W. James Cook

Persistent expression of chemokine and chemokine receptor RNAs at primary and latent sites of herpes simplex virus 1 infection

Inflammatory cytokines and infiltrating T cells are readily detected in herpes simplex virus (HSV) infected mouse cornea and trigeminal ganglia (TG) during the acute phase of infection, and certain cytokines continue to be expressed at lower levels in infected TG during the subsequent latent phase. Recent results have shown that HSV infection activates Toll-like receptor signaling. Thus, we hypothesized that chemokines may be broadly expressed at both primary sites and latent sites of HSV infection for prolonged periods of time. Real-time reverse transcriptase-polymrease chain reaction (RT-PCR) to quantify expression levels of transcripts encoding chemokines and their receptors in cornea and TG following corneal infection. RNAs encoding the inflammatory-type chemokine receptors CCR1, CCR2, CCR5, and CXCR3, which are highly expressed on activated T cells, macrophages and most immature dendritic cells (DC), and the more broadly expressed CCR7, were highly expressed and strongly induced in infected cornea and TG at 3 and 10 days postinfection (dpi). Elevated levels of these RNAs persisted in both cornea and TG during the latent phase at 30 dpi. RNAs for the broadly expressed CXCR4 receptor was induced at 30 dpi but less so at 3 and 10 dpi in both cornea and TG. Transcripts for CCR3 and CCR6, receptors that are not highly expressed on activated T cells or macrophages, also appeared to be induced during acute and latent phases; however, their very low expression levels were near the limit of our detection. RNAs encoding the CCR1 and CCR5 chemokine ligands MIP-1α, MIP-1β and RANTES, and the CCR2 ligand MCP-1 were also strongly induced and persisted in cornea and TG during the latent phase. These and other recent results argue that HSV antigens or DNA can stimulate expression of chemokines, perhaps through activation of Toll-like receptors, for long periods of time at both primary and latent sites of HSV infection. These chemokines recruit activated T cells and other immune cells, including DC, that express chemokine receptors to primary and secondary sites of infection. Prolonged activation of chemokine expression could provide mechanistic explanations for certain aspects of HSV biology and pathogenesis.

Latent Herpes Simplex Virus Infection of Sensory Neurons Alters Neuronal Gene Expression

ABSTRACT The persistence of herpes simplex virus (HSV) and the diseases that it causes in the human population can be attributed to the maintenance of a latent infection within neurons in sensory ganglia. Little is known about the effects of latent infection on the host neuron. We have addressed the question of whether latent HSV infection affects neuronal gene expression by using microarray transcript profiling of host gene expression in ganglia from latently infected versus mock-infected mouse trigeminal ganglia. 33P-labeled cDNA probes from pooled ganglia harvested at 30 days postinfection or post-mock infection were hybridized to nylon arrays printed with 2,556 mouse genes. Signal intensities were acquired by phosphorimager. Mean intensities (n = 4 replicates in each of three independent experiments) of signals from mock-infected versus latently infected ganglia were compared by using a variant of Students t test. We identified significant changes in the expression of mouse neuronal genes, including several with roles in gene expression, such as the Clk2 gene, and neurotransmission, such as genes encoding potassium voltage-gated channels and a muscarinic acetylcholine receptor. We confirmed the neuronal localization of some of these transcripts by using in situ hybridization. To validate the microarray results, we performed real-time reverse transcriptase PCR analyses for a selection of the genes. These studies demonstrate that latent HSV infection can alter neuronal gene expression and might provide a new mechanism for how persistent viral infection can cause chronic disease.

Microarrays for studying the host transcriptional response to microbial infection and for the identification of host drug targets.

It is essential to understand the molecular basis of a hosts response to microbial infection so that disease and tissue damage can be prevented. Modulation of host RNA expression is a critical set of molecular changes that occur upon infection. Global analysis of gene expression should provide an understanding of host RNA transcriptional changes that occur upon host-pathogen interaction. This series of articles focuses on the use of microarrays for analyzing the transcriptional response of a host to microbial infection and for drug target identification.

The Programmed Death-1 and Interleukin-10 Pathways Play a Down-Modulatory Role in LP-BM5 Retrovirus-Induced Murine Immunodeficiency Syndrome

ABSTRACT Pathology due to the immune systems response to viral infections often represents a delicate balance between inhibition of viral pathogenesis and regulation of protective immunity. In susceptible C57BL/6 (B6) mice, the murine retroviral isolate LP-BM5 induces splenomegaly, hypergammaglobulinemia, profound B- and T-cell immunodeficiency, and increased susceptibility to opportunistic pathogens and terminal B-cell lymphomas. Here, we report that B6.PD-1 (programmed death-1) and B6.IL-10 knockout mice are substantially more susceptible to LP-BM5-induced disease than wild-type B6 mice. LP-BM5-infected B6.PD-1−/− mice developed more severe splenomegaly, hypergammaglobulinemia, and immunodeficiency than infected B6 mice: PD-1−/− mice are more susceptible to lower doses of LP-BM5 and show more exaggerated disease early postinfection. LP-BM5-infected B6.IL-10−/− mice also develop exaggerated LP-BM5-induced disease, compared to B6 mice, without a significant change in the retroviral load. By reciprocal reconstitution experiments, comparing wild-type versus PD-1−/− sources of the requisite cells for LP-BM5 pathogenesis—CD4 T and B cells, PD-1+ B cells appear to be crucial in the normal limitation of LP-BM5-induced disease in B6 mice. Also, infected B6 mice have increased CD11b+ spleen cells that express interleukin-10 (IL-10). However, PD-1−/− mice, though showing an even greater expansion of CD11b+ cells after LP-BM5 inoculation, did not show an equivalent increase in IL-10-producing cells. Thus, it appears that PD-1/PD-L interactions and IL-10 are primarily important in moderating the effects of LP-BM5-induced disease in B6 mice.

The CD154/CD40 Interaction Required for Retrovirus-Induced Murine Immunodeficiency Syndrome Is Not Mediated by Upregulation of the CD80/CD86 Costimulatory Molecules

ABSTRACT C57BL/6 (B6) mice infected with LP-BM5 retroviruses develop disease, including an immunodeficiency similar to AIDS. This disease, murine AIDS (MAIDS), is inhibited by in vivo anti-CD154 monoclonal antibody treatment. The similar levels of insusceptibility of CD40−/− and CD154−/− B6 mice indicate that CD154/CD40 molecular interactions are required for MAIDS. CD4+ T and B cells, respectively, provide the CD154 and CD40 expression needed for MAIDS induction. Here, the required CD154/CD40 interaction is shown to be independent of CD80 and CD86 expression: CD80/CD86−/− B6 mice develop MAIDS after LP-BM5 infection.

CD40-associated TRAF 6 signaling is required for disease induction in a retrovirus-induced murine immunodeficiency.

ABSTRACT LP-BM5 retrovirus-infected C57BL/6 mice develop splenomegaly, lymphadenopathy, hypergammaglobulinemia, and immunodeficiency; thus, this disease has been named mouse AIDS. In this syndrome, CD154/CD40 interactions are required for but do not mediate disease by upregulation of CD80 or CD86. We report here that there is nonetheless a necessity for CD40 signaling competence, specifically an intact tumor necrosis factor receptor-associated factor 6 (TRAF 6) binding site.