Gastroenterology 2014;147:1106 BASIC AND TRANSLATIONAL ... 2014_Criscimanna.pdf · Methods Mice Mice used in this study were maintained according to protocols approved by the University

Gastroenterology 2014;147:1106–1118

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BASIC AND TRANSLATIONAL—PANCREAS

Activated Macrophages Create Lineage-SpecificMicroenvironments for Pancreatic Acinar- andb-Cell Regeneration in Mice
Angela Criscimanna,1 Gina M. Coudriet,1 George K. Gittes,1 Jon D. Piganelli,1
and Farzad Esni1,2,3,4

1Department of Surgery, Division of Pediatric General and Thoracic Surgery, Children’s Hospital of Pittsburgh, University ofPittsburgh Medical Center, Rangos Research Center, Pittsburgh, Pennsylvania; 2Department of Developmental Biology,University of Pittsburgh, Pittsburgh, Pennsylvania; 3Department of Microbiology and Molecular Genetics, University ofPittsburgh, Pittsburgh, Pennsylvania; and 4University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania

BACKGROUND & AIMS: Although the cells that contribute topancreatic regeneration have been widely studied, little isknown about the mediators of this process. During tissueregeneration, infiltrating macrophages debride the site of injuryand coordinate the repair response. We investigated the role ofmacrophages in pancreatic regeneration in mice. METHODS:We used a saporin-conjugated antibody against CD11b toreduce the number of macrophages in mice following diph-theria toxin receptor–mediated cell ablation of pancreatic cells,and evaluated the effects on pancreatic regeneration. Weanalyzed expression patterns of infiltrating macrophages aftercell-specific injury or from the pancreas of nonobese diabeticmice. We developed an in vitro culture system to study theability of macrophages to induce cell-specific regeneration.RESULTS: Depletion of macrophages impaired pancreaticregeneration. Macrophage polarization, as assessed by expres-sion of tumor necrosis factor�a, interleukin 6, interleukin 10,and CD206, depended on the type of injury. The signals pro-vided by polarized macrophages promoted lineage-specificgeneration of acinar or endocrine cells. Macrophage fromnonobese diabetic mice failed to provide signals necessary forb-cell generation. CONCLUSIONS: Macrophages produce celltype–specific signals required for pancreatic regeneration inmice. Additional study of these processes and signals mightlead to new approaches for treating type 1 diabetes orpancreatitis.

Keywords: Macrophage Polarization; Tissue Damage; Pancrea-titis; Diabetes; NOD.

acrophages are monocyte-derived myeloid cells

Abbreviations used in this paper: ADM, acinar-to-ductal metaplasia; DTR,diphtheria toxin receptor; Fizz1, found in inflammatory zone 1; IL, inter-leukin; Mac1-SAP, saporin-conjugated anti-Mac1 antibody; NOD, non-obese diabetic; T1D, type 1 diabetes; Tgf, transforming growth factor;TNF, tumor necrosis factor.

© 2014 by the AGA Institute0016-5085/$36.00

http://dx.doi.org/10.1053/j.gastro.2014.08.008

Mthat develop from a common myeloid progenitorcell residing within the bone marrow of adult mammals.1

Upon tissue damage or infection, monocytes are rapidlyrecruited to the injured tissue, where they differentiate intomacrophages.1 Infiltrating macrophages exist across anM1�M2 polarization state in which M1 cells are implicatedin initiating and sustaining inflammation through produc-tion of high levels of pro-inflammatory cytokines, reactivenitrogen, and oxygen intermediates, while the more het-erogeneous M2 cells are characterized by alternative argi-nine metabolism, exhibit a different chemokine expression

profile, and are associated with resolution or smolderingchronic inflammation.2,3 Accumulating data suggest thatmacrophages produce different mediators during normaldevelopment or regeneration after injury in several organs,such as liver, kidney, and heart.4–14 In addition, recent re-ports indicate that in the mouse pancreas, macrophagesmight be involved in inducing acinar-to-ductal metaplasia(ADM), as well as promoting b-cell regeneration.15–17

In a previous work we used diphtheria toxin receptor(DTR)�mediated conditional and targeted cell ablation tostudy acinar and endocrine regeneration in the absence ofautoimmunity.18 In these models, the internalization of DTleads to rapid apoptotic cell death of the targeted cells,followed by massive infiltration of macrophages. This led usto speculate that macrophages might contribute to pancre-atic regeneration by providing the proper signals to theductal cell progenitor niche.

Here we first examine the role of macrophages followingdifferent cell type-specific ablation models in the absence ofautoimmunity. Specifically, we show with both in vivo andin vitro approaches that uptake of apoptotic cells by mac-rophages determines a progressive switch in macrophagephenotype in an injury-specific manner. Using a saporin-conjugated monoclonal antibody to kinetically depletemacrophages in the pancreas, we then demonstrate thatpolarized macrophages are required for proper pancreaticregeneration. Likewise, we find that the decreased ability toregenerate observed in old animals is also related toinsufficient polarization. Finally, we show that this pro-regenerative function is impaired in nonobese diabetic(NOD) mice, strongly suggesting that this aspect should beaddressed in the search of novel therapies for patients withtype 1 diabetes (T1D).

http://crossmark.crossref.org/dialog/?doi=10.1053/j.gastro.2014.08.008&domain=pdf


November 2014 Macrophages and Pancreatic Regeneration 1107

MethodsMice

Mice used in this study were maintained according toprotocols approved by the University of Pittsburgh InstitutionalAnimal Care and Use Committee. Description of additionalmethods and details for reagents are provided inSupplementary Materials and Methods.

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ResultsInfiltrating Macrophages Polarize to an M2-Phenotype in a Successful NonautoimmuneModel of Global Pancreatic Regeneration

We have recently reported that after extensive ablationof both acinar and endocrine cells in adult PdxCre;R26DTR

mice, epithelial cells within the ductal network are capableof contributing to organ regeneration through recapitulationof the embryonic pancreatic developmental program.18 Inthis model, during the first week after injury (early stage),the pancreas is massively infiltrated by macrophagesengulfing the apoptotic cells (Figure 1A–C).

As the regenerative process proceeds (mid stage), thefrequency of apoptosis among the epithelial cells, as well asthe number of macrophages, drops significantly (Figure 1D)and becomes almost nonexistent during the late stage.18

It is known that phagocytosis of apoptotic cells regulatesmacrophage immune response by shifting the cellularphenotype from classical M1 to alternative M2 activation af-ter a critical number of cells have contributed to an overallchange in the microenvironment.19–21 To confirm the dy-namic change in macrophage activation in the regeneratingpancreas, we analyzed F4/80-positive macrophages (pan-macrophagemarker) for expression of theM2-marker Fizz1.8

Macrophages present during the early stage were mainlyFizz1� (Figure 1E). By contrast, duringmid stage, the numberof F4/80þ/Fizz1þ cells in the pancreas progressivelyincreased (Figure 1E). To further characterize phenotypicallythemacrophage infiltrates, we isolated F4/80þ/CD11bþ cells(Figure 2A) from the pancreas of DT-treated PdxCre;R26DTR

mice. Flow cytometry analysis confirmed that macrophagesprogressively switch from a classic tumor necrosis factor(TNF)�aþ/interleukin (IL)6þ M1 to an alternative IL10þ/CD206þ M2 phenotype during the transition from early tomid stage (Figure 2C).

Polarized Macrophages Exhibit a DifferentCytokine Phenotype After Cell-Specific Injury

M2 is a generic name for various forms of macrophageactivation other than the classic M1, expressing heteroge-neous repertoires of cytokines.2 To investigate whether cell-specific ablation (rather than global) induces a differentpolarization profile, F4/80þ/CD11bþ cells were isolatedfrom PdxCreERT;R26DTR mice (apoptotic b-cells), or Ela-CreERT2;R26DTR mice (apoptotic acinar cells) at differenttime points and analyzed for M1 (TNFa or IL6) and M2(IL10 or CD206) markers (Figure 2B–E). Interestingly, wefound that macrophages obtained from DT-treated

PdxCreERT;R26DTR mice displayed initially a M1 signature(TNFaþ/IL6þ); however, they did not switch completely to aclassic M2 signature and instead remained in a M1/M2(TNFaþ/IL6þ/IL10þ) state (Figure 2D). On the other hand,phagocytosis of apoptotic acinar cells after acinar-specificablation in ElaCreERT2;R26DTR mice, induced a prominentM1/M2 phenotype (TNFaþ/IL6þ/IL10þ), which persistedthroughout the regenerative process (Figure 2E).

Macrophages Are Required for Both Acinar andb-Cell Regeneration

The existence of different macrophage activation statesimplies distinct roles during the phases of the immunologicresponse, ie, inflammation vs resolution and tissue remod-eling. We speculated that the initial function of macrophageswould be that of debriding themassive apoptotic cell residue,while their persistence during active regeneration could berelated to their role as mediators of regenerative signals. Tounequivocally assess the role of macrophages in vivo duringtissue repair in our DT/DTR-mediated cell ablation models,we used a saporin-conjugated antibody against CD11b/Mac1(Mac1-SAP) to deplete infiltrating pancreatic macrophagesduring regeneration. Compared with clodronate, whichmainly depletes circulating monocytes, this antibody iseffective to deplete tissue macrophages in both the spleenand the injured pancreas 48 hours after administration(Figure 3A). To evaluate the effect of macrophage depletionon pancreatic regeneration, we first allowed the macro-phages to debride the injured pancreas after DT treatment,and subsequently administered Mac1-SAP on day 7 post-DT,when the M2 polarization peak happens (Figure 3C). Themice were then sacrificed either at mid stage (Figure 3B–D)or at late stage (Figure 3E). We have previously shown thatpancreatic regeneration in the PdxCre;R26DTR model isassociated with re-expression of Pdx1, and apparent reac-tivation of the embryonic program in the ducts.18 As ex-pected, in the control DT-treated mice, the vast majority ofthe regenerative duct cells were SOX9þ/PDX1þ (Figure 3D).However, macrophage ablation on day 7 post DT led to asignificant reduction in the number of PDX1-positive cellsduring the regenerative process (Figure 3D) and ultimatelycaused failed regeneration at later stages (Figure 3E).

To evaluate the role of macrophages specifically in b-cellregeneration, PdxCreERT;R26DTR mice were injected withMac1-SAP antibody on the last day of DT treatment, andweresubsequently sacrificed 3 days later (Figure 4A). The adultmouse pancreas contains few (if any) resident macrophages,however, after b-cell ablation, the islets are specificallyinfiltrated by F4/80þ cells (Figure 4B). Mac1-SAP–mediatedmacrophage depletion was confirmed by a substantialdecline in the number of F4/80þ cells within the islets of DT-treated PdxCreERT;R26DTRmice (Figure 4B). Interestingly, b-cell proliferation after b-cell ablation decreased significantlyin macrophage-depleted mice (Figure 4C and D).

Next, we depleted macrophages in DT-treated Ela-CreERT2;R26DTR mice (Figure 4E and F). As expected,18

the acinar compartment in DT-treated ElaCreERT2;R26DTR

mice was recovered on day 7 after injury. However,

Figure 1.Macrophage po-larization is required forpancreatic regeneration.(A–C) Fluorescent stainingof sections obtained fromearly-stage DT-treatedPdxCre;R26DTR pancreasusing anti-F4/80 to labelmacrophages and E-cad-herin (Ecad) to label epithe-lial cells shows abundantmacrophage infiltrationwithin the apoptotic acinarand endocrine compart-ments. (B)Note theabsenceof macrophages in theareas next to the survivingducts. (C) Phagocytosis ofthe apoptotic epithelial cellsbymacrophages (asterisks).(D) Flow cytometry analysishighlights how CD11bþ

macrophages decline innumberduring the transitionfrom early- to mid-stage inthe regenerating PdxCre;R26DTR pancreata. (E) F4/80/Fizz1 double-staining ofearly and mid-stage pan-creata reveals that ma-crophage polarizationcoincides with the peak ofthe regenerative process.Scale bars ¼ 20 mm.

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Figure 2.Macrophagespolarize in a cell type spe-cific injurymanner. (A) Flowcytometry analysis con-firms that the isolated F4/80þ macrophages areCD11bþ. (B) Gating strat-egy. Cells obtained afterdigestion of the pancreasare stained for CD11b andintracellular cytokines.Then the percentage ofcytokineþ cells is calcu-lated on gated CD11bþ

cells. (C–E) Gated CD11bþ

cells isolated from early- ormid-stage regeneratingpancreas of PdxCre;R26DTR (C), PdxCreERT;R26DTR (D), or Ela-CreERT2;R26DTR mice (E)were stained for M1 (TNFa,IL6) or M2 markers (IL10,CD206).


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ElaCreERT;R26DTR mice that had been treated with DT andMac1-Sap antibody displayed impaired acinar regeneration(Figure 4F).

Macrophages Induce Cell Type-SpecificDifferentiation In Vitro

To determine whether macrophages play only apermissive or rather an inductive role in pancreatic

regeneration, we established an in vitro system where weco-cultured embryonic pancreatic epithelium with bonemarrow–derived macrophages previously exposed todifferent types of apoptotic cells (Supplementary Figure 1).The embryonic pancreas is receptive to cues necessary fornormal pancreatic differentiation and was therefore used toevaluate whether macrophages are able to provide regen-erative signals. We first generated bone marrow–derivedmacrophages from 8- to 12-week-old C57Bl/6 mice, which

Figure 3.Macrophagedepletion leads to impairedregenerative capacity. (A)Mac1-SAP efficiently de-pletes tissue macrophagesin the injured pancreas.PdxCre;R26DTR mice wereinjected with Mac1-SAPantibody on the last day ofDT-treatment. After 48hours, flow cytometryanalysis shows significantdecrease in the CD11bþ

population both in thespleen and in the pancreas.(B) Schematic outline of themacrophage depletion ex-periments in the PdxCre;R26DTR model. (C) Signifi-cantmacrophagedepletionis observed 48 hours afterMac1SAP-injection onday 7 post-DT comparedwith control Mac1SAP-uninjected mice. (D) Thereactivation of the embry-onic program associatedwith regeneration in theDT-treated PdxCre;R26DTR

pancreas does not occurafter macrophage ablation.Tissues obtained from theDT-treated PdxCre;R26DTR

pancreas and harvestedfrom uninjected or Mac1-SAP–injected mice showlack of PDX1 expression inSOX9þ ductal cells. (E) Thepancreas of DT-treatedPdxCre;R26DTR mice injec-ted with Mac1-SAP duringmid stage never recoversand contains only Ck19þ/Ecadþ ductal structureswhen harvested at latestage. Ck19, cytokeratin19; Ecad, E-cadherin. Scalebars ¼ 20 mm.


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we subsequently exposed overnight to apoptotic acinarcells, endocrine cells, or a mix of both. On the following day,we removed the cell debris, added fresh media, and startedthe co-culture with E11.5 pancreatic epithelium on aTranswell system to avoid any direct contact between themacrophages and the explant. As expected,22 the intactembryonic buds (with mesenchyme) grew, underwentbranching, and differentiated into both endocrine and

acinar cells (Figure 5A and F), and dorsal epitheliumdepleted of mesenchyme gave rise mainly to endocrine cells(Figure 5A and F). Similarly, the isolated epitheliumcultured with noninduced macrophages (ie, nonexposedovernight to apoptotic cells) also gave rise almost entirelyto endocrine cells (Figure 5B and F). Interestingly, the ex-plants where the epithelium was co-cultured with macro-phages that had been previously fed with apoptotic acinar

Figure 4.Macrophagesare necessary for b- andacinar cell regeneration. (A)Schematic outline of DTand Mac1-SAP treatmentof PdxCreERT;R26DTR

mice. (B, C) Comparativeanalyses of tissues ob-tained from PdxCreERT orDT-treated PdxCreERT;R26DTR mice with orwithout Mac1-SAP-injec-tion, and harvested 3 dayspost DT demonstrate thatproliferation among thesurviving b-cells is corre-lated with the presence ofmacrophages. Note thespecific homing of themacrophages to the islets.Bromodeoxyuridine (BrdU)was given via the drinkingwater 18 hours before theharvest. (D) Quantificationof the proliferation rateamong b-cells. (E) Sche-matic outline of DT andMac1-SAP treatment ofElaCreERT2;R26DTR mice.(F) Comparative analysis oftissues obtained fromDT-treated ElaCreERT2;R26DTR mice with orwithout Mac1-SAP injec-tion, and harvested on day7 post-DT shows thatmacrophage depletion re-sults in impaired acinarregeneration, with persis-tence of Sox9þ/DBAþ

structures. INS: Insulin.Scale bars ¼ 20 mm.


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(Figure 5C and F) or islet cells (Figure 5D and F) showed anincrease in the number of acinar or endocrine cells,respectively. Consistently, the epithelium co-cultured withmacrophages that had been fed with apoptotic endocrineand acinar cells, promoted differentiation of both cell types(Figure 5E and F). In addition, the ability to mediate signalsis likely to be independent of the type of cell death, assimilar results were obtained from macrophages that hadbeen fed with necrotic pancreatic cells (SupplementaryFigure 2).

Macrophages From Old Mice Display DefectivePolarization and Impaired Regeneration In VivoBut Can Induce Cell-Specific DifferentiationIn Vitro

Macrophages from old mice and humans display anage-related systemic chronic pro-inflammatory status, whichincludes defects in polarization, a process known as inflamm-ageing.23 This led us to investigate pancreatic regenerationin older animals in vivo. We noticed an overall decline inthe regenerative capacity in the 8- to 12-month-old

Figure 5.Macrophages are capable of inducing cell-specific differentiation in vitro. E11.5 dorsal pancreatic buds with intactmesenchyme (A) or epithelium depleted of mesenchyme (Epi) are co-cultured in a Transwell system without macrophages (A),with noninduced bone marrow–derived macrophages (B), or with bone marrow–derived macrophages that have been exposedovernight to apoptotic acinar (C), islet (D), or a mixture of acinar and islet (E) cells. Note that in this in vitro system, the explantnever comes into direct contact with the macrophages (due to the Transwell support) or with the apoptotic cells (which areremoved after the overnight exposure). After 7 days of co-culture, the explants are analyzed for the expression of amylase(AMY) and E-cadherin (Ecad), or insulin (INS), glucagon (GCG) and E-cadherin. (F) Quantification analysis shows significantincrease of endocrine or acinar cells in the explants co-cultured with apoptotic islets (Apo Isl) or acinar (Apo Aci) cells,respectively. Consistently, the macrophages exposed to a mix of apoptotic endocrine and acinar cells (Apo Mix), promote thedifferentiation of both cell types. Graphs show either the cell number or the number of each cell type over total counted cells ineach explant. n ¼ 5 for each condition, **P � .01; ***P � .001. e, epithelium; m, mesenchyme. Scale bar ¼ 20 mm.


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PdxCre;R26DTR mice, as assessed by impaired tissue regen-eration (Supplementary Figure 3A). Interestingly, this wasnot associated with a decreased number of infiltrating mac-rophages in the early stage, indicating the recruitment wasnot affected (Supplementary Figure 3B and C). However, the

infiltrating macrophages in older mice failed to acquire theM2 phenotype during mid stage, as shown by absence ofFizz1þ cells (Supplementary Figure 3D). These findings werealso consistent with a significant reduction in PDX1þ/SOX9þ-double positive cells within the ductal structures, confirming


that the impaired macrophage polarization affects themicroenvironment of the epithelial progenitor niche(Supplementary Figure 3E and F).

Next, we asked whether the defective polarizationobserved in vivo in older PdxCre;R26DTR mice was due tointrinsic macrophage deficiency or rather to an aging envi-ronment. To remove the macrophages from the aged milieu,we generated bone marrow–derived macrophages from 8-to 12-month-old mice and repeated the in vitro co-culturesystem described previously. As shown in SupplementaryFigure 4, macrophages originating from older mice wereable to convert injury signals into proper regenerative sig-nals in vitro, suggesting that their inability to do so in vivo islikely the result of the aging environment.

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Nonobese Diabetic Macrophages Have anInherent Inability to Provide Appropriate Signalsfor b-Cell Generation Both In Vivo and In Vitro

Macrophages derived from autoimmune-susceptible ro-dent strains or T1D patients produce abnormally high levelsof cytokines, which is thought to contribute to persistence ofthe inflammation, instead of its resolution.24–26 This exacer-bated M1 profile plays a prominent role in the pathogenesisof islet autoimmunity by promoting inefficient clearance ofapoptotic cells, epitope-spreading and progressive destruc-tion of b-cells.27,28 To explore whether this inherent macro-phage defect is associated with additional inability to provideproper regenerative signals, we analyzed the expressionprofile of macrophages isolated from NOD pancreas at theonset of diabetes. As expected, NOD macrophages display apersistent M1-signature, confirming their inability to con-tract the inflammation response and transition to a regen-erative state (Figure 6A). Transforming growth factor (Tgf)�b1/Smad signaling prevents b-cell proliferation, which canbe blocked through activation of epidermal growth factorreceptor (EGFR) pathway.17 Macrophages isolated fromdiabetic NODmice express high levels of Tgfb1, while lackingepidermal growth factor expression (EGF) (Figure 6B).

The expression profile of NOD macrophages might beheavily influenced by the cytokine milieu generated by otherinflammatory cells, primarily autoreactive T cells. To spe-cifically study the ability of NOD macrophages to produceregenerative signals outside the autoimmune setting, weperformed co-culture experiments with bone mar-row–derived macrophages harvested from young nondia-betic NOD mice, and fed them with apoptotic acinar, islets,or a mixture of acinar and endocrine cells (Figure 6C).Surprisingly, we found that when co-cultured with embry-onic E11 pancreatic epithelium, the NOD macrophagespromoted acinar differentiation, regardless of the type ofapoptotic cells they had been exposed to (Figure 6C and D).

Macrophages Exhibit Different ExpressionProfiles in Response to Cell-SpecificInjury In Vivo

Macrophages promote regeneration and tissue repair byproducing numerous growth factors and Wnt-ligands.9–11

Based on these premises and our results showing thatpolarized macrophages exhibit a different cytokine pheno-type after cell-specific injury, we investigated whether thephagocytosis of different apoptotic cell types in vivo leads todistinct expression profiles. To do so, CD11bþ cells wereisolated from the pancreas of diabetic NOD mice or DT-treated PdxCre;R26DTR, ElaCreERT;R26DTR, or PdxCreERT;R26DTR mice. The isolated macrophages were then analyzedfor the expression of selected growth factors or Wnt-ligands(Figure 7A). The growth factors andWnt-ligandswere chosenbased on their expression or function in the developingmouse pancreas.22,29–32 Quantitative reverse transcriptionpolymerase chain reaction (QRTPCR) showed macrophagesexhibit a differential expression profile, depending on thetype of injury. In addition, ligands and factors expressed bythe macrophages display a dynamic expression pattern dur-ing different phases of the same regenerative process. TheQRTPCR data were confirmed by Western blot and immu-nostaining analyses (Figure 7B, Supplementary Figure 5).Accordingly, isolated duct cells from the conditionsmentioned show a different expression profile for selectedWnt-target genes, indicating an active cross-talk between thepolarized macrophages providing the regenerative signalsand the ductal cells receiving them (Figure 7C).

NOD-derived macrophages express high levels of TNFaand IL6 (Figure 6A). Interestingly, this is associated withlack of hepatocyte growth factor (HGF) up-regulation(Figure 7A). We have previously shown that hepatocytegrowth factor suppresses the production of the pro-inflammatory cytokine IL6 in lipopolysaccharide-stimulated (LPS) macrophages from C57Bl/6 mice, at thesame time allowing for the production of anti-inflammatoryIL10.33 Here, we show that LPS NOD macrophages not onlysecrete significantly higher basal amount of the pro-inflammatory cytokine TNFa (Figure 7D), but also fail todown-regulate its synthesis after treatment with HGF(Figure 7D). These results are consistent with our in vitrofindings showing how NOD-derived macrophages possess amore prominent M1 signature during the culture periodcompared with their nonautoimmune counterparts(Supplementary Figure 6). Compared with macrophagesisolated from DT-treated PdxCreERT;R26DTR (non-autoimmune b-cell–specific injury), the NOD-derived mac-rophages displayed significantly lower expression of Wnt7b,and expressed higher levels of Tgfa and vascular endothelialgrowth factor–a (VEGFa), although they shared similarexpression profile for fibroblast growth factor�10 (FGF10),Wnt3a, and Wnt5a (Figure 7A).

DiscussionIt is well known that macrophages respond to environ-

mental cues with the acquisition of distinct functional phe-notypes, undergoing classical M1 or alternative M2activation, where the pro-inflammatory M1 macrophagesact as scavengers, and the anti-inflammatory M2 macro-phages facilitate repair and regenerative activities.2,3 Thisproperty has been a recent object of intense study4–11 as thesearch for clinical strategies that improve the body’s

Figure 6. NOD macro-phages have an inherentinability to provide appro-priate signals for b-cellgeneration in vitro. (A)Macrophages infiltratingthe pancreas of NOD miceat the onset of diabetes arecharacterizedbyhigh levelsof M1 pro-inflammatorycytokines TNFa and IL6.(B) NOD-derived macro-phages express signifi-cantly higher levels of Tgfb1compared with macro-phages isolated fromDT-treated PdxCreERT;R26DTR pancreas (day 3post DT treatment) or non-induced macrophages iso-lated from the spleen (WTC57Bl6). (C) The E11.5dorsal pancreatic epithe-lium depleted from mesen-chyme (Epi) is co-culturedwith noninduced bonemarrow–derived NODmacrophages or bonemarrow–derived NODmacrophages that havebeen fed with apoptoticacinar (Apo Aci), islet (ApoIsl), or a mixture of acinarand islet cells (Apo Mix) asdescribed in Figure 5. (D)Quantification of glucagon(GCG), insulin (INS), andacinar (AMY) cells showsthat NOD macrophagespromote acinar differentia-tion, regardless of the typeof apoptotic cells they havebeen exposed to. Graphsshow either the cell numberor the number of each celltype over total countedcells in each explant. n ¼ 5for each condition, **P�.01; ***P �.001. Scalebar ¼ 20 mm.


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endogenous repair mechanisms are topics of major publichealth concern. However, the molecular mechanisms un-derlying tissue repair or its failure are not completely un-derstood. This is particularly important for the pancreas andT1D, as the question whether b-cells can regenerate evenwhen autoimmunity is abrogated is still under debate.34,35

We have previously shown that following selective abla-tion of acinar tissue in the ElaCreERT2;R26DTR model, tissue

regeneration occurred by direct reprogramming of ductalcells into acinar cells, as opposite to recapitulation of theembryonic pancreatic program observed in the PdxCre;R26DTR mouse when both acinar and endocrine cells wereablated.18 Here, and consistent with previous reports,36,37 weshow that in the PdxCreERT;R26DTR model, the mechanismfor b-cell regeneration is self-duplication of survivingb-cells. In this study, we sought to determine how these

Figure 7.Macrophages exhibit a different profile in response to injury in vivo. (A) Macrophages isolated from the pancreas ofNOD mice, or the DT-treated PdxCre;R26DTR, ElaCreERT2;R26DTR, or PdxCreERT;R26DTR transgenic animals display differentmessenger RNA expression profiles for selected growth factors and Wnt-ligands. Bars represent relative gene expression/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (mean ± SE). n ¼ 5 independent experiments; for each experimentmacrophages were isolated from 5 pooled pancreata (for a total of 25 animals per time point). P value is calculated bycomparing each bar with spleen macrophages of C57Bl/6 (WT) mice. *P � .05; **P � .01; ***P � .001. (B) Western blot analysesfor Wnt5a, Wnt7b, and b-actin on protein lysates extracted from spleen macrophages of C57Bl/6 (WT) mice, or infiltratingmacrophages isolated from early- or mid stage of PdxCre;R26DTR pancreata (PD early, PD mid). (C) Duct cells isolated from thepancreas of wild-type or DT-treated PdxCre;R26DTR, ElaCreERT2; R26DTR, or PdxCreERT;R26DTR mice display differentexpression profiles with respect to Wnt-target genes. Bars represent relative gene expression/GAPDH (mean ± SE). Mouseembryonic pancreas (E14) was used as control. n ¼ 5 independent experiments; for each experiment macrophages wereisolated from 5 pooled pancreata (for a total of 25 animals per time point). (D) Compared with WT (C57BL/6) controls, bonemarrow–derived macrophages isolated from NOD mice secrete higher basal levels of TNFa in response to lipopolysaccharide(LPS) and fail to suppress it when pretreated with hepatocyte growth factor (HGF). (E) Schematic representation of macro-phage polarization in different injury models.


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seemingly different regenerative processes are regulated.Mounting evidence suggests that macrophages are highlyinvolved in orchestrating tissue regeneration in differentorgans.4,5,8–12,14–17 Our data confirm that regardless of theablation model, the injured pancreas is infiltrated by macro-phages. However, the subsequent phenotypes that theseinfiltrating macrophages develop seem to be heavily depen-dent on the apoptotic cell types they phagocyte. As summa-rized in Figure 7E, we could detect a gradual but clear switchfrom anM1 toM2 phenotype during the transition from earlyto mid stage in the PdxCre;R26DTR pancreas, but not inPdxCreERT;R26DTR mice, where the initial M1 macrophageslater acquired a M1/M2 phenotype. In contrast to thePdxCre;R26DTR or PdxCreERT;R26DTR mice, macrophagesisolated from DT-treated ElaCreERT2;R26DTR pancreasmaintained a M1/M2 signature throughout regeneration.Additionally, our analysis revealed that macrophages withthe same cytokine phenotype but obtained from differentablationmodels, show different growth factor and wnt ligandexpression profiles (compare M1 macrophages isolated fromPdxCre;R26DTR and PdxCreERT;R26DTR in Figure 7). Like-wise, macrophages originating from the same injury model,but isolated at different regenerative stages, despite sharingthe same polarization signature, express a different set ofgenes (compare M1/M2macrophages isolated from early- ormid-stage ElaCreERT2;R26DTR mice in Figure 7). Our resultshighlight the necessity of a revision in howwedefine differentmacrophage polarization phenotypes, at least in studiesexploring the regenerative properties of macrophages.

The data presented here on DT-mediated injury modelsindicate that macrophages undergo a phenotype switchduring regeneration. This is in line with a recent report inwhich it was shown that tissue-specific signals control areversible program of functional polarization of macro-phages.13 Our conclusion is based on the facts that i) highpercentage of cells positive for IL6, IL10, and CD206, espe-cially in the PdxCre:R26DTR model; ii) it is unlikely that aputative second wave of macrophages would display differ-entially expression profiles, depending on the apoptotic celltype that the initially infiltratingmacrophages have engulfed;and iii) in our in vitro co-culture system there is no influx ofnewly infiltrating macrophages.

In this study, we demonstrate in several independentexperiments how macrophage polarization is required forpancreatic regeneration. First, we show that depletion ofmacrophages after b-cell, acinar cell, or both endocrine andacinar cell ablation leads to impaired tissue regeneration(Figures 3 and 4). Secondly, we report that the impairedpancreatic regeneration in older PdxCre;R26DTR mice isassociated with the absence of M2 macrophages(Supplementary Figure 3). Finally, our in vitro co-culturesystem confirms that macrophages are able to translateinjury signals into cell-type–specific regenerative signals(Figure 5). Collectively, these data demonstrate that properpancreatic regeneration is dependent on precise temporallyregulated macrophage polarization.

It has been reported that defective function of macro-phages is a characteristic of T1D-prone individuals andanimals.27,28,38 Here, we show that macrophages from NOD

mice have a persistent pro-inflammatory M1 phenotype andinefficient ability to produce several molecular mediatorsinvolved in pancreatic regeneration (Figure 6 andSupplementary Figure 6). Accordingly, in vitro, they exhibitintrinsic deficiency in providing the proper signals for b-cellgeneration, suggesting that their functional impairmentmight be a key factor concurring to failure of regeneration inthe setting of autoimmunity. It was recently reported thatthe inhibitory effect of Tgf1b/Smad-signaling on b-cellproliferation could be blocked through activation of theEGFR pathway.17 Interestingly, NOD-derived macrophagesexpress significantly high levels of TGFb1, and no EGF(Figure 6). It is tempting to speculate that the excessivepresence of TGFb1, produced by macrophages may preventany attempt on b-cell proliferation in the diabetic NODpancreas.

Pancreatic regeneration is the result of the coordinatedeffects of regenerative signals on responding cells. Interest-ingly, we could detect an overall higher reactivation ofselected Wnt-target genes among ductal cells in the injurymodels compared with duct cells isolated from normalpancreas (Figure 7), which could be explained by Wnt-ligands secreted by macrophages. Other components ofregeneration that should be considered are the nature of celldeath (apoptosis vs necrosis) and also whether the damagetriggers significant inflammatory response (other than mac-rophages). It is likely that the DT approach, which leads to aapoptotic cell death, may be one of the reasons for the robustregeneration that is observed in these ablation models. Thedata presented in this study indicate that macrophages playan essential role in mediating regenerative signals. To whatextent macrophages play a similar role in acinar regenerationin pancreatitis remains to be revealed. Macrophages secreteRANTES, TNFa, and IL6 during pancreatitis, which in turninitiate ADM in acinar cells.15 Our data show that macro-phages isolated from DT-treated ElaCreERT:R26DTR micemaintain high level of TNFa and IL6 expression throughoutacinar regeneration (Figure 2). Although, the regenerativemechanism in our acinar ablationmodel does not entail ADM,it suggests that phagocytosis of acinar cells may promoteexpression of these cytokines. Similarly, NOD-derived mac-rophages display high levels of baseline TNFa and IL6expression and secretion (Figures 6 and 7), which mightexplain why acinar differentiation was primarily promoted inthe explants co-cultured with NOD macrophages (Figure 6).ADM is considered as an early step in acinar regeneration,39

therefore, it is possible that macrophages support acinarregeneration in pancreatitis by producing factors that induceADM.

In summary, we show here for the first time that thespatiotemporal secretion of signals arising from macro-phages is necessary for proper pancreatic regeneration. Thisprocess is reminiscent of their role in wound healing, wheremacrophages regulate the resolution of inflammation andpromote tissue repair.8 In the absence of this temporallydefined activity, such as after macrophage depletion, as wellas in the context of an “aged” environment, the regenerationprocess is impaired. The inability of NOD-derived macro-phages to specifically promote b-cell generation strongly


suggests a potential, but as yet unexplored, link betweendefective macrophage polarization and impaired b-cellregeneration in the pathogenesis of T1D. Although moredetailed studies are required to identify the specific factorsthat are necessary for cell type–specific regeneration, ourfindings provide a framework with which to test these keyquestions. The identification of the factors that regulate andpromote the macrophage-derived environment could besuccessfully exploited for macrophage-centered therapeuti-cally approaches.

Supplementary MaterialNote: To access the supplementary material accompanyingthis article, visit the online version of Gastroenterology atwww.gastrojournal.org, and at http://dx.doi.org/10.1053/j.gastro.2014.08.008.

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IONA

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8. Lucas T, Waisman A, Ranjan R, et al. Differential roles ofmacrophages in diverse phases of skin repair. J Immunol2010;184:3964–3977.

9. Stefater JA 3rd, Lewkowich I, Rao S, et al. Regulation ofangiogenesis by a non-canonical Wnt-Flt1 pathway inmyeloid cells. Nature 2011;474:511–515.

10. Boulter L, Govaere O, Bird TG, et al. Macrophage-derived Wnt opposes Notch signaling to specify hepaticprogenitor cell fate in chronic liver disease. Nat Med2012;18:572–579.

11. Stefater JA 3rd, Rao S, Bezold K, et al. Macrophage Wnt-Calcineurin-Flt1 signaling regulates mouse woundangiogenesis and repair. Blood 2013;121:2574–2578.

12. Yang L, Kwon J, Popov Y, et al. Vascular endothelialgrowth factor promotes fibrosis resolution and repair inmice. Gastroenterology 2014;146:1339–1350.e1.

13. Okabe Y, Medzhitov R. Tissue-specific signals controlreversible program of localization and functional polari-zation of macrophages. Cell 2014;157:832–844.

14. Aurora AB, Porrello ER, Tan W, et al. Macrophages arerequired for neonatal heart regeneration. J Clin Invest2014;124:1382–1392.

15. Liou GY, Doppler H, Necela B, et al. Macrophage-secreted cytokines drive pancreatic acinar-to-ductalmetaplasia through NF-kappaB and MMPs. J Cell Biol2013;202:563–577.

16. Brissova M, Aamodt K, Brahmachary P, et al. Isletmicroenvironment, modulated by vascular endothelialgrowth factor-A signaling, promotes beta cell regenera-tion. Cell Metab 2014;19:498–511.

17. Xiao X, Gaffar I, Guo P, et al. M2 macrophages promotebeta-cell proliferation by up-regulation of SMAD7. ProcNatl Acad Sci U S A 2014;111:E1211–E1220.

18. Criscimanna A, Speicher JA, Houshmand G, et al. Ductcells contribute to regeneration of endocrine and acinarcells following pancreatic damage in adult mice.Gastroenterology 2011;141:1451–1462. 1462 e1–6.

19. Voll RE, Herrmann M, Roth EA, et al. Immunosuppressiveeffects of apoptotic cells. Nature 1997;390:350–351.

20. Erwig LP, Kluth DC, Walsh GM, et al. Initial cytokineexposure determines function of macrophages and ren-ders them unresponsive to other cytokines. J Immunol1998;161:1983–1988.

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23. Mahbub S, Deburghgraeve CR, Kovacs EJ. Advancedage impairs macrophage polarization. J Interferon Cyto-kine Res 2012;32:18–26.

24. Alleva DG, Pavlovich RP, Grant C, et al. Aberrantmacrophage cytokine production is a conserved featureamong autoimmune-prone mouse strains: elevatedinterleukin (IL)-12 and an imbalance in tumor necrosisfactor-alpha and IL-10 define a unique cytokine profile inmacrophages from young nonobese diabetic mice. Dia-betes 2000;49:1106–1115.

25. Plesner A, Greenbaum CJ, Gaur LK, et al. Macrophagesfrom high-risk HLA-DQB1*0201/*0302 type 1 diabetesmellitus patients are hypersensitive to lipopolysaccharidestimulation. Scand J Immunol 2002;56:522–529.

26. Khanna S, Biswas S, Shang Y, et al. Macrophagedysfunction impairs resolution of inflammation in thewounds of diabetic mice. PLoS One 2010;5:e9539.

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34. Ablamunits V, Sherry NA, Kushner JA, et al. Autoimmu-nity and beta cell regeneration in mouse and human type1 diabetes: the peace is not enough. Ann N Y Acad Sci2007;1103:19–32.

35. Herold KC, Bluestone JA. Type 1 diabetes immuno-therapy: is the glass half empty or half full? Sci TranslMed 2011;3:95fs1.

36. Dor Y, Brown J, Martinez OI, et al. Adult pancreatic beta-cells are formed by self-duplication rather than stem-celldifferentiation. Nature 2004;429:41–46.

37. Nir T,MeltonDA,Dor Y. Recovery fromdiabetes inmice bybeta cell regeneration. J Clin Invest 2007;117:2553–2561.

38. Hayashi T, Faustman DL. Role of defective apoptosis intype 1 diabetes and other autoimmune diseases. RecentProg Horm Res 2003;58:131–153.

39. Morris JP 4th, Cano DA, Sekine S, et al. Beta-cateninblocks Kras-dependent reprogramming of acini intopancreatic cancer precursor lesions in mice. J Clin Invest2010;120:508–520.

Author names in bold designate shared co-first authorship.

Received December 2, 2013. Accepted August 12, 2014.

Reprint requestsAddress requests for reprints to: Farzad Esni, PhD, Department of Surgery,University of Pittsburgh, John G. Rangos Research Center, One Children’sHospital Drive, Rangos Floor 6, Room 6119, Pittsburgh, PA 15224. e-mail:[email protected]; fax: (412)-692-3466.

AcknowledgmentsThe authors would like to thank the Gittes’ laboratory members for usefuldiscussions, and Alexis Styche for helping with flow cytometry.

Conflicts of interestThe authors disclose no conflicts.

FundingThis work was supported by The Cochrane-Weber endowed Fund in DiabetesResearch (F.E., A.C.), National Pancreas Foundation (F.E.), American DiabetesAssociation (J.D.P.), Juvenile Diabetes Research Foundation (J.D.P., G.M.C.),and The Children’s Hospital of Pittsburgh of UPMC (F.E.).











































mailto:[email protected]

Supplementary MaterialMice, Tamoxifen, and Diphtheria Toxin Treatment

The Rosa26DTR and NOD mice were purchased from TheJackson Laboratory (Bar Harbor, ME). The PdxCre1,2 micewere obtained from the Mouse Models of Human CancerConsortium. The ElaCreERT22 and PdxCreERT strains weregenerated in the laboratories of Drs Craig Logsdon (Uni-versity of Texas MD Anderson Cancer Center) and DouglasMelton (Harvard Medical School). Mice were treated withtamoxifen and DT, as described previously.2 Briefly, Ela-CreERT2; R26DTR and PdxCreERT; R26DTR mice were firstinjected intraperitoneally with 2 mg tamoxifen for 2consecutive days to activate Cre. All animals were thentreated intraperitoneally with 0.5 ng/g body weight of DTfor 5 consecutive days and killed at different time points. Inthe PdxCre; R26DTR model, the early stage of regenerationrefers to the first week post DT injections, when thepancreas sustains a massive loss of the epithelial cell pop-ulation and is infiltrated by macrophages. Mid and latestages of regeneration correspond approximately to days7–21 and 14–35, when the actual regeneration processstarts and is completed, respectively.

In the ElaCreERT2;R26DTR and PdxCreERT; R26DTR

models, early corresponds to days 4–5 of DT injections andmid corresponds to day-3 post-DT. PdxCre;R26DTR, Ela-CreERT; R26DTR, and PdxCreERT;R26DTR mice described as“young” throughout the article were sacrificed at 8–10weeks of age. PdxCre;R26DTR mice described as “old” weresacrificed at 8–12 months of age. Diabetic NOD mice atonset are females about 12–16 weeks of age with bloodglucose �16.5 mmol/L in 2 consecutive measurements. Forthe in vivo experiments, n ¼ 5 represents 5 independentexperiments, where in each experiment pooled samplesfrom macrophages isolated from several mice (similarconditions) have been used.

For the in vitro experiments, NOD mice were sacrificedat 8–10 weeks for bone marrow harvesting (nondiabetic).Eight- to 10- week-old C57Bl/6 mice were used as controlanimals for the transgenic strains in all experiments,included in vivo macrophage isolation and in vitro genera-tion of bone marrow–derived macrophages.

Macrophage DepletionTo specifically deplete infiltrating pancreatic macro-

phages, we administered, by tail vein injection 20 ugsaporin-conjugated antibody against the pan-macrophagesurface marker Mac1/CD11b3–6 (Mac-1-SAP mouse/hu-man; Advanced Targeting Systems, San Diego, CA; cat. no.IT-06-25).

Targeted SAP conjugates are powerful and specificlesioning agents used in the technique known as molecularsurgery. The ribosome-inactivating protein saporin is boundto a targeting agent (in this case anti-Mac1 antibody). Therequirement for the target is that it should be on the cellsurface and also be able to get internalized. The targetedconjugate is administered to cells and binds to its target onthe cell surface, and gets internalized. Upon internalization,

saporin breaks away from the targeting agent, inactivatesthe ribosomes, which causes protein synthesis inhibitionand, ultimately, leads to cell death. Mac1 (also known asCD11b) is specifically expressed in the macrophage lineageand is localized to the cell surface.

PdxCre; R26DTR model. To test the efficacy ofmacrophage depletion in the injured pancreas, Mac-1-SAPwas administered on day 5 of DT injections, and then themice were harvested on day 2 post-DT (see Figure 3A). Todeplete macrophages at the beginning of the mid stage,Mac-1-SAP was administered on day 7 post-DT injections,and then the mice were harvested on days 14 and 35 postDT injections (see Figure 3B), respectively.

ElaCreERT2;R26DTR and PdxCreERT;R26DTR

models. To deplete macrophages during the regenerativephase, Mac-1-SAP was administered on day 5 of DT in-jections, and mice were then sacrificed on day 7 post-DT(ElaCreERT2;R26DTR) or day 3 post DT (PdxCreERT;R26DTR),respectively (see Figure 4A and E). For the evaluation ofb-cell proliferation in the PdxCreERT;R26DTR model, bro-modeoxyuridine (BrdU) was provided in the drinking waterfor 18 hours before sacrifice (see Figure 4A), as describedpreviously.2

Isolation of Macrophages and Ductal Cells FromInjured Pancreata

After harvesting, pancreata were finely minced and putinto a 50-mL conical tube in a solution containing 2 mg/mLCollagenase type V (Sigma-Aldrich, St Louis, MO; cat. no.C9263) in Hank’s balanced salt solution with Ca2þ andMg2þ. Digestion was carried out in a shaking water bath at37�C for 8–12 minutes and stopped with ice-cold Hank’sbalanced salt solution without Ca2þ and Mg2þ. The digestedtissue was subsequently filtered through a 40-mm pre-wetcell strainer in order to remove large acinar clumps andislets. Cells were then centrifuged for 5 minutes at 1300rpm and filtered into polystyrene round-bottom tubes with35-mm cell strainer caps. If necessary, red cell lysis wasperformed (Red Blood Cell Lysis Buffer Hybri-Max, Sigma-Aldrich; cat. no. R7757) according to the manufacturer’sinstructions. Cells were then washed, counted, and resus-pended at 107 cells/100 uL Separation/Sorting Buffer(phosphate-buffered saline without Ca2þ, 0.5% bovineserum albumin and 2 mM EDTA) and subjected to magneticsorting in order to isolate macrophages or ductal cells.Briefly, cells were first treated with 10 mL FcR BlockingReagent (Miltenyi Biotec, Bergisch Gladbach, Germany; cat.no. 130-092-575) for 10 minutes at 4�C, and then incubatedfor 20 minutes at 4�C in the dark with the primary antibody.For macrophage isolation, cells were incubated witheFluor660-conjugated anti-mouse F4/80 antibody (eBio-science, San Diego, CA; cat. no. 50-4801-82). After washing,cells were magnetically labeled and sorted with anti-Cy5/anti-Alexa Fluor 647 Microbeads (Miltenyi Biotec; cat. no.130-091-395), according to the manufacturer’s instructions.Alternatively, macrophages were isolated with CD11bmicrobeads (Miltenyi Biotec; cat. no. 130-049-601), ac-cording to the manufacturer’s instructions. For ductal cell

November 2014 Macrophages and Pancreatic Regeneration 1118.e1

isolation, cells were incubated with a biotinylated dolichosbiflorus agglutinin lectin (DBA) and then magnetically sor-ted with anti-biotin microbeads (Miltenyi Biotec; cat. no.130-090-485). Cell purity (�90%) after magnetic sortingwas confirmed by flow cytometry. Isolated macrophagesand ductal cells were then subsequently used for down-stream analyses. Antibodies and dilutions used are listed inSupplementary Table 1.

Flow Cytometry AnalysisFor surface staining, cells were treated with 10 mL FcR

Blocking Reagent (Miltenyi Biotec; cat. no. 130-092-575) for10 minutes at 4�C, and then incubated with primary con-jugated antibodies for 20 minutes at 4�C in the dark. Forintracellular cytokine staining, BD Cytofix/Cytoperm Fixa-tion/Permeabilization solution Kit (BD Bioscience, San Jose,CA; cat. no. 554714) was used according to the manufac-turer’s instructions. Cells were then incubated with conju-gated intracellular antibodies for 20 minutes at 4�C in thedark. Data were acquired with a BD FACSAria Cell Sorter,analyzed with FACSDiva Software (BD Bioscience). Gatingstrategy is shown in Figure 2A. Antibodies and dilutionsused are listed in Supplementary Table 1.

Generation of Bone Marrow–DerivedMacrophages

Femurs and tibiae were harvested from 8- to 12-week-old C57Bl/6 (young), 8- to 12-month-old C57Bl/6 (OLD) or8-week-old NOD/LtJ mice and the bones were flushed withComplete Macrophage Media using a 26-gauge needle into a50-mL conical tube. The collected bone marrow wascentrifuged at 1100 RPM for 7 minutes at 4�C. The pelletwas resuspended in 10 mL Complete Macrophage Media(high-glucose Dulbecco’s modified Eagle medium supple-mented with 10% fetal bovine serum, 10 % L929 condi-tioned media, 2% MEM nonessential amino acids, 1%L-glutamine, 1% HEPES, 1% penicillin-streptomycin, and0.1% 2-mercaptoethanol) and filtered into a new tube. Thecells were seeded at 2 � 106 per well in 24-well plates andcultured in a 37�C humid CO2 (5%) incubator. The cellswere fed with fresh media every other day for 7 days. Onday 7, the cells were replenished with L929-free media andwere ready for subsequent assays.

Co-Culture of Bone Marrow–DerivedMacrophages With explants

Islets and acinar cells were isolated from 8- to 12-week-old C57Bl/6 mice as described previously.7 For induction ofapoptosis, freshly isolated hand-picked islets and acinarcells were resuspended in phosphate-buffered saline andirradiated in ultra low-attachment plates with a dose of160 kV, 19 mA for 6 minutes (XRAD-160, Precision X-rayInc., North Branford, CT). Necrosis was achieved by sub-jecting cells to multiple cycles of freezing/thawing. Bonemarrow–derived macrophages obtained at day 7 were thenincubated overnight with apoptotic or necrotic acinar cells,islets, or a mixture of islets and acinar cells. Each 24-well

received 20 islets/well, 40 uL acinar cell suspension/well,or 20 islets þ 20 uL acinar cell suspension, resuspended in500 uL Pancreatic Culture Media (M199 media supple-mented with 10% fetal bovine serum, 1% penicillin-streptomycin) (Supplementary Figure 1).

After 18–24 hours, the dead cells were removed andfresh Pancreatic Culture Media was added to the wellscontaining the macrophages. At the same time, E11.5 dorsalpancreatic epithelium (depleted of the mesenchyme) wasdissected and put on top of Transwell inserts and culturedin 700 uL Pancreatic Culture Media, as described pre-viously.8–10 To ensure the constant presence of the factorsproduced and released by the macrophages during the7-day culture period, 100 uL fresh media was added everyother day to compensate for the evaporation. Control E11.5dorsal buds, with or without mesenchyme, were cultured inthe absence of macrophages or with unfed macrophages.

Minimum number of cells counted for each conditionwas: intact buds: 1200 cells, epithelium: 300 cells, epithe-lium with noninduced wt macrophages: 300 cells, epithe-lium with wt macrophages fed with apoptotic acinar cells:800 cells, epithelium with wt macrophages fed withapoptotic islets: 380 cells, epithelium with wt macrophagesfed with a mixture of apoptotic acinar cells and islets: 750cells, epithelium with noninduced NOD macrophages: 240cells, epithelium with NOD macrophages fed with apoptoticacinar cells: 780 cells, epithelium with NOD macrophagesfed with apoptotic islets: 700 cells, epithelium with NODmacrophages fed with a mixture of apoptotic acinar cellsand islets: 700 cells.

Quantitative Real-Time Polymerase ChainReaction and Western Blotting

Quantitative real-time polymerase chain reaction andWestern Blotting were carried out on in vivo infiltratingmacrophages isolated from injured pancreata or on bonemarrow–derived macrophages cultured in vitro in differentconditions. For quantitative reverse transcription polymer-ase chain reaction, messenger RNA isolation and subse-quent complementary DNA synthesis were performed usingmMACS One-step cDNA kit (Miltenyi Biotec, Cat. No. 130-091-902) according to the manufacturer’s instructions.Polymerase chain reaction primers were purchased fromQiagen (QuantiTect Primer Assays, Qiagen) and are listed inSupplementary Table 2. Reactions were performed withPerfeCTa SYBR Green SuperMix for IQ (Quanta Biosciences,Cat. No. 95053) on a BioRad IQ5 Instrument (Biorad). Re-actions were performed at least in triplicates and specificityof the amplified products was determined by melting peakanalysis. Quantification for each gene of interest was per-formed with the 2-DDCt method. Quantified values werenormalized against the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase, which proved to be stableacross the samples. Primers are listed in SupplementaryTable 2. For Western Blotting, cells were lysated in Com-plete Lysis-M Reagent supplemented with protease in-hibitors (Roche, Cat. No. 04719964001). Proteinquantification was performed with Pierce BCA protein

1118.e2 Criscimanna et al Gastroenterology Vol. 147, No. 5

Assay Kit (Thermo Scientific, Cat. No. 23227). Protein ly-sates were separated on 4%�20% gradient sodium dodecylsulfate polyacrylamide gel electrophoresis gels (Mini-PRO-TEAN TGX Gels, BioRad, Cat. No. 456-1094) and transferredonto 0.45-mm charged polyvinylidene difluoride membranesusing a Mini Trans-Blot electrophoretic apparatus (Bio-Rad).11 All buffers were purchased from BioRad. Antibodiesand dilutions used are listed in Supplementary Table 1.

Immunofluorescence and Quantification AnalysisTissue processing, immunostaining, and quantification

analysis were performed as described previously.2 Anti-bodies and dilutions used are listed in SupplementaryTable 1.

Enzyme-Linked Immunosorbent AssayBone marrow–derived macrophages from both C57Bl/6

and NOD mice were treated with 10 ng/mL recombinantmouse hepatocyte growth factor (R&D Systems) for 24 hoursat 37�C before stimulation with 100 ng/mL lipopolysaccha-ride from Escherichia coli (055:B5) (Sigma Aldrich) for 6hours. Only lipopolysaccharide treatment was performed incontrols. Supernatants were collected from triplicate wellsand TNFa was measured by enzyme-linked immunosorbentassay using Mouse TNF-alpha DuoSet (R&D Systems).

References1. Hingorani SR, Petricoin EF, Maitra A, et al. Preinvasive

and invasive ductal pancreatic cancer and its earlydetection in the mouse. Cancer Cell 2003;4:437–450.

2. Criscimanna A, Speicher JA, Houshmand G, et al. Ductcells contribute to regeneration of endocrine and acinarcells following pancreatic damage in adult mice. Gastro-enterology 2011;141:1451–1462. 1462 e1–6.

3. Kanai T, Watanabe M, Okazawa A, et al. Macrophage-derived IL-18-mediated intestinal inflammation in themurine model of Crohn’s disease. Gastroenterology2001;121:875–888.

4. Kanai T, Uraushihara K, Totsuka T, et al. Amelioratingeffect of saporin-conjugated anti-CD11b monoclonalantibody in a murine T-cell-mediated chronic colitis.J Gastroenterol Hepatol 2006;21:1136–1142.

5. Heldmann U, Mine Y, Kokaia Z, et al. Selective depletionof Mac-1-expressing microglia in rat subventricular zonedoes not alter neurogenic response early after stroke.Exp Neurol 2011;229:391–398.

6. Ferrini F, Trang T, Mattioli TA, et al. Morphine hyper-algesia gated through microglia-mediated disruption ofneuronal Cl(-) homeostasis. Nat Neurosci 2013;16:183–192.

7. Li DS, Yuan YH, Tu HJ, et al. A protocol for islet isolationfrom mouse pancreas. Nat Protoc 2009;4:1649–1652.

8. Esni F, Stoffers DA, Takeuchi T, et al. Origin of exocrinepancreatic cells from nestin-positive precursors indeveloping mouse pancreas. Mech Dev 2004;121:15–25.

9. Esni F, Miyamoto Y, Leach SD, et al. Primary explantcultures of adult and embryonic pancreas. Methods MolMed 2005;103:259–271.

10. Esni F, Ghosh B, Biankin AV, et al. Notch inhibits Ptf1function and acinar cell differentiation in developingmouse and zebrafish pancreas. Development 2004;131:4213–4224.

11. Criscimanna A, Duan LJ, Rhodes JA, et al. PanIN-Spe-cific regulation of Wnt Signaling by HIF2alpha duringEarly pancreatic tumorigenesis. Cancer Res 2013;73:4781–4790.

Author names in bold designate shared co-first authorship.





















































Supplementary Table 1.List of Antibodies Used in this Study

Antigen Species Company Cat. No. Dilution

E-cadherin Goat R&D AF748 1:200 (IF)E-cadherin Rat Invitrogen 13-1900 1:200 (IF)panCK Rabbit Dako Z0622 1:100 (IF)Ck19 Rat DSHB TROMA III 1:100 (IF)Insulin Guinea pig Millipore 4011-01F 1:1000 (IF)Glucagon Guinea pig Millipore 4031-01F 1:1000 (IF)Glucagon Rabbit Millipore 4030-01F 1:2000 (IF)Pdx1 Rabbit Abcam AB47267 1:2000 (IF)Pdx1 Goat Abcam AB47383 1:10,000 (IF)Sox9 Rabbit Millipore AB5535 1:1000 (IF)Amylase Rabbit Sigma A8273 1:300 (IF)Amylase Goat Santa Cruz Biotechnology Sc-12821 1:250 (IF)Fizz1 (RELM alpha) Rabbit Abcam AB39626 1:100 (IF)F4/80 Rat Invitrogen MF48000 1:100 (IF)F/480 eFluor 660 Rat eBioscience 50-4801-82 1:100 (Flow)CD11b APC Rat Miltenyi Biotech 130-091-241 1:10 (Flow)CD11b PE Rat BD Pharmingen 557397 1:100 (Flow)IL6 PE Rat BD Pharmingen 554401 1:100 (Flow)TNFa PE Rat BD Pharmingen 554419 1:100 (Flow)IL10 PE Rat BD Pharmingen 554467 1:100 (Flow)CD206 FITC Mouse BD Pharmingen 551135 1:10 (Flow)Biotinylated DBA Vector Laboratories B-1035 1:10 (Flow)Wnt5a Rabbit Abcam Ab72583 1:250 (WB)Wnt7b Rabbit Novus Biologicals NBP1-59564 1:500 (WB)

1:1000 (IF)b-actin Mouse Sigma A5441 1:20,000 (WB)

NOTE. All secondary antibodies used for immunostaining were purchased from Jackson ImmunoResearch Laboratories:biotin-conjugated anti-rabbit (1:500), biotin-conjugated anti-rat (1:500), biotin-conjugated anti-guinea pig (1:500), biotin-conjugated anti-goat (1:250); Cy2-conjugated streptavidin 1:500; Cy3-conjugated streptavidin 1:500; Cy5-conjugated strep-tavidin 1:100; and Cy2- and Cy3-conjugated donkey anti-guinea pig, anti-rabbit, anti-rat, anti-goat (all 1:300). All isotypes forflow cytometry (Flow) were purchased from BD Pharmingen. Secondary antibodies for Western blotting (WB) were purchasedfrom Santa Cruz Biotechnology (Santa Cruz, CA) and used 1:2000 for 1 hour at room temperature.APC, allophycocyanin; Ck, cytokeratin; DBA, dolichos biflorus agglutinin; FITC, fluorescein isothiocyanate; Fizz1, Found ininflammatory zone 1; Pdx1, pancreatic and duodenal homeobox 1; PE, phycoerythrin; Sox9, SRY (sex determining region Y)-box 9; TGF, transforming growth factor; IF, immunofluorescence.


Supplementary Table 2.List of Primers Used in This Study

Gene Qiagen QuantiTect primer assay Catalog number

GAPDH Mm_Gapdh_3_SG QT01658692iNOS Mm_Nos2_1_SG QT00100275IL6 Mm_Il6 _1_SG QT00098875IL10 Mm_Il10 _1_SG QT00106169Arg1 Mm_Arg1_1_SG QT00134288FGF10 Mm_Fgf10_1_SG QT00164157TGFa Mm_Tgfa_1_SG QT00133539VEGFa Mm_Vegfa_1_SG QT00160769HGF Mm_Hgf_1_SG QT00158046Wnt3a Mm_Wnt3a_1_SG QT00250439Wnt5a Mm_Wnt5a_1_SG QT00164500Wnt7b Mm_Wnt7b_1_SG QT00168812EGF Mm_Tgfb1_1_SG QT00145250TGFb1 Mm_Egf_1_SG QT00151018Lef1 Mm_Lef1_1_SG QT00148834Axin2 Mm_Axin2_1_SG QT00126539CyD1 Mm_Ccnd1_1_SG QT00154595c-Myc Mm_Myc_1_SG QT00096194

Arg1, arginase 1; EGF, epidermal growth factor; FGF10,fibroblast growth factor 10; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HGF, hepatocyte growth factor;iNOS, inducible nitric oxide synthase; VEGF, vascular endo-thelial growth factor.


Supplementary Figure 1. Schematic outline of the co-culture experiments. Full description has been provided in the Materialsand Methods section. Aci, acinar; Apo, apoptotic; BM, bone marrow; CSF-1, colony-stimulating factor 1; Isl, islet; Nec,necrotic.


Supplementary Figure 2.Macrophages induce cell-specific differentiation in vitro independent of the type of cell death. E11.5dorsal pancreatic bud depleted of mesenchyme (Epi) were co-cultured with macrophages that had been “fed” with necroticacinar (Nec Aci) (A), islet (Nec Isl) (B), or a mixture of acinar and islet cells (Nec Mix) (C). The day-7 explants were analyzed forthe expression of amylase (AMY) and E-cadherin (Ecad), or insulin (INS), glucagon (GCG), and E-cadherin, and quantified (D).n ¼ 5 for each condition, *P � .05; **P � .01. Scale bar ¼ 20 mm.


Supplementary Figure 3. Impairedpancreatic regeneration in older mice isassociated with defective macrophagepolarization in vivo. (A) The late stagepancreas in old PdxCre;R26DTR miceafterDT treatmentdoesnot recover andconsists entirely of Ck19þ/Ecadherinþ

ductal structures. (B) The number ofmacrophages in the pancreas of DT-treated old PdxCre;R26DTR mice is notreduced during early stage, indicatingthat the recruitment of macrophages isnormal. (C, D) Older mice lack propermacrophage polarization during themid stage of regeneration. Tissuesharvested at early (C) ormid stage (D) ofDT-treated old PdxCre;R26DTR pan-creata show decreased number ofF/80/Fizz1 double-positive cells. (E, F)The reactivation of embryonic programassociated with regeneration in the DT-treated PdxCre;R26DTR pancreas doesnot occur in the old mice. Tissues ob-tained from DT-treated old PdxCre;R26DTR pancreas lack PDX1 re-expression in SOX9þ ductal cells.Ck19, cytokeratin 19, Ecad, Ecadherin.Scale bars ¼ 20 mm.


Supplementary Figure 4.Macrophages isolated from old mice are capable of inducing cell-specific differentiation in vitro.E11.5 dorsal pancreatic bud depleted of mesenchyme (Epi) were co-cultured with noninduced macrophages (A), or withmacrophages that had been “fed” with apoptotic acinar (Apo Aci) (B), islet (Apo Isl) (C), or a mixture of acinar and islet cells(Apo Mix) (D). The day 7 explants were analyzed for the expression of amylase (AMY) and E-cadherin (Ecad), or insulin (INS),glucagon (GCG) and E-cadherin, and quantified (E). n ¼ 5 for each condition, *P � .05; **P � .01. Scale bar ¼ 20 mm. Aci,acinar; Apo, apoptotic; Isl, islet; epi, epithelium.


Supplementary Figure 5.WNT7b expression in macro-phages. Tissues obtained from DT-treated PdxCre;R26DTR (A)or diabetic NOD mice (B) were stained for Wnt7b, Ecadherin(Ecad), and F4/80. WNT7b is detected in a subset of F4/80þ

macrophages in the regenerating PdxCre;R26DTR pancreas,but it is hardly detected in NOD macrophages at the onset ofdiabetes. Arrows and asterisk highlight the ducts or peri-insulitis, respectively. Scale bars ¼ 20 mm.


Supplementary Figure 6. NOD-derivedmacrophages display a different growthfactor profile when fed with apoptotic cellsin vitro. Quantitative reverse transcriptionpolymerase chain reaction analysis ofbone marrow–derived macrophages origi-nating from C57Bl/6 control or NOD micethat had been fed with apoptotic acinar(Apo Aci), islets (Apo Isl), or a mixture ofendocrine and acinar cells (Apo Mix) andharvested on days 2 or 7 in culture. TheNOD-derived macrophages exhibit anexacerbated M1-signature, and expressdifferent expression profile than theircontrol counterparts. Bars represent rela-tive gene expression/glyceraldehyde-3-phosphate dehydrogenase (GAPDH)(mean ± SE) expressed as fold over non-induced C57Bl/6 control macrophages.n ¼ 5. *P � .05; **P � .01; ***P � .001.


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Gastroenterology 2014;147:1106 BASIC AND TRANSLATIONAL ... 2014_Criscimanna.pdf · Methods Mice Mice used in this study were maintained according to protocols approved by the University