PRISM :: Browsing by Author "McCafferty, Donna-Marie"

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Browsing by Author "McCafferty, Donna-Marie"

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Open Access
Assessment of the Rat Tapeworm Hymenolepis diminuta and its Antigens in the Treatment of DNBS Colitis
(2015) Campbell, Alec; McKay, Derek; Rioux, Kevin; Buret, Andre; McCafferty, Donna-Marie; Chadee, Khrisandreth
The concept of helminth therapy for autoinflammatory disorders is well-established. We utilized the H. diminuta-mouse model system to explore novel therapeutic options. We hypothesized that an optimal treatment regimen for H. diminuta existed which would be ideal for further study. Mice were infected with H. diminuta, or injected with one of two H. diminuta protein extracts, and the impact of this on dinitrobenzene sulphonic acid (DNBS)-induced colitis assessed. We found infection with a single H. diminuta significantly protected mice from DNBS. A single intraperitoneal injection of H. diminuta soluble products was also protective. Selective degradation of H. diminuta soluble products revealed a glycosylated, heat-stable immunomodulator. Finally, treatment with H. diminuta excretory-secretory products was effective at blocking DNBS. We conclude that infection with H. diminuta could be developed therapeutically for IBD and that further studies investigating the mechanism of H. diminuta’s immunomodulation should focus on the study of its excretory-secretory products.
Open Access
Characterization of Bacterial Depletion and Colitis-Associated Changes in the Gut Microbiota on Behaviour and Intestinal Physiology
(2021-05-10) Vicentini, Fernando Augusto; Sharkey, Keith Alexander; Hirota, Simon Andrew; Pittman, Quentin Jerome; McCafferty, Donna-Marie; DeVinney, Rebekah; Gareau, Melanie
The role of the microbiota in modulating host physiology is now widely accepted. Acting via the microbiota-gut-brain (MGB) axis, intestinal bacteria have been associated with the regulation of gastrointestinal tract and the central nervous system function, in both physiological and pathophysiological conditions, such as in inflammatory bowel disease (IBD). Patients with IBD have an increased incidence of anxiety and depression, which is accompanied by alterations in the microbiota composition. However, the link between changes in microbiota composition and behavioural abnormalities in the context of intestinal inflammation is unknown. Mechanisms that mediate the MGB axis in homeostasis have emerged but are still incomplete. We sought to characterize the effects of bacterial depletion on intestinal physiology and animal behaviour, and the role of colitis-associated microbiota in the modulation of behavioural abnormalities. Using mouse models of bacterial depletion (antibiotic treatment) and colitis (administration of dextran sulfate sodium), we investigated the role of the microbiota in intestinal physiology and behavioural outputs, and the potential mechanisms of these interactions. The major findings were: (1) bacterial depletion induced a reduction in despair behaviour in a sex-specific manner. The altered behaviour observed in male mice was normalized by activation of the Aryl hydrocarbon receptor, suggesting that this receptor is involved in modulation of behaviour via host-microbe interactions. (2) Bacterial depletion was linked to sex-independent changes in intestinal physiology, including reduced motility, altered secretion, and increased permeability. These changes were accompanied by a reduction in the numbers of enteric neurons, suggesting that microbiota influences the enteric nervous system integrity. Administration of bacterial-derived molecules, lipopolysaccharide and short-chain fatty acids, was associated with neuronal survival and neurogenesis, respectively. (3) Colitis induced changes in the intestinal bacteria composition, which was associated with behavioural abnormalities. Microbiota transfer of colitis-associated microbiota into recipient healthy mice was sufficient to induce behavioural alterations. Remarkably, no intestinal or neuroinflammation were observed in recipient mice. Collectively, these findings provide support to expand our understanding of the MGB axis under homeostatic and intestinal inflammatory conditions. Thus, a better understanding of this integrated network provides a rationale for the development of microbiota-centred therapeutic approaches for the improvement of health.
Open Access
Dynamic Alterations of the Mesenteric Lymphatic System in Murine Ileitis: Impications for Crohn’s Disease Pathogenesis
(2017) Rehal, Sonia; von der Weid, Pierre-Yves; MacNaughton, Wallace; Eksteen, Bertus; McCafferty, Donna-Marie
Inflammatory bowel disease (IBD) remains a challenging disease to treat due to its complex pathophysiology. It encompasses Crohn’s Disease and ulcerative colitis, both characterized by chronic inflammation. Exaggerated inflammatory responses in IBD are thought to be partly mediated by over-reactive immune cells. In particular, dendritic cells (DCs) have been shown to be over-recruited and hyper-responsive in clinical and experimental IBD tissue. DCs require trafficking through mucosal and mesenteric lymphatic vessels to drain into mesenteric lymph nodes (MLNs). Remodeling of these vessels during inflammation could have dire effects on DC effector function and immune responses in the inflamed intestine. Lymphatic vessel plasticity is also an important concept during acute intestinal injury for proper resolution of inflammation. Human and animal studies demonstrate pathological remodeling of mucosal lymphatic changes during IBD, including lymphangiogenesis and lymphatic vessel dilation. However, no studies have addressed the role of mesenteric lymphatic remodeling in replicable models of acute or chronic ileitis. These objectives were investigated using both the acute, dextran sodium sulphate (DSS) mouse model of ileitis and the chronic, transgenic TNFΔARE mouse model of ileitis. Plasticity of this remodeling was further assessed in recovery DSS mice. Our research findings demonstrate that chronic CD-like ileitis in TNFΔARE mouse model displays many lymphatic abnormalities. These include intestinal lymphangiectasia and MLN lymphadenopathy, reminiscent of human CD. TNFΔARE vessels have increased lipid transport, lymphangiogenesis and critically the development of mesenteric tertiary lymphoid organs (TLOs), actively involved in immune modulation; all of which we believe cause altered DC trafficking in these mice. Many of these changes were replicated in the acute DSS ileitis mouse model, albeit at a lesser extent. However, decreased lymph flow was observed during acute injury. Despite having recovered from DSS-induced intestinal inflammation, mice continued to display marked mesenteric lymphatic dysfunctions, such as lymphadenopathy and lymphangiogenesis. Lamina propria (LP), lymphatic vessels and mesenteric lymph nodes (MLNs) also continued to exhibit enhanced DC populations. This study contributes intriguing findings on remodeling of the lymphatic system within the context of small intestinal inflammation, commonly found in CD patients. Modulating lymphatic function during inflammation might offer new therapeutic avenues for IBD.
Open Access
Enhancing Oncolytic Rhabdovirus Infection With Sunitinib In An IFN Responsive Tumour Model
(2016) Dastidar, Himika; Mahoney, Douglas; Morris, Don; McCafferty, Donna-Marie
Oncolytic virus (OV) therapy for cancer is an emerging biotherapeutic strategy that employs replicating viruses to selectively infect and kill cancer cells. While promising, host innate immunity, namely type I IFN signaling, remains a barrier to OV therapy as it eliminates the virus before it spreads efficiently through the tumour. Sunitinib (Su), a receptor tyrosine kinase inhibitor, was recently shown to enhance OV infection by inhibiting IFN signaling in tumour cells. We therefore hypothesized that Su might enhance oncolytic rhabdovirus (ORV) infection in tumours. Indeed, Su treatment improved ORV productivity, tumour regression and overall survival in an IFN responsive tumour model. Su reduced the number and function of IFN producing myeloid cells such as cDCs and MΦ and thereby improved tumour infection with ORV. Collectively, our findings provide further support for the clinical evaluation of Su/ORV co-therapy in OV refractory tumors.
Open Access
Entamoeba histolytica-induced Activation of Caspase-4 Regulates Gasdermin D Cleavage to Mediate IL-1β Secretion in Macrophages
(2022-04) Wang, Shanshan; Chadee, Kris; McCafferty, Donna-Marie; DeVinney, Rebekah
The parasite Entamoeba histolytica (Eh) is named for its capability to lyse healthy host tissues. A hallmark of Eh invasion in the gut is acute intestinal inflammation dominated by the secretion of pro-inflammatory cytokines. Eh in direct contact with macrophages activates caspase-1 by the assembly of the NLRP3 inflammasome complex in a Gal-lectin and EhCP-A5-dependent manner, resulting in the maturation and secretion of IL-1β and IL-18. Pyroptosis, an inflammatory mode of lytic programmed cell death, is essential for host defence and other danger signals. Eh activates caspase-4/1 in macrophage that cleaved GSDMD, the executioner of cell pyroptosis, leading to the insertion of N-terminal effector domain into the cell membrane, driving both pore formation and IL-1β secretion without causing significant cell death. In this study, I interrogated the requirements and molecular mechanism for Eh-induced caspase-4 activation in cleaving GSDMD and in initiating a robust pro-inflammatory response. The noncanonical caspase-4 is considered inextricably connected to the NLRP3 inflammasome, however, the concise mechanism of this interaction remains unclear. To determine if Eh-induced activation of caspase-4 interacted with the canonical NLRP3 inflammasome to regulate GSDMD-mediated pro-inflammatory cytokine release, CRISPR-Cas9 gene editing of CASP4/1, ASC, NLRP3, GSDMD THP-1 cells were utilized. In the absence of caspase-1, caspase-4 activation was significantly upregulated and enhanced GSDMD pore formation to trigger robust IL-1β secretion as quantified by HEK-BlueTM reporter cell. The fundamental function of pyroptosis is to trigger vigorous inflammatory responses to defend against pathogens. However, excessive and prolonged pyroptosis can lead to severe inflammatory diseases or immunological conditions, including sepsis and autoimmune disorders. The most outstanding finding of this work revealed that Eh did not trigger extensive cell pyroptosis for robust IL-1β secretion, rather Eh induced “hyperactivated macrophages” that led to caspase-4 dependent GSDMD cleavage and IL-1β release. This was in marked contrast to the positive control, LPS + Nigericin that induced high expression of caspase-1, enhanced GSDMD cleavage, IL-1β secretion, and massive pyroptotic cell death. Edman degradation and in vitro caspase cleavage assays showed that caspase-4 cleaved GSDMD at the same position as caspase-1 (human) to form pores in the plasma membrane. This work sheds new light on the functional role of caspase-4 in regulating GSDMD pore forming activity in innate host defenses against Eh.
Open Access
Entamoeba histolytica-Induced Caspase-4 Activation Regulates IL-1β Secretion Through Caspase-1
(2018-04-27) Quach, Jeanie; Chadee, Kris C.; McCafferty, Donna-Marie; Yates, Robin Michael
Entamoeba histolytica (Eh) is the causative agent of amebiasis, one of the top four parasitic causes of mortality worldwide. In 90% of infected individuals, Eh harmlessly colonizes the large intestine and results in a non-invasive and asymptomatic infection. In the remaining 10% of infected individuals, the parasite breaches the intestinal barrier causing amebic colitis and in rare cases, it can cause extra-intestinal lesions, mainly liver abscesses. During invasion, Eh encounter macrophages in the lamina propria and this intricate host-parasite interaction is critical in eliciting a tissue damaging raging pro-inflammatory response. When Eh binds macrophages via the Gal-lectin, surface EhCP-A5 ligates α5β1 integrin to activate caspase-1 in a complex known as the NLRP3 inflammasome. In this study, we investigated the parasite requirements underlying macrophage caspase-4 and -1 activation and the role caspase-4 play in augmenting pro-inflammatory cytokine responses. Surprisingly, caspase-4 activation was similar to caspase-1 requiring live Eh attachment via the Gal-lectin, EhCP-A5 and cellular stresses such as K+ efflux and ROS. However, unlike caspase-1, caspase-4 activation was independent of ASC and NLRP3. Using CRISPR/Cas9 gene editing of caspase-4 and caspase-1 in human macrophages, we determined that caspase-1 and bioactive IL-1β release was highly dependent on caspase-4 activation in response to Eh. Formaldehyde cross-linking to stabilize protein-protein interactions in transfected COS-7 cells stimulated with Eh revealed that caspase-4 specifically interacted with caspase-1 in a protein complex that enhanced the cleavage of caspase-1 CARD domains to augment IL-1β release. The mouse ortholog caspase-11, displayed similar requirements for its activation, however, it was not involved in regulating caspase-1 activation in the same way as caspase-4. These findings reveal a novel role for human caspase-4 as a critical sensor molecule to amplify downstream pro-inflammatory responses when macrophage encounters live Eh.
Open Access
Equine Glandular Gastric Disease: Prevalence and Potential Mechanisms of Formation
(2017) Pedersen, Sarah Kate; Banse, Heidi; Cribb, Alastair; Read, Emma; French, Daniel; McCafferty, Donna-Marie
The pathophysiology of and risk factors for equine glandular gastric disease (EGGD) formation are poorly understood. The present studies aimed to explore risk factors for spontaneous EGGD, and evaluate the role of prostaglandin synthesis in NSAID-induced gastric glandular disease formation. In a population of 86 show jumping Warmblood horses, prevalence of EGGD grade ≥2 was 49%. Competing at the national level was the risk factor identified in the multivariable analysis for grade ≥2 EGGD. In order to evaluate the role of prostaglandin concentration in NSAID-induced EGGD, twelve horses were treated with either phenylbutazone or placebo for one week. Glandular disease score increased in the phenylbutazone group. Prostaglandin concentrations in glandular gastric biopsies increased over time in both treatment groups. In summary, competition level appears to be a risk factor for spontaneous EGGD. Decreased prostaglandin concentration does not appear to be a primary contributor to NSAID-induced EGGD.
Open Access
Examining the activation of xenobiotic receptors using microbial metabolites and chemical ligands
(2024-09-18) Shenoda, Eva Ibrahim Gorgy; Hirota, Simon; Nasser, Yasmin; McCafferty, Donna-Marie
The aryl hydrocarbon receptor (AhR) and pregnane X receptor (PXR) are key xenobiotic receptors involved in regulating chemical metabolism and detoxification. Traditionally, these receptors were known for mediating toxic responses by sensing and responding to chemicals. Recent research shows their role in maintaining gut homeostasis and regulating inflammation. However, the mechanisms by which they induce these responses are not clear. This thesis examines whether the activation of AhR and PXR in epithelial cells by microbial metabolites versus chemical ligands drives unique transcriptional responses and could explain differences in beneficial versus deleterious biological outcomes in the host. PXR and AhR were activated with indole-3-propionic acid (IPA) and indole-3-pyruvic acid (IPyA) as microbial metabolites, and pregnenolone 16α-carbonitrile (PCN) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as chemical ligands, respectively. We compared responses to these ligands, revealing significant gene expression differences. Further analysis showed both IPyA and TCDD activated genes involved in hypoxia-inducible factor (HIF) signaling and metabolism. IPyA upregulated genes for ATP synthesis and purine biosynthesis, while TCDD promoted genes related to cell cycle, cancer, and apoptosis. Given that AhR translocates to the nucleus upon activation, we hypothesized that the differences in transcriptomic responses and gene induction might be attributed to differences in nuclear translocation of AhR, with TCDD potentially inducing greater nuclear translocation compared to IPyA. To test this hypothesis, we established a cellular fractionation protocol for organoids. However, our results revealed no significant difference in AhR nuclear translocation between the two treatments, suggesting that other factors may affect gene expression in the AhR pathway. These findings highlight AhR and PXR's complex roles in gut health and inflammation, suggesting receptor activation can have both adverse and beneficial effects. This research enhances our understanding of AhR and PXR mechanisms and their potential for therapeutic strategies targeting gut disorders and inflammation.
Embargo
FCGBP Maintains MUC2 Mucus Structural Integrity By Stabilizing The Mucus Layer In Innate Host Defense In The Colon
(2023-10-12) Gorman, Hayley; Chadee, Kris; McCafferty, Donna-Marie; DeVinney, Rebekah
The gastrointestinal tract is lined with a mucus layer that protects against ingested toxins and pathogens from contacting the underlying epithelium. In the colon, the major protein that forms the mucus layer is MUC2, a glycoprotein that creates a protective gel and provides a food source for resident microbiota. Mucus also contain other proteins, including IgG Fc gamma binding protein (FCGBP) that is heavily N-linked glycosylated with no known function. Based on this knowledge gap, FCGBP was studied to explore its role in host defense and epithelial barrier function. Basally, FCGBP was co-ordinately expressed with MUC2 mucin in human goblet-cell like LS174T cells. Both FCGBP and MUC2 were coordinately biosynthesized, packaged, and stored in mucin granules. Secreted mucus contained both MUC2 and FCGBP and the proteins interacted through non-covalent interactions. Wound healing assays revealed that MUC2 and FCGBP were strongly expressed and co-localized at the wound margin, but this pattern was lost in the absence of MUC2. FCGBP expression was decreased in a mouse model of colonic inflammation, suggesting a role for FCGBP in wound healing. LS174T cells with a missense mutation in FCGBP had an altered mucus and cell glycomics profile from WT cells that correlated with increased intracellular stress and tumorgenicity. Even though FCGBP-Mut cells had higher expression and secretion of MUC2, the mucus was more penetrable to microbeads and susceptible to Salmonella enterica infection with increased adhesion, invasion, and cell death as compared to WT cells. In addition to bacterial invasion, the mucus degrading colonic parasite, Entamoeba histolytica (Eh) was also examined to elucidate how FCGBP-MUC2 mucin protein complex was altered and/or degraded. In response to Eh, both MUC2 and FCGBP expression in WT cells were markedly upregulated and degraded by Eh secreted proteinases. The degradation of FCGBP preceded MUC2 cleavage at the C-terminus. FCGBP-Mut cells constitutively secreted mucus was more adherent to Eh associated with high cell death as compared to WT. These results demonstrate that FCGBP is coordinately regulated and expressed with MUC2 mucin to form a protein complex that constitute the structural building blocks of colonic mucus. Alterations in FCGBP by a single missense mutation rendered the mucus layer more penetrable to pathogens associated with cell death, highlighting the importance of FCGBP together with MUC2 to form the protective mucus layer.
Open Access
High MUC2 Biosynthesis and Production Augment Pro-Inflammatory Responses in Colonic Goblet Cells
(2023-09-11) Kim, Ariel Jayu; Chadee, Kris; McCafferty, Donna-Marie; Dufour, Antoine
Goblet cells are specialized secretory epithelial cells whose primary function is to secrete mucins into the lumen of the gastrointestinal tract to form the mucus layer, which acts as a physical barrier separating luminal contents from the underlying epithelium. During amoebiasis, caused by the human intestinal protozoan parasite, Entamoeba histolytica, goblet cells hypersecrete and become depleted of MUC2 mucin, impairing the protective mucus barrier to allow the parasite to contact the colonic epithelium and induce a robust pro-inflammatory cytokine response. While colonic absorptive epithelial and immune cells have been shown to produce pro-inflammatory cytokines, it is not known if goblet cells do so as well in addition to mucin production. In this study, I investigated if the pro-inflammatory chemokine, CXCL8, was altered in WT and CRISPR/Cas9 MUC2KO human colonic goblet cells in response to E. histolytica. CXCL8 (IL-8) is a potent chemokine that recruits neutrophils to sites of inflammation, and symptomatic E. histolytica infections are marked by robust production of CXCL8 and subsequent neutrophil tissue infiltration. RT-qPCR and ELISA were used to analyze mRNA and protein expression in response to various stimuli. Specific pro-inflammatory pathway pharmacological inhibitors were used to enumerate defects in intracellular signaling, actinomycin D to inhibit transcription, and cycloheximide to inhibit translation. Diphenyleneiodonium chloride (DPI) was used to alleviate reactive oxygen species (ROS)-induced endoplasmic reticulum (ER) stress and ROS production was measured by the 2’,7’-dichlorodihydrofluorescein diacetate (DCFDA) assay. WT goblet cells produced more CXCL8 and ROS than MUC2KO cells in response to amoebic stimulation. Whereas CXCL8 mRNA was reduced by inhibiting multiple pro-inflammatory signaling pathways in WT, only the MAPK/ERK pathway reduced transcription in MUC2KO cells. CXCL8 mRNA from WT was more stable than MUC2KO cells, and this stability was reduced when ER stress was alleviated. This study identified that metabolically stressed goblet cells regulate the expression of CXCL8 in response to E. histolytica by a ROS-dependent mechanism, activating multiple signalling pathways to stabilize transcripts and maximize pro-inflammatory responses against E. histolytica. Importantly, these results unraveled a previously uncharacterized dualistic role for goblet cells- mucus secretion and pro-inflammatory chemokine production- in innate host defence against E. histolytica.
Open Access
Impact of the Group IV Protein Tyrosine Kinase, Fer, on Intestinal Inflammation and Neutrophil Function
(2009) Khajah, Maitham A.; McCafferty, Donna-Marie
Open Access
New Insights into the Immunological Changes in IL-10-Deficient Mice during the Course of Spontaneous Inflammation in the Gut Mucosa
(2012-02-01) Gomes-Santos, Ana Cristina; Moreira, Thais Garcias; Castro-Junior, Archimedes Barbosa; Horta, Bernardo Coelho; Lemos, Luisa; Cruz, Deborah Nogueira; Guimarães, Mauro Andrade Freitas; Cara, Denise Carmona; McCafferty, Donna-Marie; Faria, Ana Maria Caetano
IL-10 is a regulatory cytokine that plays a major role in the homeostasis of the gut and this is illustrated by the fact that IL-10−/− mice develop spontaneous colitis. In this study, IL-10−/− mice were analyzed for immunological changes during colitis development. We found a reduced frequency of regulatory T cells and higher frequency of activated T cells in the colon that precedes the macroscopic signs of the disease. Production of IL-17 and IFN-γ was higher in the colon. Colitis progression culminates with the reduction of regulatory T cells in the intestine. Frequency of B1 cells and the secretory IgA production were both elevated. Despite these alterations, 16-week-old IL-10−/− mice could be rendered tolerant by a continuous feeding protocol. Our study provides detailed analysis of changes that precede colitis and it also suggests that oral tolerance could be used to design novel alternative therapies for the disease.
Open Access
New Insights into the Immunological Changes in IL-10-Deficient Mice during the Course of Spontaneous Inflammation in the Gut Mucosa
(Hindawi Publishing Corporation, 2011-10-15) Gomes-Santos, Ana Cristina; Moreira, Thais Garcias; Castro-Junior, Archimedes Barbosa; Horta, Bernardo Coelho; Lemos, Luisa; Cruz, Deborah Nogueira; Guimarães, Mauro Andrade Freitas; Cara, Denise Carmona; McCafferty, Donna-Marie; Faria, Ana Maria Caetano
Open Access
Pregnane X Receptor (PXR) modulates NLRP3 Inflammasome
(2017-12-18) Hudson, Grace; Hirota, Simon; McCafferty, Donna-Marie; Chadee, Khrisendath
Compounds released from the intestinal microbiota may play a role in maintaining mucosal homeostasis, but little is known about the receptors that sense and respond to these compounds. Recently, a cytosolic xenobiotic sensor, the pregnane X receptor (PXR), was identified as a receptor for microbial metabolites in the gastrointestinal (GI) tract. The PXR has been shown to play a protective role in the gut, with gene variants associated with IBD risk. Recent data suggest that the PXR may regulate innate immune signaling platforms, in a variety of tissues. In vascular endothelial cells, the PXR was shown to stimulate the expression of NLRP3, and initiate NLRP3 inflammasome activation. Interestingly, alterations in NLRP3 inflammasome function have been linked to IBD susceptibility. In the current thesis, we sought to characterize the role of the PXR in modulating the function of the NLRP3 inflammasome in macrophages, a key innate immune cell that contributes to host-defense and the regulation of intestinal mucosal homeostasis. Using the THP-1 cell line and mouse peritoneal macrophages, we found that PXR agonists stimulated caspase-1 activation, along with IL-1β processing and release. These responses were lost in cells lacking NLRP3 and blocked by selective inhibition of caspase-1. Furthermore, PXR-deficient cells failed to activate caspase-1 and release IL-1β in response to PXR agonist stimulation. Lastly, we found that PXR activation triggered ATP release, an effect that was responsible for inflammasome activation, as these responses were abolished by apyrase and P2X7 inhibition. Through this thesis, we demonstrated that the PXR activates the NLRP3 inflammasome through stimulating ATP release within a macrophage.
Open Access
Regulation of colonic mucus and epithelial cell barrier function by cathelicidin
(2022-05) Blyth, Graham; Cobo, Eduardo; Chadee, Kris; DeVinney, Rebekah; McCafferty, Donna-Marie
The gastrointestinal tract has the physiologic function of digestion and absorption of food and nutrients. These functions must be balanced with prevention of injury, infection, or inflammation associated with physical, chemical, or pathogenic challenge. Due to its large surface area and exposure to a variety of pathogens, the gastrointestinal tract is an important target for pathogen colonization. Several immune mechanisms exist in the colon to resist against or clear established intestinal infections. Secretion of the MUC2 mucin glycoprotein by colonic goblet cells generates a mucus layer that acts as a physical barrier to pathogen penetration. Equally important, a continuous layer of intestinal epithelial cells, held together by tight junction proteins, prevents the penetration of microbial pathogens into the underlying lamina propria. The mucus layer is expected to contain an array of antimicrobial peptides secreted by intestinal epithelial cells, including cathelicidin. Cathelicidin possesses both direct antibacterial and immunomodulatory functions. Although cathelicidin has well defined immunomodulatory roles in leukocytes, its role in modulating intestinal epithelial cell responses is less understood. Mice deficient in cathelicidin (Camp-/-) had impaired goblet cell secretory responses during infection with C. rodentium, corresponding to increased bacterial load and colonization of the epithelium. Colonic goblet cells in Camp-/- mice were swollen with a retained number of mucin granules, and showed altered mucus secretion during C. rodentium infection. Human cathelicidin, LL-37, induced secretion of two goblet cell proteins, TFF3 and RELMβ, in human colonic epithelial goblet-like (LS174T) cells, an effect that was dependent on reactive oxygen species production. While Camp-/- mice did not display altered intestinal barrier function, stimulation of human colonic epithelial (T84) cells with LL-37 resulted in a temporary increased permeability via the endocytosis and degradation of the tight junction proteins occludin and claudin-2. Collectively, these data establish immunomodulatory functions of cathelicidin acting on the colonic epithelium that contribute to clearance of intestinal bacterial infections.
Open Access
Regulation of Intestinal Epithelial Thymic Stromal Lymphopoietin Gene Expression by Retinoic Acid Receptor Alpha
(2021-08-23) Mahmood, Ramsha; Jijon, Humberto; Beck, Paul; Hirota, Simon; McCafferty, Donna-Marie; Dufour, Antoine
Inflammatory bowel disease is characterized by chronic inflammation of the gastrointestinal tract. The pathogenesis is thought to be due to a dysregulated immune response to intestinal microbiota. Approximately 15% of the risk is genetically linked and approximately 85% is attributed to environmental exposures. Dietary factors like retinoic acid (RA), a vitamin A metabolite, have been linked to the onset of IBD by influencing intestinal immune function. RA induces Tregs and inhibits the actions of proinflammatory Th-17 cells. We have previously described decreased CD103+ DCs numbers in the intestinal compartments of RARα-deficient mice (RARαvillin mice). We sought to generate an experimental system to identify signaling pathway(s) or mechanism(s) that might be governing these effects, specifically RA signaling, as this data suggested there might be contributing factors intrinsic to IECs. We chose to establish a knockout cell line using the CRISPR/Cas9 system, given its affordability and efficiency compared to other in vitro models, and used it to study the effects of RAR⍺ ablation in IECs. Thus, our hypothesis was that RA signaling regulates the expression of lymphokines and other immune mediators (e.g., TSLP) by IECs, which then modulate the intestinal immune compartment. We hypothesized TSLP could be contributing to the decrease in CD103+ DCs as it is an important cytokine involved in TH2-type immunity and plays a key role in the maintenance of peripheral CD4+ T cell homeostasis by modulating the activation/maturation of myeloid cells. We used the CRISPR/Cas9 system to examine the effects of RARα ablation and its role in regulating intestinal epithelial TSLP expression. We found that TSLP expression is controlled by RARα in IECs where it may act as a repressor of TSLP promoter transactivation. This suggests an important role for RA signaling on myeloid/T cell function via effects on TSLP gene expression.
Open Access
Role of High MUC2 Production in Colonic Goblet Cell Apoptosis and Wound Healing
(2017) Tawiah, Adelaide Ama; Chadee, Kris; McCafferty, Donna-Marie; DeVinney, Rebekah
MUC2 mucin is produced by goblet cells, which forms the protective mucus blanket overlying the intestinal epithelium as the first line of innate host defense. MUC2 forms a physical barrier between the luminal contents and epithelium as a protective shield. Under normal physiological conditions, goblet cells produce MUC2 continuously to replenish the mucus blanket and maintain epithelial barrier function. However, in inflammatory bowel disease (IBD) and infectious colitis, MUC2 mucin is hyper secreted that leads to a phenomenon commonly referred to as ‘goblet cell depletion’ and eventual injury to the epithelium. It remains unclear however, how MUC2 production affects this phenomenon and whether goblet cells undergo increased endoplasmic reticulum (ER) stress and apoptosis in response to high MUC2 biosynthesis and secretion, which could explain how ‘goblet cell depletion’ occurs. In this study, I investigated the role of high MUC2 mucin production in goblet cell-induced ER stress, susceptibility to apoptosis and restitution following injury. Two cells lines were used; a high and low MUC2-producing human goblet cell-like cell lines designated HT29-H and HT29-L, respectively. Goblet cell ER stress and apoptosis were also quantified during early onset of DSS-induced colitis in C57BL/6 and Math1M1GFP mice. Compared to HT29-L, HT29-H cells showed significant increase in ER stress and apoptosis in response to ER stressors and apoptosis-inducing agents. Increased ER stress and apoptosis were dependent on reactive oxygen species (ROS) as inhibition of ROS significantly alleviated basal and tunicamycin- and thapsigargin-induced ER stress and rescued cells from apoptosis. DSS-induced colitis caused early hyper secretion of mucus and severe ER stress and apoptosis of goblet cells at the early onset of colitis. Increased MUC2 production in HT29-H cells inhibited production of growth factors and enzymes (FGF1, FGF2, VEGF-C, MMP-1) and intracellular signalling pathways (MMP-1, MAPK/ERK) critical for wound repair. As a result, HT29-H cells migrated significantly slower than HT29-L cells in epithelial restitution. This study have unravelled that high MUC2 biosynthesis during intestinal inflammatory diseases increases goblet cell susceptibility to ER stress and apoptosis, leading to goblet cell depletion, mucus barrier dysfunction and impaired goblet cell restitution and mucosal repair.
Open Access
The Role of Protease-Activated Receptor-2 in Colonic Epithelial Wound Healing
(2019-06-27) Yatigalpoththe, Mahesha N. S.; MacNaughton, Wallace Keith; McCafferty, Donna-Marie; Hirota, Simon Andrew; von der Weid, Pierre Yves
The intestinal barrier function relies on the presence of a single layer of epithelial cells that selectively absorb nutrients and water while limiting intestinal content and gut microbiota into the lumen. When epithelial barrier function is compromised, unregulated translocation of microbiota and microbial products into the mucosa can result in intestinal inflammation as seen in IBD patients. Effective resolution of inflammation requires elimination of inflammatory triggers and this is partly dependant on epithelial wound healing. The intestine is constantly exposed to proteases originating from a variety of sources and these proteases can activate protease-activated receptors (PARs). The mechanism of intestinal epithelial wound healing is not completely understood, particularly in the context of proteases and PARs. It was recently shown that PAR2 activation transactivates epidermal growth factor receptor (EGFR) and also induces COX-2 expression in colonic epithelial cells. Based on this, it was hypothesized that PAR2-induced EGFR transactivation and COX-2 expression drive colonic epithelial wound healing. In the first part of the study, CMT-93 cells were characterized for PAR2 expression and PAR-2 induced COX-2 expression. PAR2 is primarily expressed in the plasma membrane with punctate immunoreactivity in the cytoplasm. Furthermore, the majority of PAR2 expression appeared to be basolateral. PAR2 activation by 2fLI (2-furoyl-LIGRLO-NH2) induced Ca2+ signaling. COX-2 expression was characterized using western blotting and PAR2 activation did not induce COX-2 expression in CMT93 cells. The effect of PAR2 activation in wound healing was investigated in the second part of the study. PAR2 activation induced wound healing and the treatment with the COX-2 selective inhibitor (NS-398) did not affect the PAR-2 induced wound healing, indicating that PAR-2 induced wound healing does not depend on COX-2 activity. The treatments with the EGFR tyrosine kinase inhibitor (PD153035), broad-spectrum matrix metalloproteases (MMP) inhibitor (GM6001) or Src tyrosine kinase inhibitor (PP2) inhibited PAR2-induced wound healing. These results suggest EGFR activity is required for PAR2-induced wound healing and PAR2 activation possibly transactivates EGFR via-matrix metalloproteinases and Src tyrosine kinase activity. In conclusion, this study shows the contribution of PAR-2 in epithelial wound healing, which could subsequently aid in the resolution of intestinal inflammation.
Open Access
The role of TOR kinase signaling in responses to bacterial infection
(2021-08-10) Deshpande, Rujuta Shailesh; Grewal, Savraj; Brook, William; Huang, Peng; McCafferty, Donna-Marie; Jean, Steve
Animals in their natural ecology are often exposed to environmental stressors (e.g., starvation, extreme temperature, hypoxia, pathogens) that can affect their physiology, development, and lifespan. An important question in biology is how animals sense these stresses and, in response, adapt their metabolism to maintain homeostasis and survival. In some cases, specific tissues function as stress sensors to control whole body adaptive responses. One well-studied example is the Drosophila intestine. Along with performing absorptive, digestive, and endocrine functions, the intestine also functions as both a stress sensor and signaling hub, to regulate systemic metabolic changes. Upon encountering enteric pathogenic bacteria, Drosophila adults mount organism-wide immune and physiological responses in order to provide infection resistance and promote tolerance. The Drosophila intestine controls both local and systemic anti-bacterial immune responses. Recent work shows that the gut also signals to other tissues to control whole-body metabolic changes to promote infection tolerance. However, the mechanisms underlying how these stress sensing tissues link the stressors such as infection to metabolic adaptations is not well understood. In my thesis, I show that one way by which the fly intestine mediates these adaptive metabolic responses is via induction of target-of-rapamycin (TOR) kinase signaling. TOR is a well-established regulator of metabolism that has classically been shown to be activated by growth cues and suppressed by stress conditions. Interestingly however, I found a rapid increase in TOR activity in the fly gut in response to enteric gram-negative bacterial infection stress, independent of the classic innate immune response. Furthermore, I showed that blocking this TOR induction reduced survival upon infection. My data suggest that these protective effects of gut TOR signaling on organismal survival may be mediated through altered whole-body lipid metabolism. Lipid stores are an important metabolic fuel source. They can be synthesized and stored in specific tissues and then mobilized, transported to other tissues, and used to fuel metabolism, particularly in stress conditions. Infection leads to transient loss of lipids which is perhaps needed to fuel the immune response. This transient loss and restoration of lipids was further exacerbated by TOR inhibition. Infection also induced TOR dependent systemic expression of transcription factors and enzymes that promote de novo lipid biogenesis, indicating one way by which TOR inhibits excess lipid loss is by promoting de novo lipid synthesis upon infection. Moreover, genetic upregulation of intestinal TOR was sufficient to induce the expression of some of these lipid synthesis genes. In addition to systemic effects, enteric infection also induced TOR dependent local intestinal lipolysis and beta oxidation genes, and endocrine signaling peptides which have previously been implicated in whole body lipid homeostasis. I propose a model in which induction of intestinal TOR signaling is an infection stress sensor that leads to local intestinal changes such as lipolysis and secretion of signaling peptides, which perhaps non autonomously signal to the rest of the animal to upregulate lipid synthesis upon infection. TOR upregulation represents a host adaptive response to counteract infection mediated loss of whole-body lipid stores in order to promote survival. While only a handful of studies have investigated a role for TOR signaling upon infection with varying results, my thesis supports the idea of TOR activity being beneficial for the host to survive enteric infection. I propose TOR signaling as a link between infection and metabolic adaptations which contributes to infection tolerance.
Open Access
The Adoptive Transfer of Helminth Antigen-Pulsed Dendritic Cells as a Novel Therapy for Colitis
(2016) Matisz, Chelsea; McKay, Derek; Sharkey, Keith; McCafferty, Donna-Marie; Jirik, Frank
The incidence of autoimmune, inflammatory, and allergic diseases—such as inflammatory bowel disease (IBD)—is increasing in westernized societies at alarming rates. Similarly, dramatically reduced exposure to infectious organisms, including helminth parasites, has been observed. The important role that pathogens play in the shaping and functioning of our immune systems, both evolutionarily and developmentally speaking, has led to the hygiene hypothesis: that reduced exposure to organisms can predispose to immune dysregulation under certain contexts. Infection with helminth parasites has therefore been explored as a potential treatment for certain autoimmune diseases, with mixed success. This work extends upon the traditional paradigm of helminth therapy, and explores the potential of using helminth antigens to develop a cellular therapy as a novel treatment for colitis. The adoptive transfer of bone marrow-derived dendritic cells (DC) treated with an antigen extract from the rat tapeworm Hymenolepis diminuta (HD) significantly attenuated the severity of the dinitrobenzene sulfonic acid (DNBS) model of colitis in mice: these HD-DC cells localized in the spleen, mesenteric lymph nodes (MLN), and colons of recipient mice. HD-DCs evoked significant increases in IL-4 and IL-10 in secondary lymphoid organs. Mechanistically, HD-DCs suppressed colitis through mobilization of recipient IL-10, and IL-4Ra signaling; further, HD-DCs drove the proliferation of CD4+ splenocytes, which also suppressed colitis in recipients via IL-10. HD-DCs that lacked IL-10 or IL-4Ra were unable to suppress colitis, suggesting these cells are a source of IL-10, and must respond to IL-4. In the absence of MHC II signaling HD-DCs were unable to drive splenic IL-4 or IL-10, but were still able to suppress disease, suggesting an alternative mechanism of DNBS suppression in the absence of TCR engagement: increased TGF-B produced by splenocytes of MHC II knock out (KO) HD-DC recipients, relative to WT HD-DC recipients, points to this anti-inflammatory cytokine as a potential mechanism. Further, MHC II KO HD-DCs were still capable of inducing an anti-colitic CD4+ splenocyte, suggesting a non-canonical means of T cell activation at play. Combined, these proof-of-principle studies highlight the potential for helminth antigen-based cellular therapy as a novel treatment strategy for IBD.