28 results on '"Kirstie M. Bertram"'
Search Results
2. Optimal Isolation Protocols for Examining and Interrogating Mononuclear Phagocytes From Human Intestinal Tissue
- Author
-
Chloe M. Doyle, Erica E. Vine, Kirstie M. Bertram, Heeva Baharlou, Jake W. Rhodes, Suat Dervish, Martijn P. Gosselink, Angelina Di Re, Geoffrey P. Collins, Faizur Reza, James W. T. Toh, Nimalan Pathma-Nathan, Golo Ahlenstiel, Grahame Ctercteko, Anthony L. Cunningham, Andrew N. Harman, and Scott N. Byrne
- Subjects
dendritic cells (DC) ,flow cytometry ,human tissue ,intestine ,enzymatic digestion ,macrophage – cell ,Immunologic diseases. Allergy ,RC581-607 - Abstract
The human intestine contains numerous mononuclear phagocytes (MNP), including subsets of conventional dendritic cells (cDC), macrophages (Mf) and monocytes, each playing their own unique role within the intestinal immune system and homeostasis. The ability to isolate and interrogate MNPs from fresh human tissue is crucial if we are to understand the role of these cells in homeostasis, disease settings and immunotherapies. However, liberating these cells from tissue is problematic as many of the key surface identification markers they express are susceptible to enzymatic cleavage and they are highly susceptible to cell death. In addition, the extraction process triggers immunological activation/maturation which alters their functional phenotype. Identifying the evolving, complex and highly heterogenous repertoire of MNPs by flow cytometry therefore requires careful selection of digestive enzyme blends that liberate viable cells and preserve recognition epitopes involving careful selection of antibody clones to enable analysis and sorting for functional assays. Here we describe a method for the anatomical separation of mucosa and submucosa as well as isolating lymphoid follicles from human jejunum, ileum and colon. We also describe in detail the optimised enzyme digestion methods needed to acquire functionally immature and biologically functional intestinal MNPs. A comprehensive list of screened antibody clones is also presented which allows for the development of high parameter flow cytometry panels to discriminate all currently identified human tissue MNP subsets including pDCs, cDC1, cDC2 (langerin+ and langerin-), newly described DC3, monocytes, Mf1, Mf2, Mf3 and Mf4. We also present a novel method to account for autofluorescent signal from tissue macrophages. Finally, we demonstrate that these methods can successfully be used to sort functional, immature intestinal DCs that can be used for functional assays such as cytokine production assays.
- Published
- 2021
- Full Text
- View/download PDF
3. Identification of HIV transmitting CD11c+ human epidermal dendritic cells
- Author
-
Kirstie M. Bertram, Rachel A. Botting, Heeva Baharlou, Jake W. Rhodes, Hafsa Rana, J. Dinny Graham, Ellis Patrick, James Fletcher, Toby M. Plasto, Naomi R. Truong, Caroline Royle, Chloe M. Doyle, Orion Tong, Najla Nasr, Laith Barnouti, Mark P. Kohout, Andrew J. Brooks, Michael P. Wines, Peter Haertsch, Jake Lim, Martijn P. Gosselink, Grahame Ctercteko, Jacob D. Estes, Melissa J. Churchill, Paul U. Cameron, Eric Hunter, Muzlifah A. Haniffa, Anthony L. Cunningham, and Andrew N. Harman
- Subjects
Science - Abstract
Composition and function of immune populations at barrier surfaces is crucial for response to infection. Here, the authors identify a population of dendritic cells in human epidermis, abundant in anogenital epithelia and distinct from Langerhans cells by surface phenotype and by high capacity for HIV infection and transmission.
- Published
- 2019
- Full Text
- View/download PDF
4. Characterising plasmacytoid and myeloid AXL+ SIGLEC-6+ dendritic cell functions and their interactions with HIV.
- Author
-
Freja A Warner van Dijk, Orion Tong, Thomas R O'Neil, Kirstie M Bertram, Kevin Hu, Heeva Baharlou, Erica E Vine, Kate Jenns, Martijn P Gosselink, James W Toh, Tim Papadopoulos, Laith Barnouti, Gregory J Jenkins, Gavin Sandercoe, Muzlifah Haniffa, Kerrie J Sandgren, Andrew N Harman, Anthony L Cunningham, and Najla Nasr
- Subjects
Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Abstract
AXL+ Siglec-6+ dendritic cells (ASDC) are novel myeloid DCs which can be subdivided into CD11c+ and CD123+ expressing subsets. We showed for the first time that these two ASDC subsets are present in inflamed human anogenital tissues where HIV transmission occurs. Their presence in inflamed tissues was supported by single cell RNA analysis of public databases of such tissues including psoriasis diseased skin and colorectal cancer. Almost all previous studies have examined ASDCs as a combined population. Our data revealed that the two ASDC subsets differ markedly in their functions when compared with each other and to pDCs. Relative to their cell functions, both subsets of blood ASDCs but not pDCs expressed co-stimulatory and maturation markers which were more prevalent on CD11c+ ASDCs, thus inducing more T cell proliferation and activation than their CD123+ counterparts. There was also a significant polarisation of naïve T cells by both ASDC subsets toward Th2, Th9, Th22, Th17 and Treg but less toward a Th1 phenotype. Furthermore, we investigated the expression of chemokine receptors that facilitate ASDCs and pDCs migration from blood to inflamed tissues, their HIV binding receptors, and their interactions with HIV and CD4 T cells. For HIV infection, within 2 hours of HIV exposure, CD11c+ ASDCs showed a trend in more viral transfer to T cells than CD123+ ASDCs and pDCs for first phase transfer. However, for second phase transfer, CD123+ ASDCs showed a trend in transferring more HIV than CD11c+ ASDCs and there was no viral transfer from pDCs. As anogenital inflammation is a prerequisite for HIV transmission, strategies to inhibit ASDC recruitment into inflamed tissues and their ability to transmit HIV to CD4 T cells should be considered.
- Published
- 2024
- Full Text
- View/download PDF
5. AFid: a tool for automated identification and exclusion of autofluorescent objects from microscopy images.
- Author
-
Heeva Baharlou, Nicolas P. Canete, Kirstie M. Bertram, Kerrie J. Sandgren, Anthony Cunningham, Andrew N. Harman, and Ellis Patrick
- Published
- 2021
- Full Text
- View/download PDF
6. HIV transmitting mononuclear phagocytes; integrating the old and new
- Author
-
Erica E. Vine, Jake W. Rhodes, Freja A. Warner van Dijk, Scott N. Byrne, Kirstie M. Bertram, Anthony L. Cunningham, and Andrew N. Harman
- Subjects
Immunology ,Immunology and Allergy - Published
- 2022
7. Defining the landscape of human epidermal mononuclear phagocytes
- Author
-
Kirstie M. Bertram, Thomas R. O’Neil, Erica E. Vine, Heeva Baharlou, Anthony L. Cunningham, and Andrew N. Harman
- Subjects
Infectious Diseases ,Immunology ,Immunology and Allergy - Published
- 2023
8. An in situ quantitative map of initial human colorectal HIV transmission
- Author
-
Heeva Baharlou, Nicolas Canete, Erica E Vine, Kevin Hu, Di Yuan, Kerrie J Sandgren, Kirstie M Bertram, Najla Nasr, Jake W Rhodes, Martijn P Gosselink, Angelina Di Re, Faizur Reza, Grahame Ctercteko, Nimalan Pathma-Nathan, Geoff Collins, James Toh, Ellis Patrick, Muzlifah A Haniffa, Jacob D. Estes, Scott N Byrne, Anthony L Cunningham, and Andrew N Harman
- Abstract
The initial immune response to HIV is critical in determining transmission. However, due to technical limitations we still do not have a comparative map of early mucosal transmission events. We combined RNAscope, cyclic-immunofluorescence and novel image analysis tools to quantify HIV transmission dynamics in intact human colorectal tissue. We mapped HIV enrichment to mucosal dendritic cells (DC) and submucosal macrophages, but not CD4+ T-cells, the primary targets of downstream infection. DCs appeared to funnel virus to lymphoid aggregates which acted as early sanctuaries of high viral titres whilst facilitating HIV passage to the submucosa. Finally, HIV entry induced rapid recruitment and clustering of target cells, facilitating DC and macrophage mediated HIV transfer and enhanced infection of CD4+ T-cells. These data demonstrate a rapid response to HIV structured to maximise the likelihood of mucosal infection, and provide a framework for in situ studies of host pathogen interactions and immune mediated pathologies.Highlights-in situ quantification of host cellular microenvironment response to pathogen invasion in human colorectal tissue.-HIV first localises to mucosal DCs and submucosal macrophages, but not CD4+ T cells.-Viral enrichment first occurs in lymphoid aggregates which is associated with passage into the submucosa.-Early localisation of HIV to CD4+ T cells is associated with interactions with DCs and macrophages.Graphical Abstract
- Published
- 2022
9. HIV transmitting mononuclear phagocytes; integrating the old and new
- Author
-
Erica E, Vine, Jake W, Rhodes, Freja A, Warner van Dijk, Scott N, Byrne, Kirstie M, Bertram, Anthony L, Cunningham, and Andrew N, Harman
- Subjects
CD4-Positive T-Lymphocytes ,Phagocytes ,Langerhans Cells ,Macrophages ,Humans ,HIV Infections ,Dendritic Cells ,Mononuclear Phagocyte System - Abstract
In tissue, mononuclear phagocytes (MNP) are comprised of Langerhans cells, dendritic cells, macrophages and monocyte-derived cells. They are the first immune cells to encounter HIV during transmission and transmit the virus to CD4 T cells as a consequence of their antigen presenting cell function. To understand the role these cells play in transmission, their phenotypic and functional characterisation is important. With advancements in high parameter single cell technologies, new MNPs subsets are continuously being discovered and their definition and classification is in a state of flux. This has important implications for our knowledge of HIV transmission, which requires a deeper understanding to design effective vaccines and better blocking strategies. Here we review the historical research of the role MNPs play in HIV transmission up to the present day and revaluate these studies in the context of our most recent understandings of the MNP system.
- Published
- 2021
10. OMIP 082: A 25-color phenotyping to define human innate lymphoid cells, natural killer cells, mucosal-associated invariant T cells, and γδ T cells from freshly isolated human intestinal tissue
- Author
-
Chloe M. Doyle, Nicole L. Fewings, Grahame Ctercteko, Scott N. Byrne, Andrew N. Harman, and Kirstie M. Bertram
- Subjects
Intestines ,Killer Cells, Natural ,Histology ,Humans ,Cell Biology ,Flow Cytometry ,Biomarkers ,Immunity, Innate ,Mucosal-Associated Invariant T Cells ,Pathology and Forensic Medicine - Abstract
We developed a 25-color flow cytometry panel to comprehensively interrogate innate lymphoid cells (ILC), mucosal-associated invariant T (MAIT) cells, natural killer (NK) cells and γδ T cells in human tissues. The ability to isolate and interrogate these cells from fresh human tissue is crucial in understanding the role these cells play at immune-privileged mucosal surfaces like the intestine in health and disease settings. However, liberating these cells from tissue is extremely challenging as many key surface identification markers are susceptible to enzymatic cleavage. Choosing the correct enzyme-antibody clone combination within a high-parameter panel is, therefore, a critical consideration. Here, we present a comprehensive, in-depth analysis of the effect different common digestive enzyme blends have on key surface markers used to identify these cell types. In addition, we compared multiple antibody clones for surface markers that are highly susceptible to enzymatic cleavage, such as CD127 and NKp44, to achieve the most consistent and superior staining patterns among donors.
- Published
- 2021
11. Relay of herpes simplex virus between Langerhans cells and dermal dendritic cells in human skin.
- Author
-
Min Kim, Naomi R Truong, Virginia James, Lidija Bosnjak, Kerrie J Sandgren, Andrew N Harman, Najla Nasr, Kirstie M Bertram, Norman Olbourne, Shailandra Sawleshwarkar, Kaylene McKinnon, Ralph C Cohen, and Anthony L Cunningham
- Subjects
Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Abstract
The mechanism by which immunity to Herpes Simplex Virus (HSV) is initiated is not completely defined. HSV initially infects mucosal epidermis prior to entering nerve endings. In mice, epidermal Langerhans cells (LCs) are the first dendritic cells (DCs) to encounter HSV, but it is CD103(+) dermal DCs that carry viral antigen to lymph nodes for antigen presentation, suggesting DC cross-talk in skin. In this study, we compared topically HSV-1 infected human foreskin explants with biopsies of initial human genital herpes lesions to show LCs are initially infected then emigrate into the dermis. Here, LCs bearing markers of maturation and apoptosis formed large cell clusters with BDCA3(+) dermal DCs (thought to be equivalent to murine CD103(+) dermal DCs) and DC-SIGN(+) DCs/macrophages. HSV-expressing LC fragments were observed inside the dermal DCs/macrophages and the BDCA3(+) dermal DCs had up-regulated a damaged cell uptake receptor CLEC9A. No other infected epidermal cells interacted with dermal DCs. Correspondingly, LCs isolated from human skin and infected with HSV-1 in vitro also underwent apoptosis and were taken up by similarly isolated BDCA3(+) dermal DCs and DC-SIGN(+) cells. Thus, we conclude a viral antigen relay takes place where HSV infected LCs undergo apoptosis and are taken up by dermal DCs for subsequent antigen presentation. This provides a rationale for targeting these cells with mucosal or perhaps intradermal HSV immunization.
- Published
- 2015
- Full Text
- View/download PDF
12. An in situ analysis pipeline for initial host-pathogen interactions reveals signatures of human colorectal HIV transmission
- Author
-
Heeva Baharlou, Nicolas Canete, Erica E. Vine, Kevin Hu, Di Yuan, Kerrie J. Sandgren, Kirstie M. Bertram, Najla Nasr, Jake W. Rhodes, Martijn P. Gosselink, Angelina Di Re, Faizur Reza, Grahame Ctercteko, Nimalan Pathma-Nathan, Geoff Collins, James Toh, Ellis Patrick, Muzlifah A. Haniffa, Jacob D. Estes, Scott N. Byrne, Anthony L. Cunningham, and Andrew N. Harman
- Subjects
CD4-Positive T-Lymphocytes ,Host-Pathogen Interactions ,HIV-1 ,Humans ,HIV Infections ,Dendritic Cells ,Colorectal Neoplasms ,General Biochemistry, Genetics and Molecular Biology - Abstract
The initial immune response to HIV determines transmission. However, due to technical limitations we still do not have a comparative map of early mucosal transmission events. By combining RNAscope, cyclic immunofluorescence, and image analysis tools, we quantify HIV transmission signatures in intact human colorectal explants within 2 h of topical exposure. We map HIV enrichment to mucosal dendritic cells (DCs) and submucosal macrophages, but not CD4
- Published
- 2022
13. Human anogenital monocyte-derived dendritic cells and langerin+cDC2 are major HIV target cells
- Author
-
Anneliese S. Ashhurst, Peter A. Haertsch, Martijn P. Gosselink, J. Dinny Graham, Grant P Parnell, Laith Barnouti, Kirstie M. Bertram, Andrew N. Harman, Faizur Reza, Najla Nasr, James Fletcher, Ellis Patrick, Thomas R. O’Neil, Jake J. K. Lim, Eric Hunter, Peter Vegh, Scott N. Byrne, Grahame Ctercteko, Rachel A. Botting, Hafsa Rana, Gregory Jenkins, Heeva Baharlou, Jake W. Rhodes, Andrew J. Brooks, Erica E. Vine, Anthony L. Cunningham, Angelina Di Re, and Muzlifah Haniffa
- Subjects
0301 basic medicine ,CD4-Positive T-Lymphocytes ,Sexual transmission ,Langerin ,Receptors, CCR5 ,Transcription, Genetic ,Sialic Acid Binding Ig-like Lectin 1 ,Science ,CD14 ,Antigen presentation ,Lipopolysaccharide Receptors ,General Physics and Astronomy ,Anal Canal ,Inflammation ,HIV Infections ,Virus-host interactions ,General Biochemistry, Genetics and Molecular Biology ,Virus ,Article ,Monocytes ,03 medical and health sciences ,0302 clinical medicine ,Antigen ,Antigens, CD ,medicine ,Humans ,Lectins, C-Type ,Collagenases ,Genitalia ,Receptor ,Cell Shape ,Phagocytes ,Multidisciplinary ,Retrovirus ,Mucous Membrane ,biology ,General Chemistry ,Dendritic Cells ,Dermis ,Innate immune cells ,030104 developmental biology ,Mannose-Binding Lectins ,Phenotype ,Immunology ,biology.protein ,HIV-1 ,medicine.symptom ,030215 immunology - Abstract
Tissue mononuclear phagocytes (MNP) are specialised in pathogen detection and antigen presentation. As such they deliver HIV to its primary target cells; CD4 T cells. Most MNP HIV transmission studies have focused on epithelial MNPs. However, as mucosal trauma and inflammation are now known to be strongly associated with HIV transmission, here we examine the role of sub-epithelial MNPs which are present in a diverse array of subsets. We show that HIV can penetrate the epithelial surface to interact with sub-epithelial resident MNPs in anogenital explants and define the full array of subsets that are present in the human anogenital and colorectal tissues that HIV may encounter during sexual transmission. In doing so we identify two subsets that preferentially take up HIV, become infected and transmit the virus to CD4 T cells; CD14+CD1c+ monocyte-derived dendritic cells and langerin-expressing conventional dendritic cells 2 (cDC2)., Epithelial tissue mononuclear phagocytes (MNP) can transmit HIV to CD4 T cells, but less is known about sub-epithelial cells. Here, the authors describe MNPs in human anogenital and colorectal tissues and find that CD14+CD1c+ monocyte-derived dendritic cells and langerin-expressing conventional dendritic cells 2 preferentially take up and transmit HIV.
- Published
- 2021
14. Herpes Simplex Virus type 1 infects Langerhans cells and the novel epidermal dendritic cell, Epi-cDC2s, via different entry pathways
- Author
-
Monica Miranda-Saksena, Ellis Patrick, Jake W. Rhodes, Kevin Danastas, Kirstie M. Bertram, Kerrie J Sandgren, Hafsa Rana, Naomi R. Truong, Min Kim, Steven Merten, Konrad L. Feng, Andrew N. Harman, Jake Lim, Ralph C. Cohen, Jason J. Herbert, Anthony L. Cunningham, and Jacinta B. Smith
- Subjects
Cultured tumor cells ,Apoptosis ,Herpesvirus 1, Human ,Pathology and Laboratory Medicine ,medicine.disease_cause ,White Blood Cells ,Spectrum Analysis Techniques ,0302 clinical medicine ,Animal Cells ,Chlorocebus aethiops ,Medicine and Health Sciences ,HaCaT Cells ,Biology (General) ,Child ,Cells, Cultured ,Skin ,0303 health sciences ,education.field_of_study ,Cell Death ,integumentary system ,T Cells ,Dermis ,Flow Cytometry ,Cell biology ,Cell Processes ,Spectrophotometry ,Medical Microbiology ,Viral Pathogens ,Child, Preschool ,Viruses ,Herpes Simplex Virus-1 ,Cell lines ,Cytophotometry ,Anatomy ,Integumentary System ,Cellular Types ,Pathogens ,Biological cultures ,Research Article ,Signal Transduction ,Herpesviruses ,Cell type ,Adolescent ,QH301-705.5 ,Immune Cells ,Immunology ,Population ,Biology ,Research and Analysis Methods ,Microbiology ,03 medical and health sciences ,Immune system ,Virology ,Genetics ,medicine ,Animals ,Humans ,HeLa cells ,education ,Microbial Pathogens ,Vero Cells ,Molecular Biology ,030304 developmental biology ,Blood Cells ,Epidermis (botany) ,Organisms ,Biology and Life Sciences ,Infant ,Herpes Simplex ,Cell Biology ,Dendritic cell ,Virus Internalization ,RC581-607 ,Cell cultures ,Herpes Simplex Virus ,Herpes simplex virus ,Langerhans Cells ,Vero cell ,Parasitology ,Epidermis ,Immunologic diseases. Allergy ,DNA viruses ,030215 immunology - Abstract
Skin mononuclear phagocytes (MNPs) provide the first interactions of invading viruses with the immune system. In addition to Langerhans cells (LCs), we recently described a second epidermal MNP population, Epi-cDC2s, in human anogenital epidermis that is closely related to dermal conventional dendritic cells type 2 (cDC2) and can be preferentially infected by HIV. Here we show that in epidermal explants topically infected with herpes simplex virus (HSV-1), both LCs and Epi-cDC2s interact with HSV-1 particles and infected keratinocytes. Isolated Epi-cDC2s support higher levels of infection than LCs in vitro, inhibited by acyclovir, but both MNP subtypes express similar levels of the HSV entry receptors nectin-1 and HVEM, and show similar levels of initial uptake. Using inhibitors of endosomal acidification, actin and cholesterol, we found that HSV-1 utilises different entry pathways in each cell type. HSV-1 predominantly infects LCs, and monocyte-derived MNPs, via a pH-dependent pathway. In contrast, Epi-cDC2s are mainly infected via a pH-independent pathway which may contribute to the enhanced infection of Epi-cDC2s. Both cells underwent apoptosis suggesting that Epi-cDC2s may follow the same dermal migration and uptake by dermal MNPs that we have previously shown for LCs. Thus, we hypothesize that the uptake of HSV and infection of Epi-cDC2s will stimulate immune responses via a different pathway to LCs, which in future may help guide HSV vaccine development and adjuvant targeting., Author summary Here we describe the first interactions of herpes simplex virus type 1 (HSV-1) with the human immune system as it enters the human genital skin. It was previously thought that this initial interaction of HSV-1 was only with ‘Langerhans cells’ (LCs) but we describe another important interaction with a new immune cell, Epi-cDC2s, which can also be infected by HIV and much more efficiently than LCs. Thus, there are now two of these types of immune cells resident in human epidermis. Therefore the way in which sexually transmitted and skin infecting viruses establish infection needs to be re-examined, including for HSV. We compared how these two cell types interacted with HSV-1 and showed Epi-cDC2s are more susceptible to HSV-1 infection than LCs and take up the virus via a different entry pathway. We speculate these cells may then carry HSV fragments to the dermis and then to lymph nodes to stimulate a protective immune response. These findings suggest similar routes may apply for chickenpox and cowpox viruses which also enter via the skin. Studying this pathway to establishment of natural immunity to HSV, may help guide the development of a successful vaccine.
- Published
- 2021
15. Identification of HIV transmitting CD11c+ human epidermal dendritic cells
- Author
-
Andrew N. Harman, Eric Hunter, Chloe M Doyle, Najla Nasr, Hafsa Rana, Jake W. Rhodes, Jake Lim, Caroline Royle, James Fletcher, Naomi R. Truong, Mark P. Kohout, Paul U. Cameron, Laith Barnouti, Jacob D. Estes, Michael Wines, Andrew J. Brooks, Ellis Patrick, Peter A. Haertsch, Rachel A. Botting, J. Dinny Graham, Kirstie M. Bertram, Toby M. Plasto, Anthony L. Cunningham, Heeva Baharlou, Muzlifah Haniffa, Orion Tong, Grahame Ctercteko, Martijn P. Gosselink, and Melissa J Churchill
- Subjects
Male ,0301 basic medicine ,T-Lymphocytes ,Sialic Acid Binding Ig-like Lectin 3 ,General Physics and Astronomy ,HIV Infections ,02 engineering and technology ,lcsh:Science ,Cells, Cultured ,Antigen Presentation ,education.field_of_study ,Multidisciplinary ,integumentary system ,virus diseases ,hemic and immune systems ,Mononuclear phagocyte system ,021001 nanoscience & nanotechnology ,Healthy Volunteers ,3. Good health ,Cell biology ,Mucosal immunology ,Female ,0210 nano-technology ,Sexual transmission ,Receptors, CCR5 ,Science ,Primary Cell Culture ,Population ,Antigen presentation ,CD11c ,chemical and pharmacologic phenomena ,Biology ,Article ,General Biochemistry, Genetics and Molecular Biology ,Virus ,03 medical and health sciences ,Humans ,education ,Epidermis (botany) ,Dendritic Cells ,General Chemistry ,Virus Internalization ,CD11c Antigen ,030104 developmental biology ,Epidermal Cells ,Cell culture ,HIV-1 ,lcsh:Q ,Epidermis - Abstract
Langerhans cells (LC) are thought to be the only mononuclear phagocyte population in the epidermis where they detect pathogens. Here, we show that CD11c+ dendritic cells (DCs) are also present. These cells are transcriptionally similar to dermal cDC2 but are more efficient antigen-presenting cells. Compared to LCs, epidermal CD11c+ DCs are enriched in anogenital tissues where they preferentially interact with HIV, express the higher levels of HIV entry receptor CCR5, support the higher levels of HIV uptake and replication and are more efficient at transmitting the virus to CD4 T cells. Importantly, these findings are observed using both a lab-adapted and transmitted/founder strain of HIV. We also describe a CD33low cell population, which is transcriptionally similar to LCs but does not appear to function as antigen-presenting cells or acts as HIV target cells. Our findings reveal that epidermal DCs in anogenital tissues potentially play a key role in sexual transmission of HIV., Composition and function of immune populations at barrier surfaces is crucial for response to infection. Here, the authors identify a population of dendritic cells in human epidermis, abundant in anogenital epithelia and distinct from Langerhans cells by surface phenotype and by high capacity for HIV infection and transmission.
- Published
- 2019
16. AFid: A tool for automated identification and exclusion of autofluorescent objects from microscopy images
- Author
-
Kerrie J Sandgren, Nicolas P Canete, Heeva Baharlou, Anthony L. Cunningham, Ellis Patrick, Kirstie M. Bertram, and Andrew N. Harman
- Subjects
Statistics and Probability ,Computer science ,Image Processing ,Image processing ,Image Analysis ,Biochemistry ,Workflow ,03 medical and health sciences ,Microscopy ,Image Processing, Computer-Assisted ,Fluorescence microscope ,Humans ,Computer vision ,Instrumentation (computer programming) ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,Pixel ,business.industry ,Histological Techniques ,030302 biochemistry & molecular biology ,Computer Science Applications ,Staining ,Computational Mathematics ,Autofluorescence ,Identification (information) ,Microscopy, Fluorescence ,Computational Theory and Mathematics ,Artificial intelligence ,business ,Software - Abstract
Motivation Autofluorescence is a long-standing problem that has hindered the analysis of images of tissues acquired by fluorescence microscopy. Current approaches to mitigate autofluorescence in tissue are lab-based and involve either chemical treatment of sections or specialized instrumentation and software to ‘unmix’ autofluorescent signals. Importantly, these approaches are pre-emptive and there are currently no methods to deal with autofluorescence in acquired fluorescence microscopy images. Results To address this, we developed Autofluorescence Identifier (AFid). AFid identifies autofluorescent pixels as discrete objects in multi-channel images post-acquisition. These objects can then be tagged for exclusion from downstream analysis. We validated AFid using images of FFPE human colorectal tissue stained for common immune markers. Further, we demonstrate its utility for image analysis where its implementation allows the accurate measurement of HIV–Dendritic cell interactions in a colorectal explant model of HIV transmission. Therefore, AFid represents a major leap forward in the extraction of useful data from images plagued by autofluorescence by offering an approach that is easily incorporated into existing workflows and that can be used with various samples, staining panels and image acquisition methods. We have implemented AFid in ImageJ, Matlab and R to accommodate the diverse image analysis community. Availability and implementation AFid software is available at https://ellispatrick.github.io/AFid. Supplementary information Supplementary data are available at Bioinformatics online.
- Published
- 2020
- Full Text
- View/download PDF
17. Identification of HIV-Transmitting Sub-Epithelial Mononuclear Phagocytes in Human Anogenital and Colorectal Tissues
- Author
-
Najla Nasr, Andrew N. Harman, Grant P Parnell, Rachel A. Botting, Martijn P. Gosselink, Thomas R. O’Neil, Peter Vegh, Ellis Patrick, Erica E Longmuir-Vine, Scott N. Byrne, Grahame Ctercteko, Heeva Baharlou, Hafsa Rana, Gregory Jenkins, Peter A. Haertsch, J. Dinny Graham, Laith Barnouti, Kirstie M. Bertram, James Fletcher, Jake J. K. Lim, Muzlifah Haniffa, Andrew J. Brooks, Jake W. Rhodes, Anthony L. Cunningham, and Angelina Di Re
- Subjects
Immune system ,Sexual transmission ,Transmission (medicine) ,CD14 ,Antigen presentation ,Immunology ,medicine ,CD11c ,Inflammation ,Biology ,medicine.symptom ,Virus - Abstract
Tissue mononuclear phagocytes (MNP) are specialised in pathogen detection and antigen presentation. They are the first cells of the immune system to encounter HIV and play a key role in transmission as they deliver the virus to CD4 T cells, which are the primary HIV target cell in which the virus undergoes replication. Most studies have investigated the role that epithelial MNPs play in HIV transmission but, as mucosal trauma and inflammation are strongly associated with HIV transmission, it is also important to examine the role that sub-epithelial MNPs play. Sub-epithelial MNPs are present in a diverse array of subsets which differ in their function and the pathogens they detect. Understanding how specific subsets interact with HIV and deliver the virus to CD4 T cells is therefore of key importance to vaccine and microbicide development. In this study we have shown that, after topical application, HIV can penetrate to interact with sub-epithelial resident myeloid cells in anogenital explants and defined the full array of MNP subsets that are present in all the human anogenital and colorectal sub-epithelial tissues that HIV may encounter during sexual transmission. In doing so we have identified two subsets that preferentially take up HIV, become infected and transmit the virus to CD4 T cells; CD14+CD1c+CD11c+monocyte-derived dendritic cells and langerin-expressing dendritic cells 2 (DC2).
- Published
- 2020
18. Vaccines for Herpes Simplex: Recent Progress Driven by Viral and Adjuvant Immunology
- Author
-
Naomi R. Truong, Kerrie J Sandgren, Kirstie M. Bertram, Jacinta B. Smith, and Anthony L. Cunningham
- Subjects
0301 basic medicine ,Innate immune system ,biology ,business.industry ,viruses ,medicine.medical_treatment ,Vaccination ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Immune system ,Antigen ,Viral entry ,Immunology ,biology.protein ,Medicine ,030212 general & internal medicine ,Viral shedding ,Antibody ,business ,Adjuvant - Abstract
Herpes simplex viruses (HSV) types 1 and 2 are ubiquitous. They both cause genital herpes, occasionally severe disease in the immunocompromised, and facilitate much HIV acquisition globally. Despite more than 60 years of research, there is no licensed prophylactic HSV vaccine and some doubt as to whether this can be achieved. Nevertheless, a previous HSV vaccine candidate did have partial success in preventing genital herpes and HSV acquisition and another immunotherapeutic candidate reduced viral shedding and recurrent lesions, inspiring further research. However, the entry pathway of HSV into the anogenital mucosa and the subsequent cascade of immune responses need further elucidation so that these responses could be mimicked or improved by a vaccine, to prevent viral entry and colonization of the neuronal ganglia. For an effective novel vaccine against genital herpes the choice of antigen and adjuvant may be critical. The incorporation of adjuvants of the vaccine candidates in the past, may account for their partial efficacy. It is likely that they can be improved by understanding the mechanisms of immune responses elicited by different adjuvants and comparing these to natural immune responses. Here we review the history of vaccines for HSV, those in development and compare them to successful vaccines for chicken pox or herpes zoster. We also review what is known of the natural immune control of herpes lesions, via interacting innate immunity and CD4 and CD8 T cells and the lessons they provide for development of new, more effective vaccines.
- Published
- 2019
19. A NOVEL LANGERIN EXPRESSING TYPE 2-CONVENTIONAL DENDRITIC CELL IS SIGNIFICANTLY DECREASED IN CROHN’S DISEASE
- Author
-
Scott N. Byrne, Kirstie M. Bertram, Jake W. Rhodes, Grahame Ctercteko, Chloe M Doyle, Anthony L. Cunningham, and Andrew N. Harman
- Subjects
Crohn's disease ,Hepatology ,Langerin ,biology ,Gastroenterology ,Mucous membrane ,Transforming growth factor beta ,medicine.disease ,Interleukin 22 ,medicine.anatomical_structure ,Immune system ,Immunology ,medicine ,biology.protein ,Interleukin 17 ,Conventional Dendritic Cell - Published
- 2021
20. Digital removal of autofluorescence from microscopy images
- Author
-
Heeva Baharlou, Andrew N. Harman, Kirstie M. Bertram, Ellis Patrick, Anthony L. Cunningham, Nicolas P Canete, and Kerrie J Sandgren
- Subjects
In situ ,Autofluorescence ,Materials science ,Microscopy ,Fluorescence microscope ,Digital removal ,Protein expression ,Biomedical engineering - Abstract
Autofluorescence is a long-standing problem that has hindered fluorescence microscopy image analysis. To address this, we have developed a method that identifies and removes autofluorescent signals from multi-channel images post acquisition. We demonstrate the broad utility of this algorithm in accurately assessing protein expression in situ through the removal of interfering autofluorescent signals.Availability and implementationhttps://ellispatrick.github.io/AFremoverContactellis.patrick@sydney.edu.auSupplementary informationSupplementary Figs. 1–13
- Published
- 2019
- Full Text
- View/download PDF
21. FOXP3+TREGS THAT EXPRESSED CD127 EXISTING MEMORY TREGS FEATURE IN A TOLERANT MOUSE MODEL AND IN HUMAN SKIN
- Author
-
Thomas R. O’Neil, Leigh Nicholson, Andrew N. Harman, Natasha M. Rogers, Shounan Yi, Stephen I. Alexander, Yuanfei Zhao, Philip J. O'Connell, Min Hu, Kirstie M. Bertram, and Wayne J. Hawthorne
- Subjects
Transplantation ,Feature (computer vision) ,Computer science ,FOXP3 ,Human skin ,Computational biology ,Interleukin-7 receptor - Published
- 2020
22. Herpes Simplex Virus Type 2–Infected Dendritic Cells Produce TNF-α, Which Enhances CCR5 Expression and Stimulates HIV Production from Adjacent Infected Cells
- Author
-
Najla Nasr, Andrew R. Lloyd, Elizabeth Keoshkerian, Valerie Marsden, Heather Donaghy, Anthony L. Cunningham, Andrew N. Harman, Steven Merten, and Kirstie M. Bertram
- Subjects
Receptors, CCR5 ,Herpesvirus 2, Human ,medicine.medical_treatment ,Immunology ,HIV Infections ,Stratified squamous epithelium ,Biology ,Virus Replication ,medicine.disease_cause ,Models, Biological ,Microbiology ,Dermis ,medicine ,Humans ,Immunology and Allergy ,Herpes Genitalis ,Epidermis (botany) ,Coinfection ,Tumor Necrosis Factor-alpha ,Dendritic Cells ,medicine.disease ,Up-Regulation ,medicine.anatomical_structure ,Herpes simplex virus ,Cytokine ,Gene Expression Regulation ,Viral replication ,Culture Media, Conditioned ,HIV-1 ,Cytokines ,Tumor necrosis factor alpha - Abstract
Prior HSV-2 infection enhances the acquisition of HIV-1 >3-fold. In genital herpes lesions, the superficial layers of stratified squamous epithelium are disrupted, allowing easier access of HIV-1 to Langerhans cells (LC) in the epidermis and perhaps even dendritic cells (DCs) in the outer dermis, as well as to lesion infiltrating activated T lymphocytes and macrophages. Therefore, we examined the effects of coinfection with HIV-1 and HSV-2 on monocyte-derived DCs (MDDC). With simultaneous coinfection, HSV-2 significantly stimulated HIV-1 DNA production 5-fold compared with HIV-1 infection alone. Because
- Published
- 2015
23. Phenotypic and functional consequences of different isolation protocols on skin mononuclear phagocytes
- Author
-
Rachel A, Botting, Kirstie M, Bertram, Heeva, Baharlou, Kerrie J, Sandgren, James, Fletcher, Jake W, Rhodes, Hafsa, Rana, Toby M, Plasto, Xin Maggie, Wang, Jake J K, Lim, Laith, Barnouti, Mark P, Kohout, Tim, Papadopoulos, Steve, Merten, Norman, Olbourne, Anthony L, Cunningham, Muzlifah, Haniffa, and Andrew N, Harman
- Subjects
Phagocytes ,Phenotype ,Cell Movement ,ex vivo ,Humans ,Cell Separation ,human ,macrophage ,Host Defense & Pathophysiology ,DC ,Monocytes ,Enzymes ,Skin - Abstract
Comparison of skin mononuclear phagocyte isolation techniques on function and subset definition, and the effects of collagenase blends on pathogen binding receptor cleavage., Mononuclear phagocytes are present in skin and mucosa and represent one of the first lines of defense against invading pathogens, which they detect via an array of pathogen-binding receptors expressed on their surface. However, their extraction from tissue is difficult, and the isolation technique used has functional consequences on the cells obtained. Here, we compare mononuclear phagocytes isolated from human skin using either enzymatic digestion or spontaneous migration. Cells isolated via enzymatic digestion are in an immature state, and all subsets are easily defined. However, cells isolated by spontaneous migration are in a mature state, and CD141 cross-presenting DCs (cDC1) are more difficult to define. Different pathogen-binding receptors are susceptible to cleavage by blends of collagenase, demonstrating that great care must be taken in choosing the correct enzyme blend to digest tissue if carrying out pathogen-interaction assays. Finally, we have optimized mononuclear phagocyte culture conditions to enhance their survival after liberation from the tissue.
- Published
- 2016
24. Langerhans cells and sexual transmission of HIV and HSV
- Author
-
Najla Nasr, Kirstie M. Bertram, Anthony L. Cunningham, Heeva Baharlou, Andrew N. Harman, Hafsa Rana, Jake W. Rhodes, and Rachel A. Botting
- Subjects
0301 basic medicine ,Sexual transmission ,Cell ,Stratified squamous epithelium ,HIV Infections ,HSL and HSV ,Biology ,03 medical and health sciences ,0302 clinical medicine ,Immune system ,Virology ,medicine ,Humans ,Antigen-presenting cell ,Herpes Genitalis ,integumentary system ,Transmission (medicine) ,030104 developmental biology ,Infectious Diseases ,medicine.anatomical_structure ,Langerhans Cells ,Immunology ,Host-Pathogen Interactions ,Function (biology) ,030215 immunology - Abstract
Langerhans cells (LCs) situated in stratified squamous epithelium of the skin and mucosal tissue are amongst the first cells that sexually transmitted pathogens encounter during transmission. They are potent antigen presenting cells and play a key role in the host mounting an appropriate immune response. As such, viruses have evolved complex strategies to manipulate these cells to facilitate successful transmission. One of best studied examples is HIV, which manipulates the natural function of these cells to interact with CD4 T cells, which are the main target cell for HIV in which rapid replication occurs. However, there is controversy in the literature as to the role that LCs play in this process. Langerhans cells also play a key role in the way the body mounts an immune response to HSV, and there is also a complex interplay between the transmission of HSV and HIV that involves LCs. In this article, we review both past and present literatures with a particular focus on a few very recent studies that shed new light on the role that LCs play in the transmission and immune response to these 2 pathogens.
- Published
- 2016
25. Understanding natural herpes simplex virus immunity to inform next-generation vaccine design
- Author
-
Anthony L. Cunningham, Kirstie M. Bertram, and Kerrie J Sandgren
- Subjects
0301 basic medicine ,Partially successful ,Innate immune system ,Effector ,medicine.medical_treatment ,Immunology ,Review ,Immune control ,Biology ,medicine.disease_cause ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Herpes simplex virus ,Immune system ,Immunity ,medicine ,Immunology and Allergy ,Adjuvant ,General Nursing ,030215 immunology - Abstract
Incremental advances in our knowledge of how natural immune control of herpes simplex virus (HSV) develops have yielded insight as to why previous vaccine attempts have only been partially successful, however, our understanding of these pathways, particularly in humans, is still incomplete. Further elucidation of the innate immune events that are responsible for stimulating these effector responses is required to accurately inform vaccine design. An enhanced understanding of the mechanism of action of novel adjuvants will also facilitate the rational choice of adjuvant to optimise such responses. Here we review the reasons for the hitherto partial HSV vaccine success and align these with our current knowledge of how natural HSV immunity develops. In particular, we focus on the innate immune response and the role of dendritic cells in inducing protective T-cell responses and how these pathways might be recapitulated in a vaccine setting.
- Published
- 2016
26. LB1.5 Initial interactions of herpes simplex virus with human skin dendritic cells
- Author
-
Naomi R. Truong, Najla Nasr, S Sawleshwarkar, V James, Anthony L. Cunningham, Lidija Bosnjak, Kirstie M. Bertram, Min Kim, Andrew N. Harman, Kerrie J Sandgren, Norman Olbourne, Ralph C. Cohen, and K McKinnon
- Subjects
integumentary system ,Antigen presentation ,hemic and immune systems ,chemical and pharmacologic phenomena ,Human skin ,Stratified squamous epithelium ,Dermatology ,Biology ,medicine.disease_cause ,Foreskin ,Infectious Diseases ,medicine.anatomical_structure ,Herpes simplex virus ,Antigen ,Dermis ,Immunology ,medicine ,CD80 - Abstract
Introduction The mechanism by which immunity to herpes simplex virus (HSV) is initiated is not completely defined. HSV initially infects the stratified squamous epithelium of the anogenital mucosa prior to entering nerve endings. We have recently reported that topical application of HSV-1 to human foreskin explants results in infection of epidermal Langerhans cells (LCs) which then emigrate into the dermis where they expressed the maturation marker CD80 and formed large cell clusters with BDCA3+ subsets of DC-SIGN+ and dermal dendritic cells (DCs) and. HSV-expressing LC fragments were observed inside the dermal DCs/macrophages. No other infected epidermal cells interacted with the dermal DCs (Kim et al. Plos Pathogens, 2015). Methods Therefore, we isolated LCs and dermal DCs from large abdominal skin specimens by collagenase digestion and flow sorting. LCs were pulsed with fluorescent tagged HSV and co-cultured with a subset of (BDCA3+) dermal DCs. Results All infected LCs showed markers of apoptosis at 18 hr p.i. Approximately 50% of BDCA3+ DCs co-localised with infected LCs and in some cases fragments of infected LCs were observed within the dermal DC cytoplasm. Such colocalizaton of HSV antigen bearing LCs and dermal DC subsets, was also detected within biopsies of initial genital herpes lesions. The mechanism of interaction of apoptotic LCs and dermal DCs, and uptake by phagocytosis are being determined. Conclusion Thus, a viral antigen relay takes place where HSV infected LCs slowly die by apoptosis during migration to the dermis and are taken up by dermal DCs by phagocytosis for subsequent antigen presentation. This provides a rationale for targeting these dermal DCs for mucosal or perhaps intradermal HSV immunisation. Disclosure of interest statement No pharmaceutical grants were received in the development of this study.
- Published
- 2015
27. Relay of Herpes Simplex Virus between Langerhans Cells and Dermal Dendritic Cells in Human Skin
- Author
-
Kaylene McKinnon, Kerrie J Sandgren, Andrew N. Harman, Anthony L. Cunningham, Najla Nasr, Naomi R. Truong, Lidija Bosnjak, Kirstie M. Bertram, Norman Olbourne, Virginia James, Ralph C. Cohen, Shailandra Sawleshwarkar, and Min Kim
- Subjects
lcsh:Immunologic diseases. Allergy ,Simplexvirus ,food.ingredient ,viruses ,Immunology ,Antigen presentation ,chemical and pharmacologic phenomena ,Human skin ,Herpesvirus 1, Human ,Biology ,medicine.disease_cause ,Microbiology ,03 medical and health sciences ,Foreskin ,0302 clinical medicine ,food ,Dermis ,Cell Movement ,Virology ,Genetics ,medicine ,Humans ,lcsh:QH301-705.5 ,Molecular Biology ,Skin ,030304 developmental biology ,0303 health sciences ,integumentary system ,Epidermis (botany) ,hemic and immune systems ,Dendritic Cells ,Flow Cytometry ,3. Good health ,medicine.anatomical_structure ,Herpes simplex virus ,Microscopy, Fluorescence ,lcsh:Biology (General) ,Apoptosis ,Langerhans Cells ,Parasitology ,lcsh:RC581-607 ,Research Article ,030215 immunology - Abstract
The mechanism by which immunity to Herpes Simplex Virus (HSV) is initiated is not completely defined. HSV initially infects mucosal epidermis prior to entering nerve endings. In mice, epidermal Langerhans cells (LCs) are the first dendritic cells (DCs) to encounter HSV, but it is CD103+ dermal DCs that carry viral antigen to lymph nodes for antigen presentation, suggesting DC cross-talk in skin. In this study, we compared topically HSV-1 infected human foreskin explants with biopsies of initial human genital herpes lesions to show LCs are initially infected then emigrate into the dermis. Here, LCs bearing markers of maturation and apoptosis formed large cell clusters with BDCA3+ dermal DCs (thought to be equivalent to murine CD103+ dermal DCs) and DC-SIGN+ DCs/macrophages. HSV-expressing LC fragments were observed inside the dermal DCs/macrophages and the BDCA3+ dermal DCs had up-regulated a damaged cell uptake receptor CLEC9A. No other infected epidermal cells interacted with dermal DCs. Correspondingly, LCs isolated from human skin and infected with HSV-1 in vitro also underwent apoptosis and were taken up by similarly isolated BDCA3+ dermal DCs and DC-SIGN+ cells. Thus, we conclude a viral antigen relay takes place where HSV infected LCs undergo apoptosis and are taken up by dermal DCs for subsequent antigen presentation. This provides a rationale for targeting these cells with mucosal or perhaps intradermal HSV immunization., Author Summary Herpes Simplex Virus (HSV) is a highly prevalent virus that causes cold sores and genital herpes but also increases the chance of contracting HIV by several folds. In fact, most new cases of HIV in Africa occur in people infected with HSV. Thus, a protective HSV vaccine would have a large impact on public health. Currently, the process by which immunity to HSV is generated is incompletely understood. Paradoxically, the first immune cells to become infected, Langerhans cells in the epidermis, are not the cells that initiate the immune response, while the dermal dendritic cells thought to be responsible for initiating the immune response are not likely to be infected. Here, we have shown, in human skin models and genital herpes lesion biopsies, an interaction between these dendritic cells that could relay HSV to the lymph node. HSV is taken up by the epidermal Langerhans cells that then migrate into the dermis, die and are taken up by another subset of dermal dendritic cells—the homologs of those in mice which stimulate HSV-specific T cells in the lymph node. Thus, a mucosal or intradermal vaccine targeting these two dendritic cells may be required.
- Published
- 2015
28. Herpes Simplex Virus type 1 infects Langerhans cells and the novel epidermal dendritic cell, Epi-cDC2s, via different entry pathways.
- Author
-
Kirstie M Bertram, Naomi R Truong, Jacinta B Smith, Min Kim, Kerrie J Sandgren, Konrad L Feng, Jason J Herbert, Hafsa Rana, Kevin Danastas, Monica Miranda-Saksena, Jake W Rhodes, Ellis Patrick, Ralph C Cohen, Jake Lim, Steven L Merten, Andrew N Harman, and Anthony L Cunningham
- Subjects
Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Abstract
Skin mononuclear phagocytes (MNPs) provide the first interactions of invading viruses with the immune system. In addition to Langerhans cells (LCs), we recently described a second epidermal MNP population, Epi-cDC2s, in human anogenital epidermis that is closely related to dermal conventional dendritic cells type 2 (cDC2) and can be preferentially infected by HIV. Here we show that in epidermal explants topically infected with herpes simplex virus (HSV-1), both LCs and Epi-cDC2s interact with HSV-1 particles and infected keratinocytes. Isolated Epi-cDC2s support higher levels of infection than LCs in vitro, inhibited by acyclovir, but both MNP subtypes express similar levels of the HSV entry receptors nectin-1 and HVEM, and show similar levels of initial uptake. Using inhibitors of endosomal acidification, actin and cholesterol, we found that HSV-1 utilises different entry pathways in each cell type. HSV-1 predominantly infects LCs, and monocyte-derived MNPs, via a pH-dependent pathway. In contrast, Epi-cDC2s are mainly infected via a pH-independent pathway which may contribute to the enhanced infection of Epi-cDC2s. Both cells underwent apoptosis suggesting that Epi-cDC2s may follow the same dermal migration and uptake by dermal MNPs that we have previously shown for LCs. Thus, we hypothesize that the uptake of HSV and infection of Epi-cDC2s will stimulate immune responses via a different pathway to LCs, which in future may help guide HSV vaccine development and adjuvant targeting.
- Published
- 2021
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.