Human intestinal epithelial cells respond to β-glucans via Dectin-1 and Syk

Eur. J. Immunol. 2014. 00: 1–12 Innate immunityDOI: 10.1002/eji.201444876 1

Human intestinal epithelial cells respond to β-glucansvia Dectin-1 and Syk

Sarit Cohen-Kedar1,2,5, Liran Baram1,2,5, Hofit Elad1,2,5, Eli Brazowski3,5,Hanan Guzner-Gur4,5 and Iris Dotan1,2,5

1 Inflammatory Bowel Diseases Center, Department of Gastroenterology and Liver Diseases,Tel Aviv Sourasky Medical Center, Tel Aviv, Israel

2 Research Center for Digestive Tract and Liver Diseases, Tel Aviv Sourasky Medical Center,Tel Aviv, Israel

3 Department of Pathology, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel4 Internal Medicine B, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel5 Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

Intestinal epithelial cells (IECs) are the first to encounter luminal antigens and may beinvolved in intestinal immune responses. Fungi are important components of the intesti-nal microflora. The potential role of fungi, and in particular their cell wall componentβ-glucan, in modulating human intestinal epithelial responses is still unclear. Here weexamined whether human IECs are capable of recognizing and responding to β-glucans,and the potential mechanisms of their activation. We show that human IECs freshlyisolated from surgical specimens, and the human IEC lines HT-29 and SW480, expressthe β-glucan receptor Dectin-1. The β-glucan-consisting glycans curdlan and zymosanstimulated IL-8 and CCL2 secretion by IEC lines. This was significantly inhibited by aDectin-1 blockade using its soluble antagonist laminarin. Spleen tyrosine kinase (Syk),a signaling mediator of Dectin-1 activation, is expressed in human IECs. β-glucans andCandida albicans induced Syk phosphorylation, and Syk inhibition significantly decreasedβ-glucan-induced chemokine secretion from IECs. Thus, IECs may respond to β-glucansby the secretion of pro-inflammatory chemokines in a Dectin-1- and Syk-dependent path-way, via receptors and a signaling pathway described to date only for myeloid cells. Thesefindings highlight the importance of fungi–IEC interactions in intestinal inflammation.

Keywords: Dectin-1 � Epithelial cells � Inflammation � Intestinal immunity � Mucosal immunity

� Additional supporting information may be found in the online version of this article at thepublisher’s web-site

Introduction

The epithelium facing the gut lumen is the first line of encounterbetween the host and the intestinal microflora. Beyond its roleas a physical barrier, the intestinal epithelium is an active

Correspondence: Dr. Iris Dotane-mail: [email protected]

participant in mucosal immune responses, including pathogenbinding, IgA processing, cytokine and chemokine secretion, andlymphocyte activation [1–5]. Fungi are important components ofthe intestinal microflora [6, 7]. C-type lectin receptors (CLRs)are the main group of PRRs involved in antifungal responses [8].Dectin-1, the CLR prototype, uniquely recognizes β-1,3 and β-1,6glucans [9, 10] expressed on the surface of most fungi and playsa nonredundant role in host defense against a number of fungalspecies [8, 11].

C© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

2 Sarit Cohen-Kedar et al. Eur. J. Immunol. 2014. 00: 1–12

Figure 1. Dectin-1 is expressed by humanIECs. (A–D) Dectin-1 expression in humanintestinal mucosa was assessed by immuno-histochemistry. Formalin-fixed paraffin-embedded sections of mucosa from healthy(A) colon and (B, C) ileum of the sameindividual were stained with anti-Dectin-1Ab at 10 μg/mL. (D) Staining with isotype-matched control Ab served as a negativecontrol. Filled arrowheads indicate epithe-lium and open arrowheads indicate laminapropria. Original magnification ×20, insets×40, scale bar 200 and 100 μm, respectively.Data shown are representative of stainedmucosa from more than ten individuals.(E) EpCAM-based flow cytometric gating onIECs. Freshly isolated human IEC-enrichedpreparation was stained with EpCAMand CD45 antibodies to identify IECs andleukocytes, respectively. (F and G) HumanIEC cell-surface expression of Dectin-1.IECs generated from the (F) ileum and(G) colon of the same donor were stainedas above, as well as with Dectin-1 Ab.Dectin-1 expression by IECs (gated EpCAM+

population) was assessed by flow cytometry(filled gray histograms) and was comparedwith isotype-matched control Ab (openhistograms). Data shown are representativeof at least eight independent experimentsperformed.

Recently, it was reported that Dectin-1 recognizes fungi in themouse intestine, and its deficiency appears to increase suscepti-bility to experimental murine colitis [6]. In humans, Dectin-1 wasshown to be expressed by bronchial epithelial cells, and its activa-tion by Aspergillus fumigatus resulted in the generation of ROS andthe expression of inflammatory cytokines [12]. Human intestinalepithelial cells (IECs, primary and IEC lines) express various PRRs[4, 13], including TLR2 and TLR4, which have been implicatedin myeloid-cell antifungal responses [14–17] as well as in intesti-nal inflammation such as inflammatory bowel diseases (IBD) andexperimental colitis [1, 18]. Nevertheless there are scant data onDectin-1 expression and function in human IECs [19, 20]. Further-more, the putative effects of glycans, and specifically β-glucans, onhuman IECs are still unclear.

We hypothesized that human IECs may express receptors rec-ognizing fungal cell-wall glycans such as the β-glucan receptorDectin-1, and may play a role in fungal induction of intesti-nal inflammation. Specifically, the aim of this study was to testwhether β-glucans can activate human IECs, and to define themechanisms of activation. We show that human IECs expressfunctional Dectin-1, and that β-glucans induce Dectin-1- and

spleen tyrosine kinase (Syk)-dependent pro-inflammatorychemokine secretion by IECs in a signaling pathway comparableto that described in cells of the myeloid lineage.

Results

Dectin-1 is expressed by human IECs

We first assessed whether Dectin-1 is expressed by human IECs.Paraffin-embedded sections generated from healthy ileum andcolon were stained with anti-Dectin-1 (Fig. 1A–C) and a controlAb (Fig. 1D). Interestingly, Dectin-1 was detected not only in thelamina propria where it was expressed by mononuclear cells, butalso in the colonic (Fig. 1A) and ileal (Fig. 1B and C) epithe-lium. We next tested Dectin-1 cell-surface expression by freshlyisolated human IECs generated from intestinal mucosal samples.Autologous ileal (Fig. 1F) and colonic (Fig. 1G) IECs, identifiedusing EpCAM staining (Fig. 1E), expressed cell-surface Dectin-1.Thus, Dectin-1 is expressed by normal human IECs along theintestinal tract (from the ileum to the colon). To evaluate the


Eur. J. Immunol. 2014. 00: 1–12 Innate immunity 3

Figure 2. Dectin-1 is expressed by human IEC lines. (A) Dectin-1expression by IECs was detected by immunofluorescence. SW480 andHT-29 cells were stained with anti-Dectin-1 Ab and with DAPI. (Rightpanels) Staining with isotype control Ab. Dectin-1 expression wasassessed by confocal microscopy. Original magnification ×20, inset ×63,scale bars 20 and 10 μm, respectively. (B) Western blot analysis of celllysates from primary human PBMCs, HT-29 and SW480 cells revealedsignals referring to the two major isoforms of Dectin-1: isoform A withits glycosylated forms migrating as 35 KDa and above, and isoform B at23 KDa. Blots were reacted with Dectin-1 rabbit polyclonal Ab, and withmouse α-tubulin or rabbit β-actin (on a duplicate blot) antibodies asloading controls. A representative example of at least ten experimentsis presented. Similar results were obtained when using goat polyclonalAb as well as mouse monoclonal Ab (clone GE2, not shown).

expression level of Dectin-1 on IECs compared with that on lam-ina propria macrophages (representing myeloid cells), we deter-mined the mean fluorescence intensity of Dectin-1 (normalized tothe isotype control) in IECs (EpCAM+ cells) and CD45+ CD13+

cells isolated from the same colonic mucosal samples. Dectin-1expression in IECs and CD45+ CD13+ cells was similar (Support-ing Information Fig. 1). We next identified Dectin-1 expression bythe human IEC lines HT-29 and SW480 by immunofluorescence(Fig. 2A). Finally, Dectin-1 expression by IEC lines and primaryPBMCs (positive control) was confirmed by western blot (Fig. 2B).Of the eight human Dectin-1 isoforms, the functional β-glucansensing and signaling ones were shown to be isoforms A and B[10]. These were identified by bands migrating at approximately35 KDa and above, corresponding to Dectin-1 isoform A, and itsglycosylated forms and a band of approximately 23 KDa, corre-sponding to Dectin-1 isoform B [21] (Fig. 2B). Taken together,these findings show that human IECs express the β-glucan receptorDectin-1.

IECs respond to β-glucans by chemokine secretion

We next investigated the functional consequences of IEC acti-vation with Dectin-1 ligands. The Dectin-1 agonists zymosan (ayeast cell wall preparation composed mainly of β-glucan andmannan) and curdlan (pure β-glucan) were used to activateIEC lines. HT-29 and SW480 cells responded to zymosan andcurdlan by enhanced IL-8 secretion in a dose-dependent manner(Fig. 3A–D); for example HT-29 cells secreted 1806 ± 461.4and 7484 ± 592.9 pg/mL IL-8, upon activation with zymosanat 1 and 10 μg /mL, respectively. Dose-dependent secretion ofthe pro-inflammatory chemokine CCL2 (MCP-1) was induced aswell (SW480 cells, Fig. 3E and F). No β-glucan-induced CCL2secretion by HT-29 cells was observed. This is in accordance with aprevious report where TNF-α-induced CCL2 secretion was noticedin SW480 but not HT-29 cells, while IL-8 secretion was inducedin both cell lines [22]. Subsequent studies were conductedwith submaximal doses of zymosan and curdlan, which inducedchemokine secretion from both cell lines (i.e. 10 and 100 μg/mLfor HT-29 and SW480, respectively). No IL-1β, TNF-α, IL-6, IL-10,or IL-12p70 secretion by β-glucan-activated IECs was detected.Hence, IECs responded to β-glucan activation by the secretion ofthe pro-inflammatory chemokines IL-8 and CCL2. Interestingly,we detected zymosan- and curdlan-induced mRNA expressionof the antimicrobial peptide human β-defensin-2 by both SW480and HT-29 cells (Supporting Information Fig. 2 and data notshown), as reported for bronchial epithelial cells [12].

Dectin-1 blockade decreases β-glucan-inducedchemokine secretion

In the myeloid lineage, β-glucan recognition is mediated solely byDectin-1. Hence we tested whether the observed IEC response toβ-glucans was Dectin-1-dependent. Laminarin, a soluble β-glucancommonly used as a Dectin-1 antagonist [6, 9, 23, 24], was incu-bated with IEC lines prior to their activation with zymosan. Prein-cubation of HT-29 cells with 100 μg/mL laminarin resulted in61% inhibition of zymosan-induced IL-8 secretion (p < 0.01 vs.no laminarin), and with 500 μg/mL laminarin IL-8 secretion wascomparable to the basal secretion (Fig. 4A). Similar results wereobtained when HT-29 cells were activated with curdlan (Fig. 4B),and in SW480 cells (Fig. 4C). Laminarin also inhibited CCL2 secre-tion from zymosan-activated SW480 cells (Fig. 4D). This suggeststhat the IEC response to β-glucan-containing glycans is Dectin-1-mediated.

β-Glucan-induced chemokine secretion isSyk-dependent

We next tested whether Syk, the cell signaling mediator of Dectin-1activation [8, 25–28], is expressed in primary human IECs.Healthy ileal and colonic mucosa exhibited pronounced epithe-lial as well as lamina propria Syk expression (Fig. 5A–F). Syk



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Figure 3. β-Glucans dose-dependently induce IL-8 andCCL2 secretion by IECs. (A and B) HT29 cells and (C–F)SW480 cells were incubated for 20 h in the absence (NT) orpresence of (A, C, E) zymosan or (B, D, F) curdlan, added atthe indicated concentrations. The secretion of (A–D) IL-8and (E and F) CCL2 in the supernatants was assessed byELISA. Data are shown as mean ± SD of triplicate wellsfrom one experiment representative of three independentexperiments performed. ND, not detected.

expression was specific to the intestinal mucosa, since the sub-mucosa was not stained (Fig. 5C and F). Furthermore, intracellu-lar Syk was identified in freshly isolated IECs by flow cytometry(Fig. 5G). Syk expression in HT-29 and SW480 cells was identi-fied as well using western blot (Fig. 5H) and immunofluorescence(HT-29, Fig. 5I–K).

To determine whether β-glucan activation of human IECsinvolves Syk signaling, β-glucan-induced chemokine secretion fol-lowing Syk inhibition was assessed. A significant decrease in IL-8secretion was observed when HT-29 cells were incubated withSyk inhibitor (574711, Calbiochem) prior to their activation withzymosan or curdlan. Specifically, pre-incubation with 1 or 5 μMSyk inhibitor resulted in a 55% (p < 0.01 vs. no inhibitor) and 76%inhibition (p < 0.001 vs. no inhibitor) respectively of zymosan-induced IL-8 secretion (Fig. 6A), and a 79% inhibition (at bothconcentrations) of curdlan-induced IL-8 secretion (p < 0.01 vs.no inhibitor, Fig. 6B). Similarly, pre-incubation with 1 μM or 5μM Syk inhibitor resulted in a significant decrease (p < 0.001vs. no inhibitor) in IL-8 secretion (Fig. 6C) in CCL2 secretionin zymosan-activated SW480 cells (Fig. 6D). The Syk inhibitorR406 significantly decreased zymosan-induced IL-8 secretion as

well (p < 0.01, Fig. 6E). As Dectin-1 signaling may involveRaf1 [29, 30] which is functional in IECs [31, 32], we nexttested whether Raf-1 inhibition would decrease glycan-inducedIL-8 secretion. However, preincubation of HT-29 cells with theRaf-1 inhibitor GW5074 (at 1 and 5 μM) prior to the addition ofzymosan or curdlan had no effect on IL-8 secretion (not shown),suggesting that Raf-1 signaling is not involved in β-glucan-inducedchemokine secretion from IECs. Taken together, these results sug-gest that the IEC response to Dectin-1 ligands is Syk- mediated.

β-Glucans induce Dectin-1-dependent Sykphosphorylation

Since our results suggested that β-glucan-induced IL-8 andCCL2 secretion was Syk-dependent, we next assessed whetherβ-glucans could activate epithelial Syk. Syk activation was evalu-ated using an Ab targeting only Tyr525/526 phosphorylated humanSyk. Zymosan- and curdlan-induced Syk phosphorylation wasdetected in HT-29 and SW480 cells by western blot (Fig. 7A)


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Figure 4. Dectin-1 blockade by laminarinreduces β-glucan-induced chemokine secretionby IEC lines. (A and B) HT-29 cells and (C andD) SW480 cells were incubated with laminarin(100–500 μg/mL, as indicated) for 30 min priorto the addition of (A) 10 or (C and D) 100 μg/mLof zymosan or (B) 10 μg/mL curdlan. Super-natants of β-glucan-activated as well as non-treated control cells (NT) were collected after20 h and (A–C) IL-8 and (D) CCL2 secretionwas determined by ELISA. Data are shown asmean + SD of triplicate wells from a singleexperiment representative of three indepen-dent experiments performed. ND, not detected;+zym, with zymosan; +curd, with curdlan.*p < 0.05, **p < 0.01, ***p < 0.001 vs. no lam-inarin; Student’s t-test. Comparable resultswere obtained in SW480 cells when activationwith curdlan followed preincubation with lam-inarin.

and immunofluorescence (Fig. 7C), providing direct evidence forSyk signaling involvement in β-glucan-induced IEC activation.Furthermore, after preincubation with laminarin, there wasdecreased Syk-phosphorylation in zymosan-activated HT-29 cells(Fig. 7C). Thus, our results suggest that β-glucans are capable ofactivating human IECs, and that β-glucan-induced IEC activationis mediated by Dectin-1 and Syk-signaling. Importantly, we identi-fied Syk-phosphorylation upon activation of HT-29 cells with heat-killed, UV-inactivated, and a live WT strain of Candida albicans(Fig. 7B). However, IL-8 secretion in response to 20 h activationby these C. albicans preparations was not observed. Similar resultswere obtained when heat-killed Saccharomices cerevisiae was usedfor activation, and upon activation of SW480 cells (not shown).Overall, the results suggest that IECs recognize fungi, with discor-dant consequences—that is Syk-phosphorylation without cytokinesecretion, compared with those observed with β-glucans.

It should be noted that although mucosal Syk expression wasfound uniformly in the lamina propria as well as in IECs (Fig. 8Aand Fig. 5A–F), phospho-Syk was detected mainly in IECs and incells of the germinal center in biopsies taken from healthy colonic(Fig. 8 B–D) and small bowel mucosa (not shown, n = 5), sug-gesting that in vivo Syk signaling can occur in human IECs.

Discussion

Fungi are an important component of the intestinal microflora,and an antifungal immune response was recently suggested to be

a factor contributing to gut homeostasis and inflammation [6]. Wehypothesized that IECs, which are the first to encounter intestinalantigens, may recognize and respond to β-glucans such as thoseexpressed by fungi. We hence examined whether fungal glycansare capable of activating human IECs, in an attempt to beginunraveling the mechanism of such activation.

Here, we show that the β-glucan receptor Dectin-1 is expressedby both primary human IECs and in IEC cell lines. We also showthat β-glucans can activate IECs to secrete the pro-inflammatorychemokines IL-8 and CCL2 in a Dectin-1- and Syk-dependent man-ner. More importantly, our data support the notion of active IECinvolvement in mucosal responses to β-glucans via Dectin-1, andsuggest that human IECs may recognize and respond to luminalfungi.

IECs express various PRRs recognizing microbial components,and have been shown to play an active role in microbial sensingand responses [1, 4, 13, 17]. However, CLRs, a class of PRRs pri-marily responsible for antifungal immune responses, have so farnot been found to be functional in IECs. Unlike murine Dectin-1,human Dectin-1 is not myeloid-lineage-restricted and has beenidentified in B cells, in a subpopulation of T-cells, and recently inthe bronchial epithelium [12, 33]. Here, we show for the first timethat there is cell-surface expression of Dectin-1 by primary humanIECs in both the normal ileum and colon. This expression was com-parable to that of mucosal myeloid cells (such as CD45+ CD13+

cells, Fig. 1 and Supporting Information Fig. 1). Dectin-1 expres-sion along the intestinal tract implies that in vivo fungal recogni-tion by IECs is possible. We found Dectin-1 expression in IEC lines



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Figure 5. Syk is expressed by human IECs.(A–F) Syk expression in the healthy intesti-nal mucosa was assessed by immuno-histochemistry. Formalin-fixed paraffin-embedded sections of mucosa from healthy(A–C) colon and (D–F) ileum were stainedwith anti-Syk Ab (clone 4D10), at 2 μg/mL (B,C, E, and F). (A and D) Staining of sectionsfrom the same blocks with isotype-matchedcontrol Ab served as a negative con-trol. Original magnification ×20, scale bar200 μm (A, B, D, and E); original mag-nification ×4, scale bar 1 mm (C and F).Filled arrowheads indicate epithelium andopen arrowheads indicate lamina propria.Data are representative of five individuals.(G) Intracellular Syk expression in primaryhuman IECs was detected by flow cytom-etry. Syk expression by freshly isolatedIECs generated from surgical specimens(gated EpCAM+ population) was assessedby flow cytometry using staining with FITC-conjugated Syk Ab (filled gray histograms)and was compared to isotype-matched con-trol Ab (open histograms). Data are repre-sentative of three individuals. (H) Syk pro-tein (72 kDa) was detected in lysates fromHT-29 and SW480 cells as well as from pri-mary PBMCs (positive control) by westernblot using mouse monoclonal anti-Syk Ab(4D10). (I–K) HT-29 cells seeded on coverslips were stained with mouse monoclonalanti-Syk Ab at 2 μg/mL, followed by stain-ing with (I) AF488-conjugated donkey anti-mouse secondary Ab, and (J) with DAPI.Magnification ×20, scale bar 20 μm. (H–K)Data shown are from single experimentsrepresentative of three experiments per-formed.

as well (Fig. 2). Recent studies have reported the induction ofDectin-1 expression in the human IEC line Caco-2 by OVA encap-sulated in yeast β-glucan particles [19] and in bronchial epithelialcells exposed to Aspergillus [12]. Hence, it is reasonable to assumethat Dectin-1 might be functional in cell types other than myeloid-lineage cells, and could participate in β-glucan-induced responsesin human mucosal surfaces. We found a marked secretion of IL-8and CCL2 in response to β-glucans (Fig. 3). This was significantlyreduced using the Dectin-1 antagonist laminarin, indicating thatDectin-1 is necessary for β-glucan-induced chemokine secretion(Fig. 4).

There are conflicting data concerning the expression and func-tion of Dectin-1 by human enterocytes. Using RT-PCR and westernblot, Volman et al. reported Dectin-1 expression by the humanembryonic IEC line INT-407 [20]; however, Dectin-1 cell-surfaceexpression could not be detected. These authors also failed toinhibit IL-8 production using anti-Dectin-1 antibodies, possiblydue to the modest IL-8 production induced by β-glucan-enrichedfecal water [20]. In contrast, our use of freshly isolated humanIECs as well as paraffin blocks of human intestinal mucosa in

addition to cell lines enabled the detection of Dectin-1 expression.This was further supported by our mechanistic studies of its rolein β-glucan-induced IEC activation.

Marked secretion of both IL-8 and CCL2, which are knownmicrobial-induced IEC chemokines [22, 34–36], as well asincreased mRNA levels of the antimicrobial peptide humanβ-defensin-2 was detected in response to β-glucan activation ofIEC lines (Fig. 3 and Supporting Information Fig. 2). Our datasuggest that β-glucan-induced chemokine secretion depends onDectin-1. However, we cannot rule out the possibility that otherreceptors cooperate with Dectin-1, as has been reported in humanand mouse cells of the myeloid lineage [15, 16, 28, 37]. Interest-ingly, whole yeast activation of IECs resulted in discordant conse-quences. While Syk phosphorylation was observed following expo-sure of IECs to heat-killed or UV-inactivated C. albicans, indicatingthat fungi were recognized by these cells (Fig. 7B), IECs activationby C. albicans (heat-killed, UV-inactivated WT, or live nonhyphalmutant) and heat-killed S. cerevisiae did not result in IL-8 secretion(not shown). This is in accordance with a previous report whereCaco-2 cells did not respond to heat-killed C. albicans and



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Figure 6. Zymosan-induced IL-8 and CCL2 secretion is Syk-dependent. (A, B, and E) HT-29 cells and (C and D) SW480 cells were incubatedwith/without (A–D) Syk inhibitor (574711, Calbiochem, 1 or 5 μM) or (E) R406 (5 or 10 μM) for 30 min prior to the addition of zymosan (10 μg/mL(A and E) or 100 μg/mL (C and D)) or curdlan (10 μg/mL (B)). Supernatants of β-glucan-activated as well as nontreated control cells (NT) were collectedafter 20 h and IL-8 (A, B, C, and E) and CCL2 (D) secretion was assessed. Data are shown as mean + SD of triplicate wells from a single experimentrepresentative of three independent experiments performed. ND, not detected. *p < 0.05, **p < 0.01, ***p < 0.001 vs. no inhibitor; Student’s t-test.

S. cerevisiae, and secreted low levels of IL-8 in response to live fungionly in the presence of butyrate [38]. Fungal particles exhibit avariety of ligands to the PRRs of mucosal cells. It was recentlyreported that different CLRs may differentially affect cellularresponses to fungi. Specifically, the CLR Mincle, which is involvedin fungal recognition, interferes with the Dectin-1-mediated anti-fungal response in human DCs [39]. Furthermore, it was suggestedthat the final outcome of fungal recognition may vary across dif-ferent cells [40]. Since IECs express functional TLRs [17] andDectin-1, it is reasonable to speculate that they may express addi-tional CLRs, and hence the outcome of their response to fungi suchas C. albicans would represent the sum of signals representing acti-vation of different receptors including pro- or anti-inflammatorysignals. We propose that, in vivo, IECs may participate in mucosalrecognition and response to fungi such as C. albicans andS. cerevisiae, possibly in concert with DCs and residentmacrophages. Under normal conditions, the lack or minor pro-inflammatory response of IECs to C. albicans may contribute tointestinal homeostasis, while when skewed, pro-inflammatory sig-nals such as IL-8 secretion in response to β-glucans may promotefurther inflammation.

In immune cells, the canonical signaling pathway of Dectin-1involves the intracellular mediator Syk, which becomes phospho-rylated upon Dectin-1 activation [8, 25–27]. Here we found Sykexpression in primary human IECs as well as in HT-29 and SW480cell lines (Fig. 5). The cytoplasmic and nuclear localization of Syk

in HT-29 is consistent with its cellular localization in B cells andin bronchial epithelial cells [41, 42]. Although there have beenreports of Syk expression in HT-29 cells [41] and Syk mRNA iden-tification using array studies of SW480 [43], this is the first accountof Syk expression by primary human IECs, as well as functionalSyk in human IECs. β-glucan activation of IECs resulted inDectin-1-dependent Syk phosphorylation, as revealed by its block-ade by laminarin (Fig. 7). This provides evidence for Dectin-1 sig-naling in human IECs comparable to that reported in cells of themyeloid lineage. We also found that IL-8 and CCL2 secretion uponβ-glucan-induced IEC activation was Syk-dependent, as specificinhibitors abrogated most of this secretion. This finding impliesthat Syk signaling is the main pathway involved in chemokinesecretion (Fig. 6). In addition, inhibition of Raf1, which is involvedin an alternative Dectin-1 responsive pathway in myeloid cells[29], did not affect β-glucan-induced chemokine secretion. Itwould be important to determine whether downstream signalingmediators such as Card9/Bcl10/Malt1 are involved in Dectin-1pathway in IECs.

Finally, phospho-Syk could be readily detected in vivo inhealthy human epithelium (Fig. 8), suggesting that Syk signal-ing occurs in IECs. P-Syk staining in human intestinal mucosamay indicate that Syk is constantly active. It should be notedthat in addition to activation by Dectin-1, Syk phosphoryla-tion can be triggered by several signals and receptors includ-ing CD74, the Fc receptor, integrins, and the CLRs Dectin-2 and



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Figure 7. Syk is phosphorylated in β-glucan-activated IECs. (A) HT-29(left) and SW480 cells (right) were stimulated with 100 μg/mL zymosanor curdlan for 30 min. Syk phosphorylation was assessed by westernblot of cell lysates using anti-phospho-Syk (Y525/526) monoclonal Aband compared with total Syk content and β-actin as a loading con-trol. NT, no treatment; zym, zymosan; curd, curdlan. Data shown arerepresentative of at least six independent experiments. (B) Syk is phos-phorylated in C. albicans activated IECs. HT-29 cells were stimulatedwith heat-killed (HK), UV-inactivated (UV), or live C. albicans cells at afinal concentration of 107/mL for 30 or 60 min. Syk phosphorylation wasassessed as above. Data shown are representative of three independentexperiments. (C) Zymosan-induced Syk phosphorylation is inhibited bylaminarin. HT-29 cells were seeded on cover slips in 6-well plates at5 × 105 cells/well. The next day, cells were left untreated (control) ortreated with zymosan (100 μg/mL) for 30 min with or without 30 minof preincubation with laminarin (500 μg/mL). Following fixation andpermeabilization cells were stained with anti-phospho-Syk polyclonalAb followed by AF-647-conjugated donkey anti-rabbit secondary Ab(p-Syk). Original magnification ×20, scale bar 20 μm. Data shown arefrom single experiments representative of three performed.

Mincle [8, 25, 44, 45]. Thus, exclusive Dectin-1 involvement inthe observed Syk phosphorylation in vivo cannot be concluded.Nevertheless, our in vitro studies suggest that β-glucan activationof Dectin-1 might trigger Syk signaling in IECs.

Loss of tolerance of the intestinal immune system to lumi-nal microorganisms is a hallmark of IBD. It may be reflectedby the occurrence of antimicrobial antibodies, including thosedirected against various glycans that characterize fungal cellwalls, including that of residential yeast such as S. cerevisiae andC. albicans. For example, anti-S. cerevisiae antibodies (ASCAs),detected in up to 70% of Crohn’s disease (CD) patients, andanti-laminaribioside carbohydrate antibodies (ALCAs), detected in�30% of CD patients, are directed against mannan and β-glucanmoieties, respectively [46–52].

The exact mechanism responsible for the presence of anti-glycan serological responses in CD patients is still unclear. Wesuggest that distorted stimulation of the intestinal immune sys-tem, including IECs, by glycans expressed mainly by fungi may bean early step in this process.

In conclusion, we presented evidence for β-glucan- and fungal-induced activation of human IECs and its potential mechanisminvolving the Dectin-1/Syk pathway. The recognition and responseof IECs to commensal fungi and to fungal components may play arole in intestinal homeostasis, and when skewed, may be an earlyevent in the pathogenesis of IBD.

Materials and methods

Mucosal samples

Mucosal samples were taken from surgical specimens of patientsundergoing bowel resection for colonic tumors (normal mucosawas taken from a distance of at least 10 cm from the tumor). Whereindicated, immunohistochemical studies were performed on biop-sies generated from the normal mucosa of patients undergoingcolonoscopies. Local Institutional Ethical Committee approval wasreceived for the studies and the informed consent of all participat-ing subjects was obtained.

Isolation of primary human IECs

IECs were freshly isolated as previously described [53, 54] withmodifications. Briefly, mucosa and submucosa were separated.The tissue was dissected and incubated in 30 mL of HBSS withoutCa2+ and Mg2+, containing 1 mM dithiothreitol, 5% FBS, and2 mM EDTA, for 30 min at 37°C, while agitating at 250 rpm.The suspension was filtered through nylon mesh (70 mm porediameter) and the pellet containing IECs was used for furtherstudies.

Cell lines

Human colon epithelial cell lines HT-29 and SW480 were pur-chased from ATCC (Manassas, VA, USA). HT-29 cells were grownin DMEM (Gibco, Life Technologies, Carlsbad, CA, USA) andSW480 cells in RPMI medium, supplemented with 10% fetalbovine serum (both from Biological Industries, Beit Haemek,Israel). All growth media contained 100 units/mL penicillin G, and100 μg/mL streptomycin. All cells were maintained in a humidi-fied incubator at 37°C with 5% CO2.

C. albicans growth conditions

C. albicans WT strain SC5314 was kindly provided by JudithBerman (Tel Aviv University). Cells were grown overnight in YPAD



A

DC

B

p-Sykp-Syk

Syk p-SykFigure 8. Syk signaling occurs in primaryIECs. Phospho-Syk expression in intestinalmucosa was assessed by immunohistochem-istry. Formalin-fixed paraffin-embedded sec-tions of mucosal biopsies from healthy colonwere stained with (A) anti-Syk Ab at 2 μg/mL,as a control for Syk distribution or with (B–D)anti phospho-Syk Ab at 20 μg/mL. (C–D) Imagesare higher magnifications of the field shown in(B). Original magnification ×4 (A and B) and ×20(C and D), scale bars 1 mm and 200 μm, respec-tively. Filled arrowheads indicate epitheliumand white-headed arrows indicate a germinalcenter. The results are representative of ilealand colonic mucosal samples from at least fiveindividuals.

medium at 30ºC. Heat-killed and UV-inactivated C. albicans cellswere prepared as previously described [55]. Briefly, heat-killedcells were prepared by 30 min incubation at 95ºC, and for UVinactivation cells were exposed in a thin liquid suspension to fourdoses of UV radiation (100 mJ/cm2) in a UV cross-linker (CL-1000UVP, Upland, CA, USA). Cells were counted and resuspended inPBS. Killing was verified by seeding onto YPAD-agar plates.

Immunohistochemistry

Immunohistochemistry was performed as previously described[56] on paraffin-embedded histopathological slides. Mouseanti-human Dectin-1 monoclonal Ab (clone 259931, MAB1859,R&D Systems, Minneapolis, MN, USA) was used at 10 μg/mL,mouse anti-human Syk monoclonal Ab (clone 4D10, sc-1240,Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used at2 μg/mL, and rabbit polyclonal anti-human phospho-Syk (Y-525,ab53133, Abcam, Cambridge, UK) was used at 20 μg/mL. Anisotype-matched control Ab was used at equivalent concentra-tions. All images within each experiment were taken under thesame conditions.

Flow cytometry studies

Cell-surface immunostaining of IECs was performed using FITC-conjugated anti-human Dectin-1 mAb (clone GE2, MCA4661F,AbD Serotec, Oxford, UK). Intracellular staining was performedwith FITC-conjugated human Syk mAb (clone 4D10, 552476,BD Biosciences, Franklin Lakes, NJ, USA) according to the

manufacturer’s instructions. All studies were carried out usingmatched-isotype controls. Primary IECs were identified bycostaining with APC-conjugated human EpCAM mAb (clone 9C4,324208, Biolegend, San Diego, CA, USA). Data acquisition wasperformed using a FACSCanto flow cytometer (Becton Dickinson,San Jose, CA, USA) and data were analyzed with FlowJo software(Treestar, Inc. Ashland, OR, USA).

Chemokine secretion

Cells were seeded in 12-well plates at a density of 106 (HT-29)or 7.5 × 105 (SW480) cells/well. After overnight incubation, themedium was replaced with a serum-free medium and zymosan(tlrl-zyn, InvivoGen, San Diego, CA, USA) or curdlan (C7821Sigma-Aldrich, St. Louis, MO, USA) was added at the indicatedconcentrations, based on previously reported doses [6, 23, 24]. Toavoid possible contaminating endotoxin, we used endotoxin-free(<0.001 EU) zymosan, and all experiments with curdlan werecarried out in the presence of polymyxin B (P4932, Sigma) at10 μg/mL. Where indicated, laminarin (L9634, Sigma) or theSyk inhibitors [3-(1-methyl-1H-indol-3-yl-methylene)-2-oxo-2,3-dihydro-1H-indole-5-sulfonamide] (574711, Calbiochem, Merck-Millipore, Darmstadt, Germany) and R406 (S2194, Selleckchem,Houston, TX), or c-Raf1 inhibitor GW5074 (G6416 Sigma) wereadded 30 min prior to the addition of zymosan or curdlan. Alltreatments were performed in triplicate. After 20 h, supernatantswere collected, centrifuged for 5 min at 10 000 g and maintainedat −70ºC until analysis. IL-8 and CCL2 concentrations were deter-mined using ELISA (DY208 and DY271, respectively, from R&Dsystems) according to the manufacturer’s instructions.



Cell lysis and western blot

Confluent cells were exposed to the indicated stimuli. Aftertreatment, cells were solubilized in lysis buffer (50 mM HEPESpH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X, 1 mM EDTApH 8, 1 mM EGTA pH 8, 1.5 mM MgCl2, 200 μM Na3VO4), anda protease inhibitor cocktail (539131, Calbiochem) consisting of150 nM aprotinin, 1 μM leupeptin, 500 μM AEBSF, and 1 μME64 protease inhibitor. Lysates were cleared by centrifugation(21 000 g for 10 min at 4ºC). For direct electrophoretic analysis,gel sample buffer was added to the cell lysates, and the lysateswere boiled for 5 min. Human PBMCs were isolated by Ficoll-Hypaque (GE-Healthcare, Uppsala, Sweden) density gradientcentrifugation, and cell lysates were prepared as above.

Proteins were resolved by SDS-polyacrylamide gel elec-trophoresis (PAGE) through 8–16% gradient precast Precise Tris-Glycine Gels (PIR-25263, Thermo Scientific, Waltham, MA, USA)and electrophoretically transferred to a nitrocellulose membrane.Membranes were blocked for 1 h in a TBST buffer containing6% nonfat milk, and reacted with the following primary antibod-ies according to the manufacturer’s instructions: rabbit polyclonalhuman Dectin-1 antibody (NBP1–25514, Novus Biologicals, Little-ton, CO, USA), antibodies specific for human Syk phosphorylatedon Tyr 525/526 (rabbit polyclonal 2711 and monoclonal 2710,clone C87C1, Cell Signaling, Danvers, MA, USA), mouse anti-human Syk monoclonal Ab (clone 4D10, sc-1240, Santa Cruz),rabbit β-actin polyclonal Ab (ab8227, Abcam), mouse α-tubulinmonoclonal Ab (Ab7291 Abcam). One hour incubation with HRP-linked secondary antibodies (goat-anti-rabbit (111-035-144) andgoat-anti-mouse (111-035-003), both from Jackson ImmunoRe-search, West Grove, PA, USA) was performed at room tempera-ture at 1:5000 dilution. Immunoreactive bands were detected withSuperSignal West Pico Chemiluminescent Substrate (PIR-34080,Thermo Scientific), using a MicroChemi imager (DNR BioimagingSystems, Jerusalem, Israel).

Immunofluorescence

Cells were seeded on cover slips in 6-well plates at a density of3–4×105 cells/well. After overnight incubation, cells were treatedas indicated in the figure legends, or directly fixed with4% paraformaldehyde for 30 min, washed with PBS, and per-meabilized with 0.1% Triton for 10 min at room temperature.Following fixation and permeabilization, the cells were stainedwith either mouse anti-human Dectin-1 (clone 259931, MAB0042,R&D systems), mouse anti-human Syk (4D10, sc-1240 SantaCruz), or rabbit polyclonal anti p-Syk (2711, Cell Signaling)overnight at 4°C, followed by staining with comparable secondaryAb (donkey-anti-mouse AF-488 (A21202), or donkey-anti-rabbitAF-647 (A31573, Molecular probes Life Technologies) for 1 hat room temperature and counterstaining with DAPI, which wasincluded in the mounting medium (GBI Labs, E-19-18, Mukilteo,WA, USA). Cells were visualized by confocal microscope (LSM700, Zeiss, Oberkochen, Germany), original magnification ×20.

All images within each experiment were taken under the sameconditions.

Statistical analysis

Data are reported as the mean of triplicates ± SD of a repre-sentative example of at least three experiments. Significance wasdetermined using Student’s t-test (GraphPad Prism software, SanDiego, CA, USA). Differences were noted as significant by thefollowing conventions: *p < 0.05, **p < 0.01, ***p < 0.001, asspecifically indicated in the legend of each figure.

Acknowledgments: We thank Judith Berman for C. albicansstrains, Oxana Elman for technical assistance, and Keren Rabi-nowitz for critical comments. This work was partially supportedby a generous grant from The Leona M. and Harry B. HelmsleyCharitable Trust to I.D. I.D. and S.C.K. conceived the projectand wrote the manuscript. I.D., S.C.K., and L.B. designed theexperiments and interpreted the data; S.C.K. and L.B. performedthe experiments. H.E. assisted in collecting samples and dataacquisition. E.B. designed and interpreted the immunohistochem-istry tests; H.G.G. interpreted the data and critically advisedthroughout the project.

Conflict of interest: The authors declare no commercial or finan-cial conflict of interest.

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Abbreviations: CD: Crohn’s disease · CLR: C-type lectin receptor · IBD:

inflammatory bowel disease · IEC: intestinal epithelial cell · Syk: spleen

tyrosine kinase

Full correspondence: Dr. Iris Dotan, Inflammatory Bowel DiseasesCenter, Department of Gastroenterology and Liver Diseases, Tel AvivSourasky Medical Center, 6 Weizmann Street, Tel Aviv 64239, IsraelFax: +972-3-6974184e-mail: [email protected]

Received: 27/5/2014Revised: 4/9/2014Accepted: 22/9/2014Accepted article online: 24/9/2014


Human intestinal epithelial cells respond to β-glucans via Dectin-1 and Syk

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Transcript of Human intestinal epithelial cells respond to β-glucans via Dectin-1 and Syk