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Giardia duodenalis cathepsin B proteases degrade intestinal epithelial interleukin-8 and attenuate

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IAI Accepts, published online ahead of print on 14 April 2014 Infect. Immun. doi: /iai Copyright 2014, American Society for Microbiology. All Rights Reserved. 1 2 Giardia duodenalis cathepsin
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IAI Accepts, published online ahead of print on 14 April 2014 Infect. Immun. doi: /iai Copyright 2014, American Society for Microbiology. All Rights Reserved. 1 2 Giardia duodenalis cathepsin B proteases degrade intestinal epithelial interleukin-8 and attenuate interleukin-8-induced neutrophil chemotaxis James A. Cotton a,b,c, Amol Bhargava a,b,c,, Jose G. Ferraz b,d Robin M. Yates e,f Paul L. Beck b,d, Andre. G Buret a,b,c # Department of Biological Sciences a, Inflammation Research Network b, Host-Parasite Interactions c, Department of Medicine d, Department of Comparative Biology and Experimental Medicine e, Department of Biochemistry and Molecular Biology f, University of Calgary, Calgary, AB, Canada Running Head: Giardia duodenalis cathepsin B proteases degrade CXCL #Address correspondence to Andre G. Buret, 18 Abstract Giardia duodenalis (syn. G. intestinalis, G. lamblia) infections are a leading cause of waterborne diarrheal disease that can also result in the development of post-infectious functional gastrointestinal disorders via mechanisms that remain unclear. Parasite numbers exceed 10 6 trophozoites per centimeter of gut at the height of an infection. Yet, the intestinal mucosa of G. duodenalis-infected individuals is devoid of signs of overt inflammation. G. dudoenalis infections can also occur concurrently with other pro-inflammatory gastrointestinal pathogens. Little is known of whether and how this parasite can attenuate host inflammatory responses induced by other pro-inflammatory stimuli, such as a gastrointestinal pathogen. Identifying hitherto unrecognized parasitic immune-modulatory pathways, the present studies demonstrate that G. duodenalis trophozoites attenuate secretion of the potent neutrophil chemoattractant interleukin-8 (CXCL8); these effects were observed in human small intestinal mucosal biopsy tissues, and from intestinal epithelial monolayers, activated through administration of proinflammatory interleukin-1β or Salmonella typhimurium. This attenuation is caused by the secretion of G. duodenalis cathepsin B cysteine proteases that degrade CXCL8 posttranscriptionally. Furthermore, the degradation of CXCL8 via G. duodenalis cathepsin B cysteine proteases attenuates CXCL8-induced chemotaxis of human neutrophils. Taken together, these data demonstrate for the first time that G. duodenalis trophozoite cathepsins are capable of attenuating a component of their host s pro-inflammatory response induced by a separate proinflammatory stimulus. 2 38 Introduction Giardia duodenalis (syn. G. intestinalis, G. lamblia) is a non-invasive protozoan parasite of the upper small intestine of many animals, including humans, which is now included on WHO s Neglected Disease Initiative (1). Giardiasis is a leading cause of waterborne diarrheal disease worldwide, and the infection is known to result in the development of post-infectious functional gastrointestinal disorders, as well as in extraintestinal complications (2, 3). G. duodenalis is currently subdivided into eight distinct genetic assemblages designated A through H with assemblages A and B isolates being infective to humans (4, 5). There is an ongoing discussion as to whether at least some G. duodenalis assemblages may represent distinct Giardia species (6, 7). At the height of Giardia infections, millions of trophozoites closely associate with the apical surface of the intestinal epithelium and induce pathophysiological responses that can culminate in malabsorptive diarrheal disease (reviewed in (8)). With the exception of the small but significant increase in numbers of intra-epithelial lymphocytes (9, 10), acute infection with G. duodenalis is not associated with the infiltration of inflammatory cells, for reasons that remain obscure. These observations represent a counter-intuitive observation not only in view of the direct presence of large numbers of parasites, but also because G. duodenalis breaks the epithelial barrier via direct effects on tight junctional proteins (11-14), and therefore likely facilitates the translocation of potent pro-inflammatory luminal antigens. In addition, G. duodenalis infections can occur concurrently with other pro-inflammatory gastrointestinal pathogens, such as Cryptosporidium parvum (15) Helicobacter pylori (16), rotavirus (17, 18), and Salmonella (19) To date, little research has focused on G. duodenalis ability to modulate host intestinal pro-inflammatory responses and the recruitment of pro-inflammatory immune cells induced by 3 other, co-infecting pro-inflammatory gastrointestinal pathogens. Previous studies have demonstrated that mast-cell hyperplasia occurs in the late stages of a Giardia infection or following parasite clearance (20, 21) and eosinophil accumulation may occur in vivo in a isolatedependent manner (22). Furthermore, G. duodenalis parasite products have been shown to modulate dendritic cell responses to lipopolysaccharide (23, 24), while separate studies have demonstrated IECs exposed to Giardia trophozoites produce a unique chemokine profile (25). The need to investigate G. duodenalis ability to modulate its host s pro-inflammatory responses is now apparent due to data collected from in vivo animal and human studies. Microarray analysis of jejunal tissues collected from assemblage E Giardia-infected calves revealed decreased mrna expression of several pro-inflammatory mediators and increased expression of anti-inflammatory transcription factors (26). Several human studies have suggested Giardia infections in children may reduce the incidence or severity of diarrheal disease (17, 27, 28). One study demonstrated that Tanzanian children infected with Giardia had a reduced likelihood developing fever and lower levels of serum C-reactive protein, a classic marker of inflammation, when compared to their non-infected counterparts (27). A separate study suggested children coinfected with rotavirus and Giardia displayed a marked reduction in the severity of diarrheal disease when compared to children only infected with rotavirus (17). However, findings from the latter study directly conflict with other authors who were unable to find a reduction in the severity of diarrheal disease when children co-infected with rotavirus and Giardia (18). These contrasting studies may suggest that Giardia-mediated protection from diarrheal disease may be isolate-specific, reliant on host genetics, or other yet-to-be determined factors As G. duodenalis trophozoites are non-invasive, the intestinal epithelium represents the primary point of contact between parasite and host; this structure is comprised of a single, 4 polarized layer of intestinal epithelial cells that function to separate the external environment of the intestinal lumen from underlying host tissues (reviewed in (29)). The intestinal epithelium is also involved in the induction of acute inflammatory responses within the intestinal mucosa, with an important function being the secretion of pro-inflammatory chemokines (reviewed in (30, 31)). In response to a variety of pro-inflammatory stimuli, including direct exposure to translocated bacterial antigens, intestinal epithelial cells secrete different classes of chemokines including the potent neutrophil/polymorphonuclear leukocyte (PMN) chemoattractant interleukin-8 (CXCL8) (32-34); this intermediary chemokine recruits extravasated PMNs to the basolateral membrane of the intestinal epithelium so that subsequent signals can, if necessary, drive their migration across to the apical surface of the intestinal epithelium (35-37). During an acute intestinal inflammatory response, PMNs are typically the first leukocyte to exit the vasculature and be recruited to the inflammatory site (reviewed in (38)) Cathepsin cysteine proteases are defined by a catalytic dyad comprised of active site cysteine (Cys) and histidine (His) residues and classified as clan CA cysteine proteases; these are further subdivided into superfamilies including cathepsin L (catl)-like or cathepsin B (catb)-like proteases (reviewed in (39)). CatB proteases contain an additional 20 amino acid insertion referred to as the occluding loop containing two characteristic His residues that enables their function as an endo- or exopeptidase (40). The G. duodenalis genome contains genes for numerous cathepsin cysteine proteases, the majority of which have no described function (41, 42). However, several G. duodenalis cathepsin proteases are upregulated upon exposure to intestinal epithelial cells (IECs) (43). While some parasites may use cathepsin cysteine proteases to evade or modulate their host s immune responses (reviewed in (44, 45)), previous research has demonstrated that Entamoeba histolytica cysteine protease 2 (EhCP2) cleaves CXCL8 into a 5 more potent isoform that enhances PMN chemotaxis (46). We hypothesized that Giardia cathepsin cysteine proteases may be involved in modulating acute pro-inflammatory responses within the intestinal epithelium. Specifically, we hypothesized that G. duodenalis cathepsin proteases are capable of attenuating IEC-induced CXCL8 secretion and the resulting PMN chemotaxis, which explains at least in part the lack of overt inflammation in the intestine during infection. Our study is the first to describe a role for G. duodenalis catb proteases in degrading intestinal epithelial pro-inflammatory secretion of CXCL8 following exposure to host- or pathogen-derived pro-inflammatory stimuli. Furthermore, degradation of CXCL8 by G. duodenalis catb proteases resulted in attenuation of CXCL8-induced PMN chemotaxis 117 Materials and Methods 118 Ethics statement All studies involving human small intestinal mucosal biopsy tissues were approved by the Conjoint Health Research Ethics Board (CHREB) at the University of Calgary and the Calgary Health Region. In accordance with CHREB guidelines, adult subjects used in this study provided informed, written consent and a parent or guardian of any child participant provided informed, written consent on their behalf. 124 Reagents Recombinant human interleukin-1β (IL-1β), and CXCL8, and all corresponding ELISAs were purchased from R&D Systems. The broad-spectrum, clan CA membrane permeable cysteine protease inhibitor (2S,3S)-trans-Epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester (E-64d) (47) was purchased from Sigma-Aldrich. The membrane permeable, catb-specific inhibitor L-3-trans-(Propylcarbamoyl)Oxirane-2-Carbonyl)-L-Isoleucyl-L-Proline Methyl Ester (Ca-074Me) (40), the catb/l fluorgenic substrate benzyloxycarbonyl-l-phenylalanyl-l-arginine 4-Methyl-Coumaryl-7-Amide (ZFR-AMC), and the catb fluorogenic substrate benzyloxycarbonyl-l-arginine-l-arginine 4-Methyl-Coumaryl-7-Amide (ZRR-AMC) were purchased from Peptides International (48, 49). QIAZol, RNEasy RNA extraction kits, QuantiTect Reverse Transcription Kit, QuantiFast SYBR Green PCR kits, and human CXCL8 (Accession no.: NM_000584) (Qiagen catalog # PPH00568A) and β-2 microglobulin (β2m) (Accession no.: NM_004048) (Qiagen catalog # PPH01094E) qpcr primers were purchased from Qiagen. The validity of these primers has been validated previously (50, 51). 138 Human biopsy tissues and cell lines 7 Adapting a previous protocol (52), small intestinal mucosal biopsy tissues were obtained from the terminal ileum of patients with Crohn s disease (CD) in remission or, in separate experiments, areas of active inflammation. Samples were washed three times in Dulbecco s PBS (Sigma-Aldrich) containing 0.016% 1,4-Dithioerythritol (Sigma-Aldrich) to remove loosely adherent mucous and bacteria followed by one wash in PBS. Washed biopsy tissues were placed into 96 well plates and incubated in 300 μl of OptiMEM (Life Technologies) at 37 0 C, 5% CO 2, and 96% humidity. Biopsy tissue weights on average were approximately 30 mg. The human adenocarcinoma Caco-2 cell line (ATCC HTB-37) was grown in ATCC complete growth medium comprised of Minimum Essential Medium Eagle (MEME) (Sigma-Aldrich M5650) supplemented with 100 g/ml streptomycin, 100 U/mL penicillin, 200 mm L-glutamine, 5mM sodium pyruvate, and 20% heat-inactivated FBS (VWR). Cells were passaged at 80% confluence with 2x Trypsin-EDTA and seeded onto 6-well plates, 12-well plates with 0.4 or 3.0 µm transwells (Corning) or small petri dishes pre-treated with poly-l-ornithine (Sigma-Aldrich). Cells were maintained at 37 0 C, 5% CO 2, and 96% humidity and media replaced every 2 to 3 days. Cells were used between passages 22 and 34. Transepithelial resistance Caco-2 cells were grown to confluence ( 500 Ω) on 3.0 µm transwell filter units and treated according to experimental design. Transepithelial electrical resistance was recorded with an electrovoltometer (World Precision Instruments). Parasites All Giardia duodenalis trophozoite isolates used in this study were previously obtained and isolated. Giardia duodenalis NF trophozoites were obtained from a water sample during an outbreak of giardiasis in Newfoundland, Canada (53), WB trophozoites (ATCC 30957) were 8 obtained from a symptomatic patient with chronic giardiasis (54), and GS/M clone H7 trophozoites (ATCC 50581) were isolated during a previous study (55). Trophozoites were grown axenically at 37 0 C in Keister s modified TYI-S-33 medium (56, 57) supplemented with piperacillin (Sigma-Aldrich) in 15 ml polystryene tubes (Becton-Dickinson Falcon) and used at peak density culture. For sonication, trophozoites were re-suspended in 1mL PBS, and sonicated three times on ice with three bursts of 30 seconds each (550 Versonic Dismembranator, Fisher Scientific). 169 Salmonella typhimurium Salmonella typhimurium (ATCC 14028) was a gift from Dr. Kenneth Sanderson, University of Calgary. A non-agitated microaerophilic culture of log-phase S. typhimurium was generated by inoculating 10 μl of an overnight stationary phase culture into 10 ml of Luria Broth and incubated at 37 0 C until log-phase was attained. The number of colony forming units (CFUs) per ml was determined by measuring the optical density at 600 nm (OD 600 ). Cultures were subsequently centrifuged at 1000x g and re-suspended in a volume of Caco-2 growth media without antibiotics. As previously described (58), Caco-2 cells were infected at a multiplicity of infection (MOI) of 100:1 for 5 or 7 hours to induce CXCL8 secretion. Therefore, 100 CFUs of S. typhimurium were added per Caco-2 cell. To determine the number of S. typhimurium associated with Caco-2 monolayers following 5-hour incubation, Caco-2 supernatants were decanted and monolayers were lysed in sterile RIPA buffer, serial diutes, and spot-plated onto LB agar plates. 181 Giardia duodenalis trophozoite infection Confluent tubes of G. duodenalis trophozoites were harvested by cold shock on ice for 30 minutes and subsequently pooled into 50 ml polypropylene tubes (Falcon) and centrifuged at 9 x g for 10 minutes. Resulting pellets were collectively re-suspended in 10 ml of ice cold PBS (Sigma-Aldrich) and centrifuged at 500x g for 10 minutes. The pellet was re-suspended in 3 ml of fresh PBS and trophozoites were enumerated with a hemocytometer and adjusted to the appropriate concentration. For ex vivo human biopsy experiments, G. duodenalis trophozoites were adjusted to a concentration of 5.0x10 6 trophozoites/well, while trophozoites were coincubated with in vitro Caco-2 monolayers at a MOI of 1:1, 10:1, or 50:1. For all experiments involving co-incubation of ex vivo human small intestinal biopsy tissues or Caco-2 monolayers in vitro with G. duodenalis trophozoites, cells were maintained at 37 0 C, 5% CO 2, and 96% humidity for the experimental duration. 193 Giardia modulation of intestinal epithelial CXCL8 secretion Following a previously established protocol (52), ex vivo human small intestinal mucosal biopsy tissues were co-incubated with G. duodenalis NF trophozoites in OptiMEM (Life Technologies) for 2 hours and subsequently administered 1.0 ng/ml of pro-inflammatory IL-1β or vehicle (0.05% bovine serum albumin in PBS) control for 4 hours. In separate experiments, ex vivo inflamed human small intestinal biopsy tissues collected from the terminal ileum of patients with active Crohn s disease were co-incubated with G. duodenalis NF trophozoites in OptiMEM for 6 hours. Similarly, G. duodenalis trophozoites (NF, WB, or GS/M) were co-incubated in the presence or absence of Caco-2 monolayers in ATCC Caco-2 complete growth media for 2 hours and subsequently administered IL-1β (1.0 ng/ml) or CXCL8 (1.0 ng/ml) for 4 hours or S. typhimurium (MOI 100:1) for 5 or 7 hours. Positive control groups were administered IL-1β (1.0 ng/ml), CXCL8 (1.0 ng/ml), or S. typhimurium (MOI 100:1) for appropriate incubation times. In all experiments, supernatants were collected and centrifuged at 500x g for 10 minutes at 4 0 C. Resulting supernatants were decanted and stored at C. Biopsy tissues were homogenized in 1 10 ml of RIPA buffer (1% Igepal, 0.1% SDS, and 0.5% sodium deoxycholate in PBS) containing a protease inhibitor tablet (Roche ), centrifuged at 10,000x g, aliquoted, and stored at C. CXCL8 and IL-1β levels in supernatants and/or biopsy tissues were determined using a commercially available human CXCL8 and IL-1β ELISAs (R&D Systems) with samples assayed in triplicate. 212 CXCL8 mrna expression Caco-2 monolayers were co-incubated with G. duodenalis NF or GS/M trophozoites at an MOI of 10:1 for 2 hours in Caco-2 growth media and subsequently administered 1.0 ng/ml IL- 1β. After 1 and 4 hours post IL-1β administration, cell monolayers were washed with PBS, lysed in 1 ml of QIAZol, and stored at C in RNAse free tubes. mrna was isolated using a modified RNEasy protocol from Qiagen. Briefly, 0.2 ml of chloroform was added to tubes and samples were shaken for 15 seconds. After 3-minute incubation at room temperature, samples were centrifuged at 12,000x g for 15 minutes at 4 0 C. The top mrna-containing layer was mixed with an equal volume of 70% ethanol and applied to RNEasy spin columns; at this point, the Qiagen RNEasy mrna isolation protocol was followed. Samples were assessed with a NanoDrop to determine mrna concentration and ensure that 260/280 ratios were greater than 1.8. Following this, 1 μg of mrna from each sample was reverse-transcribed into cdna via a QuantiTect Reverse Transcription Kit and a PCR thermal cycler (BioRad). qpcr was run on cdna samples using QuantiFast SYBR Green PCR Kit using a RotorGeneQ qpcr machine (Qiagen). A positive control sample was used to generate a relative standard curve that consisted of six 1 in 10 dilutions. All samples were diluted 1 in 10 to fit within the standard curve. Levels 11 of CXCL8 were normalized against loading control β2m. All primers were pre-designed and purchased from Qiagen. 230 Cathepsin cysteine protease inhibition Previous reports have suggested that 1μM E-64 is the maximal inhibitory dose that does not affect G. duodenalis trophozoite viability (59). The minimal inhibitory concentration of Ca- 074Me was determined by performing experiments with increasing concentrations of Ca-074Me. Therefore, Caco-2 supernatants in ATCC growth medium were pre-treated with 1 μm E-64d, increasing concentrations of Ca-074Me (1, 10, or 50 μm), or vehicle control (dimethyl sulfoxide; DMSO) for ~10-15 minutes. After this incubation period, G. duodenalis trophozoites were added to supernatants, incubated for 2 hours, and IL-1β was subsequently added for 4 hours. Cell supernatants were collected and assayed for cathepsin cysteine protease activity (see below). In separate experiments, confluent tubes of G. duodenalis trophozoites were pre-treated with 1 μm E-64d, increasing concentrations of Ca-074Me (1, 10, or 50 μm), or vehicle control (DMSO) for 30 minutes. Following this, trophozoites were harvested via cold shock on ice for 30 minutes. G. duodenalis trophozoites were then co-incubated with Caco-2 monolayers for 2 hours, and administered recombinant CXCL8 for 4 hours. In addi
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