19 results found
Reddi D, Durant L, Bernardo D, et al., 2021, In vitro priming of human T cells by dendritic cells provides a screening tool for candidate vaccines for Burkholderia pseudomallei, Vaccines, Vol: 9, Pages: 1-10, ISSN: 2076-393X
Murine dendritic cells, when pulsed with heat-killed Burkholderia pseudomallei and used to immunise naïve mice, have previously been shown to induce protective immunity in vivo. We have now demonstrated the in vitro priming of naïve human T cells against heat-killed B. pseudomallei, by co-culture with syngeneic B. pseudomallei-pulsed dendritic cells. Additionally, we have enriched the DC fraction such that a study of the differential response induced by pulsed DCs of either myeloid or plasmacytoid lineage in syngeneic human T cells was achievable. Whilst both mDCs and pDCs were activated by pulsing, the mDCs contributed the major response to B. pseudomallei with the expression of the migration marker CCR7 and a significantly greater secretion of the proinflammatory TNFα and IL1β. When these DC factions were combined and used to prime syngeneic T cells, a significant proliferation was observed in the CD4+ fraction. Here, we have achieved human T cell priming in vitro with unadjuvanted B. pseudomallei, the causative organism of melioidosis, for which there is currently no approved vaccine. We propose that the approach we have taken could be used to screen for the human cellular response to candidate vaccines and formulations, in order to enhance the cell-mediated immunity required to protect against this intracellular pathogen and potentially more broadly against other, difficult-to-treat intracellular pathogens. To date, the polysaccharide capsule of B. pseudomallei, fused to a standard carrier protein, e.g., Crm, looks a likely vaccine candidate. Dendritic cells (DCs), providing, as they do, the first line of defence to infection, process and present microbial products to the immune system to direct downstream immune responses. Here, we have sought to use DCs ex vivo to identify immunogenic products from heat-killed B. pseudomallei. Using practical volumes of fresh human donor blood, we show that heat-killed B. pseudomallei activated and stimula
Dilke SM, Durant LR, Stentz R, et al., 2021, DIRECT MANIPULATION OF THE INTESTINAL MICROBIOME TO INFLUENCE POSTOPERATIVE OUTCOMES, Publisher: OXFORD UNIV PRESS, ISSN: 0007-1323
Durant L, Stentz R, Noble A, et al., 2020, Bacteroides thetaiotaomicron-derived outer membrane vesicles promote regulatory dendritic cell responses in health but not in inflammatory bowel disease, Microbiome, Vol: 8, ISSN: 2049-2618
BackgroundBacteroides thetaiotaomicron (Bt) is a prominent member of the human intestinal microbiota that, like all Gram-negative bacteria, naturally generates nanosized outer membrane vesicles (OMVs) which bud off from the cell surface. Importantly, OMVs can cross the intestinal epithelial barrier to mediate microbe-host cell crosstalk involving both epithelial and immune cells to help maintain intestinal homeostasis. Here we have examined the interaction between Bt OMVs and blood or colonic mucosa-derived dendritic cells (DC) from healthy individuals and patients with Crohn’s disease (CD) or ulcerative colitis (UC). ResultsIn healthy individuals, Bt OMVs stimulated significant (p<0.05) IL-10 expression by colonic DC, whereas in peripheral blood-derived DC they also stimulated significant (p<0.001 and p<0.01, respectively) expression of IL-6 and the activation marker CD80. Conversely, in UC Bt OMVs were unable to elicit IL-10 expression by colonic DC. There were also reduced numbers of CD103+ DC in the colon of both UC and CD patients compared to controls, supporting a loss of regulatory DC in both diseases. Furthermore, in CD and UC, Bt OMVs elicited a significantly lower proportion of DC which expressed IL-10 (p<0.01 and p<0.001, respectively) in blood compared to controls. These alterations in DC responses to Bt OMVs were seen in patients with inactive disease, and thus are indicative of intrinsic defects in immune responses to this commensal in inflammatory bowel disease (IBD). ConclusionsOverall, our findings suggest a key role for OMVs generated by the commensal gut bacterium Bt in directing a balanced immune response to constituents of the microbiota locally and systemically during health which is altered in IBD patients.
Noble A, Durant L, Reddi D, et al., 2020, Deficient resident memory T-cell and CD8 T-cell response to commensals in inflammatory bowel disease, Journal of Crohn's and Colitis, Vol: 14, Pages: 525-537, ISSN: 1873-9946
Background and AimsThe intestinal microbiota is closely associated with resident memory lymphocytes in mucosal tissue. We sought to understand how acquired cellular and humoral immunity to the microbiota differ in health versus inflammatory bowel disease [IBD].MethodsResident memory T cells [Trm] in colonic biopsies and local antibody responses to intraepithelial microbes were analysed. Systemic antigen-specific immune T and B cell memory to a panel of commensal microbes was assessed.ResultsSystemically, healthy blood showed CD4 and occasional CD8 memory T cell responses to selected intestinal bacteria, but few memory B cell responses. In IBD, CD8 memory T cell responses decreased although B cell responses and circulating plasmablasts increased. Possibly secondary to loss of systemic CD8 T cell responses in IBD, dramatically reduced numbers of mucosal CD8+ Trm and γδ T cells were observed. IgA responses to intraepithelial bacteria were increased. Colonic Trm expressed CD39 and CD73 ectonucleotidases, characteristic of regulatory T cells. Cytokines/factors required for Trm differentiation were identified, and in vitro-generated Trm expressed regulatory T cell function via CD39. Cognate interaction between T cells and dendritic cells induced T-bet expression in dendritic cells, a key mechanism in regulating cell-mediated mucosal responses.ConclusionsA previously unrecognised imbalance exists between cellular and humoral immunity to the microbiota in IBD, with loss of mucosal T cell-mediated barrier immunity and uncontrolled antibody responses. Regulatory function of Trm may explain their association with intestinal health. Promoting Trm and their interaction with dendritic cells, rather than immunosuppression, may reinforce tissue immunity, improve barrier function, and prevent B cell dysfunction in microbiota-associated disease and IBD aetiology.
Vora R, Bernardo D, Durant L, et al., 2016, Age-related alterations in blood and colonic dendritic cell properties, Oncotarget, Vol: 7, Pages: 11913-11922, ISSN: 1949-2553
Background Dendritic cells (DC) determine initiation, type and location of immuneresponses and, in adults, show decreased Toll-like receptors and some increasedcytokine levels on ageing. Few studies in children have characterised DC orexplored DC-related mechanisms producing age-related immune changes.Methods Blood and colonic DC phenotypes were determined in healthy adults andchildren by flow cytometry and correlated with aging. Blood DC were divided intoplasmacytoid (pDC) and myeloid (mDC) while only mDC were identified in colon.Serum cytokine levels were determined by multiplex cytokine assays and correlatedwith DC properties.Results The pDC marker BDCA2 (but not CD123) was absent in pre-pubertalchildren and numbers of pDC decreased with age. Blood and colonic DC were moremature and activated in adults. Decrease in pDC numbers correlated with reducedGM-CSF levels with aging, but increasing IL-4 and IL-8 levels correlated with a moreactivated DC profile in blood. CXCL16 levels decreased with age.Conclusions In children, lack of BDCA2, a receptor mediating antigen capture andinhibiting interferon induction, may be immunologically beneficial during immunedevelopment. Conversely, reduced pDC numbers, probably secondary todecreasing GM-CSF and increasing cytokine-induced maturation of DC are likely todetermine deteriorating immunity with ageing.
Goritzka M, Pereira C, Makris S, et al., 2015, T cell responses are elicited against Respiratory Syncytial Virus in the absence of signalling through TLRs, RLRs and IL-1R/IL-18R, Scientific Reports, Vol: 5, ISSN: 2045-2322
Pattern recognition receptors (PRRs) and cytokine receptors are key players in the initiation of immune responses to infection. PRRs detecting viral RNA, such as toll like receptor (TLR)-3, -7/8, and RIG-I like receptors (RLRs; RIG-I and MDA-5), as well as cytokine receptors such as interleukin 1 receptor (IL-1R), have been implicated in responses to RNA viruses that infect the airways. The latter includes respiratory syncytial virus (RSV), a human pathogen that can cause severe lower respiratory tract infections, especially in infants. To evaluate the collective contribution of PRRs and IL-1R signalling to RSV immunity, we generated Myd88/Trif/Mavs−/− mice that are deficient in signalling by all TLRs, RLRs and IL-1R, as well as other cytokine receptors such as IL-18 receptor. Early production of pro-inflammatory mediators and lung infiltration by immune cells were completely abrogated in infected Myd88/Trif/Mavs−/− mice. However, RSV-specific CD8+ T cells were elicited and recruited into the lungs and airways. Consistent with these findings, Myd88/Trif/Mavs−/− mice survived RSV infection but displayed higher viral load and weight loss. These data highlight an unappreciated level of redundancy in pathways that couple innate virus sensing to adaptive immunity, providing the host with remarkable resilience to infection.
Bernardo D, Mann ER, Montalvillo E, et al., 2015, CCR2 mediates dendritic cell recruitment to the human colon but is not responsible for differences observed in dendritic cell subsets, phenotype and function between the proximal and distal colon, Cellular and Molecular Gastroenterology and Hepatology, Vol: 2, Pages: 22-39.e5, ISSN: 2352-345X
Background & aimsMost knowledge about gastrointestinal (GI)-tract dendritic cells (DC) relies on murine studies where CD103+ DC specialize in generating immune tolerance with the functionality of CD11b+/- subsets being unclear. Information about human GI-DC is scarce, especially regarding regional specifications. Here, we characterized human DC properties throughout the human colon.MethodsPaired proximal (right/ascending) and distal (left/descending) human colonic biopsies from 95 healthy subjects were taken; DC were assessed by flow cytometry and microbiota composition assessed by 16S rRNA gene sequencing.ResultsColonic DC identified were myeloid (mDC, CD11c+CD123-) and further divided based on CD103 and SIRPα (human analog of murine CD11b) expression. CD103-SIRPα+ DC were the major population and with CD103+SIRPα+ DC were CD1c+ILT3+CCR2+ (although CCR2 was not expressed on all CD103+SIRPα+ DC). CD103+SIRPα- DC constituted a minor subset that were CD141+ILT3-CCR2-. Proximal colon samples had higher total DC counts and fewer CD103+SIRPα+ cells. Proximal colon DC were more mature than distal DC with higher stimulatory capacity for CD4+CD45RA+ T-cells. However, DC and DC-invoked T-cell expression of mucosal homing markers (β7, CCR9) was lower for proximal DC. CCR2 was expressed on circulating CD1c+, but not CD141+ mDC, and mediated DC recruitment by colonic culture supernatants in transwell assays. Proximal colon DC produced higher levels of cytokines. Mucosal microbiota profiling showed a lower microbiota load in the proximal colon, but with no differences in microbiota composition between compartments.ConclusionsProximal colonic DC subsets differ from those in distal colon being more mature. Targeted immunotherapy using DC in T-cell mediated GI-tract inflammation may therefore need to reflect this immune compartmentalization.
Goritzka M, Makris S, Kausar F, et al., 2015, Alveolar macrophage-derived type I interferons orchestrate innate immunity to RSV through recruitment of antiviral monocytes, Journal of Experimental Medicine, Vol: 212, Pages: 699-714, ISSN: 0022-1007
Type I interferons (IFNs) are important for host defense from viral infections, acting to restrict viral production in infected cells and to promote antiviral immune responses. However, the type I IFN system has also been associated with severe lung inflammatory disease in response to respiratory syncytial virus (RSV). Which cells produce type I IFNs upon RSV infection and how this directs immune responses to the virus, and potentially results in pathological inflammation, is unclear. Here, we show that alveolar macrophages (AMs) are the major source of type I IFNs upon RSV infection in mice. AMs detect RSV via mitochondrial antiviral signaling protein (MAVS)–coupled retinoic acid–inducible gene 1 (RIG-I)–like receptors (RLRs), and loss of MAVS greatly compromises innate immune restriction of RSV. This is largely attributable to loss of type I IFN–dependent induction of monocyte chemoattractants and subsequent reduced recruitment of inflammatory monocytes (infMo) to the lungs. Notably, the latter have potent antiviral activity and are essential to control infection and lessen disease severity. Thus, infMo recruitment constitutes an important and hitherto underappreciated, cell-extrinsic mechanism of type I IFN–mediated antiviral activity. Dysregulation of this system of host antiviral defense may underlie the development of RSV-induced severe lung inflammation.
Goritzka M, Durant L, Pereira C, et al., 2014, Alveolar macrophage-derived type I IFNs orchestrate immune responses to RSV through recruitment of antiviral monocytes, IMMUNOLOGY, Vol: 143, Pages: 104-104, ISSN: 0019-2805
Durant LR, Pereira C, Boakye A, et al., 2014, DNGR-1 is dispensable for CD8(+) T-cell priming during respiratory syncytial virus infection, European Journal of Immunology, Vol: 44, Pages: 2340-2348, ISSN: 1521-4141
During respiratory syncytial virus (RSV) infection CD8+ T cells both assist in viral clearance and contribute to immunopathology. CD8+ T cells recognize viral peptides presented by dendritic cells (DCs), which can directly present viral antigens when infected or, alternatively, “cross-present” antigens after endocytosis of dead or dying infected cells. Mouse CD8α+ and CD103+ DCs excel at cross-presentation, in part because they express the receptor DNGR-1 that detects dead cells by binding to exposed F-actin and routes internalized cell debris into the cross-presentation pathway. As RSV causes death in infected epithelial cells, we tested whether cross-presentation via DNGR-1 is necessary for CD8+ T-cell responses to the virus. DNGR-1-deficient or wild-type mice were intranasally inoculated with RSV and the magnitude of RSV-specific CD8+ T-cell induction was measured. We found that during live RSV infection, cross-presentation via DNGR-1 did not have a major role in the generation of RSV–specific CD8+ T-cell responses. However, after intranasal immunization with dead cells infected with RSV, a dependence on DNGR-1 for RSV-specific CD8+ T-cell responses was observed, confirming the ascribed role of the receptor. Thus, direct presentation by DCs may be the major pathway initiating CD8+ T-cell responses to RSV, while DNGR-1-dependent cross-presentation has no detectable role.
Goritzka M, Durant LR, Pereira C, et al., 2014, Alpha/Beta Interferon Receptor Signaling Amplifies Early Proinflammatory Cytokine Production in the Lung during Respiratory Syncytial Virus Infection, Journal of Virology, Vol: 88, Pages: 6128-6136, ISSN: 0022-538X
Type I interferons (IFNs) are produced early upon virus infection and signal through the alpha/beta interferon (IFN-α/β) receptor (IFNAR) to induce genes that encode proteins important for limiting viral replication and directing immune responses. To investigate the extent to which type I IFNs play a role in the local regulation of inflammation in the airways, we examined their importance in early lung responses to infection with respiratory syncytial virus (RSV). IFNAR1-deficient (IFNAR1−/−) mice displayed increased lung viral load and weight loss during RSV infection. As expected, expression of IFN-inducible genes was markedly reduced in the lungs of IFNAR1−/− mice. Surprisingly, we found that the levels of proinflammatory cytokines and chemokines in the lungs of RSV-infected mice were also greatly reduced in the absence of IFNAR signaling. Furthermore, low levels of proinflammatory cytokines were also detected in the lungs of IFNAR1−/− mice challenged with noninfectious innate immune stimuli such as selected Toll-like receptor (TLR) agonists. Finally, recombinant IFN-α was sufficient to potentiate the production of inflammatory mediators in the lungs of wild-type mice challenged with innate immune stimuli. Thus, in addition to its well-known role in antiviral resistance, type I IFN receptor signaling acts as a central driver of early proinflammatory responses in the lung. Inhibiting the effects of type I IFNs may therefore be useful in dampening inflammation in lung diseases characterized by enhanced inflammatory cytokine production.
Goritzka M, Durant LR, Pereira C, et al., 2014, Interferon-a/b receptor signalling is amplifying early proinflammatory cytokine production in the lung during Respiratory Syncytial Virus (RSV) infection, Annual Congress of the British-Society-for-Immunology, Publisher: Wiley, Pages: 6128-6136, ISSN: 0019-2805
Type I interferons (IFNs) are produced early upon virus infection and signal through the alpha/beta interferon (IFN-/) receptor(IFNAR) to induce genes that encode proteins important for limiting viral replication and directing immune responses. Toinvestigate the extent to which type I IFNs play a role in the local regulation of inflammation in the airways, we examined theirimportance in early lung responses to infection with respiratory syncytial virus (RSV). IFNAR1-deficient (IFNAR1 / ) mice displayedincreased lung viral load and weight loss during RSV infection. As expected, expression of IFN-inducible genes was markedlyreduced in the lungs of IFNAR1 / mice. Surprisingly, we found that the levels of proinflammatory cytokines and chemokinesin the lungs of RSV-infected mice were also greatly reduced in the absence of IFNAR signaling. Furthermore, low levels ofproinflammatory cytokines were also detected in the lungs of IFNAR1 / mice challenged with noninfectious innate immunestimuli such as selected Toll-like receptor (TLR) agonists. Finally, recombinant IFN- was sufficient to potentiate the productionof inflammatory mediators in the lungs of wild-type mice challenged with innate immune stimuli. Thus, in addition to itswell-known role in antiviral resistance, type I IFN receptor signaling acts as a central driver of early proinflammatory responsesin the lung. Inhibiting the effects of type I IFNs may therefore be useful in dampening inflammation in lung diseases characterizedby enhanced inflammatory cytokine production.
Goritzka M, Durant LR, Pereira CR, et al., 2013, Alveolar macrophages modulate early immune responses to Respiratory Syncytial Virus (RSV) infection through MAVS signalling and type I interferons, Annual Congress of the British-Society-for-Immunology, Publisher: WILEY-BLACKWELL, Pages: 87-87, ISSN: 0019-2805
Durant LR, Makris S, Voorburg CM, et al., 2013, Regulatory T Cells Prevent Th2 Immune Responses and Pulmonary Eosinophilia during Respiratory Syncytial Virus Infection in Mice, JOURNAL OF VIROLOGY, Vol: 87, Pages: 10946-10954, ISSN: 0022-538X
Loebbermann J, Durant L, Thornton H, et al., 2013, Defective immunoregulation in RSV vaccine-augmented viral lung disease restored by selective chemoattraction of regulatory T cells, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 110, Pages: 2987-2992, ISSN: 0027-8424
Loebbermann J, Thornton H, Durant L, et al., 2012, Regulatory T cells expressing granzyme B play a critical role in controlling lung inflammation during acute viral infection, MUCOSAL IMMUNOLOGY, Vol: 5, Pages: 161-172, ISSN: 1933-0219
Loebbermann J, Schnoeller C, Thornton H, et al., 2012, IL-10 Regulates Viral Lung Immunopathology during Acute Respiratory Syncytial Virus Infection in Mice, PLOS ONE, Vol: 7, ISSN: 1932-6203
Durant L, Watford WT, Ramos HL, et al., 2010, Diverse Targets of the Transcription Factor STAT3 Contribute to T Cell Pathogenicity and Homeostasis, Immunity, Vol: 32, Pages: 605-615, ISSN: 1074-7613
Watford WT, Hissong BD, Durant LR, et al., 2008, Tpl2 kinase regulates T cell interferon-γ production and host resistance to Toxoplasma gondii, Journal of Experimental Medicine, Vol: 205, Pages: 2803-2812, ISSN: 0022-1007
<jats:p>Tpl2 (Tumor progression locus 2), also known as Cot/MAP3K8, is a hematopoietically expressed serine-threonine kinase. Tpl2 is known to have critical functions in innate immunity in regulating tumor necrosis factor–α, Toll-like receptor, and G protein–coupled receptor signaling; however, our understanding of its physiological role in T cells is limited. We investigated the potential roles of Tpl2 in T cells and found that it was induced by interleukin-12 in human and mouse T cells in a Stat4-dependent manner. Deficiency of Tpl2 was associated with impaired interferon (IFN)-γ production. Accordingly, Tpl2−/− mice had impaired host defense against Toxoplasma gondii with reduced parasite clearance and decreased IFN-γ production. Furthermore, reconstitution of Rag2−/− mice with Tpl2-deficient T cells followed by T. gondii infection recapitulated the IFN-γ defect seen in the Tpl2-deficient mice, confirming a T cell–intrinsic defect. CD4+ T cells isolated from Tpl2−/− mice showed poor induction of T-bet and failure to up-regulate Stat4 protein, which is associated with impaired TCR-dependent extracellular signal-regulated kinase activation. These data underscore the role of Tpl2 as a regulator of T helper cell lineage decisions and demonstrate that Tpl2 has an important functional role in the regulation of Th1 responses.</jats:p>
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