, Kühn R, Rajewsky K (1991) A B cell-deficient mouse by targeteddisruption of the membrane exon of the immunoglobulin mu chain gene. Nature350(6317):423–426.

34. Nojima T, et al. (2011) In-vitro derived germinal centre B cells differentially generatememory B or plasma cells in vivo. Nat Commun 2:465.

35. Yoshizaki A, et al. (2012) Regulatory B cells control T-cell autoimmunity through IL-21-dependent cognate interactions. Nature 491(7423):264–268.

36. Swirski FK, et al. (2009) Identification of splenic reservoir monocytes and their de-ployment to inflammatory sites. Science 325(5940):612–616.

37. Robbins CS, et al. (2012) Extramedullary hematopoiesis generates Ly-6C(high) mon-ocytes that infiltrate atherosclerotic lesions. Circulation 125(2):364–374.

38. Dutta P, et al. (2012) Myocardial infarction accelerates atherosclerosis. Nature487(7407):325–329.

39. Cipolletta D, et al. (2012) PPAR-γ is a major driver of the accumulation and phenotypeof adipose tissue Treg cells. Nature 486(7404):549–553.

40. Nguyen MT, et al. (2007) A subpopulation of macrophages infiltrates hypertrophicadipose tissue and is activated by free fatty acids via Toll-like receptors 2 and 4 andJNK-dependent pathways. J Biol Chem 282(48):35279–35292.

41. Alexaki VI, et al. (2009) Adipocytes as immune cells: Differential expression of TWEAK,BAFF, and APRIL and their receptors (Fn14, BAFF-R, TACI, and BCMA) at differentstages of normal and pathological adipose tissue development. J Immunol 183(9):5948–5956.

42. Hamada M, et al. (2011) B cell-activating factor controls the production of adipokinesand induces insulin resistance. Obesity 19(10):1915–1922.

43. Yang M, et al. (2010) Novel function of B cell-activating factor in the induction ofIL-10-producing regulatory B cells. J Immunol 184(7):3321–3325.

44. Singh A, et al. (2008) Regulatory role of B cells in a murine model of allergic airwaydisease. J Immunol 180(11):7318–7326.

45. Flores-Borja F, et al. (2013) CD19+CD24hiCD38hi B cells maintain regulatory T cellswhile limiting TH1 and TH17 differentiation. Sci Trans Med 5(173):173ra123.

46. Wu L, et al. (2012) Activation of invariant natural killer T cells by lipid excess promotestissue inflammation, insulin resistance, and hepatic steatosis in obese mice. Proc NatlAcad Sci USA 109(19):E1143–E1152.

47. Lee PH, et al. (2014) The transcription factor E74-like factor 4 suppresses differenti-ation of proliferating CD4+ T cells to the Th17 lineage. J Immunol 192(1):178–188.

Wu et al. PNAS | Published online October 13, 2014 | E4647

IMMUNOLO

GYAND

INFLAMMATION

PNASPL

US

Dow

nloa

ded

by g

uest

on

Dec

embe

r 24

, 202

0