In this study, we found that DENV-3 infected mice developed lethal encephalitis. Behavioral changes occurred at the peak of inflammatory changes in the CNS of infected animals and preceded death. To our knowledge, behavioral changes associated with leukocyte trafficking, the population of immune cells recruited into the brain, and cytokine/chemokine expression in DENV-3 encephalitis have not been previously reported.
In the present model, non-adapted DENV-3 was inoculated by an intracranial route to evaluate the CNS immune response to the infection. This route allows the study of viral-associated CNS lesions which are not seen in mice inoculated by an intraperitoneal route . The observed CNS lesions were similar to those seen in human dengue encephalitis . Using a similar approach, we have demonstrated that the intracranial route allows investigation of specific immune mechanisms involved in the CNS response to viral infection which are not evident when HSV-1 is inoculated in the periphery .
Our results also provide evidence of CNS compromise using a standardized protocol which demonstrated changes in muscle tone and strength, and motor behavior. Similar neurological deficits have been reported in patients with dengue infection . We also found that these behavioral changes occurred after the increase of the inflammatory response and tissue destruction in CNS. Furthermore, the infected animals presented neurological symptoms of apathy, stereotyped behavior, and seizures, characterized by forelimb clonus with rearing and falling. At day 6 p.i. we observed greater pathologic brain scores with loss of more than 30% of neurons in the hippocampus, mainly in the CA3 region. Apoptotic cell death has been described in several flaviviral infections, such as dengue viruses, Japanese encephalitis virus and West Nile Virus (WNV). DENV-2 triggers apoptotic signaling to kill infected cells and initiate survival signaling to hold the cells in a favorable condition for longer virus progeny production. Similar pathways could also be involved in DENV-3 encephalitis . DENV-2 activates the PI3K/Akt pathway as an antiapoptotic pathway to protect infected cells from early apoptotic cell death . Infection with WNV in the CNS causes limbic seizures with participation of IFN-γ in the development of excitatory glutamate receptor-responsive circuits in the CNS, especially involving the N-methyl-D-aspartate (NMDA) receptor. NMDA inhibition not only abrogates limbic seizures, but also prolongs survival of infected animals. The modulation of these neurological pathways has not been evaluated here but may have important implications for patients suffering from dengue encephalitis in the clinical setting . Further studies should investigate this possibility.
DENV-3-infected mice exhibited progressive meningoencephalitis characterized by infiltration of neutrophils and mononuclear cells. Before migrating to the brain parenchyma, leukocytes must roll and adhere to the brain microvasculature . We assessed leukocyte rolling and adhesion in pia-mater vessels at days 3 and 6 p.i. Leukocyte recruitment was up-regulated in the microvasculature, leading to increased infiltration of inflammatory cells into brain tissue. Recruitment of these cells into the CNS may be the result of chemoattraction exerted by increased levels of cytokines and/or chemokines .
CXCL2, a chemokine related to preferential recruitment of neutrophils, was increased at 3 and 6 days p.i., indicating the presence of stimulus for leukocyte recruitment during the course of the infection. CXCL2 has been associated with exacerbation of brain damage via neutrophil-dependent mechanisms . It is highly expressed in sera of dengue fever and dengue hemorrhagic fever patients, as well as in in vitro models of DENV infection [26, 27]. In the lethal encephalitis induced by the JHM strain of mouse hepatitis virus (JHMV), neutrophils enter the CNS at least 24 h before other infiltrating mononuclear cells and remain the dominant inflammatory cell population throughout infection. Infection with JHMV in neutropenic animals results in increased levels of virus replication and mortality. Furthermore, neutropenia is associated with reduced infiltration of all inflammatory cells, showing that neutrophils play a key role in promoting infiltration of mononuclear inflammatory cell populations in response to CNS viral infection . A paradoxical role for neutrophils has been reported in the pathogenesis of WNV . Neutrophils have a biphasic role in WNV infection, serving as a reservoir for replication and dissemination in early infection and later contributing to viral clearance. Also, the immune response to dengue virus may involve cell infiltration at two moments, since neutrophils are visualized in early and late stages of brain inflammation after DENV-3 infection. Further studies are needed to dissect the role of neutrophils along the disease course.
DENV-3-infected mice presented increased brain levels of CCL2, CCL5, CXCL1, as well as of the cytokines TNF-α and IFN-γ, at 6 days p.i. The production of these proteins possibly created a more favorable milieu for cell migration and may themselves be produced by migrated cells. The early and late expression of CXCL1 and CXCL2 suggests that there is a continuous recruitment of neutrophils to the brain, which was confirmed by histopathology, MPO and FACS results. CCL5 is a member of the CC chemokine family and recruits monocytes and T cells via the chemokine receptors CCR1, CCR3, and CCR5. CCL5 has also been reported to be up-regulated during dengue infection . We recently demonstrated that CCR1, CCR2 and CCR4 have minor effects in the pathogenesis of disease in a model of DENV-2 in mice inoculated by i.p. route. It appears that these receptors do not play an essential role in protection against primary infection, suggesting that the chemokine storm that follows severe primary dengue infection correlates primarily with development of disease rather than protection against severe infection .
We found increased levels of TNF-α in mouse brain tissue just before the onset of clinical signs of encephalitis. It is well known that higher concentrations of TNF-α correlate with severe dengue disease in vivo and high viral titers in vitro and in vivo . Higher levels of TNF-α in infected mice have been associated with endothelial activation [33, 34]. During viral encephalitis, MMPs can affect inflammatory responses by processing molecules like TNF-α, mediating transmigration of leukocytes and the development of CNS damage . In DENV-3 infected mice, enhanced levels of TNF-α may reflect activation of endothelial cells, which in turn leads to an increase in the number of rolling and adhered leukocytes seen by intravital microscopy. These results are in accordance with in vitro studies that have demonstrated TNF-α up-regulation of polymorphonuclear cell adhesion to cerebral endothelium . Increased levels of TNF-α may also be involved in the behavioral signs detected on day 6 p.i. The cytokines IL-1-β, IL-6, and TNF-α have been associated with cognitive processes such as synaptic plasticity, neurogenesis, and neuromodulation .
Another important cytokine up-regulated in DENV-3 infected mice was IFN-γ. IFN-γ plays a crucial role in the ability of the murine host to deal with dengue infection. High levels of IFN-γ are observed in patients with dengue and are associated with severity of the disease [37, 38]. Dengue hemorrhagic fever induced by DENV-3 has been associated with higher viremia early in illness and earlier peak plasma IFN-γ levels; maximum plasma viremia levels correlate with degree of plasma leakage and thrombocytopenia . Some researchers have demonstrated that IFN-deficient mice are more susceptible to dengue infection . We have previously detected high levels of IFN-γ, together with IL-6 and CCL2, in brain homogenates and sera of mice infected with DENV-3 genotype I, confirming its virulence and immunogenicity in the brain . The secretion of the cytokine IFN-γ by CD8+ T-cells plays a role in the defense against DENV-2 . We also observed a progressive increase of IFN-γ associated with high numbers of CD8+ T-cells in brain tissue, showing also the importance of this cytokine during infection with DENV-3. Some experimental studies have provided evidence that glucocorticoid treatment can have beneficial effects, but this may also exacerbate the pathogenesis of viral encephalitis. The use of glucocorticoids during viral encephalitis can restrain inflammation and microglial reactivity, and decrease cerebral damage due to viral replication. However, such therapy initiated before the establishment of an appropriate acute-phase response may increase neurovirulence and CNS damage .
Other cytokines may also be involved in the pathogenesis of DENV-3 infection. IFN type I (α/β), which is produced by many cells, is crucial for the immediate control of initial viral replication and powerfully initiates innate and specific immune responses . Studies with mice infected with DENV-2 by intravenous route have demonstrated that IFN-α/β receptor-mediated action limits initial virus replication in extraneural sites and controls subsequent viral spread into the CNS. In contrast, IFN-γ receptor-mediated responses seem to act at later stages of dengue disease by restricting viral replication in the periphery and eliminating virus from the CNS . The mechanisms by which the IFN system mediates the antiviral response in mice after intracranial inoculation with DENV-3 have not yet been elucidated. Further experiments are necessary to clarify the role of IFNs during meningoencephalitis caused by DENV.
In conclusion, the present study shows that neuroinflammatory changes lead to alterations in motor behavior and muscle tone and strength in DENV-3-infected mice. The neuroinflammatory process is marked by up-regulation of the chemokines CCL2, CCL5, CXCL1, and CXCL2, and of the cytokines TNF-α and IFN-γ, which occurs in parallel with increased leukocyte rolling and adhesion in meningeal vessels and infiltration of immune cells into the brain. The inflammatory response may play a key role in the development of severe neurological manifestations in dengue disease.