LN medullary macrophages are recognized for their ability to capture, phagocytose and destroy pathogens draining from sites of infection, a process that helps prevent systemic spread [64
]. Therefore it has come as something of a surprise to learn that SSMs and their CD169+ counterparts in the spleen, MMMs, are prone to infection by a range of viruses [35
]. These observations suggest a permissivity of SSMs and MMMs for viral infection [66
]. The physiological basis for and benefits of this permissivity are beginning to come to light. In the case of systemic vesicular stomatitis virus (VSV) infection, many blood-exposed macrophage types become rapidly infected. However, viral replication is only detected in the spleen and here only within MMMs [69
]. Building from evidence that type I IFN receptor signaling is highly restrictive for VSV replication, Honke et al., hypothesized that removing Usp18, a negative regulator of IFNαR1 signaling, would reduce the extent of viral replication [69
]. Consistent with their model, they found that MMMs expressed Usp18 and that viral replication in the spleen was prevented in Usp18−/− mice, suggesting that MMMs, and thus perhaps SSMs, depend on Usp18-mediated negative regulation of IFNR1 to sustain a viral replication permissive state. This specialized property of MMMs and SSMs may account for the apparently selective replication of other viruses within these macrophages: vaccinia virus (VV) [66
] and cow pox virus [67
] replicate preferentially in SSMs after subcutaneous inoculation; murine cytomegalovirus infected both SSMs in the mediastinal LN and splenic MMMs following peritoneal inoculation [67
]; lymphocytic choriomeningitis virus (LCMV) replicated in MMMs (and possibly MZMs) in the spleen after intravenous infection [68
]. In addition, dengue virus replicated in SSMs following skin infection [70
] though mice lacking IFNαR1 and IFNγR were studied here because of dengue’s ability to inhibit human but not mouse IFNR signaling; given the Usp18 findings it would be interesting to revisit this analysis and ask whether early dengue virus replication can be detected in SSMs of wild-type mice.
The permissivity of SSMs to infection may not be limited to viruses. Subcutaneously inoculated Toxoplasma gondii
preferentially infects and replicates within SSMs [45
] and IFN-I inhibits T. gondii
growth in monocyte-derived macrophages [71
]. Whether other properties of SSMs (and MMMs) make them unusually permissive to infection by intracellular pathogens, such as low lysosomal activity or repression of intracellular pathogen sensing pathways remains an interesting area for future investigation. Of course, these cells are unlikely to lack all anti-pathogen defense mechanisms. Indeed, following Leishmania donovani
infection, splenic MZMs and MMMs showed IRF7 recruitment to phagosomes and IRF7 deficiency was associated with a small increase in intracellular parasite load [72
]. Finally, the expression of CD4 by SSMs in rodents [9
][and unpubl. obs.] raises the question of whether CD4 is expressed by human SSMs and, if so, what role these cells play during HIV propagation in human LNs.
Viral replication in SSMs may augment LN immune responses in a number of ways. In the case of subcutaneous VSV infection, viral replication in SSMs was necessary for the production of sufficient IFN-I to protect intranodal nerves from infection [35
]. The IFN-I appeared to come both from the SSMs and from plasmacytoid DCs that were recruited into their proximity [35
]. Similarly, splenic MMMs and MZMs are the major IFN-I producers following intravenous HSV infection [73
]. A second function of permissive viral replication in SSMs may be to generate intact viral particles to provoke antibody responses, since viral particles are more effective in promoting B cell activation than free antigens [74
]. In the case of systemic VSV infection, ablation of CD169+ cells by diphtheria toxin (DT) treatment of mice expressing the DT receptor in CD169+ cells (CD169-DTR mice), or removal of Usp18 and thus suppression of viral replication in CD169+ cells, led to a reduced antibody (and T cell) response [69
]. However, following subcutaneous VSV infection, macrophage ablation by CLL treatment led to an augmented antibody response [35
]. This different outcome might indicate that the less selective CLL-based macrophage-ablation tool led to a more drastic loss of viral clearance capability, thus increasing exposure of adaptive immune cells to virus or virally-induced inflammatory mediators. Future studies of the LN response to VSV in mice in which CD169+ cells have been selectively ablated may help address this discrepancy.
A third benefit of SSM viral replication permissivity may be to facilitate the development of effector CD8 T cell responses, either directly, by presenting antigen to T cells, or indirectly, by antigen transfer to nearby DCs. Following subcutaneous VV infection, 85% of the infected LN cells were CD169+ macrophages, with the majority of these being F4/80− SSMs [75
]. Antigen specific CD8 T cells accumulated in proximity to these macrophages in interfollicular regions, but most CD8 T cell contacts were with DCs, and priming was more successful via DCs [66
]. Presentation by the macrophages alone was able to stimulate some T cell division, but was not good at generating effector CD8 T cells. One factor to keep in mind in this context was the use of DT treatment of CD11c-DTR mice to study the antigen presenting contribution of DCs versus macrophages. Many LN macrophages, as well as splenic MZ macrophages, express sufficient amounts of CD11c to be ablated by DT treatment of CD11c-DTR mice [35
][and unpubl. obs.] and consistent with this, a ~10-fold reduction in VV infected macrophages was noted in the DT treated CD11c-DTR mice [75
]. Thus, the functional properties being studied following DT treatment are of the toxin-resistant fraction of macrophages.
Perhaps the replication of virus in CD169+ macrophages permits subsequent infection of nearby DCs, facilitating their full activation, recruitment of CD8 T cells and antigen presentation. Further evidence for antigen transfer from sinus associated macrophages to T cell-priming DCs comes from the finding that antibody-mediated targeting of antigen to CD169+ MMMs in the spleen led to cross-presentation by CD8+ DCs to achieve effective CTL activation [77
]. Similarly, CD169+ macrophages participated in apoptotic cell capture in the spleen but presentation to T cells was by neighboring CD8+CD103+ DCs [78
]. SSMs in LNs captured subcutaneously injected apoptotic cells and when SSMs and other CD169+
cells were ablated by treating CD169-DTR mice with DT, CD8 T cell priming against an apoptotic cell antigen failed [36
cells were involved in antigen presentation to the CD8 T cells, and we suspect that these cells acquired apoptotic cell-derived antigen from SSMs or CD169+
interfollicular macrophages [36
]. This study suggests a further mechanism by which SSMs might contribute to anti-viral CD8 T cell responses is by capturing apoptotic cells or cell debris draining to lymph nodes from infected tissues and making viral antigen available for the subsequent priming of CD8 T cell responses.
During the splenic response to LCMV there was marked upregulation of CXCR3 ligands in the MZ, likely made by MMMs, and CXCR3-mediated guidance of CD8 T cells to the MZ was critical for generation of short-term effector cells [68
]. These data suggested that recruitment of cognate T cells into the region of high viral antigen density is important for generating short-term effector cells. Although the study did not determine whether the macrophages were directly involved in antigen presentation to the CD8 T cells or whether a DC intermediary was involved, it seems likely that the CXCR3 receptor-ligand system will operate along with the CCR5 receptor-ligand system [75
] during LN macrophage infections that result in effector CD8 T cell recruitment and activation.
The ability of infected SSMs to present antigens in the context of MHC class I is indicated by their ability to be killed by CD8 cells during Toxoplasma infection [79
]. However, whether they express sufficient amounts of costimulatory molecules to support naïve T cell priming is less clear, and most studies to date suggest they are not well equipped for this function, though they are apparently competent to promote activation of the effector type iNKT cell [80
The ability of SSMs to present CD1d-restricted lipid antigens to iNKT cells was tested by immunizing mice with 200nm silica microspheres coated with αGalCer containing liposomes [80
]. These particles colocalized with CD169 staining cells in mediastinal LNs 2 hr after intraperitoneal immunization and real-time imaging showed that iNKT cells made increased numbers of contacts with SSMs 6 hr after antigen administration. Depletion of macrophages (but not DCs or B cells) using CLLs led to a loss of iNKT cell activation. Given that lipid loading on CD1d is thought to require passage through endocytic compartments, these data suggest that SSMs internalized some of the lipid. However, whether the cells actively phagocytose the 200nm microspheres or instead absorb some of the surface displayed liposomes remains to be assessed. Recent evidence has highlighted the importance of CD1d presentation of the endogenous lipid βGlcCer and provides evidence that upregulation of lipid presentation occurs following exposure to innate stimuli such as LPS [81
]. Given the ability of SSMs to bind lymph borne LPS [82
], endogenous lipid presentation may be another pathway of SSM-mediated iNKT cell activation.