28 results on '"Milling, Simon W. F."'
Search Results
2. The mannose receptor (CD206) identifies a population of colonic macrophages in health and inflammatory bowel disease
- Author
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Wright, Pamela B., McDonald, Elizabeth, Bravo-Blas, Alberto, Baer, Hannah M., Heawood, Anna, Bain, Calum C., Mowat, Allan M., Clay, Slater L., Robertson, Elaine V., Morton, Fraser, Nijjar, Jagtar Singh, Ijaz, Umer Z., Milling, Simon W. F., and Gaya, Daniel R.
- Published
- 2021
- Full Text
- View/download PDF
3. Regulatory T cells control the dynamic and site-specific polarization of total CD4 T cells following Salmonella infection
- Author
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Clay, Slater L., Bravo-Blas, Alberto, Wall, Daniel M., MacLeod, Megan K. L., and Milling, Simon W. F.
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- 2020
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4. Ankylosing spondylitis patients display altered dendritic cell and T cell populations that implicate pathogenic roles for the IL-23 cytokine axis and intestinal inflammation
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Wright, Pamela B., McEntegart, Anne, McCarey, David, McInnes, Iain B., Siebert, Stefan, and Milling, Simon W. F.
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- 2016
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5. Targeted Delivery of Narrow-Spectrum Protein Antibiotics to the Lower Gastrointestinal Tract in a Murine Model of Escherichia coli Colonization.
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Carpena, Nuria, Richards, Kerry, Bello Gonzalez, Teresita D. J., Bravo-Blas, Alberto, Housden, Nicholas G., Gerasimidis, Konstantinos, Milling, Simon W. F., Douce, Gillian, Malik, Danish J., and Walker, Daniel
- Subjects
BACTERIAL colonies ,WATER-soluble polymers ,CONTROLLED release drugs ,DRUG delivery systems ,HUMAN microbiota ,GASTROINTESTINAL system - Abstract
Bacteriocins are narrow-spectrum protein antibiotics that could potentially be used to engineer the human gut microbiota. However, technologies for targeted delivery of proteins to the lower gastrointestinal (GI) tract in preclinical animal models are currently lacking. In this work, we have developed methods for the microencapsulation of Escherichia coli targeting bacteriocins, colicin E9 and Ia, in a pH responsive formulation to allow their targeted delivery and controlled release in an in vivo murine model of E. coli colonization. Membrane emulsification was used to produce a water-in-oil emulsion with the water-soluble polymer subsequently cross-linked to produce hydrogel microcapsules. The microcapsule fabrication process allowed control of the size of the drug delivery system and a near 100% yield of the encapsulated therapeutic cargo. pH-triggered release of the encapsulated colicins was achieved using a widely available pH-responsive anionic copolymer in combination with alginate biopolymers. In vivo experiments using a murine E. coli intestinal colonization model demonstrated that oral delivery of the encapsulated colicins resulted in a significant decrease in intestinal colonization and reduction in E. coli shedding in the feces of the animals. Employing controlled release drug delivery systems such as that described here is essential to enable delivery of new protein therapeutics or other biological interventions for testing within small animal models of infection. Such approaches may have considerable value for the future development of strategies to engineer the human gut microbiota, which is central to health and disease. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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6. Regulatory T cells control the dynamic and site-specific T helper bias following Salmonella infection
- Author
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Clay, Slater L., Bravo-Blas, Alberto, Wall, Daniel M., MacLeod, Megan K.L., and Milling, Simon W. F.
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chemical and pharmacologic phenomena ,hemic and immune systems - Abstract
FoxP3 + regulatory T cells (Tregs) control inflammation and maintain mucosal homeostasis, but their functions during infection are less well understood. Th1, Th2 and Th17 cells can be identified by their master transcription factors (TFs) T-bet, GATA3 and RORγT; Tregs also express these TFs. While T-bet + Tregs can selectively supress Th1 cells, it is unclear whether this is true of Tregs expressing other TFs, or whether such selective suppression can alter the balance of the Th cell response. To address these questions, we used Salmonella enterica serotype Typhimurium (STM) to induce non-lethal colitis. Following infection, we observed an early colonic Th17 response, followed by a Th1-dominated response. The early Th17 response parallels an increase in T-bet + Tregs. Later, Th1 cells and RORγT + Tregs dominate. This reciprocal dynamic between Th cells and Tregs expressing the same TF indicates that Tregs may selectively suppress Th subsets. To test this, Treg depletion experiments were performed. These demonstrated that Tregs enable both the early colonic Th17 response and the later Th1 response. Thus, Tregs can shape the tissue CD4 T cell response in a fine-tuned manner. This highlights the potential for subpopulations of Tregs to be used in targeted therapeutic approaches.
- Published
- 2019
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7. Mucosal CD8 T Cell Responses Are Shaped by Batf3-DC After Foodborne Listeria monocytogenes Infection.
- Author
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Imperato, Jessica Nancy, Xu, Daqi, Romagnoli, Pablo A., Qiu, Zhijuan, Perez, Pedro, Khairallah, Camille, Pham, Quynh-Mai, Andrusaite, Anna, Bravo-Blas, Alberto, Milling, Simon W. F., Lefrancois, Leo, Khanna, Kamal M., Puddington, Lynn, and Sheridan, Brian S.
- Subjects
T cells ,LISTERIOSIS ,LISTERIA monocytogenes ,DENDRITIC cells ,INTESTINAL mucosa - Abstract
While immune responses have been rigorously examined after intravenous Listeria monocytogenes (Lm) infection, less is understood about its dissemination from the intestines or the induction of adaptive immunity after more physiologic models of foodborne infection. Consequently, this study focused on early events in the intestinal mucosa and draining mesenteric lymph nodes (MLN) using foodborne infection of mice with Lm modified to invade murine intestinal epithelium (InlA
M Lm). InlAM Lm trafficked intracellularly from the intestines to the MLN and were associated with Batf3-independent dendritic cells (DC) in the lymphatics. Consistent with this, InlAM Lm initially disseminated from the gut to the MLN normally in Batf3–/– mice. Activated migratory DC accumulated in the MLN by 3 days post-infection and surrounded foci of InlAM Lm. At this time Batf3–/– mice displayed reduced InlAM Lm burdens, implicating cDC1 in maximal bacterial accumulation in the MLN. Batf3–/– mice also exhibited profound defects in the induction and gut-homing of InlAM Lm -specific effector CD8 T cells. Restoration of pathogen burden did not rescue antigen-specific CD8 T cell responses in Batf3–/– mice, indicating a critical role for Batf3 in generating anti-InlAM Lm immunity following foodborne infection. Collectively, these data suggest that DC play diverse, dynamic roles in the early events following foodborne InlAM Lm infection and in driving the establishment of intestinal Lm -specific effector T cells. [ABSTRACT FROM AUTHOR]- Published
- 2020
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8. Isolation and Identification of Conventional Dendritic Cell Subsets from the Intestine of Mice and Men.
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Scott, Charlotte L., Wright, Pamela B., Milling, Simon W. F., and Mowat, Allan McI
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- 2016
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9. Regulation of intestinal immunity: Effects of the oral adjuvant Escherichia coli heat-labile enterotoxin on migrating dendritic cells.
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Milling, Simon W. F., Yrlid, Ulf, Jenkins, Chris, Richards, Claire M., Williams, Neil A., and MacPherson, Gordon
- Abstract
Escherichia coli heat-labile enterotoxin (Etx) is an oral adjuvant in mice. We show that this is also true for rats. To understand this adjuvant activity we examined lymph dendritic cells (DC) migrating from the intestine to mesenteric lymph nodes (MLN) in animals fed Etx. These DC can prime antigen-specific antibody responses. We show that in rats the small intestine contains 7-24 million DC and 8 × 10of these migrate to MLN each day. Surprisingly, Etx does not stimulate increased migration of lymph DC. However, oral Etx affects the activation, antigen transport and localization of migratory DC. Specifically, expression of CD25 increases on the CD172a subset of lymph DC. Oral Etx also increases the number of CD172a lymph DC containing co-administered ovalbumin. CD172a lymph DC treated with Etx in vitro, or purified from the lymph of animals fed Etx, stimulate stronger proliferative responses from primed T cells. Etx also directs more of the CD172a lymph DC into the central region of the MLN T cell areas. This change in DC localization is associated with an increase in the expression of CCR7. These data help advance our understanding of the role of DC in initiating mucosal immune responses in vivo. [ABSTRACT FROM AUTHOR]
- Published
- 2007
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10. Characterization of Conventional and Atypical Receptors for the Chemokine CCL2 on Mouse Leukocytes.
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Ford, Laura B., Cerovic, Vuk, Milling, Simon W. F., Graham, Gerard J., Hansell, Chris A. H., and Nibbs, Robert J. B.
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CHEMOKINE receptors , *CHEMOKINE genetics , *CHEMOTACTIC factors , *LEUCOCYTES , *FLUORESCENT antibody technique , *PHYSIOLOGY - Abstract
Chemokine-directed leukocyte migration is crucial for effective immune and inflammatory responses. Conventional chemokine receptors (cCKRs) directly control cell movement; atypical chemokine receptors (ACKRs) regulate coexpressed cCKRs; and both cCKRs and ACKRs internalize chemokines to limit their abundance in vivo, a process referred to as scavenging. A leukocyte's migratory and chemokine-scavenging potential is determined by which cCKRs and ACKRs it expresses, and by the ligand specificity, signaling properties, and chemokine internalization capacity of these receptors. Most chemokines can bind at least one cCKR and one ACKR. CCL2 can bind to CCR2 (a cCKR) and two ACKRs (ACKR1 and ACKR2). In this study, by using fluorescent CCL2 uptake to label cells bearing functional CCL2 receptors, we have defined the expression profile, scavenging activity, and ligand specificity of CCL2 receptors on mouse leukocytes. We show that qualitative and quantitative differences in the expression of CCR2 and ACKR2 endow individual leukocyte subsets with distinctive CCL2 receptor profiles and CCL2-scavenging capacities. We reveal that some cells, including plasmacytoid dendritic cells, can express both CCR2 and ACKR2; that Ly6Chigh monocytes have particularly strong CCL2-scavenging potential in vitro and in vivo; and that CCR2 is a much more effective CCL2 scavenger than ACKR2. We confirm the unique, overlapping, ligand specificities of CCR2 and ACKR2 and, unexpectedly, find that cell context influences the interaction of CCL7 and CCL12 with CCR2. Fluorescent chemokine uptake assays were instrumental in providing these novel insights into CCL2 receptor biology, and the sensitivity, specificity, and versatility of these assays are discussed. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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11. Salmonella enterica Serovar Typhimurium Travels to Mesenteric Lymph Nodes Both with Host Cells and Autonomously.
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Bravo-Blas, Alberto, Utriainen, Lotta, Clay, Slater L., Kästele, Verena, Cerovic, Vuk, Wall, Daniel M., Milling, Simon W. F., Cunningham, Adam F., and Henderson, Ian R.
- Abstract
Salmonella infection is a globally important cause of gastroenteritis and systemic disease and is a useful tool to study immune responses in the intestine. Although mechanisms leading to immune responses against Salmonella have been extensively studied, questions remain about how bacteria travel from the intestinal mucosa to the mesenteric lymph nodes (MLN), a key site for Ag presentation. In this study, we used a mouse model of infection with Salmonella enterica serovar Typhimurium (STM) to identify changes in intestinal immune cells induced during early infection. We then used fluorescently labeled STM to identify interactions with immune cells from the site of infection through migration in lymph to the MLN. We show that viable STM can be carried in the lymph by any subset of migrating dendritic cells but not by macrophages. Moreover, approximately half of the STM in lymph are not associated with cells at all and travel autonomously. Within the MLN, STM associates with dendritic cells and B cells but predominantly with MLN-resident macrophages. In conclusion, we describe the routes used by STM to spread systemically in the period immediately postinfection. This deeper understanding of the infection process could open new avenues for controlling it. [ABSTRACT FROM AUTHOR]
- Published
- 2019
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12. CD11c identifies microbiota and EGR2-dependent MHCII + serous cavity macrophages with sexually dimorphic fate in mice.
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Bain CC, Louwe PA, Steers NJ, Bravo-Blas A, Hegarty LM, Pridans C, Milling SWF, MacDonald AS, Rückerl D, and Jenkins SJ
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- Animals, Cell Differentiation, Female, Male, Mice, Mice, Inbred C57BL, Sex Characteristics, CD11c Antigen metabolism, Early Growth Response Protein 2 metabolism, Macrophages, Peritoneal metabolism, Microbiota
- Abstract
The murine serous cavities contain a rare and enigmatic population of short-lived F4/80
lo MHCII+ macrophages but what regulates their development, survival, and fate is unclear. Here, we show that mature F4/80lo MHCII+ peritoneal macrophages arise after birth, but that this occurs largely independently of colonization by microbiota. Rather, microbiota specifically regulate development of a subpopulation of CD11c+ cells that express the immunoregulatory cytokine RELM-α, are reliant on the transcription factor EGR2, and develop independently of the growth factor CSF1. Furthermore, we demonstrate that intrinsic expression of RELM-α, a signature marker shared by CD11c+ and CD11c- F4/80lo MHCII+ cavity macrophages, regulates survival and differentiation of these cells in the peritoneal cavity in a sex-specific manner. Thus, we identify a previously unappreciated diversity in serous cavity F4/80lo MHCII+ macrophages that is regulated by microbiota, and describe a novel sex and site-specific function for RELM-α in regulating macrophage endurance that reveals the unique survival challenge presented to monocyte-derived macrophages by the female peritoneal environment., (© 2022 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)- Published
- 2022
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13. Monocytes mediate Salmonella Typhimurium-induced tumor growth inhibition in a mouse melanoma model.
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Johnson SA, Ormsby MJ, Wessel HM, Hulme HE, Bravo-Blas A, McIntosh A, Mason S, Coffelt SB, Tait SWG, Mowat AM, Milling SWF, Blyth K, and Wall DM
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- Animals, Cytokines immunology, Female, Mice, Salmonella typhimurium genetics, Immunotherapy, Melanoma, Experimental immunology, Melanoma, Experimental therapy, Monocytes immunology, Salmonella typhimurium immunology, Th1 Cells immunology
- Abstract
The use of bacteria as an alternative cancer therapy has been reinvestigated in recent years. SL7207: an auxotrophic Salmonella enterica serovar Typhimurium aroA mutant with immune-stimulatory potential has proven a promising strain for this purpose. Here, we show that systemic administration of SL7207 induces melanoma tumor growth arrest in vivo, with greater survival of the SL7207-treated group compared to control PBS-treated mice. Administration of SL7207 is accompanied by a change in the immune phenotype of the tumor-infiltrating cells toward pro-inflammatory, with expression of the T
H 1 cytokines IFN-γ, TNF-α, and IL-12 significantly increased. Interestingly, Ly6C+ MHCII+ monocytes were recruited to the tumors following SL7207 treatment and were pro-inflammatory. Accordingly, the abrogation of these infiltrating monocytes using clodronate liposomes prevented SL7207-induced tumor growth inhibition. These data demonstrate a previously unappreciated role for infiltrating inflammatory monocytes underlying bacterial-mediated tumor growth inhibition. This information highlights a possible novel role for monocytes in controlling tumor growth, contributing to our understanding of the immune responses required for successful immunotherapy of cancer., (© 2021 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)- Published
- 2021
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14. Antibiotic-induced disturbances of the gut microbiota result in accelerated breast tumor growth.
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McKee AM, Kirkup BM, Madgwick M, Fowler WJ, Price CA, Dreger SA, Ansorge R, Makin KA, Caim S, Le Gall G, Paveley J, Leclaire C, Dalby M, Alcon-Giner C, Andrusaite A, Feng TY, Di Modica M, Triulzi T, Tagliabue E, Milling SWF, Weilbaecher KN, Rutkowski MR, Korcsmáros T, Hall LJ, and Robinson SD
- Abstract
The gut microbiota's function in regulating health has seen it linked to disease progression in several cancers. However, there is limited research detailing its influence in breast cancer (BrCa). This study found that antibiotic-induced perturbation of the gut microbiota significantly increases tumor progression in multiple BrCa mouse models. Metagenomics highlights the common loss of several bacterial species following antibiotic administration. One such bacteria, Faecalibaculum rodentium , rescued this increased tumor growth. Single-cell transcriptomics identified an increased number of cells with a stromal signature in tumors, and subsequent histology revealed an increased abundance of mast cells in the tumor stromal regions. We show that administration of a mast cell stabilizer, cromolyn, rescues increased tumor growth in antibiotic treated animals but has no influence on tumors from control cohorts. These findings highlight that BrCa-microbiota interactions are different from other cancers studied to date and suggest new research avenues for therapy development., Competing Interests: All authors declare no conflicts of interest., (© 2021 The Author(s).)
- Published
- 2021
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15. Isolation and functional characterisation of lamina propria leukocytes from helminth-infected, murine small intestine.
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Webster HC, Andrusaite AT, Shergold AL, Milling SWF, and Perona-Wright G
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- Adaptive Immunity, Animals, CD4-Positive T-Lymphocytes metabolism, Cytokines chemistry, Cytokines metabolism, Dendritic Cells immunology, Disease Models, Animal, Female, Flow Cytometry methods, Intestinal Diseases, Parasitic parasitology, Intestinal Mucosa immunology, Intestinal Mucosa parasitology, Intestine, Small cytology, Intestine, Small immunology, Intestine, Small parasitology, Macrophages immunology, Mice, Nematospiroides dubius immunology, Staining and Labeling methods, Strongylida Infections parasitology, CD4-Positive T-Lymphocytes immunology, Cell Separation methods, Intestinal Diseases, Parasitic immunology, Intestinal Mucosa cytology, Strongylida Infections immunology
- Abstract
The use of helminth infections as tools to understand the type 2 immune response is a well-established technique and important to many areas of immunological research. The phenotype and function of immune cell populations at the site of infection is a key determinant of pathogen clearance. However, infections with helminths such as the murine nematode Heligomosmoides polygryrus cause increased mucus production and thickening of the intestinal wall, which can result in extensive cell death when isolating and analysing cells from the lamina propria (LP). Populations of larger immune cells such as macrophages and dendritic cells are often trapped within mucus or dying tissues. Here we describe an optimised protocol for isolating LP leukocytes from the small intestine of H.polygyrus -infected mice, and we demonstrate phenotypic and functional identification of myeloid and CD4
+ T cell subsets using cytokine staining and flow cytometry. Our protocol may provide a useful experimental method for the immunological analysis of the affected tissue site during helminth infections., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)- Published
- 2020
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16. Exposure to the antimicrobial peptide LL-37 produces dendritic cells optimized for immunotherapy.
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Findlay EG, Currie AJ, Zhang A, Ovciarikova J, Young L, Stevens H, McHugh BJ, Canel M, Gray M, Milling SWF, Campbell JDM, Savill J, Serrels A, and Davidson DJ
- Abstract
Immunization of patients with autologous, ex vivo matured dendritic cell (DC) preparations, in order to prime antitumor T-cell responses, is the focus of intense research. Despite progress and approval of clinical approaches, significant enhancement of these personalized immunotherapies is urgently needed to improve efficacy. We show that immunotherapeutic murine and human DC, generated in the presence of the antimicrobial host defense peptide LL-37, have dramatically enhanced expansion and differentiation of cells with key features of the critical CD103
+ /CD141+ DC subsets, including enhanced cross-presentation and co-stimulatory capacity, and upregulation of CCR7 with improved migratory capacity. These LL-37-DC enhanced proliferation, activation and cytokine production by CD8+ (but not CD4+ ) T cells in vitro and in vivo . Critically, tumor antigen-presenting LL-37-DC increased migration of primed, activated CD8+ T cells into established squamous cell carcinomas in mice, and resulted in tumor regression. This advance therefore has the potential to dramatically enhance DC immunotherapy protocols.- Published
- 2019
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17. Antibiotics induce sustained dysregulation of intestinal T cell immunity by perturbing macrophage homeostasis.
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Scott NA, Andrusaite A, Andersen P, Lawson M, Alcon-Giner C, Leclaire C, Caim S, Le Gall G, Shaw T, Connolly JPR, Roe AJ, Wessel H, Bravo-Blas A, Thomson CA, Kästele V, Wang P, Peterson DA, Bancroft A, Li X, Grencis R, Mowat AM, Hall LJ, Travis MA, Milling SWF, and Mann ER
- Subjects
- Animals, Butyrates pharmacology, Cytokines metabolism, Fatty Acids metabolism, Gastrointestinal Microbiome drug effects, Inflammation pathology, Lipopolysaccharides pharmacology, Macrophages drug effects, Mice, Inbred C57BL, Receptors, CCR2 metabolism, T-Lymphocytes drug effects, Th1 Cells drug effects, Anti-Bacterial Agents pharmacology, Homeostasis drug effects, Immunity, Innate drug effects, Intestines cytology, Macrophages metabolism, T-Lymphocytes immunology
- Abstract
Macrophages in the healthy intestine are highly specialized and usually respond to the gut microbiota without provoking an inflammatory response. A breakdown in this tolerance leads to inflammatory bowel disease (IBD), but the mechanisms by which intestinal macrophages normally become conditioned to promote microbial tolerance are unclear. Strong epidemiological evidence linking disruption of the gut microbiota by antibiotic use early in life to IBD indicates an important role for the gut microbiota in modulating intestinal immunity. Here, we show that antibiotic use causes intestinal macrophages to become hyperresponsive to bacterial stimulation, producing excess inflammatory cytokines. Re-exposure of antibiotic-treated mice to conventional microbiota induced a long-term, macrophage-dependent increase in inflammatory T helper 1 (T
H 1) responses in the colon and sustained dysbiosis. The consequences of this dysregulated macrophage activity for T cell function were demonstrated by increased susceptibility to infections requiring TH 17 and TH 2 responses for clearance (bacterial Citrobacter rodentium and helminth Trichuris muris infections), corresponding with increased inflammation. Short-chain fatty acids (SCFAs) were depleted during antibiotic administration; supplementation of antibiotics with the SCFA butyrate restored the characteristic hyporesponsiveness of intestinal macrophages and prevented T cell dysfunction. Butyrate altered the metabolic behavior of macrophages to increase oxidative phosphorylation and also promoted alternative macrophage activation. In summary, the gut microbiota is essential to maintain macrophage-dependent intestinal immune homeostasis, mediated by SCFA-dependent pathways. Oral antibiotics disrupt this process to promote sustained T cell-mediated dysfunction and increased susceptibility to infections, highlighting important implications of repeated broad-spectrum antibiotic use., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)- Published
- 2018
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18. The Transcription Factor ZEB2 Is Required to Maintain the Tissue-Specific Identities of Macrophages.
- Author
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Scott CL, T'Jonck W, Martens L, Todorov H, Sichien D, Soen B, Bonnardel J, De Prijck S, Vandamme N, Cannoodt R, Saelens W, Vanneste B, Toussaint W, De Bleser P, Takahashi N, Vandenabeele P, Henri S, Pridans C, Hume DA, Lambrecht BN, De Baetselier P, Milling SWF, Van Ginderachter JA, Malissen B, Berx G, Beschin A, Saeys Y, and Guilliams M
- Subjects
- Animals, Cell Lineage immunology, Epithelial-Mesenchymal Transition, Female, Gene Expression Regulation, Neoplastic, Kupffer Cells immunology, Liver cytology, Liver X Receptors metabolism, Lung cytology, Male, Mice, Mice, Inbred C57BL, Mice, Transgenic, Kupffer Cells cytology, Liver X Receptors genetics, Zinc Finger E-box Binding Homeobox 2 genetics
- Abstract
Heterogeneity between different macrophage populations has become a defining feature of this lineage. However, the conserved factors defining macrophages remain largely unknown. The transcription factor ZEB2 is best described for its role in epithelial to mesenchymal transition; however, its role within the immune system is only now being elucidated. We show here that Zeb2 expression is a conserved feature of macrophages. Using Clec4f-cre, Itgax-cre, and Fcgr1-cre mice to target five different macrophage populations, we found that loss of ZEB2 resulted in macrophage disappearance from the tissues, coupled with their subsequent replenishment from bone-marrow precursors in open niches. Mechanistically, we found that ZEB2 functioned to maintain the tissue-specific identities of macrophages. In Kupffer cells, ZEB2 achieved this by regulating expression of the transcription factor LXRα, removal of which recapitulated the loss of Kupffer cell identity and disappearance. Thus, ZEB2 expression is required in macrophages to preserve their tissue-specific identities., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2018
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19. Isolation and Identification of Conventional Dendritic Cell Subsets from the Intestine of Mice and Men.
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Scott CL, Wright PB, Milling SW, and Mowat AM
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- Animals, Biomarkers metabolism, Cell Separation, Humans, Intestinal Mucosa metabolism, Intestine, Large metabolism, Intestine, Small metabolism, Leukocytes cytology, Leukocytes metabolism, Male, Mice, Intestine, Large chemistry, Intestine, Small cytology, Intestines cytology
- Abstract
The identification of conventional dendritic cells (cDCs) in the intestinal mucosa has been hampered by the difficulties associated with isolating cells from the intestine and by the fact that overlapping markers have made it complicated to discriminate them accurately from other intestinal mononuclear phagocytes such as macrophages (MFs). Here we detail the protocols we have developed to isolate live leukocytes from both murine and human small and large intestines and describe reliable strategies which can be used to identify bona fide cDCs in such preparations.
- Published
- 2016
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20. Intestinal macrophages and dendritic cells: what's the difference?
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Cerovic V, Bain CC, Mowat AM, and Milling SW
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- Animals, Cell Differentiation, Dendritic Cells metabolism, Homeostasis, Humans, Immunity, Inflammation immunology, Inflammation metabolism, Inflammation microbiology, Intestinal Mucosa immunology, Intestinal Mucosa metabolism, Intestinal Mucosa microbiology, Intestines microbiology, Macrophages metabolism, Phagocytes immunology, Phagocytes metabolism, T-Lymphocyte Subsets cytology, T-Lymphocyte Subsets immunology, T-Lymphocyte Subsets metabolism, Dendritic Cells immunology, Intestines immunology, Macrophages immunology
- Abstract
Mononuclear phagocytes (MPs) in the murine intestine, comprising dendritic cells (DCs) and macrophages (Mϕs), perform disparate yet complementary immunological functions. Functional analyses of these distinct MP subsets have been complicated by the substantial overlap in their surface phenotypes. Here, we review recent findings that have enabled more accurate definition of these MP subsets. We discuss these recent advances in the context of the current understanding of the functions of DCs and Mϕs in the maintenance of intestinal homeostasis, and how their functions may alter when homeostasis is disrupted., (Copyright © 2014 Elsevier Ltd. All rights reserved.)
- Published
- 2014
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21. Hyporesponsiveness of intestinal dendritic cells to TLR stimulation is limited to TLR4.
- Author
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Cerovic V, Jenkins CD, Barnes AG, Milling SW, MacPherson GG, and Klavinskis LS
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- Animals, Bacillus subtilis immunology, Bone Marrow Cells immunology, Enzyme-Linked Immunosorbent Assay, Flow Cytometry, Immunity, Mucosal immunology, Intestines cytology, Lipopolysaccharides immunology, RNA, Messenger analysis, Rats, Reverse Transcriptase Polymerase Chain Reaction, Toll-Like Receptors immunology, Dendritic Cells cytology, Dendritic Cells immunology, Intestines immunology, Toll-Like Receptor 4 immunology
- Abstract
Dendritic cells (DCs) are crucial to intestinal immune regulation because of their roles in inducing protective immunity against pathogens while maintaining tolerance to commensal bacteria. Nonetheless, relatively little is known about intestinal DC responsiveness to innate immune stimuli via TLRs. We have previously shown that DCs migrating from the rat intestine in lymph (iLDCs) are hyporesponsive to LPS stimulation, thus possibly preventing harmful immune responses being induced to commensal flora. In this study, to understand how iLDC function is regulated by innate immune stimuli, we have characterized the expression and function of TLRs in iLDCs isolated from the thoracic duct lymph of mesenteric lymphadenectomized rats and compared these with DCs grown from bone marrow in the presence of Flt3 ligand. We show that iLDCs express mRNAs for all TLRs, but express significantly less TLR4 mRNA than bone marrow-derived DCs. Functionally, iLDCs could be activated by TLR agonists representing intestinal pathogen-associated molecular patterns, with the important exception of the TLR4 agonist LPS. Furthermore, we show that DCs in the intestinal wall interact directly with noninvasive bacteria (Bacillus subtilis spores), leading to an increase in the output of activated iLDCs into lymph, and that DCs containing spores are activated selectively. These data highlight a functional difference between TLR4 and other TLRs. As iLDCs can respond to TLR stimulation in vitro, there must be other mechanisms that prevent their activation by commensal bacteria under steady-state conditions.
- Published
- 2009
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22. New insights into the roles of dendritic cells in intestinal immunity and tolerance.
- Author
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Cerovic V, McDonald V, Nassar MA, Paulin SM, Macpherson GG, and Milling SW
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- Animals, CD4-Positive T-Lymphocytes immunology, CD8-Positive T-Lymphocytes immunology, Dendritic Cells cytology, Humans, Inflammatory Bowel Diseases microbiology, Intestines immunology, Mucous Membrane cytology, Mucous Membrane immunology, Parasitic Diseases immunology, Cell Movement physiology, Dendritic Cells immunology, Immune Tolerance immunology, Inflammatory Bowel Diseases immunology, Intestines cytology, Virus Diseases immunology
- Abstract
Dendritic cells (DCs) play a critical key role in the initiation of immune responses to pathogens. Paradoxically, they also prevent potentially damaging immune responses being directed against the multitude of harmless antigens, to which the body is exposed daily. These roles are particularly important in the intestine, where only a single layer of epithelial cells provides a barrier against billions of commensal microorganisms, pathogens, and food antigens, over a huge surface area. In the intestine, therefore, DCs are required to perform their dual roles very efficiently to protect the body from the dual threats of invading pathogens and unwanted inflammatory reactions. In this review, we first describe the biology of DCs and their interactions with other cells types, paying particular attention to intestinal DCs. We, then, examine the ways in which this biology may become misdirected, resulting in inflammatory bowel disease. Finally, we discuss how DCs potentiate immune responses against viral, bacterial, parasitic infections, and their importance in the pathogenesis of prion diseases. We, therefore, provide an overview of the complex cellular interactions that affect intestinal DCs and control the balance between immunity and tolerance.
- Published
- 2009
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23. Regulation of intestinal dendritic cell migration and activation by plasmacytoid dendritic cells, TNF-alpha and type 1 IFNs after feeding a TLR7/8 ligand.
- Author
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Yrlid U, Milling SW, Miller JL, Cartland S, Jenkins CD, and MacPherson GG
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- Animal Feed, Animals, Cell Movement immunology, Cells, Cultured, Dendritic Cells metabolism, Imidazoles pharmacology, Interferon Type I genetics, Interferon Type I immunology, Intestinal Mucosa metabolism, Intestines cytology, Ligands, Mice, Mice, Knockout, Plasma Cells cytology, Plasma Cells immunology, Plasma Cells metabolism, Rats, Tumor Necrosis Factor-alpha immunology, Dendritic Cells cytology, Dendritic Cells immunology, Interferon Type I metabolism, Intestines immunology, Toll-Like Receptor 7 immunology, Toll-Like Receptor 8 immunology, Tumor Necrosis Factor-alpha metabolism
- Abstract
Dendritic cells (DCs) migrating via lymph are the primary influence regulating naive T cell differentiation, be it active immunity or tolerance. How DCs achieve this regulation in vivo is poorly understood. Intestinal DCs are in direct contact with harmless or pathogenic luminal contents, but may also be influenced by signals from epithelial cells, macrophages, or other resident or immigrant cells. To understand the role of TLR7 and TLR8 in regulating intestinal DC function, we fed a TLR7/8 ligand (resiquimod (R-848)) to rats and mice and examined DC in pseudoafferent lymph (rat) and mesenteric lymph nodes (MLNs). Oral R-848 induced a 20- to 30-fold increase in DC output from the intestine within 10 h due to a virtually total release of lamina propria DCs. This resulted in an accumulation of DCs in the MLNs that in mice was completely TNF-alpha dependent. Surprisingly, intestinal lymph DCs (iL-DCs) released by R-848 did not up-regulate CD86, but did up-regulate CD25. In contrast, MLN-DCs from R-848-stimulated rats and mice expressed high levels of CD86. This DC activation in MLNs was dependent on type 1 IFNs. The major source of these rapidly released cytokines is plasmacytoid DCs (pDCs) and not classical DCs, because depletion of pDCs significantly reduces the R-848-stimulated increase in serum cytokine levels as well as the accumulation and activation of DCs in MLNs. These experiments show that TLR-mediated regulation of iL-DC functions in vivo is complex and does not depend only on direct iL-DC stimulation, but can be regulated by pDCs.
- Published
- 2006
- Full Text
- View/download PDF
24. Collection of lymph-borne dendritic cells in the rat.
- Author
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Milling SW, Jenkins C, and MacPherson G
- Subjects
- Animals, Intestines immunology, Lymph Node Excision, Male, Rats, Thoracic Duct, Catheterization methods, Dendritic Cells, Lymph cytology
- Abstract
Dendritic cells (DCs) are crucial in immune induction. Not only do they collect antigens in peripheral tissues, and transport and process them for presentation to lymphocytes in draining lymph nodes, but they also regulate the immune response by modulating T-cell differentiation. Intestinal and hepatic DCs migrating in lymph can be collected from rats under near-physiological conditions. Initially, the mesenteric or celiac lymph nodes are removed from young rats (30 min). The afferent and efferent lymph vessels subsequently heal, permitting DCs to enter the thoracic duct. After at least 6 wk, the duct is cannulated (40 min). Lymph can be collected for up to 48 h. DCs can subsequently be identified, enriched and sorted to high degrees of purity. This two-stage technique generates large numbers of immunologically relevant DCs under near-physiological conditions. Lymph collection requires 2-3 h per animal over 6 wk.
- Published
- 2006
- Full Text
- View/download PDF
25. How do DCs interact with intestinal antigens?
- Author
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Milling SW, Cousins L, and MacPherson GG
- Subjects
- Animals, Antigens administration & dosage, Antigens, Bacterial administration & dosage, Dendritic Cells microbiology, Immune Tolerance, Intestines microbiology, Lymph Nodes immunology, Lymph Nodes microbiology, Salmonella immunology, Salmonella pathogenicity, Dendritic Cells immunology, Intestines immunology
- Abstract
Recent evidence demonstrates that dendritic cells (DCs) can insert dendrites between the epithelial cells that form the barrier protecting the body from the gut contents. Although first observed almost a decade ago, this is a controversial area of DC biology and the physiological importance of this phenomenon is only now being clarified. A recent study by Niess and colleagues shows that this behaviour enables efficient sampling of both invasive and non-invasive bacteria and might enhance the ability of an organism to resist infections by a pathogenic strain of Salmonella.
- Published
- 2005
- Full Text
- View/download PDF
26. Inhibition of melanoma growth after treatment with dendritic cells in a Tyr-SV40E murine model requires CD4+ T cells but not CD8+ T cells.
- Author
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Milling SW, Sai T, Silvers WK, and Mintz B
- Subjects
- Animals, Antigens, Neoplasm, Cell Proliferation, Cells, Cultured, Cytokines metabolism, Cytotoxicity Tests, Immunologic, Dendritic Cells metabolism, Immunoglobulin G blood, Lymphocytes, Tumor-Infiltrating immunology, MART-1 Antigen, Macrophages, Melanoma, Experimental immunology, Melanoma, Experimental pathology, Mice, Mice, Inbred C57BL, Mice, Transgenic, Monophenol Monooxygenase immunology, Monophenol Monooxygenase metabolism, Neoplasm Proteins metabolism, Peptide Fragments metabolism, Simian virus 40 immunology, Simian virus 40 metabolism, Skin Neoplasms immunology, Skin Neoplasms pathology, Skin Neoplasms therapy, T-Lymphocytes, Cytotoxic, CD4-Positive T-Lymphocytes immunology, CD8-Positive T-Lymphocytes immunology, Dendritic Cells immunology, Melanoma, Experimental therapy, Monophenol Monooxygenase genetics, Peptide Fragments immunology, Simian virus 40 genetics
- Abstract
Melanomas are promising targets for immunotherapy, as they express a number of tissue-specific antigens against which immune responses can be elicited. We have previously described transgenic mice in which malignant cutaneous melanomas are produced. The 1042 melanoma cell line, derived from a primary melanoma in one of these mice, was used here to generate tumours by subcutaneous inoculation in syngeneic animals. All mice injected with 1 x 10(6) cells of the 1042 cell line developed a tumour. CD4+ T cells, CD8+ T cells and macrophages infiltrated the tumours. Treatment with dendritic cells pulsed with peptides from melanogenic proteins slowed tumour growth and resulted in increased numbers of infiltrating lymphocytes and macrophages, expansion of CD4+ T cells specific for 1042 cell antigens, and increased levels of 1042-specific immunoglobulin G1 (IgG1) and IgM in serum. The frequency of cytotoxic T lymphocytes (CTLs) specific for the MART-1 melanocytic antigen did not increase after dendritic cell treatment. Indeed, the presence of CD8+ T cells was apparently not required for the anti-tumour effects: slowing of tumour growth was not abrogated in animals depleted of CD8+ T cells using antibodies, or in syngeneic CD8-/- animals. In contrast, treatment with dendritic cells + peptides was ineffective after depletion of CD4+ T cells and in syngeneic CD4-/- mice. This experimental system therefore provides an opportunity to investigate CD4-dependent anti-tumour effector mechanisms, and for studies designed to activate the quiescent CTLs which infiltrate melanomas.
- Published
- 2004
- Full Text
- View/download PDF
27. Freezing and thawing of bone marrow-derived murine dendritic cells with subsequent retention of immunophenotype and of antigen processing and presentation characteristics.
- Author
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Sai T, Milling SW, and Mintz B
- Subjects
- Animals, Bone Marrow Cells metabolism, Cell Movement immunology, Cell Survival immunology, Cells, Cultured, Culture Media, Dendritic Cells metabolism, Dendritic Cells transplantation, Epitopes, T-Lymphocyte immunology, Epitopes, T-Lymphocyte metabolism, Female, Fetal Blood, Hybridomas, Injections, Intravenous, Lymphocyte Culture Test, Mixed methods, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Ovalbumin immunology, Ovalbumin metabolism, Protein Denaturation, Spleen cytology, Spleen immunology, T-Lymphocyte Subsets cytology, T-Lymphocyte Subsets immunology, Antigen Presentation, Bone Marrow Cells cytology, Bone Marrow Cells immunology, Cell Culture Techniques methods, Dendritic Cells cytology, Dendritic Cells immunology, Freezing, Immunophenotyping
- Abstract
Murine dendritic cells (DCs) are widely used for experimental vaccinations in mouse models. A high-yield method for freezing and thawing batches of these cells, if compatible with retention of cell immunophenotype, would reduce the time required for repeated preparations from DC precursors in bone marrow (BM), as well as variability among lots. Following depletion of specific lineages, murine bone marrow cells from C57BL/6 inbred-strain mice were grown in medium containing 10% fetal calf serum (FCS) and granulocyte/macrophage colony-stimulating factor (GM-CSF); after 6 days, large numbers of immature DCs were obtained. The immature cells were frozen in complete medium with GM-CSF and 10% DMSO, at a cell density of 5x10(6) DCs/ml. After thawing, 80% of DCs survived; they were induced to mature by addition of lipopolysaccharide (LPS). In comparison with fresh DCs, the thawed DCs had similar morphology, purity, and expression of class I (H-2D(b) and H-2K(b)) and class II major histocompatibility complex (MHC) proteins, as well as CD11b, CD11c, CD40, CD80, and CD86 molecules. Freeze-thawing did not affect trafficking to T cell areas of spleen, nor reduce the capacity to stimulate an alloresponse. Frozen-thawed cells were also proficient at uptake, processing, and presentation of native or denatured ovalbumin (OVA) protein to a peptide-specific T cell hybridoma, and were able to induce T cell responses in vivo after being loaded with denatured OVA protein. The ability to freeze and thaw DCs, and to obtain high yields without altering their essential properties, will facilitate future immunotherapy experiments in laboratory mouse models.
- Published
- 2002
- Full Text
- View/download PDF
28. OVCA2 is downregulated and degraded during retinoid-induced apoptosis.
- Author
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Prowse AH, Vanderveer L, Milling SW, Pan ZZ, Dunbrack RL, Xu XX, and Godwin AK
- Subjects
- Amino Acid Sequence, Animals, Apoptosis genetics, COS Cells, Cell Division drug effects, Down-Regulation, Evolution, Molecular, Fenretinide pharmacology, Genes, Tumor Suppressor, HeLa Cells, Humans, Leukemia, Promyelocytic, Acute, Lung Neoplasms, Minor Histocompatibility Antigens, Models, Molecular, Molecular Sequence Data, Proteins chemistry, RNA, Messenger analysis, RNA, Messenger genetics, Sequence Homology, Tissue Distribution, Tretinoin pharmacology, Tumor Cells, Cultured, Apoptosis drug effects, Proteins genetics, Proteins metabolism, Retinoids pharmacology, Tumor Suppressor Proteins
- Abstract
Retinoids, the natural and synthetic derivatives of vitamin A, have been shown to regulate the growth and differentiation of a wide variety of cell types and consequently have enormous potential as chemotherapeutic agents. We have previously identified 2 genes, termed OVCA1 and OVCA2, which are located in a small region showing a high frequency of allelic loss in breast and ovarian tumors and share a common exon. Recent studies have suggested that expression of OVCA1 may be influenced by retinoids. Therefore, we analyzed the expression of OVCA1 and OVCA2 in cells in response to treatment with all-trans retinoic acid (RA) and N-(4-hydroxyphenyl)retinamide (4HPR), or under conditions of low serum and confluence, to determine further the roles of OVCA1 and OVCA2 in cell growth, apoptosis and differentiation. We show that OVCA2 mRNA and protein are ubiquitously expressed and that they are downregulated in the lung cancer cell line Calu-6 after treatment with RA and 4HPR. In addition, we observed that OVCA2 protein is proteolytically degraded in response to RA and 4HPR treatment in a time- and dose-dependent manner in the promyelocytic leukemia cell line HL60. In contrast, expression of the candidate tumor suppressor OVCA1 was not downregulated by these treatments. Furthermore, we demonstrate that OVCA2 is evolutionarily conserved and shows regional homology with dihydrofolate reductases (DHFRs), specifically with hydrolase folds found in alpha-beta hydrolases. Our results are in contrast to a previous report and show that OVCA2, not OVCA1 mRNA and protein, is downregulated in response to RA and 4HPR., (Copyright 2002 Wiley-Liss, Inc.)
- Published
- 2002
- Full Text
- View/download PDF
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