Douglas M. Durrant

A complement–microglial axis drives synapse loss during virus-induced memory impairment

Over 50% of patients who survive neuroinvasive infection with West Nile virus (WNV) exhibit chronic cognitive sequelae. Although thousands of cases of WNV-mediated memory dysfunction accrue annually, the mechanisms responsible for these impairments are unknown. The classical complement cascade, a key component of innate immune pathogen defence, mediates synaptic pruning by microglia during early postnatal development. Here we show that viral infection of adult hippocampal neurons induces complement-mediated elimination of presynaptic terminals in a murine WNV neuroinvasive disease model. Inoculation of WNV-NS5-E218A, a WNV with a mutant NS5(E218A) protein leads to survival rates and cognitive dysfunction that mirror human WNV neuroinvasive disease. WNV-NS5-E218A-recovered mice (recovery defined as survival after acute infection) display impaired spatial learning and persistence of phagocytic microglia without loss of hippocampal neurons or volume. Hippocampi from WNV-NS5-E218A-recovered mice with poor spatial learning show increased expression of genes that drive synaptic remodelling by microglia via complement. C1QA was upregulated and localized to microglia, infected neurons and presynaptic terminals during WNV neuroinvasive disease. Murine and human WNV neuroinvasive disease post-mortem samples exhibit loss of hippocampal CA3 presynaptic terminals, and murine studies revealed microglial engulfment of presynaptic terminals during acute infection and after recovery. Mice with fewer microglia (Il34−/− mice with a deficiency in IL-34 production) or deficiency in complement C3 or C3a receptor were protected from WNV-induced synaptic terminal loss. Our study provides a new murine model of WNV-induced spatial memory impairment, and identifies a potential mechanism underlying neurocognitive impairment in patients recovering from WNV neuroinvasive disease.

IL-1R1 is required for dendritic cell–mediated T cell reactivation within the CNS during West Nile virus encephalitis

IL-1R1 signaling drives T cell activation in the CNS via effects on DC activation.

Viral Pathogen-Associated Molecular Patterns Regulate Blood-Brain Barrier Integrity via Competing Innate Cytokine Signals

ABSTRACT Pattern recognition receptor (PRR) detection of pathogen-associated molecular patterns (PAMPs), such as viral RNA, drives innate immune responses against West Nile virus (WNV), an emerging neurotropic pathogen. Here we demonstrate that WNV PAMPs orchestrate endothelial responses to WNV via competing innate immune cytokine signals at the blood-brain barrier (BBB), a multicellular interface with highly specialized brain endothelial cells that normally prevents pathogen entry. While Th1 cytokines increase the permeability of endothelial barriers, type I interferon (IFN) promoted and stabilized BBB function. Induction of innate cytokines by pattern recognition pathways directly regulated BBB permeability and tight junction formation via balanced activation of the small GTPases Rac1 and RhoA, which in turn regulated the transendothelial trafficking of WNV. In vivo, mice with attenuated type I IFN signaling or IFN induction (Ifnar−/− Irf7−/−) exhibited enhanced BBB permeability and tight junction dysregulation after WNV infection. Together, these data provide new insight into host-pathogen interactions at the BBB during neurotropic viral infection. IMPORTANCE West Nile virus (WNV) is an emerging pathogen capable of infecting the central nervous system (CNS), causing fatal encephalitis. However, the mechanisms that control the ability of WNV to cross the blood-brain barrier (BBB) and access the CNS are unclear. In this study, we show that detection of WNV by host tissues induces innate immune cytokine expression at the BBB, regulating BBB structure and function and impacting transendothelial trafficking of WNV. This regulatory effect is shown to happen rapidly following exposure to virus, to occur independently of viral replication within BBB cells, and to require the signaling of cytoskeletal regulatory Rho GTPases. These results provide new understanding of host-pathogen interactions at the BBB during viral encephalitis. West Nile virus (WNV) is an emerging pathogen capable of infecting the central nervous system (CNS), causing fatal encephalitis. However, the mechanisms that control the ability of WNV to cross the blood-brain barrier (BBB) and access the CNS are unclear. In this study, we show that detection of WNV by host tissues induces innate immune cytokine expression at the BBB, regulating BBB structure and function and impacting transendothelial trafficking of WNV. This regulatory effect is shown to happen rapidly following exposure to virus, to occur independently of viral replication within BBB cells, and to require the signaling of cytoskeletal regulatory Rho GTPases. These results provide new understanding of host-pathogen interactions at the BBB during viral encephalitis.

Development of Allergen-Induced Airway Inflammation in the Absence of T-bet Regulation Is Dependent on IL-17

Dysfunctional expression of T-bet, a transcription factor that is critical for IFN-γ production, has been implicated in the development of asthma. To investigate in detail the mechanisms responsible for exacerbated disease in the absence of T-bet expression, BALB/c wild-type (WT) and T-bet−/− mice were used in a murine model of OVA-induced allergic lung inflammation. Following OVA challenge, T-bet−/− mice displayed increased histological inflammation in the lungs as well as greater thickening of the bronchiole linings, increased numbers of eosinophils and neutrophils in the lung, and enhanced airway hyperresponsiveness, compared with WT mice. However, the production of Th2 cytokines in T-bet−/− mice did not appear to be significantly greater than in WT mice. Interestingly, a marked increase in the levels of the proinflammatory cytokine IL-17 was observed in T-bet−/− mice. Neutralization of pulmonary IL-17 in T-bet−/− mice by anti-IL-17 mAb treatment during OVA challenge resulted in decreased levels of neutrophilic infiltration into the airways and decreased airway inflammation, essentially reversing the development of allergic asthma development. These findings indicate that IL-17 is a key mediator of airway inflammation in the absence of T-bet. The results of this study suggest a possible target for therapeutic intervention of human asthma.

IL-1R1 Signaling Regulates CXCL12-Mediated T Cell Localization and Fate within the Central Nervous System during West Nile Virus Encephalitis

Immune cell entry into the virally infected CNS is vital for promoting viral clearance yet may contribute to neuropathology if not rigorously regulated. We previously showed that signaling through IL-1R1 is critical for effector T cell reactivation and virologic control within the CNS during murine West Nile virus (WNV) encephalitis. WNV-infected IL-1R1−/− mice also display increased parenchymal penetration of CD8+ T cells despite lack of CD4-mediated full activation, suggesting dysregulation of molecular components of CNS immune privilege. In this study, we show that IL-1 signaling regulates the CNS entry of virus-specific lymphocytes, promoting protective immune responses to CNS viral infections that limit immunopathology. Analysis of blood–brain barrier function in the WNV-infected IL-1R1−/− mice revealed no alterations in permeability. However, parenchymal proinflammatory chemokine expression, including CCL2, CCL5, and CXCL10, was significantly upregulated, whereas microvasculature CXCL12 expression was significantly decreased in the absence of IL-1 signaling. We show that during WNV infection, CD11b+CD45hi infiltrating cells (macrophages) are the primary producers of IL-1β within the CNS and, through the use of an in vitro blood–brain barrier model, that IL-1β promotes CXCR4-mediated T cell adhesion to brain microvasculature endothelial cells. Of interest, IFNγ+ and CD69+ WNV-primed T cells were able to overcome CXCL12-mediated adhesion via downregulation of CXCR4. These data indicate that infiltrating IL-1β–producing leukocytes contribute to cellular interactions at endothelial barriers that impart protective CNS inflammation by regulating the parenchymal entry of CXCR4+ virus-specific T cells during WNV infection.

Regional astrocyte IFN signaling restricts pathogenesis during neurotropic viral infection

Type I IFNs promote cellular responses to viruses, and IFN receptor (IFNAR) signaling regulates the responses of endothelial cells of the blood-brain barrier (BBB) during neurotropic viral infection. However, the role of astrocytes in innate immune responses of the BBB during viral infection of the CNS remains to be fully elucidated. Here, we have demonstrated that type I IFNAR signaling in astrocytes regulates BBB permeability and protects the cerebellum from infection and immunopathology. Mice with astrocyte-specific loss of IFNAR signaling showed decreased survival after West Nile virus infection. Accelerated mortality was not due to expanded viral tropism or increased replication. Rather, viral entry increased specifically in the hindbrain of IFNAR-deficient mice, suggesting that IFNAR signaling critically regulates BBB permeability in this brain region. Pattern recognition receptors and IFN-stimulated genes had higher basal and IFN-induced expression in human and mouse cerebellar astrocytes than did cerebral cortical astrocytes, suggesting that IFNAR signaling has brain region–specific roles in CNS immune responses. Taken together, our data identify cerebellar astrocytes as key responders to viral infection and highlight the existence of distinct innate immune programs in astrocytes from evolutionarily disparate regions of the CNS.

CCR5 limits cortical viral loads during West Nile virus infection of the central nervous system

BackgroundCell-mediated immunity is critical for clearance of central nervous system (CNS) infection with the encephalitic flavivirus, West Nile virus (WNV). Prior studies from our laboratory have shown that WNV-infected neurons express chemoattractants that mediate recruitment of antiviral leukocytes into the CNS. Although the chemokine receptor, CCR5, has been shown to play an important role in CNS host defense during WNV infection, regional effects of its activity within the infected brain have not been defined.MethodsWe used CCR5-deficient mice and an established murine model of WNV encephalitis to determine whether CCR5 activity impacts on WNV levels within the CNS in a region-specific fashion. Statistical comparisons between groups were made with one- or two-way analysis of variance; Bonferroni’s post hoc test was subsequently used to compare individual means. Survival was analyzed by the log-rank test. Analyses were conducted using Prism software (GraphPad Prism). All data were expressed as means ± SEM. Differences were considered significant if P ≤ 0.05.ResultsAs previously shown, lack of CCR5 activity led to increased symptomatic disease and mortality in mice after subcutaneous infection with WNV. Evaluation of viral burden in the footpad, draining lymph nodes, spleen, olfactory bulb, and cerebellum derived from WNV-infected wild-type, and CCR5−/− mice showed no differences between the genotypes. In contrast, WNV-infected, CCR5−/− mice exhibited significantly increased viral burden in cortical tissues, including the hippocampus, at day 8 post-infection. CNS regional studies of chemokine expression via luminex analysis revealed significantly increased expression of CCR5 ligands, CCL4 and CCL5, within the cortices of WNV-infected, CCR5−/− mice compared with those of similarly infected WT animals. Cortical elevations in viral loads and CCR5 ligands in WNV-infected, CCR5−/− mice, however, were associated with decreased numbers of infiltrating mononuclear cells and increased permeability of the blood-brain barrier.ConclusionsThese data indicate that regional differences in chemokine expression occur in response to WNV infection of the CNS, and that cortical neurons require CCR5 activity to limit viral burden in this brain region.

The Olfactory Bulb: An Immunosensory Effector Organ during Neurotropic Viral Infections.

In 1935, the olfactory route was hypothesized to be a portal for virus entry into the central nervous system (CNS). This hypothesis was based on experiments in which nasophayngeal infection with poliovirus in monkeys was prevented from spreading to their CNS via transection of olfactory tracts between the olfactory neuroepithelium (ONE) of the nasal cavity and the olfactory bulb (OB). Since then, numerous neurotropic viruses have been observed to enter the CNS via retrograde transport along axons of olfactory sensory neurons whose cell bodies reside in the ONE. Importantly, this route of infection can occur even after subcutaneous inoculation of arboviruses that can cause encephalitis in humans. While the olfactory route is now accepted as an important pathway for viral entry into the CNS, it is unclear whether it provides a way for infection to spread to other brain regions. More recently, studies of antiviral innate and adaptive immune responses within the olfactory bulb suggest it provides early virologic control. Here we will review the data demonstrating that neurotropic viruses gain access to the CNS initially via the olfactory route with emphasis on findings that suggest the OB is a critical immunosensory effector organ that effectively clears virus.

Chemokines Referee Inflammation within the Central Nervous System during Infection and Disease.

The discovery that chemokines and their receptors are expressed by a variety of cell types within the normal adult central nervous system (CNS) has led to an expansion of their repertoire as molecular interfaces between the immune and nervous systems. Thus, CNS chemokines are now divided into those molecules that regulate inflammatory cell migration into the CNS and those that initiate CNS repair from inflammation-mediated tissue damage. Work in our laboratory throughout the past decade has sought to elucidate how chemokines coordinate leukocyte entry and interactions at CNS endothelial barriers, under both homeostatic and inflammatory conditions, and how they promote repair within the CNS parenchyma. These studies have identified several chemokines, including CXCL12 and CXCL10, as critical regulators of leukocyte migration from perivascular locations. CXCL12 additionally plays an essential role in promoting remyelination of injured white matter. In both scenarios we have shown that chemokines serve as molecular links between inflammatory mediators and other effector molecules involved in neuroprotective processes.

Chemokines and Viral Infections of the CNS

A critical factor in the host immune response to invading pathogens, such as viral infections, is the recruitment and infiltration of immune cells to infected tissues. Although the goal of the recruited leukocytes is to eliminate the invading pathogens, collateral tissue damage may be induced in the process, and, in certain circumstances, may pose a serious threat to the survival of the host. The central nervous system (CNS) is a unique site with limited regenerative potential and therefore a low threshold for inflammation-induced tissue damage. However, as the CNS may become the target of life-threatening viral infections, it is imperative that immunological surveillance and efficient effector responses occur in this organ to aid in pathogen clearance. Although traditionally characterized as a site of “immune privilege,” evidence suggests that immune surveillance and antiviral immunity does occur in the CNS (Carson et al., 2006). Understanding how the local inflammatory response within the CNS is regulated is a key to understanding the pathogenesis of viral infections in the CNS and developing therapies that promote protective and limit pathogenic responses. Leukocyte recruitment to any organ site is generally a complex, multistep process. Under normal conditions, leukocyte migration into the CNS is maintained at low levels (Hickey, 2001). During virus-induced inflammation, however, the extravasation of leukocytes is increased and targeted to specific compartments of the CNS depending on the inflammatory stimulus and the infected region. Chemokines and chemokine receptors have been identified as pivotal players in regulating immune cell trafficking into the CNS. Chemokines consist of a large family of small, structurally related, chemotactic cytokines that are involved in regulating the normal lymphocytic traffic to both the lymphoid and nonlymphoid organs and leukocyte emigration into sites of injury and infection (Rossi & Zlotnik, 2000). Chemokines select leukocytes for tissue entry based on their expression of chemokine receptors, G-protein-coupled cell surface receptors, which have a characteristic seven transmembrane structure (Premack & Schall, 1996). In addition to targeting distinct leukocyte populations during inflammation, chemokines and their receptors have emerged as crucial mediators of a variety of biological processes including development and tissue homeostasis. With regard to virus-induced inflammation in the CNS, chemokines and chemokine receptors are in a strategic position to coordinate immune responses through both the regulation of leukocyte extravasation and also in the final positioning and activation of infiltrating cells. Immune surveillance of the CNS and the effector function of infiltrating leukocytes into the CNS dictate the host-pathogen relationship during viral pathogenesis of the CNS. The