Dendritic Cells Promote Pancreatic Viability in Mice With Acute Pancreatitis

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GASTROENTEROLOGY 2011;141:1915–1926

BASIC AND TRANSLATIONAL—PANCREAS

Dendritic Cells Promote Pancreatic Viability in Mice With AcutePancreatitisANDREA S. BEDROSIAN,* ANDREW H. NGUYEN,‡ MICHAEL HACKMAN,* MICHAEL K. CONNOLLY,*

SHIM MALHOTRA,* JUNAID IBRAHIM,* NAPOLEON E. CIEZA–RUBIO,* JUSTIN R. HENNING,* ROCKY BARILLA,*DEEL REHMAN,* H. LEON PACHTER,* MARCO V. MEDINA–ZEA,* STEVEN M. COHEN,* ALAN B. FREY,‡

DEVRIM ACEHAN,‡ and GEORGE MILLER*,‡

S. Arthur Localio Laboratory, Departments of *Surgery and ‡Cell Biology, New York University School of Medicine, New York, New York

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BACKGROUND & AIMS: The cellular mediators ofcute pancreatitis are incompletely understood. Dendriticells (DCs) can promote or suppress inflammation, de-ending on their subtype and context. We investigated theoles of DC in development of acute pancreatitis. METH-

ODS: Acute pancreatitis was induced in CD11c.DTRmice using caerulein or L-arginine; DCs were depletedby administration of diphtheria toxin. Survival wasanalyzed using Kaplan–Meier method. RESULTS: Num-bers of major histocompatibility complex II�CD11c� DCsncreased 100-fold in pancreata of mice with acute pancre-titis to account for nearly 15% of intrapancreatic leukocytes.ntrapancreatic DCs acquired a distinct immune phenotypen mice with acute pancreatitis; they expressed higher levelsf major histocompatibility complex II and CD86 and in-reased production of interleukin-6, membrane cofactorrotein–1, and tumor necrosis factor–�. However, rather

than inducing an organ-destructive inflammatory process,DCs were required for pancreatic viability; the exocrinepancreas died in mice that were depleted of DCs andchallenged with caerulein or L-arginine. All mice withpancreatitis that were depleted of DCs died from acinarcell death within 4 days. Depletion of DCs from mice withpancreatitis resulted in neutrophil infiltration and in-creased levels of systemic markers of inflammation. How-ever, the organ necrosis associated with depletion of DCsdid not require infiltrating neutrophils, activation of nuclearfactor–�B, or signaling by mitogen-activated protein kinaseor tumor necrosis factor–�. CONCLUSIONS: DCs are re-quired for pancreatic viability in mice with acute pan-creatitis and might protect organs against cell stress.

Keywords: Immune Response; Regulation; Suppression;MCP-1; IL-6.

Acute pancreatitis has a considerable medical and eco-nomic impact. In the United States, this disease re-

ults in �200,000 hospital admissions annually, at a costf �$2 billion.1 While most patients will have an uncom-

plicated course, 10%–20% of cases are categorized as se-vere, resulting from organ necrosis, and are often associ-
ated with localized infection, systemic sepsis, and
multiorgan failure. Acute pancreatitis carries a 2% overallmortality rate, which increases to 10%–30% in cases ofpancreatic necrosis.2 These data demand an enhancedunderstanding of the pathophysiology of acute pancreati-tis in order to implement more effective treatments.

Acute pancreatitis usually results from either alcoholabuse or obstructing gallstones, which are postulated tocause intra-acinar activation of proteolytic enzymes leadingto intense pancreatic inflammation. The inflammatory me-diators in this disease have been partially delineated in ani-mal models mimicking human pancreatitis.3,4 Massive leu-kocyte infiltration is one of the early events of pancreatitis,and neutrophils play a distinctly important role in its patho-physiology.5 Neutrophil recruitment worsens pancreatic in-ury6 and, conversely, neutrophil depletion lessens tissuenjury associated with acute pancreatitis.7 Similarly, CD4� T

cells are required for pancreatic acinar injury in rodent mod-els of acute pancreatitis.8 Importantly, Gukovsky et al9 dem-

nstrated that the nuclear transcription factor nuclear fac-or–�B (NF-�B), a key regulator of inflammation, is activated

early in pancreatitis. Furthermore, NF-�B has recently beenhown to regulate expression of activator protein–1, which

odulates the chemokine cascade after pancreatic insult.10

The CXC-ELR family of chemoattractants, including CXCL1and CXCL2, further regulate the influx of inflammatory cellsin acute pancreatitis.11

Dendritic cells (DCs) have emerged as important partici-pants in inflammation. As instructors of the T-cell response,DCs are the most powerful antigen-presenting cell in theimmune system. Upon exposure to bacterial or viral infec-tion or inflammatory stimuli, immature DCs become acti-vated and drive both adaptive and innate immune re-sponses.12 Pattern recognition receptors on the DC surface,he most well-understood being Toll-like receptors, recog-

Abbreviations used in this paper: DCs, dendritic cells; IL, interleukin;MAPK, mitogen-activated protein kinase; MCP-1, membrane cofactorprotein–1; MHC, major histocompatibility complex; MIP–1�, macro-phage inflammatory protein–�; NF-�B, nuclear factor–�B; pDC, plas-

acytoid DC; TNF-�, tumor necrosis factor–�; Treg, regulatory T cell.© 2011 by the AGA Institute

0016-5085/$36.00
doi:10.1053/j.gastro.2011.07.033

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1916 BEDROSIAN ET AL GASTROENTEROLOGY Vol. 141, No. 5

nize pathogen-associated molecular patterns as well as dam-age-associated molecular patterns released by cellular in-jury.13 Immature DCs become activated to maturity by the

inding of pathogen-associated molecular patterns or dam-ge-associated molecular patterns, which may be releasedecondary to tissue damage or by cytokines generated duringhe inflammatory response. Mature DCs process and presentntigen to T cells via major histocompatibility complexMHC) complexes. This classical model dictates very tightlyegulated pathways to DC activation, as mature DCs areentral to development of inflammation and effective immu-ogenic responses.Recent work by our group and others has shown a central

ole for DCs in a number of organ-specific inflammatoryiseases. We reported that DCs tightly regulate the extent of

ntrahepatic inflammation after liver insult from hepatotox-ns via their production of tumor necrosis factor–� (TNF-

�).14 Bamboat et al15 also reported that DCs modulate thextent of liver injury after ischemic insult via their produc-ion of interleukin (IL)-10. DCs have also been assigned aentral role in modulating the severity of systemic sepsis.16,17

Our preliminary investigations in the current study revealeda marked increase in the numbers of intrapancreatic DCs inacute pancreatitis. Based on this, we postulated an impor-tant role for DCs in modulating intrapancreatic inflamma-tion and thus determining the extent of endocrine andexocrine pancreatic injury.

MethodsAnimalsMale C57BL/6 (H-2Kb) and CD45.1 (B6.SJL-Ptprca/

BoyAiTac) mice (6–8 weeks old) were purchased from TaconicFarms (Germantown, NY). CD11b.DTR18 and CD11c.DTR19 micewere purchased from Jackson Laboratory (Bar Harbor, ME). Micewere housed in a pathogen-free environment and fed standardchow. Mouse serum was collected by retro-orbital bleeding. Toaffect macrophage or DC depletion, respectively, CD11b.DTR andCD11c.DTR mice were treated with a single intraperitoneal dose ofdiphtheria toxin (4 ng/g; Sigma-Aldrich, St Louis, MO). Animalprocedures were approved by the New York University School ofMedicine Institutional Animal Care and Use Committee.

Models of Acute Pancreatitis and In VivoExperimentsPancreatitis was induced using a regimen of 7 hourly

intraperitoneal injections of caerulein (50 �g/kg; Sigma-Aldrich)for 2 consecutive days before sacrifice 12 hours later, unlessotherwise specified. Alternatively, intraperitoneal administrationof 2 doses of L-arginine (40 mg/kg; Sigma-Aldrich) at hourlyintervals was used to induce pancreatic injury.3,4 In selectedxperiments, bone marrow chimeric animals were created byrradiating C57BL/6 mice (100 Gy) before intravenous bone

arrow transfer (1 � 107) from CD45.1 or CD11c.DTR donors.n vivo TNF-� blockade was accomplished using MP6-XT22200 �g/day). IL-6 blockade was accomplished using MP5-20F3

(200 �g/day). Macrophage inflammatory protein–� (MIP-1�)lockade was accomplished using clone 39624 (300 �g/day; all

R&D Systems, Minneapolis, MN). NF-�B blockade was accom-
plished using both cell permeable inhibitors of the NF-�B p50
omain, which prevents its nuclear translocation (NF-�B SN50)nd the NEMO binding domain inhibitor (both 1 mg/kg/day),hich prevents binding of NEMO to the IKK (I�B)-kinase com-

plex (both EMD4Biosciences, Gibbstown, NJ ). Mitogen-acti-vated protein kinase (MAPK) blockade was accomplished usingPD98059 (2.5 mg/kg/day; Invivogen, San Diego, CA). To depleteGr1� cells or CD4� T cells, RB6-8C5, or GK1.5 (MonoclonalAntibody Core Facility, Sloan-Kettering Institute, New York,NY) were employed, respectively, as described.20,21 In vivo plas-

acytoid DC (pDC) depletion was accomplished using eithernti–mPDCA-1 (500 �g; Miltenyi, Bergisch Gladbach, Germany)r 120G8 (200 �g; Imgenex, San Diego, CA).22,23 Serum levels ofancreatic enzymes and glucose were measured using the Olym-us AU400 Chemistry Analyzer (Center Valley, PA).

StatisticsData are presented as mean � standard error of mean.

urvival was measured according to the Kaplan-Meier method.tatistical significance was determined by the Student’s t testnd or log-rank test using GraphPad Prism 5 (GraphPad Soft-are, La Jolla, CA). P values �.05 were considered significant.See additional Supplemental Materials and Methods.

ResultsDCs Expand in Acute PancreatitisTo assess the significance of DCs in acute pancreati-

tis, we first tested whether the intrapancreatic MHCII�CD11c� DC population expands after pancreatic insultrom 14 caerulein injections during a 36-hour period. Weound that while DCs were rare in the normal pancreas, theotal number of intrapancreatic DCs increased markedly incute pancreatitis, reaching a peak at 72 hours after begin-ing caerulein challenge (Figure 1A). Intrapancreatic DCumbers returned to normal by 7 days after beginningaerulein injections. The total number of other leukocyteubgroups also increased markedly in acute pancreatitis;owever, there was a disproportional increase in DCs (FigureB). In particular, the fraction of intrapancreatic MHCI�CD11c� DCs expanded from a baseline of 1%–3% to

nearly 15% of all CD45� intrapancreatic leukocytes (Figure1C–E). Conversely, the number of splenic DCs remainedonstant in acute pancreatitis, suggesting that DC expansions a pancreas-specific phenomenon (Figure 1C).

The origins of intrapancreatic DCs in acute pancreatitisre not entirely certain, given the experimental limitationsf tracking DCs in situ. However, we found that 48 hoursfter adoptive transfer of carboxyfluorescein succinimidylster–stained bone marrow– derived DCs to mice under-oing caerulein challenge, a large number of transferredone marrow– derived DCs were found in the pancreas,uggesting that DCs derived from the bone marrow mayave a tendency to migrate to the pancreas in inflamma-ory conditions (Figure 1F). To further explore the issue ofCs origins in pancreatitis, we made C57BL/6 mice chi-eric using bone marrow from CD45.1 mice. Two weeks

ater, when bone marrow leukocytes were almost entirelyD45.1� (Supplementary Figure 1A), but only approxi-

mately 50% of intrapancreatic antigen-presenting cells
were CD45.1� (Supplementary Figure 1B), acute pancre-

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November 2011 DCs IN ACUTE PANCREATITIS 1917

atitis was induced. We found that in caerulein-chal-lenged animals, the vast majority of expanded intra-pancreatic DCs were CD45.1�, suggesting a bonemarrow origin of pancreatic DCs in acute pancreatitis(Supplementary Figure 1C).

Pancreatic DCs Gain a Distinct ImmunePhenotype in Acute PancreatitisTo determine whether intrapancreatic DCs are in

an activated state in acute pancreatitis, we compared the

Figure 1. Intrapancreatic DCs expand in acute pancreatitis. (A) Mice wenumber of intrapancreatic DCs was determined at various time pointsleukocytes from caerulein-treated mice were characterized at 48 hours asaline-treated controls. (C) The fraction of DCs among intrapancreatic aC) treated animals. (D) Frozen pancreatic sections were stained using anntrapancreatic CD45�CD11c� cells in control and caerulein-treatedintrapancreatic carboxyfluorescein succinimidyl ester (CFSE)� cells wamarrow–derived DCs. Experiments were repeated more than 3 times u

surface phenotype of pancreatic DCs from caerulein-in-

duced pancreatitis to those of saline-treated mice. Pancre-atic DCs in acute pancreatitis were markedly more maturethan controls, expressing higher levels of MHC II, CD40,CD80, and CD86 (Figure 2A). In addition, the DC popu-lation underwent a mild myeloid shift expressing slightlyhigher levels of CD11b and lower CD8� (Figure 2A).Conversely, spleen DC surface phenotype was unaltered inacute pancreatitis (Figure 2B). In acute pancreatitis, pan-creatic inflammatory cells expressed TNF-� and IL-6 (Sup-plementary Figure 2A). To determine whether pancreatic

hallenged with caerulein at hourly intervals 7 times daily for 2 days. Theting from immediately before the first injection. (B) Pancreas-infiltratinginitiation of caerulein treatment and their numbers were compared with

splenic CD45� leukocytes was compared in saline- (Ctl) and caerulein-dies directed against MHC II and visualized by immunofluorescence. (E)

e were measured at 48 hours by flow cytometry. (F) The number ofetermined at 48 hours after injection of saline or CFSE-labeled bone2–5 mice per data point (***P � .001).

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DCs become proinflammatory after pancreatic insult, we

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measured their production of cytokines. Pancreatic DCsproduced markedly elevated levels of activating cytokinesin acute pancreatitis, including TNF-� (Figure 2C and D),membrane cofactor protein–1 (MCP-1) (Figure 2C), IL-6(Figure 2C and D), IL-17a (Figure 2D), and interferon-gamma (Figure 2D). Conversely, pancreatic DC produc-tion of IL-10 and transforming growth factor–�, a reg-

latory cytokine, was unchanged in pancreatitis (FigureC). Furthermore, in consort with our previous find-

ngs, spleen DC production of inflammatory mediatorsas unchanged in acute pancreatitis (Supplementaryigure 2B).

DCs Are Necessary for Pancreas ViabilityAfter InjuryBecause DCs expand, mature, and become proin-

flammatory in acute pancreatitis, we postulated that theycontribute significantly to intrapancreatic inflammationand end-organ injury. To test this hypothesis, we em-ployed CD11c.DTR mice,19 in which transient DC deple-

Figure 2. Intrapancreatic DCs are mature and proinflammatory in acutemarker expression by flow cytometry. Median fluorescence index is indicinflammatory mediators was measured (C) in cell culture supernatantexperiments repeated 3 times and performed in triplicate using 2–5 mic

tion can be effected for 48 hours (Supplementary Figure t

3). Mice were either depleted of DC (C-DC) or mock-depleted before caerulein challenge. Remarkably, ratherthan lessening the severity of pancreatitis, DC depletionresulted in almost complete death of the exocrine pan-creas (Figure 3A and B). Furthermore, all C-DC mice died

f acinar cell death within 4 days of commencing caeru-ein injections (Figure 3C). Importantly, the severe effectsn the pancreas associated with DC depletion were notelated to either diphtheria toxin treatment or theD11c.DTR transgenic model, as diphtheria toxin–

reated C57BL/6 or CD11b.DTR (macrophage-depleted)ice did not develop exacerbated injury (Figure 3D). Sim-

larly, CD11c.DTR mice challenged with caerulein but notepleted of DC did not progress to acinar cell death

Figure 3D).Inflammation can result in expansion of immature

DCs.24 We found that approximately 10% of intra-ancreatic DCs in pancreatitis had the B220� plasma-ytoid phenotype (Supplementary Figure 4A). To inves-

ncreatitis. (A) Pancreatic and (B) splenic DCs were assayed for surfacebelow each histogram. (C, D) Intrapancreatic DC production of various

d (D) using intracellular cytokine analysis. Data are representative ofer group (*P � .05, **P � .01, ***P � .001).

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protection, we selectively depleted pDCs using eitheranti–mPDCA-1 or 120G8. However, regardless of modelof depletion, pDC depletion did not result in exacer-bated pancreatic injury in acute pancreatitis (Supple-mentary Figure 4B). Notably, adoptive transfer of BM-

Cs or spleen DCs was unable to rescue the pancreataf caerulein-challenged DC-depleted mice (Supplemen-ary Figure 5).

To confirm that pancreatic parenchymal cells were not

Figure 3. DC depletion in acute pancreatitis results in pancreatic necroor mock depletion. (A) Pancreata were analyzed using H&E. (B) Thehigh-powered fields (HPF) per mouse (mean, 5 mice per group). (C) Cosurvival according to the Kaplan-Meier method. (D) Diffuse acinar cell d(DPT) treatment. Notably, treatment of CD11b.DTR mice with caerulein astrain is indicated in parentheses preceded by treatment (***P � .001).

nadvertently targeted in depleting DC in CD11c.DTR

mice, we tested their expression of CD11c. However,CD45� pancreatic cells did not express CD11c (Supple-mentary Figure 6A). To further exclude direct targeting ofpancreatic acini or ducts in CD11c.DTR mice, we createdCD11c.DTR bone marrow chimerics by irradiatingC57BL/6 mice and transferring CD11c.DTR bone marrowcells. For controls, we created C57BL/6 chimerics by trans-ferring wild-type bone marrow to irradiated mice. Sixweeks later, chimeric mice were treated with DPT and

(A–C) Mice were challenged for 2 days with caerulein after DC depletionrcentage of nonviable acinar cells was determined by examining 10ts of mice (4–5 per group) were not sacrificed but instead analyzed for

was specific to DC depletion and not mouse strain or diphtheria toxinPT to deplete macrophages did not result in exacerbated injury. Mouse

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merics developed severely exacerbated pancreatitis, ex-cluding direct targeting of parenchymal cells in theCD11c.DTR model (Supplementary Figure 6B–D).

Effects of DC Depletion Are Not Model-SpecificTo confirm the relevance of our findings, we next

investigated whether the nonviable pancreatic paren-chyma resulting from DC depletion in the context ofacute pancreatitis was restricted to the caerulein model.To test this, we employed L-arginine to induce acutepancreatitis in DC-depleted CD11c.DTR mice or con-trols. We again observed diffusely nonviable pancreataafter DC depletion in the L-arginine model of acutepancreatitis, confirming that this finding is not model-specific (Figure 4).

Time Course of Acinar Cell DeathTo determine the time course of acinar death

upon induction of acute pancreatitis in the context ofDC depletion, we harvested pancreata at serial intervalsafter beginning caerulein treatment. Approximately20% of acini were nonviable after 7 caerulein doses inmice depleted of DCs. The fraction of nonviable aciniincreased to nearly 80% after 14 treatments by histo-logic examination. Acinar death was nearly completeafter an additional 12-hour delay and was associatedwith a robust inflammatory infiltrate (Figure 5A). Ter-

inal deoxynucleotidyl transferase–mediated deoxyuri-ine triphosphate nick-end labeling staining also re-

Figure 4. DC depletion in L-arginine–induced acute pancreatitis resuDC depletion or mock depletion (3 mice per group). Percent organ n
pancreas (***P � .001).
vealed that acinar cell apoptosis increased significantlyduring the 12-hour lag period after cessation of caeru-lein treatment (Figure 5B).

DCs have recently been assigned an important role inthe clearance of cellular debris in inflammatory dis-ease.25,26 Because DCs expand most sharply during theegeneration phase of acute pancreatitis (Figure 1A), andC depletion results in an marked increase in apoptotic

ells and necrotic elements, we postulated that DCs mayave a primary role in clearance of cellular debris inancreatitis and, consequently, DC depletion may result

n expansion of sterile inflammation in acute pancreatitis.n support of this notion, we found that C-DC pancreataave markedly higher levels of HMGB-1 (Supplementaryigure 7A), which is also consistent with our histologicndings showing increased apoptotic and necrotic acini.e tested the relative capacity of pancreatic DCs to cap-

ure antigen as well as necrotic and apoptotic cells incute pancreatitis. CD11c�MHCII� DCs from acute pan-reatitis pancreata captured fluorescein isothiocyanate–extran at a higher rate than CD11c�MHCII� antigen-

presenting cells from the same pancreata (SupplementaryFigure 7B). In addition, we found that pancreatic DCcaptured 7-aminoactinomycin D� necrotic cells at a far

reater rate than other antigen-presenting cells in APSupplementary Figure 7C). Moreover, the in vivo uptakef 7-aminoactinomycin D� necrotic cellular debris (Sup-lementary Figure 7D) and Annexin� apoptotic bodies

Supplementary Figure 7E) by MHC II� cells was severely

n severe injury. Mice were challenged with 2 doses of L-arginine afterosis was calculated by examining 10 high-powered fields (HPF) per

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deficient in pancreata depleted of DCs compared withpancreata with a normal complement of DCs. Takentogether, these data suggest that DCs may exert theirprotective effects by clearance of necrotic and apoptoticcells thereby limiting sterile inflammation.

Systemic Effects of DC Depletion in AcutePancreatitisConsistent with our findings of pancreatic end

organ destruction, by 48 hours serum amylase levels werelower in C-DC mice than in animals challenged withcaerulein alone (Figure 6A). Serum levels of inflammatorycytokines, including MCP-1, IL-6, and TNF-� were ele-

Figure 5. Time course of pancreatic injury. (A) Mice were depleted of Dsacrificed either 1 hour after the final dose or after a 12-hour delay and p

ice were treated with 1, 3, 7, or 14 doses of caerulein and sacrifiaraffin-embedded pancreata were then examined by terminal deoxynutaining (*P � .05; **P � .01; ***P � .001).

vated in C-DC mice, consistent with a severe inflamma- p

tory process (Figure 6B). Notably, however, tissue necrosiswas specific to the pancreas, as other organs were pre-served when examined at 36 hours (Supplementary Figure8) or 72 hours (not shown). Furthermore, cell death waslimited to the exocrine pancreas. The endocrine pancreaswas entirely protected in C-DC–treated mice as there wasno evident islet cell destruction (Figure 6C) and serumglucose levels were not elevated in C-DC mice (Figure 6D).

Elevated Expression of IL6, MIP1A, andEGR1 in C-DC PancreataBecause TNF-� was up-regulated in C-DC mice, we

and treated with various doses of caerulein over 1–2 days. Mice werereata examined by H&E staining (average of 3 mice per data point). (B)either 1 hour or 12 hours after the final treatment. Formalin-fixed,

tidyl transferase–mediated deoxyuridine triphosphate nick-end labeling

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ing in pancreatitis in the context of DC depletion. Con-sistent with this hypothesis, we found lower I�B� andlevated pI�B� in C-DC pancreata (Supplementary Figure

9A), which is consistent with increased signaling via NF-�B.27 We performed additional experiments investigatingdifferential expression of genes that are known to modu-late pancreatic injury in C-DC pancreata compared withcaerulein alone. We found that pancreatic expression ofIL6 and EGR1, both positive regulators of pancreaticinjury28,29 as well as MIP1A, were markedly increased inthe pancreata of C-DC mice (Figure 6E and F). Becausethe MAPK pathway is known to regulate EGR1 via thephosphorylation of the transcription factor Elk-1, we in-vestigated MAPK signaling in C-DC mice. We found asmall elevation in pElk-1 in C-DC pancreata at early timepoints; however, there was no detectable increase inErk1/2 phosphorylation (Supplementary Figure 9B andC). Because there is increased acinar cell death in C-DCpancreata, we also examined the relative expression of cell

Figure 6. Effects of DC depletion in pancreatitis. Mice were challengedof (A) amylase and (B) TNF-�, IL-6, and MCP-1 were measured at 48 houf glucose were also measured. (E, F) RNA was harvested from whole panere performed in triplicate and repeated 3 times (*P � .05; *** P � .00

cycle regulator, BCL2; however, there was no change (Sup-

plementary Figure 9D). We similarly did not find differ-ences in expression in additional genes related to cell cycleregulation, metabolism, or heat shock proteins, whichhave been shown to regulate the severity of pancreatitis30

(Supplementary Figure 9E–G).

Effects of DC Depletion Are Not Contingenton Secondary Changes in Cellular ImmunityTo determine whether a baseline level of endoge-

nous DCs directly protects the stressed pancreas fromnecrosis or whether DC depletion results in a secondarysequence of events leading to organ necrosis, we investi-gated the alterations in cellular immunity in acute pan-creatitis upon DC depletion. We found that in C-DCmice, the total number of CD45� pancreatic leukocytes

as elevated (Figure 7A). Moreover, this infiltrate wasttributable to a disproportionate increase in pancre-tic Gr1� neutrophils, which increased in step with

progressive acinar necrosis (Figure 7B and C). Never-

days with caerulein after DC depletion or mock depletion. Serum levelsC) The fraction of pancreatic area occupied by islets and (D) serum levelsata and analyzed for expression of (E) IL6, MIP1A, and (F) EGR1. Assays

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theless, whereas neutrophil depletion partially pro-

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tected caerulein-treated and C-DC mice from edemaand inflammation (Supplementary Figures 10 and 11)as expected,31 it did not protect C-DC pancreata fromcellular death (Figure 7D), suggesting that the neutro-phil infiltration was a consequence of the exacerbatedinjury associated with DC depletion rather than a caus-ative factor. Furthermore, because CD4� T cells have

een implicated in exacerbating acute pancreatitis,8 weostulated that CD4� T-cell depletion, in association

Figure 7. Additional cellular depletion or cytokine blockade does notharvested from C-DC mice and controls and (A) the number of intrapainfiltrating neutrophils were examined by both immunohistochemistrydetermined by immunohistochemistry. Mice were sacrificed either 1 houin C-DC mice was studied in mice concurrently depleted of neutrophneutralizing antibodies targeting IL-6, MIP-1�, or TNF-� (mean, 4 mice/

with DC depletion, may protect pancreata from necro-

sis. However, whereas depletion of CD4� T cells aloneimproved pancreatic edema in non–DC-depleted mice(Supplementary Figure 10) and mitigated inflammationin C-DC–treated mice (Supplementary Figure 11), it didnot protect against complete exocrine destruction (Fig-ure 7D).

To further investigate whether DC depletion in pancre-atitic animals results in necrosis as a result of secondaryeffects of DC depletion, rather than the specific absence of

gate the effects of DC depletion on pancreas viability. Pancreata wereeatic CD45� leukocytes and (B) the number and fraction of pancreas

flow cytometry. (C) The time course of neutrophil recruitment wasfter the final dose of caerulein or after a 12-hour delay. (D) Acinar deathCD4� T cells, mice with blockaded NF-�B signaling, or treated withup; *P � .05; **P � .01; ***P � .001).

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DC, we selectively blocked TNF-�, IL-6, and MIP-1�, each

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of which is markedly elevated in C-DC–treated animals(Figures 4B and 6E), or NF-�B, a key regulator in pancre-atitis.9,11 Blockade of TNF-� was not protective (Figure

D and Supplementary Figures 10 and 11). IL-6 andIP-1� blockade had variable effects on pancreatitis in

absence of DC depletion (Supplementary Figure 10); how-ever, selective cytokine or chemokine blockade offered noprotection against acute pancreatitis in the context of DCdepletion (Figure 7D and Supplementary Figure 11). In-hibition of NF-�B using either a NEMO binding domainnhibitor or a p50 inhibitor mitigated pancreatic edemand inflammation as expected32 (Supplementary Figures0 and 11). However, mice were not protected from acinareath in the context of DC depletion despite abatement in

nflammation (Figure 7D and Supplementary Figure 11).imilarly, MAPK blockade in vivo partially improved pan-reatic edema and inflammation, but did not protect-DC–treated mice from organ destruction (Supplemen-

ary Figure 12).

DiscussionPancreatitis is a disease with significant medical

and economic impact, yet its specific immunologicalpathogenesis is uncertain. We have shown here that DCs,principal modulators of the immune response, play a keyrole in the progression of acute pancreatitis and the extentof organ-specific injury. Using mouse models of acutepancreatitis that mimic human disease, we revealed anexpansion of the DC population specific to the pancreas.DCs also expressed a mature phenotype and producedhigh levels of proinflammatory cytokines without a com-mensurate increase in the regulatory cytokine IL-10.Again, these changes were noted to be pancreas-specific;cytokine levels of spleen DCs remained constant in pan-creatitic mice. The recognition of a discrete, mature, andimmunogenic body of DCs in the pancreas led us toinvestigate whether DCs contribute to the destructiveinflammatory response in acute pancreatitis.

A very surprising and novel finding was that depletionof DCs using CD11c.DTR mice treated with caerulein orL-arginine resulted in a massive increase in the severity ofpancreatitis, with near-complete pancreatic exocrine celldeath and subsequent mortality of all animals treated inthis manner. Markers of injury and inflammation, includ-ing IL-6, EGR1, MCP-1, and TNF-�, were elevated in

ancreatitic mice depleted of DC. Tissue necrosis wasimited to the exocrine pancreas and other organs werereserved. This was in considerable contrast to our previ-us work in chronic liver disease, in which DC depletionbrogated inflammatory markers in the fibrotic liver,14

suggesting that distinct regulatory mechanisms governDC function in acute pancreatitis.

We next investigated the pancreas’ immunological pro-file to shed light on the deleterious effects of DC deple-tion on acute pancreatitis. The severe necrosis observed incaerulein-treated, DC-depleted mice could be attributable
to either a protective influence of endogenous DCs on the H
injured/inflamed pancreas, or a secondary destructivepathway in which DC depletion promotes other eventsculminating in organ necrosis. In the DC-depleted acutepancreatitis model, there was a disproportionate increasein Gr1� neutrophils among the pancreatic leukocyte pop-

lation with progressive acinar necrosis. However, thiseutrophil infiltration, which was confirmed by myeloper-xidase staining, appeared to be merely a sequela of DC-epletion–induced necrosis, as neutrophil depletion hado protective effect on necrosis in DC-depleted pancre-titic mice. Demols et al8 established that CD4� T lym-hocytes are key players in tissue injury in rodent modelsf acute pancreatitis. However, in the present study, CD4�

T-cell depletion did not mitigate the damage seen inDC-depleted pancreatitic mice. Selective blockade of theproinflammatory mediators IL-6, MIP-1�, and TNF-�,

nd NF-�B and MAPK—all known to play a role in pan-reatitis and showing differential activity in C-DC mice—id not protect from severe organ destruction.Another notable finding was that macrophage-depletedice treated with caerulein did not exhibit the adverse

ffects associated with DC depletion (Figure 3D). LikeCs, macrophages are antigen-presenting cells with the

apability of mobilizing other leukocytes against patho-enic insults. However, macrophages and DCs appear toave contrasting roles in acute pancreatitis. Pancreaticnd peritoneal resident macrophages release TNF-� and

IL-1� during the initial pancreatic insult,33,34 and thuspropagate local and systemic inflammation as well asorgan damage by recruitment of monocytes and neutro-phils and positive regulation of other proinflammatorychemokines, including MIP-1� and regulated upon acti-ation, normal T cell expressed and secreted to RAN-ES.35 In this manner, peritoneal macrophages can mea-

surably expand the inflammatory response to pancreaticinjury and contribute to pancreatic necrosis. Administra-tion of the macrophage-pacifying compound CNI-1493prior to the induction of severe pancreatitis in rats re-sulted in an increased survival and a reduction in theseverity of pancreatitis as exemplified by lower levels ofcirculating pancreatic enzymes and proinflammatory cy-tokines.36,37 A protective effect was also observed by de-pleting macrophages with the injection of liposome-en-capsulated dichloromethylenediphosphonate in a mousemodel of virus-induced pancreatitis.38 In the context of

ur work, we have found that, like their macrophagecousins,” DCs are proinflammatory in acute pancreatitisy releasing TNF-�, IL-6, and MCP-1. However, DCs also

protect the pancreas from severe injury. The mechanismunderlying these paradoxical effects is unclear. It can bespeculated that DC depletion in acute pancreatitis pre-vents induction of regulatory T cells (Treg), allowing in-flammation to go unchecked. Recent work by Tiemessenet al39 has shown that CD4�CD25�Foxp3� Tregs can

oth inhibit the proinflammatory function of macro-hages and direct their differentiation toward the “alter-atively activated,” or M2, anti-inflammatory phenotype.
owever, we did not appreciate a diminution in the in-

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trapancreatic Treg population upon DC depletion (notshown). Furthermore, the fact that depletion of CD4� Tcells did not exacerbate pancreatitis suggests that theDC-Treg axis is not a central modulator in acute pancre-atitis.

Our findings implicating endogenous pancreatic DCsin an innate protective mechanism against pancreatic ne-crosis are, in part, analogous to the role of DCs in sepsis.40

Development of shock and multiple organ system failurein sepsis has been attributed not to the inciting infectiousagent or the hyperinflammatory response, but to progres-sive immunosuppression.41 In consort with these find-ings, studies in rodents have identified a marked deple-tion in splenic DC 12–36 hours after initiation of septicinsult.42,43 This immune depletion results from apoptosisof DCs, seen with increased active caspase 3 and AnnexinV staining in lymph nodes of mice undergoing cecalligation and puncture, simulating polymicrobial sepsis.44

Similarly, our study showed that a lack of DCs severelyworsened organ injury in murine acute pancreatitis andresulted in frank pancreatic necrosis. Thus, in acute pan-creatitis, DCs appear to both galvanize the inflammatoryresponse to injury by producing high levels of proinflam-matory cytokines, but also protect the pancreas uponcellular stress. The physiologic or molecular switch thatgoverns DC functionality has yet to be determined inacute pancreatitis. However, given the apparent increasein sterile inflammation in acute pancreatitis associatedwith DC depletion (Supplementary Figure 7A), the emerg-ing role of DCs in clearance of the byproducts of injury,26

as well as our findings that DCs are uniquely capable ofclearance of antigen as well as apoptotic and necroticdebris (Supplementary Figure 7B and C), our work sug-

ests that DCs may limit sterile inflammation in pancre-titis via this exact role. In support of this hypothesis, wehow that, in the context of DC depletion, intrapancreaticntigen-presenting cells have poor capabilities in thelearance of the byproducts of pancreatic injury (Supple-entary Figure 7D and E), suggesting that DCs are re-

quired for optimal clearance of cellular debris. Furtherwork to elucidate the regulatory function of DCs in pan-creatitis may be a key step in immune-directed therapyagainst acute pancreatitis and its sequelae.

Supplementary Material

Note: To access the supplementary materialaccompanying this article, visit the online version ofGastroenterology at www.gastrojournal.org, and at doi:10.1053/j.gastro.2011.07.033.

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2. Dominguez PM, Ardavin C. Differentiation and function of mousemonocyte-derived dendritic cells in steady state and inflammation.Immunol Rev 2010;234:90–104.

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5. Bamboat ZM, Ocuin LM, Balachandran VP, et al. ConventionalDCs reduce liver ischemia/reperfusion injury in mice via IL-10secretion. J Clin Invest 2010;120:559–569.

6. Kerschen E, Hernandez I, Zogg M, et al. Activated protein Ctargets CD8� dendritic cells to reduce the mortality of endotox-emia in mice. J Clin Invest 2010;120:3167–3178.

7. Pene F, Zuber B, Courtine E, et al. Dendritic cells modulate lungresponse to Pseudomonas aeruginosa in a murine model of sep-sis-induced immune dysfunction. J Immunol 2008;181:8513–8520.

8. Cailhier JF, Partolina M, Vuthoori S, et al. Conditional macrophageablation demonstrates that resident macrophages initiate acuteperitoneal inflammation. J Immunol 2005;174:2336–2342.

9. Jung S, Unutmaz D, Wong P, et al. In vivo depletion of CD11c�dendritic cells abrogates priming of CD8� T cells by exogenouscell-associated antigens. Immunity 2002;17:211–220.

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42. Tinsley KW, Grayson MH, Swanson PE, et al. Sepsis inducesapoptosis and profound depletion of splenic interdigitating andfollicular dendritic cells. J Immunol 2003;171:909–914.

43. Flohe SB, Agrawal H, Schmitz D, et al. Dendritic cells duringpolymicrobial sepsis rapidly mature but fail to initiate a protectiveTh1-type immune response. J Leukoc Biol 2006;79:473–481.

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Received November 15, 2010. Accepted July 18, 2011.

Reprint requestsAddress requests for reprints to: George Miller, MD, Departments

of Surgery and Cell Biology, New York University School of Medicine,Medical Science Building 601, 550 First Avenue, New York, NewYork 10016. e-mail: [email protected]; fax: (212) 263-8139.

AcknowledgmentsDrs Bedrosian and Nguyen contributed equally to this work.

Conflicts of interestThe authors disclose no conflicts.

FundingThis work was supported in part by grants from National Pancreas

Foundation (AM), the Society of University Surgeons (GM), andNational Institute of Health Awards CA108573 (ABF), DK085278
(GM), CA155649 (GM).
mailto:[email protected]

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November 2011 DCs IN ACUTE PANCREATITIS 1926.e1

Supplementary Methods

Cellular Isolation and AnalysisTo isolate mononuclear cells from the pancreas or

spleen, organs were harvested via postmortem laparot-omy and processed using both mechanical and enzymaticdigestion with Collagenase IV (Sigma–Aldrich). Cell sur-face marker analysis was performed by flow cytometryusing the FACSCalibur (Beckman Coulter; Brea, CA) af-ter incubating 5 � 105 cells with 1 �g anti-Fc�RIII/II

ntibody (2.4G2, Fc block; Monoclonal Antibody Coreacility, Sloan-Kettering Institute) and then labeling with�g fluorescein isothiocyanate, phycoerythrin, peridinin

chlorophyll protein complex, or allophycocyanin-conju-gated antibodies directed against MHC class II (I-Ab),B220 (RA3– 6B2), CD3� (17A2), CD4 (RM4-5) CD8�(53– 6.7), CD11b (M1/70), CD11c (HL3), CD40 (HM40-3), CD45.2 (104), CD80 (16-10A1), CD86 (GL1), Gr1(RB6-8C5), and NK1.1 (PK136) (all BD Biosciences).Dead cells were identified and excluded from analysisby staining with 7-aminoactinomycin D and apoptoticcells were identified using Annexin V (both BD Biosci-ences). Cellular uptake of antigen was tested using fluores-ein isothiocyanate-Dextran (Sigma–Aldrich). MHC II�

CD11c� DCs were isolated by fluorescence-activated cellsorting using a MoFlo cell sorter (Beckman Coulter).Bone marrow– derived DCs were generated as described.20

Briefly, bone marrow aspirates were cultured for 8 days incomplete RPMI (RPMI 1640 with 10% heat-inactivatedfetal bovine serum, 2 mM L-glutamine, and 0.05 mM2-ME) supplemented with murine granulocyte macro-phage colony-stimulating factor (20 ng/mL; MonoclonalAntibody Core Facility, Sloan-Kettering Institute). DCswere cultured in vitro at a concentration of 1 � 106

cells/mL. In selected experiments, supernatant was har-vested at 24 hours for cytokine assay using a cytometricbead array (BD Biosciences). Transforming growth fac-tor–� was measured by enzyme-linked immunosorbentassay according to manufacturer’s protocol (R&D Sys-tems). For polymerase chain reaction (PCR) assays, RNAwas isolated from pancreata using the Qiagen RNeasyisolation kit (Qiagen, Germantown, MD). QuantitativePCR was performed using a standardized preconfiguredPCR array (SA Biosciences, Frederick, MD) on the Strat-agene MX3000P (Stratagene, La Jolla, CA) according tothe respective manufacturers’ protocols. Alternatively, in-
dividual primers for BCL2 and ERK2 were obtained from
Qiagen and PCR amplification was performed using theSYBR Green/ROX RT-PCR Master Mix (Qiagen).

Western BlotsWestern blotting was performed as described.1

Liquid nitrogen frozen pancreata were powdered thenhomogenized in RIPA buffer with Complete ProteaseInhibitor cocktail (Roche, Pleasanton, CA) and pro-teins were separated from larger fragments by centrif-ugation at 14,000g. After determination of total pro-tein by the Lowry protein assay, 10% polyacrylamidegels were equiloaded with samples, electrophoresed at90 V, electrotransferred to polyvinylidene difluoridemembranes and probed with monoclonal antibodiesto Erk1, Erk2, pErk1/2, Elk1, pElk1, I�B�, pI�B�,

MGB-1, and �-actin (Santa Cruz Biotechnology,anta Cruz, CA). Blots were developed by enhan-ed chemoluminescence (Thermo Scientific, Asheville,C).

Histology and ImmunohistochemistryFor histological analysis, pancreatic specimens

were fixed with 10% buffered formalin, dehydrated inethanol, and then embedded with paraffin and stainedwith H&E. Acinar cell viability was calculated by exam-ining 10 high-powered fields per slide. Apoptotic celldeath was determined by terminal deoxynucleotidyltransferase–mediated deoxyuridine triphosphate nick-end labeling staining using the ApopTag Peroxidase InSitu Apoptosis Detection Kit (Millipore, Billerica, MA).Immunohistochemistry was performed using antibodiesdirected against CD45 (BD Biosciences), myeloperoxidase(Lifespan Biosciences, Seattle, WA), IL-6, and TNF-�(both Abbiotec, San Diego, CA). Photographs were takenwith Axiovert 40 microscope (Carl Zeiss, Oberkochen,Germany). For immunofluorescence studies, frozen sec-tions of liver were stained with anti-CD11c antibody (BDBiosciences). Images were captured on a Axiovert 200Mfluorescence microscope (Carl Zeiss, Oberkochen, Ger-many).

Reference

1. Xu Y, Malhotra A, Ren M, et al. The enzymatic function of tafazzin.J Biol Chem 2006;281:39217–39224.

oo was CD45.1� suggesting that bone marrow–derived expansion is likely.

1926.e2 BEDROSIAN ET AL GASTROENTEROLOGY Vol. 141, No. 5

Supplementary Figure 1. DCs expand from the bone marrow in pancf CD45.2� cells. (B) Conversely, MHC II� intrapancreatic cells had a suverwhelming majority of the expanded intrapancreatic DC population

reatitis. (A) Bone marrow cells in day 14 CD45.1 chimeric mice were devoidbstantial fraction expressing CD45.2. (C) However, in acute pancreatitis, the

r


Supplementary Figure 2. Pancreas-specific inflammation in pancreatitis. (A) Immunohistochemistry was performed on pancreata using antibod-ies directed against IL-6 and TNF-�. (B) Spleen DCs were tested for production of inflammatory mediators in cell culture supernatant. Experiments
epresent averages of triplicates and were repeated 3 times.


Supplementary Figure 3. DC depletion in CD11c.DTR mice. CD11c.DTR mice were treated with diphtheria toxin. Splenocytes were harvested
and CD45� cells were gated and examined for CD11c expression.


Supplementary Figure 4. pDC depletion does not result in exacerbated pancreatic injury. (A) B220 expression was examined in MHC II�CD11c�

intrapancreatic DC in caerulein-treated mice. (B) Mice were treated with caerulein alone, C-DC, caerulein � 120G8, or caerulein � anti–mPDCA-1.
Pancreata were stained using H&E and the fraction of nonviable acinar cells was calculated.

Cm


Supplementary Figure 5. DC adoptive transfer cannot rescue DC-depleted mice from exacerbated injury. Pancreata from C-DC–treated mice or-DC–treated mice adoptively transferred with bone marrow–derived DC were examined by H&E. The fraction of nonviable acinar cells was
easured at 48 hours.


Supplementary Figure 6. Diphtheria toxin (DPT) does not target pancreatic parenchymal cells. (A) CD11c expression was compared inCD45�MHCII�CD11c� DC and in CD45� pancreatic parenchymal cells. (B) Representative H&E-stained sections of pancreata from caerulein andDPT-treated C57BL/6 bone marrow chimeric mice and CD11c.DTR bone marrow chimeric mice are shown. (C) CD45 and (D) myeloperoxidase
immunohistochemistry were quantified by examining 10 high-powered fields per mouse. Experiments were repeated twice using 2 mice per group.

C

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Supplementary Figure 7. DC depletion is associated with an augmentation in sterile inflammation and decreased capacity for clearance of antigenor cellular debris. (A) HMGB-1 and �-actin levels were measured in pancreatic lysates using Western blotting. (B) CD11c�MHC II� and

D11c�MHCII� cells were sorted from the pancreas of mice with acute pancreatitis and incubated with fluorescein isothiocyanate–dextran for 30minutes. The fraction of FL1� cells and the mean fluorescent index (MFI) were calculated by flow cytometry. (C) Sorted CD11c�MHC II� and CD11c�

MHC II� cells as well as F480� pancreatic macrophages from pancreatitic mice were co-incubated with fluorescence-activated cell-sorted7-aminoactinomycin D� (7AAD�) necrotic pancreatic cells from additional pancreatitic animals. Capture of necrotic cells was determined by flowytometry. (D, E) Similarly, uptake of fluorescent (D) 7AAD� necrotic cells and (E) Annexin� apoptotic cells by bulk MHC II� cells was compared in

pancreatitic mice and pancreatitic mice depleted of DCs.

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Supplementary Figure 8. Tissue injury is limited to the pancreas. Pancreas, kidney, liver, and lung tissue were harvested from C and C-DC mice
nd examined using H&E staining of paraffin-embedded sections.


Supplementary Figure 9. Gene expression changes in pancreatitis. (A) Western blotting was performed to measure whole pancreas expressionof I�B�, pI�B�, and �-actin (48 hours), (B) Elk, pElk, (C) Erk1, Erk2, pErk1/2, and �-actin. (D) Conventional and quantitative polymerase chain reaction(PCR) were performed for BCL2. (E–G) A PCR array was used to measure expression of genes involved in (E) cell cycle regulation, (F) metabolism,
and (G) heat shock proteins.


Supplementary Figure 9. Continued

Hd


Supplementary Figure 10. CD4 depletion, Gr1 depletion, IL-6, MIP-1�, and NF-�B blockade are partially protective in caerulein-treated mice. (A)&E-stained sections of pancreata are shown from mice treated with saline, caerulein, or caerulein along with either neutrophil depletion, CD4 T-cellepletion, IL-6 blockade, TNF-� blockade, MIP-1� blockade, or NF-�B blockade using either a p50 inhibitor or NEMO binding domain peptide

inhibitor. (B) Pancreatic edema was measured for each group as were (C) CD45 leukocyte infiltration and (D) myeloperoxidase staining by examining
10 high-powered fields per pancreata (*P � .05; **P � .01; ***P � .001).

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Supplementary Figure 11. Selective cellular, cytokine, or cell signaling blockade can mitigate inflammation but fails to protect C-DC pancreatafrom acinar cell death. (A) Pancreatic edema was measured, as were (B) CD45 and (C) myeloperoxidase-stained sections of pancreata of micetreated with C-DC, or C-DC along with neutrophil depletion, CD4� T-cell depletion, IL-6 blockade, TNF-� blockade, MIP-1� blockade, or NF-�B

lockade using either a p50 inhibitor or NEMO binding domain peptide inhibitor. (D) Serum amylase was measured in each group and in controls.
xperiments were repeated twice using an average of 3 mice per group (*P � .05; **P � .01; ***P � .001).

bg


Supplementary Figure 12. MAPK blockade partially improves pancreatic inflammation and edema but does not protect C-DC pancreata fromorgan destruction. (A) H&E staining and CD45 and myeloperoxidase immunohistochemistry of pancreata from mice treated with C, C � MAPKblockade, C-DC, and C-DC � MAPK blockade. (B) MAPK blockade did not protect C-DC pancreata from acinar destruction. (C) However, MAPK

lockade improved pancreatic edema, (D) CD45� leukocyte infiltration, and (E) neutrophil infiltration. Experiments were performed using 3 mice per
roup (*P � .05; **P � .01).

Dendritic Cells Promote Pancreatic Viability in Mice With Acute Pancreatitis

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Transcript of Dendritic Cells Promote Pancreatic Viability in Mice With Acute Pancreatitis