Thesis (MScMedSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Introduction:
Chronic HIV-1 infection is characterized by immune activation and dysregulation of immune
homeostasis, which impacts on multiple immune cell types. The B-cell compartment, which
plays an important role in the producing neutralizing antibodies, is also dysregulated in HIV-
1 infection. In this study we investigated peripheral blood B-cell subset distribution, and
changes in expression of cellular activation, inhibition, and apoptosis signaling markers in
both untreated chronic HIV-1 infected individuals and healthy uninfected controls. The
neuropeptide immune modulator, vasoactive intestinal peptide (VIP) is known to selectively
down-regulate activation of CD4+ T-cells in various disease settings including HIV-1,
however to our knowledge, no studies have investigated the effect of VIP inhibition on B-cell
activation.
Materials & Methods:
A total of 21 HIV+ve (CD4 count >250 cells/µl), and 19 HIV-ve individuals were recruited from
the Emavundleni voluntary testing and counseling clinic in Crossroads, Western Province,
South Africa. Whole blood was stained to distinguish B-cell subsets (activated memory (AM:
CD21-CD27+), resting memory (RM: CD21+CD27+), mature naïve (MN: CD21+CD27-), or
tissue-like memory (TLM: CD21loCD27lo). In addition expression of markers of B-cell
activation (CD126, CD86, CD38, CD284, CD287), inhibition (CD72, CD85j, CD300a,
CD305, CD307d), and apoptosis signaling (CD95), was assessed ex vivo by flow cytometry
(BD FACSCanto II). For determination of functional responsiveness isolated B-cells
(RosetteSep, Stemcell Technologies) were cultured for 18h (37°C, 5%CO2) without
stimulation or stimulated with TLR ligands (LPS or R848). Stimulation experiments were also
performed in the presence or absence of VIP.
Results:
Chronic HIV-1 infection affected B-cell subset distribution. The percentage (%) of TLM was
increased by 59.24%, and %RM was decreased by 22.73% (both p<0.01). Total expression
of the VIP receptor VPAC2 was decreased by 47.35% (p=0.0296). Subsets had a mixed
phenotype ex vivo; HIV infection upregulated CD38 (by 59.56%, p=0.0004), CD72 (by
60.70%, p=0.0396), CD307d (by 68.63%, p=0.0015) on AM, while RM B-cells had increased
expression of TLR4 (by 107.04%, p=0.0057) and TLR7 (by 208.14%, p=0.0199). TLM B
cells (i.e. exhausted phenotype) displayed upregulated TLR7 (by 550%, p=0.0128) and
CD307d (by 72.40% p=0.045) expression. MN B-cells had increased CD72 expression (by
70.98%, p=0.0026). R848 upregulated CD86 expression by 42.20% on AM (p<0.01), and by
56.06% on RM B-cells (p<0.01), which was significantly downregulated with VIP inhibition
(both p<0.05). Similarly, CD95 expression on RM, TLM, and MN B-cells increased by
31.10% (p<0.001), 21.46% (p<0.01), and 39.92% (p<0.01) with R848 stimulation
respectively, which was also significantly downregulated with VIP inhibition.
Conclusion:
These data indicate that B-cells in untreated HIV infection display increased levels of
activation, and also the potential for increased susceptibility to apoptosis as evidenced by
increased FAS (CD95) expression. VIP significantly down-regulated markers of activation,
inhibition, and apoptosis signaling. Dysregulation of B-cells is thus apparent in asymptomatic
stable chronic HIV-1 infection, which may impact on both inefficient neutralizing antibody
production and hypergammaglobulinemia. The ability of VIP to prevent stimulationassociated
marker upregulation may indicate that VIP is a potential therapeutic agent. Its
immuno-modulatory properties were demonstrated to limit B-cell hyperactivation, and
selectively down-regulate apoptosis and mark it out for further investigation. / AFRIKAANSE OPSOMMING: Inleiding:
Immunaktivering en ongekoppelde immuun-homeostase is kenmerke van chroniese MIVinfeksie.
Ons het perifere bloed B-sel subgroep-verspreiding, en veranderinge in die
uitdrukking van merkers van aktivering, inhibisie, en apoptose in 'n onbehandelde MIV-1
besmettende groep ondersoek (in vergelyk met 'n gesonde onbesmettende kontrole). Die
immuun-moduleerder, vasoaktiewe intestinale peptied (VIP) is bekend om aktivasie van
geaktiveerde CD4+ T-selle te verminder, maar tot ons kennis, is daar geen studies wat die
effek van VIP-inhibisie op B-sel aktivering ondersoek het, in die konteks van MIV-1 infeksie.
Materiaal & Metodes:
MIV+we individue (CD4-telling >250 selle/µl) , en MIVwe
kontroles is gewerf uit die vrywillige
toetsing en berading Emavundleni kliniek, Crossroads, Westelike Provinsie, Suid-Afrika. Bsel
subgroepe is gedefinieer as geaktiveerde geheue (AM: CD21-
CD27+
), rusende geheue (RM: CD21+
CD27+
), volwasse naïef (MN: CD21+
CD27-
), of weefsel-agtige geheue (TLM:
CD21loCD27lo). Merkers van aktivering (CD126, CD86, CD38, CD284, CD287), inhibisie
(CD72, CD85j, CD300a, CD305, CD307d), en apoptose signalering (CD95) is via
vloeisitometrie (BD FACSCanto II) op B-selle ex vivo en ook op geïsoleerde B-selle
(RosetteSep, Cell Technologies) ondersoek. Vir die bepaling van funksionele responsiwiteit,
geïsoleerde B-selle (RosetteSep, StemCell Technologies) was vir 18h (37°C, 5%CO2)
gekweek, sonder stimulasie of gestimuleer met TLR ligande (LPS of R848). Stimulasie
eksperimente het ook in die teenwoordigheid of afwesigheid van VIP plaasgevind.
Resultate:
Chroniese MIV-1 infeksie het B-sel subset verspreiding geraak. Die persentasie (%) van
TLM is verhoog deur 59,24%, en% RM het met 22.73% afgeneem (beide p <0,01). Totale
uitdrukking van die VIP reseptor VPAC2 het met 47,35% afgeneem (p = 0,0296). Subgroepe
het 'n gemengde ex vivo fenotipe; MIV-infeksie het CD38 (deur 59,56%, p=0,0004), CD72
(deur 60,70%, p=0,0396), CD307d (deur 68,63%, p=0,0015) op AM verhoog, terwyl RM Bselle
het verhoogde uitdrukking van TLR4 (deur 107,04%, p=0,0057) en TLR7 (deur
208,14%, p=0,0199). TLM B-selle (die uitgeputtende fenotiep) het verhoogde TLR7 (deur
550%, p=0,0128) en CD307d (deur 72,40% p=0.045) uitdrukking gewys. MN B-selle het
verhoogde uitdrukking van CD72 (deur 70,98%, p = 0,0026). R848 het CD86 uitdrukking op
AM deur 42,20%, en op RM deur 56,06% toegeneem (beide p <0,01). Dit het met VIP
inhibisie beduidend afgeneem (beide p <0.05). CD95 uitdrukking was soortgelyk verhoog op
RM, TLM, en MN B-selle met 31.10% (p <0.001), 21,46% (p <0,01), en 39,92% (p <0,01)
met R848 stimulasie. Al drie het beduidend afgeneem met VIP inhibisie.
Gevolgtrekking:
Hierdie data dui daarop dat B-selle in onbehandelde MIV-infeksie vertoon verhoogde
aktiveringsvlakke, en ook die potensiaal vir verhoogde vatbaarheid vir apoptose soos bewys
deur verhoogde uitdrukking van FAS (CD95). VIP het beduidend merkers van aktivering,
inhibisie, en apoptose af-gereguleer. Wanfunksie van B-selle is dus in asimptomatiese
stabiele chroniese MIV-1 infeksie duidelik, wat impak kan hê op beide oneffektiewe
neutraliserende teenliggaampie produksie, en hiepergammaglobulinimie. Die vermoë van
VIP stimulasie-verwante merker opregulasie te voorkom kan aandui dat VIP 'n potensiële
terapeutiese agent is. VIP se immuno-moduleerende eiendomme is gedemonstreer om Bsel
hieperaktiveering te beperk, en selektief apoptose afreguleer, en merk dit vir verdere
ondersoek.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/96898 |
| Date | 04 1900 |
| Creators | Reid, Timothy Dawson |
| Contributors | Glashoff, Richard H., Ipp, Hayley, Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | xv, 108 pages : colour illustrations |
| Rights | Stellenbosch University |
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