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Cellular Microbiology (2009) 11(12), 1802–1815
doi:10.1111/j.1462-5822.2009.01372.x First published online 8 September 2009
Internalization-dependent recognition of Mycobacterium avium ssp. paratuberculosis by intestinal epithelial cells Johanna Pott,1 Tina Basler,2 Claudia U. Duerr,1 Manfred Rohde,3 Ralph Goethe2 and Mathias W. Hornef1* 1 Institute for Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany. 2 Institute for Microbiology, Department of Infectious Diseases, University of Veterinary Medicine, Hannover, Germany. 3 Helmholtz Center for Infection Biology, Braunschweig, Germany. Summary Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of Johne’s disease, a highly prevalent chronic intestinal infection in domestic and wildlife ruminants. The microbial pathogenesis of MAP infection has attracted additional attention due to an association with the human enteric inflammatory Crohn’s disease. MAP is acquired by the faecal–oral route prompting us to study the interaction with differentiated intestinal epithelial cells. MAP was rapidly internalized and accumulated in a late endosomal compartment. In contrast to other opportunistic mycobacteria or M. bovis, MAP induced significant epithelial activation as indicated by a NF-kBindependent but Erk-dependent chemokine secretion. Surprisingly, MAP-induced chemokine production was completely internalizationdependent as inhibition of Rac-dependent bacterial uptake abolished epithelial activation. In accordance, innate immune recognition of MAP by differentiated intestinal epithelial cells occurred through the intracellularly localized pattern recognition receptors toll-like receptor 9 and NOD1 with signal transduction via the adaptor molecules MyD88 and RIP2. The internalization-dependent
Received 28 May, 2009; revised 5 August, 2009; accepted 6 August, 2009. *For correspondence. E-mail hornef.mathias@ mh-hannover.de; Tel. (+49) 511 532 4540; Fax (+49) 511 532 4366.
innate immune activation of intestinal epithelial cells is in contrast to the stimulation of professional phagocytes by extracellular bacterial constituents and might significantly contribute to the histopathological changes observed during enteric MAP infection.
Introduction Mycobacterium avium ssp. paratuberculosis (MAP) infects domestic and wild ruminant animals via the faecal– oral route. It causes Johne’s disease, a highly prevalent chronic intestinal infection that after a long preclinical phase ultimately leads to overt chronic enteritis with diarrhoea and weight loss. In contrast to other M. avium subspecies, MAP appears not to be able to proliferate in the environment. Shedding via the faeces of infected animals and the high tenacity in soil and water results in a widespread presence of MAP in the environment (Cocito et al., 1994). MAP belongs to the group of opportunistic M. avium subspecies that cause human infections particularly in immunodeficient individuals. In addition, an association of MAP infection with the chronic inflammatory bowel disease Crohn’s disease has been proposed (Chiodini et al., 1984; Feller et al., 2007; Pierce, 2009). Studies comparing the presence of MAP in clinical samples of Crohn’s disease patients and appropriate controls have given contradictory results, possibly due to the extraordinary difficulties to cultivate MAP from clinical material. The requirement to incubate cultures for months to even years before classifying them as culture-negative has been proposed due to the fastidious nature and extremely slow growth of MAP (Bull et al., 2003). In addition, MAP might be able to constitute infective but noncultivable vegetative forms. The application of alternative detection methods such as PCR or serology has indicated a specific association of MAP with Crohn’s disease, but the question of an aetiological role is still unresolved (Feller et al., 2007; Pierce, 2009). The pathogenic potential of MAP and the molecular basis for its intestinal organ tropism are still unclear. After crossing the mucosal barrier, subepithelial macrophages are the major target cells, in which they are able to persist
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cellular microbiology
Intraepithelial innate immune recognition of MAP 1803 and multiply (Momotani et al., 1988; Sangari et al., 2001). Experiments with macrophage cell lines suggest that the host cell interaction of MAP differs from that of other M. avium subspecies. MAP specifically inhibits the antigen-specific stimulatory capacity of macrophages for antigen-specific CD4+ T cells (Zur Lage et al., 2003) and restricts the macrophage pro-inflammatory response (Basler et al., 2008). There is growing evidence that also the intestinal epithelium is actively involved in the antibacterial host defence and maintenance of the mucosal homeostasis (Zaph et al., 2007; Nenci et al., 2007). Indeed, in vivo studies demonstrated infiltration of MAP into the intestinal mucosa and a strong inflammatory reaction of the enteric mucosal tissue (Sigurdardottir et al., 2005; Wu et al., 2007; Golan et al., 2009; Khare et al., 2009). Yet, detailed in vivo studies have been hampered by the lack of suitable animal models and the in vitro analysis of the interaction of MAP with epithelial cells has largely been restricted to cell lines that are not of intestinal origin (Hines et al., 2007). Therefore, we focused on the cellular and molecular mechanisms of MAP internalization and epithelial activation using a well-established model of differentiated and polarized intestinal epithelial cells (Bens et al., 1996; Hornef et al., 2002). Our results illustrate the pro-inflammatory potential of MAP. They demonstrate for the first time internalization-dependent activation of intestinal epithelial cells and identify the molecular and cellular mechanisms involved.
Results MAP is rapidly internalized and persists in intestinal epithelial cells MAP enters the host via the intestinal tract and exhibits a marked intestinal organ tropism indicating a direct interaction of the bacterium or bacterial components with the apical surface of epithelial cells. Scanning electron microscopy images of MAP-exposed m-ICcl2 cells confirmed an intense contact between MAP and the apical cell surface of the polarized epithelium (Fig. 1A). Also, transmission electron microscopy clearly demonstrated rapid internalization of MAP by intestinal epithelial cells (Fig. 1B). Although a significant number of intracellular bacteria was detected and despite a careful analysis at various time points after infection was performed, the structural characteristics of the internalization process could not be visualized by electron microscopy. The viability of internalized bacteria was confirmed using a modified internalization (amikacin-kill) assay. A significant fraction (4%) of the added bacteria was cultured as soon as 30 min after infection from epithelial cell lysate following removal of extracellular bacteria. Internalized MAP persisted intracellularly in a cultivable form over 7 days © 2009 Blackwell Publishing Ltd, Cellular Microbiology, 11, 1802–1815
(Fig. 1C). Bacterial internalization was quantified using flow cytometric analysis of intestinal epithelial cells infected with fluorophore-labelled MAP. This method illustrated the efficiency of bacterial uptake with approximately 50% MAP-positive epithelial cells after exposure at a multiplicity of infection (moi) of 100:1. Incubation in the presence of the inhibitor of actin polymerization latrunculin B significantly reduced bacterial internalization from 55% to 22% MAP-positive cells confirming a cell-mediated, actin-dependent internalization process (Fig. 1D). Also, no bacterial uptake was observed in MAP-infected m-ICcl2 cells incubated at 4°C excluding significant staining of epithelial cells by adherent bacteria (data not shown). Subsequent immunofluorescence microscopy characterization of the epithelial internalization process revealed MAP-positive actin-enriched cellular compartments (left panel, Fig. 1E) with significant colocalization of the late endosomal marker LAMP2 (right panel, Fig. 1E). Together, these results are consistent with a rapid cell-mediated internalization process leading to significant numbers of viable intraepithelial MAP and intracellular persistence. MAP-induced activation of intestinal epithelial cells A marked inflammatory response of the intestinal mucosa upon MAP exposure in vivo has been described (Golan et al., 2009). We therefore analysed the epithelial response of differentiated intestinal epithelial m-ICcl2 cells to apical exposure with MAP. A pronounced epithelial response illustrated by a time-dependent increase of the pro-inflammatory chemokine MIP-2 in the cell culture supernatant was noted. Although chemokine levels reached similar concentrations as compared with stimulation with lipopolysaccharide (LPS), MAP-induced epithelial activation appeared to be significantly delayed. Diminished epithelial activation was observed in response to heat-inactivated bacteria (Fig. 2A). Importantly, exposure with the MAP strains 6783 and K10 induced significant MIP-2 secretion, whereas only marginal epithelial activation was observed in response to infection with M. avium ssp. avium, M. avium ssp. hominissuis, or the vaccine strain M. bovis BCG (Fig. 2B). Subsequently, MAP-induced signalling pathways leading to chemokine secretion were analysed. Mitogenactivated protein (MAP) kinase signalling is frequently observed following bacterial stimulation. Inhibition of p38 (with 3-O-acetyl-b-boswellic acid) or c-Jun N-terminal kinase (JNK) signalling (with JNK inhibitor 1), however, did not alter MAP-induced chemokine secretion. Also, an only weak increase of p38 phosphorylation and no JNK phosphorylation was detected after MAP infection whereas both p38 and JNK phosphorylation was observed following LPS stimulation (Fig. 2C and data not shown). In contrast, the extracellular signal-regulated
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Fig. 1. Internalization of MAP by intestinal epithelial cells. A and B. (A) Scanning electron microscopy and (B) transmission electron microscopy of polarized and differentiated intestinal epithelial m-ICcl2 cells 1, 6 and 24 h after MAP-infection (moi 100:1) respectively. Bar, 1 mm. C. Determination of viable intraepithelial MAP by bacterial culture after intestinal epithelial infection (moi 10:1) for the indicated time. The number of intracellular MAP was determined by serial dilution of cell lysate following removal of extracellular bacteria by extensive washing and amikacin treatment. The number of cultured bacteria is shown in per cent of the inoculum. D. Flow cytometric analysis of intestinal epithelial m-ICcl2 cells 4 h after infection with PKH26-labelled MAP (moi 100:1) in the absence or presence of latrunculin B (1 mM). E. Coimmunostaining of MAP and actin (left panel) or MAP and the late endosomal marker LAMP2 (right panel) in intestinal epithelial m-ICcl2 cells 4 h after infection. Bar, 10 mm.
kinases (Erk) inhibitor UO126 significantly diminished MAP-mediated epithelial activation in a dose-dependent fashion and a marked phosphorylation of Erk was noted 15 min following MAP challenge (Fig. 2D). In contrast, no significant NF-kB activation was observed in epithelial cells upon MAP stimulation. No increase in the phosphorylation of the p65 NF-kB subunit nor degradation of IkB-a was observed in epithelial cells in response to MAP whereas LPS stimula-
tion of m-ICcl2 cells or MAP stimulation of macrophagelike RAW264.7 cells resulted in p65 phosphorylation and IkB-a degradation (Fig. 3A). Similarly, m-ICcl2 cells stably carrying a NF-kB luciferase construct were stimulated by LPS exposure, but no reporter activity was noted in response to MAP (Fig. 3B). Analysis of p65 nuclear translocation by immunofluorescent staining and immunoblotting of nuclear extracts further confirmed that NF-kB p65 activation is not initiated after MAP challenge © 2009 Blackwell Publishing Ltd, Cellular Microbiology, 11, 1802–1815
Intraepithelial innate immune recognition of MAP 1805
Fig. 2. MAP-induced activation of intestinal epithelial cells. A. Kinetic of intestinal epithelial m-ICcl2 cell activation by apical exposure to viable or heat killed MAP (moi 10:1) as compared with LPS (1 ng ml-1). Supernatants were taken at the indicated time points and analysed for MIP-2 concentration. h.i., heat-inactivated. B. m-ICcl2 cell activation by several opportunistic mycobacterial species and M. bovis BCG (moi 100:1). MIP-2 secretion was quantified after 6 h. C. LPS (10 ng ml-1) and MAP (moi 100:1) induced epithelial MIP-2 secretion after 6 h in the absence or presence of p38 or JNK inhibitors. Immunoblot for phosphorylated p38 or JNK in epithelial lysate after MAP stimulation for the indicated time period. D. LPS (10 ng ml-1) and MAP (moi 100:1) induced epithelial MIP-2 secretion after 6 h in the absence or presence of the Erk inhibitor UO126. Immunoblot for phosphorylated ERK in epithelial lysate after MAP stimulated for the indicated time.
in epithelial cells (Fig. 3C and D). In accordance, chemokine secretion by epithelial cells in response to MAP was NF-kB p65/p50-independent as the selective NF-kB p50 inhibitor SN50 failed to alter MAP-mediated MIP-2 secretion. In contrast, the LPS-induced chemokine secretion was significantly reduced in the presence of SN50 (Fig. 3E). Yet, SN50 treatment only led to a partial inhibition of LPS-induced MIP-2 secretion in accordance with a contribution of MAP kinase signalling © 2009 Blackwell Publishing Ltd, Cellular Microbiology, 11, 1802–1815
also in LPS-induced chemokine secretion (data not shown). Finally, also heat-killed bacteria, shown to induce MIP-2 secretion, failed to induced NF-kB reporter activity making an active interference of MAP with epithelial NF-kB activation unlikely (Fig. 3F). Thus, MAP harbours a marked stimulatory potential on intestinal epithelial cells and induces pro-inflammatory chemokine secretion in a NF-kB p65/p50-independent fashion via Erk-mediated signal transduction.
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Fig. 3. Absence of NF-kB p65/p50-activation in MAP-stimulated intestinal epithelial cells. A. Immunoblot for phosphorylated p65 and total IkB-a in cell lysate of epithelial cells and macrophages stimulation with MAP (moi 100:1 for mICcl2 cells; 10:1 for RAW264.7 cells) or LPS (10 ng ml-1) for the indicated time. B. m-ICcl2 cells stably expressing a NF-kB luciferase reporter construct were exposed to MAP (moi 100:1) and luciferase synthesis was determined after the indicated time points. Epithelial NF-kB activation in response to LPS (10 ng ml-1) was included as control. C and D. Analysis of p65 nuclear translocation after MAP infection (moi 100:1) or LPS stimulation (100 ng ml-1) analysed by immunofluorescent staining 30 min after infection (C) and immunoblot of nuclear extracts obtained after the indicated time (D). Bar, 10 mm. E. LPS (10 ng ml-1) and MAP (moi 100:1)-induced epithelial MIP-2 secretion after 6 h in the absence or presence of the NF-kB inhibitor SN50 (16 mM). F. NF-kB-induced luciferase activity in response to LPS (10 ng ml-1), MAP and heat-inactivated (h.i.) MAP 4 h after infection. © 2009 Blackwell Publishing Ltd, Cellular Microbiology, 11, 1802–1815
Intraepithelial innate immune recognition of MAP 1807 MAP-mediated activation of intestinal epithelial cells depends on internalization Similar to other enteropathogenic bacteria, MAP is efficiently internalized by intestinal epithelial cells. The specific cellular localization of microbial recognition receptors at the plasma membrane, in endosomal vesicles, or within the cytosol indicates a potentially important role of bacterial internalization for cellular stimulation. To differentiate epithelial stimulation by extracellular bacterial stimuli and internalization-dependent cell activation, the use of PKH26-labelled MAP was combined with quantitative analysis of epithelial activation on the single-cell level by intracellular chemokine staining for MIP-2 or surface expression of the intercellular adhesion molecule (ICAM)-1. With this method, MAP-positive and negative cells were clearly discriminated and could be analysed separately (Fig. 4A). MIP-2 staining of control versus total MAP-infected cells revealed a significant increase of chemokine-producing or ICAM-1-expressing epithelial cells following MAP infection (Fig. 4B and C). Importantly, flow cytometric discrimination of MAP-positive and MAPnegative epithelial cells with simultaneous determination of the proportion of MIP-2 producing cells or the intensity of epithelial ICAM-1 expression revealed a markedly enhanced chemokine or adhesion molecule expression in the fraction of MAP-positive cells. In contrast, MAPnegative epithelial cells exhibited only marginal activation (Fig. 4B and C). To confirm internalization-dependent activation the internalization pathway involved in epithelial MAP uptake was analysed. Both inhibitors of the phosphoinositide 3-kinase (PI3K) and the small GTPase Rac but not of Rho significantly reduced bacterial internalization (Fig. 5A). Flow cytometry indicated reduced numbers of MAP-
positive cells from 55% in untreated controls to 25% in the presence of the PI3K inhibitor LY294002 and to 19% in the presence of wortmannin (Fig. 5B left panel). Consistently, inhibition of PI3K also significantly affected the secretion of MIP-2 (Fig. 5B right panel). Similarly, epithelial internalization of MAP depended on the small GTPase Rac as inhibition of Rac by the clostridial toxin B serotype F significantly reduced bacterial uptake from 54% in untreated controls to 47%, 30% and 19% in the presence of 0.05, 0.5 and 1.0 mg ml-1 toxin respectively (Fig. 5C left panel). Again, in the absence of MAP internalization by Rac inhibition, no epithelial activation was observed (Fig. 5C right panel). A direct effect of PI3K or Rac inhibition on epithelial activation and chemokine secretion was excluded by the finding that wortmannin, LY294002 or Toxin B serotype F had no effect on LPS- or TNFmediated epithelial stimulation (Fig. 5B and C). Of note, internalization-dependent activation was not observed in macrophages: inhibition of bacterial uptake by latrunculin B had no significant influence on MAP-induced activation of RAW264.7 macrophages (Fig. 5D). Thus, MAPmediated activation of intestinal epithelial cells is dependent on bacterial uptake, indicating that epithelial activation requires endosomal alteration of the bacterial surface for ligand release or accessibility to intracellularly localized innate immune receptors.
Toll-like receptor 9/MyD88 and NOD1/Rip2 contribute to epithelial innate immune recognition Studies on macrophages indicated an important role of toll-like receptor (TLR)2 and TLR4 for MAP recognition (Ferwerda et al., 2007). Proposed ligands are the mycobacterial cell membrane constituents lipomannan (LM)
Fig. 4. Restriction of cellular activation to MAP-positive intestinal epithelial cells. A. Flow cytometric analysis of intestinal epithelial m-ICcl2 cells infected with PKH26-labelled MAP for 6 h. B and C. Intraepithelial MIP-2 staining (B) and ICAM-1 surface expression (C) of MAP-infected intestinal epithelial m-ICcl2 cells. The numbers indicate the proportion of MIP-2-positive cells or the mean fluorescence intensity (MFI) of ICAM-1 expression in control cells, total infected cells or MAP-negative, and MAP-positive infected epithelial cells discriminated as indicated in (A). The data presented are representative for three independent experiments. © 2009 Blackwell Publishing Ltd, Cellular Microbiology, 11, 1802–1815
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Fig. 5. Characterization of MAP internalization and internalization-dependent activation. A. Effect of various inhibitors of internalization on MAP-positive intestinal epithelial cells as measured by flow cytometry. The numbers indicate the reduction of positive cells as compared with the proportion of MAP-positive cells obtained after infection of untreated m-ICcl2 cells. B. The influence of PI3-kinase inhibition on MAP internalization (left panel) and epithelial activation (right panel). Flow cytometric analysis of m-ICcl2 cells infected with PKH26-labelled MAP in the absence or presence of the PI3-kinase inhibitor LY294002 (50 mM) or wortmannin (0.5 mM). MIP-2 secretion by m-ICcl2 cells stimulated with MAP (moi 100:1) or LPS (10 ng ml-1) in the absence or presence of the PI3-kinase inhibitor LY294002 (10 mM) or wortmannin (0.2 mM). C. The influence of Rac inhibition on MAP internalization (left panel) and epithelial activation (right panel). Flow cytometric analysis of m-ICcl2 cells infected with PKH26-labelled MAP in the absence or presence of various concentrations of toxin B serotype F. MIP-2 secretion by m-ICcl2 cells stimulated with MAP (moi 100:1), TNF (50 ng ml-1), or LPS (10 ng ml-1) in the absence or presence of the indicated concentration of toxin B serotype F. D. The influence of uptake inhibition on MAP internalization (left panel) and activation (right panel) by macrophage cells. Flow cytometric analysis of RAW264.7 cells infected with PKH26-labelled MAP in the absence or presence of latrunculin B (5 mM). MIP-2 secretion by RAW264.7 infected with MAP in the absence or presence of latrunculin B (5 mM).
and lipoarabinomannan (LAM). Whereas exposure to lipoarabinomannan had no significant stimulatory effect, exposure of polarized intestinal epithelial cells to LM indeed induced secretion of the chemokine MIP-2 (Fig. 6A). However, high LM concentrations were required for epithelial stimulation and the amount of LM-induced MIP-2 secretion as compared with the epithelial response to infection with viable MAP was moderate. As expected, the LM-induced activation of intestinal epithelial cells was abolished following siRNA-induced downregulation of TLR2 or the adaptor molecule myeloid differentiation factor 88 (MyD88) and no contribution of TLR4 was detected (Fig. 6B). To analyse the contribution of TLR2 for
epithelial stimulation by whole viable bacteria and identify additional receptors involved in epithelial MAP recognition, a systematic screen of siRNA-mediated downregulation of additional innate immune receptors such as TLR2, 4, 5, 9, and NOD1 and NOD2 as well as the respective adaptor molecules MyD88 and receptorinteracting protein (Rip)2 was performed. This approach indicated that both TLR- and NOD-mediated signal transduction pathways are involved: siRNA-mediated downregulation of MyD88 and Rip2 expression caused a highly significant (P < 0.01) impairment of epithelial MAP recognition. Furthermore, TLR9 and NOD1 receptor molecules were identified to significantly (P < 0.05) contribute to © 2009 Blackwell Publishing Ltd, Cellular Microbiology, 11, 1802–1815
Intraepithelial innate immune recognition of MAP 1809
Fig. 6. Intracellularly localized innate immune receptors mediate intestinal epithelial MAP recognition. A. MIP-2 secretion by intestinal epithelial m-ICcl2 cells 6 h after exposure to various concentrations of the cell wall constituents LM or LAM. B. MIP-2 secretion by intestinal epithelial m-ICcl2 cells treated with control siRNA or siRNA directed against Myd88, Tlr2 and Tlr4 (10 nM) prior to exposure with LM (5 mg ml-1) for 6 h. C. MIP-2 secretion by intestinal epithelial m-ICcl2 cells treated with control siRNA or siRNA directed against the indicated innate immune receptors (10 nM) prior to infection with MAP (moi 100:1) for 6 h. D. RT-PCR amplification of mRNAs targeted by siRNA as control for siRNA treatment efficiency. E. Schematic illustration of the MAP-induced activation of intestinal epithelial cells.
epithelial stimulation in contrast to what has been reported on MAP-induced macrophage activation (Fig. 6C). Notably, both NOD1 and TLR9 are localized intracellularly consistent with an internalization-dependent recognition process. The efficacy of the siRNA-mediated inhibition of gene expression was confirmed by RT-PCR (Fig. 6D). Although the ligand for NOD1, meso-diaminopimelic acid is a characteristic motiv of the Gram-negative peptidoglycan structure, it is also found in some Gram-positive bacteria such as Bacillus or Mycobacteria (Uehara et al., 2006). No significant reduction of MAP-induced activation was noted using siRNA treatment to downregulate TLR2, TLR4, TLR5 or NOD2. © 2009 Blackwell Publishing Ltd, Cellular Microbiology, 11, 1802–1815
Discussion The primary route of MAP infection is through ingestion of milk or faecal material containing high numbers of viable bacteria. Once within the intestinal lumen, MAP has the ability to penetrate through the intestinal epithelial barrier and to enter the sterile subepithelial lamina propria to cause invasive infection. In accordance, recent in vivo studies suggest that luminal exposure leads to rapid bacterial internalization by the small intestinal epithelium and subsequent spread (Sangari et al., 2001; Sigurdardottir et al., 2005; Wu et al., 2007). Clearly, M cells overlaying enteric Peyer’s patches are able to internalize MAP after
1810 J. Pott et al. infection (Momotani et al., 1988; Secott et al., 2002). In addition, other epithelial cell types such as goblet cells and absorptive enterocytes have been proposed to be involved in bacterial uptake (Sigurdardottir et al., 2005; Golan et al., 2009). The importance of individual cell types for MAP-induced disease manifestation, however, has not been elucidated. Interestingly, mucosal internalization was associated with marked tissue inflammation illustrated by significant recruitment of professional inflammatory cells (Golan et al., 2009; Khare et al., 2009). So far, work on the host response to MAP has mainly focused on the involvement of professional immune cells such as macrophages and various subsets of lymphoid cells. These studies have successfully determined the underlying mechanisms causing macrophage activation and inhibition of phagosome maturation by MAP and characterized the development of a protective adaptive immune response (Kuehnel et al., 2001; Stabel, 2007; Allen et al., 2009). Yet also intestinal epithelial cells express receptors of the innate immune system and are able to recognize and respond to microbial exposure (Lotz et al., 2007). The present study therefore focuses on the process of small intestinal epithelial activation by MAP and the characterization of the cellular and molecular mechanisms involved as summarized in Fig. 6E. Internalization of MAP by epithelial cells has been observed in epithelial cell lines such as bovine kidney (MDBK) cells or human colon HT29 and Caco-2 or laryngeal Hep-2 cells (Secott et al., 2002; Miltner et al., 2005; Patel et al., 2006; Harriff et al., 2009). A systematic screen identified several mycobacterial genes involved in epithelial internalization (Miltner et al., 2005). Further analysis indicated bacteria-induced epithelial internalization through bacterial surface membrane molecules and active interference with cellular traffic (Bannantine et al., 2003; Harriff et al., 2009). Our results significantly extend these studies characterizing the precise internalization pathway and identifying the association of bacterial internalization with epithelial activation. Using the highly specific clostridial toxin B serotype F we clearly demonstrate that bacterial uptake by small intestinal epithelial cells depends on the small GTPase Rac potentially in combination with Cdc42 (Alonso-Hearn et al., 2008; Harriff et al., 2009). No significant importance of the small GTPase Rho for bacterial internalization was observed in contrast to the conclusion drawn by Sangari et al. (2000) studying M. avium ssp. avium internalization by human colon epithelial cells. MAP is efficiently internalized by intestinal epithelial cells by inducing bacterial uptake and survives intracellularly in a cultivable form. Of note, internalization by epithelial cells has also been described for other mycobacteria such as, e.g. M. tuberculosis and intraepithelial survival has been linked to pathogenicity (Bermudez et al., 2002; García-Pérez et al., 2008).
Bacterial recognition is mediated by innate immune receptors that initiate signal transduction and cellular activation upon exposure to specific microbial structures. Whereas members of the family of TLRs are transmembrane molecules and survey the extracellular space or endosomal compartments, NOD receptors are situated in the cytosol and sense the presence of cytosolic bacteria. NOD receptors and TLRs appear to be involved in MAP recognition by both epithelial and myeloid cells. Myeloid cells have been described to recognize MAP predominantly via TLR2 and NOD2 (Ferwerda et al., 2007). In contrast, suppression of MAP-induced epithelial activation was observed using siRNA treatment against TLR9 and NOD1. This finding is consistent with constitutive expression of NOD1 but low expression of NOD2 in naïve non-myeloid cells (Gutierrez et al., 2002). The ligands of TLR2 are phospho- and glycolipids such as the mycobacterial cell wall constituent LM (Vignal et al., 2003). LM is a well-known potent immunostimulus and significantly contributes to the inflammatory consequences of mycobacterial infection induced by professional immune cells such as macrophages (Vignal et al., 2003). Although high concentrations of isolated LM induced TLR2-mediated activation also of intestinal epithelial cells, TLR2 contributed only to a minor degree to the overall stimulation of epithelial cells by viable MAP. Instead, a significant effect of TLR9 siRNA treatment on MAP-induced epithelial stimulation was noted. TLR9 has not been associated with MAP recognition so far. Interestingly, TLR9 activation of macrophages by M. bovis antigen has recently been shown to enhance delta-like 4 Notch ligand expression, Th17 effector stimulation and granuloma formation (Ito et al., 2009). The identification of TLR9-mediated MAP recognition within the intestinal epithelium might therefore significantly contribute to a better understanding of the pathogenesis of MAP infection. Yet additional receptor molecules might contribute to total MAP-induced epithelial activation. Also, mycobacterial constituents have been shown to actively interfere with cellular signalling pathways (Doz et al., 2009). MAP is highly virulent for ruminants and has been detected in patients with the inflammatory bowel disease Crohn’s disease (Bull et al., 2003; Feller et al., 2007). Here we demonstrate a strikingly potent innate immune activation of intestinal epithelial cells by MAP in contrast to other mycobacteria of the M. avium group. Importantly, innate immune activation of epithelial cells is dependent on bacterial internalization similar to what has been observed in cells infected with the enteroinvasive human pathogen Listeria monocytogenes (Kayal et al., 1999). Internalization by epithelial cells was recently also described for obligate pathogenic mycobacterial species (Bermudez et al., 2002; García-Pérez et al., 2008). The identification of NOD1 and TLR9 suggest that both, cyto© 2009 Blackwell Publishing Ltd, Cellular Microbiology, 11, 1802–1815
Intraepithelial innate immune recognition of MAP 1811 solic as well as endosomal recognition, take place, but the molecular processes responsible for intraepithelial receptor activation remain to be elucidated. Interestingly, TLR9 expression has been described to be strongly upregulated in intestinal tissue of asymptomatic but not symptomatic MAP-infected animals, suggesting a protective role of TLR9 signalling for mucosal antibacterial host defence (Nalubamba et al., 2008). Also, TLR9 activation induces Th17 effector stimulation and granuloma formation, both observed in patients with Crohn’s disease (Kleinschek et al., 2009). In contrast to internalization-dependent recognition in epithelial cells, strong macrophage activation has been noted in the absence of intracellular MAP. Indeed, whereas the innate immune receptors involved in intestinal epithelial activation are restricted to the cytosol and endosomal compartments, TLRs localized to the plasma membrane of myeloid cells were shown to contribute to macrophage activation (Ferwerda et al., 2007). In addition, cell type-dependent differences in the MAP-induced cell signalling were noted. Whereas stimulation of macrophages with MAP led to significant p65 phosphorylation and IkB-a degradation, no detectable NF-kB activation was observed in intestinal epithelial cells despite a marked chemokine response. Interestingly, Erkdependent but NF-kB-independent activation was previously described in human epithelial cells in response to CpG (Akhtar et al., 2003). These results might therefore indicate cell type-specific differences in the downstream signalling pathways of certain innate immune receptors. In conclusion, we demonstrate the potent immunostimulatory activity of MAP for intestinal epithelial cells. Epithelial activation requires Erk-signalling leading to strong pro-inflammatory chemokine secretion. In contrast to what has been described for macrophages, epithelial innate immune recognition depends on bacterial internalization via the small GTPase Rac and is mediated via NOD1 and TLR9. Our results illustrate functional differences in the innate immune recognition of epithelial versus myeloid cells and corroborate the need to analyse different cell types in order to understand the complex cellular interplay involved in mucosal host defence and inflammation.
from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and the mouse anti-histone H3 antibody from BioLegend (San Diego, CA, USA). The NF-kB reporter construct pBIIX-luciferase carrying two copies (2¥ NF-kB) of the kB sequences from the Igk enhancer was provided by S. Ghosh (Yale University Medical School, New Haven, CT). Escherichia coli K12 D31m4 LPS was ordered from List Biological Laboratories (Campbell, CA, USA). The MAP strain 6783 (DSM 44135) was isolated from a cow with progressive paratuberculosis (Jark et al., 1997). This strain was used for all experiments if not stated otherwise. Also, the MAP K10 strain (ATCC BAA-968), the M. avium ssp. avium DSM44156 strain and the M. avium ssp. hominissuis strain 04A/ 1287, a field isolate from deer kindly provided by Irmgard Moser (Friedrich-Loeffler-Institut, Jena, Germany), were used in this study. M. bovis BCG was kindly provide by F. Bange (Hannover Medical School, Hannover, Germany). All strains were cultured in Watson Reid’s medium as described previously (Basler et al., 2008;), MAP strains were cultured in the presence of Mycobactine J (Synbiotics, Stuttgart, Germany).
Preparation of the LM from M. avium ssp. paratuberculosis LM preparations were performed according to the procedures of Chatterjee and coworkers (Chatterjee et al., 1992a,b; Khoo et al., 1996) with some modifications. Briefly, the MAP strain 6783 was grown to confluence on Watson Reid medium (supplemented with Mycobactin J) in 600 ml tissue culture flasks (6–12 weeks). Bacteria were collected by centrifugation and washed with PBS (pH 7.5). Approximately 30 g (wet weight) was suspended in PBS containing 4% Triton X-114, protease inhibitor cocktail, DNaseI and RNaseA (Sigma). Bacteria were disrupted by French press and lipids were extracted for 18 h at 4°C and over head shaking. The insoluble material was removed by centrifugation for 1 h at 27 000 g at 4°C and was subsequently extracted again with PBS containing 4% Triton X-114. The Triton X-114 extracts were combined, incubated at 37°C until biphasic partitioning and centrifuged at 27 000 g for 30 min at 25°C. The detergent layer was collected and macromolecules including LM were recovered by precipitation at -20°C with 95% ethanol (10 vols). The precipitates were suspended in endotoxin-free H2O and proteins were removed by phenolic treatment. Then the material was dialysed against H2O. The crude carbohydrate mixture was fractionated by size exclusion chromatography through Sephacryl S-200/S-100 columns (GE-healthcare, Freiburg, Germany). Elution profiles were monitored by SDS-PAGE and silver staining (Tsai and Frasch, 1982). The LM-containing fractions were pooled and buffer contaminants removed by extensive dialysis against endotoxin-free H2O. The solution was lyophilized and stored at -80°C.
Experimental procedures Reagents and bacterial strains
Cell culture and stimulation assays
The rabbit monoclonal anti-phospho SAPK/JNK (T138/Y185), phospho-p38 MAPK (T180/Y182), rabbit polyclonal antiphospho–NF-kB p65 (Ser536) antibody, rabbit IkB-a antibody and monoclonal phospho-p44/42 MAPK (Thr202/Tyr204) were obtained from Cell Signalling Technology (Danvers, MA, USA). The rabbit polyclonal anti-actin antiserum was from SigmaAldrich Chemie GmbH (Munich, Germany), the rabbit anti-p65
Murine small intestinal epithelial m-ICcl2 cells were cultured as described (Bens et al., 1996). m-ICcl2 cells were chosen due to the lack of a suitable human or bovine small intestinal epithelial cell line. They represent a well-established model of innate immune recognition by polarized and differentiated intestinal epithelial cells (Hornef et al., 2002; Lotz et al., 2006). RAW264.7 cells were obtained from the American Type Culture Collection
© 2009 Blackwell Publishing Ltd, Cellular Microbiology, 11, 1802–1815
1812 J. Pott et al. and propagated in RPMI 1640 medium (Invitrogen) supplemented with 20 mM Hepes, 2 mM l-glutamine and 10% FCS. Stimulation was performed by the addition of 50 ng ml-1 TNF, 10 ng ml-1 LPS or bacteria at the indicated concentration. For heat inactivation of MAP, the bacteria were incubated for 15 min at 85°C. To allow timely contact between bacteria and epithelial cells, cell culture plates for incubation times less than 4 h were centrifuged for 5 min at 300 g. The following inhibitors were used: JNK inhibitor 1 (Alexis, Lörrach, Germany), 3-O-Acetyl-bboswellic acid (Alexis), latrunculin B (Sigma), wortmannin (Alexis), LY294002 (Cell Signalling Technology), SN50 (Alexis), MEK inhibitor UO126 (Promega, Mannheim, Germany). m-ICcl2 cells were incubated for 30 min in the presence of the indicated inhibitors prior to stimulation or infection. Clostridial toxin B serotype F and exoenzyme C3 were generously provided by Ingo Just and Ralf Gerhard (Hannover Medical School, Hannover, Germany). Validated siRNAs directed against Myd88, Rip-2, Tlr2, Tlr4 and Tlr9 mRNA or control siRNA were purchased from Qiagen (Hilden, Germany). Cells were transfected as described by Chassin et al. (2007) with a final concentration of 10 nM 48 h prior to stimulation. Cell culture supernatants were harvested after 6 h and stored at -20°C until further analysis. The chemokine MIP-2 was quantified using a commercial ELISA from Nordic Biosite (Täby, Sweden). Variation of the chemokine levels observed between individual experiments resulted from: (i) slight differences in the seeding density together with the long incubation time required to reach confluency, polarization and cell differentiation of m-ICcl2 cells, (ii) stimulation of subconfluent cells after siRNA treatment to allow efficient translocation of the siRNA and (iii) some variation in the MAP preparations due to the very long culture time of approximately 2 months. To determine NF-kB activation, m-ICcl2 cells stably transfected with a NF-kB reporter construct (Hornef et al., 2003) were stimulated as described above. Luciferase activity was determined with luciferin substrate (PJK GmbH, Kleinblittersdorf, Germany) and quantified using a Victor3 luminometer (Perkin Elmer, Waltham, MA, USA). To determine bacteria internalization, confluent and polarized m-ICcl2 cells were infected with MAP at an moi of 10:1 for 30 min or 4 h, washed three times with PBS and incubated with amikacin-containing culture medium (200 mg ml-1) for 2 h at 37°C to kill extracellular bacteria. Supernatant was plated to confirm that no living bacteria remained. For 4 and 7 day persistence, amikacin treatment was performed after 4 h incubation and subsequently the cells were kept under culture conditions with medium change every second day. The cells were lysed with Triton-X (0.1% in water) and serial dilutions were plated to determine colony count, following the amikacin treatment or after indicated culture time.
Flow cytometry and immunoblotting Bacteria were fluorescently labelled using PKH26 from Sigma. 6 ¥ 108 cfu MAP were incubated in 5% Glucose solution with 0.4% (v/v) PKH26 for 3 min. Excess dye was blocked by the addition of 1% FCS and labelled bacteria were thoroughly washed before infection. m-ICcl2 and RAW264.7 cells were infected at an moi of 100:1 and 10:1 respectively. Analysis of extracellular ICAM-1 was carried out by staining fixed cells (CytoFix, BD) with a phycoerythrin-conjugated rat anti-ICAM-1 antibody (Abcam). Intracellular staining for MIP-2 was performed after a 6 h incubation in the presence of 0.5 mg ml-1 brefeldin A
(Sigma) followed by fixation with CytoFix (BD) and permeabilization with 0.5% saponin. Bound MIP-2 antibodies (Nordic Biosite) were detected using a goat anti-rabbit Cy5-conjugated antibody (Jackson ImmunoResearch Laboratories) and cells were analysed with a BD FACSCalibur. Nuclear extracts were prepared as described (Schreiber et al., 1989). For immunoblotting, m-ICcl2 cells were resuspended in lysis buffer (3:1 WB/SB v/v; SB: 250 mM Tris, pH 6.5, 8% SDS, 40% glycerol; WB: 50 mM Tris, pH 7.4, 120 mM NaCl) supplemented with a proteinase inhibitor cocktail (Complete Mini; Roche Diagnostics). Samples were sonified and the protein concentration was determined (DC Protein Assay; Bio-Rad Laboratories). For Western blot analysis 5–20 mg of total cell lysate was used. Protein was eluted in loading buffer and separated on a 10% acrylamide gel and blotted on nitrocellulose. Membranes were incubated overnight at 4°C with the primary antibody at the recommended concentration. Detection was performed using peroxidase-labelled goat anti-mouse or goat anti-rabbit secondary antibodies (Jackson ImmunoResearch Laboratories) in combination with the ECL kit (GE Healthcare).
Immunostaining and electron microscopy Bacteria were covalently labelled with biotin by incubation of 2 mg Ez-Link Sulfo-NHS-LC-Biotin (Pierce) with 6 ¥ 108 cfu MAP for 2 h at 4°C followed by repeated washing. Epithelial cells were cultured for 6 days on eight-well chamber slides before infection. After fixation in 3.5% paraformaldehyde and permeabilization with 0.5% saponin, MAP was visualized with Streptavidin-Cy3 (Jackson ImmunoResearch Laboratories). To visualize nuclear translocation of the NF-kB subunit p65, cells were permeabilized in methanol at -20°C. Counterstaining was performed with MFP488 phalloidin (MoBiTec, Göttingen, Germany). Immunostaining for LAMP-2 was performed using a rat antibody purchased from Santa Cruz Biotechnology (Santa Cruz, CA) in combination with a FITC-conjugated donkey anti-rat antibody (Jackson ImmunoResearch Laboratories). Vectashield-mounted slides (Vector Laboratories) were visualized using a Leica Inverted-2 confocal microscope. Images were analysed with LCS Lite Leica Confocal Software (Leica Microsystems, Heidelberg GmbH). For scanning electron microscopy samples were fixed with 5% formaldehyde and 2% glutaraldehyde at 4°C, washed with TE-buffer (20 mM TRIS, 2 mM EDTA, pH 6.9), dehydrated with a graded series of acetone (10%, 30%, 50%, 70%, 90% on ice; 100% at room temperature), followed by critical-point drying with liquid CO2 (Bal-Tec CPD030) and sputter-coated with gold (Bal-Tec SCD500). Samples were then examined in a Zeiss field emission scanning electron microscope DSM 982 Gemini at an acceleration voltage of 5 kV applying the Everhart-Thornley SE-detector and the inlens SE-detector at a 50:50 ratio. Images were stored on MO disks and contrast and brightness were adjusted with Adobe Photoshop 8.0. For ultrastructural analysis of intracellular residing MAP samples were treated as previously described (Goldmann et al., 2004).
RNA extraction, reverse transcription and PCR Total RNA was isolated from siRNA-treated m-ICcl2 cells with Trizol (Invitrogen) according to manufacturer’s instruction and 2 mg of total RNA was reversely transcribed using MVL-reverse © 2009 Blackwell Publishing Ltd, Cellular Microbiology, 11, 1802–1815
Intraepithelial innate immune recognition of MAP 1813 transcriptase (Invitrogen). cDNA was analysed by PCR using primer pairs for the following genes: Tlr2 forward 5′-CTC CCACTTCAGGCTCTTTG-3′ and reverse 5′-TTATCTTGCGC AGTTTGCAG-3′; Myd88 forward 5′-AGAGCTGCT GGCCTTGT TAG-3′ and reverse 5′-CCACCTGTAAAGGCTTCTCG-3′; Tlr4 forward 5′-ATCTGAGCTTCAACCCCTTG-3′ and reverse 5′-AGA GGTGGTGTAAGCCATGC-3′; Tlr9 forward 5′-CATGGACGGGA ACTGCTACT-3′ and reverse 5′-GGCACCTTT-GTGAGGTTGTT3′; Rip2 forward 5′-CTGCACCCGAAGGAGGAACAATCA-3′ and reverse 5′-GCGCCCATCCACTCTGTAT-TAGA-3′; Nod1 forward 5′-CCTGGACAAC-AACAACCTCA-3′ and reverse 5′-CCACATA CCTGGCTCCGATA-3′; Nod2 forward 5′-TTGAGTGTGCTCT TCGCTGT-3′ and reverse 5′-CGGCTGTGATGTGATTGTTC-3′; Hprt forward 5′-TGCTGACCTGCTGGATTACA-3′ and reverse 5′-GCTTAACCAGGG-AAAGCAAA-3′. The annealing temperature was 56°C and 59°C for Rip-2 and Myd88, as well as Tlr2, Tlr4, Tlr9, Nod1, Nod2 and Hprt respectively. Thirty PCR cycles were run for Myd88, Rip2, Tlr4, Nod1 and Hprt and 35 cycles for Tlr2, Tlr9 and Nod2. Products were analysed in SybrSafe (Invitrogen) stained 2% agarose gels.
Statistics All experiments were performed at least three times and results are expressed as mean ⫾ SD of one representative experiment. Significant differences were analysed using the unpaired Student’s t-test. P < 0.05 was considered significant.
Acknowledgements J.P., T.B., R.G. and M.W.H. were supported by the Germany Ministry for Science and Education (BMBF, 01 KI 0752). M.W.H. and R.G. were additionally supported by individual grants from the German Research Foundation (DFG, Ho2236/5–2 and Go983/1 respectively). C.U.D. was supported by the DFGfounded European Research Training Group 1273. The clostridial toxins were generously provided by Ingo Just and Ralf Gerhard (Hannover Medical School, Hannover, Germany) and the LM preparation was provided by Jens Abel (University of Veterinary Medicine, Hannover, Germany). We thank Julian Sander for help with the animals and Karen Stevenson (Moredun Research Institute, Penicuik, UK) and Tim Bull (Division of Cellular and Molecular Medicine, St. George’s University, London, UK) for discussions.
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