Inadequate Clearance of Translocated Bacterial Products in HIV-Infected Humanized Mice

Bacterial translocation from the gut and subsequent immune activation are hallmarks of HIV infection and are thought to determine disease progression. Intestinal barrier integrity is impaired early in acute retroviral infection, but levels of plasma lipopolysaccharide (LPS), a marker of bacterial translocation, increase only later. We examined humanized mice infected with HIV to determine if disruption of the intestinal barrier alone is responsible for elevated levels of LPS and if bacterial translocation increases immune activation. Treating uninfected mice with dextran sodium sulfate (DSS) induced bacterial translocation, but did not result in elevated plasma LPS levels. DSS-induced translocation provoked LPS elevation only when phagocytic cells were depleted with clodronate liposomes (clodrolip). Macrophages of DSS-treated, HIV-negative mice phagocytosed more LPS ex vivo than those of control mice. In HIV-infected mice, however, LPS phagocytosis was insufficient to clear the translocated LPS. These conditions allowed higher levels of plasma LPS and CD8+ cell activation, which were associated with lower CD4+/CD8+ cell ratios and higher viral loads. LPS levels reflect both intestinal barrier and LPS clearance. Macrophages are essential in controlling systemic bacterial translocation, and this function might be hindered in chronic HIV infection

Humanized Mice Recapitulate Key Features of HIV-1 Infection: A Novel Concept Using Long-Acting Anti-Retroviral Drugs for Treating HIV-1

BACKGROUND: Humanized mice generate a lymphoid system of human origin subsequent to transplantation of human CD34+ cells and thus are highly susceptible to HIV infection. Here we examined the efficacy of antiretroviral treatment (ART) when added to food pellets, and of long-acting (LA) antiretroviral compounds, either as monotherapy or in combination. These studies shall be inspiring for establishing a gold standard of ART, which is easy to administer and well supported by the mice, and for subsequent studies such as latency. Furthermore, they should disclose whether viral breakthrough and emergence of resistance occurs similar as in HIV-infected patients when ART is insufficient. METHODS/PRINCIPAL FINDINGS: NOD/shi-scid/γ(c)null (NOG) mice were used in all experimentations. We first performed pharmacokinetic studies of the drugs used, either added to food pellets (AZT, TDF, 3TC, RTV) or in a LA formulation that permitted once weekly subcutaneous administration (TMC278: non-nucleoside reverse transcriptase inhibitor, TMC181: protease inhibitor). A combination of 3TC, TDF and TMC278-LA or 3TC, TDF, TMC278-LA and TMC181-LA suppressed the viral load to undetectable levels in 15/19 (79%) and 14/14 (100%) mice, respectively. In successfully treated mice, subsequent monotherapy with TMC278-LA resulted in viral breakthrough; in contrast, the two LA compounds together prevented viral breakthrough. Resistance mutations matched the mutations most commonly observed in HIV patients failing therapy. Importantly, viral rebound after interruption of ART, presence of HIV DNA in successfully treated mice and in vitro reactivation of early HIV transcripts point to an existing latent HIV reservoir. CONCLUSIONS/SIGNIFICANCE: This report is a unique description of multiple aspects of HIV infection in humanized mice that comprised efficacy testing of various treatment regimens, including LA compounds, resistance mutation analysis as well as viral rebound after treatment interruption. Humanized mice will be highly valuable for exploring the antiviral potency of new compounds or compounds targeting the latent HIV reservoir

RAG2−/−γc−/− Mice Transplanted with CD34+ Cells from Human Cord Blood Show Low Levels of Intestinal Engraftment and Are Resistant to Rectal Transmission of Human Immunodeficiency Virus▿

Rectal transmission is one of the main routes of infection by human immunodeficiency virus type 1 (HIV-1). To efficiently study transmission mechanisms and prevention strategies, a small animal model permissive for rectal transmission of HIV is mandatory. We tested the susceptibility of RAG2−/−γc−/− mice transplanted with human cord blood hematopoietic stem cells to rectal infection with HIV. We rectally exposed these humanized mice to cell-free and cell-associated HIV. All mice remained HIV negative as assessed by plasma viral load. The same mice infected intraperitoneally showed high levels of HIV replication. In the gut-associated lymphatic tissue, we found disproportionately smaller numbers of human cells than in other lymphoid organs. This finding may explain the observed resistance to rectal transmission of HIV. To increase the numbers of local HIV target cells and the likelihood of HIV transmission, we treated mice with different proinflammatory stimuli: local application of interleukin-1β, addition of seminal plasma to the inoculum, or induction of colitis with dextran sodium sulfate. These procedures attracted some human leukocytes, but the transmission rate was still very low. The humanized mice showed low levels of human engraftment in the intestinal tract and seem to be resistant to rectal transmission of HIV, and thus they are an unsuitable model for this application

Epineural adipose-derived stem cell injection in a sciatic rodent model

The aim was to evaluate the regenerative effect of epineural injection of rat ASCs (rASCs) in three different settings of acute and chronic compression in a rat sciatic nerve model

Triggering TLR7 in mice induces immune activation and lymphoid system disruption, resembling HIV-mediated pathology

Chronic immune activation is a major cause for progressive immunodeficiency in human immunodeficiency virus type-1 (HIV) infection. The underlying trigger, however, remains largely unknown. HIV single-stranded RNA is a potent immune activator by triggering Toll-like receptor (TLR) 7/8. Thus, we hypothesized that sustained TLR7 triggering induces chronic immune activation and thereby contributes to progressive immunodeficiency. We used the synthetic compound R848 or a mixture of uridine-rich HIV single-stranded (ss) RNA oligonucleotides - both are potent TLR7/8 agonists - to explore the effects of sustained TLR7 triggering on the murine lymphoid system. Sustained TLR7 triggering induced an immunopathology reminiscent of progressive lymphoid destruction in HIV disease; we observed lymphopenia, elevated proinflammatory cytokines, splenomegaly, contracted lymphoid subsets, and lymphoid microarchitecture alteration with reduced marginal zone B-lymphocytes. Upon exposure to inactivated vesiculo-stomatitis virus, antibody production was abolished, although splenic lymphocytes were activated and total IgG was elevated. Our data imply that HIV itself may directly contribute to immune activation and dysfunction by stimulating TLR7. Thus, manipulation of TLR7 signalling may be a potential strategy to reduce chronic hyper-immune activation and, thereby, disease progression in HIV infection

HIV RNA load at the terminal bleeding in mice on ART or on double long-acting drugs.

*<p>n.d.  =  non-detectable.</p><p>- Humanized mice were sacrificed 151 days after HIV infection and 114 days after starting ART or double long-acting drugs.</p><p>- Detection limit: the volume of plasma available was slightly different for the mice euthanized and thus the lower detection limit varied accordingly between 40 and 60 copies/ml.</p

Emergence of resistance in the mice under monotherapy with TMC278-LA.

*<p>S = susceptible (wildtype strain).</p>**<p>#192 showed suppressed HIV RNA under TMC278-LA monotherapy.</p>***<p>#191, 224 showed viral failure under the ART regimen of 3TC, TDF and TMC278-LA. #190 gave a positive signal for HIV RNA but below the limit of detection (<800 copies/ml)</p><p>#221, 245 only baseline analyses have been done, and therefore data from these mice were not integrated in the table.</p><p>¶#n.d. = not done.</p

Recovery of cell-associated HIV DNA (A) and increase of HIV mRNA transcripts in vitro from splenic tissue obtained from HIV-infected mice with suppressed HIV RNA following activation.

<p>(A) DNA from infected HeLa cells (HeLa inf) and from the spleen of HIV-infected mice served as positive controls, DNA from an uninfected humanized mouse (uninf) served as negative control. The specimens of the treated and HIV-infected mice were from the experiments investigating the antiviral potency of the double long-acting drugs; (MNE = mean normalized expression). B) Splenic tissue specimens from either HIV infected ART naïve hu mice (HIV), ART treated mice (ART) or mice treated with the two long acting drugs (Double-LA) were subjected to mitogens (PMA, PHA) in concert with anti-CD3/28 and IL-7. 18 hours later RNA was extracted and real-time PCR done for quantifying HIV Gag transcripts. Specimens of two mice which were treated with double LA drugs did not show any HIV transcript at all (data not shown in the graph); in five mice we did not detect any HIV transcripts prior to stimulation. The real-time PCRs were done in duplicates. *this specimen is from a mouse (#417) with detectable HIV RNA at the time of euthanization (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038853#pone-0038853-t002" target="_blank">Table 2</a>).</p