Discussion
Recent studies have reported increased viral titers in the CNS of mice treated with CSF1R antagonists [36, 60, 61]; however, the mechanisms underlying this loss of virologic control and the effects on peripheral immune responses have not been well characterized. We sought to determine the role of CSF1R signaling in viral clearance of both systemic and neurotropic infection. Our results indicate that antagonism of CSF1R signaling limits virologic control within the CNS via reduction of critical APCs that express co-stimulatory B7 molecules, CD80 and CD86, necessary for local reactivation of antiviral CD8+ T cells. Our data also indicate that CSF1R antagonism reduces expression of co-stimulatory B7 molecules on peripheral APCs, contributing to loss of virologic control in the periphery and dissemination of virus to the CNS. As previously reported, WNV-NY inoculated into mice via f.p. is detected first within the olfactory bulb then spreads caudally through the cerebral cortex to the brain stem with significant virus not detected in the cerebellum until 8 dpi [20]. CSF1R antagonism with PLX5622 leads to increased viral replication within each of these CNS regions and also in the peripheral organs: the spleen, kidney, and serum. In contrast to CNS titers, the virus is cleared from the spleen and serum by 8 dpi in WNV-infected, control, and PLX5622-treated mice. Notably, we detected expansion of viral tropism with significant viral replication in the kidney of PLX5622-treated mice at 4 and 6 dpi compared with control-treated animals. These data suggest that renal macrophages may be sensitive to CSF1R antagonism, which is consistent with a study utilizing a model of recovery from acute kidney injury in which CSF1R inhibition with GW2580 reportedly decreased both kidney macrophage proliferation and their polarization towards wound-healing phenotypes [62]. In order to better isolate the role of microglia during neurotropic infection, we examined the effect of PLX5622 in an established murine model of CNS infection using an attenuated strain of WNV (WNV-NS5-E218A), which lacks 2′-O-methyltransferase activity, thus increasing sensitivity to IFIT-mediated suppression and limiting virulence in peripheral organs with intact immunity [8, 45, 49]. Mice infected i.c. with WNV-NS5-E218A exhibited increased viral titers in the CNS and increased lethality with PLX5622 treatment compared with control treatment. These data suggest that loss of CNS immunity increases lethality of WNV infection.
Clearance of WNV within the CNS requires local reactivation of antiviral CD8+ T cells for efficient T cell-mediated adaptive immune responses [55, 63]. Under homeostatic conditions, microglia express very low levels of co-stimulatory B7 molecules (CD80/CD86); however, microglia upregulate expression of these molecules in response to inflammatory cytokines [54, 64]. Our results show PLX5622 treatment decreased expression of these molecules by depleting their cellular sources in the CNS, which include both resident microglia and infiltrating monocytes/macrophages. In the periphery, PLX5622 also reduced cellular expression of B7 molecules on splenic, blood, and pLN APCs. Under certain conditions, other co-stimulatory molecules are able to compensate for the loss of B7 signals in T cell reactivation. For example, expansion of lymphocytic choriomeningitis virus-specific antiviral T cells can be driven by either alternative co-stimulatory TNFR superfamily members or by enhanced expression of antiviral cytokines, such as high levels of type I IFNs [65]. However, during WNV encephalitis, PLX5622 treatment also significantly decreased transcriptional expression of both TNF and IFNβ. Together, our results indicate that CSF1R antagonism contributes impaired local reactivation of antiviral T cells within the CNS by depleting co-stimulatory signals including both B7 molecules and inflammatory cytokines.
In addition to augmenting antiviral T cell activation, many cytokines have essential protective roles during WNV infection. Infected human monocyte-derived macrophages release IL-8, IFNα, IFNβ, and TNF [66, 67]. Microglia become activated in response to neurotropic WNV infection and upregulate expression of CXCL10, CXCL1, CCL5, CCL3, CCL2, TNF, and IL-6 [18]. Neurons and astrocytes in WNV-infected brains also release proinflammatory mediators including CCL2, CCL5, CXCL10, IL1β, IL-6, IL-8, and TNF, which promote lymphocyte trafficking to the CNS [19–21]. In fact, results from a recent study demonstrated that peripheral infection with WNV increased CNS expression of chemokines important for lymphocyte trafficking, including CCL2, CCL7, CXCL9, and CXCL10, in PLX5622-treated mice compared with control-treated mice [61]. Type I IFN response is widely accepted as the most immediate antiviral host response, essential for controlling viral replication during the initial infection. Mice lacking type I IFN signaling are highly vulnerable to uncontrolled WNV replication, exhibiting 100% mortality [68]. Clearance of viral infections, however, requires additional immune response including type II IFN, i.e., IFNγ, which is produced by activated CD8+ T cells. Mice deficient in IFNγ show higher peripheral viral load, increased CNS infection, and increased lethality [69]. Data described here show significantly decreased cytokine expression in PLX5622-treated mice, which may also contribute to the loss of virologic control via the above-described mechanisms.
The exact role of microglia during viral infection has been difficult to define because infiltrating monocytes can mask the impact of resident microglia. In a previous report, IL34−/− mice, which have significantly reduced development of microglia, are more susceptible to lethal infection after WNV-NS5-E218A i.c. inoculation [36]. Similar to results reported here, IL34−/− mice exhibited increased neuronal death compared with wildtype controls. In contrast with PLX5622-treated mice, IL34−/− mice exhibited similar levels of viral burden compared with wildtype controls in the brain at 3 and 6 dpi. Numbers of infiltrating monocytes/macrophages and WNV-specific T cells were also similar between IL34−/− and wildtype controls; however, the authors did not investigate antiviral T cell activation in this model [36]. In another model of flavivirus encephalitis, in which suckling mice were infected with dengue virus, depletion of microglia with liposome-encapsulated clodronate resulted in increased viral replication and reduced infiltration of IFNγ+CD8+ T cells [70]. Here, we report no reduction in CD8+ T cell infiltration; however, this difference may be due to the age of mice, virus, and/or method of microglial depletion. Another recent study reported use of PLX5622 in a model of neuroattenuated murine corona virus, mouse hepatitis virus (MHV). Similar to our results, PLX5622 treatment resulted in increased viral replication and lethality. In contrast to our results, PLX5622 treatment reduced expression of MHCII on microglia and macrophages and reduced CD4+ T cell response [60]. While CD4+ T cells can improve CD8+ T cell response, the dependence of CD8+ T cells on CD4+ T cells varies by virus, antigen exposure, tissue environment, and effector sites [71]. In models of MHV encephalitis, control of viral replication requires a collaborative effort between CD4+ and CD8+ T cells [71]. However, when virus replication induces activation of APCs, activation of CD8+ T cells is relatively CD4+ T cell-independent [71].
Recently, chronic activation of innate immune response by microglia is believed to be a major contributor to neurodegenerative conditions [72]. Genetic studies have confirmed the relevance of inflammatory response in common neurodegenerative diseases, such as Alzheimer’s disease and multiple sclerosis, indicating that neuroinflammation is likely implicated in the primary pathogenesis, rather than a secondary response [73]. We and others have reported that microglia impact neurocognitive function by executing complement-mediated synapse elimination [8, 74, 75]. Because of the neurological consequences of microglial activation, researchers have attempted to use CSF1R antagonism to deplete microglia in a variety of models to improve neurological function, including traumatic brain injury [38, 39], brain irradiation [50], myelin-induced catatonia [76], experimental autoimmune encephalomyelitis [40], and Alzheimer’s disease [41]. However, our results indicate that CSF1R antagonism may ultimately be detrimental to brain health due to loss of innate and adaptive immune response in the CNS, as well as decreased peripheral APC activation. Thus, in assessing the benefit of CSF1R antagonism, it will be important to consider the diminished immune response in both the CNS and peripheral immune compartments.