Systemic inflammation and liver damage in HIV/hepatitis C virus coinfection.

OBJECTIVES: Chronic hepatitis C virus (HCV) and HIV viral infections are characterized by systemic inflammation. Yet the relative levels, drivers and correlates of inflammation in these settings are not well defined. METHODS: Seventy-nine HIV-infected patients who had been receiving antiretroviral therapy (ART) for more than 2 years and who had suppressed plasma HIV levels (< 50 HIV-1 RNA copies/mL) were included in the study. Two patient groups, HCV-positive/HIV-positive and HCV-negative/HIV-positive, and a control group comprised of healthy volunteers (n = 20) were examined. Markers of systemic inflammation [interleukin (IL)-6, interferon gamma-induced protein (IP)-10, soluble tumour necrosis factor receptor-I (sTNF-RI) and sTNF-RII], monocyte/macrophage activation [soluble CD163 (sCD163), soluble CD14 and neopterin], intestinal epithelial barrier loss [intestinal fatty acid binding protein (I-FABP) and lipopolysaccharide (LPS)] and coagulation (d-dimers) were analysed. CD4 naïve T cells and CD4 recent thymic emigrants (RTEs) were enumerated. RESULTS: Plasma levels of IP-10, neopterin and sCD163 were higher in HCV/HIV coinfection than in HIV monoinfection and were positively correlated with indices of hepatic damage [aspartate aminotransferase (AST), alanine aminotransferase (ALT) and the AST to platelet ratio index (APRI)]. Levels of I-FABP were comparably increased in HIV monoinfection and HIV/HCV coinfection but LPS concentrations were highest in HCV/HIV coinfection, suggesting impaired hepatic clearance of LPS. Plasma HCV levels were not related to any inflammatory indices except sCD163. In coinfected subjects, a previously recognized relationship of CD4 naïve T-cell and RTE counts to hepatocellular injury was defined more mechanistically by an inverse relationship to sCD163. CONCLUSIONS: Hepatocellular injury in HCV/HIV coinfection is linked to elevated levels of certain inflammatory cytokines and an apparent failure to clear systemically translocated microbial products. A related decrease in CD4 naïve T cells and RTEs also merits further exploration.

Productive HIV-1 infection of human cervical tissue ex vivo is associated with the secretory phase of the menstrual cycle.

Cervical tissue explants (CTEs) from 22 HIV-1 seronegative women were exposed to R5 HIV-1 ex vivo. Eight CTEs were productively infected in terms of HIV-1 p24Gag release in culture supernatants, whereas 14 were not. Nonetheless, both accumulation of HIV-1gag DNA and of p24Gag(+) CD4(+) T cells and macrophages occurred in both productive and, at lower levels, in nonproductive CTEs. Nonproductive CTEs differed from productive CTEs for higher secretion of C-C motif chemokine ligand 3 (CCL3) and CCL5. A post-hoc analysis revealed that all productive CTEs were established from women in their secretory phase of the menstrual cycle, whereas nonproductive CTEs were derived from women either in their secretory (28%) or proliferative (36%) menstrual cycle phases or with an atrophic endometrium (36%). Thus, our results support the epidemiological observation that sexual HIV-1 transmission from males to women as well as from women to men is more efficient during their secretory phase of the menstrual cycle.

HIV-1 sexual transmission: early events of HIV-1 infection of human cervico-vaginal tissue in an optimized ex vivo model.

Infection and dissemination of human immunodeficiency virus (HIV)-1 through the female body after vaginal intercourse depends on the activation/differentiation status of mucosal CD4 T cells. In this study, we investigated this status and the susceptibility to HIV-1 infection of human cervico-vaginal tissue ex vivo. We found that virtually all T cells are of the effector memory phenotype with broad CC chemokine receptor 5 (CCR5) expression. As it does in vivo, human cervico-vaginal tissue ex vivo preferentially supports the productive infection of R5 HIV-1 rather than that of X4 HIV-1 in spite of the broad expression of CXC chemokine receptor 4 (CXCR4). X4 HIV-1 replicated only in the few tissues that were enriched in CD27(+)CD28(+) effector memory CD4 T cells. Productive infection of R5 HIV-1 occurred preferentially in activated CD38(+)CD4 T cells and was followed by a similar activation of HIV-1-uninfected (bystander) CD4 T cells that may amplify viral infection. These results provide new insights into the dependence of HIV-1 infection and dissemination on the activation/differentiation of cervico-vaginal lymphocytes.

Human immunodeficiency virus type 1 induces apoptosis in CD4(+) but not in CD8(+) T cells in ex vivo-infected human lymphoid tissue.

Progression of human immunodeficiency virus (HIV) disease is associated with massive death of CD4(+) T cells along with death and/or dysfunction of CD8(+) T cells. In vivo, both HIV infection per se and host factors may contribute to the death and/or dysfunction of CD4(+) and CD8(+) T cells. Progression of HIV disease is often characterized by a switch from R5 to X4 HIV type 1 (HIV-1) variants. In human lymphoid tissues ex vivo, it was shown that HIV infection is sufficient for CD4(+) T-cell depletion. Here we address the question of whether infection of human lymphoid tissue ex vivo with prototypic R5 or X4 HIV variants also depletes or impairs CD8(+) T cells. We report that whereas productive infection of lymphoid tissue ex vivo with R5 and X4 HIV-1 isolates induced apoptosis in CD4(+) T cells, neither viral isolate induced apoptosis in CD8(+) T cells. Moreover, in both infected and control tissues we found similar numbers of CD8(+) T cells and similar production of cytokines by these cells in response to phorbol myristate acetate or anti-CD3-anti-CD28 stimulation. Thus, whereas HIV-1 infection per se in human lymphoid tissue is sufficient to trigger apoptosis in CD4(+) T cells, the death of CD8(+) T cells apparently requires additional factors.

Breakdown of tolerance to a neo-self antigen in double transgenic mice in which B cells present the antigen.

Transgenic (Tg) mice expressing a foreign Ag, hen egg lysozyme (HEL), under control of the alphaA-crystallin promoter ("HEL-Tg" mice) develop immunotolerance to HEL attributed to the expression of HEL in their thymus. In this paper we analyzed the immune response in double (Dbl)-Tg mice generated by mating the HEL-Tg mice with Tg mice that express HEL Abs on their B cells ("Ig-Tg" mice). The B cell compartment of the Dbl-Tg mice was unaffected by the HEL presence and was essentially identical to that of the Ig-Tg mice. A partial breakdown of tolerance was seen in the T cell response to HEL of the Dbl-Tg mice, i.e., their lymphocyte proliferative response against HEL was remarkably higher than that of the HEL-Tg mice. T-lymphocytes of both Dbl-Tg and Ig-Tg mice responded to HEL at concentrations drastically lower than those found stimulatory to lymphocytes of the wild-type controls. Cell mixing experiments demonstrated that 1) the lymphocyte response against low concentrations of HEL is due to the exceedingly efficient Ag presenting capacity of the Ab expressing B cells and 2) breakdown of tolerance in Dbl-Tg mice can also be attributed to the APC capacity of B cells, that sensitize in vivo and stimulate in vitro populations of T cells with low affinity toward HEL, assumed to be escapees of thymic deletion. These results thus indicate that T cell tolerance can be partially overcome by the highly potent Ag presenting capacity of Ab expressing B cells.

Preferential coreceptor utilization and cytopathicity by dual-tropic HIV-1 in human lymphoid tissue ex vivo.

Many HIV-1 isolates at the late stage of disease are capable of using both CXCR4 and CCR5 in transfected cell lines, and are thus termed dual-tropic. Here we asked whether these dual-tropic variants also use both coreceptors for productive infection in a natural human lymphoid tissue microenvironment, and whether use of a particular coreceptor is associated with viral cytopathicity. We used 3 cloned dual-tropic HIV-1 variants, 89.6 and its chimeras 89-v345.SF and 89-v345.FL, which use both CCR5 and CXCR4 in transfected cell lines. In human lymphoid tissue ex vivo, one variant preferentially used CCR5, another preferentially used CXCR4, and a third appeared to be a true dual-tropic variant. The 2 latter variants severely depleted CD4(+) T cells, whereas cytopathicity of the virus that used CCR5 only in lymphoid tissue was mild and confined to CCR5(+)/CD4(+) T cells. Thus, (a) HIV-1 coreceptor usage in vitro cannot be unconditionally extrapolated to natural microenvironment of human lymphoid tissue; (b) dual-tropic viruses are not homogeneous in their coreceptor usage in lymphoid tissue, but probably comprise a continuum between the 2 polar variants that use CXCR4 or CCR5 exclusively; and (c) cytopathicity toward the general CD4(+) T cell population in lymphoid tissue is associated with the use of CXCR4.

Efficient presentation of multivalent antigens targeted to various cell surface molecules of dendritic cells and surface Ig of antigen-specific B cells.

To study the relation between the form of an Ag and the response to it, we compared presentation in vitro with hen egg lysozyme (HEL)-specific T cells from TCR transgenic mice of free HEL and liposome-encapsulated HEL by different APC. HEL-specific splenic B cells or bone marrow-derived dendritic cells were incubated with free HEL or HEL-containing liposomes targeted by Ab to either surface Ig, the Fc receptor, or MHC class I and II molecules. Ag presentation by HEL-specific B cells was at least 100-fold more efficient for HEL in surface Ig-targeted liposomes than free HEL taken up by the same receptor or HEL in liposomes targeted to class I or II molecules. Ag presentation by dendritic cells from Fc receptor-targeted vesicles was augmented 1,000-10,000-fold compared with free Ag or nontargeted liposomes, but presentation was also efficient when Ag was targeted to class I or II molecules. These results indicate that Ag-specific B cells and dendritic cells can be equally efficient in stimulating IL-2 production by Ag-specific T cells from unimmunized TCR transgenic mice when the Ag is multivalent and taken up by appropriate receptors. In contrast to B cells, which require engagement of surface Ig for optimal presentation, dendritic cells may present Ag by means of several different cell surface molecules.

Evidence for the HIV-1 phenotype switch as a causal factor in acquired immunodeficiency.

Both cellular and humoral immunodeficiency develop in vivo after prolonged infection with HIV-1, but the mechanisms are unclear. Initial infection with HIV-1 is transmitted by macrophage (M)-tropic/non-syncytia-inducing (NSI) viruses, which hyperactivate the immune system, and, in one view, cause immunodeficiency by "exhaustion" of lymphoid tissue. An alternative hypothesis is that immunodeficiency is caused by the replacement of M-tropic viruses by T cell (T)-tropic/syncytia-inducing (SI) viruses, which are known to be highly cytopathic in vitro and emerge late in infected individuals around the time of transition to AIDS (refs. 1, 7-9). To test these two possibilities, we have developed an ex vivo model of humoral immunity to recall antigens using human lymphoid tissue. This tissue supports productive infection with both M- and T-tropic HIV-1 isolates when cultured ex vivo. We found that specific immune responses were enhanced by productive infection of the tissue with M-tropic/NSI HIV-1 isolates, but were blocked by T-tropic/SI HIV-1 isolates. The mechanism involves specific irreversible effect on B-cell activity. Our results support the hypothesis that the phenotype switch to T-tropic viruses is a key determinant of acquired humoral immunodeficiency in patients infected with HIV.

Different endocytic compartments are involved in the tight association of class II molecules with processed hen egg lysozyme and ribonuclease A in B cells.

The processing of exogenous antigens and the association of peptides with class II molecules both occur within the endocytic pathway. 2A4 B lymphoma cells of the H-2k haplotype were grown in the presence or the absence of two different exogenous antigens (hen egg lysozyme and ribonuclease A) internalized by fluid-phase endocytosis. Using subcellular fractionation techniques, we demonstrate that, in the presence of hen egg lysozyme, newly synthesized SDS-stable class II molecules are detected in a dense endocytic compartment which does not have the characteristics of neither early and late endosomes nor lysosomes. In contrast, no SDS-stable class II molecules are observed between ribonuclease A and newly synthesized class II molecules. Interestingly, when class II molecules are analyzed at steady state, SDS-stable class II molecules induced by ribonuclease A are found in a compartment cosedimenting with late endosomes. These results suggest that the tight associations between ribonuclease A or hen egg lysozyme with class II molecules occur in distinct endocytic compartments and that these associations may depend on the sensitivity of antigens to proteolysis.

Endocytosis and presentation of liposome-associated antigens by B cells.

B cells have limited endocytic capacity and are reported to endocytose and present liposome-encapsulated antigens poorly. B cells also endocytose soluble antigens poorly, except those for which their surface immunoglobulin is specific, which are taken up and presented efficiently. We present results indicating that, in vitro, B cells endocytose small liposomes bearing antigen with affinity for their surface immunoglobulin. Antigen encapsulated in liposomes targeted by antibody specific for surface immunoglobulin is presented to T cells as efficiently as specific antigen in soluble form. These studies provide a rational basis for the design of liposomes optimized to stimulate T-dependent B-cell responses.