SII | SMA | SMAMS | SMB | SCMB
| Main Page
MICROBIAL PATHOGENS
IN HUMAN LYMPHOID TISSUE
Leonid Margolis, PhD, Head, Section on Intercellular Interactions Jean-Charles Grivel, PhD, Staff Scientist Angelique Biancotto, PhD, Visiting Fellow Yoshinori Ito, MD, PhD, Visiting Fellow Wendy Fitzgerald, Special
Volunteer Yana Elke Rucker, Contractor |
|
Critical events of human immunodeficiency
virus (HIV) disease occur in lymphoid tissue, where infection leads to T cell
depletion and immunodeficiency. Therefore, we study HIV pathogenesis in human
lymphoid tissue ex vivo. We found that noninfectious virions are
immunosuppressive, indicating that defective virions in vivo that
constitute the majority of viral particles may contribute to
immunodeficiency. In contrast, T cell depletion ex vivo requires
productive infection. Work conducted in our laboratory has suggested that
triggering in vivo viral production in latently infected cells in
combination with the delivery of therapies may be a meaningful strategy to
purge latent viral reservoirs. To identify adequate activators of latent
infection, we showed that prostratin, which reactivates HIV-1 from latency
and restricts HIV infection but does not stimulate cell cycling, is a
promising activator. We also investigated the role of HIV accessory genes in
CD4 T cell depletion. Using HIV constructs deleted of individual accessory
genes or their combinations, we found that nef, vpr, and vpu
are relevant for efficient viral infection and for CD4 T cell depletion in
HIV-1-infected human lymphoid tissues. Effect of accessory genes on HIV-1 replication
and CD4+ T cell depletion in human lymphoid tissue ex vivo Rücker,
Grivel, Margolis; in collaboration with Kirchhoff HIV-1 and simian immunodeficiency virus (SIV) contain
several genes that are not required for viral spread in cell lines and are
therefore called accessory genes. However, work has suggested that these
genes might play important roles in infected hosts in vivo as well as
in primary cells. The relevance of the accessory vpr, vpu, and
nef genes for HIV-1 replication in human lymphoid tissue (HLT),
the major site of viral replication in vivo, is largely unknown. We
investigated the role of accessory genes by infecting blocks of human tonsillar
tissue with HIV-1 mutants containing single or combined deletions of vpu,
vpr, and nef and evaluating virus replication and CD4+
T cell depletion. A single deletion of any of the accessory genes
nef, vpr, and vpu decreased viral replication in ex
vivo–infected human lymphoid tissues to the levels of 13 ± 7
percent, 38 ± 9 percent, and 32 ± 16 percent, respectively, of that
of the wild-type parental virus. Combined deletion of vpr and
vpu impaired HIV-1 replication even more severely, and additional
deletion of either vpr or vpu further attenuated replication
of the nef-deleted HIV-1 variant. However, only the combined
deletion of all three accessory genes completely disrupted HIV-1 replication.
The progressive loss of CD4+ T
lymphocytes is a major characteristic of HIV-1 infection and AIDS. We
evaluated this loss in tissues infected with wild-type virus and accessory gene–deleted
HIV-1 variants by means of flow cytometry. Consistent with findings in a
previous study, parental NL4-3 HIV-1 depleted ex vivo–infected
tissues of 40 to 50 percent of these cells within 15 days of infection. In
contrast, the deletion mutants caused less CD4+ T cell depletion
with a maximum of about 15 percent, with a strong correlation between the
number of productively infected CD4+ T cells and the level of
their depletion. Thus, although molecular mechanisms for low viral
infectivity and virus production may differ for different mutants, they all
result in a lower number of infected cells and hence less CD4+ T
cell depletion in infected tissues. Many of the suggested mechanisms underlying
facilitation of HIV-1 infection by accessory genes are related to their
involvement in cell activation. Therefore, we tested whether these genes
change the sensitivity of the system to IL-2. Our results demonstrate that
although IL-2 stimulates replication of both wild-type NL4-3 and its mutants,
exogenous IL-2 increased, on average, NL4-3 wild-type virus production about
13-fold, whereas production of the vpr or vpu deletion mutants
was enhanced only 2.5- to 5.4-fold. Thus, all three accessory genes, nef,
vpr, and vpu, are important for efficient replication and CD4+
T cell depletion in ex vivo–infected human lymphoid tissues. Some
effects of the genes may be related to cell activation. Most important, our
data suggest that, similarly to Nef, Vpr and Vpu are relevant for efficient viral
infection and for CD4 T cell depletion in HIV-1–infected individuals. Rücker E, Grivel J-C, Münch J, Kirchhoff F,
Margolis L. Vpr and Vpu are important for efficient HIV-1 replication and
CD4+ T cell depletion in human lymphoid tissue ex vivo. J Virol
2004;78:12689-12693. Rücker E, Münch J, Wildum S, Brenner M,
Eisemann J, Margolis L, Kirchhoff F. A naturally occurring variation in the
proline-rich region does not attenuate human immunodeficiency virus type 1
Nef function. J Virol 2004;78:10197-10201. Immunosuppressive activity
of noninfectious HIV-1 virions in human lymphoid tissue ex vivo Fitzgerald,
Sylwester, Grivel,a Margolis; in collaboration with Lifson Ex vivo HIV-1 infection of human
lymphoid tissue recapitulates some aspects of in vivo HIV-1 infection,
including a severe depletion of CD4+ T cells and suppression of
humoral immune responses to recall antigens or to polyclonal stimuli. These
effects are induced by infection with X4 HIV-1 variants, whereas infection
with R5 variants results in only mild depletion of CD4+ T cells
and no suppression of immune responses. The mechanisms of immunosuppression
observed both in vivo and ex vivo and the contribution of
various factors to disease progression are not fully understood. The vast majority
of HIV-1 virions circulating in HIV-1–infected patients are not infectious.
Therefore, cells are more likely to interact with noninfectious particles
than with their infectious counterparts, raising the question of the
pathogenesis of noninfectious virions in human lymphoid tissue. To answer
this question, we used HIV-1 virions rendered noninfectious by treatment with
aldrithiol-2 (AT-2). The AT-2–treated virions retain both the structure and
function of the viral envelope glycoproteins, allowing authentic interactions
with a variety of target cells, but are not infectious and, in human tissues ex
vivo, do not induce CD4+ T cell loss. However, we found that
the inactivated HIV-1 virions impair the ability of ex vivo human
lymphoid tissue to produce IgG in response to stimulation. In particular, we found that exposure of the cultures
to AT-2–inactivated X4LAV.04 resulted in inhibition of antibody
responses to tetanus toxoid (TT) and/or pokeweed mitogene (PWM) similarly to
productive infection of lymphoid tissues by X4LAV.04. In contrast,
no inhibition of anti-TT IgG or total IgG was observed when stimulated
tissues were exposed to comparable or higher amounts of AT-2–inactivated R5SF162
virions. Inactivated R5SF162 thus behaved similarly to its
infectious counterpart, which does not inhibit antibody responses in ex
vivo human lymphoid tissues. Furthermore, we found that virion-free
medium conditioned by tissues exposed to AT-2–inactivated X4LAV.04
inhibited anti-TT IgG and total IgG responses of fresh cultures, indicating
that AT-2–inactivated X4LAV.04 virus induced the secretion of an
immunosuppressive factor. This factor(s) was inactivated by heat, whereas
freezing and thawing did not affect its immunosuppressive activity. To
evaluate the size of this factor, or factor-containing complex, we
size-fractionated virion-free conditioned medium. The inhibitory activity of
the media conditioned by tissue exposed to AT-2–inactivated X4LAV.04 is
associated with the fraction containing molecules of MW greater than 50 kDa.
To identify the cellular source of the factor, we fractionated tonsillar
cells. The supernatants of cultures of the CD4+ T cell–enriched
fraction exposed to AT-2–inactivated X4LAV.04, inhibited immune
responses, whereas the fraction depleted of CD4+ T cells was not
immunosuppressive. Finally, we found that the immunosuppressive factor
secreted by tissues treated with AT-2–inactivated X4LAV.04
significantly suppressed production of IgG in B cells isolated from lymphoid
tissue. In conclusion, neither productive viral
infection nor CD4+ T cell depletion seems to be necessary to mediate
HIV-induced inhibition of antibody production in human lymphoid tissue in ex
vivo. Moreover, the virus itself is only required to trigger suppression
of B cell responses; its suppressive activity is maintained without its
continuous presence and can be transferred by virion-free conditioned medium.
We conclude that medium conditioned by tissue incubated with AT-2–inactivated
X4 HIV-1 contains soluble factor(s) that are responsible for suppression of B
cell responses. Given that the majority of the virions circulating in vivo
are not detectably infectious, it is reasonable to speculate that some of the
defective HIV-1 virions may suppress immune responses, similar to what we have
shown for AT-2–inactivated virus. Identification and isolation of the
immunosuppressive factor and elucidation of the exact mechanism(s) by which
it inhibits humoral immune responses may help to explain the
immunopathogenesis of HIV-1 infection and AIDS and may suggest avenues for
therapeutic intervention. Ito Y, Grivel J-C, Kiselyeva Y, Reichelderfer P,
Margolis L. CXCR4-tropic HIV-1 suppresses replication of CCR5-tropic HIV-1 in
human lymphoid tissue by selective induction of CC-chemokines. J Infect
Dis 2004;189:506-514. Fitzgerald W, Sylwester AW, Grivel J-C, Lifson
D, Margolis L. Non-infectious X4 but not R5 HIV-1 virions inhibit humoral
immune responses in human lymphoid tissue ex vivo. J Virol 2004;78:13:7061-7068. Reactivation of HIV-1 replication and CD4+
T lymphocyte depletion and in human lymphoid tissue infected ex vivo with
doxycycline-dependent HIV-1 Kiselyeva,
Biancotto, Ito, Grivel, Margolis; in collaboration with Das, Berkhout, Hirsch
The hallmark of HIV infection is the depletion
of CD4+ T lymphocytes. It is well established that HIV productive
infection kills CD4+ T cells. However, it is believed that CD4+
T cells that do not produce virus but nonetheless reside in infected tissues (infected
latently or “bystanders�) are killed as well. To address this problem, we
used human lymphoid tissues infected ex vivo with HIV-rtTA, a newly
developed virus construct that can
infect cells and establish an integrated provirus in the absence of
doxycycline but whose transcription and replication depend on the presence of
the antibiotic doxycycline. To evaluate T cell depletion in latently and
productively infected tissues and to study the effect of free virions on cell
depletion, we compared CD4+ T cell numbers in tissues inoculated
with HIV-rtTA in the presence and absence of doxycycline. In the productively
infected tissues (that is, those inoculated with HIV-rtTA and cultured in the
constant presence of doxycycline), more than 60 percent of CD4+ T
cells were depleted after 12 days of culture. In contrast, we observed no
significant CD4+ T cell depletion in matched tissues that were
similarly inoculated but cultured in the absence of doxycycline (and thus
without viral replication). Moreover, when the amount of virus present in
latently infected cultures was matched to that in productively infected
(doxycycline-treated) tissues by multiple inoculation of tissue (13 times
over the nine days), we observed no significant depletion of CD4+
T cells in the absence of doxycycline. It seems that nonproductive infection
does not contribute significantly to the death of CD4+ T cells. In
conclusion, HIV is sufficient to deplete tissue of productively infected CD4+
T cells but is not sufficient to cause a significant death of uninfected or
latently infected CD4+ T cells in the context of human lymphoid
tissue; thus, additional factors seem to contribute to bystander cell death in
vivo. Given that, unlike in vivo–infected tissues, ex vivo–infected
tissues do not respond to HIV infection by general immunostimulation, such an
immunostimulation may be the main additional factor for CD4+ T cell
depletion in HIV-infected individuals. Nevertheless, despite general stimulation,
some of the infected CD4+ T cells in HIV-infected individuals
continue to harbor a replication-competent HIV but do not constantly produce virus.
These cells constitute a major obstacle for anti–HIV therapy. To design
strategies to purge latent HIV reservoir, we investigated mechanisms by which
a non–tumor-promoting phorbol ester, prostratin
(12-deoxyphorbol-13-acetate, which was originally extracted from the Samoan
medicinal plant Homalanthus nutans), stimulates cells. First, we
investigated the effects of prostratin on cellular susceptibility to the
virus by using ex vivo human lymphoid tissue. Prostratin induced
expression of the activation markers CD25 and CD69 but inhibited cell
cycling. HIV-1 uptake was reduced in prostratin-stimulated CD4 T cells
consistent with a down-modulation of CD4 and CXCR4 receptors in PBMC and
lymphoid tissue. At post-entry level, prostratin reduced completion of
reverse transcription of the viral genome in lymphatic tissue. However,
prostratin facilitated integration of the reverse-transcribed HIV-1 genome in
nondividing CD4+ T cells and expression of already integrated
HIV-1, including latent forms. Thus, while prostratin stimulation restricts
susceptibility of primary resting CD4 T cells to HIV infection at the virus
cell-entry and at the reverse-transcription levels, it efficiently
reactivates HIV-1 from pre- and post-integration latency in resting CD4+
T cells. This dual role makes prostratin an excellent candidate for the
elimination of persistent HIV infection from latent reservoirs. A system of
human lymphoid tissue infected ex vivo with doxycycline-dependent
HIV-1 provides a model for studying viral latency under controlled
experimental conditions. Biancotto A, Grivel J-C, Gondois-Rey F,
Bettendroffer L, Vigne R, Brown S, Margolis L, Hirsch I. Dual role of
prostratin in inhibition of infection and reactivation of human
immunodeficiency virus from latency in primary blood lymphocytes and in
lymphoid tissue. J Virol 2004;78:10507-10515. Grivel J-C, Biancotto A, Ito Y, Kiselyeva Y, Ito I, aAndrew Sylwester, PhD,
former Postdoctoral Fellow COLLABORATORS Atze T. Das, PhD, Ben Berkhout, MD, Ivan
Hirsch, PhD, Institut National de la Santé et de la Recherche Médicale,
Marseille, France Frank Kirchhoff, PhD, Jeffrey D. Lifson, PhD,
AIDS Vaccine Program, SAIC Frederick, Inc., NCI, Frederick, MD For
further information, contact margolis@helix.nih.gov |