Single cell ribonucleic acid sequencing in Tuberculosis research.
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Date
2021
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Abstract
Tuberculosis (TB) remains a global challenge, with approximately 1,5 million deaths annually.
Addressing deficits in our understanding of disease pathology and treatment is needed for the
development of new treatment modalities. Despite much effort, prevalence of this
disease remains high in resource limited regions, where research capacity is not sufficient to
successfully combat the endemic. Research in developed countries has generally been
constrained to animal models due lack of access to clinical samples from the site of TB
disease, the human lung. Although these animal models have their utility, it is essential that
findings from these systems be tested and validated in human tissue. In this thesis, I leveraged
a relatively new technology called Seq-well, which is highly portable and low-tech single cell
ribonucleic acid sequencing (scRNAseq) platform and access to TB infected lung
tissue obtained from lung resections, to generate a single cell atlas of TB affected lung tissue.
This involved processing the human tissue immediately post-surgery and loading
unprocessed/neat cells or FACS sorted cells (tissue resident t cells) onto a microarray that
allowed capture and subsequent sequencing of the cell transcriptomes. In the first part of the
thesis, I identified and profiled cellular subsets from TB infected tissue, focussing on a subset
of FAP+PDPN + fibroblasts associated with the organisation of tertiary lymphoid organs. I
also demonstrated that this dataset can be useful in evaluating current and future TB
biomarkers, by superimposing signatures from the literature onto the cellular subsets and
localizing them to different parenchymal, stromal and immune cell types. I also profiled tissue
resident CD4 T cells from the same lung tissue, identifying canonical marker genes (ITGA1,
PRF1) in one specific cluster, together with naive (CCR7, SELL), regulatory (RORA) and
activated/myeloid-like T cells (LYZ, S100A9) in separate clusters. Finally, I demonstrated the
applicability of this dataset in research involving other pulmonary diseases, by identifying
ACE2+ TMPRSS2+ type 2 pneumocytes, a target of the SARS-CoV-2. Taken together, these
findings provide new insights into the immunopathology of TB in the human lung together
with the impact of HIV on specific immune subsets. It serves as a resource for cross validation
of lung immune signatures generated in experimental infections of both mice and non-human
primates, which is beneficial for scientists lacking access to the technology and/or tissue.
Iqoqa
Isifo sofuba (i-TB) silokhu siyinselelo emhlabeni jikelele, ngokufa okuhlobene naso okucishe
kufike esigidini esi-1.5 njalo ngonyaka. Ukubhekana nokushoda ekuqondeni kwethu
umumosakhiwo wesifo bese kuncishiswa ukufa. Ngaphandle kwemizamo emikhulu,
ukudlanga kwalesi sifo kusalokhu kuphezulu ezifundeni ezintula imithombokusiza, lapho
umthamokwenza wocwaningo unqindekile. Ucwaningo emazweni asethuthukile, ngakolunye
uhlangothi, belwenzeka kuphela kumamodeli asebenzisa izilwane ngenxa yokuntuleka
kokufinyelela amasampuleni okwelapha engxenyeni okuqubuke kuyo isifo sofuba,
okuyiphaphu lomuntu. Nakuba kunamamodeli ezilwane anomsebenzi, kubalulekile ukuba
okutholakele kulezo zinhlelo kuyohlolwa bese kuqinisekiswa ngesigqa somuntu ukuqinisekisa
ubunjalo. Kule thesisi, ngiveze ubuchwepheshe obusha obungenayo obubizwa nge-Seq-well,
iseli eyodwa e-low-tech ephathekayo ene-ribonucleic acid sequencing (scRNASeq)
okuyindawo kanye nokufinyelela esicutshini sephaphu esitheleleke ngesifo sofuba esitholakale
ekuhlukanisweni kabusha kwamaphaphu okukhonjwe ngokokwelapha, ukwakha iseli eyodwa
yesicutshana sephaphu elitheleleke ngesifo sofuba.
Lokhu kwafaka ukusebenzakuhlola isicubu somuntu ngokushesha emva kokuhlinza nokufaka
amaseli ahlanzekile angasetshenziwe noma amaseli ahleliwe angama-FACS (ama-T cells
asesicutshini) ohlelweni lolibofuzo olwavumela ukufaka ohlwini nokulandelanisa
okulandelayo womumofuzo oqondene nezicubu. Engxenyeni yokuqala yethesisi, amaqoqwana
ahlonziwe nafakwe kwiphrofayli esicubini esitheleleke ngesifo sofuba kugxilwe eqoqweni le
FAP+PDPN + amafayibhroplasti ahlobene nokuhlelwa kwezingxenye zomzimba ezinkulu
zamalimfoyidi kanye nemichilwana yamafayibhrodi kanye noma igranyuloma yesifo sofuba.
Ngivezile ukuthi lamadathasethi angaba nomsebenzi omkhulu ekuhlaziyeni amabhayomakha
amanje nawasesikhathini esizayo esifo sofuba, ngokufaka izinkombabunjalo emaqoqweni
amancane nokuwabeka ezinhlotsheni ezehlukene zamaseli angamapharenikhayma
nangamastroma.
Ngiphinde ngachaza esizindeni sezicutshana ze-CD4 T esicutshini sephaphu elifanayo
okuchaza ulibofuzo olukala amakhenoni (i-ITGA1, PRF1) eqoqweni elilodwa eliqondile,
kanye namaseli angachazi lutho (CCR7, SELL), alawulayo (RORA) nama-T cell aqaliswe
ukusebenza/efana ne-myeloid (LYZ, S100A9) emaqoqweni aseceleni. Okokugcina, ngiveze
ukungena kwedathasethi ocwaningweni olufaka izifo zamaphaphu nokuphefumula
ngokuhlonza i- ACE2+ TMPRSS2+ type 2 wama-pneumocytes, okuhlosiwe kwe-SARS-CoV-
2. Uma kuhlanganisiwe, lokhu okutholakele kuletha imibono emisha yomumobugciwane
bokutheleleka ngesifo sofuba ephashini lomuntu, umthelela we-HIV kokutholakele
emumwenikuphila kwephaphu ekuthelelekeni okuyilinga kwakho kokubili amagundane kanye
nalokho okungebona abantu.
Description
Doctoral Degree. University of KwaZulu-Natal, Durban.