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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 21, 2002 as doi:10.1096/fj.01-0947fje. |
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* CNRS UPR 0415 and
|| INSERM U380, Institut Cochin de Génétique Moléculaire, 75014 Paris, France; and
¶ Université Paris 7, Place Jussieu, 75251 Paris, France
4Correspondence: CNRS UPR 0415, Institut Cochin de Génétique Moléculaire, 22 rue Méchain, 75014 Paris, France. E-mail: pietri-rouxel{at}cochin.inserm.fr
SPECIFIC AIMS
The aim of this study is to present evidence of the expression of CD4, CXCR4, and CCR5 receptors by human adipocytes and to test whether adipose cells support HIV infection.
PRINCIPAL FINDINGS
We have investigated the presence of HIV-1 receptors in vivo in embedded human adipose tissue or in highly purified primary cultures of human white preadipocytes that were differentiated in vitro into mature differentiated cells.
1. Expression of the CD4 and CXCR4, CCR5 coreceptors
Expression of all necessary receptors to allow HIV entry (CD4 and CXCR4 and CCR5 coreceptors) was demonstrated in vitro by semiquantitative RT-PCR and immunocytochemical experiments on preadipocytes and adipocytes (Fig. 1
A). Similar results were obtained using the immortalized adipose cell line PAZ6 (not shown). Moreover, immunohistochemical detection of the three receptors on sections of human white adipose tissue slices demonstrated their in vivo expression in adipose cells (Fig. 1B
).
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2. Exposure of adipose cells to HIV-1
As adipose cells express the receptors required for HIV-1 infection, we investigated whether preadipocytes supported HIV-1 infection. Primary preadipocytes were exposed for 5–18 h with laboratory-adapted HIV-1 NDK (X4), NL43 (X4), primary VEN, Mitch, THI, or AMI HIV-1 R5 strains.
Analysis of viral output
Preadipocytes were exposed to different amounts of X4 HIV-1 for 5–18 h. Early viral output was monitored 24 h after infection by the kinetic measurement of p24 antigen release (Fig. 2
A). Between 24 and 30 h postinfection with NDK and NL43 X4 viruses, p24 antigen could be detected. Aspects of kinetics were similar whatever viral inputs were used and levels of p24 antigen correlated with initial viral amounts. These levels progressively increased on the first 48 h. This suggested either a weak viral production or a passive release. However, as early as 48 h postinfection, p24 amounts progressively decreased to reach a low level. Nevertheless, 5–10 days after infection (depending on experiments), supernatants from X4-infected-preadipocytes were added to the CD4-positive human T cell line CEM (Fig. 2B
). Indeed, infection of the CEM cells resulted in the occurrence of a massive cytopathic effect. Coculture of these CEM cells with indicator target cells showed a massive syncytia formation clearly evidenced by an Xgal test. These data demonstrated that efficient infectious viral particles were present in the preadipocytes culture medium 10 days postinfection.
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Relationship between receptor expression and viral entry
Inhibition experiments were performed to analyze the relationship between receptor expression and viral entry. The CXCR4-specific ligand SDF1
was added to the cell culture medium before exposure to the HIV-1 NDK. Under these conditions, a strong inhibition of p24 production was observed until days 8 and 10 postinfection. p24 antigen production was dosed in culture supernatants of days 8 and 10 postinfection with values of 145 ± 2 pg/mL and 215 ± 5 pg/mL, respectively, and 35 ± 10 pg/mL (76% inhibition) and 48 ± 13 pg/mL (77% inhibition) in the presence of SDF1 (Fig. 2C
). These data suggest that the presence of SDF1 before infection inhibited the viral entry. Infection experiments performed using a receptor-negative cell line (U373 MG) did not result in p24 production under the same conditions (not shown).
Analysis of viral DNA presence
We performed PCR experiments on total DNA 10–15 days after infection and characterized viral-specific DNA. HIV-1-specific DNA sequences could be amplified using gag-specific primers in X4- and R5-infected preadipocytes and PAZ6 cells. We systematically controlled our viral input preparations to demonstrate the lack of detectable viral DNA in infectious stocks, and no DNA amplification was detected in viral preparations used for cell infections (data not shown). Complementary inhibition experiments were done using the CXCR4-specific ligand SDF1 and/or the RT inhibitor AZT before incubation with the virus. PCR analysis using gag-specific primers revealed that amplification yields were strongly diminished on preincubation in the presence of HIV entry or RT inhibitors (not shown). These results strongly suggest HIV-1 X4 and R5 entry and retro-transcription of viral RNA.
Analysis of viral Env protein expression
HIV-1 env expression was evaluated by a fusion assay between HIV-1-infected human adipose cells and specific indicator target cell lines at days 5 and 10 postinfection. Syncytia formation was clearly evidenced by an X-Gal test and revealed that NDK and NL43- or HIV-1 VEN-infected-preadipocytes expressed Env protein, allowing fusion between susceptible and indicator cells. Depending on the donor, the number of syncytia ranged between 1 and 5 to > 100 (not shown).
CONCLUSION AND SIGNIFICANCE
Taken altogether, these results strongly suggest entry of X4 or R5 strains in preadipocytes. Entry with the X4 HIV-1 strains would be dependent on the use of the CXCR4 receptor; finally, preadipocytes could exhibit a weak release of infectious viral particles.
1. Expression of HIV-1 receptors
Immunocytochemical analysis clearly showed expression of CD4, CXCR4, and CCR5 proteins on fully mature adipocytes containing fat droplets (Fig. 1A
). The fact that we obtained similar results using the PAZ6 cell line allowed us to consider that detection of the three receptors in primary culture was not due to the presence of CD4-positive contaminating cell types (CD4 lymphocytes or macrophages). Immunohistochemical detection of the three receptors on sections of human fat tissue confirmed their in vivo expression in adipose cells (Fig. 1B
).
2. Adipocytes as HIV-1 target cells?
Infection experiments clearly revealed a low and increasing p24 release (Fig. 2A
). The amounts obtained barely reached those of viral inputs and raised the question of an efficient infection of preadipocytes. It is noteworthy that during the time count experiment, no evidence of cytopathic effect could be observed in host-infected cells. Indeed, our results may reflect a low efficiency of infection and/or a low efficiency of viral replication compared to either lymphocytic or monocytic cells. It has recently been shown that adipose cells display macrophage-like characteristics and monocytes/macrophages do not allow efficient entry of X4 strains although they express both CXCR4 and CD4. Viral production yields obtained after exposure of preadipocytes to high viral inputs did not clearly demonstrated an efficient viral production. In fact, we cannot exclude the hypothesis of a release of particles as consequence of virus uptake as previously shown for dendritic cells. The fusion efficiencies observed after cocultures of infected preadipocytes with indicator cells may also be associated with a ‘fusion from without’ mechanism, thus accounting for the low number of blue syncytia (not shown). Nonetheless, even though p24 production, infection of CEM cells, and fusion experiments could sustain the hypothesis of a dendritic cell-like and/or ‘fusion from without’ behavior, characterization of specific viral DNA 10–15 days after infection of preadipocytes strongly suggested that viral entry and retro-transcription had occurred (not shown). Besides, passive uptake of viral particle and low infection efficiency mechanisms may not be exclusive. Work is in progress to investigate this hypothesis.
Finally, adipose cells represent the first cell type that does not belong to the immune system (T CD4 lymphocytes and cells from the myelomonocytic lineage), expressing all specific HIV receptors, and may be susceptible to being weakly infected or to capture viral particles. Considering the implications of adipose tissue in the pathogenesis of AIDS (cachectic state and lipodystrophy syndrome after anti-protease treatment), the relevance of our in vitro observations may be of great interest in the context of HIV-1 pathogenesis. However, evidence of viral production after completion of virus cycle and/or of passive release after viral capture is of great interest and needs to be further investigated.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0947fje; to cite this article, use FASEB J. (June 21, 2002) 10.1096/fj.01-0947fje 
2 These authors contributed equally to the work.
3 Present address: Department of Biological Sciences, The Open University, Walton Hall, Milton Keynes MK7 6AA, UK.
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