Arthropod-borne viruses (arboviruses) are important human pathogens for which there are no specific antiviral medicines. The abundance of genetically distinct arbovirus species, coupled with the unpredictable nature of their outbreaks, has made the development of virus-specific treatments challenging. Instead, we have defined and targeted a key aspect of the host innate immune response to virus at the arthropod bite that is common to all arbovirus infections, potentially circumventing the need for virus-specific therapies. Using mouse models and human skin explants, we identify innate immune responses by dermal macrophages in the skin as a key determinant of disease severity. Post-exposure treatment of the inoculation site by a topical TLR7 agonist suppressed both the local and subsequent systemic course of infection with a variety of arboviruses from the Alphavirus, Flavivirus, and Orthobunyavirus genera. Clinical outcome was improved in mice after infection with a model alphavirus. In the absence of treatment, antiviral interferon expression to virus in the skin was restricted to dermal dendritic cells. In contrast, stimulating the more populous skin-resident macrophages with a TLR7 agonist elicited protective responses in key cellular targets of virus that otherwise proficiently replicated virus. By defining and targeting a key aspect of the innate immune response to virus at the mosquito bite site, we have identified a putative new strategy for limiting disease after infection with a variety of genetically distinct arboviruses.

The mechanisms by which the neurophysiological and inflammatory responses to brain injury contribute to memory impairments are not fully understood. Recently, we reported that the innate immune receptor, toll-like receptor 4 (TLR4) enhances AMPA receptor (AMPAR) currents and excitability in the dentate gyrus after fluid percussion brain injury (FPI) while limiting excitability in controls. Here, we examine the cellular mediators underlying TLR4 regulation of dentate excitability and its impact on memory performance. In ex vivo slices, astrocytic and microglial metabolic inhibitors selectively abolished TLR4 antagonist modulation of excitability in controls, but not in rats after FPI, demonstrating that glial signaling contributes to TLR4 regulation of excitability in controls. In glia-depleted neuronal cultures from naïve mice, TLR4 ligands bidirectionally modulated AMPAR charge transfer consistent with neuronal TLR4 regulation of excitability, as observed after brain injury. In vivo TLR4 antagonism reduced early post-injury increases in mediators of MyD88-dependent and independent TLR4 signaling without altering expression in controls. Blocking TNFα, a downstream effector of TLR4, mimicked effects of TLR4 antagonist and occluded TLR4 agonist modulation of excitability in slices from both control and FPI rats. Functionally, transiently blocking TLR4 in vivo improved impairments in working memory observed one week and one month after FPI, while the same treatment impaired memory function in uninjured controls. Together these data identify that distinct cellular signaling mechanisms converge on TNFα to mediate TLR4 modulation of network excitability in the uninjured and injured brain and demonstrate a role for TLR4 in regulation of working memory function.

The microbiome-gut-brain (MGB) axis is a bi-directional route of communication that exists between the brain and the microbes that reside in the gut. The MGB axis is becoming of increasing importance as significant alterations in the gut microbiota are now linked to numerous neurological conditions, however, little is currently known about the microbiome derived mediators of communication. Here we used mass spectrometry imaging (MSI), a label free imaging technique, to identify bacterial products that cross the blood brain barrier in specific pathogen free (SPF) mice. We identified two bacterial molecules abundant in white matter regions of the murine that were absent in the brain and gut in germ free (GF) mice. We have identified the primary gut microbial producers of these metabolites to be members of the Lachnospiraceae family. Both molecules were found to be structurally similar to carnitine and localise with carnitine in the SPF mouse brain. Using a primary murine cell culture model of the central nervous system white matter we show that these molecules are capable of significantly impairing mitochondrial basal respiration. Given their systemic presence in the mouse and their presence in human biological samples, these metabolites may have significant implications for diseases associated with mitochondrial dysfunction and an altered gut microbiota. These results are the first to describe a direct molecular inter-kingdom communication between prokaryotes and the mammalian brain that can facilitate functional inhibition in mammalian brain cells.

Zika virus infection was recently linked to microcephaly and peripheral neuropathy (GBS) in Zika virus epidemic areas. Building on our previous work (Cumberworth et al, 2017) we investigated, in a time-course study, how the viral infection and the injury of cell processes of oligodendrocytes and neurons are related to each other in the same in vitro model. We generated CNS myelinating cultures from a reporter mouse (Thy1-YFP) on the Ifnar1 -/-background. A proportion of neurons and their processes are positive for YFP in those cultures, which enabled us to visualise single neurons. Cultures were infected with the Brazilian Zika virus strain (PE243) at an MOI of 0.3 and cultured for up to 6 days post infection. We observed that the neuronal cell processes were affected as early as the appearance of the first clusters of infected glial cells. To analyse the interrelation of neurons and myelin further, cultures were labeled with an antibody recognising proteolipid protein. We found that the myelin got injured as early as neuronal processes. These results suggest that the injury to neuronal processes might be a consequence of the infection of the primary target of Zika virus: oligodendroglia. These data help us to understand disease pathogenesis of Zika virus infection of the CNS, and whether there is a time-window to intervene therapeutically. Furthermore, this gives an insight as to how viral infection of glial cells can affect neuronal processes such as axons.

We report a custom epi-fluorescent microscope setup using GRIN lens microendoscopes for minimally invasive microscopic imaging in rodents. A simple scanning system and deconvolution provides high quality wide field-of-view images through these highly aberrated endoscopes.