Impact of Duffy Antigen Receptor for Chemokines (DARC)-null linked Neutropenia on Neutrophil and Natural Killer cell Function in HIV-1 Infection Kewreshini K. Naidoo Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy in Immunology at the Nelson R. Mandela School of Medicine, College of Health Sciences University of KwaZulu-Natal, Durban. 2018 PREFACE This project represents original work done by the author and where others have made contributions it has been acknowledged in the text. This work has not been submitted in any form for any degree or diploma at another tertiary institution. The experimental work described in this thesis was performed in the HIV Pathogenesis Programme Laboratory at the Doris Duke Medical Research Institute at the Nelson R. Mandela School of Medicine, University of KwaZulu-Natal in Durban, South Africa. The work was supervised by Dr Christina F. Thobakgale-Tshabalala and Professor Thumbi Ndung’u. Kewreshini K. Naidoo Signed: Date: 04 December 2018 Christina F. Thobakgale-Tshabalala Signed: Date: 04 December 2018 As the candidate’s supervisor I agree to the submission of this thesis. Christina F. Thobakgale-Tshabalala Signed: Date: 04 December 2018 i DECLARATION I, Kewreshini K. Naidoo, declare that (i) The research reported in this thesis, except where otherwise indicated, is my original work. (ii) This thesis has not been submitted for any degree or examination at any other university. (iii) This thesis does not contain other person’s data, pictures, graphs or other information, unless specifically acknowledged as being sourced from other persons. (iv) This thesis does not contain other persons’ writing, unless specifically acknowledged as being sourced from other researchers. Where other written sources have been quoted, then: (a) their words have been re-written but the general information attributed to them has been referenced; (b) where their exact words have been used, their writing has been placed inside quotation marks, and referenced. (v) Where I have reproduced a publication of which I am author, co-author or editor, I have indicated in detail which part of the publication was actually written by myself and have fully referenced such publications. (vi) This thesis does not contain text, graphics or tables copied and pasted from the Internet, unless specifically acknowledged, and the source being detailed in the thesis and in the References section. Signed: Date: 04 December 2018 ii PUBLICATIONS AND PRESENTATIONS Peer-reviewed Publications Thobakgale, C., Naidoo, K., Mckinnon, L. R., Werner, L., Samsunder, N., Karim, S. A., Ndung'u, T., Altfeld, M. & Naidoo, K. 2017. Interleukin 1-Beta (IL-1beta) Production by Innate Cells Following TLR Stimulation Correlates With TB Recurrence in ART-Treated HIV- Infected Patients. Journal of Acquired Immune Deficiency Syndromes, 74, 213-220. Zulu, M. Z., Naidoo, K. K., Mncube, Z., Jaggernath, M., Goulder, P. J. R., Ndung'u, T., Altfeld, M. & Thobakgale, C. F. 2017. Reduced Expression of Siglec-7, NKG2A, and CD57 on Terminally Differentiated CD56(-)CD16(+) Natural Killer Cell Subset Is Associated with Natural Killer Cell Dysfunction in Chronic HIV-1 Clade C Infection. AIDS Res Hum Retroviruses, 33, 1205-1213 (shared first author). Naidoo, K.K., Ngubane, A., Gaza, P., Moodley, A., Ndung'u, T., Thobakgale, C.F. Neutrophil Effector Functions are not Impaired in Duffy Antigen Receptor for Chemokines (DARC)-null Black South Africans. Frontiers in Immunology (in press). Presentations Naidoo, K.K., Mbhele, N., Ngubane, A., Dong, K.L., Ndung’u, T. and Thobakgale-Tshabalala, C.F. NK Cell Responses in a DARC-null Zulu/Xhosa African HIV-1 Infected Population. Keystone Symposia on Molecular and Cellular Biology: HIV Vaccines, 20-24 March 2016, Olympic Valley, California, USA. Abstract X8 3047 (poster presentation). Naidoo, K.K., Mbhele, N., Rashid, T., Ngubane, A., Dong, K.L., Ndung’u, T. and Thobakgale- Tshabalala, C.F. NK Cell Responses in a DARC-null Zulu/Xhosa African HIV-1 Infected Population. University of KwaZulu-Natal College of Health Sciences Research Symposium, 8-9 September 2016, Durban, KwaZulu-Natal, South Africa (oral presentation). Naidoo, K.K., Ngubane, A., Gaza, P., Moodley, A., Ndung'u, T., Thobakgale, C.F. Neutrophil Effector Functions are not Impaired in Duffy Antigen Receptor for Chemokines (DARC)-null Black South Africans. European Society for Clinical Investigation (ESCI) Congress, 22-24 May 2019, Coimbra, Portugal (poster presentation). iii ACKNOWLEDGEMENTS I would like to thank: Dr Christina Thobakgale-Tshabalala and Professor Thumbi Ndung’u for their supervision, encouragement and mentorship throughout this study. Professor Arturo Zychlinsky for hosting my time at the Max Planck Institute for Infection Biology. Christian Goosmann and Falko Apel for training of neutrophil isolation and functional assays. Nokuzola Mbhele and Tabassum Rashid for their exceptional technical assistance in the laboratory. The participants of the FRESH and HPP Acute Infection Cohorts and our dedicated clinic staff. The South African Medical Research Council, the National Research Foundation and Sullivan Funding for financial support of this study. iv ABSTRACT Sub-Saharan Africa carries a disproportionate burden of the HIV-1 epidemic. The Duffy Antigen Receptor for Chemokines (DARC)-null polymorphism is a predictor of ethnic neutropenia which commonly occurs in persons of African ethnicity and is thought to account for up to 11% of HIV-1 infections on the African continent. Neutrophils are recognised for their killing mechanisms and have been noted for their regulatory mechanisms in recent years. For example, a role for neutrophils in natural killer (NK) cell priming in the periphery has been suggested, and neutrophil deficiency has been implicated in contributing to NK cell immaturity and dysfunction. While the DARC-null genotype is well associated with lower absolute neutrophil counts (ANCs), studies that assess the effects of the polymorphism on neutrophil functionality are lacking and the impact of DARC-null linked neutropenia on HIV disease progression is debatable. Furthermore, the influence of DARC-null neutropenia on the NK cell compartment is unknown. In this cross sectional pilot study, we assessed the impact of the DARC-null trait on neutrophil effector functions and also characterised NK cell profiles in Zulu/Xhosa African individuals from a high incidence HIV setting in Durban, South Africa. We hypothesised that in the context of the DARC-null genotype and lower ANCs in our cohort participants, neutrophils would have impaired functionality and would be unable to efficiently prime NK cells; thus affecting NK cell maturation and function, and altering NK cell homeostatic activities such as survival and proliferation. We further hypothesised that the impaired cellular responses in DARC-null individuals would be more prominent in HIV-1 infected individuals compared to HIV negative individuals. Neutrophil killing mechanisms were measured in HIV-1 chronically infected (n=22) individuals and HIV negative (uninfected) controls (n=20). For assessment of key neutrophil effector functions, isolated neutrophils were evaluated for Fc receptor-mediated phagocytosis following uptake of IgG opsonised beads using flow cytometry; reactive oxygen species (ROS) emission was measured by chemi-luminesce after activation of neutrophils with phorbol 12- myristate 13-acetate (PMA). Activated neutrophils were also visualised by fluorescent microscopy for neutrophil extracellular trap (NET) quantification. Assessment of the NK cell compartment in chronically HIV-1 infected (n=18) and uninfected (n=20) individuals using multi-parametric flow cytometry determined NK cell subsets, maturation profiles, cytokine production and degranulation. Annexin V and propidium iodide assays were used to determine NK cell survival, whilst CFSE staining was used to examine cytokine-activated NK cell v proliferation. Study subjects were genotyped for the DARC trait using TaqMan allelic discrimination assays and ANCs were measured by full blood count. Our findings confirmed a high prevalence of the DARC-null allele in the African population and the polymorphism was significantly associated with lower ANCs. Neutrophil functional analysis detected rapid and higher phagocytic activity in the absence of DARC at 10 minutes (p=0.05 and p=0.009) and 60 minutes (p=0.05 and p=0.07) in HIV negative and HIV-1 infected subjects respectively. ROS and NET production in neutrophils were mostly unaffected by DARC negativity irrespective of HIV status. The only exception to this was a reduction in NET production in neutrophils from DARC-null HIV infected subjects (p=0.04) following prolonged in vitro stimulation. In the NK cell compartment, individuals showed similar NK cell counts irrespective of HIV status. In HIV negative individuals, a marked reduction of total NK cell counts was noted in the absence of DARC (p=0.006) and this correlated with lower ANCs (p=0.002) and a weak trend towards higher CD56 bright subset proportions was noted in DARC-null individuals (p=0.08). HIV negative DARC-null subjects also displayed a less mature NK cell phenotype with higher proportions of hypo-responsive KIR-NKG2A- NK cells (p=0.06) and lower frequencies of terminally differentiated CD57 (p=0.02) expressing NK cells. However, this immature phenotype did not translate to differences in expression of NK cell activation markers CD69 or HLA-DR and exhaustion marker PD-1 by DARC state. Furthermore,