The Type I IFN Response to MCMV in B6 Mice Is Biphasic
Infection of B6 mice with MCMV induced a biphasic IFNβ and INFα response in the spleen as detected by quantitative PCR (). The initial splenic IFNβ and INFα mRNA induced by infection reached maximal accumulation by 8 hours postinfection and then declined over the next 16 hr (). At the peak response, the relative induction of mRNA was 104-fold increased for IFNβ and 105-fold for INFα over baseline expression in naive mice. A second, more sustained accumulation of IFNβ and INFα mRNA occurred between 36−72 hr. The IFNβ mRNA levels that accumulated in the initial phase was substantially greater than achieved in the sustained phase (587 mRNA relative units/day versus 17 mRNA units/day) indicating the initial phase accounts for the majority of the IFNβ transcriptional activity at these infection conditions.
Analysis of the IFNαβ Response during MCMV Infection
Expression of MCMV transcripts for immediate early (ie)-1 and 3, and the envelope glycoprotein B (gB, an early/late gene product) were readily detected in the spleen 4−8 hr postinfection (). The levels of ie1 mRNA decreased over the next 16 hr (~100-fold), paralleling the expression profile of INFα and IFNβ mRNA. In contrast, ie3 and gB mRNA remained relatively constant over the first 24 hr (). As the infection progressed, viral gene expression dramatically increased, reaching maximum abundance after two days, corresponding closely to the peak production of infectious virus, which occurs in the spleens of B6 mice 3 to 4 days postinfection (Scalzo et al., 1990
). This biphasic pattern of viral ie3 and gB mRNA expression in the spleen suggests that the initial replication cycle of MCMV was probably completed by ~30−36 hr postinoculation (consistent with the replication cycle in cultured fibroblasts), followed by spread and additional rounds of virus replication over the next 2−4 days. The initial INFαβ response in the spleen was proportional to the pfu in the inoculum of MCMV, as revealed by the identical ratio of INFαβ mRNA to ie1 mRNA at different doses of virus (Figure S1
The liver, also a major site of MCMV replication, showed a similar biphasic INFαβ response, although ie1 mRNA levels remained relatively constant over the first 24 hr and increased dramatically from 2 to 4 days postinfection (). In comparison to the spleen, the accumulation of IFNβ mRNA was ~100-fold lower at the initial times of infection (). Serum levels of INFα also showed a biphasic response with maximum serum levels occurring by 8−12 hr and a second peak at 48 hr concurrent with the first round of virus spread within organs. Serum INFα was not detected during peak virus production over the 72−96 hr period (). BALB/c mice also displayed a biphasic INFαβ mRNA response to MCMV infection (Figure S2
). These data suggest that the bulk of INFαβ transcriptional activity and serum INFαβ response to MCMV infection occur during the initial phase.
The LTαβ-LTβR System Is Required for Initial IFNαβ Response to MCMV Infection in the Spleen
mice exhibit a defect in the induction of IFNβ mRNA in the spleen at 8 hr postinfection (Banks et al., 2005
), it was not clear whether LTβR signaling was needed for both phases of the INFαβ response in B6 mice. Mice deficient in both ligands for LTβR, LTβ, and LIGHT (LTβ/LIGHT−/−
) were used to attenuate physiological signaling while retaining the option to activate LTβR signaling using a specific agonist antibody. LTβ/LIGHT−/−
mice exhibited reduced IFNβ mRNA levels in the spleen at 8 hr postinfection, consistent with results previously observed in LTα−/−
mice (Banks et al., 2005
) (). Notably, a minor decrease in ie1 mRNA expression at this time was also observed in LTβ/LIGHT−/−
mice (~2-fold) (), and an analogous decrease in ie1 mRNA levels was also seen in LTα−/−
mice (Figure S3
). By contrast, hepatic expression of IFNβ and ie1 mRNA in LTβ/LIGHT−/−
mice was comparable to wild-type B6 mice, indicating that LTβR signaling was dispensable in the liver (). Moreover, splenic IFNβ and ie1 mRNA levels measured 48 hr after infection in LTβ/LIGHT−/−
mice were not significantly different from B6 mice (). These results indicate that LTβR-dependent signaling is required for the initial INFαβ response in the spleen. Serum levels of INFα were also dramatically reduced (~50-fold) in the LTβ/LIGHT−/−
-deficient mice 8 hr postinfection () even though INFαβ mRNA levels were not compromised in the liver. This result implicates the spleen as the major source of INFαβ during the initial response to MCMV infection.
LTβ/LIGHT−/− Mice Are Defective in Their Initial IFNαβ Response to MCMV
Administration of an agonistic anti-LTβR antibody to the LTβ/LIGHT−/− mice at the time of MCMV infection partially restored IFNβ mRNA and serum INFα (). These results indicated that the LTαβ/LIGHT-LTβR pathway regulates the initial INFαβ response to MCMV in the spleen, but not in the liver. Furthermore, the ability of an agonist anti-LTβR mAb to partially restore the splenic IFNβ response suggests that signaling through the LTβR is required during the infection to promote an optimal INFαβ response.
The Initial IFNαβ Response to MCMV Is TLR Independent
Toll-like receptor (TLR) sensing of pathogens plays a key role in innate immune defenses. Importantly, mice deficient in both TLR9 and TLR3 signaling display increased susceptibility to MCMV infection with reduced serum levels of INFαβ during the sustained phase (Krug et al., 2004
; Tabeta et al., 2004
mice, which are unresponsive to unmethylated CpG-oligodeoxynucleotides (Tabeta et al., 2004
), showed normal INFαβ mRNA accumulation in the spleen 8 hr post-MCMV infection. Additionally, TLR9Cpg1
had near normal levels of serum INFα () and IFNβ (data not shown) after initial infection. MCMV infection of TRIFLps2/Lps2
-deficient mice, which cannot activate the TLR3 pathway (Hoebe et al., 2003
; Yamamoto et al., 2003
), resulted in a slightly higher induction of IFNβ mRNA in the spleen and in serum INFα 8 hr postinfection (). INFα mRNA levels paralleled serum protein levels for both TLR9Cpg1
- and TRIFLps2/Lps2
-deficient mice (data not shown). To determine whether other TLRs might function redundantly in TLR9Cpg1
mice, mice deficient in both MyD88 and TRIF signaling pathways were infected (). These mice showed no decrease in either IFNβ or INFα mRNA levels, and ie1 levels were also normal 8 hr postinfection. Taken together, these results indicate that the LTβR-dependent, initial INFαβ response to MCMV in the spleen is independent of TLR signaling and suggests a specific LTαβ-expressing cell population promotes the initial INFαβ response.
The Initial Splenic IFNαβ Response to MCMV Infection is TLR Independent
The Adaptive Immune System Promotes the Initial IFNαβ Response to MCMV
The initial INFαβ response to MCMV is rapid, suggesting that either constitutive or quickly activated LTβR signaling may be required for promoting this response. A variety of cell types including activated T cells, B-lymphocytes, and NK cells express LTβ as well as DC and lymphoid tissue inducer (LTi) cells. However, naive B cells and CD4+ T cells in the spleen constitutively express LTβ on their cell surface, as detected by flow cytometry (). To determine the cellular source of LTβ, we analyzed MCMV in RAG−/− (which lack T- and B cells) and RAG/γc−/− mice (which, in addition, lack NK and LTi cells, due to the absence of the common γ chain of the IL-2/4/7/9/15/21 receptors). Additionally, splenic DC subpopulations in RAG−/− mice are normal, while DC in RAG/γc−/− mice are skewed, with less CD4+ DC (C.D.T., unpublished data). However, both RAG−/− and RAG/γc−/− mice showed a substantial reduction in the accumulation of IFNβ mRNA compared to wild-type mice (). Splenic ie1 mRNA levels in both RAG−/− and RAG/γc−/− mice at eight hr postinfection trended lower but were not statistically different from wild-type (). However, normalization of IFNβ to ie1 mRNA revealed a defective initial IFNβ mRNA response (~6-fold) in RAG−/− and RAG/γc−/− mice, very similar in magnitude to LTβ/LIGHT−/− mice. Mice deficient in both RAG and LTβR were severely compromised in their initial INFαβ response (>50-fold), which was commensurate with a substantial reduction in ie1 mRNA levels (~6-fold). However, the IFNβ/ie1 mRNA ratio revealed a comparable, viral “dose-dependent” INFαβ defect to that seen in both RAG−/− and LTβ/LIGHT−/− mice (). Serum INFα levels in RAG−/−, RAG/γc−/−, and RAG/LTβR−/− mice during the initial response to MCMV were undetectable by the ELISA ().
The Initial Splenic IFNαβ Expression during MCMV Infection Is Dependent upon B-Lymphocytes
B Cells Expressing LTβ Are Required for the Initial IFNαβ Response to MCMV
The results observed in RAG−/− mice raised the possibility that naive T and/or B cells provide the stimulus for the initial INFαβ response to MCMV infection, either directly or indirectly. As both T and B lymphocytes lack expression of LTβR, it is likely that lymphocytes deliver the ligand to LTβR-expressing cells. To delineate which of these lymphocyte populations were the responsible subset, mice genetically deficient in either B cells (Igh-6−/−), CD4+ (Cd4−/−), or CD8+ T lymphocytes (β2μ−/−) were infected with MCMV. B cell-deficient mice showed a 4-fold reduction in IFNβ mRNA at 8 hr post-MCMV infection and a drastic reduction in serum INFα (). In contrast, neither CD4 T cell nor CD8 T cell-deficient mice showed a reduction in splenic IFNβ mRNA or serum INFα (). The expression level of ie1 mRNA was similar in all groups tested.
To directly test whether the lack of LTβ expression by B cells was responsible for the compromised initial INFαβ response to MCMV, mice conditionally deficient in ltβ
in B- or (B-LTβ) or T cells (T-LTβ) were utilized (Tumanov et al., 2003
). Strikingly, B-LTβ mice, but not T-LTβ mice, displayed a dramatic reduction in IFNβ (7-fold) and INFα mRNA (12-fold) (). B-LTβ-deficient mice also showed ~3-fold reduction in ie1 mRNA in the spleen that when normalized to ie1 mRNA yielded an ~5-fold decrease in INFαβ mRNA, a result virtually identical to that seen in mice lacking the entire B lymphocyte compartment or deficient in LTβ/LIGHT. Taken together, this result highlights the critical importance of LTβ expressed in B cells to promote the initial INFαβ response to MCMV infection.
LTβ Expressed by B Cells Is Required for the Initial IFNαβ Response to MCMV
LTβR-Expressing Stromal Cells Produce the Initial IFNαβ during MCMV Infection via a NIK-Dependent Pathway
The identification of B-lymphocytes as the critical, LTβ-expressing cells promoting the initial INFαβ response to MCMV in the spleen was surprising given that naive B cells are not infected by MCMV (Banks et al., 2005
) and lack expression of the LTβR (Force et al., 1996
). To determine whether the hematopoietic or radio-resistant stromal compartments require expression of the LTβR, bone marrow chimeric mice were generated between LTβR−/−
or wild-type mice. LTβR−/−
recipients reconstituted with wild-type B6 bone marrow (B6→LTβR) exhibited reduced IFNβ mRNA accumulation (~10-fold) when compared to wild-type controls (B6→B6) at 8 hr postinfection (). This IFNβ deficiency was comparable to that seen in mice defective for LTβR expression in both cellular compartments (LTβR→LTβR). In contrast, wild-type mice that received LTβR-deficient bone marrow (LTβR→B6) produced normal levels of IFNβ mRNA. Thus, stromal cells require expression of LTβR to mount the initial INFαβ response to MCMV ().
LTβR-Expressing Splenic Stromal Cells Promote IFNαβ Production during MCMV Infection via a NIK-Dependent Pathway
Separation of the spleen into stroma and hematopoietic fractions revealed that IFNβ and INFα mRNA were 25- to 30-fold enriched in the stromal cell fraction from mice infected for 8 hr with MCMV (). Moreover, viral ie1 mRNA expression also preferentially localized to the stromal compartment (~15-fold enriched) consistent with previous immunohistochemical (Benedict et al., 2006
) and electron microscopy studies (Mercer et al., 1988
) that stromal cells are the major site of MCMV infection in the spleen. Together, these results indicate that radioresistant stromal cells expressing the LTβR are both the target of initial infection and the major producer of INFαβ in response to MCMV.
LTβR activation of the NF-κB pathway requires the activity of the NF-κB-inducing kinase (NIK) (Dejardin et al., 2002
; Basak et al., 2007
), and LTβR-mediated, canonical NF-κB signaling is also dependent upon NIK (Basak et al., 2007
). To test whether NIK is required for the initial INFαβ response in the splenic stroma, aly/aly
mice (functional mutation in NIK) (Xiao et al., 2001
) were infected with MCMV. Strikingly, aly/aly
mice showed a reduction in both IFNβ and INFα mRNA at 8 hr postinfection (~26- and 104-fold, respectively), with a commensurate reduction in ie1 mRNA (~11-fold).
The type I Interferon response is a key contributor to successful host defense against viral pathogens, such as MCMV (Banks et al., 2005
; Dalod et al., 2002
; Salazar-Mather et al., 2002
). Here, we define the cellular interactions required to initiate the earliest innate INFαβ response to a β-herpesvirus. In response to MCMV, the LTαβ-LTβR pathway is required for the induction of INFαβ in the spleen, which constitutes a major source of initial INFαβ production. Our results reveal that B-lymphocytes provide the source of LTβ required to promote the initial innate INFαβ response in LTβR-expressing stromal cells, and it is cells in the stromal compartment that are both infected and producers of INFαβ during MCMV infection. Induction of initial INFαβ mRNA and protein regulated by the LTβR pathway is independent of TLR-signaling pathways, indicating that a significant difference exists in the molecular mechanisms promoting the initial and sustained phases of the INFαβ response to MCMV infection. Together, the results reveal a frontline innate host defense strategy directed by B cells of the adaptive immune system communicating with stromal cells via the LTαβ-LTβR system.
The biphasic pattern of INFαβ induction in the spleen and liver reflects the initial host response to the primary inoculum, with the subsequent sustained phase resulting from the secondary response to viral spread and continued cycles of replication at later times. The initial INFαβ mRNA response to MCMV peaked at 8 hr, paralleled by a rise in serum INFα, rapidly declined and was followed by sustained expression for the next few days. The accumulated peak of IFNβ mRNA levels indicated the bulk of the INFαβ transcriptional activity occurred during the initial phase of the infection, and the magnitude of the response in the spleen was substantially higher than in the liver. In addition, the level of INFα in the serum, reflecting accumulation from all tissue sources, was dramatically reduced in the LTβ/LIGHT−/−-deficient mice, despite an uncompromised INFαβ mRNA response in the liver. This evidence, together with the relative abundance of INFαβ mRNA in the spleen, supports the idea that the LTβR pathway regulates the major source of type I IFN during MCMV infection.
Our results and those of others indicate that TLR-dependent defense pathways control INFαβ expression in response to MCMV during the later, sustained phase. This conclusion is based on the uncompromised INFαβ response to MCMV during the initial phase in TLR9CpG
, and MyD88/TRIFLps2/Lps2
mice. The complexity of the sustained phase of the INFαβ response is apparent from studies showing TLR, MyD88, and plasmacytoid DC are important at 36 hr post-MCMV infection (Andoniou et al., 2005
; Delale et al., 2005
; Krug et al., 2004
; Tabeta et al., 2004
), but additional results indicate their influence may decline at 44 hr postinfection (Andoniou et al., 2005
; Delale et al., 2005
; Krug et al., 2004
). These data further support the idea that the initial INFαβ response regulated by the LTβR pathway is distinct from the TLR-dependent pathways functioning in plasmacytoid DC. These results indicate the LTαβ-IFN axis is an independent mechanism from conventional paradigms in which myeloid lineage cells largely promote innate defenses to pathogens. Additionally, that LTβ/LIGHT−/−
mice fail to control acute MCMV replication in both the spleen and liver at 72 hr postinfection (Banks et al., 2005
) indicates mice lacking LTβR-signaling are compromised in additional aspects of innate defenses to MCMV infection.
The conditional deletion of ltβ
in B cells, as well as mice lacking B lymphocytes (RAG−/−
), demonstrated B cell expression of LTβ is the key factor for promoting the initial INFαβ response in the spleen. B cell-expressed LTβ is required for maturation of the normal splenic architecture (Tumanov et al., 2002
) although B-LTβ mice develop a normal complement of lymph nodes and Peyer's patches, which depends on expression of LTβ in lymphoid tissue inducer cells. However, the splenic architecture (marginal zone, segregation of T and B cells) in B-LTβ mice is not as severely disrupted as in mice harboring the null genotype, but the IFNβ/ie1 mRNA ratio in the spleens of B-LTβ and LTβ/LIGHT−/−
mice were similar in response to MCMV infection. Additionally, although dendritic cell subpopulations are altered in the spleen LTβR→B6 bone marrow chimeric mice (Kabashima et al., 2005
; De Trez et al., 2008
), the initial INFαβ response to MCMV was normal in these mice. Finally, although LTβ/LIGHT−/−
mice lack both marginal zone and marginal metallophillic macrophage populations, they mount a normal INFαβ response to MCMV at 48 hr, and our studies in LTβR→B6 mice indicate LTβR expression by these macrophages is not required for the initial response. Together, these results indicate that alterations in myeloid cell subsets or the lymphatic vasculature are not likely to account for the reduced initial INFαβ production in LTβ/LIGHT−/−
mice. Moreover, the partial restoration of the IFNβ mRNA response in LTβ/LIGHT−/−
mice treated with an agonist anti-LTβR indicates that LTβR signaling contributes during the virus infection to induce IFNβ. The limited effect of acutely delivered anti-LTβR suggests that differentiative effects on stromal cells may be necessary (Drayton et al., 2004
) to fully restore the INFαβ response. The agonist LTβR antibody may also enhance the regulation of cellular trafficking across the high endothelial venules, which is regulated by LTβR signaling, perhaps increasing the initial dissemination of MCMV to the spleen from the peritoneal cavity (Browning et al., 2005
; Drayton et al., 2003
). Strikingly, the bone marrow chimeric mice indicated that LTβ-expressing B cells exclusively communicate with, or regulate the differentiation of, LTβR expressing stromal cells, which produce the majority (>95%) of the INFαβ and IE1 mRNA following MCMV infection. Along these lines, a role for constitutively expressed LTβ by B cells in regulating the microarchitecture of the spleen was recently shown to play an important role in containing VSV and LCMV infection (Junt et al., 2006
). LTαβ is exclusively positioned in the membrane indicating the conversation between B cells and stromal cells occurs through direct cell contact. The radioresistant stromal compartment includes many distinct cell types that could be infected, including endothelial cells, myocytes, and fibroblasts in addition to specialized parenchymal cells identified by specific chemokine expression, such as CXCL13 (Cyster, 2003
). The exact lineage of stromal cells infected by MCMV in the spleen is currently unknown.
The importance of this B cell-stromal cell interaction is evident in LT deficient mice, which are highly susceptible to infection with MCMV (for review see Ware, 2005
). Increased morbidity occurs in LTα-, LTβ-, or LTβR-deficient mice or wild-type mice treated with LTβR-Fc decoy in response to MCMV (Banks et al., 2005
; Benedict et al., 2001
). Mice deficient in INFαβ receptor phenocopy LT-deficient mice, which undergo massive apoptosis of T and B lymphocytes during infection that are not directly infected by the virus (Banks et al., 2005
). LTβR expression was required in both the hematopoietic and stromal compartments of the spleen to completely protect against this apoptosis induced by MCMV, implicating crosstalk occurs between the myeloid and stromal compartments to induce INFαβ and promote survival of lymphocytes (Banks et al., 2005
). Our results in aly/aly
mice strongly indicate this LT-dependent crosstalk requires NIK, likely involving activation of NF-κB, a known key player in the regulation of IFNβ transcription (Thanos and Maniatis, 1995
). Together, these results indicate that the initial B cell directed INFαβ response contributes to viral control, and ultimately helps to promote the survival of the adaptive immune response.
B lymphocytes play a critical role in the post natal maturation and homeostasis of the splenic microarchitecture, a structural feature designed to promote highly efficient immune responses (Cupedo et al., 2004
). LTαβ-LTβR signaling pathway is required to form the marginal zone, wherein antigen-capturing macrophages, B cells and dendritic cells reside (Kraal, 1992
) and regulate expression of the lymphoid tissue chemokines (CCL21, CXCL13) in specialized stromal cells creating discrete micro-niches that compartmentalize T cells to the periarteriole region surrounded by the B cell follicles (Cyster, 2003
). MCMV targets the splenic microarchitecture by specifically suppressing expression of CCL21 mRNA, evident two to three days after infection (Benedict et al., 2006
). The specific loss of CCL21 leaves T cells without sufficient cues to localize around the arteriole. However, MCMV did not significantly alter the B cell follicle chemokine CXCL13, unlike infection with lymphocytic choriomeningitis virus, which reduces expression of both these splenic chemokines (Mueller et al., 2007
) suggesting the LT-IFN pathway might protect the B cell micro-niche during MCMV infection.
The commitment of genomic information encoding resistance to MCMV (estimated at 1%–3% of the host genome) suggests herpesviruses provide powerful selective pressures for diversification of host resistance mechanisms (Beutler et al., 2005
). Likewise, the accumulation of numerous immune modulating mechanisms in the viral genome, many derived from “captured” cellular genes, indicates a long coevolutionary history. That two distinct human herpesviruses, HSV-1 and HCMV, target receptors (HVEM and BTLA) involved in LT-dependent signaling, underscores this coevolutionary relationship (Cheung et al., 2005
). Indeed, resistance to other pathogens is provided to the host as a consequence of infection with either γ- and β-herpesviruses, suggesting a strong symbiotic relationship has emerged from the host-virus interplay (Barton et al., 2007
). In the context of evolution, the LTαβ-LTβR pathway, functioning as a developmental and homeostatic regulator, may have evolved as an adaptive countermeasure to promote an early IFN response in the cells initially infected by the virus, perhaps in response to the functional paralysis of macrophages and DC by MCMV (Andrews et al., 2001
; Popkin et al., 2003
). The discovery of this B cell-dependent pathway also suggests an unrecognized route to INFαβ production that may operate in autoimmune diseases, such as lupus erythematosus, where both infection and genetic factors contribute to pathogenesis (Baccala et al., 2007