Downregulation of the Syk Signaling Pathway in Intestinal Dendritic Cells

Downregulation of the Syk Signaling Pathway in Intestinal Dendritic Cells Is Sufficient To Induce Dendritic Cells That Inhibit Colitis This information is current as of September 25, 2021. Long Hang, Arthur M. Blum, Sangeeta Kumar, Joseph F. Urban, Jr., Makedonka Mitreva, Timothy G. Geary, Armando Jardim, Mary M. Stevenson, Clifford A. Lowell and Joel V. Weinstock

J Immunol published online 24 August 2016 Downloaded from http://www.jimmunol.org/content/early/2016/08/24/jimmun ol.1600063 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published August 24, 2016, doi:10.4049/jimmunol.1600063 The Journal of Immunology

Downregulation of the Syk Signaling Pathway in Intestinal Dendritic Cells Is Sufﬁcient To Induce Dendritic Cells That Inhibit Colitis

Long Hang,* Arthur M. Blum,* Sangeeta Kumar,* Joseph F. Urban, Jr.,† Makedonka Mitreva,‡ Timothy G. Geary,x Armando Jardim,x Mary M. Stevenson,{,‖ Clifford A. Lowell,# and Joel V. Weinstock*

Helminthic infections modulate host immunity and may protect people in less-developed countries from developing immunological diseases. In a murine colitis model, the helminth Heligmosomoides polygyrus bakeri prevents colitis via induction of regulatory dendritic cells (DCs). The mechanism driving the development of these regulatory DCs is unexplored. There is decreased expression of the intracellular signaling pathway spleen tyrosine kinase (Syk) in intestinal DCs from H. polygyrus bakeri–infected Downloaded from mice. To explore the importance of this observation, it was shown that intestinal DCs from DC-specific Syk2/2 mice were powerful inhibitors of murine colitis, suggesting that loss of Syk was sufficient to convert these cells into their regulatory phenotype. DCs sense gut flora and damaged epithelium via expression of C-type lectin receptors, many of which signal through the Syk signaling pathway. It was observed that gut DCs express mRNA encoding for C-type lectin (CLEC) 7A, CLEC9A, CLEC12A, and CLEC4N. H. polygyrus bakeri infection downmodulated CLEC mRNA expression in these cells. Focusing on CLEC7A, which encodes for the dectin-1 receptor, flow analysis showed that H. polygyrus bakeri decreases dectin-1 expression on the intestinal DC subsets that http://www.jimmunol.org/ drive Th1/Th17 development. DCs become unresponsive to the dectin-1 agonist curdlan and fail to phosphorylate Syk after agonist stimulation. Soluble worm products can block CLEC7A and Syk mRNA expression in gut DCs from uninfected mice after a brief in vitro exposure. Thus, downmodulation of Syk expression and phosphorylation in intestinal DCs could be important mechanisms through which helminths induce regulatory DCs that limit colitis. The Journal of Immunology, 2016, 197: 000–000.

any diseases caused by immune dysregulation are like inflammatory bowel disease (IBD). Several clinical and epi- frequent in developed nations but are uncommon in demiologic studies support this concept (1). M less-developed countries. Helminths are worm-like par- Animal models of colitis showed that helminths modulate in- by guest on September 25, 2021 asitic organisms that frequently infect humans in geographic re- testinal inflammation through enhancement of immune regulation. gions with low prevalence of these diseases. Helminthic infections Cells implicated in this regulation include regulatory T cells (2–4), are strong inducers of immune-regulatory circuits. This suggests macrophages (5–9) and dendritic cells (DCs) (10, 11). that loss of helminthic infections in developed countries as a result Alterations in DC function may be particularly important, as of improved sanitation and a clean food supply could be one of demonstrated in a Rag/T cell transfer model of IBD. T and B cell– the factors promoting the increase in immune-mediated diseases deficient Rag mice reconstituted with IL102/2 T cells develop colitis. Rag mice infected with Heligmosomoides polygyrus bakeri before IL102/2 T cell reconstitution are protected from the dis- *Division of Gastroenterology–Hepatology, Department of Internal Medicine, Tufts † ease (10). The mechanism underlying this protection involves Medical Center, Boston, MA 02111; Beltsville Human Nutrition Research Center, Diet, Genomics and Immunology Laboratory, Agricultural Research Service, United induction of regulatory DCs in the intestinal mucosa (10). Com- States Department of Agriculture, Beltsville, MD 20705; ‡Genome Institute, Wash- pared with DCs from uninfected animals, intestinal DCs isolated ington University School of Medicine, St. Louis, MO 63108; xInstitute of Parasitol- ogy, McGill University, Sainte-Anne-de-Bellevue, Quebec H9X 3V9, Canada; after H. polygyrus bakeri infection only weakly support Ag-driven {Department of Microbiology and Immunology, McGill University, Montreal, Que- IFN-g and IL-17 secretion. Furthermore, DCs isolated from the ‖ bec H3A 2B4, Canada; Department of Medicine, McGill University, Montreal, intestines of H. polygyrus bakeri–infected Rag mice transferred Quebec H4A 3J1, Canada; and #Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA 94143 into colitis-susceptible mice block colitis (11). How these regu- ORCIDs: 0000-0002-1590-8869 (J.F.U.); 0000-0001-9572-3436 (M.M.); 0000-0002- latory DCs quell colitis is only partly characterized (11). 9619-6663 (A.J.); 0000-0002-4766-1047 (M.M.S.); 0000-0002-0467-7073 (C.A.L.). DCs are the critical link between innate and adaptive immunity. Received for publication January 11, 2016. Accepted for publication July 14, 2016. They sample intestinal luminal contents and present Ags to T cells, This work was supported by National Institutes of Health Grants DK38327 and inducing their differentiation and proliferation or perhaps rendering DK058755, the Schneider Family, and the Gilman Family. them inert (12, 13). Address correspondence and reprint requests to Dr. Joel V. Weinstock, Division of Intestinal DCs sense threats coming from the gut through display Gastroenterology–Hepatology, Tufts Medical Center, 800 Washington Street, #233, of pattern recognition receptors. These are inherited, germline- Boston, MA 02111. E-mail address: [email protected] encoded receptors that instinctively engage classes of molecules Abbreviations used in this article: CLEC, C-type lectin; CTLR, C-type lectin receptor; DC, dendritic cell; HES, H. polygyrus bakeri excretory/secretory product; IBD, common to many types of bacteria, fungi, viruses, helminths, or inflammatory bowel disease; LP, lamina propria; LPMC, LP mononuclear cell; MLN, host dead or dying cells. One family of such receptors is the mesenteric lymph node; RPMI, RPMI 1640 medium; rt-PCR, real-time PCR; Syk, C-type lectin receptors (CTLRs). Many groups of CTLRs also are spleen tyrosine kinase; TI, terminal ileum; WT, wild-type. called C-type lectins (CLECs), which are mostly transmembrane Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 receptors that engage their ligands to induce intracellular signaling

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600063 2 H. BAKERI INHIBITS Syk SIGNALING IN GUT DCs that alters cellular function (14). Many CLECs activate DCs via according to the instructions. The beads used to isolate CD11c+ cells from the gut recovered ∼85% of these cells at ∼95% purity, as determined by phosphorylation of the spleen tyrosine kinase (Syk) signaling hi pathway (15). FACS. The beads exhibited equal efficiency at isolating the CD11c and CD11clo subsets. In earlier experiments, microarray analysis was performed on intestinal DCs isolated from H. polygyrus bakeri–infected and rt-PCR uninfected control mice to better understand how H. polygyrus Total RNA was isolated from individual samples using Quick-RNA Mini bakeri infection affects gene expression in gut DCs. The micro- Prep (Zymo Research, Irvine, CA), as per the manufacturer’s instructions. array data suggested that H. polygyrus bakeri infection profoundly RNA quality and quantity were determined using an Agilent Bioanalyzer inhibited gene expression for nearly all of the CLECs expressed (Agilent Technologies, Santa Clara, CA). RNA was converted to cDNA using qScript cDNA SuperMix (Quanta Bioscience, Gaithersburg, MD). rt- by the intestinal DCs and Syk (15). PCR was performed using the Eco Real-Time PCR System (Illumina, San Using real-time PCR (rt-PCR), the current study confirmed the Diego, CA). GAPDH levels were used to normalize the data. TaqMan real- microarray data showing that DCs from the intestine express time primers for CLEC7A, CLEC9A, CLEC12A, CLEC4N, Syk, GAPDH, mRNA for Syk, as well as for CLEC7A, CLEC9A, CLEC12A, and HPRT, and Reg3 were obtained from Applied Biosystems (Grand Island, CLEC4N, whose levels of expression were substantially decreased NY). 2 2 by H. polygyrus bakeri infection. Syk / DCs transferred into a Curdlan preparation murine model of IBD inhibited colitis and induced regulatory DCs Curdlan (cat. no. tlrl-cur; InvivoGen, San Diego, CA) (10 mg) is a linear in the intestines that could block an Ag-induced T cell response in b-1,3-glucan that was dissolved in 1 ml of 0.1 N NaOH. This was then vitro. Focusing on CLEC7A mRNA, which encodes for the receptor diluted to 1 mg/ml in complete medium (see “Induction and modulation of dectin-1, flow analysis showed a marked decrease in the expression colitis and cell culture experiments”) and used in cultures at 100 mg/ml. Downloaded from of this receptor on intestinal DC subsets after H. polygyrus bakeri Production of H. polygyrus bakeri excretory/secretory product infection. Cells with diminished dectin-1 failed to respond to the dectin-1 agonist curdlan with Syk phosphorylation or enhanced Ag- H. polygyrus bakeri excretory/secretory product (HES) was from Dr. Mary induced T cell IFN-g/IL-17 secretion. Moreover, H. polygyrus Stevenson (18). H. polygyrus bakeri were collected from the gut of infected mice and cultured in serum-free RPMI with 2% glucose and anti- bakeri secretes molecules that directly inhibit Syk and CLEC ex- biotics (penicillin, streptomycin, gentamicin, polymyxin). The supernatant pression in these cells. Thus, it appears that downmodulation of the was collected after 36 h of culture and concentrated using a 3-kDa con- http://www.jimmunol.org/ Syk signaling pathway is an important mechanism through which centrator. H. polygyrus bakeri helps to promote the development of regulatory Sandwich ELISAs DCs that control colitis. ELISAs were performed using paired Abs. To measure IFN-g, plates were coated with a mAb to IFN-g (HB170; American Type Culture Collection) Materials and Methods and incubated with supernatant. IFN-g was detected with polyclonal rabbit Mice anti–IFN-g (gift from Dr. Mary Wilson, University of Iowa), followed by biotinylated goat anti-rabbit IgG (AXcell, Westbury, NY). IL-17 ELISA 2 2 This study used Rag1, wild-type (WT), IL-10 / , and OT2 CD45.1 mice was done using primary capture mAb and biotinylated anti–IL-17A mAb f/f f/f

(The Jackson Laboratory, Bar Harbor, ME), as well as Syk and Syk (R&D Systems, Minneapolis, MN). by guest on September 25, 2021 CD11c-cre mice (gift of Dr. Clifford A. Lowell). Syk mice were cross-bred to get Syk2/2 CD11c mice (16). All mice were on the C57BL/6 back- Flow cytometry ground. Breeding colonies were maintained in specific pathogen–free fa- LPMCs were washed twice and adjusted to 1 3 107 cells/ml in RPMI cilities at Tufts University. Animals were housed and handled following 25 containing 10% FCS, 25 mM HEPES buffer, 2 mM L-glutamine, 5 3 10 M national guidelines and as approved by Tufts University Animal Review 2-ME, 1 mM sodium pyruvate, 100 U/ml penicillin, 5 mg/ml gentamicin, Committee. and 100 mg/ml streptomycin (complete medium) (all from Life Technol- H. polygyrus bakeri infection ogies, Gaithersburg, MD) and stained with saturating amounts of conjugated mAb for 30 min at 4˚C. Following staining, cells were washed twice For some experiments, 5–6-wk-old mice were colonized with 125 H. polygyrus and resuspended in complete medium for analysis on the BD LSR II flow bakeri third-stage larvae by oral gavage, and infected mice were used 2 cytometer using FACSDIVA V6.1.1 software (BD Bioscience, Mountain wk later. Infective, ensheathed H. polygyrus bakeri L3 (U.S. National View, CA). Before adding labeled mAb, each tube received 1 mg of anti-Fc Helminthological Collection no. 81930) were obtained from fecal cul- mAb to block nonspecific binding of conjugated Abs to Fc receptors. The tures of eggs by the modified Baermann method and stored at 4˚C. mAbs used for staining or cell sorting were anti-CD11c–FITC, anti- CD103–PE, anti-CD103–allophycocyanin, anti-dectin-1–PE (all from Histological assessment of colitis severity eBioscience, San Diego, CA), and CD11b–allophycocyanin–Cy7 (BD Stained histological sections of colon tissue were examined by two blind Pharmingen). observers to assess the severity of inflammation. Tissue preparation and the Induction and modulation of colitis and cell culture four-point scoring system were described previously (17). experiments Dispersion of splenocytes and mesenteric lymph node cells and The first series of experiments determined whether intestinal DCs from isolation of splenic T cells WT or Syk2/2 mice could inhibit colitis in the Rag IL102/2 T cell– Single-cell suspensions of splenocytes and mesenteric lymph node (MLN) reconstituted/piroxicam-induced model of IBD. Rag mice of similar age 3 6 2/2 cells were prepared by gentle teasing in RPMI 1640 medium (RPMI; Life (5–6 wk old) were reconstituted i.p. with 4 10 IL10 splenic T cells. 3 4 2/2 Technologies, Grand Island, NY). The cells were washed three times in Some mice also received 4 10 TI DCs i.p. from WT or Syk mice. RPMI. Splenic T cells were isolated by negative selection using the EasySep After 1 wk, mice were fed food with the nonsteroidal anti-inflammatory Mouse T Cell Enrichment Kit (cat. no. 19751; STEMCELL Technologies, drug piroxicam for 2 wk to induce colitis. Mice were sacrificed 1 wk after Vancouver, BC, Canada), as outlined by the manufacturer. Viability was stopping the piroxicam (11). The colons were removed, opened, rolled determined using exclusion of trypan blue dye. onto glass rods, fixed in formalin, cut into 4-mm-thick sections, and stained with H&E for histological scoring of inflammation using a four-point 2 2 Lamina propria mononuclear cell isolation and lamina propria scale. Also, intestinal LPMCs from IL10 / T cell–reconstituted or 2/2 5 cell fractionation IL10 T cell/DC–reconstituted Rag mice were cultured (2 3 10 cells/ well) for 48 h in 96-well round-bottom plates along with OT2 cells Gut lamina propria (LP) mononuclear cells (LPMCs) were isolated from the (105 cells per well). Cells were cultured in complete medium alone or with terminal ileum (TI), as described (2). Cell viability was 90%, as determined OVA (50 mg/ml) (cat no. 3054; Worthington Biochemicals, Lakewood, NJ) by trypan blue exclusion. DCs (CD11c+) were isolated from dispersed LPMCs to stimulate cytokine secretion. After culture, the supernatants were by CD11c positive selection (cat. no. 18758; STEMCELL Technologies), assayed for IFN-g and IL-17A using ELISAs (as described above). In The Journal of Immunology 3 some experiments, curdlan (100 mg/ml) and OVA were added to the cell cultures to determine whether curdlan (CLEC7A agonist) enhanced cytokine release. In other experiments, intestinal DCs isolated from piroxicam-treated Rag mice that received WT or Syk2/2 DCs were mixed in vitro with LPMCs isolated from colitic Rag mice that received no supplemental DCs to determine whether these DCs could modulate the in vitro OVA response. Rag mice were reconstituted with IL102/2 T cells and either WT or Syk2/2 DCs and then treated with piroxicam, as described above. Then, intestinal CD11c DCs were isolated and cultured in complete medium with intestinal LPMCs derived from Rag mice reconstituted just with IL102/2 T cells and treated with piroxicam. These cultures also contained OT2 cells, and some wells received OVA for the 48-h cultures before measuring the cytokines in the supernatants. The final series of experiments involved culturing or not intestinal DCs isolated from Rag mice with HES to determine whether HES could directly FIGURE 1. H. polygyrus bakeri infection inhibits Syk mRNA expres- downmodulate Syk or CLEC7A expression in these cells. DCs (2 3 105) sion in intestinal DCs. Mice were infected with H. polygyrus bakeri (Hpb). were cultured or not for 2 h with HES (50 mg/ml). Then, RNA was Control mice of similar age were not infected (No-Hpb). Two weeks later, extracted and analyzed for Syk and CLEC7A mRNA expression using rt- DCs were isolated from the TI, MLN, or spleen (SPL) of groups of three or PCR. four mice. Then, RNA was extracted and converted to cDNA. Quantitative rt-PCR was used to assess the levels of Syk mRNA expression. Data show Intracytoplasmic staining for phosphorylated Syk that Syk mRNA expression in intestinal DCs from H. polygyrus bakeri– CD11c+CD103+ intestinal DCs from H. polygyrus bakeri–infected or uninfected mice was lower than in DCs from uninfected mice (p , 0.01). Downloaded from infected Rag mice were isolated by FACS. DCs (5 3 104) were placed in Data were normalized relative to the expression of the housekeeping gene complete medium and cultured for 5 min at 37˚C in the presence or ab- GAPDH. Data are mean 6 SE from three separate experiments. sence of the dectin-1 agonist curdlan (100 mg/ml). Then DCs were fixed, permeabilized, and intracellularly stained for phosphorylated Syk, following the eBioscience intracellular staining protocol, using anti-Syk- phosph-PE or allophycocyanin (from eBioscience). responsiveness of isolated LPMCs to OVA stimulation after OT2

cell supplementation (Fig. 2A, experimental design). http://www.jimmunol.org/ Treatment of mice with Syk inhibitor Fig. 2B and 2C show, as expected, that severe colitis developed Mice were treated with the Syk inhibitor Cerdulatinib (cat. no. S7634; in Rag mice receiving no DCs. Mice receiving intestinal DCs Selleckchem, Houston, TX) to determine whether it could inhibit colitis in from WT mice fared no better. Intestinal Syk2/2 DC murine recip- 2/2 the Rag/IL10 T cell transfer model. The inhibitor was dissolved in 5% ients exhibited a reduction in the intensity of the inflammatory DMSO and 95% corn oil, as recommended by Selleckchem Technical response. Services. The inhibitor was started after the first week of piroxicam treatment and continued on weekdays for 2 wk, for a total of 10 d of LPMCs isolated from Rag mice that received WT DCs or no DCs treatment. The drug was dosed at 5 mg/kg body weight. Mice were given produced substantial amounts of IFN-g and IL-17 when cultured the inhibitor dissolved in 0.1 ml via oral gavage once in the morning and in vitro with OVA. However, LPMCs from mice receiving Syk2/2 once in the late afternoon. The last inhibitor treatment was given the day DCs showed blunted responses to OVA stimulation (Fig. 2D). by guest on September 25, 2021 before sacrifice. 2/2 Statistical analysis The intestines of Rag mice protected from colitis by Syk DC transfer contain regulatory DCs 6 Data are mean SE of multiple determinations. The difference between 2 2 two groups was compared using the Student t test. Multiple-group com- The above experiments suggest that Syk / intestinal DCs from parisons used ANOVA and the Dunnett t test. The p values , 0.05 were mice never infected with H. polygyrus bakeri can protect mice considered significant. from colitis. To further test the significance of this observation, we next determined whether animals protected from colitis by Syk2/2 Results DC transfer have regulatory-type DCs within their intestines. H. polygyrus bakeri infection leads to a downmodulation of Rag mice were reconstituted with splenic IL102/2 T cells and Syk expression in intestinal DCs then treated with piroxicam to induce colitis. One week after Rag mice, postinfection with H. polygyrus bakeri, develop regu- stopping the piroxicam, the mice were sacrificed, and the severity latory DCs in their intestines that can control colitis when trans- of colitis was scored by examining histological tissue sections. ferred into a murine IL102/2 T cell model of IBD (11). Previous Also, intestine was dissociated to isolate LPMCs, which were microarray analyses showed that H. polygyrus bakeri infection cultured with OT2 cells in vitro, with or without OVA, to stimulate caused a marked decreased in Syk expression in these gut DCs. IFN-g and IL-17 release. This was confirmed by rt-PCR analysis of RNA extracted from In parallel, a second group of age-matched Rag mice received 2/2 2/2 intestinal DCs isolated from Rag mice infected with H. polygyrus similar IL10 T cells along with gut WT or Syk DCs. They bakeri (Fig. 1). H. polygyrus bakeri infection also substantially also were treated with piroxicam. One week after stopping the reduced Syk expression in DCs isolated from MLNs, but not piroxicam, the mice were sacrificed, and their colitis also was spleens, of infected mice. scored for intensity of inflammation by examining histological sections. In these mice, intestine was dissociated to isolate intes- DCs deficient in Syk are sufficient to inhibit colitis tinal DCs, which were added to some of the cultures of LPMCs The next series of experiments determined whether the Syk sig- from the severely colitic mice who received no supplement DCs to naling pathway was important for development of DCs that control determine whether these DCs would affect the OVA response colitis. These studies used intestinal DCs from uninfected WT mice (Fig. 3, experimental design). and transgenic mice with a Syk deficiency limited to the CD11c+ Once again, IL102/2 T cell–reconstituted Rag mice developed DCs. Rag mice received IL102/2 T cells administered i.p. Some severe colitis after piroxicam treatment, unless they received mice also received WT or Syk2/2 DCs. After nonsteroidal anti- Syk2/2 DCs (data not shown). Isolated LPMCs from colitic mice inflammatory drug administration to induce colitis, the animals produced IFN-g and IL-17 after OVA stimulation (Fig. 3B). Only were sacrificed 4 wk later to assess the severity of colitis and the intestinal DCs from Rag mice protected from colitis by Syk2/2 4 H. BAKERI INHIBITS Syk SIGNALING IN GUT DCs

DC transfer diminished OVA-induced, cytokine secretion when they were added in vitro to LPMCs from colitic animals. There was little or no IL-4 or IL-10 in the culture supernatants (,,100 pg). The amount of IL-4 and IL-10 did not increase with the addition of DCs. We showed previously that DCs block colitis independently of IL-10 (4). H. polygyrus bakeri infection decreases CLEC receptor expression in TI DCs Microarray analysis suggested that intestinal DCs strongly express four types of transmembrane CLEC receptors (CLEC7A, CLEC9A, CLEC4N, and CLEC12A), all of which affect Syk signaling and whose expression was inhibited by H. polygyrus bakeri infection. To conﬁrm this observation, DCs were isolated from the intestines of H. polygyrus bakeri–infected mice or their uninfected littermate controls. Their RNA was extracted, converted to cDNA, and checked for CLEC mRNA expression using rt-PCR. Fig. 4 shows that gut DCs express mRNA for CLEC7A,

CLEC9A, CLEC4N, and CLEC12A. Moreover, H. polygyrus Downloaded from bakeri infection substantially reduced the level of CLEC mRNA expression in gut DCs from H. polygyrus bakeri–infected mice compared with those from uninfected control mice. H. polygyrus bakeri infection did not alter the relative proportion of LPMCs expressing the DC marker CD11c (Fig. 5A). +

CD11c DCs in the intestinal LP can be subdivided into several http://www.jimmunol.org/ functionally distinct subsets based on their differential expression of CD11b and CD103 (12, 13). Further analysis of CD11c+ DCs from the TI showed no change in the relative proportion of cells expressing CD11b, CD103, or both markers during H. polygyrus bakeri infection (Fig. 6A). Thus, the effect of H. polygyrus bakeri infection on DC CLEC receptor expression did not simply result from the ingress of CLEC receptor-negative DCs into the LP that diluted the resident DC population. by guest on September 25, 2021 H. polygyrus bakeri infection decreases dectin-1 expression on intestinal DCs CLEC7A encodes for a cell surface receptor called dectin-1. Availability of various reagents allowed for the analysis of dectin-1. To further explore the importance of the above observation, experiments focused on the consequence of downmodulation of CLEC7A receptor mRNA expression with regard to dectin-1 expression. Flow analysis showed that intestinal DCs expressed dectin-1 and that fewer cells expressed this receptor after H. polygyrus bakeri

photographed (original magnification 3400). The intense lymphocytic infiltration in the mucosa of No DC and WT DC recipients was not seen in mice receiving Syk2/2 DC. (C) Colitis was scored for the severity of inflammation on a four-point scale in stained histology sections, again showing that Syk2/2 DC transfer inhibited the intensity of colitis. Data are mean 6 SE from three separate experiments, each containing four or five mice per group. p , 0.01, No DCs or WT DCs transferred versus Syk2/2 DCs transferred. (D) Effect of DC transfer on mucosal cytokine production. LPMCs were isolated from TI at the time of sacrifice for histological FIGURE 2. Syk2/2 DCs transferred into Rag mice reduces colonic in- analysis. These cells were cultured in vitro, at 2 3 105 cells per well, in flammation. (A) Experimental design. DCs were isolated from the TI of WT complete RPMI for 48 h with OT2 cells (ratio 2:1). Some wells contained or Syk2/2 mice and transferred (4 3 104/mouse) into Rag mice that received OVA (10 mg/ml) to stimulate cytokine secretion. Cell culture supernatants splenic IL102/2 T cells (4 3 106) i.p. A third group of Rag mice was were assayed for IFN-g and IL-17 using ELISAs after the 48-h culture reconstituted with IL102/2 T cells but did not receive DCs. Animals were period. LPMCs from Rag mice reconstituted with Syk2/2 secrete less treated with piroxicam, as described, to induce colitis. At the end of the ex- cytokines compared with LPMCs from Rag mice that received no DCs or periment, colonic tissue was examined microscopically to score the severity of DCs from WT mice. Data are mean 6 SE of three independent experi- colitis using a four-point scale. (B and C) Severity of colitis in mice receiving ments, each consisting of triplicate determinations. p , 0.01, LPMCs from no DCs (No DC), DCs from WT mice (WT DC), or DCs from Syk2/2 No DC– or WT DC–transferred mice + OVA versus LPMCs from Syk2/2 mice (Syk2/2 DC). (B) Colonic tissue was sectioned, stained with H&E, and DC–transferred mice + OVA. The Journal of Immunology 5

infection substantially reduced dectin-1 expression only on CD103+CD11b+CD11c+ and CD103+CD11b2CD11c+ DC subsets (Fig. 6B). Also, the relative degree of ﬂuorescence was lower on these dectin-1+ DCs during H. polygyrus bakeri infection, suggesting a lower density of receptor expression. DCs with diminished dectin-1 expression do not respond to curdlan DCs are the major initiators and regulators of mucosal T cell responses. Engagement of CLECs by molecules derived from micro- organisms or dying host cells often transmit molecular signals leading to DC activation and enhanced adaptive immune responses. Dectin-1 recognizes b-1,3-glucans that is often found in the cell walls of fungi (19). Curdlan, a b-1,3-glucan, is a dectin-1 agonist. It was determined whether this dectin-1 agonist would facilitate intestinal DC interactions with effector T cells and whether H. polygyrus bakeri infection altered this response. Splenic T cells from OT2-transgenic mice, which respond to OVA Ag, were used

in these experiments. Downloaded from First, intestinal DCs from uninfected mice were mixed with splenic OT2 T cells and cultured or not with OVA in the present or absence of curdlan to determine whether this dectin-1 agonist would enhance IFN-g and IL-17 secretion. We chose to measure these cytokines because IFN-g and IL-17 drive colitis in most + + models of IBD. Also, CD11b CD103 DCs are particularly http://www.jimmunol.org/ noteworthy for their ability to drive mucosal Th17 cell differentiation (20), whereas CD11c+CD103+CD11b2 DCs can drive Th1 polarization and IFN-g production (21). OT2 cells mixed with intestinal DCs from uninfected mice produced IFN-g and IL-17 in response to OVA (Fig. 7). The cells stimulated with OVA secreted substantially more IFN-g and IL-17 when curdlan was added to the culture mix. Cells stimulated with curdlan only did not release cytokines. Similar experiments were performed using gut DCs isolated by guest on September 25, 2021 from H. polygyrus bakeri–infected mice. When OT2 T cells were cultured with intestinal DCs from H. polygyrus bakeri–infected mice, OVA still stimulated IFN-g and IL-17 release; however, curdlan failed to enhance cytokine production (Fig. 7). Engagement of dectin-1 with its ligand initiates intracellular

2/2 signaling via phosphorylation of Syk. It was determined whether FIGURE 3. Rag mouse recipients of Syk DCs acquire DCs in their curdlan, the dectin-1 agonist, induced Syk phosphorylation in intestines that prevent LPMC from secreting pro-inflammatory cytokines. 2 2 intestinal DCs and whether H. polygyrus bakeri infection impeded (A) Rag mice were reconstituted with 4 3 106 splenic IL10 / T cells i.p. and given piroxicam for 2 wk to induce colitis, as described in Materials this process. Intestinal DCs from H. polygyrus bakeri–infected or and Methods. One week after stopping the piroxicam, LPMCs were iso- uninfected mice were exposed briefly to curdlan. Cells were per- lated from the TI. A second group of Rag mice received intestinal DCs meabilized and intracellularly stained with mAb to detect phos- from WT or Syk2/2 mice together with IL102/2 splenic T cells before phorylated Syk using flow cytometry. It was interesting to note administration of piroxicam. One week after stopping the piroxicam that phosphorylation of Syk before curdlan stimulation was lower treatment, DCs were isolated from the TI. To study the effect of these DCs in DCs from H. polygyrus bakeri–infected mice (Fig. 8A). Fig. 8B on cytokine secretion from LPMCs isolated from mice with colitis who did shows that curdlan stimulation induced Syk phosphorylation in not receive DCs, DCs (4 3 104) were added to some of the LPMCs (2 3 5 5 DCs from uninfected mice; however, it failed to enhance Syk 10 ) along with OT2 T cells (10 ) and cultured for 48 h. Some wells also phosphorylation in DCs from H. polygyrus bakeri–infected mice. contained OVA (10 mg/ml) to stimulate IFN-g and IL-17 release, which was measured in the culture supernatants at the end of the incubation. (B) H. polygyrus bakeri secretes molecules that directly inhibit Syk OVA induced cytokine release (IFN-g and IL-17) from these cell cultures. and CLEC7A expression in intestinal DCs Only intestinal DCs from Rag mice reconstituted with Syk2/2 DCs inhibited the in vitro LPMC OVA-induced cytokine response. Data are H. polygyrus bakeri cultured in vitro releases biologically active mean 6 SE from three independent experiments each performed in trip- molecules into the culture medium. Preparations derived from licate. p , 0.01, LPMCs + OVA or LPMCs + WT DCs + OVA versus these supernatants are known as HES. It was ascertained whether 2 2 LPMCs + Syk / DCs + OVA. HES could inhibit Syk or CLEC7A expression in intestinal DCs. DCs were isolated from the intestines of mice and were cultured in medium alone or with HES for 2 h. Then, RNA was extracted from infection (Fig. 5B). We next determined which subsets of intestinal the cells and analyzed for Syk and CLEC7A expression using DCs expressed dectin-1. Flow analysis showed that dectin-1 was quantitative rt-PCR. Fig. 9 shows that HES rapidly inhibits Syk expressed on the CD11c+CD103+CD11b+, CD11c+CD103+CD11b2, and CLEC7A expression in gut DCs. Also examined was the and CD11c+CD1032CD11b+ DC subsets. H. polygyrus bakeri effect of HES on three housekeeping genes (actin, HPRT and 6 H. BAKERI INHIBITS Syk SIGNALING IN GUT DCs

relative number of DCs or DC subsets in the LP of this region of the gut. Thus, the effect of H. polygyrus bakeri infection on DC CLEC and Syk mRNA expression likely does not reﬂect ingress into the LP of DCs simply expressing low levels of these mRNAs and diluting the resident DC population. Exposing DCs isolated from the gut of uninfected mice to HES quickly and selectively inhibits Syk and CLEC expression, further supporting the supposition that H. polygyrus bakeri deactivates Syk signaling in gut DCs. Also, this latter observation suggests that H. polygyrus bakeri produces one or more biologically active molecules that accomplish this task. It is tempting to speculate that H. polygyrus bakeri has acquired the ability to regulate Syk signaling in host DCs to weaken immune responses injurious to the worm. Other investigators showed that various immunoregu- latory molecules, such as TGF-b (22), IL-10 (23, 24), vitamin D3 FIGURE 4. H. polygyrus bakeri infection inhibits CLEC mRNA ex- (25), and dexamethasone (26), can also induce regulatory DCs. pression in intestinal DCs. Mice were infected with H. polygyrus bakeri Previous studies showed that different species of helminths pro- (Hpb). Control mice of similar age were not infected (No Hpb). Two weeks duce factors that induce DCs that block inﬂammation in various later, DCs were isolated from the TI of groups of three or four mice. The animal models of immune-mediated disease and/or enhance immune Downloaded from RNA was extracted and converted to cDNA. Quantitative rt-PCR was used to assess the level of CLEC7A, CLEC9A, CLEC12A, and CLEC4N mRNA expression. Data were normalized relative to expression of the housekeeping gene GAPDH. DCs from H. polygyrus bakeri–infected mice expressed less CLEC mRNA compared with DCs from uninfected mice (p , 0.01). Data are mean 6 SE from four independent experiments, each performed in triplicate. http://www.jimmunol.org/

GAPDH). Their expression was not affected by HES (data not shown). We also transferred some of these HES-exposed DCs (2 h) into our Rag colitis model of IBD. They did not block colitis, perhaps because such a brief exposure was insufﬁcient to maintain the regulatory phenotype.

Treatment of mice with a Syk inhibitor blocks colitis by guest on September 25, 2021 Using the Rag/IL102/2 T cell transfer model of colitis, mice were treated for 2 wk with a Syk inhibitor to determine whether it could inhibit colitis. The drug was started 2 wk after IL102/2 T cell transfer. Stained histological sections of colons were assessed for colitis severity using the usual four-point scale. As expected, mice receiving no drug developed severe colitis (3.4 6 0.2, two separate experiments using 12 mice). The colitis was substantially less severe in drug-treated animals (1.5 6 0.2, two separate experiments using eight mice; p , 0.01, treatment versus control).

Discussion Previous studies showed that infection with H. polygyrus bakeri induces changes in gut DCs, enabling them to suppress intestinal DC–driven Ag-specific T cell responses in vitro and to inhibit colitis in vivo in a murine model of IBD (11). The above experiments demonstrate that, during H. polygyrus bakeri infection, the DCs located in the intestines express substantially less Syk, which is part of an important proinflammatory intracellular signaling pathway, and fewer CLECs capable of phosphorylating Syk. Us- ing transgenic mice, it was shown that selective disruption of the FIGURE 5. H. polygyrus bakeri infection decreases dectin-1 receptor Syk signaling pathway in intestinal DCs is sufficient to induce expression on intestinal DCs. (A) Representative FACS plots showing the regulatory DCs that can block colitis and inhibit Ag-induced percentage of LPMCs isolated from the TI that express CD11c (upper T cell responses in the gut. Thus, it appears that down- panel). The percentages of LPMCs expressing CD11c in the TI were modulation of the Syk signaling pathway within gut DCs is an similar in H. polygyrus bakeri–infected (Hpb) mice and uninfected control mice (No Hpb) (lower panel). (B) Overlapping flow cytometry graph gated important mechanism leading to the formation of these regulatory on CD11c+ DCs in LPMCs isolated from the TI of H. polygyrus bakeri– cells. infected (Hpb) (shaded area) and uninfected (no Hpb) control (open area) It is likely that H. polygyrus bakeri infection downmodulates mice (upper panel). The bracket indicates the dectin-1+ cell gate. The activity of the Syk signaling pathway in resident intestinal intestine contains fewer dectin-1+ DCs after H. polygyrus bakeri infection. DCs from infected mice. H. polygyrus bakeri infection induced Data in the lower panels of (A) and (B) are mean percentage 6 SE from no inflammation in the distal intestines and did not change the eight independent experiments containing three or four mice per group. The Journal of Immunology 7

collagen-induced arthritis (30). Bone marrow–derived DCs stimulated with LPS and ES-62 produce less TNF-a, IL-6, and IL-23 and are less able to induce IL-17 production by OT-II T cells in vitro (31). DCs exposed to HES from the helminth H. polygyrus bakeri can induce differentiation of IL-10–producing CD4+ regulatory T cells, which suppress bystander T cell proliferation (32). Taenia crassiceps is a helminth used to study cysticercosis. Its excretory/secretory molecules impair DC responses to LPS and CpG. These worm-derived molecules bind mannose and galactose C-type lectin receptors, triggering the cRAF intracellular signaling pathway. This blocks Th1 proinflammatory TLR signaling in DCs favoring Th2 polarization (33). Fasciola hepatica, a liver fluke, expresses a tegmental coat Ag that inhibits DC function. It inhibits LPS-induced NF-kB and MAPK pathways in these cells (34). Syk is a nonreceptor tyrosine kinase. It is rapidly phosphorylated when receptors linked to Syk are engaged. Syk-mediated down- stream signaling can lead to activation of proinflammatory pathways, including NF-kB, NFAT, and CARD9 (35). Syk has a role in

cell adhesion because it is important for several integrin- and Downloaded from selectin-mediated functions. Many pattern recognition receptors that recognize potential pathogens or dead/dying host cells use the Syk signaling pathway to promote adaptive immunity and strong Th1/ Th17 responses (15). This includes a number of CLECs (15), as well as TLR5 (36). The latter recognizes ﬂagellin, which is the pri-

mary protein component of bacterial ﬂagella. This study thoroughly http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 6. H. polygyrus bakeri infection decreases dectin-1 expression on specific intestinal DC subsets. (A) Representative FACS plots showing the percentage of CD11c+ DCs expressing CD103 and/or CD11b in LPMCs isolated from the TI of H. polygyrus bakeri–infected (Hpb)or uninfected (No Hpb) control mice (upper panel). The intestinal DCs from H. polygyrus bakeri–infected and uninfected mice contain similar percentages of the three DC subsets (lower panel). (B) Representative overlapping flow cytometry graphs gated on CD11c+ DC subsets in LPMCs isolated from the TI of H. polygyrus bakeri–infected (shaded areas) and uninfected control (open areas) mice. The brackets indicate the dectin-1+ cell gate. The percentage of DCs expressing dectin-1 decreased in the CD103+CD11b+CD11c+ and CD103+CD11b2CD11c+ DC subsets after H. polygyrus bakeri infection (p , 0.05) (lower panel). Data in the lower panels of (A) and (B) are mean 6 SE derived from four independent experiments, each using three mice per group. regulation in vitro. For instance, adoptive transfer of bone marrow– derived DCs exposed to excretory/secretory products from cul- FIGURE 7. The dectin-1 agonist, curdlan, promotes cytokine secretion tured Trichinella spiralis muscle cyst larvae protects against EAE only from intestinal DCs isolated from uninfected mice. DCs isolated from (27). Splenocytes from rats that received DCs exposed to this the TI of uninfected mice (No Hpb) or from H. polygyrus bakeri–infected excretory/secretory product before EAE challenge have more mice (Hpb) were mixed with OT2 T cells and cultured or not with OVA Foxp3+ T cells. Hymenolepis diminuta Ag–pulsed DCs transferred (50 mg/ml) for 48 h in the presence or absence of curdlan (Curd; 100 mg/ml), into mice with dinitrobenzene sulfonic acid–induced colitis block as outlined in Materials and Methods. After the incubations, cytokine concentrations in the supernatants were assayed by ELISAs. OVA stimu- inflammation in an IL-10–dependent manner (28). Human PBMC- lated IFN-g and IL-17 secretion in cultures containing OT2 T cells and derived DCs exposed to soluble schistosome egg Ags and then 2 DCs from either source. However, curdlan enhanced cytokine secretion cocultured with autologous CD25 T cells increase T cell only in the presence of DCs from uninfected mice (p , 0.001). Data are SMAD3 activation and Foxp3 expression (29). A 62-kDa mean 6 SE from three independent experiments for which each condition phosphocholine-containing glycoprotein (ES-62) isolated from was cultured in triplicate. DCs were isolated from the TI of three or four Acanthocheilonema viteae prevents and inhibits established mice for each experimental group. 8 H. BAKERI INHIBITS Syk SIGNALING IN GUT DCs

colitis or Crohn’s disease (40) and appear to have a role in the disease process. Thus, downmodulation of Syk may decrease Th1/ Th17 responsiveness, leading to inhibition of IBD. Other mechanisms must be considered. Blocking of Syk in WT DCs in vitro reduces their expression of costimulatory markers (CD80, CD86) and MHC class II as well as secretion of proin- ﬂammatory cytokines (e.g., IL-12p40) (41). Regulatory DCs from the gut of H. polygyrus bakeri–infected mice have a similar phenotype (10, 42). A known effect of regulatory DCs with impaired MHC complex expression is induction of T cell anergy due to de- livery of defective costimulation to CD8+ and CD4+ T cells (43). They also restrain NK cell IFN-g production (44). Thus, induction of T cell anergy could be one of the mechanisms through which they work to limit the T cell responses that induce colitis. This study also characterized the CLEC subsets highly expressed in intestinal DCs from Rag mice. CLEC7A, CLEC9A, CLEC4N, and CLEC12A were expressed at high levels in gut DCs of uninfected healthy Rag mice (42). H. polygyrus bakeri infection

strongly inhibited expression of these four CLECs without in- Downloaded from ducing the expression of other membrane-bound CLEC subtypes. CLEC7A, CLEC9A, and CLEC4N encode for Syk-coupled C- type lectin receptors. The various membrane-bound CLECs, expressed on several cell types, affect immune responses following recognition of their

various ligands. Studies using other model systems show that http://www.jimmunol.org/ CLECs affect DC behavior. CLEC7A encodes for the receptor dectin-1, which engages b-1,3-glucans present on several fungal by guest on September 25, 2021

FIGURE 8. H. polygyrus bakeri infection decreases curdlan-induced Syk phosphorylation. CD11c+CD103+ DCs were isolated from dispersed LPMCs derived from the TI of Rag mice by FACS and subjected to intracellular staining (Materials and Methods) to determine the level of Syk phosphorylation in each cell. (A) and (B) Representative overlapping flow cytometry graphs showing the degree of Syk phosphorylation in these cells. The bracket in each FACS plot indicates the window of phosphorylated Syk-specific staining. (A) Intestinal DCs from H. polygyrus bakeri– infected mice (Hpb) have lower constitutive expression of phosphorylated Syk than do DCs from uninfected mice (No Hpb). (B) The dectin-1 agonist curdlan induces Syk phosphorylation in DCs from uninfected mice (No- Hpb) (upper panel), whereas it fails to do so in DCs from infected mice (Hpb) (lower panel). As shown in the histograms, the results in (C) and (D) in the table show the mean of three individual experiments. demonstrated that engagement of dectin-1, the receptor encoded by CLEC7A, induces Syk phosphorylation in intestinal DCs and enhances an Ag-induced T cell–dependent IFN-g and IL-17 response. Syk also is reported to induce the production of other cytokines, such as IL-1b (37), and several chemokines. It remains unknown how inactivation of the Syk signaling pathway in intestinal DCs leads to development of powerful regulatory DCs in the intestine that block colitis and mucosal Ag- FIGURE 9. HES inhibits Syk and CLEC7A mRNA expression in intestinal DCs. DCs isolated from the TI of Rag mice were cultured in vitro induced T cell responses. Syk signaling in DCs appears to be for 2 h in the presence or absence of HES (50 mg/ml). DC RNA was important for Th1/Th17 responses (38). In many murine models of extracted and converted to cDNA. Quantitative rt-PCR was used to assess IBD, poorly regulated effector T cells located in the gut respond to the levels of Syk and CLEC7A mRNA expression in these samples. Data luminal Ags and release proinflammatory molecules like IFN-g were normalized relative to expression of the housekeeping gene GAPDH. and IL-17 that drive the colitis (39). These two cytokines are com- Data are mean 6 SE from three separate experiments, and each experiment monly expressed at high levels in many patients with ulcerative was performed in duplicate. The Journal of Immunology 9 species and at least another unidentified ligand on mycobacteria. 2010. Helminth secretions induce de novo T cell Foxp3 expression and regulatory function through the TGF-b pathway. J. Exp. Med. 207: 2331–2341. Activation of this receptor promotes Th1 and Th17 T cell devel- 4. Hang, L., A. M. Blum, T. Setiawan, J. P. Urban, Jr., K. M. Stoyanoff, and opment (45). This was demonstrated for the first time, to our J. V. Weinstock. 2013. Heligmosomoides polygyrus bakeri infection activates knowledge, in this study using intestinal DCs mixed with OT2 colonic Foxp3+ T cells enhancing their capacity to prevent colitis. J. Immunol. 191: 1927–1934. cells that were stimulated with an Ag and a dectin-1 agonist. After 5. Smith, P., N. E. Mangan, C. M. Walsh, R. E. Fallon, A. N. McKenzie, N. van H. polygyrus bakeri infection, intestinal DCs no longer responded Rooijen, and P. G. Fallon. 2007. Infection with a helminth parasite prevents to CLEC7A agonist. This loss of response is assumed to be the experimental colitis via a macrophage-mediated mechanism. J. Immunol. 178: 4557–4566. consequence of the diminution of dectin-1 on the surface of these 6. Schnoeller, C., S. Rausch, S. Pillai, A. Avagyan, B. M. Wittig, C. Loddenkemper, cells, as demonstrated using FACS. Also shown was that a dectin- A. Hamann, E. Hamelmann, R. Lucius, and S. Hartmann. 2008. A helminth 1 agonist could no longer induce Syk phosphorylation in gut DCs immunomodulator reduces allergic and inflammatory responses by induction of IL-10-producing macrophages. J. Immunol. 180: 4265–4272. after H. polygyrus bakeri infection. Syk is the critical intracellular 7. Johnston, M. J., A. Wang, M. E. Catarino, L. Ball, V. C. Phan, J. A. MacDonald, signaling pathway for many CLECs. Dectin-1 can also collaborate and D. M. McKay. 2010. Extracts of the rat tapeworm, Hymenolepis diminuta, with MyD88-coupled TLRs to modulate their function (46, 47). suppress macrophage activation in vitro and alleviate chemically induced colitis in mice. Infect. Immun. 78: 1364–1375. CLEC9A encodes for DNGR1, which is expressed on plasma- 8. Hunter, M. M., A. Wang, K. S. Parhar, M. J. Johnston, N. Van Rooijen, cytoid DCs and CD8a+ DCs. This receptor recognizes F-actin P. L. Beck, and D. M. McKay. 2010. In vitro-derived alternatively activated macrophages reduce colonic inflammation in mice. Gastroenterology 138: 1395– coming from dead or dying cells, and it helps to prime cytotoxic 1405. T cells (48). Loss of this receptor and the Syk signaling pathway 9. Leung, G., A. Wang, M. Fernando, V. C. Phan, and D. M. McKay. 2013. Bone could conceivably affect how the mucosal immune system responds marrow-derived alternatively activated macrophages reduce colitis without promoting fibrosis: participation of IL-10. Am. J. Physiol. Gastrointest. Liver to damaged mucosal epithelium or virally infected cells (49). Physiol. 304: G781–G792. Downloaded from CLEC4N (dectin-2) binds a-mannan and zymosan expressed by 10. Hang, L., T. Setiawan, A. M. Blum, J. Urban, K. Stoyanoff, S. Arihiro, potentially many pathogens (50). It also participates in Th2 (51, H. C. Reinecker, and J. V. Weinstock. 2010. Heligmosomoides polygyrus infection can inhibit colitis through direct interaction with innate immunity. 52) and Th17 (53, 54) cell development. J. Immunol. 185: 3184–3189. 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