Tumor Necrosis Factor-Mediated Survival of CD169+ Cells Promotes Immune Activation during Vesicular Stomatitis Virus Infection

ABSTRACT Innate immune activation is essential to mount an effective antiviral response and to prime adaptive immunity. Although a crucial role of CD169+ cells during vesicular stomatitis virus (VSV) infections is increasingly recognized, factors regulating CD169+ cells during viral infections remain unclear. Here, we show that tumor necrosis factor is produced by CD11b+ Ly6C+ Ly6G+ cells following infection with VSV. The absence of TNF or TNF receptor 1 (TNFR1) resulted in reduced numbers of CD169+ cells and in reduced type I interferon (IFN-I) production during VSV infection, with a severe disease outcome. Specifically, TNF triggered RelA translocation into the nuclei of CD169+ cells; this translocation was inhibited when the paracaspase MALT-1 was absent. Consequently, MALT1 deficiency resulted in reduced VSV replication, defective innate immune activation, and development of severe disease. These findings indicate that TNF mediates the maintenance of CD169+ cells and innate and adaptive immune activation during VSV infection. IMPORTANCE Over the last decade, strategically placed CD169+ metallophilic macrophages in the marginal zone of the murine spleen and lymph nodes (LN) have been shown to play a very important role in host defense against viral pathogens. CD169+ macrophages have been shown to activate innate and adaptive immunity via “enforced virus replication,” a controlled amplification of virus particles. However, the factors regulating the CD169+ macrophages remain to be studied. In this paper, we show that after vesicular stomatitis virus infection, phagocytes produce tumor necrosis factor (TNF), which signals via TNFR1, and promote enforced virus replication in CD169+ macrophages. Consequently, lack of TNF or TNFR1 resulted in defective immune activation and VSV clearance.

factors regulating CD169 ϩ cells during viral infections remain unclear. Here, we show that tumor necrosis factor is produced by CD11b ϩ Ly6C ϩ Ly6G ϩ cells following infection with VSV. The absence of TNF or TNF receptor 1 (TNFR1) resulted in reduced numbers of CD169 ϩ cells and in reduced type I interferon (IFN-I) production during VSV infection, with a severe disease outcome. Specifically, TNF triggered RelA translocation into the nuclei of CD169 ϩ cells; this translocation was inhibited when the paracaspase MALT-1 was absent. Consequently, MALT1 deficiency resulted in reduced VSV replication, defective innate immune activation, and development of severe disease. These findings indicate that TNF mediates the maintenance of CD169 ϩ cells and innate and adaptive immune activation during VSV infection. IMPORTANCE Over the last decade, strategically placed CD169 ϩ metallophilic macrophages in the marginal zone of the murine spleen and lymph nodes (LN) have been shown to play a very important role in host defense against viral pathogens. CD169 ϩ macrophages have been shown to activate innate and adaptive immunity via "enforced virus replication," a controlled amplification of virus particles. However, the factors regulating the CD169 ϩ macrophages remain to be studied. In this paper, we show that after vesicular stomatitis virus infection, phagocytes produce tumor necrosis factor (TNF), which signals via TNFR1, and promote enforced virus replication in CD169 ϩ macrophages. Consequently, lack of TNF or TNFR1 resulted in defective immune activation and VSV clearance. KEYWORDS TNF, MALT1, innate immunity, interferon, NF-B, innate immunity, interferons, tumor necrosis factor I nnate immune activation is crucial for inducing antiviral immunity through cytokine production and adaptive immune priming (1). Splenic marginal-zone macrophages and metallophilic marginal-zone macrophages play an important role in eliminating blood-borne bacteria, parasites, and viral pathogens (2,3). Metallophilic macrophages were originally described when rat splenic marginal-zone macrophages were stained by iron and silver impregnation (4). These metallophilic macrophages express the lectin-like hemagglutinin CD169, which was identified using a monoclonal antibody, MOMA-1 (5)(6)(7). CD169 ϩ macrophages are increasingly recognized to play a pivotal role in host defense (8). CD169 ϩ macrophages (referred to here as CD169 ϩ cells), specifically allow early viral replication to promote innate immune recognition and antigen presentation (9). The absence of CD169 ϩ cells results in reduced type I interferon (IFN-I) production, reduced B-cell activation, and development of severe disease during viral infection (10,11). B-cell-derived lymphotoxin alpha (Lt␣) and lymphotoxin beta (Lt␤) drive the maintenance of CD169 ϩ cells in spleen and lymph node tissue (10,12,13). Consequently, B-cell-deficient mice exhibit fewer CD169 ϩ cells and limited immune activation, including the production of IFN-I (13,14). However, the factors promoting survival and the presence of CD169 ϩ cells after viral infection have not yet been sufficiently studied.
IFN-I triggers strong inhibitory effects on viral replication and is crucial for preventing severe infections with the vesicular stomatitis virus (VSV) model system (1,15). This system can be used as a laboratory system for immune recognition during viral infection, as a vaccine vector system, as a tool for viral transduction, and as an oncolytic virus (16,17). Clearance of VSV depends heavily on IFN-I and the presence of neutralizing antibodies (15,18). VSV has been used as a murine model of viral infections to study the innate immune response and virus replication in secondary lymphoid organs and the central nervous system (CNS) (19)(20)(21). Pathology in VSV infection is seen particularly during infection of the CNS; this pathology includes paralysis and death after infection with VSV (22). Accordingly, mice deficient in IFN-␣/␤ receptor (IFNAR) signaling exhibit paralysis and the presence of VSV in the CNS (15). Consistently, IFN-I can inhibit VSV replication in neurons, and defects in IFN-stimulated genes (ISGs) in the CNS tissue trigger pathology during VSV infection (23,24). During infection with low doses of VSV, replication of VSV in CD169 ϩ cells in the spleen and lymph node tissue is important for inducing protective immunity and preventing CNS infection (9,10). The VSV backbone is also used during vaccination to induce protective immunity against viruses such as Ebola virus (25).
The role of tumor necrosis factor (TNF) in marginal-zone development and marginalzone function is controversial. Although reports show that marginal-zone development is impaired and fewer marginal-zone macrophages are present in TNF-deficient and p55-TNFR (tumor necrosis factor receptor 1 [TNFR1])-deficient mice (26), other reports suggest that TNF triggers marginal-zone macrophage depletion after infection (27,28). It has also been shown that TNFR1-deficient mice are less susceptible to West Nile virus infection as a result of an uncompromised blood-brain barrier (29). However, other studies utilizing herpes simplex virus 1 as an infection model showed that TNFR1deficient mice are more susceptible to virus infection (30,31). It is clear that TNFdeficient mice exhibit CD169 ϩ cells in the spleen, whereas this cell population is absent in Lt␣ Ϫ/Ϫ mice (26,27). Furthermore, the production of neutralizing antibodies and the proliferation of antiviral T cells can be induced in TNF-deficient animals (28,32). These findings suggest that TNF, which is crucial for overcoming bacterial infections (33)(34)(35)(36), plays a minor role in antiviral immunity.
In this study, we found that absence of TNF reduced the number of CD169 ϩ cells; inhibited IFN-I production; and, consequently, led to a severe disease outcome during infection with VSV. These effects were mainly transmitted by TNFR1 and were dependent on canonical nuclear factor B (NF-B).

RESULTS
TNF production by CD11b ؉ Ly6C ؉ Ly6G ؉ cells following VSV infection. TNF can be detected during an infection with VSV (32,37). Consistently, we found that TNF expression levels were higher in the spleen after infection with VSV than in uninfected controls (Fig. 1A). Backgating of intracellular-TNF-producing cells showed that TNFproducing cells are a heterogeneous CD11b ϩ CD19 Ϫ population ( Fig. 1B and C). Therefore, we hypothesized that TNF was likely not expressed by B or T cells during infection. Accordingly, we observed TNF mRNA expression levels in Cd8 Ϫ/Ϫ , B-celldeficient Jh Ϫ/Ϫ , and Rag1 Ϫ/Ϫ mice that were comparable to those in wild-type (WT) mice (Fig. 1D). TNF-producing cells could be predominantly characterized as CD11b ϩ CD11c Ϫ Ly6C ϩ Ly6G ϩ major histocompatibility complex class II negative (MHC-II Ϫ ) (Fig.  1E). Consistent with reports that neutrophils (38,39) and CD11b ϩ Ly6C ϩ Ly6G ϩ cells (40) are important during early defense against bacterial and viral infections via production of proinflammatory cytokines, such as interleukin 1b (IL-1b), IL-6, TNF, and IFN-I, we found a significant increase of TNF ϩ CD11b ϩ Ly6C ϩ Ly6G ϩ cells (Fig. 1F). Treatment with clodronate encapsulated in liposomes (clodronate liposomes) can deplete phagocytic cells in mice (Fig. 1G) (41,42). Accordingly, clodronate depletion reduced TNF expression after VSV infection, suggesting a role of these phagocytic cells in the production of TNF (Fig. 1H). However, when we employed diphtheria toxin receptor (DTR)-induced specific depletion of CD169 ϩ cells and CD11c ϩ cells, we did not observe a reduction in TNF production (Fig. 1H). Taken together, these findings indicate that TNF production following intravenous VSV infection is triggered by CD11b ϩ CD11c Ϫ Ly6C ϩ Ly6G ϩ phagocytes.
TNF triggers the maintenance of CD169 ؉ cells during viral infection to protect animals against the development of severe disease. To determine whether TNF affects the outcome after VSV infection, we infected WT and TNF-deficient mice. TNF-deficient mice developed severe VSV infection in comparison to WT mice ( Fig. 2A). A neutralizing antibody titer was achieved later in TNF-deficient mice than in WT mice after infection with low doses of VSV (Fig. 2B). Since IFN-I is critical to overcome an infection with VSV (15), we measured IFN-␣ and IFN-␤ in the sera of infected animals. IFN-␣ production was impaired in TNF-deficient mice compared to control animals (Fig.  2C). However, IFN-␤ was undetectable in the sera of animals infected with 10 5 PFU VSV (Fig. 2C). Previous findings showed that CD169 ϩ cells contribute to innate immune activation in mice, not only by allowing viral replication, but also by producing IFN-I (10,43). When we depleted CD169 ϩ cells expressing diphtheria toxin receptor (CD169-DTR cells) by administering diphtheria toxin (DT) (44), we observed reduced IFN-I concentrations in the sera of infected animals (Fig. 2D). To exclude the possibility of defective innate Toll-like receptor (TLR) activation, we administered the TLR3 agonist poly(I·C). We found that the IFN-I production was intact in both WT and TNF-deficient mice (Fig. 2E). Hence, we speculated that TNF promoted the function of CD169 ϩ cells and thus contributed to IFN-I production following VSV infection. Shortly after infection with VSV, the number of CD169 ϩ cells in spleen tissue decreased in TNF-deficient mice compared to spleen tissue harvested from WT animals ( Fig. 2F to H). To understand the reduced production of IFN-I in the absence of TNF, we monitored the virus replication in spleen tissue of WT and Tnfa Ϫ/Ϫ mice. The expression of VSV glycoprotein (VSV-G) was detected in smaller quantities in spleen tissue harvested from TNF-deficient animals than in spleen tissue harvested from WT mice after VSV infection ( Fig. 2I and J). Consistently, early VSV titers after infection were lower in Tnfa Ϫ/Ϫ mice than in control mice, a condition that negatively affected antiviral immune activation (Fig. 2K). Injection of UV light (UV)-inactivated virus could increase TNF mRNA expression in WT mice (Fig. 2L). However, the decrease of CD169 ϩ cells was dependent on live virus, because UV-inactivated virus did not affect CD169 ϩ cells in spleen tissue of Tnfa Ϫ/Ϫ mice (Fig. 2M). These findings indicate that TNF is necessary to sustain virus replication in the early hours of infection but is dispensable for sterile innate immune activation. Notably, CD169 Ϫ/Ϫ mice exhibited VSV-G expression in spleen tissue, a finding indicating that downregulation of the protein CD169 would not cause absence of virus replication (Fig. 2N). Taken together, these findings indicate that the absence of TNF results in defective antiviral innate immune activation after infection with VSV.
CD169 ؉ cell maintenance via TNFR1 results in productive VSV replication and immune activation. To further characterize the role of TNF during viral infection, we infected TNFR1-and TNFR2-deficient mice with VSV. In line with findings from TNFdeficient animals, the absence of TNFR1, but not that of TNFR2, resulted in a decrease in the number of CD169 ϩ cells in spleen tissue ( Fig. 3A and B). Furthermore, VSV-G production was lower in Tnfrsf1a Ϫ/Ϫ animals than in WT or Tnfrsf1b Ϫ/Ϫ mice (Fig. 3A). Consistently, VSV titers were reduced in spleen tissue shortly after infection in Tnfrsf1a Ϫ/Ϫ animals, in sharp contrast to the findings in WT and Tnfrsf1b Ϫ/Ϫ mice (Fig.  3C). Interestingly, IFN-I production was defective in Tnfrsf1a Ϫ/Ϫ mice but was also lower in Tnfrsf1b Ϫ/Ϫ animals than in WT control mice (Fig. 3D). IFN-I is necessary for the expression of antivirally active ISGs (1). Consistently, we found reduced expression of ISGs in the CNS of Tnfrsf1a Ϫ/Ϫ mice after infection with VSV (Fig. 3E). Defective ISG expression was not found to the same extent in Tnfrsf1b Ϫ/Ϫ CNS tissue (Fig. 3F). VSV can drive neuropathological symptoms by infecting the CNS (22). When we measured viral titers in the spinal cord and brain tissue of mice exhibiting hind leg paralysis, we found infectious VSV in tissue from TNFR1-deficient mice (Fig. 3G). Consequently, Tnfrsf1a Ϫ/Ϫ mice developed clinical signs of CNS infection, unlike WT and Tnfrsf1b Ϫ/Ϫ mice (Fig. 3H). Taken together, these findings suggest that TNFR1 drives antiviral defense by promoting CD169 ϩ cell survival.
TNFR1 triggers the survival of CD169 ؉ cells. Next, we opted to determine which factors drive the maintenance of CD169 ϩ cells and enforced viral replication after viral infection. B-cell-mediated Lt␤ production is important for splenic CD169 ϩ cells. Hence, we wondered whether the defects in the absence of TNF were triggered by B cells.  Notably, we did not observe any major changes of B-cell subsets in TNF-, TNFR1-, or TNFR2-deficient mice (Fig. 4A). Consistently, we did not see differential expression of Lt␣, Lt␤, or Lt␤ receptor (LtbR) in TNFR1-deficient mice (Fig. 4B). Additionally, we found no major differences in B-cell subsets between WT and Tnfrsf1a Ϫ/Ϫ mice after infection (Fig. 4C). Furthermore, we reconstituted lethally irradiated C57BL/6 mice with mixed bone marrow (BM) from Rag1 Ϫ/Ϫ and Tnfrsf1a Ϫ/Ϫ and from Rag1 Ϫ/Ϫ and WT donors at a ratio of 1:1. Mice reconstituted with Rag1 Ϫ/Ϫ -Tnfrsf1a Ϫ/Ϫ bone marrow exhibited no significant reduction in IFN-␣ in the serum compared to mice reconstituted with Rag1 Ϫ/Ϫ -WT bone marrow (Fig. 4D). Furthermore, there was no difference between these mice in neutralizing antibody production (Fig. 4E). To elucidate if TNFR1 deficiency specifically on CD169 ϩ cells has a role in virus replication, we reconstituted lethally irradiated C57BL/6 mice with mixed bone marrow from CD169-DTR ϩ and Tnfrsf1a Ϫ/Ϫ donors, as well as CD169-DTR ϩ and WT donors, at a ratio of 1:1. We observed that the production of IFN-␣ was lower in the mice reconstituted with CD169-DTR ϩ and Tnfrsf1a Ϫ/Ϫ bone marrow compared to control mice reconstituted with CD169-DTR ϩ and WT bone marrow after infection with VSV and DT treatment (Fig.  4F). Furthermore, we found slightly delayed presence of VSV neutralizing antibody titers in CD169-DTR ϩ -Tnfrsf1a Ϫ/Ϫ recipients compared to corresponding CD169-DTR ϩ -WT recipients (Fig. 4G). CD169 ϩ cells can be depleted in CD11c-DTR mice, because CD169 ϩ cells exhibit intermediate expression of CD11c (10,45). Consistently, lethally irradiated mice reconstituted with mixed bone marrow from CD11c-DTR ϩ and Tnfrsf1a Ϫ/Ϫ mice exhibited reduced concentrations of IFN-␣ after VSV infection compared to CD11c-DTR ϩ -WT bone marrow recipients (Fig. 4H). These findings suggest that TNFR1 triggers cell-intrinsic effects on CD169 ϩ cells.
We speculated that TNF delivers an important survival signal for CD169 ϩ cells. To determine if TNF is involved in protection against VSV-induced apoptosis, we measured caspase 3 activity on whole spleen tissue lysates. After VSV infection, caspase 3 activity was significantly higher in Tnfa Ϫ/Ϫ mice than in control animals (Fig. 5A). VSV is known to induce apoptosis and inactivates Mcl-1 and Bcl-Xl (46). To elucidate if TNF plays a role in promoting expression of antiapoptotic genes, we measured mRNA expression of Bcl2, Bcl-Xl, and xIAP in spleen tissue of mice after VSV infection (Fig. 5B). After VSV infection, Bcl2 and Bcl-Xl expression was significantly reduced in Tnfa Ϫ/Ϫ mice compared to WT mice (Fig.  5B). To enumerate the mechanism that reduces CD169 ϩ cells in TNF-deficient mice after infection, we made use of terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assays. The number, as well as the mean fluorescence intensity, of TUNEL-positive CD169 ϩ cells was higher in spleen tissue from TNF-deficient mice than in tissue from corresponding WT control mice ( Fig. 5C and D). The proportion of CD169 ϩ cells that stained positive for 7-aminoactinomycin D (7-AAD) was higher in TNFR1-deficient mice than in WT control mice 8 h after infection (Fig. 5E). Next, we wondered if we could rescue the CD169 ϩ cells by injecting the pancaspase inhibitor Z-Val-Ala-Asp-fluoromethylketone (Z-VAD). Z-VAD treatment restored the presence of CD169 ϩ cells in TNFdeficient animals, a finding indicating that CD169 ϩ cells depend on TNF-mediated survival ( Fig. 5F and G). Although treatment of TNF-deficient mice with Z-VAD-rescued CD169 ϩ cells, it failed to rescue the IFN-I response, suggesting the role of TNF signaling is essential not only to prevent apoptosis, but also for IFN-I production (Fig. 5H). In summary, these findings indicate that TNF delivers a survival signal that is important for the maintenance of CD169 ϩ cells in the spleen after viral infection and for IFN-I production.  The NF-B regulator MALT1 promotes canonical NF-B expression, VSV replication in CD169 ؉ cells, and immune activation during viral infection. TNF can induce NF-B activation via TNFR1 and can promote the expression of genes driving survival and of proinflammatory cytokines (47). Furthermore, TNF is known to promote IFN-I production (48). Consistently, RelA expression was increased in the marginal zone of spleen tissue after VSV infection (Fig. 6A). We quantified cytoplasmic and nuclear expression of RelA in CD169 ϩ cells. The nuclear presence of RelA in CD169 ϩ cells was higher in VSV-infected mice than in naive controls (Fig. 6B). We wondered whether nuclear RelA protein expression was dependent on TNF. As expected, compared with WT control mice, VSV-infected mice exhibited reduced expression of RelA in the nuclear compartments of CD169 ϩ cells in the absence of TNF (Fig. 6C). Notably, the presence of RelA was reduced in TNFR1-deficient mice, but we observed no difference in RelA expression between TNFR2-deficient mice and corresponding control mice ( Fig. 6D and E). It has been reported that one of the major regulators of RelA signaling is RelB, which acts through sequestration of RelA in the cytoplasm and competitive binding of DNA (49). It has also been reported that the paracaspase MALT1 can promote canonical NF-B signaling by cleaving RelB (50,51). Hence, we stained spleen sections of Malt1 ϩ/Ϫ and Malt1 Ϫ/Ϫ mice for RelB. Ablation of MALT1 resulted in increased levels of RelB in CD169 ϩ cells in the marginal zone of the spleen (Fig. 7A and B). In turn, nuclear RelA levels were lower in CD169 ϩ cells in Malt1 Ϫ/Ϫ spleen tissue than in control tissue (Fig. 7C). Consistently, mouse embryonic fibroblasts (MEFs) derived from Malt1 Ϫ/Ϫ mice showed reduced translocation of p65 into the nucleus after stimulation with TNF but higher expression of RelB in the nucleus (Fig. 7D and E). These findings indicate that MALT1 destabilizes RelB in marginal-zone macrophages to promote canonical NF-B signaling. The presence of CD169 ϩ cells in spleen tissue was not affected by Malt1 before or after infection with VSV (Fig. 8A). However, the expression of VSV-G was lower in Malt1 Ϫ/Ϫ mice than in control mice ( Fig. 8B and C). Consistently, the numbers of infectious VSV particles were lower in spleen tissue harvested from Malt1 Ϫ/Ϫ mice than in spleen tissue from control mice (Fig. 8D). Hence, serum IFN-I concentrations after VSV infection were lower in MALT1-deficient mice than in control mice (Fig. 8E). A previous report suggested that MALT1 is not required for RIG-I activation (52). Consistently, when we injected poly(I·C) into Malt1 ϩ/Ϫ and Malt1 Ϫ/Ϫ mice, we found similar serum IFN-I levels in both groups (Fig. 8F). Hence, we concluded that defective IFN-I production during VSV infection was caused by reduced VSV replication early during infection. Consequently, MALT1-deficient mice succumbed to the infection, in sharp contrast to control animals (Fig. 8G).
Taken together, these findings indicate that absence of MALT1 results in reduced canonical NF-B signaling in response to VSV infection. Malt1-deficient mice exhibit reduced VSV replication and immune activation.

DISCUSSION
In this study, we found that TNF plays a crucial role in the maintenance of CD169 ϩ cells early after infection with VSV. Consequently, TNF-, TNFR1-, and MALT1-deficient animals exhibited reduced immune activation and limited IFN-I production, which consequently led to severe VSV infection.
The role of TNF during viral infection is controversial and not sufficiently under-    stood. Although reports describe activating polymorphisms in TNF, which are associated with the establishment of a chronic viral infection (53), other reports state that the same mutations are protective against chronic hepatitis B virus (HBV) infection (54). In vitro, TNF can propagate the replication of hepatitis C virus (HCV) (55), although HCV increases the incidence of TNF-induced apoptosis (56). On the other hand, TNF strongly inhibits influenza virus replication in porcine lung epithelial cells (57). Consistently, the attenuation of TNF signaling in a murine T-cell-independent model of HBV infection results in viral persistence (58). In turn, the application of second mitochondrionderived activator of caspases (SMAC) mimetics enhances TNF signaling and is associated with increased clearance of HBV in this model system (59). During infection with VSV, the production of neutralizing antibodies is not defective in the absence of TNFR1 (32). Moreover, TNF can induce T-cell dysfunction and therefore promote chronic viral infection (60). Our finding that TNF is crucial for the maintenance of CD169 ϩ cells in spleen tissue may be important for infections with lower doses of virus, because allowing viral replication in CD169 ϩ cells is particularly important for protective adaptive immunity (9,13). This may be crucial for the maintenance of CD169 ϩ cells in spleen tissue during vaccination with attenuated virus strains or VSV vector-based vaccines (25). These findings may not be specific only for splenic CD169 ϩ cells, since intranasal infection with recombinant TNF overexpressing rabies virus (RV) reduced the RV load and mortality (61). Viral replication in CD169 ϩ cells, which is promoted by TNF, contributes to improved antigen presentation. CD169 ϩ cells in the marginal zone are in close contact with pathogens and are ideally situated to induce an immune response (62). Furthermore, CD169 ϩ cells have been shown not only to present antigens to B cells in the lymph nodes, but also to prime T cells (11,63). Moreover, CD169 ϩ cells are important for virus-mediated IFN-I production, which prevents severe CNS infection in mice (64). Our findings show that TNF promotes maintenance of CD169 ϩ cells and IFN-I production following VSV infection. Furthermore, our findings show that the translocation of RelA to the nuclei of CD169 ϩ cells after VSV infection is dependent on TNF. It has been postulated that canonical NF-B can contribute to the production of IFN-␣ (65,66). However, RelA-deficient and p50-deficient MEFs can produce IFN-␣ after viral infection, whereas only early IFN-I transcription is reduced (67,68). Furthermore, RelA-deficient plasmacytoid dendritic cells (pDCs) exhibited reduced IFN production after exposure to Sendai virus (69). Our findings indicate that canonical NF-B activation can also promote early viral replication and consequently contribute to the production of IFN-I. Consistently, noncanonical NF-B, which can inhibit canonical NF-B signaling, is a potent inhibitor of IFN-I production (70). Hence, the paracaspase MALT1, which can cleave RelB and consequently promote canonical NF-B signaling (50,51), is necessary for the sufficient propagation of VSV replication and IFN-I production.
Taken together, we have found that TNF-TNFR1 signaling is crucial for protecting CD169 ϩ cells and their function in innate immune activation during VSV infection.

MATERIALS AND METHODS
Mice, viruses, and virus titration. Tnfa Ϫ/Ϫ , Tnfrsf1b Ϫ/Ϫ , CD8 Ϫ/Ϫ , and Rag1 Ϫ/Ϫ mice were purchased from Jackson Laboratories (United States). Tnfrsf1a Ϫ/Ϫ mice have been previously described (34). Malt1 Ϫ/Ϫ , CD169 Ϫ/Ϫ , CD169-DTR, and CD11c-DTR mice have also been previously described (71)(72)(73)(74). All the mice were maintained on a C57BL/6 genetic background. VSV Indiana serotype (Mudd-Summers strain) was originally obtained from D. Kolakofsky (University of Geneva, Geneva, Switzerland). VSV was propagated and titrated as previously described (13). Mice were infected with VSV via tail vein injection. In survival experiments, mice exhibiting symptoms of hind leg paralysis were considered "severe," taken out of the experiment, and counted as dead. Blood was collected at the indicated time points after infection. VSV neutralizing antibody titers were measured by plaque reduction neutralization test (PRNT) as previously described (9,13). Briefly, serum samples were diluted 1:40 and incubated at 56°C for 30 min. To evaluate immunoglobulin G (IgG), the serum was pretreated with 0.1 M ␤-mercaptoethanol. Serial 2-fold dilutions were performed for 12 steps, and the serum was incubated with 5,000 PFU of VSV. The virus and serum mixture was incubated on a Vero cell monolayer. The plates were stained with crystal violet after 24 h. To inhibit caspase activity in vivo, we administered three doses (2 g/g of body weight each) of Z-VAD (Abcam, Cambridge, United Kingdom) (75,76). For chimera experiments, mice were lethally irradiated with 10.2 Gy. After 24 h, mixed bone marrow from WT or Tnfrsf1a Ϫ/Ϫ and CD169-DTR, CD11c-DTR, and Rag1 Ϫ/Ϫ mice was transplanted into the irradiated mice as indicated. All mice were maintained under specific-pathogen-free conditions under the authorization of the Landesamt für Natur, Umwelt und Verbraucherschutz of North Rhine-Westphalia (LANUV NRW) in accordance with the German laws for animal protection.
Depletion of cells. To deplete macrophages, 200 l clodronate liposomes was injected intravenously, and 24 h later, the mice were infected with VSV. Liposomes containing phosphate-buffered saline (PBS; PBS liposomes) were used as a control. Clodronate and PBS liposomes were provided by Nico van Rooijen (Vrije University Medical Center, Netherlands) and used as previously described (41,42). CD169and CD11c-expressing cells in CD169-DTR and CD11c-DTR mice were depleted by injecting 2 doses of 100 ng DT (Sigma) 1 day before and on the day of infection.
Histology and ELISA. Histological analysis of snap-frozen tissue was performed as previously described (9). Briefly, snap-frozen tissue sections were cut at 7-m thickness, air dried, and fixed with acetone for 10 min. The sections were blocked with 2% fetal calf serum in phosphate-buffered saline (PBS) for 1 h and stained with anti-CD169 (final concentration, 4 g/ml; Acris, Germany; clone MOMA-1), anti-VSV-G (final concentration, 1 g/ml; produced in house; clone Vi10), anti-RelA (final concentration, 1 g/ml; Santa Cruz Biotechnology, USA), anti-F4/80 (final concentration, 2 g/ml; eBioscience; clone BM8), and anti-RelB (final concentration, 1 g/ml; Cell Signaling, USA; polyclonal) for 1 h. Then, the sections were washed with PBS containing 0.05% Tween 20 (Sigma). The secondary antibodies, phycoerythrin (PE) streptavidin (final concentration, 1 g/ml; eBioscience), anti-rabbit fluorescein isothiocyanate (FITC) (final concentration, 1 g/ml; Thermo Fisher), and anti-goat FITC (final concentration, 1 g/ml; Santa Cruz Biotechnology, USA), were incubated for 1 h. Then, the sections were washed with PBS containing 0.05% Tween 20 (Sigma) and mounted using fluorescence mounting medium (Dako). A caspase 3 activity assay was performed with a fluorescence assay (Cell Signaling) according to the manufacturer's instructions. TUNEL staining was performed on formalin-fixed spleen sections according to the manufacturer's instructions (Thermo Scientific, USA). Images were obtained with an LSM510 confocal microscope and an Axio Observer Z1 fluorescence microscope (Zeiss, Germany). Analysis of the fluorescence images was performed with ImageJ software. IFN-␣ and IFN-␤ (PBL Biosciences, New Jersey, USA) concentrations were determined using an enzyme-linked immunosorbent assay (ELISA) according to the manufacturers' instructions.
Reverse transcription (RT)-PCR analyses. RNA purification (Qiagen RNeasy kit or TRIzol) was performed according to the manufacturer's instructions. Gene expression of Bcl2, Bcl-xl, Xiap, Lta, Ltb, Ifit1, Ifit2, Ifit3, Irf7, Isg15, Oasl1, and Tnfa was performed using 6-carboxyfluorescein (FAM) and VIC probes (Applied Biosystems) and an iTAQ one-step PCR kit (Bio-Rad). For analysis, the expression levels of all the target genes were normalized to ␤-actin/GAPDH (glyceraldehyde-3-phosphate dehydrogenase) expression (ΔC T ). Gene expression values were then calculated based on the ΔΔC T method, using naive WT mice as a control to which all other samples were compared. Relative quantities (RQ) were determined using the following equation: RQ ϭ 2 ϪΔΔCT .
Statistical analyses. Data are represented with standard errors of the mean (SEM). Statistically significant differences between two groups were determined with Student's t test. Statistically significant differences between several groups were determined by one-way analysis of variance (ANOVA) with additional Bonferroni or Dunnett post hoc tests. Statistically significant differences between groups in experiments involving more than one time point were determined with two-way ANOVA (repeated measurements).