INTERACTIONS OF HUMAN
PATHOGENSIN HUMAN LYMPHOID TISSUE
Jean-Charles Grivel, Ph.D., Staff Scientist
Yoshinori Ito, M.D., Ph.D., Postdoctoral Fellow
Silvia Chen, Ph.D., Guest Researcher
Wendy Fitzgerald, Guest Researcher
Yana Kiselyeva, Guest Researcher
Critical events of HIV disease occur in lymphoid tissue. One such event is HIV-1 divergence into a “swarm” of viral quasi-species whose dominant forms change in the course of the disease. In particular, CCR5-utilizing (R5) HIV-1 variants predominate in the early stages of HIV-1 disease, whereas CXCR4-utilizing (X4) HIV-1 variants may dominate later. When such a switch occurs, it is followed by an acceleration of disease progression to AIDS. The general aim of this project is to investigate the mechanisms underlying the R5-to-X4 switch and the immunodeficiency development associated with such a switch. We postulate that R5 and X4 HIV variants affect each other and that various microbes can selectively inhibit or stimulate R5 or X4 replication by modulating the chemokine/cytokine network, thus contributing to the switch of viral dominance. We addressed mechanisms of HIV-1 immunopathogenesis in human lymphoid tissue ex vivo, tissue that preserves tissue cytoarchitecture and supports productive HIV infection without exogenous stimulation or activation. These tissues are infected ex vivo with a mixture of HIV-1 quasi-species differing in co-receptor usage and with HIV co-pathogens, in particular human herpesvirus 6 (HHV-6).
CXCR4-Tropic HIV-1 Suppression of CCR5-Tropic HIV-1 Replication in Human Lymphoid Tissue ex vivo by Selective Induction of CC-Chemokines
Ito, Grivel, Chen, Kiselyeva, Margolis; in collaboration with Reichelderfer
To evaluate replication of individual CCR5- and CXCR4-utilizing variants, we developed a new real-time RT PCR–based assay that accurately estimates the number of viral-specific RNA copies even when their relative abundance is two orders of magnitude. We designed primers that recognize only target sequences of gp120 that are specific for R5 and X4 HIV-1 variants and that do not give any non-specific amplification. We further improved the assay so that variations were small and the standard curves were linear, allowing a reliable determination of the number of RNA copies in experimental samples. We verified the accuracy of the assay by comparing the results obtained with viral-specific primers and those obtained with a primer common to all the viruses. Moreover, the amounts of HIV-1 RNA copies in various experimental conditions correlated with the concentrations of p24, another conventional assay to determine virus replication. We applied the newly developed assay to an ex vivo model consisting of blocks of human lymphoid tissue infected with mixtures of R5 and X4 HIV-1 variants.
To study whether the R5 and X4 viruses interfere with each other in the context of lymphoid tissue, we used two pairs of HIV-1, one pair consisting of the prototypic X4 and R5 HIV-1 variants, LAV.04 and SF162, respectively, and the other pair consisting of the “X4-skewed” dual-topic (R5X4) 89.6 and its R5 chimera 89-v345.SF. On average, LAV.04 infection suppressed SF162 replication to about 7 percent of the level of tissues infected with SF162 alone. In contrast, the levels of LAV.04 replication in tissues co-infected with SF162 did not differ from those found in controls infected only with LAV.04. Further, 89.6 replicated in human lymphoid tissue to a level similar to that found in singly infected tissue, whereas replication of the R5-chimera 89-v345.SF in tissues co-infected with 89.6 was suppressed about five-fold compared with matched tissues infected with 89-v345SF only. To study whether X4 merely prevents the establishment of R5 infection or affects ongoing R5 infection as well, we delayed tissue inoculation with LAV.04 for 3, 24, 72, 96, or 144 hours to allow the establishment of SF162 infection. In all experiments, LAV.04 significantly inhibited SF162 replication in co-infected tissues compared with singly infected tissues.
Given that CC-chemokines are potent inhibitors of replication of R5 HIV-1 variants in both isolated cells and human lymphoid tissues, we investigated whether X4 infection up-regulates CC-chemokines. Using a multiplex Luminex assay, we evaluated the concentrations of a panel of cytokines in the culture medium bathing the lymphoid tissue. In SF162-infected tissues, the average concentrations of all measured chemokines/cytokines were similar to those in uninfected controls. In contrast, in tissues infected with LAV.04 either alone or together with SF162, the levels of MIP-1a, MIP-1b, and RANTES increased four- to seven-fold compared with those in matched uninfected controls. Flow cytometry revealed that X4 infection augments both the number of chemokine-producing cells and cell productivity. A cocktail of exogenous CC-chemokines at concentrations similar to those induced by LAV.04 simulated the effects of the X4 virus on SF162 replication.
We conclude that X4 and R5 HIV-1 variants interfere with each other in human lymphoid tissues and that such interference is associated with an up-regulation of CC-chemokines. X4 inhibition of R5 replication may result in a shift of dominance among the HIV-1 variants, thus contributing to the R5-X4 switch.
Selective Suppression of CCR5 HIV-1 Variants by Co-Infection with HHV-6 in Human Lymphoid Tissue ex vivo
Grivel, Ito, Fitzgerald, Margolis; in collaboration with Fagà, Lusso, Malnati, Santoro
In vivo, HIV disease progression is associated with the emergence of concurrent infections that may affect the course of the disease by mechanisms that remain unknown. A lymphotropic agent frequently reactivated in HIV-infected patients is human herpesvirus 6 (HHV-6), which has been proposed as a co-factor in AIDS progression. As multiple interactions have been documented between HHV-6 and HIV-1 both in vitro and in vivo, we investigated whether the two viruses affect each other within the context of human lymphoid tissue.
We developed a new model system that uses human lymphoid tissue cultured ex vivo to study the cellular tropism and pathogenic mechanisms of HHV-6. Viral strains belonging to both subgroup A (strain GS) and subgroup B (strain PL1) were able to infect human tonsillar tissue fragments productively. The majority of productively infected cells were CD4+ T lymphocytes expressing a memory phenotype (CD45RA-62L-) while infection of CD8+ T cells was efficient only with HHV-6 A. Little, if any, HHV-6 replication was documented in B cells and non-lymphoid cells. Expression of CD4 was up-regulated, whereas both CD46, which serves as a cellular receptor for HHV-6, and CD3 were down-regulated. The results provide a novel ex vivo model for investigating the complex interactions between HHV-6 and other pathogens of the immune system, in particular HIV.
Strikingly, co-infection with HHV-6 differen-tially affected the replication of the R5 and X4 HIV-1 variants. A dramatic (five-fold) suppres-sion of HIV-1 replication was observed in tissues co-infected with HHV-6 and the CCR5-using variant SF162. In contrast, we noted a slight enhancement in the replication of the CXCR4-using variant LAV.04 in matched tissue blocks. To prove conclusively that the differential effect of HHV-6 on HIV-1 replication was related to HIV-1 co-receptor tropism, we used two iso-genic dual-tropic molecular clones, 89.6 and 89-v345.SF, described above, that behave in ex vivo human lymphoid tissue as CXCR4- or CCR5-tropic variants. In agreement with our results with uncloned HIV-1 isolates, HHV-6 co-infection of lymphoid tissue resulted in a marked (five-fold) inhibition of 89-v345.SF replication but did not affect 89.6 replication. To investigate the potential mechanisms underlying the effects of HHV-6 on HIV-1 infection, we evaluated the production of RANTES, MIP-1a, and MIP-1b, the three CC chemokines that bind to CCR5, as they were reported to cause similar effects on the replication of different HIV-1 strains. In all the tissues infected with HHV-6, either alone or in combination with HIV-1, we documented a dramatic increase in the production of RANTES, whereas the levels of MIP-1a and MIP-1b were not significantly augmented. Regression analysis demonstrated a significant correlation between HHV-6 replication and RANTES up-regulation. Moreover, we observed a significant correlation between HHV-6–induced RANTES up-regula-tion and suppression of SF162 replication. Flow cytometry of cells isolated from HHV-6–infected tissue blocks also documented increased numbers of RANTES-producing cells; interest-ingly, about one-third of the RANTES-producing cells were infected by HHV-6. Adding RANTES to the culture medium in amounts similar to those induced by HHV-6 causes inhibition of R5, but not X4 replication, to a level similar to that caused by HHHV-6 co-infection.
Our results provide the first demonstration that another viral agent can selectively affect HIV-1 infection in the context of human lymphoid tissue. Our observations suggest that HHV-6 replication, an event that frequently occurs in vivo during the progression of HIV-1 disease, selectively suppresses CCR5-utilizing HIV-1 variants while not affecting or even stimulating the replication of CXCR4-tropic variants. Thus, it is possible that HHV-6 favors the latter variants in co-infected tissues, from which they can spread throughout the entire organism and become dominant, as frequently occurs during AIDS progression. Exploitation of the ability of HHV-6 to induce RANTES in lymphoid tissue might lead to novel therapeutic and preventive strategies against HIV infection.
PUBLICATIONS
- Glushakova S, Munch J, Carl S, Greenough TC, Sullivan JL, Margolis
L, Kirchhoff F. CD4 down-modulation by human immunodeficiency virus
type 1 nef correlates
with the efficiency of viral replication and with CD4(+) T-cell depletion in
human lymphoid tissue ex vivo. J Virol. 2001;75:10113-10117.
- Gondois-Rey F, Grivel JC, Biancotto A, Pion M, Vigne R, Margolis L, Hirsch
I. Segregation of R5 and X4 HIV-1 variants to memory T cell subsets differentially
expressing CD62L in ex vivo infected human lymphoid tissue. AIDS. 2002;16:1245-1249.
- Grivel JC, Ito Y, Fagá G, Santoro F, Shaheen F, Malnati M,
Fitzgerald W, Lusso P, Margolis L. Suppression of CCR5- but not CXCR4-tropic
HIV-1 in
lymphoid tissue by human herpesvirus 6. Nat Med. 2001;7:1232-1235.
- Malkevitch N, McDermott DH, Yi Y, Grivel JC, Schols D, De Clercq
E, Murphy PM, Glushakova S, Collman R, Margolis L. Coreceptor choice
and T cell depletion
by R5, X4 and R5X4 HIV-1 variants in CCR5-deficient (CCR5D32)
and normal human lymphoid tissue. Virology. 2001;281:239-247.
- Malkevich N, Womack C, Pandya P, Grivel JC, Fauci AS, Margolis L. Human immunodeficiency virus type 1 (HIV-1) non-B subtypes are similar to HIV-1 subtype B in that coreceptor specificity is a determinant of cytopathicity in human lymphoid tissue infected ex vivo. J Virol. 2001;75:10520-10522.
COLLABORATORS
Giovanni Fagà, Ph.D., San Raffaele Institute, Milan, Italy
Paolo Lusso, M.D., San Raffaele Institute, Milan, Italy
Mauro S. Malnati, Ph.D., San Raffaele Institute, Milan, Italy
Patricia Reichelderfer, Ph.D., Contraception and Reproductive Health Branch,
NICHD, Bethesda, MD
Fabio Santoro, Ph.D., San Raffaele Institute, Milan, Italy