New mouse model of HHV-6A CNS infection developed

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HHV-6A infection results in persistent infection and neuroinflammation via TLR9 in CD46 transgenic mice

An article published in the Journal of Virology this month details novel findings from a newly developed transgenic mouse model of HHV-6A infection. Dr. Branka Horvat’s team at the International Center for Infectiology Research in Lyon, France, developed the model by using transgenic mice expressing human CD46, identified as a receptor for HHV-6A. The study suggests that the presence of HHV-6A in the CNS, even if only a latent or abortive infection, can have a strong impact on inflammation through upregulation of CCL5, CCL2, CXCL10 and CCL1.

HHV-6A-induced chemokines in the glial cultures were inhibited when toll-like receptor 9 (TLR9) was blocked. This finding may have important clinical implications, as TLR9 inhibitors may now be useful in therapeutic approaches to decrease the production of pro-inflammatory cytokines (*See further discussion of TLR9 inhibitors below).

The group found that HHV-6A infection results in the expression of viral transcripts in primary brain glial cultures from CD46-expressing mice. HHV-6A DNA persisted for up to 9 months in the brain of CD46-expressing mice but not in the non-transgenic littermates. Infiltrating lymphocytes and monocytes were found in the periventricular areas of the brain of HHV-6A-infected mice. In addition, HHV-6A was found to stimulate the production of pro-inflammatory chemokines in primary brain glial cultures, including CCL2, CCL5 and CXCL10. It also induced the expression of CCL5 in brains of HHV-6A infected CD46 transgenic mice.

HHV-6A has long been associated with several neuroinflammatory diseases and pathologies, however the absence of a small animal model of HHV-6A has inhibited detailed investigation of the specific pathological processes in question. While laboratories at the NINDS have studied non-human primate models of HHV-6 infection for several years, and in fact recently demonstrated that common marmosets infected with HHV-6A exhibit significant neurological symptoms, primate models are difficult as well as costly to maintain. In 2013, a group from Brigham Young University showed that Rag2-/-γc-/- mice humanized with cord blood-derived human hematopoietic stem cells can develop significant immune dysfunction. The HHV-6A infected cells caused depletion of specific thymocyte population. However, the French group’s model is the first to allow study of the impact of HHV-6 infection in brain tissues of a small immunocompetent animal model, for which many experimental tools are available, opening thus new avenues for the further study of HHV-6-induced neuropathogenesis.

Interestingly, the investigators found evidence of non-productive infection in CD46 expressing murine cells. Genes associated with U94, known as the “latency-associated” gene, as well as several other intermediate and early genes were expressed, but there was an absence in expression of viral proteins, unless particular culture conditions with infected human cells were provided. Inactivated HHV-6 virus also caused inflammation. The authors speculate that HHV-6 entry receptor CD46 mediates capture of viral particles and allows the access of viral DNA to the endosomal compartment, containing TLR9, and consequent activation of inflammatory pathways.

Although marmosets infected with HHV-6A demonstrated limb weakness and sensory deficits, no neurological symptoms were noticed in the HHV-6A infected mice. The authors were surprised to find that IFN-I signaling does not play any role in HHV-6A infection in mice.

These mice could not support HHV-6B infection. It was recently reported by Yasuko Mori’s lab that HHV-6B uses the CD134 receptor for cell entry (Mori 2013). Measles virus also uses CD46 for cellular entry, infecting both oligodendritic and neuronal cells (Lawrence 1999).

For more information, read the full article.

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*NOTE: Toll-Like Receptor 9 Inhibition by Antimalarials, Imidazoquinolines, and Aspirin

Toll-like receptors (TLRs) are transmembrane proteins that are characterized by leucine-rich repeats (LRR) in their extracellular domain and by cytoplasmic Toll/Interleukin-1 Receptor (TIR) signaling domains [1]. There are thirteen TLRs that allow the innate immune system to recognize pathogen-associated molecular patterns (PAMPs) and mount a proper immune response [1].  Upon activation by PAMPs, such as peptidoglycan, lipopolysaccharide (LPS), and flagellin, TLRs induce a signaling pathway, which results in the activation of transcription factors that code for costimulatory proteins and pro-inflammatory cytokines [1]. One of these TLRs, TLR9, activates macrophages and monocytes after recognizing unmethylated bacterial CpG-DNA [1]. After being activated, TLRs trigger NF-κB activation, which results in the expression of various proinflammatory cytokines [2].

In addition to being activated by peptidoglycan, LPS, and flagellin, TLR9 has been shown to be activated by human herpesvirus-6 (HHV-6) [3].

Recent studies have shown that antimalarials, such as artesunate, chloroquine, and quinacrine, act as TLR9 inhibitors. In 2008, Bin Li and colleagues studied the effects of artesunate (AS), an artemisinin derivative, on TLR9 expression. It was found that AS decreased TLR9 mRNA expression upon stimulation by CpG ODN and heat-killed Escherichia coli (E. coli) [2]. It was also found that pretreatment with AS led to a decrease in TLR9 protein expression after stimulation by CpG ODN 1826 [2].   Pretreatment with AS also resulted in a significant decrease in NF-κB activation when later stimulated with heat-killed E. coli [2]. These findings suggest that AS decreases TLR9 signaling by inhibiting NF-κB activation [2]. The antimalarials chloroquine and quinacrine have been shown to disrupt the action of endosomal hydrolytic enzymes and the trafficking of receptors because of their ability to partition into acidic vesicles as weak bases [4]. In a study performed by Rutz and colleagues, it was found that both chloroquine and quinacrine inhibit CpG-DNA-driven cellular activation of TLR9 [1]. This find is supported by the data collected by Kuznik et al showing that chloroquine inhibits TLR9 activation in a dose-dependent manner [5]. In addition, it was found that binding of TLR9 to CpG-DNA was dose-dependently inhibited by chloroquine and quinacrine [1].

In 2011, Kuznik and colleagues also studied the effects of imidazoquinolines on TLR9 inhibition. It was found that when imidazoquinolines were present, there was a decrease in TLR9 activity [5]. The mechanism by which this inhibition of TLR9 occurs is similar to that by which antimalarials act [5].

In 2009, Imaeda and colleagues studied acetaminophen (APAP)-induced hepatotoxicity in mice.  It was found that APAP-induced liver toxicity acted through a two signals pathway: first, TLR9 signals for transcription of pro-IL-1β and pro-IL-18, and second, the Nalp3 inflammasome signals for the cleavage and activation of these two pro-cytokines [6]. In low doses, aspirin inhibits the Nalp3 inflammasome-mediated inflammatory response, thus inhibiting this TLR9-mediated pathway [6].

1.         Rutz, M., et al., Toll-like receptor 9 binds single-stranded CpG-DNA in a sequence- and pH-dependent manner. Eur J Immunol, 2004. 34(9): p. 2541-50.

2.         Li, B., et al., Antimalarial artesunate protects sepsis model mice against heat-killed Escherichia coli challenge by decreasing TLR4, TLR9 mRNA expressions and transcription factor NF-kappa B activation. Int Immunopharmacol, 2008. 8(3): p. 379-89.

3.         Reynaud, J.M., et al., Human Herpesvirus 6A infection in CD46 transgenic mice: viral persistence in the brain and increased production of proinflammatory chemokines via TLR9. J Virol, 2014.

4.         Macfarlane, D.E. and L. Manzel, Antagonism of immunostimulatory CpG-oligodeoxynucleotides by quinacrine, chloroquine, and structurally related compounds. J Immunol, 1998. 160(3): p. 1122-31.

5.         Kuznik, A., et al., Mechanism of endosomal TLR inhibition by antimalarial drugs and imidazoquinolines. J Immunol, 2011. 186(8): p. 4794-804.

6.         Imaeda, A.B., et al., Acetaminophen-induced hepatotoxicity in mice is dependent on Tlr9 and the Nalp3 inflammasome. J Clin Invest, 2009. 119(2): p. 305-14.