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Effects of HAART on the gut mucosal immune system in HIV-infected patients
No full text0 Titelblatt und Inhaltsverzeichnis
1.0 Einleitung 1
1.1
1.2
1.3
1.4 Die besondere Rolle der intestinalen Mukosa bei der HIV-Infektion
Zytokine
Zytokinexpression in der Mukosa während der HIV-Infektion
Zielsetzung 1
3
7
8
2..0 Material 9
2.1
2.2
2.2.1
2.2.2
2.3
2.4
2.5
2.6
2.7
2.8
2.9
2.10
2.11 Charakterisierung von Patienten und Kontrollpersonen
HIV-Beladung und CD4+T-Zellzahl>
HIV-Beladung im Blut und in der intestinalen Mukosa unter HAART
CD4+T-Zellzahlen im Blut und in der intestinalen Mukosa unter HAART
Chemikalien und Reagenzien
Kits
Enzyme mit Puffern
Primer und Hybridisierungssonden
Zellen.
Puffer und Stammlösungen
Kulturmedien..
Geräte
Verbrauchsmaterialien 9
10
10
11
11
12
14
15
16
17
18
19
19
3.0 Methoden 20
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
3.8.1.
3.8.2.
3.8.3.
3.8.4.
3.8.5.
3.8.6.
3.8.7.
3.9.
3.9.1.
3.9.2.
3.9.3.
3.10. RNA-Extraktion
Bestimmung von Konzentration und Reinheit der RNA
DNase-Verdauung
Reverse Transkription.
Primerdesign
Amplifikation von cDNA mittels konventioneller PCR
Detektion der Amplifikate mittels Gelelektrophorese.
Herstellung quantitativer Standardreihen für die Echtzeit-PCR
PCR-Produktreinigung.
Klonierung, Transformation und Kultivierung.
Plasmidextraktion.
Restriktionsverdauung.
Bestimmung der Konzentration des Plasmids im Eluat.
Berechnung der Kopienzahl pro Mikroliter
Erstellung der Verdünnungsreihen mit definierter Kopienzahl
Echtzeit-PCR mit dem LightCycler
Der LightCycler.....
SYBR Green.......
Spezifische Hybridisierungssonden.
Statistische Auswertung... 20
21
22
23
24
25
27
28
28
29
31
32
34
35
35
36
36
39
41
43
4.0 Ergebnisse 44
4.1.
4.2
4.3
4.3.1.
4.3.2.
4.3.3.
4.3.4.
4.3.5.
4.3.6.
4.4 RNA-Isolierung.
Wiederfindung
PCR-Etablierung.
Methodische Nachweisgrenze
LightCycler-PCR: SYBR Green oder spezifische Hybridisierungssonden
Amplifikation der quantitativen Standardreihen am LightCycler
Detektion der PCR-Produkte am LightCycler
Mit Antikörpern geblockte Taq- versus herkömmliche Taq-Polymerase
Variation der LightCycler-Messungen bei verdünnten Proben von GAPDH
Veränderungen der mukosalen Zytokin-mRNA-Expression von HIV-infizierten
Patienten unter HAART versus Kontrollpersonen 44
45
46
46
47
48
51
54
56
57
5.0 Diskussion 62
5.1.
5.2
Diskussion der Methoden
Mukosale Zytokin-mRNA-Expression ... 62
65
Zusammenfassung 69
Literaturverzeichnis 71Die intestinale Mukosa ist für die Pathogenese der HIV-Infektion von
besonderer Bedeutung. Hier findet eine hohe HIV-Replikation und ein schneller
CD4+T-Zellverlust statt. Die Ursachen hierfür sind bisher unbekannt. Lokale
Zytokine könnnen hierbei eine besondere Rolle spielen. Ziele dieser Arbeit
waren daher a) die Bestimmung der Expression mukosaler Zytokine, die für die
HIV-Replikation von Bedeutung sind und b) die Erfassung von Veränderungen der
Zytokinexpression unter HAART. Hierfür wurde eine Methode entwickelt, die es
erlaubt, aus geringen Mengen bioptischen Untersuchungsmaterials Zytokin-
Expressionsmuster zu analysieren. Mittels Echtzeit-RT-PCR wurde in
Sigmabiopsien von 7 Kontrollpersonen und zehn HIV- infizierten Patienten vor,
sowie einen, vier und neun Monate nach Einleitung einer HAART die Zytokin-
mRNA-Expression von IL-2, IL-4, IL-6, IL-10, IL-16, IFN-γ, TNF-α, CCL3 und
CCL5 basierend auf der LightCycler-Technik mit spezifischen Hybridisierungs-
Detektionssonden gemessen und zur mRNA-Expression des Housekeeping-Gens GAPDH
in Beziehung gesetzt. Im Vergleich zu Kontrollpersonen zeigten HAART-naive
Patienten eine erhöhte mukosale mRNA-Expression für IL-4, IL-6, IL-10, IFN-?,
TNF-?, CCL3 und CCL5, keine Unterschiede ergaben sich für IL-2 und IL-16.
Diese Befunde zeigen eine erhöhte Aktivierung im mukosalen Immunsystem bei der
HIV-Infektion, die allerdings keinem etablierten Th1- bzw. Th2-Muster
entspricht. Nach Einleitung einer HAART kam es bei allen untersuchten
Patienten zu einem schnellen Anstieg der mukosalen CD4+T-Zellzahl und einem
deutlichen Abfall der mukosalen Viruslast unter die Nachweisgrenze, dennoch
zeigte sich für HIV-infizierte Patienten im Vergleich zu Kontrollen weiterhin
eine erhöhte mukosale mRNA- Expression für IL-4, IL-6, IL-10, IFN-γ, TNF-α,
CCL3 und CCL5. Diese trotz effektiver Inhibition der Virusvermehrung
gesteigerte Aktivierung des mukosalen Immunsystems spricht gegen eine
entscheidende ursächliche Rolle der Virusreplikation oder viraler Antigene für
die mukosale Immunaktivierung. Die hohe Virusreplikation in der intestinalen
Mukosa bei unbehandelten Patienten ist daher eher Folge der gesteigerten
Immunaktivierung, ein Effekt, der gerade für die hoch exprimierten
inflammatorischen Zytokine TNF-α, IFN-γ und IL-6 plausibel erscheint. Die hohe
Expression von CCL3 und CCL5 in Verbindung mit einer hohen HIV-Produktion bei
unbehandelten Patienten deutet darauf hin, dass mögliche HIV- supprimierende
Effekte dieser Zytokine durch die Rekrutierung und Aktivierung potentieller
Zielzellen in der Mukosa aufgehoben werden. Ihre anhaltend hohe Expression
unter HAART könnte weiterhin die schnelle und überschießende Repopulation des
Dickdarms mit CD4+T- Zellen verursachen. HIV-infzierte T-Zellen und
Makrophagen sezernieren inflammatorische Zytokine, die die epitheliale
Barriere schädigen können. Auch nach Elimination des auslösenden Antigens
durch HAART könnte eine solche durch HIV induzierte Barrierestörung über
vermehrten Einstrom luminaler Antigene eine mukosale Entzündung
aufrechterhalten. Ob und zu welchem Zeitpunkt sich die beschriebene
Immunaktivierung unter effektiver Virussuppression zurückbildet, ist derzeit
unklar. Zumindest neun Monate nach effektiver HAART finden sich in der
intestinalen Mukosa eine im Vergleich zu Kontrollen sehr hohe Anzahl von
CD4+T-Zellen und eine hohe Expression inflammatorischer Zytokine, die unter
anderem auch die Expression der HIV-Korezeptoren CCR5 und CXCR4 stimulieren.
Es liegen damit zahlreiche Zielzellen unter für die HIV-Infektion und
-Replikation optimalen Bedingungen vor, die bei Therapieversagen oder
Therapiepausen Ausgangspunkt für einen schnellen und ausgeprägten Rebound des
HIV sein können.Background: Diarrhoea and other gastrointestinal disorders are some of the
most common manifestations of HIV-infection and AIDS. The mucosa-associated
immune system (MALT) pertaining to the gastrointestinal tract probably houses
the largest accumulation of immunocompetent cells in the body. Previous
research has shown that the loss of CD4+T- cells during HIV-infection is much
more pronounced in the mucosa than in the peripheral blood. This may be a
consequence of the high level of viral replication seen in the mucosa. The
reasons for these findings have been unclear. It has been suggested that the
presence of HIV antigen may trigger the production of proinflammatory
cytokines, which results in CD4+T-cell activation making these cells prone to
HIV infection. In this scenario HIV enhances its own replication by
stimulating cytokine production. Objectives: 1.) to quantitate the mRNA
expression of selected proinflammatory, regulatory and HIV-inhibitory
cytokines in the mucosa of HIV-infected patients and to compare it to healthy
controls. 2.) to study the changes in cytokine expression in the course of
highly active antiretroviral therapy (HAART). Methods: HIV-infected patients
underwent endoscopic biopsy of colonic mucosa before and one, four and nine
months after the start of HAART. Healthy volunteers, who underwent a single
endoscopic biopsy served as controls. HIV-RNA and CD4+T-cell counts were
measured in the blood and in the biopsy specimens. Quantitative real time PCR
assays were developed to determine the mRNA expression of TNF-α, IFN-γ, IL-6,
IL-2, IL-4, IL-10, IL- 16, CCL3 and CCL5 in the biopsy specimens. Specific
hybridisation probes were used for the detection of the amplification
products. Cytokine mRNA levels were normalised to mRNA levels of the
housekeeping gene GAPDH. Results: The sensitivity of the RT-PCR assay for each
cytokine and GAPDH was high with a limit of quantification of ten template
copies/capillary. The calibration curve was linear over 5 orders of magnitude
with R2 values > 0,998. For each of the cytokine PCRs the mean efficiency as
expressed by the slope of the standard calibration curve was < 3,7 showing
very little inter-assay variability (C.V. 2% - 14%). The intra-assay precision
was satisfactory with C.V. < 10% throughout the whole calibration range. Nine
HIV-1 infected males and one female were included in the study. The median
(range) age was 42 (27 53). The median (range) plasma HIV RNA and CD4 cell
count at baseline was 4,9 log-10 copies/ml (3,2 6,3) and 210 cells/µl (23
1217) respectively. The control group consisted of four males and three
females with a median (range) age of 56 (48 70). The expression of the
proinflammatory cytokines in the colonic mucosa was significantly higher in
the HIV-infected patients as compared to the healthy controls: median (IQR)
[copies cytokine mRNA/1000 copies GAPDH mRNA]; TNF-?: 1,67 (0,35 3,21) vs.
0,08 (0,06 0,11) p<0,05; IFN-?: 0,11 (0,09 0,72) vs. 0,02 (0,01 0,02)
p<0,05; IL-6: 0,24 (0,14 0,95) vs. 0,01 (0,00 0,01) p<0,05. The expression
level of the regulatory cytokines IL-4 and IL-10 was also higher in the HIV-
infected study population: IL-4: 0,06 (0,03 0,41) vs. 0,01 (0,01 0,02),
p<0,05; IL-10: 0,35 (0,11 0,84) vs. 0,01 (0,01 0,02) p<0,05. Accordingly
the chemokines CCL3 and CCL5 were elevated in the HIV-infected study
population: CCL3: 1,02 (0,69 3,81) vs. 0,11 (0,10 0,35) p<0,05; CCL5:
25,68 (15,24 132,10) vs. 8,80 (6,40 10,72); p<0,05. Despite a dramatic
decline of virus load in both the blood and the mucosa and a rise of
CD4+T-cells in the blood and the mucosa the high level of expression of all
studied cytokines remained unchanged throughout nine months of HAART.
Discussion: With the elimination of the HIV antigen from the mucosa we
expected the expression of proinflammatory cytokines to fall. As cytokine
production remained high despite fully suppressive antiviral therapy factors
other than HIV must be responsible for causing mucosal inflammation. We
suggest that the barrier of the colonic mucosa may have been disrupted earlier
in the course of the infection leading to a continuous influx of luminal
antigen, which maintains the inflammatory response even in the absence of the
offending agent. The rapid repopulation of the mucosa with CD4+T-cells shown
in this study could be explained by the chemotactic properties of CCL3 and
CCL5 whose expression has been shown to be more than ten fold that of
controls. Both the presence of a large number of CD4+T-cells and the continued
high levels of inflammatory cytokines leading to T-cell activation could be
the basis for the explosive virus replication often seen in the case of
treatment interruption or failure
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