Lymph nodes (LNs) capture microorganisms that breach the body's external barriers and enter draining lymphatics, limiting the systemic spread of pathogens 1 . Recent work has shown that CD11b + CD169 + macrophages, which populate the subcapsular sinus (SCS) of LNs, are critical for clearance of viruses from the lymph and for initiating antiviral humoral immune responses 2 , 3 , 4 . Using vesicular stomatitis virus (VSV), a relative of rabies virus transmitted by insect bites, we show here that SCS macrophages perform a third vital function: they prevent lymph-borne neurotropic viruses from infecting the CNS. Upon local depletion of LN macrophages, ~60% of mice developed ascending paralysis and died 7-10 days after subcutaneous infection with a small dose of VSV, while macrophage-sufficient animals remained asymptomatic and cleared the virus. VSV gained access to the nervous system via peripheral nerves in macrophage-depleted LNs. In contrast, within macrophage-sufficient LNs VSV replicated preferentially within SCS macrophages but not in adjacent nerves. Removal of SCS macrophages did not compromise adaptive immune responses against VSV, but reduced type I interferon (IFN-I) production within infected LNs. VSV-infected macrophages recruited IFN-I producing plasmacytoid dendritic cells to the SCS and additionally were a major source of IFN-I themselves. Experiments in bone marrow chimeric mice revealed that IFN-I must act on both hematopoietic and stromal compartments, including the intranodal nerves, to prevent lethal VSV infection. These results Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms To explore how neurotropic viruses spread from their entry site to the CNS, we studied VSV, an arthropod-borne rhabdovirus that causes fatal paralytic disease in mammals, including mice 5 . While numerous studies have investigated immune responses to intravenous VSV infection 6 , the immunological consequences elicited by the more natural subcutaneous (sc) route are incompletely understood. Previous work has shown that, following peripheral inoculation, VSV is captured by macrophages in draining LNs, preventing hematogenous dissemination 2 . Here, we asked whether this macrophage filter affects the ability of neurotropic viruses to access the CNS.C57BL/6 mice were injected sc with clodronate liposomes (CLL) into one hind footpad, which selectively eliminated CD11b + CD169 + macrophages in the draining popliteal LN, but not in distal LNs or the spleen 2 . Six days later, mice were challenged in the ipsilateral or contralateral footpad using a low dose (10 4 pfu) of VSV-Indiana. While this dose was cleared by virtually all untreated and contralaterally-infected mice, ~60% of CLL-treated ipsilaterally-infected animals developed ascending CNS pathology starting with ipsilateral hindleg paralysis and progressing to death 7-10 ...

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SUMMARY Deciphering the signaling networks that underlie normal and disease processes remains a major challenge. Here, we report the discovery of signaling components involved in the Toll-like receptor (TLR) response of immune dendritic cells (DCs), including a previously unkown pathway shared across mammalian antiviral responses. By combining transcriptional profiling, genetic and small molecule perturbations, and phosphoproteomics, we uncover 35 signaling regulators, including 16 known regulators, involved in TLR signaling. In particular, we find that Polo-like kinases (Plk) 2 and 4 are essential components of antiviral pathways in vitro and in vivo, and activate a signaling branch involving a dozen proteins among which is Tnfaip2, a gene associated with autoimmune diseases but whose role was unknown. Our study illustrates the power of combining systematic measurements and perturbations to elucidate complex signaling circuits and discover potential therapeutic targets.

SUMMARY Neutralizing antibodies have been thought to be required for protection against acutely cytopathic viruses, such as the neurotropic vesicular stomatitis virus (VSV). Utilizing mice that possess B cells but lack antibodies, we show here that survival upon subcutaneous (s.c.) VSV challenge was independent of neutralizing antibody production or cell-mediated adaptive immunity. However, B cells were absolutely required to provide lymphotoxin (LT) α1β2, which maintained a protective subcapsular sinus (SCS) macrophage phenotype within virus draining lymph nodes (LNs). Macrophages within the SCS of B cell-deficient LNs, or of mice that lack LTα1β2 selectively in B cells, displayed an aberrant phenotype, failed to replicate VSV, and therefore did not produce type I interferons, which were required to prevent fatal VSV invasion of intranodal nerves. Thus, although B cells are essential for survival during VSV infection, their contribution involves the provision of innate differentiation and maintenance signals to macrophages, rather than adaptive immune mechanisms.