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.

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.

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.

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

BACKGROUND: Cell-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. METHODS: We 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. RESULTS: As 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. CONCLUSIONS: These 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.

Border Patrol Gone Awry: Lung NKT Cell Activation by Francisella tularensis Exacerbates Tularemia-Like Disease.

The respiratory mucosa is a major site for pathogen invasion and, hence, a site requiring constant immune surveillance. The type I, semi-invariant natural killer T (NKT) cells are enriched within the lung vasculature. Despite optimal positioning, the role of NKT cells in respiratory infectious diseases remains poorly understood. Hence, we assessed their function in a murine model of pulmonary tularemia--because tularemia is a sepsis-like proinflammatory disease and NKT cells are known to control the cellular and humoral responses underlying sepsis. Here we show for the first time that respiratory infection with Francisella tularensis live vaccine strain resulted in rapid accumulation of NKT cells within the lung interstitium. Activated NKT cells produced interferon-γ and promoted both local and systemic proinflammatory responses. Consistent with these results, NKT cell-deficient mice showed reduced inflammatory cytokine and chemokine response yet they survived the infection better than their wild type counterparts. Strikingly, NKT cell-deficient mice had increased lymphocytic infiltration in the lungs that organized into tertiary lymphoid structures resembling induced bronchus-associated lymphoid tissue (iBALT) at the peak of infection. Thus, NKT cell activation by F. tularensis infection hampers iBALT formation and promotes a systemic proinflammatory response, which exacerbates severe pulmonary tularemia-like disease in mice.

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(+)CD45(hi) infiltrating cells (macrophages) are the primary producers of IL-1ß within the CNS and, through the use of an in vitro blood-brain barrier model, that IL-1ß 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-1ß-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.

Viral pathogen-associated molecular patterns regulate blood-brain barrier integrity via competing innate cytokine signals.

UNLABELLED: 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.

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.

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

Infections of the central nervous system (CNS) with cytopathic viruses require efficient T cell responses to promote viral clearance, limit immunopathology, and enhance survival. We found that IL-1R1 is critical for effector T cell reactivation and limits inflammation within the CNS during murine West Nile virus (WNV) encephalitis. WNV-infected IL-1R1(-/-) mice display intact adaptive immunity in the periphery but succumb to WNV infection caused by loss of virologic control in the CNS with depressed local Th1 cytokine responses, despite parenchymal entry of virus-specific CD8(+) T cells. Ex vivo analysis of CD4(+) T cells from WNV-infected CNS of IL-1R1(-/-) mice revealed impaired effector responses, whereas CD8(+) T cells revealed no cell intrinsic defects in response to WNV antigen. WNV-infected, IL-1R1(-/-) mice also exhibited decreased activation of CNS CD11c(+)CD11b(-)CD103(+) and CD11c(+)CD11b(-)CD8α(+)Dec-205(+) cells with reduced up-regulation of the co-stimulatory molecules CD80, CD86, and CD68. Adoptive transfer of wild-type CD11c-EYFP(+) cells from WNV-infected CNS into WNV-infected IL-1R1(-/-) mice trafficked into the CNS restored T cell functions and improved survival from otherwise lethal infection. These data indicate that IL-1R1 signaling promotes virologic control during WNV infection specifically within the CNS via modulation of CD11c(+) cell-mediated T cell reactivation at this site.

Emerging roles of T helper subsets in the pathogenesis of asthma.

The cardinal features of asthma include pulmonary inflammation and airway hyperresponsiveness (AHR). Classically, asthma, specifically allergic asthma, has been attributed to a hyperactive Th2 cell immune response. However, the Th2 cell-mediated inflammation model has failed to adequately explain many of the clinical and molecular aspects of asthma. In addition, the outcomes of Th2-targeted therapeutic trials have been disappointing. Thus, asthma is now believed to be a complex and heterogeneous disorder, with several molecular mechanisms underlying the airway inflammation and AHR that is associated with asthma. The original classification of Th1 and Th2 pathways has recently been expanded to include additional effector Th cell subsets. These include Th17, Th9 and Treg cells. Emerging data highlight the involvement of these new Th cell subsets in the initiation and augmentation of airway inflammation and asthmatic responses. We now review the roles of these recently classified effector Th cell subsets in asthmatic inflammation and the insights they may provide in addition to the traditional Th2 paradigm. The hope is that a clearer understanding of the inflammatory pathways involved and the mediators of inflammation will yield better targeted therapeutics.

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-gamma 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.

Intranasal interleukin-12 treatment for protection against respiratory infection with the Francisella tularensis live vaccine strain.

Francisella tularensis is a gram-negative intracellular bacterium that can induce lethal respiratory infection in humans and rodents. However, little is known about the role of innate or adaptive immunity in protection from respiratory tularemia. In the present study, the role of interleukin-12 (IL-12) in inducing protective immunity in the lungs against intranasal infection of mice with the live vaccine strain (LVS) of F. tularensis was investigated. It was found that gamma interferon (IFN-gamma) and IL-12 were strictly required for protection, since mice deficient in IFN-gamma, IL-12 p35, or IL-12 p40 all succumbed to LVS doses that were sublethal for wild-type mice. Furthermore, exogenous IL-12 treatment 24 h before intranasal infection with a lethal dose of LVS (10,000 CFU) significantly decreased bacterial loads in the lungs, livers, and spleens of wild-type BALB/c and C57BL/6 mice and allowed the animals to survive infection; such protection was not observed in IFN-gamma-deficient mice. The resistance induced by IL-12 to LVS infection was still observed in NK cell-deficient beige mice but not in CD8-/- mice. These results demonstrate that exogenous IL-12 delivered intranasally can prevent respiratory tularemia through a mechanism that is at least partially dependent upon the expression of IFN-gamma and CD8 T cells.