Changes in CD4+ cells mirna expression following exposure to HIV 1 Claudio Casoli
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1 Changes in CD4+ cells mirna expression following exposure to HIV 1 Claudio Casoli University of Milan
2 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, ; 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, ; 2007). However, cellular mirnas expression may also favour HIV 1 infection (Han Y. et al. Nature Medicine; 2007).
3 Objectives of this study To study if mirna change is related to immune response or HIV-1 replication. To propose new approaches in investigating HIV- 1/host interactions.
4 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.
5 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).
6 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.
7 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.
8 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.
9 Are the differences in mirna profiles significant? 29 mirnas are differentially expressed between groups. These findings confirm the results shown in the hierarchical analysis.
10 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. Each class of individuals is characterized by similar levels of Dicer and Drosha. None of them seem to play a predominant role.
11 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.
12 Does in vitro exposure of healthy CD4+ T lymphocytes to gp120 induce changes in mirna expression? + mab r gp120 LAI gp UP 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.
13 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?
14 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.
15 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 San Raffaele Fondation Lucia Lopalco 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
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