The results presented here contradict the current view that B cell-derived neutralizing antibodies are absolutely required to survive a primary cytopathic viral infection, such as that caused by VSV. This paradigm arose originally from experiments in B cell-deficient mice (Bachmann et al., 1994
; Bründler et al., 1996
; Gobet et al., 1988
), which lack antibodies, but also have abnormal lymphoid tissue architecture and altered macrophage phenotype. Our experiments in mice that lack antibodies but possess B cells and normal lymphoid tissues confirm that both B cells and antibodies are critical to survive a systemic infection after i.v. bolus administration of VSV. However, only B cells are essential when VSV is encountered via the more “natural” s.c. route, whereas antibodies are neither needed nor sufficient for protection. Our data collectively indicate that immunity to s.c. VSV infection relies on B cell-derived LTα1β2, rendering SCS macrophages capable of replicating VSV and producing neuroprotective IFN-I.
Although VSV infections are typically self-limiting in mammals, rabies virus, a close relative, is responsible for >55,000 human deaths every year. Neutralizing antibodies are also believed to be required to survive rabies infections, as shown by the fact that passive antibody transfer and active vaccination to elicit humoral immunity are standard of care. Although neutralizing antibodies are undoubtedly effective prophylaxis against rhabdoviruses, our findings indicate that antibody therapy may be insufficient to treat existing rhabdoviral infections in nonimmune subjects, at least in the case of VSV. It is unclear whether this caveat applies also to rabies virus infection, but failures of both passive and active vaccination after exposure to rabies are known to occur (Anonymous, 1988
). Thus, it will be important to further dissect the role of antibodies and interferon in this disease. In addition, recent years have seen the emergence and/or spread of other arthropod-borne neurotropic viral infections, such as West Nile virus, Japanese encephalitis virus, and Eastern and Western equine encephalitis virus, to name a few (Weaver and Barrett, 2004
). It remains to be determined whether the cellular and molecular immunological events that occur upon inoculation of these pathogens in the skin are similar to the ones identified here.
Most viral pathogens, including rabies and VSV, must breach the body's external barriers to cause disease. Viruses deposited in the interstitial space of peripheral tissues are readily drained via lymph vessels to downstream LNs. Once a lymph-borne virus enters the intranodal lymph conduits, it runs a gauntlet across two phenotypically and functionally distinct LN macrophage populations within the lymphatic space. Initial encounter is with SCS macrophages, which populate the superficial cortex above B cell follicles, often with macrophages penetrating across the sinus floor such that they contact both follicular B cells and afferent lymph (Junt et al., 2007
). Subsequently, lymph-borne viruses are exposed to macrophages within medullary sinuses where lymph is collected to be discharged into efferent lymphatics. Together, SCS and medullary macrophages filter the lymph and capture particulate matter, including viruses (Junt et al., 2007
). The recognition mechanism(s) mediating this “flypaper” activity is not fully understood, but this process is extremely effective, as shown by the fact that >99% of infectious virions are retained in LNs, preventing systemic dissemination (Junt et al., 2007
Once a lymph-borne virus has been captured by LN macrophages, at least three distinct fates await the surface-bound virion: (1) phagocytosis and destruction in lysosomal compartments, (2) presentation and handover to follicular B cells for the induction of humoral responses, or (3) productive infection of the capturing cells (Iannacone et al., 2010
; Junt et al., 2007
). Paradoxically, our findings indicate that the latter event is indispensable for host resistance to VSV; infected mice survived efficiently when SCS macrophages supported VSV replication. Although both SCS and medullary macrophages captured VSV, only SCS macrophages support VSV replication, which is required for IFN-I production (Iannacone et al., 2010
). In settings where SCS macrophage differentiation was compromised, as in μMT mice, the cells failed to replicate VSV or to produce IFN-I, which is required to protect intranodal nerves from serving as viral conduits to the CNS.
Several phenotypic and functional features of SCS and medullary macrophages could regulate their difference in VSV replication capacity. For example, medullary macrophages are more phagocytic and express higher levels of endosomal degradative enzymes than their SCS counterparts (Delemarre et al., 1990b
; Fossum, 1980
; Phan et al., 2009
; Sainte-Marie and Peng, 1985
; Szakal et al., 1983
). The two subsets differ also in their expression of antiviral sensing receptors. Compared to SCS macrophages, published microarray data (Phan et al., 2009
) suggest that the medullary subset expresses higher mRNA levels for several TLRs, including TLR4 and TLR13, which both recognize VSV (Georgel et al., 2007
; Shi et al., 2011
). By contrast, SCS macrophages express more RIG-I, which enables intracellular VSV recognition (Gerlier and Lyles, 2011
; Kato et al., 2006
). Thus, medullary macrophages are well equipped to detect and destroy extracellular virus whereas SCS macrophages appear to be relatively inefficient at eliminating surface-bound virions, which may facilitate productive VSV infection. However, SCS macrophages may be preferentially responsive to viral patterns in their cytoplasm, inducing IFN-I production. Similar to differences between SCS and medullary macrophages in WT animals, our data would predict that SCS macrophages from DH
LMP2A and μMT mice are similarly divergent in their viral sensing and phagocytic capacity.
LTβR is expressed on both LN macrophage subsets, but SCS macrophage maintenance and function appear to be more sensitive to LTβR signaling (Phan et al., 2009
). Moreover, SCS but presumably not medullary macrophages are in intimate contact with LTα1β2-expressing follicular B cells. Consequently, in vivo LTα1β2 inhibition minimally affects the medullary compartment, but alters the phenotype of SCS macrophages and compromises their ability to capture lymph-borne immune complexes (Phan et al., 2009
). The present findings indicate that LTα1β2 also regulates SCS macrophage susceptibility to VSV and the ensuing IFN-I response. In the absence of B cell-expressed LTα1β2, SCS macrophages assume a phenotype resembling medullary macrophages and no longer support VSV replication. One possible explanation for this observation is that tonic exposure to LTα1β2 on follicular B cells attenuates macrophage responsiveness to autocrine IFN-I. Without LTβR signaling, as is physiologically the case in the medulla or may be experimentally achieved in the SCS by deletion of B cells or treatment with LTβR-Ig, macrophages may gain IFN-I responsiveness and VSV resistance.
It remains to be determined whether there are additional mechanisms by which B cell-derived LTα1β2 renders SCS macrophages physiologically susceptible to VSV infection. Nonetheless, the net effect is that LTα1β2 transforms SCS macrophages into unique sentinels that execute a rapid antiviral cytokine response. It seems counterintuitive that this defense mechanism should depend upon the amplification of a potentially lethal virus, but beyond jump-starting the IFN-I response, increased viral antigen availability may facilitate adaptive memory in LNs (Hickman et al., 2008
). Thus, SCS macrophage infection appears to be a “necessary evil” to ensure short-term survival and to promote long-term immunity of the host.
Defects in LTα1β2 signaling have been implicated previously in impaired antiviral immune responses (Berger et al., 1999
; Spahn et al., 2005
). Efficient IFN-I production upon cytomegalo-virus infection requires lymphotoxin signaling in splenic stromal cells (Benedict et al., 2001
; Schneider et al., 2008
), and Ltb–/–
mice are vulnerable to death after i.v. VSV infection resulting from defective viral capture by splenic marginal zone macrophages (Junt et al., 2006
). However, these earlier studies performed by systemic viral challenge were focused on the role of LTα1β2 in antiviral responses mediated by adaptive immune cells. Our findings highlight a previously underappreciated role for lymphotoxin in innate antiviral immunity in LNs draining a local infection site.
While LTα1β2 confers antiviral resistance to blood-borne or lymph-borne VSV in the spleen or peripheral LNs, respectively, our observations reveal the critical role that the route of infection plays in determining the molecular and cellular requirements for immune protection. Both i.v. and s.c. VSV infections elicit robust IFN-I and neutralizing antibody production, and both infection routes rely on macrophages for survival (Ciavarra et al., 2005
; Iannacone et al., 2010
). Consistent with earlier findings (Thomsen et al., 1997
), our experiments confirm that antibodies are critical to survive i.v. VSV infection; however, a humoral response is neither required nor sufficient to prevent fatal neuroinvasion when VSV is given subcutaneously. In the s.c. setting, VSV gains access to the CNS via peripheral nerves in draining LNs (Iannacone et al., 2010
). It is unclear how VSV accesses the CNS from the blood; however, it appears to do so rapidly, at least when given at a lethal dose, because subsequent treatment with neutralizing antibodies is protective only within 3 hr of i.v. infection (Steinhoff et al., 1995
VSV infection of SCS macrophages, although required for production of neuroprotective IFN-I, results in death of the host cells within 12–18 hr. Through this act of self-sacrifice, SCS macrophages establish a local antiviral state that prevents further viral replication. However, once depleted, SCS and medullary macrophages are slow to return. When CLL is used to eliminate essentially all LN macrophages, LNs require as long as 6 months to re-establish normal cell numbers (Delemarre et al., 1990a
). This lag time is markedly shorter after VSV infection, where SCS macrophages return to preinfection numbers within 1 month (data not shown). Nevertheless, the void of SCS macrophages for several weeks after VSV infection probably renders LNs transiently hypervulnerable to reinfection. In these cases the induction of protective memory responses by adaptive immune cells may be critical for preventing early reinfection.
In contrast to the likely benefit of adaptive immunity during reinfection, our results demonstrate that during a primary s.c. infection, recognition of viral epitopes by either antibody or TCR is neither necessary nor sufficient to prevent fatal VSV neuroinvasion. This observation runs counter to the commonly held view that during viral infections, innate immunity must orchestrate the induction of antiviral adaptive responses to achieve sterilizing immunity. Given the rapid replication of some viruses, a proliferating pathogen may overwhelm its host before adaptive immune countermeasures can be mobilized. Innate defenses like complement, type I interferon, and others are believed to provide stopgap measures, lowering pathogen burden and buying time for adaptive immune responses to develop. Although this concept may apply to other viral infections, our findings with VSV turn this view upside down, indicating that during a primary infection with this cytopathic virus, innate immunity can be sterilizing without adaptive immune contributions. Thus, the essential contribution to VSV immunity by adaptive immune cells appears to be 2-fold. (1) During primary infection, B cells are critical enablers of innate immune responses by inducing and maintaining SCS macrophage phenotype through LTα1β2 presentation. (2) During secondary infection in the presence of established immunological memory, antiviral antibodies neutralize infectious virions before host cells can be infected.
In summary, we demonstrate that naive mice can survive a s.c. VSV challenge without requiring antigen-specific adaptive immunity. Efficient protection against VSV is provided by SCS macrophages in the draining LNs that rely on contact with follicular B cells expressing LTα1β2 on their surface. The constant exposure to LTα1β2 induces and maintains the protective SCS macrophage phenotype. Consequently, SCS macrophages in B cell-deficient mice or in mice that lack B cell-expressed LTα1β2 display an altered phenotype that resembles that of medullary macrophages, which are not protective in VSV infection. Like medullary macrophages, SCS macrophages that are deprived of LTα1β2 capture lymph-borne VSV but fail to replicate it. Without replication, SCS macrophages do not produce IFN-I that is required to prevent VSV invasion of intranodal nerves. These findings establish a critical innate function for B cells in antiviral immunity. This setting requires B cells not as a source of antibodies, but as providers of an anatomically restricted maintenance signal and as the day-to-day custodians of macrophage differentiation.