Pia Hartmann

Relevance of the Organic Cation Transporters 1 and 2 for Antiretroviral Drug Therapy in Human Immunodeficiency Virus Infection

Carrier-mediated transport across cell membranes is an important determinant of activity, resistance, and toxicity of chemotherapeutic agents including antiretroviral (ARV) drugs (ARDs). The organic cation transporters (OCTs) 1 and 2 have been implicated in the translocation of different cationic drugs but so far were insufficiently tested for interactions with ARDs. Here, we assessed among cationic drugs commonly used in human immunodeficiency virus (HIV) therapy inhibitors and substrates of OCTs, and analyzed the tissue distribution of OCTs and their expression in lymph nodes (LNs), the primary intracellular target of HIV and ARDs. Inhibitors were identified by measuring the attenuated uptake of the radiolabeled model substrate 1-methyl-4-phenylpyridinium into OCT-transfected human embryonic kidney-293 cells in the presence of ARDs. Substrates were identified by measuring OCT-specific intracellular accumulation using liquid chromatography/tandem mass spectrometry. Inhibitory drugs were (in order of increasing potency): nelfinavir < ritonavir < saquinavir < indinavir < trimethoprim < pentamidine, with consistently lower IC50 values determined for OCT1. Substrates with highest transport efficacy (Vmax/Km) were lamivudine (OCT1, 8 μl/mg protein/min; OCT2, 4.4 μl/mg protein/min) and zalcitabine (OCT1, 4.1 μl/mg protein/min; OCT2, 2.6 μl/mg protein/min). Using quantitative real-time polymerase chain reaction, a marked expression level of OCT1 was detected in human samples of liver, ovary, prostate, and testis, and of OCT2 in kidney, colon, heart, skeletal muscle, and testis. Expression of OCTs in LNs was low in HIV-negative control individuals but dramatically increased in HIV-infected persons. These data suggest that drug interactions about the OCTs may be relevant for the ARV therapy, in particular by influencing drug accession to infected tissues and hepatic or renal elimination.

Plasmacytoid dendritic cells accumulate and secrete interferon alpha in lymph nodes of HIV-1 patients.

Circulating plasmacytoid dendritic cells (pDC) decline during HIV-1 infection, but at the same time they express markedly higher levels of interferon alpha (IFNα), which is associated with HIV-1 disease progression. Here we show an accumulation of pDC in lymph nodes (LN) of treatment-naïve HIV-1 patients. This phenomenon was associated with elevated expression of the LN homing marker, CCR7, on pDC in peripheral blood of HIV-1 patients, which conferred increased migratory capacity in response to CCR7 ligands in ex vivo functional assays. LN-homed pDC of HIV-1 patients presented higher CD40 and lower BDCA2 levels, but unchanged CD83 and CD86 expression. In addition, these cells expressed markedly higher amounts of IFNα compared to uninfected individuals, and were undergoing faster rates of cell death. These results demonstrate for the first time that in asymptomatic, untreated HIV-1 patients circulating pDC up-regulate CCR7 expression, accumulate in lymph nodes, and express high amounts of IFNα before undergoing cell death. Since IFNα inhibits cell proliferation and modulates immune responses, chronically high levels of this cytokine in LN of HIV-1 patients may impair differentiation and immune function of bystander CD4+ T cells, thus playing into the mechanisms of AIDS immunopathogenesis.

Increased Interferon Alpha Expression in Circulating Plasmacytoid Dendritic Cells of HIV-1-Infected Patients

Background:The role of plasmacytoid dendritic cells (pDC) and interferon alpha (IFNα) in HIV-1 infection is still unclear. On one hand, HIV-1 disease is associated with a progressive decline of pDC, which displays reduced ability to produce IFNα after in vitro challenge. On the other hand, high IFNα serum levels in HIV-1-infected individuals have been proposed to promote immune hyper-activation and disease progression. Methods:We sought to determine whether disappearance of pDC in HIV-1 disease is due to homing in lymphoid tissues. We also studied IFNα and myxovirus resistance protein A (MxA) expression in unstimulated pDC and correlated these results with selected clinical and laboratory parameters. Results:We found that pDC decline markedly in peripheral blood of patients progressing to disease but at the same time express much higher levels of IFNα and MxA compared to control individuals. On the other hand, we observed steady pDC counts in lymph nodes of HIV-1 patients. The frequency of circulating pDC correlated directly with CD4 cell counts and inversely with viral load. However, we found no correlation between IFNα and MxA expression levels, CD4 counts, and viral load. Conclusions:Circulating pDC decline sharply in the course of HIV-1 disease, but express high levels of IFNα, which may represent a hallmark of systemic immune dysfunction.

Insights into the function of the WhiB‐like protein of mycobacteriophage TM4 – a transcriptional inhibitor of WhiB2

WhiB‐like proteins of actinomycetes are known to co‐ordinate iron‐sulfur (Fe‐S) clusters and are believed to have regulatory functions in many essential bacterial processes. The systematic determination of the genome sequences of mycobacteriophages has revealed the presence of several whiB‐like genes in these viruses. Here we focussed on the WhiB‐like protein of mycobacteriophage TM4, WhiBTM4. We provide evidence that this viral protein is capable of co‐ordinating a Fe‐S cluster. The UV‐visible absorption spectra obtained from freshly purified and reconstituted WhiBTM4 were consistent with the presence of an oxygen sensitive [2Fe‐2S] cluster. Expression of WhiBTM4 in the mycobacterial host led to hindered septation resembling a WhiB2 knockout phenotype whereas basal expression of WhiBTM4 led to superinfection exclusion. The quantification of mRNA‐levels during phage infection showed that whiBTM4 is a highly transcribed early phage gene and a dominant negative regulator of WhiB2. Strikingly, both apo‐WhiB2 of Mycobacterium tuberculosis and apo‐WhiBTM4 were capable of binding to the conserved promoter region upstream of the whiB2 gene indicating that WhiB2 regulates its own synthesis which is inhibited in the presence of WhiBTM4. Thus, we provide substantial evidence supporting the hypothesis of viral and bacterial WhiB proteins being important Fe‐S containing transcriptional regulators with DNA‐binding capability.

Toll‐like receptor 2–mediated innate immune response in human nonparenchymal liver cells toward adeno‐associated viral vectors

Adeno‐associated viral vectors (rAAV) are frequently used in gene therapy trials. Although rAAV vectors are of low immunogenicity, humoral as well as T cell responses may be induced. While the former limits vector reapplication, the expansion of cytotoxic T cells correlates with liver inflammation and loss of transduced hepatocytes. Because adaptive immune responses are a consequence of recognition by the innate immune system, we aimed to characterize cell autonomous immune responses elicited by rAAV in primary human hepatocytes and nonparenchymal liver cells. Surprisingly, Kupffer cells, but also liver sinusoidal endothelial cells, mounted responses to rAAV, whereas neither rAAV2 nor rAAV8 were recognized by hepatocytes. Viral capsids were sensed at the cell surface as pathogen‐associated molecular patterns by Toll‐like receptor 2. In contrast to the Toll‐like receptor 9–mediated recognition observed in plasmacytoid dendritic cells, immune recognition of rAAV in primary human liver cells did not induce a type I interferon response, but up‐regulated inflammatory cytokines through activation of nuclear factor κB. Conclusion: Using primary human liver cells, we identified a novel mechanism of rAAV recognition in the liver, demonstrating that alternative means of sensing rAAV particles have evolved. Minimizing this recognition will be key to improving rAAV‐mediated gene transfer and reducing side effects in clinical trials due to immune responses against rAAV. (Hepatology 2012;55:287–297)

Mycobacterial Phenolic Glycolipid Inhibits Phagosome Maturation and Subverts the Pro‐inflammatory Cytokine Response

Inhibition of phagosome maturation is an important hallmark of mycobacterial pathogenesis. A variety of genomic, transcriptomic and proteomic approaches have been used to pin down the molecule responsible for this pathogenic principle. We in this study characterize a glycolipid of Mycobacterium marinum identified through a screen of mutants disabled in inhibiting phagosome maturation to be phenolphthiocerol diester (phenolic glycolipid, PGL). This molecule is sufficient to impart its ability to inhibit phagosome maturation onto other microbial cells and even inert beads that are used as model pathogens. In addition, it abrogates pro‐inflammatory cytokine secretion induced by strong inducers such as heat‐killed Mycobacterium bovis bacille Calmette–Guérin. This strong dual agonistic effect of PGL overrides pro‐inflammatory and pro‐lysosomal delivery impulses set not only by mycobacteria but also by other pathogens and thus provides convincing evidence that this molecule is a vital mycobacterial virulence factor.

Incidence and prognosis of CMV disease in HIV-infected patients before and after introduction of combination antiretroviral therapy

Background:Highly active antiretroviral therapy (HAART) has improved the prognosis of HIV–infected patients. We studied the changes in the incidence and prognosis of cytomegalovirus (CMV) disease preceding and during the first few years of HAART in a clinic cohort.Patients and Methods:All patients with CMV disease diagnosed between 1993 and 1999 from a clinic cohort in Cologne, Germany, were included. The patients were followed until death or until December 31, 2001. The time period from 1993–1996 was classified as pre–HAART, the period from 1997–1999 as the HAART era. Survival was analyzed with a Cox–proportional hazard model.Results:From a total of 1,279 HIV–infected patients, 127 patients with CMV disease were enrolled. The incidence of CMV disease declined rapidly and significantly from 7.34 cases per 100 patient years (py) in the pre–HAART era to 0.75 cases per 100 py in the HAART era. The median survival time in the pre–HAART era was 9.5 months; the median survival was not yet reached at 4 years of follow–up in the HAART era. The only risk factors influencing survival were CD4–cell count and antiretroviral therapy before and after diagnosis of CMV disease. Treatment naive patients had a better prognosis than pretreated patients and patients treated with triple combination therapy survived longer than patients with other treatment modalities.Conclusion:A rapid decline in the incidence of new CMV manifestations and a better prognosis of patients with CMV disease, especially if they were treatment naive and treated with triple combination therapy, were observed in the HAART era.

Longitudinal Analysis of Distribution and Function of Plasmacytoid Dendritic Cells in Peripheral Blood and Gut Mucosa of HIV infected patients

Aberrant activation of plasmacytoid dendritic cells (pDCs) with excessive production of interferon alpha (IFNα) represents one of the hallmarks of immune activation during chronic phase of human immunodeficiency virus (HIV) infection. A number of studies have shown that disruption of mucosal integrity in the gut is a cause of persistent immune activation. However, little is known about the role that pDCs play in this process, and our current understanding comes from the simian immunodeficiency virus macaque model. Thus, in the present study we sought to investigate the frequency and function of pDCs in peripheral blood and gut samples from HIV-infected individuals before and 6 months after initiation of antiretroviral therapy (ART). We show that circulating pDCs were depleted in ART-naive HIV+ patients, and upregulated the gut-homing receptor CD103 compared with uninfected controls. By converse, pDCs accumulated in the terminal ileum of ART-naive HIV individuals compared with controls. Baseline levels of IFNα production and markers of immune activation in gut samples of ART-naive HIV subjects were elevated. All these parameters declined after 6 months of ART. Our results suggest that in chronic HIV infection, pDCs migrate from peripheral blood to the gut-associated lymphatic tissue, where they may contribute to immune activation.

MHC class I chain-related protein A shedding in chronic HIV-1 infection is associated with profound NK cell dysfunction

Natural killer (NK) cells play a critical role in host defense against viral infections. However chronic HIV-1 infection is associated with an accumulation of dysfunctional NK cells, that poorly control viral replication. The underlying mechanisms for this NK cell mediated dysfunction are not understood. Certain tumors evade NK cell mediated detection by dampening NK cell activity through the downregulation of NKG2D, via the release of soluble NKG2D-ligands, resulting in a potent suppression of NK cell function. Here we show that chronic HIV-1 infection is associated with a specific defect in NKG2D-mediated NK cell activation, due to reduced expression and transcription of NKG2D. Reduced NKG2D expression was associated with elevated levels of the soluble form of the NKG2D-ligand, MICA, in patient sera, likely released by HIV+CD4+ T cells. Thus, like tumors, HIV-1 may indirectly suppress NK cell recognition of HIV-1-infected CD4+ T cells by enhancing NKG2D-ligand secretion into the serum resulting in a profound impairment of NK cell function.

Preferential Upregulation of Interferon-α Subtype 2 Expression in HIV-1 Patients

Humans tailor virus-specific immune responses through modulated expression of 12 different interferon (IFN)-alpha subtypes. However, exacerbated expression of certain IFN-alpha subtypes causes immunopathology in the context of autoimmune conditions and chronic viral infections. We showed that progression to AIDS is associated with elevated expression of IFN-alpha in unstimulated peripheral blood mononuclear cells. Here, we sought to determine whether distinct IFN-alpha subtypes are involved in this phenomenon. We used quantitative RT-PCR to assess expression levels of 12 IFN-alpha subtypes in peripheral blood mononuclear cells from normal donors and HIV-1 patients at CDC stage A and stage C of the disease. Three patterns of IFN-alpha subtype expression emerged. First, IFN-alpha2 and IFN-alpha6 mRNA levels were elevated in both patient groups. Second, IFN-alpha1/13, IFN-alpha8, IFN-alpha14, IFN-alpha16, IFN-alpha17, and IFN-alpha21 were upregulated in stage C but not stage A patients. Third, expression levels of IFN-alpha4, IFN-alpha5, IFN-alpha7, and IFN-alpha10 did not change among the three groups of volunteers. Among all other subtypes, IFN-alpha2 was preferentially upregulated, showing >60-fold higher levels in stage A and >400-fold in stage C patients compared with controls, which correlated with declining CD4 counts. Our results demonstrate that distinct IFN-alpha subtypes are sequentially activated during HIV-1 infection, which may be predictive of disease progression.