Subcapsular sinus macrophages prevent CNS invasion on peripheral infection with a neurotropic virus

Abstract

Lymph nodes (LNs) capture microorganisms that breach the body's external barriers and enter draining lymphatics, limiting the systemic spread of pathogens. Recent work has shown that CD11b(+)CD169(+) macrophages, which populate the subcapsular sinus (SCS) of LNs, are critical for the clearance of viruses from the lymph and for initiating antiviral humoral immune responses. Here we show, using vesicular stomatitis virus (VSV), a relative of rabies virus transmitted by insect bites, that SCS macrophages perform a third vital function: they prevent lymph-borne neurotropic viruses from infecting the central nervous system (CNS). On local depletion of LN macrophages, about 60% of mice developed ascending paralysis and died 7-10 days after subcutaneous infection with a small dose of VSV, whereas macrophage-sufficient animals remained asymptomatic and cleared the virus. VSV gained access to the nervous system through peripheral nerves in macrophage-depleted LNs. In contrast, within macrophage-sufficient LNs VSV replicated preferentially in SCS macrophages but not in adjacent nerves. Removal of SCS macrophages did not compromise adaptive immune responses against VSV, but decreased type I interferon (IFN-I) production within infected LNs. VSV-infected macrophages recruited IFN-I-producing plasmacytoid dendritic cells to the SCS and in addition were a major source of IFN-I themselves. Experiments in bone marrow chimaeric mice revealed that IFN-I must act on both haematopoietic and stromal compartments, including the intranodal nerves, to prevent lethal infection with VSV. These results identify SCS macrophages as crucial gatekeepers to the CNS that prevent fatal viral invasion of the nervous system on peripheral infection.

Figure 1. Lymph node macrophages confer resistance to fatal CNS invasion upon peripheral low-dose VSV infection

a, Survival curves of control mice (n=68) and mice that received ipsilateral (n=77) or contralateral (n=10) CLL injection prior to VSV infection. CLL ipsilateral versus control, P<0.0001. b, VSV titers in the brain of control and CLL-treated mice, 7d after VSV infection. Red squares identify paralytic animals. P=0.024. c, Survival curves of CD11c-DTR mice (n=8); P=0.0256. d, Survival curves of control and CLL-treated mice after intravenous VSV infection (n=10). e, Survival curves of CLL-treated VSV infected mice after ipsilateral (n=10) or contralateral (n=7) sciatic nerve resection. P=0.007. f, FACS plots of digested footpads (top) and the popliteal LN (bottom) of control and CLL footpad- or calf-injected mice. Numbers represent the percentage of CD45+ cells within each gate. Plots are representative of 2 experiments (n=3 mice/experiment). g, Survival curves in control (n=10) and calf CLL-treated mice (n=11). P=0.0411.

Figure 2. SCS macrophages are the primary targets for lymph-borne VSV and prevent infection of adjacent nerves

a–c, Representative micrographs of macrophage-sufficient (a,b) or CLL-treated (c) popliteal LNs after VSV-eGFP infection. Scale bars reflect 150μm (a,c) or 20μm (b). d–f, Representative MP-IVM micrographs of uninfected (d) or VSV-eGFP-infected LNs (e,f). VSV-eGFP infection of macrophage-sufficient LNs (e) induced GFP expression in macrophages but not in nerves (red), whereas nerves in CLL-treated LNs (f) expressed GFP (Suppl. Movies 1,2). Scale bars reflect 100μm. Blue depicts second harmonic signal from collagen in the LN capsule. g, Ratio of mean fluorescent intensities (MFI) in the green (488nm) and red channel (568nm) depicting GFP expression and β3-tubulin staining, respectively, in peripheral nerves. n=3, P<0.0001 (VSV versus CLL+VSV). h, VSV titers in popliteal LNs of control and CLL-treated mice. n=4, P=0.0010 (6h), P<0.0001 (12h), P=0.1043 (24h), P=0.0765 (36h). i, Serum neutralizing Ig titers in control and CLL-treated infected mice. n=4, P=0.0053 (d4), P=0.0138 (d7), P=0.0022 (d10), P=0.0054 (d26).

Figure 3. Regulation of VSV-induced IFN-I production by SCS macrophages

a, VSV-induced IFNα production in LNs (n=3); *, P<0.05, **, P<0.001 versus uninfected. b,c, IFNα concentrations in supernatants of FACS-sorted LN cells after VSV-eGFP infection (Suppl. Fig. 9a,b). Results are from one (of 3) representative experiment. d–g, Micrographs of popliteal LN sections from BM chimeric mice with 30% GFP⁺ pDCs (Suppl. Fig. 10) that were either left untreated (d) or sacrificed 8h after VSV infection (e–g) without (e,f) or with CLL pretreatment (g). Scale bars reflect 150μm (d, e, g) or 20μm (f). h, pDC frequency in the T cell area (n=4 mice/group). **, P<0.001 versus control. i, Relative pDC frequency distribution in LNs cross-sections (Suppl. Fig. 12). VSV versus control, P<0.05. j, Effect of depletion of LN macrophages, pDCs or both on survival upon VSV infection; a-PDCA1 versus control, ns; CLL versus CLL+a-PDCA1, ns. i, Survival curves of wild-type (WT), IFNαβR^−/− or irradiated IFNαβR^−/− mice that were reconstituted with WT BM upon VSV infection. n=8. WT BM→IFNαβR^−/− versus WT+CLL, ns; WT BM → IFNαβR^−/− versus IFNαβR^−/−, P=0.0001.