Download Introduction

Changes in CD4+ cells miRNA
expression following exposure to
HIV-1
Claudio Casoli
University of Milan
Rationale
There is evidence that miRNAs can modulate host innate immunity against
viruses.
Specifically for HIV-1, it has been demonstrated that miRNAs can regulate viral
gene expression by decreasing PCAF expression and promoting HIV-1 latency
or by directly targeting HIV-1 messengers (Triboulet R. et al. Science 315, 157982; 2007).
Until now, five cellular miRNAs have been identified to recognize HIV-1
messengers which are up-regulated in only resting CD4+ T cells (Huang J. et al.
Nature Medicine 13, 1241-7; 2007).
However, cellular miRNAs expression may also favour HIV-1 infection (Han Y.
et al. Nature Medicine; 2007).
Objectives of this study
• To study if miRNA change is related to immune
response or HIV-1 replication.
• To propose new approaches in investigating HIV1/host interactions.
Population study
•
The HIV-related miRNA expression was assessed in CD4+ cells.
•
Three patient groups were studied:
a. subjects classified as éLTNP (n° 7)
b. subjects naive for antiretroviral therapy (n° 10)
c. subjects who were multiply sexually exposed to HIV but remained
uninfected for at least 4 years, named MEU (n° 7)
•
No occurrence of a specific phenotype that restricts HIV-1
susceptibility (CCR5∆32 allele, CCR5 and CCR2 polymorphisms,
CCL3L1 copy number, HLA class II alleles).
•
None of the subjects had co-infection such as HBV, HCV, HTLV.
•
These subjects were characterized on the basis of cellular and
molecular phenotype (CD38, HLA-DR, sjTREC, Treg), proviral load
quantification and miRNA expression.
Methodology
• Expression levels of 377 miRNAs were obtained using real time
quantitative polymerase chain reaction based arrays (1 card for
1 patient, data obtained in 2 independent experiments). To
analyse data we chose 2 -∆∆Ct method , which relates the PCR
signal of patient cell miRNA transcripts to that of normal cells
collected from a pool of 6 healthy donors.
• Criteria for comparison:
- Replication in more than 70% of the individuals in at least one of
the three patient classes;
- Variation at least of 1 log10 in either direction (up or down).
Viro-immunological features of HIV-1+ patients
As expected, éLTNP had fewer peripheral
activated T lymphocytes in comparison with
those of naive patients.
No significant differences were observed
between éLTNP and naive patients by
analysis of the differentiation marker CD127
expression in either virgin or memory T cells.
Moreover, éLTNP had fewer
regulatory T cells that expressed
an activation marker such as
HLA-DR, a higher number of
sjTREC+ cells, and a lower
amount of intracellular HIV-1 DNA
within CD4+ T lymphocytes than
naive individuals.
Could miRNAs be considered as markers of HIV-1
exposure or infection?
- Analysis of 377 miRNAs expression demonstrated that 8 are altered in all groups.
- 5 of which are down-regulated and 3 are up-regulated.
- This alteration may be interpreted as a signature of HIV exposure or infection.
Could miRNAs distinguish HIV-1
exposure from infection?
This heat map of single pattern
subjects
shows
two
hierarchical groups: one is
related to uninfected MEU
subjects, while the second
group is related to infected
patients.
The MEU group shows a
prevalent
miRNAs
downregulation, whereas Naive and
éLTNP subjects show
a
prevalent
miRNAs
upregulation.
Are the differences in miRNA profiles significant?
29 miRNAs are differentially expressed between groups. These findings
confirm the results shown in the hierarchical analysis.
Can Dicer and Drosha expressions explain the miRNA
profiles observed in patients?
Both enzymes resulted
significantly
downregulated in MEU
subjects in comparison
to infected patients. Only
in MEU subjects the
miRNA expression is
strictly correlated to
enzyme expression.
Relative Fold Change Expression
0.70
0.60
Dicer
Drosha
0.56
0.60
0.50
0.39 0.38
0.40
0.30
0.16
0.20
0.11
0.10
0.00
Naive
éLTNP
MEU
Each class of individuals is
characterized by similar levels of Dicer
and Drosha. None of them seem to
play a predominant role.
Which are the miRNA involved in HIV-1 replication
or immune response?
Out of 377 miRNAs tested, only
a few are implicated in HIV
replication and immune
response.
Among these, some (like 34a
and 125a-3p) distinguish
infected from uninfected
patients in HIV replication and
immune response, respectively.
In addition, 23a and 155
miRNAs characterize Naive
from the other two groups.
In the MEU group, most of the
miRNAs involved in the control
of the immune response are
down-regulated.
Does in vitro exposure of healthy CD4+ T
lymphocytes to gp120 induce changes in miRNA
expression?
mAb
+- mAb
r-gp120
r-gp120
LAIgp120
gp120
LAI
17
11
14
1
23
15
0
14
11
68
UP
UP
DOWN
DOWN
When the gp120 action was neutralized
Exposure to recombinant and natural
with a specific mAb, many miRNAs show
molecules induced the same alteration
the same expression as in non-exposed
in 60% of miRNAs.
cells.
The different molecular structure of
Presumably the expression of these
gp120 used could explain the different
miRNAs could be altered by gp120
miRNAs expressed.
through the CD4 binding signalling.
Which miRNAs are similarly altered after gp120
exposure and in vitro infection?
5 out of 23 miRNAs, which expression
may be related to CD4/gp120 binding site,
were also found altered after in vitro
infection with X4 strain.
Of note these miRNAs (34c-5p, 518f, 452,
202, 487b) showed the same expression
levels in both conditions (gp120 exposure
and in vitro infection).
Can these miRNAs modulate HIV entry?
Conclusions
- miRNAs discriminate infected from uninfected subjects.
- A signature of HIV exposure may be defined.
- There is no difference between miRNA profiles of Naive
and éLTNP groups.
-Only exposure to HIV is enough to induce a marked
alteration of miRNA patterns and consequently may
influence immune function of CD4+ T cells.
-Thus a miRNA pattern may reflect a change in the
immune system.
Personnel and Institutions
involved in the research
University of Milan
Claudio Casoli
Fabio Bignami
Paola Ronzi
Massimo Galli
University of Parma
Mariolina Gulli
Nelson Marmiroli
Roberta Ruotolo
University of Modena
and Reggio Emilia
Andrea Cossarizza
Linda Bertoncelli
Marcello Pinti
Cristina Mussini
Arcispedale S. Maria Nuova
of Reggio Emilia
Giacomo Magnani
Anlaids Lombardia
Elisabetta Pilotti
San Raffaele
Fondation
Lucia Lopalco