Thymic Stromal Lymphopoetin-Induced Expression of the ... - Cell Press

Immunity

Article Thymic Stromal Lymphopoetin-Induced Expression of the Endogenous Inhibitory Enzyme SLPI Mediates Recovery from Colonic Inflammation Colin Reardon,1,5 Matthias Lechmann,1,5,6 Anne Bru¨stle,1 Me´lanie G. Gareau,2 Naomi Shuman,1 Dana Philpott,3 Steven F. Ziegler,4 and Tak W. Mak1,* 1The

Campbell Family Cancer Research Institute, Ontario Cancer Institute, University Health Network, Toronto, ON M5G 2C1, Canada Institute, Hospital for Sick Children, 555 University Avenue, Toronto, ON M5G 1X8, Canada 3Department of Immunology, 1 Kings College Circle, Toronto, ON M5S 1A8, Canada 4Benaroya Research Institute at Virginia Mason, 1201 Ninth Avenue, Seattle, WA 98101-2975, USA 5These authors contributed equally to this work 6Present address: Department of Dermatology, University Hospital Erlangen, Hartmannstrasse 14, D-91052 Erlangen, Germany *Correspondence: [email protected] DOI 10.1016/j.immuni.2011.05.015 2Research

SUMMARY

Thymic stromal lymphopoetin (TSLP) influences numerous immune functions, including those in the colonic mucosa. Here we report that TSLP-deficient (Tslp / ) mice did not exhibit increased inflammation during dextran sodium sulfate (DSS)-induced colitis but failed to recover from disease, resulting in death. Increased localized neutrophil elastase (NE) activity during overt inflammation was observed in Tslp / mice and was paralleled by reduced expression of an endogenous inhibitor, secretory leukocyte peptidase inhibitor (SLPI). Pharmacological inhibition of NE or treatment with rSLPI reduced DSS-induced mortality in Tslp / mice. Signaling through TSLPR on nonhematopoietic cells was sufficient for recovery from DSS-induced colitis. Expression of the receptor occurred on intestinal epithelial cells (IEC), with stimulation inducing SLPI expression. Therefore, TSLP is critical in mediating mucosal healing after insult and functions in a nonredundant capacity that is independent of restraining T helper 1 (Th1) and Th17 cell cytokine production.

INTRODUCTION Thymic stromal lymphopoetin (TSLP) is critical in the regulation of numerous immunological processes. These include, but are not limited to, conditioning of dendritic cells (DCs) to drive Th2 skewing of T cells (Ito et al., 2005), enhanced class switching in B cells (Xu et al., 2007), and induction of Foxp3+ regulatory T (Treg) cells in humans (Watanabe et al., 2005) but not mice (Sun et al., 2007). TSLP expression is increased in immunopathologies with dysregulated Th2 cell-type cytokine production including atopic dermatitis (Soumelis et al., 2002) and asthma (Ying et al., 2005). Although the pathophysiological features of these diseases can be recapitulated in mice overexpressing TSLP (Headley et al., 2009), decreased production of TSLP has

been observed in mucosal biopsies from patients with inflammatory bowel disease (IBD) (Rimoldi et al., 2005). Regulation of TSLP is dependent on the transcription factor NF-kB and is induced by exposure to bacteria (Rimoldi et al., 2005), agonists of toll-like receptor (TLR) or the intracellular sensor NOD (Lee and Ziegler, 2007; Zaph et al., 2007), and cytokines including TNF-a (Lee et al., 2008). Initially suggested as a mechanism to allow the commensal microbiota to induce tolerance, TSLP produced by intestinal epithelial cells is not required for tolerogenic mucosal DC and Treg cell generation (Iliev et al., 2009). Although the etiology of IBD has remained elusive, immunopathology results from loss of immunosuppressive or enhanced production of proinflammatory factors. Self-resolving immunopathology that recapitulates the hallmarks of colitis in patients can be induced in mice by addition of dextran sodium sulfate (DSS) to drinking water. Inflammation in this model is due to epithelial cell apoptosis, increasing recruitment, and activation of macrophages (Tlaskalova´-Hogenova´ et al., 2005), DCs (Berndt et al., 2007), and neutrophils (Okayasu et al., 1990). Although T cell activation and mucosal homing occur, T cells are not necessary for disease (Katakura et al., 2005). The resulting inflammation is associated with structural changes in the colon as a result of increased activity of proteases including neutrophil elastase (NE) and metallomatrix proteases (MMPs) (Castaneda et al., 2005). Recovery after inflammation is a complex process requiring not only suppression of immune processes but also balance between proteolytic enzymes and their endogenous inhibitors. Secretory leukocyte peptidase inhibitor (SLPI) is induced by microbial products, functioning as a serine protease inhibitor, antimicrobial peptide, and inhibitor of NF-kB (Taggart et al., 2005). SLPI is increased during resolution from DSS colitis (Mizoguchi et al., 2003) and exerts a nonredundant role after injury, as indicated by the fact that SLPI deficiency reduces cutaneous wound healing (Ashcroft et al., 2000). TSLP in the gastrointestinal tract has been described to inhibit Th1 cell type cytokine production after insult. Although in a previous publication increased IFN-g production was observed in mice deficient for the TSLP receptor (Crlf2 / ), potentiating the severity of DSS colitis (Taylor et al., 2009), here we report that deficiency in TSLP ligand did not increase colitis severity Immunity 35, 223–235, August 26, 2011 ª2011 Elsevier Inc. 223

Immunity TSLP Is Required for Recovery after Colitis

Figure 1. TSLP Deficiency Does Not Enhance Colitis Severity but Does Prevent Recovery

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Inflammation was assessed in DSS-treated Tslp / mice by (A) macroscopic disease, (B) colon length, (C) serum SAA concentration, (D and E) histological damage, (F) weight loss, and (G) survival. Graphs indicate control (open bars), DSS (solid bars); mean ± SD, n = 10–15 mice/group; scale bar represents 50 mm; *p < 0.05, ANOVA.

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as assessed by CD40, CD80, and CD86 (Figures S1C and S1D). In addition, we noted no spontaneous inflammation in mice aged for 18 months. Thus, the absence of TSLP does not favor the development of spontaneous inflammation or autoimmunity.

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Lack of TSLP Prevents Recovery from DSS-Induced Colitis -30 In the absence of spontaneous inflammation, we sought to determine whether the -40 lack of TSLP would enhance the severity E G 100 of an induced colonic inflammation. Unlike Tslp+/+ DSS Tslp-/- DSS the increased susceptibility reported in 75 Crlf2 / mice (Taylor et al., 2009), the 50 severity of DSS colitis was equivalent in Tslp / and Tslp+/+ mice. Macroscopic 25 score, colon length, concentration of the 0 acute phase protein serum amyloid A +/+ -/0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Tslp (SAA), and histological damage scores Time (Days) were not increased in Tslp / compared to Tslp+/+ mice at 8 days post-DSS but prevented recovery from disease. Recovery from colitis (Figures 1A–1E). When Tslp+/+ mice began to recover 9–10 days occurred through signaling transduced by TSLPR on radio- post-DSS, TSLP deficiency prevented recovery from inflammaresistant host cells, as indicated by the fact that mortality was tion (Figure 1F). Progressive weight loss and inability to recover not increased in Crlf2+/+ mice reconstituted with Crlf2 / bone met predefined humane endpoints, requiring sacrifice beginning marrow (BM). Our data demonstrate that TSLP functions as a on day 9, with no Tslp / mice surviving 14 days post-DSS (Figcritical mediator controlling the balance between host defense ure 1G). Peripheral blood analysis indicated no anemia or thromand wound repair, without constraining production of Th1 bocytopenia in all genotypes (Table S1). Accelerated disease cytokines. progression was also excluded; Tslp / mice assessed 4 days post-DSS did not display increased inflammation compared to Tslp+/+ (Figures S2A and S2B). We further excluded increased RESULTS susceptibility to DSS, because a lower dose (3% w/v) produced equivalent weight loss, colon shortening, and macroscopic and Deficiency in TSLP Does Not Alter Immune Cell histological damage scores in Tslp / and Tslp+/+ mice (Figures Development The requirement for TSLP in the development and maintenance S2C and S2D and data not shown). Therefore, lack of TSLP of B and T cell populations was assayed in TSLP wild-type does not result in enhancement of inflammation, but impairs (Tslp+/+), and deficient (Tslp / ) mice. Consistent with a lack of recovery from disease. To ascertain whether the lack of enhanced colonic inflammaa requirement for TSLP in murine Treg cell development (Sun et al., 2007), equivalent numbers of CD4+Foxp3+ cells were tion was unique to Tslp / mice, we subjected mice lacking found in the spleen and mesenteric lymph nodes (MLNs) (see TSLP receptor (Crlf2 / ) to DSS colitis. Onset of disease by Figure S1A available online) in Tslp+/+ and Tslp / mice. This weight loss, macroscopic score, colon length, and histological was mirrored by equivalent colonic Foxp3 expression in both examination indicated equivalent morbidity in Crlf2 / genotypes by qRT-PCR (Figure S1B). TSLP deficiency did not compared to WT or Tslp / mice (Figures S2E and S2F and alter the number of DCs (CD11b+CD11c+, CD11b CD11c+, or data not shown). However, Crlf2 / mice experience signifiCD11cloPDCA-1+) in the spleen or MLN, or increase activation, cantly increased mortality compared to DSS-treated WT mice -20

224 Immunity 35, 223–235, August 26, 2011 ª2011 Elsevier Inc.

Immunity

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TSLP Is Required for Recovery after Colitis

Figure 2. Deficiency in TSLP Signaling Does Not Enhance T Cell-Dependent Colitis

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FACS-sorted CD4+CD45RBhi (Teff) ± CD4+ CD45RBlo cells from Crlf2+/+ or Crlf2 / were transferred to Rag1 / recipients and monitored for (A) weight loss, (B) macroscopic disease, (C) colon length, and (D and E) histological damage (n = 4–5 mice/group). Scale bar represents 50 mm. (F) STAT5 phosphorylation was assessed after stimulation with rmIL-7 in CD4+ or CD8+ T cells from Crlf2 / , Tslp / , or WT (n = 3) mice (mean ± SD).

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demonstrate that absence of TSLPevoked signaling in T cells does not influence the development of a T cell-dependent colitis, that Treg cell function is not diminished by deficiency in TSLPR, and that lack of enhanced inflammation is not specific to innate or adaptive immune models of colitis.

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Prevention of Recovery in Tslp–/– Mice Is Not Mediated by Cytokine Skewing Crlf2+/+ Teff + Crlf2-/- Treg CD8+ CD4+ F Previous work conducted on Crlf2 / Crlf2 -/Tslp -/mice implied that increased Th1 type WT C57BL/6 cytokines produced during DSS-induced colitis enhanced the severity of disease. To ascertain whether delayed recovery was due to dysregulated Th1-type cytopSTAT5 Alexa647 kine production, the immune response to DSS was characterized by several complementary techniques. Cytokines (Figure S2G). Thus, lack of TSLP or TSLPR reduces the ability to characteristic of Th1, Th2, and Th17 immune responses were evaluated in the serum and colons of mice 8 days post-DSS. recover from colonic insult without enhanced inflammation. In order to determine whether these data were specific to the Mice with overt inflammation had significantly increased serum DSS model, we utilized a T cell-dependent model of colitis. concentrations (Figure 3A) and colonic mRNA (Figure 3B) for Disease was induced by adoptive transfer of FACS-sorted CD4+ IFN-g, IL-4, IL-6, IL-10, IL-17, TNF-a, and KC. No differences CD45RBhi effector T (Teff) cells into Rag1 / mice, with the effec- were observed between the two genotypes at baseline or during tiveness of Treg cell assessed by cotransfer of CD4+CD45RBlo inflammation (Figures 3A and 3B). CD4+ T cells from the spleen cells. Transfer of Teff cells from Crlf2+/+ or Crlf2 / mice resulted or MLN also produced equivalent amounts of IFN-g after in weight loss 5 weeks posttransfer (Figure 2A). Disease onset, in vitro restimulation irrespective of genotype (data not shown). macroscopic score, colon length, and histological scores were Therefore, the initial severity of intestinal inflammation and cytosimilar between recipients of Crlf2+/+ or Crlf2 / T cells (Figures kine production is not simply enhanced in the absence of TSLP. 2A–2E). Recipients of Teff + Treg cells from Crlf2+/+ or Crlf2 / We further assessed whether lack of TSLP would impact mice appeared healthy upon necropsy. Moreover, Treg cells systemic immune functions by analyzing the response after from Crlf2 / mice remain effective in vivo against WT Teff cells immunization with OVA. Injection of OVA:CFA or OVA:Alum as indicated by the fact that recipients of Crlf2+/+ Teff + Crlf2 / induced potent Th1 and Th2 cell responses, respectively. We deTreg cells did not develop disease (Figures 2A–2E). These data tected no difference in OVA-specific IgG1 (Figure S3A) or IgG2a demonstrate that TSLP signaling in T cells does not influence (Figure S3B) antibody titers in Tslp / mice compared to Tslp+/+ the development of T cell-dependent colitis and that Treg cell controls. These data clearly indicate that lack of TSLP alone does not increase the production of Th1 type cytokines, basally function is not diminished by deficiency in TSLPR. Because TSLP binds a heterodimer of TSLPR and IL-7Ra and or after the induction of an immune response. IL-7 can increase colitis severity (Okada et al., 2005), we investigated whether lack of TSLP or TSLPR altered IL-7 signaling. TSLP Deficiency Does Not Impair Intestinal Barrier Stimulation of isolated splenocytes with rmIL-7 induced equiva- Function or Alter the Colonic Microbiota lent STAT5 phosphorylation in CD4+ and CD8+ T cells from WT, Given the role of mucosal barrier dysfunction in IBD, it was Tslp / , and Crlf2 / mice (Figure 2F). Together, these data conceivable that delayed recovery may be due to prolonged Immunity 35, 223–235, August 26, 2011 ª2011 Elsevier Inc. 225


Figure 3. Lack of TSLP Does Not Increase Th1 Type Cytokines during DSS Colitis

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Cytokine production was assessed in (A) serum by cytometric bead array (*p < 0.05, ANOVA; n = 8–12 mice/group) or (B) qRT-PCR on colonic tissue after DSS colitis (mean ± SD, n = 8 mice/group, data normalized to GAPDH; control [open bars], DSS [solid bars]; *p < 0.05, t test).

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was no reduction in the numbers of IgA+ B cells in the Peyer’s patches (Figure S4D) or in luminal or serum IgA (data not shown) in Tslp / mice. No difference in the number of goblet cells was observed in -/+/+ -/+/+ -/+/+ Tslp the colon or terminal ileum of naive Tslp / mice (Figures S4E and S4F). Although DSS treatment reduced the B Colon RT-PCR number of colonic goblet cells, there was no difference between genotypes. Deficiency in TSLP does not reduce intestinal barrier function at baseline or during inflammation. To determine the impact of TSLP or TSLPR deficiency on the microbiota composition, we used qRT-PCR for selected bacterial 16S rDNA. No significant differences in the microbiota were observed in comparing naive or DSStreated WT, Tslp / , or Crfl2 / mice. Treatment with DSS colitis did, however, significantly reduce segmented filamentous bacteria (SFB) to below the limit of -/Tslp Tslp +/+ +/+ -/detection (Figure S4G). With this method, we detected no Salmonella sp. or Helicobacter sp., bacteria known to exacerbate or induce murine colitis (data not shown). TSLP ligand or receptor deficiency does not appear to alter the composition of the colonic microbiota in health or disease. +/+ -/Tslp To assess whether macrophage or neutrophil recruitment was reduced in Tslp / mice, immunohistochemistry (IHC) exposure to luminal contents, resulting in sustained immune was conducted. Similar numbers of these cells were observed in activation. Intestinal barrier function was assessed by transloca- the colon after DSS colitis in Tslp / and WT mice (data not tion of viable bacteria to the MLN and spleen or by detection of shown). Neutrophil recruitment after i.p. injection of LPS or the NOD2 agonist MDP-induced equivalent recruitment of neutrophils gavaged FITC-conjugated dextran probe in the serum. Tslp / mice did not have a basal intestinal barrier defect; naive mice into the peritoneum of WT and Tslp / mice (data not shown). displayed no increased bacterial translocation to the MLN (Fig- Together, these data indicate that the absence of TSLP does not ure S4A) or spleen (Figure S4B) compared to Tslp+/+ mice. In impair host defenses after breach of the epithelial barrier. contrast, increased bacterial translocation to the MLN and spleen was observed in all mice treated with DSS, irrespective TSLP Deficiency Reduces SLPI Expression during of genotype. Similarly, translocation of FITC-dextran from the Recovery from Disease lumen to the blood was not enhanced in naive Tslp / mice In order to determine the defect preventing Tslp / mice from compared to Tslp+/+ controls, whereas DSS significantly recovering after DSS colitis, we performed qRT-PCR on colonic samples for genes critical to resolution of inflammation. Expresenhanced permeability in both genotypes (Figure S4C). In addition to the IEC monolayer and tight junction proteins sion of Annexin A1 (ANXA1), PTGES2, iNOS (NOS2), the negative between adjacent cells, the mucosal barrier is also comprised regulators of NF-kB signaling IRAKM, and TNFAIP3 were of secreted IgA and mucous produced by goblet cells. There increased in all genotypes after DSS administration (Figure 4A). 226 Immunity 35, 223–235, August 26, 2011 ª2011 Elsevier Inc.


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Figure 4. Reduced Expression of the Protease Inhibitor SLPI in the Absence of TSLP

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(A) Gene expression in colonic tissue was assessed by qRT-PCR (mean ± SD, n = 10–15 mice/ group; control [open bars], DSS [solid bars]; *p < 0.05, ANOVA). (B) Mason’s trichrome staining for collagen (blue) on colonic tissues from control and DSS-treated Tslp+/+ and Tslp / mice. 203 magnification is from red rectangle; 6 mice/group; scale bar represents 50 mm. (C) SLPI expression was determined by immunoblotting colonic homogenates (n = 6 mice/group). (D and E) MMP (D) and NE (E) activity in colonic homogenates was assessed by a substrate cleavage assay (mean ± SD, n = 4–9 mice/group; control [open symbols], DSS [solid symbols]; *p < 0.05 by ANOVA). (F) PGRN was determined in colonic homogenates by immunobloting (n = 6 mice/group).

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DSS, but no differences between genotypes was observed (Figure 4D). In a similar fashion, use of a NE-selective substrate demonstrated significantly E increased colonic NE activity in Tslp / mice 8 days post-DSS compared to WT (Figure 4E). Increased NE activity was not due to enhanced NE expression (Elane, Figure 4A), neutrophilia, or recruitment after DSS in Tslp / mice (data not shown). CON DSS CON DSS NE is known to degrade a number of +/+ Tslp -/substrates in vivo. Included in these is F PGRN progranulin (PGRN), a protein that aids wound healing unless degraded by NE. βactin Whereas increased PGRN protein Tslp +/+ -/expression occurred during inflammation C C D D C C D D in WT mice, decreased protein but not mRNA expression was observed in Tslp / mice (Figures 4A and 4F). In the We also detected no difference in TNFAIP8, TFF3, REGIIIb and absence of TSLP, increased NE activity after colonic inflammaREGIIIg, lipoxygenases (ALOX5, ALOX12, ALOX15), and genes tion causes PGRN degradation, a protein critical to wound expressed by alternatively activated macrophages: Arg1, repair. Mrc1, Chi3l3, and Fizz1 between genotypes (Figure S5). In contrast, expression of Slpi (encoding secretory leukocyte pepti- Inhibition of NE Allows for Recovery from DSS Colitis dase inhibitor) mRNA was significantly upregulated after DSS in Tslp–/– Mice colitis in Tslp+/+ but not Tslp / mice (Figure 4A) and confirmed To assess how increased NE activity and lack of SLPI contribby immunoblot on full thickness colon homogenates (Figure 4C). uted to increased mortality in Tslp / mice, a selective NE inhibSeveral mechanisms have been ascribed to how SLPI aids in itor (NEi), SSR69071, was used. Mice were subjected to DSS resolution from injury, including the inhibition of proteases. colitis and orally gavaged with SSR69071, beginning on day 6, Consistent with increased protease activity, colonic sections prior to the recovery phase. SSR69071 treatment significantly from DSS-treated Tslp / mice showed defined regions below reduced mortality in Tslp / mice compared to vehicle control IEC with reduced collagen (blue staining, area indicated by (Figure 5A). Disease was not altered in Tslp+/+ mice, indicating asterisk in Figure 4B). Both MMPs and NE can degrade extracel- that SSR69071 does not simply reduce the severity of colitis. lular membrane (ECM) components, although SLPI has been To ascertain whether lack of SLPI alone led to increased NE demonstrated to inhibit NE. With a nonselective MMP assay, activity and mortality, Tslp / mice were treated with rSLPI or increased colonic MMP activity was detected 8 days post- vehicle at the beginning of recovery. Gavage of rSLPI

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Immunity 35, 223–235, August 26, 2011 ª2011 Elsevier Inc. 227


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Figure 5. Inhibition of NE Reduces DSS-Induced Mortality in TSLP–/– Mice (A) DSS-treated Tslp / or WT mice were gavaged with the NE inhibitor SSR69071 (3 mg/kg) or vehicle from day 6 to day 14 and monitored for (top) weight loss and (bottom) mortality. (B) In separate experiments, mice were gavaged with rSLPI (B) and assessed for (top) weight loss and (bottom) mortality. (C–E) IEC proliferation as determined by colonic Ki67 staining after 4 days of treatment (day 10 post-DSS) with vehicle or SSR69071, and (D) apoptosis in Tslp+/+ and Tslp / DSS-treated mice. (F) Colonic PGRN was determined on full thickness tissue homogenates 10 days post-DSS (mean ± SD, n = 4 mice/group; *p < 0.05, ANOVA).

significantly reduced mortality in Tslp / mice after DSS colitis (Figure 5B). These data demonstrate that enhanced NE activity in Tslp / mice, resulting from the absence of SLPI, prevents colonic healing after insult. Inhibition of NE by SSR69071 and the impact on newly synthesized (non-cross linked) collagen was assessed by the Sircol collagen assay. Nascent collagen was significantly increased 228 Immunity 35, 223–235, August 26, 2011 ª2011 Elsevier Inc.

after DSS administration of Tslp+/+ but not Tslp / mice. NE inhibition had no effect in Tslp+/+ but significantly increased collagen in Tslp / mice compared to vehicle control (Figure S6A). Expression of prominent intestinal collagen progenes assayed for by qRT-PCR indicated increased expression of Col1a1 in Tslp+/+ and Tslp / mice 8 days post-DSS, irrespective of NEi treatment (Figure S7). Significantly increased expression of


Figure 6. IEC Produce TSLP and SLPI In Situ and TSLPR on Nonhematopoietic Cells Are Sufficient for Recovery

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(A) Confocal microscopy was performed for TSLP (blue), SLPI (red), and E-cadherin+ IEC (green) in Tslp+/+ mice (asterisks, top) and Tslp / mice (daggers, bottom) (arrows, L, lumen; scale bar represents 20 mm). (B–F) WT mice reconstituted with Crlf2+/+ or Tslp / BM were subjected to colitis and assessed by (B) weight loss, (C) colon length, (D) Mason’s trichrome staining, (E) histological damage, and (F) SLPI expression (mean ± SD; scale bar represents 100 mm; n = 3–4 mice/group; control [open bars], DSS [solid bars]; *p < 0.05, **p < 0.001, ANOVA).

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Col4a2 was also observed in DSS-treated Tslp / mice receiving vehicle or SSR69071. We also noted reduced Col1a1 and Col4a1 after DSS administration, but this was again independent of genotype or NEi treatment. These changes therefore were unlikely to account for any difference in mortality or detection of nascent collagen protein. To address how NEi reduced mortality, we assessed whether SSR69071 prevented apoptosis or enhanced IEC proliferation. Apoptosis, as indicated by cleaved caspase 3 (CC3) staining, was not increased in Tslp+/+ or Tslp / mice 8 (Figure S6B) or 10 (Figure 5D) days post-DSS. Ki67 staining revealed significantly reduced proliferation 8 and 10 days post-DSS in Tslp / mice. Although this proliferation defect was not restored by SSR69071 treatment 8 days post-DSS (Figure S6C), IEC proliferation was restored 10 days post-DSS in NEi-treated Tslp / mice (Figures 5C and 5E). Inhibition of NE restored nascent

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collagen 8 and 10 days post-DSS (Figures S6A and S6E). More importantly, treatment normalized PGRN protein 8 days post-DSS (e.g., 2 days of NEi treatment) (Figure S6D) and 10 days post-DSS (4 days NEi treatment) (Figure 5F). This occurred at a posttranscriptional level, as indicated by the fact that PGRN mRNA expression did not increase with NEi treatment (Figures S6F and S7). Thus, although the NEi was effective within 2 days of treatment, at least 4 days of increased PGRN stability was required before an effect on IEC proliferation could be observed. TSLP-induced SLPI is required to regulate NE activity and subsequently PGRN availability, leading to healing and ultimately the survival of DSS-treated Tslp / mice.

TLSPR on Cells of a Nonhematopoietic Lineage Is Sufficient for Recovery from DSS CON DSS DSS +/+ -/+/+ Colitis We assessed the sites of TSLP and SLPI production in situ by performing immunofluorescence on colonic tissue. To this end, staining for epithelial cadherin (E-cadherin; green), a protein that localizes to the periphery of IEC, SLPI (red), and TSLP (blue), was performed (Figure 6A). TSLP was predominantly expressed in IEC (E-cadherin+ cells, adjacent to asterisk). In contrast, SLPI expression can be found in TSLP+ IEC (adjacent to dagger) and in cells of the lamina propria (arrowheads). To determine whether TSLP-induced signaling in immune cells was required for resolution of colitis, we constructed BM chimeras by reconstituting irradiated WT mice with BM from Crlf2+/+ or Crlf2 / mice and subjected them to DSS. The severity of colitis was not enhanced in WT recipients of Crlf2 / bone marrow (Crlf2 / / Crlf2+/+) compared to WT (Crlf2+/+/ Crlf2+/+) chimeras, 8 days post-DSS. Weight loss (Figure 6B), colon length (Figure 6C), and histological damage (Figure 6E) were similar in recipients of Crlf2+/+ or Crlf2 / BM, indicating that the absence of TSLPR on cells of hematopoietic origin Immunity 35, 223–235, August 26, 2011 ª2011 Elsevier Inc. 229


does not enhance colonic inflammation. In addition, increased mortality was not observed in recipients of Crlf2 / BM after DSS colitis compared to Crlf2+/+ BM recipients. Furthermore, no reduction in collagen deposition (Figure 6D) and equivalent SLPI mRNA expression was detected in Crlf2 / / Crlf2+/+ and Crlf2+/+/ Crlf2+/+ (Figure 6F) BM chimeras. Together, these data demonstrate that TSLP aids in resolution of colonic inflammation through cells of a nonhematopoietic lineage. TSLPR Is Expressed and Functional on the Surface of Intestinal Enterocytes IEC were isolated from the duodenum, jejunum, terminal ileum, and middistal colon and assessed for TSLPR by flow cytometry. Isolated and enriched epithelial cells from jejunum, terminal ileum, and colon were TSLPR+ (Figure 7A) and confirmed in situ by IHC (Figure 7B). TSLPR was not expressed on fibroblasts (aSMA , E-cadherin , vimentin+) or myofibroblasts (aSMA+, E-cadherin , vimentin+) isolated from the intestinal stroma (Figure 7C). TSLPR on IEC is functional, as shown by the fact that stimulation with rmTSLP induced AKT (S473) and ERK (T208) phosphorylation in Crlf2+/+ but not Crlf2 / IEC (Figure 7D). In order to determine the kinetics of signal transduction, we stimulated the mouse IEC line mICcl2 with rmTSLP. Stimulation elicited ERK1/2 and AKT phosphorylation within 10 min and the downstream target, Foxo3a, within 30 min (Figure 7E). Phosphorylation of Foxo3a resulted in increased expression of the antiapoptotic BCL2 within 2 hr poststimulation, whereas MCL-1 was not altered (Figure 7F). This suggests that TSLP can alter apoptosis in IEC by inducing antiapoptotic genes expression. This reductionist approach was further utilized to determine whether TSLP stimulation of IEC was sufficient to induce SLPI expression. mICcl2 monolayers stimulated with rmTSLPincreased SLPI mRNA was within 1 hr after stimulation (Figure 7G) and increased protein expression after 2 hr (Figure 7F). Together these data demonstrate that TSLP can directly elicit signaling in IEC and induce the expression of SLPI. DISCUSSION This study highlights the critical role for TSLP in colonic healing after transient inflammation. Our findings demonstrate that lack of TSLP ligand or receptor does not enhance disease severity, but prevents resolution, resulting in death. Increased mortality was due to unrestrained NE activity increasing turnover of progranulin, a protein that induces IEC proliferation. The underlying mechanism for this dysregulated NE activity was found to be due to an inability to increase SLPI expression after DSS-induced inflammation. TSLP-elicited signaling is not restricted to immune cells, with stimulation through TSLPR on IEC activating intracellular signaling and SLPI expression. Immunity was not altered in Tslp / mice in the steady state or after the induction of an immune response. No Th1 or Th17 cell bias was observed in naive mice or during DSS-induced colitis systemically (serum or splenocytes) or within the mucosal environment (MLN or colon). Lack of enhanced inflammation was not model specific, as shown by the fact that deficiency in TSLP signaling did not increase the severity of a T cell-dependent colitis by enhanced Teff (i.e., increased Th1 cytokine 230 Immunity 35, 223–235, August 26, 2011 ª2011 Elsevier Inc.

production) or decreased Treg cell function. It is important to note that although TSLP can potentiate Th2 cell responses (Ito et al., 2005), Tslp / or Crlf2 / mice remained capable of generating Th2 cytokines. In accordance with our data, Th2 cell responses remained intact after infection of Crlf2 / mice with an intestinal helminth (Massacand et al., 2009). Moreover, oral vaccination against OVA induces equivalent OVA-specific Th2 cell responses in WT and Crlf2 / mice (Bla´zquez et al., 2010). Therefore, TSLP is not required for Th2 cell responses, and the absence of TSLP does not necessarily result in Th1 cell-biased immune responses. To determine whether death in Tslp / mice was due to overt inflammation, downstream proinflammatory signaling cascades were studied. TSLP deficiency did not alter the expression of negative regulators of proinflammatory cytokine signaling, eliminating a role for uninhibited NF-kB-regulated gene expression (e.g., iNOS, IL-6, TNF-a). Although reduced healing of the intestinal mucosa and an inability to limit colonic inflammation has been observed in Anxa1 / (Babbin et al., 2008) and Ptges2 / (Morteau et al., 2000) mice, TSLP deficiency did not impact expression of these genes, indicating that the resolution defect is independent of these factors. As a whole, our data demonstrate that impaired recovery from DSS colitis is not simply due to aberrant cytokine production. We excluded the possibility that impaired host defenses prevented the ability to recover from DSS colitis and increased mortality in Tslp / mice. Naive and DSS-treated Tslp / mice did not have increased bacterial translocation to the MLN or spleen. Increased translocation is a common observation in mice with impaired host defenses because of ablation of genes such as Myd88 (Slack et al., 2009). Because the intestinal mucosa is under continuous immune surveillance that results in migration of DC laden with commensal bacteria to the MLN (Fukata et al., 2005; Macpherson and Uhr, 2004), it is improbable that increased bacteria within the colonic mucosa occurred without a parallel increase in MLN. Altered host defenses can also perturb the composition of the microbiota (Lupp et al., 2007). As an example of this, Nod2 / mice have reduced antibacterial peptides, increasing specific commensal populations (Petnicki-Ocwieja et al., 2009). We noted no significant effect on the microbiota in Tslp / or Crlf2 / compared to WT mice. In keeping with this, no difference was observed in the REG family of c-type lectins that function as antimicrobial peptides (Pickert et al., 2009). We also noted no deficiency in the recruitment of neutrophils to the colon after DSS colitis or into the peritoneum after challenge with pathogen-associated molecular patterns. Critically, mouse norovirus and Helicobacter sp. were not detected in our mice, because both can exacerbate the severity of intestinal inflammation (Cadwell et al., 2010). Therefore, it is unlikely that Tslp / mice have a defect in host defense, and instead the contribution of microbiota in different animal facilities (Friswell et al., 2010) may play a pivotal role in the differences between previous studies (Taylor et al., 2009). Repair of the epithelium occurs in at least two phases: the rapid restitution phase and a slower regeneration period. Whereas restitution involves spreading of epithelial cells to fill gaps in the damaged mucosa, regeneration requires cell proliferation, deposition of proteins including collagen around the wound site (Taupin and Podolsky, 2003), and contraction by


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Figure 7. TSLPR Is Expressed and Functional on IEC (A) Primary IEC were stained for flow cytometry for epithelial markers (E-cadherin+ CD3 , B220 , F4/80 ) and TSLPR. (B) IHC was performed on colonic tissue sections with TSLPR antibody. (C) TSLPR expression was assessed on fibroblasts (Vimentin+, alpha-smooth muscle actin- [a-SMA], E-cadherin CD3 CD11b B220 F4/80 ) or myofibroblasts (Vimentin+ a-SMA+, E-cadherin CD3 CD11b B220 F4/80 ) from Crlf2+/+ (gray fill, black outline) and Crlf2 / (negative control, red line) mice. (D) ERK and AKT phosphorylation in primary IEC from Crlf2+/+ and Crlf2 / mice were assessed after stimulation with rmTSLP (50 ng/mL, 30 min; mean ± SD, *p < 0.05, ANOVA). (E) AKT Foxo3A and ERK phosphorylation were assessed in mICc12 cells by immunoblotting after stimulation with rmTSLP for the indicated times (50 ng/mL). (F) Expression of antiapoptotic genes including the Foxo3a-regulated BCL2 and SLPI were determined after exposure to rmTSLP. (G) SLPI mRNA in mICcl2 cells after TSLP exposure was determined by qRT-PCR (n = 3, mean ± SD, *p < 0.05, ANOVA).

myofibroblasts. Deposition of structural proteins is a tightly controlled process; dysregulation results in fibrosis or fistula development resulting from increased or insufficient ECM protein deposition, respectively (Rieder et al., 2007). This process is regulated by the rate of collagen synthesis and the

balance between proteolytic enzymes and their endogenous inhibitors. Highlighting that this balance was altered in DSS-treated Tslp / mice, reduced collagen was detected by histological staining and by Sircol assay without concomitant decreases in collagen mRNA expression. These data Immunity 35, 223–235, August 26, 2011 ª2011 Elsevier Inc. 231


demonstrated that the reduced collagen deposition was due to enhanced, localized enzymatic activity resulting in collagen turnover. In accordance with a previous report (Castaneda et al., 2005), we observed increased MMP activity during overt colonic inflammation; however, this occurred irrespective of genotype, indicating that the increased mortality is MMP independent. Degradation of collagen can also occur by NE (Zhu et al., 2001), an enzyme found in abundance in samples from patients with IBD (Langhorst et al., 2008). Colonic tissue assayed at the peak of disease demonstrated significantly enhanced NE activity in DSS-treated Tslp / compared to WT mice, without increased NE expression. Increased enzymatic activity without a parallel increase in NE expression indicated a defect in an endogenous regulator. A profound reduction in SLPI mRNA and protein were observed in the colon of Tslp / mice post-DSS. This inability to increase SLPI expression after DSS colitis and to control NE activity decreased mucosal healing and ultimately increased mortality. Indeed, inhibition of NE with a selective inhibitor or rSLPI significantly improved survival in Crlf2 / mice. In support of this hypothesis for regulation of wound healing by SLPI, increased NE activity delayed recovery in Slpi / mice after dermal injury (Ashcroft et al., 2000). SLPI influences the balance between host defense and tissue repair by regulating NE-mediated conversion of progranulin to granulin. These proteins exert opposing physiological functions: progranulin induces epithelial cell proliferation and reduces neutrophil activity and granulin inhibits proliferation and increases IL-8 production (human cell lines), neutrophil recruitment, and activation (Kessenbrock et al., 2008; Zhu et al., 2002). To delineate the role of enhanced NE activity in the failure to recover from DSS colitis in Tslp / mice, we assessed the impact of NE inhibition during the recovery phase. IEC proliferation was significantly reduced 8 days post-DSS that was normalized after 4 days of SSR69071 treatment. This inhibition of NE activity restored nascent collagen protein and PGRN in DSS-treated Tslp / mice beginning 2 days after treatment. In accordance with previously published in vitro data, where PGRN increased epithelial cell proliferation after 3 days (Zhu et al., 2002), IEC proliferation occurred after 4 days of treatment subsequent to inhibition of NE and stabilization of the PGRN protein. Together our data indicate that TSLP-induced SLPI dictates the balance between host defense and tissue regeneration and consequently aids mucosal healing after colonic inflammation by modulating NE activity. Although TSLP has been primarily thought to exert physiological effects through TSLPR on B cells, T cells, and DCs, the contribution of this receptor on nonhematopoietic cells was evaluated in the resolution of colitis. By using BM chimeras, TSLPR on nonhematopoietic cells was sufficient for SLPI expression and survival. Supporting a role for nonhematopoietic cells, functional TSLPR is expressed on the surface of primary IEC and a mouse IEC line. Stimulation through this receptor initiates activation of both ERK and AKT signaling and SLPI expression. Resident colonic fibroblasts or myofibroblasts would seem to be excluded from this population of cells that can directly respond to TSLP, because they lack TSLPR. In assessing TSLP and SLPI production in situ, both proteins were prominently coexpressed in colonic IEC with some SLPI expression localized to cells in the lamina propria. As previously reported, these are 232 Immunity 35, 223–235, August 26, 2011 ª2011 Elsevier Inc.

probably macrophages (Nakamura et al., 2003) and DCs (Samsom et al., 2007), although it would appear that IEC are the predominant source of colonic SLPI. Therefore, TSLP can act on IEC in an autocrine manner that is beneficial to intestinal restitution and healing. Here we propose a model whereby TSLP acts as a key facilitator in wound repair after intestinal injury. Because TSLP expression by IEC is enhanced after TLR stimulation or proinflammatory cytokines (Zaph et al., 2007), it is conceivable that after DSSinduced epithelial injury, exposure to the luminal contents increases TSLP expression in adjacent IEC. TSLP produced in this manner can act directly on epithelial or stromal cells to induce SLPI gene expression, creating a local tissue environment that favors healing and resolution of inflammation. This is a nonredundant role of TSLP in the colonic mucosa and one that does not involve restraining of Th1 or Th17 cell immune responses. EXPERIMENTAL PROCEDURES Mice Tslp / mice were generated by a conventional targeting strategy by inserting an eGFP and a neomycin cassette into exon 2 five codons after the transcription start of the TSLP gene. Insertion of the targeting vector deletes a portion of exon 2 and all of exons 3 and 4. Deletion of TSLP in Tslp / mice was confirmed by Southern blotting and PCR with primers spanning the junction between exon 2 and the eGFP-NeoR insert (data not shown). Mice were backcrossed to C57BL/6 for at least 10 generations, with both males and females used. TSLPR-deficient (Crlf2 / ) mice were described previously (Al-Shami et al., 2005). Rag1 / mice (Jackson) were backcrossed to C57BL/6 mice for at least 10 generations. Hematological Analysis Blood collected by cardiac puncture was added to an EDTA-containing tube and was analyzed with a HEMAVET instrument (Abaxis, Union City, CA). Induction of Colitis Colitis was induced by addition of dextran sodium sulfate (DSS; 36,000– 50,000 MW; MP Biomedicals, Solon, OH) to the drinking water for 5 days, followed by 3 days of normal water. Blood was collected from anesthetized mice by cardiac puncture. In subsets of experiments, mice were treated with the NE inhibitor SSR69071 or vehicle (DMSO) by oral gavage twice daily (3 mg/kg; Tocris, Ellisville, MS). Mice were treated with rSLPI (R&D systems) by gavage in 0.1 M sodium borate twice daily (Sigma-Aldrich, St. Louis, MO). Adoptive Transfer Colitis was induced by the transfer of FACS-sorted CD4+CD45RBhi effector T cells (4 3 105 cells/mouse). Protection from colitis and hence the capacity of immunoregulatory CD4+CD45RBlo (2 3 105 cells/mouse) cells was assessed after cotransfer into recipient C57BL/6 Rag1 / mice. Colitis was assessed by macroscopic and histological scores published previously (Reardon et al., 2001). All procedures were approved by the University Health Network animal care committee. Immunization Mice were immunized with OVA (Grade V, Sigma-Aldrich) in CFA (Fisher Scientific Ottawa, ON) or Alum AlK(SO4)2 (Sigma-Aldrich) by i.p. injection as described previously (Yip et al., 1999). Serum was obtained 2 weeks later, with OVA-specific IgG1, and IgG2a was determined by ELISA (Southern Biotechnology, Birmingham, AL). Neutrophil Recruitment Mice were injected i.p. with 100 ng LPS (Sigma Aldrich) or 100 mg of MDP (Invivogen, San Diego, CA). The peritoneal cavity was washed with PBS, and recovered cells were counted, stained, and enumerated by flow cytometry.


Bone Marrow Chimeras Male recipient mice were lethally irradiated (Gammacell 40; MDS Nordion, Ottawa, ON) by two exposures of 550 rads prior to receiving 1 3 106 BM cells from Crlf2 / mice by i.v. and were maintained on Baytril for 2 weeks.

Bacterial Translocation Spleens or MLN were weighed and homogenized in 500 ml of sterile PBS containing 0.1% Triton X-100 (w/v). Samples were serially diluted in PBS, plated on 5% Sheep’s blood agar, and grown overnight.

Immunohistochemistry Tissues were formalin fixed and processed according to standard histological protocols. Staining with PAS reagent or masson’s trichrome were conducted according to manufacturer’s instructions (Sigma-Aldrich). Sections were stained for Ki67 (Lab Vision, Fremont, CA), EMR1 (Life Span Biologicals, Seattle, WA), NIMP (Santa Cruz Biotechnology, Santa Cruz, CA), Cleaved Caspase3 (Cell Signaling Technology, Danvers, MA) with a standard protocol for development with DAB substrate (VectorLabs, Burlington, ON).

MMP and Neutrophil Elastase Assays Snap frozen tissue was homogenized by TissueLyser (QIAGEN). MMP activity was assayed with a generic MMP assay kit (AnaSpec) according to manufacturer’s instructions. Neutrophil elastase activity was assessed in a similar fashion, except that Rhodamine 110, bis-CBZ-L-alanyl-L-alanyl-L-alanyl-Lalanine amide (Invitrogen) was used as the substrate.

Immunofluorescence Formalin-fixed paraffin-embedded tissues were stained by a previously published protocol (Kaniuk and Brumell, 2010). In brief, rehydrated sections were subjected to antigen retrieval (citrate buffer, pH 6.0, 90 C, 30 min). Slides were blocked for 1 hr and incubated with anti-E-cadherin (ECMbiosciences, Versailles, KY), -SLPI, -TSLP (Life Span Biologicals) overnight, washed, and incubated with appropriate conjugated secondary antibodies Alexa Fluor 488, 546, and 647 (1 hr, RT, Invitrogen). Images were acquired with a Fluoroview 1000 confocal microscope (Olympus, Markham, ON). Real-Time Quantitative PCR Tissue was homogenized in Trizol (Invitrogen, Burlington, ON) with a TissueLyser (QIAGEN). RNA was precipitated with LiCl2 and cDNA synthesis was performed (iScript kit; Bio-Rad, Mississauga, ON). Previously published primers (Peterson et al., 2010) or primers selected from the primerbank (Spandidos et al., 2008) (Table S2) were used in qRT-PCR with SyberGreen master mix (ABI). Data were analyzed by the 2DDCT method normalizing to gapdh. Assessment of Microbiota Microbial DNA was extracted with a QIAamp DNA stool kit (QIAGEN) according to manufacturer’s specifications. DNA eluted from the spin columns was quantified, and qRT-PCR was performed with microbial primers to 16S rDNA (Table S2). Data are presented as percentage relative to eubacteria. Flow Cytometry Spleens and lymph nodes were dissociated mechanically, and red blood cell lysis was performed on spleens with ACK buffer (Sigma-Aldrich). For splenic DC, spleens were injected with collagenase/DNase (37 C, 30 min). Cells were counted, and 1 3 106 cells were stained with anti-B220, -CD3, -CD11b, -CD40, -CD80, -CD86, -F4/80, -IgA, -MHCII (BD Biosciences, Mississauga, ON), -CD11c, -pDCA-1 (eBioscience, San Diego, CA), and -TSLPR (R&D systems, Minneapolis, MN). Intracellular staining for Foxp3 and IFN-g were conducted according to manufacturers’ instructions (Biolegend and eBioscience, respectively). Phosphoflow was performed with anti-pSTAT5, -pERK1/2 (BD Biosciences), or -pAKT (Cell Signaling Technology). Vimentin (Cell signaling Tech.) and aSmooth Muscle Actin (Milipore) were labeled with Alexa Fluor 405 and 488, respectively, with a Zenon labeling kit (Invitrogen). Data were acquired on a CantoII (BD Biosciences) and analyzed with FlowJo (Tree star, Ashland OR).

Sircol Collagen Assay Collagen was quantified with the Sircol assay according to manufacturer’s instructions. Colonic protein was incubated with Sirius red with gentle agitation (0.5 hr, RT). Dye-bound collagen was centrifuged, washed, and resolubilized with alkaline buffer. Collagen concentration was determined by measuring the absorbance at 555 nm (Flex Station). Cells Cells were stimulated with recombinant mouse TSLP (R&D systems). Enrichment of Epithelial Cells, Fibroblasts, and Myofibroblasts Intestinal tissues were opened, washed in PBS, and cut into 0.5 mm pieces. Tissues were incubated with cell recovery solution (BD Biosciences) (Perreault and Beaulieu, 1998). Epithelial cells were stained immediately for flow cytometry or enriched by negative selection with the BD Imag system (BD Biosciences) to remove CD11b+, CD11c+, CD3+, B220+ cells. For phosphoflow, cells were incubated in optiMEM for 15 min and restimulated with 50 ng/mL of rmTSLP for 30 min prior to analysis as described above. Fibroblasts and myofibroblasts were dissociated by collagenase/dispase as described previously (Evans et al., 1992; Fruchtman et al., 2005). Cells were surface stained for E-cadherin, CD11b, CD11c, F480, CD3, B220, fixed with formaldehyde (4% v/v), and treated with cytoperm (BD Biosciences) for intracellular staining of aSMA and vimentin as described above. Immunoblotting Immunoblotting was performed according to a standard protocol. Blots were incubated with anti-SLPI (Santa Cruz Biotechnology, Santa Cruz, CA), -Foxo3A, -Phospho-Foxo3A (T32), -Phospho-AKT, -Phospho-ERK, -BCL2 (Cell Signaling Technology), and -AKT, -ERK, -Tubulin, -MCL (Santa Cruz Biotechnology). Statistics and Data Presentation All statistics were calculated in Prism 5.0 (GraphPad, La Jolla, CA) and are twotailed Student’s t test or ANOVA with Tukey post-test as indicated in the respective figure legends. All data are presented as mean ± standard deviation. SUPPLEMENTAL INFORMATION Supplemental Information includes seven figures and two tables and can be found with this article online at doi:10.1016/j.immuni.2011.05.015. ACKNOWLEDGMENTS

Cytometric Bead Array Serum cytokine concentrations were evaluated with a mouse Th1/Th2/Th17 kit (BD Biosciences) according to the manufacturer’s instructions. Samples were acquired on a FACsArray and analyzed with CBA software (BD Biosciences). In vivo Permeability Mice were starved overnight with free access to water and gavaged the next morning with 4.4KDa FITC-Dextran (Sigma-Aldrich). Blood was collected 4 hr postgavage as described above and read on a Flex Station3 (Molecular Devices, Sunnyvale, CA).

This work was supported by an operating grant from the Canadian Institutes of Health Research (T.W.M.). C.R. is a recipient of a Cancer Research Institute/ Irvington Institute Fellowship. M.L. is a recipient of IZKF Erlangen TP A46. Additional support was from AR056113 and AI068731 grants from the NIH (S.F.Z.). A.B. is supported by a German Research Foundation DFG fellowship. Received: May 16, 2010 Revised: April 22, 2011 Accepted: May 16, 2011 Published online: August 4, 2011



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