Clara Lawler

Lymph Node Macrophages Restrict Murine Cytomegalovirus Dissemination

ABSTRACT Cytomegaloviruses (CMVs) establish chronic infections that spread from a primary entry site to secondary vascular sites, such as the spleen, and then to tertiary shedding sites, such as the salivary glands. Human CMV (HCMV) is difficult to analyze, because its spread precedes clinical presentation. Murine CMV (MCMV) offers a tractable model. It is hypothesized to spread from peripheral sites via vascular endothelial cells and associated monocytes. However, viral luciferase imaging showed footpad-inoculated MCMV first reaching the popliteal lymph nodes (PLN). PLN colonization was rapid and further spread was slow, implying that LN infection can be a significant bottleneck. Most acutely infected PLN cells were CD169+ subcapsular sinus macrophages (SSM). Replication-deficient MCMV also reached them, indicating direct infection. Many SSM expressed viral reporter genes, but few expressed lytic genes. SSM expressed CD11c, and MCMV with a cre-sensitive fluorochrome switch showed switched infected cells in PLN of CD11c-cre mice but yielded little switched virus. SSM depletion with liposomal clodronate or via a CD169-diphtheria toxin receptor transgene shifted infection to ER-TR7+ stromal cells, increased virus production, and accelerated its spread to the spleen. Therefore, MCMV disseminated via LN, and SSM slowed this spread by shielding permissive fibroblasts and poorly supporting viral lytic replication. IMPORTANCE HCMV chronically infects most people, and it can cause congenital disability and harm the immunocompromised. A major goal of vaccination is to prevent systemic infection. How this is established is unclear. Restriction to humans makes HCMV difficult to analyze. We show that peripheral MCMV infection spreads via lymph nodes. Here, MCMV infected filtering macrophages, which supported virus replication poorly. When these macrophages were depleted, MCMV infected susceptible fibroblasts and spread faster. The capacity of filtering macrophages to limit MCMV spread argued that their infection is an important bottleneck in host colonization and might be a good vaccine target.

Rhadinovirus Host Entry by Co-operative Infection

Rhadinoviruses establish chronic infections of clinical and economic importance. Several show respiratory transmission and cause lung pathologies. We used Murid Herpesvirus-4 (MuHV-4) to understand how rhadinovirus lung infection might work. A primary epithelial or B cell infection often is assumed. MuHV-4 targeted instead alveolar macrophages, and their depletion reduced markedly host entry. While host entry was efficient, alveolar macrophages lacked heparan - an important rhadinovirus binding target - and were infected poorly ex vivo. In situ analysis revealed that virions bound initially not to macrophages but to heparan+ type 1 alveolar epithelial cells (AECs). Although epithelial cell lines endocytose MuHV-4 readily in vitro, AECs did not. Rather bound virions were acquired by macrophages; epithelial infection occurred only later. Thus, host entry was co-operative - virion binding to epithelial cells licensed macrophage infection, and this in turn licensed AEC infection. An antibody block of epithelial cell binding failed to block host entry: opsonization provided merely another route to macrophages. By contrast an antibody block of membrane fusion was effective. Therefore co-operative infection extended viral tropism beyond the normal paradigm of a target cell infected readily in vitro; and macrophage involvement in host entry required neutralization to act down-stream of cell binding.

Subcapsular sinus macrophages limit acute gammaherpesvirus dissemination

Lymphocyte proliferation, mobility and longevity make them prime targets for virus infection. Myeloid cells that process and present environmental antigens to lymphocytes are consequently an important line of defence. Subcapsular sinus macrophages (SSMs) filter the afferent lymph and communicate with B-cells. How they interact with B-cell-tropic viruses is unknown. We analysed their encounter with murid herpesvirus-4 (MuHV-4), an experimentally accessible gammaherpesvirus related to Kaposis sarcoma-associated herpesvirus. MuHV-4 disseminated via lymph nodes, and intranasally or subcutaneously inoculated virions readily infected SSMs. However, this infection was poorly productive. SSM depletion with clodronate-loaded liposomes or with diphtheria toxin in CD169–diphtheria toxin receptor transgenic mice increased B-cell infection and hastened virus spread to the spleen. Dendritic cells provided the main route to B-cells, and SSMs slowed host colonization, apparently by absorbing virions non-productively from the afferent lymph.

Alveolar Macrophages Are a Prominent but Nonessential Target for Murine Cytomegalovirus Infecting the Lungs.

ABSTRACT Cytomegaloviruses (CMVs) infect the lungs and cause pathological damage there in immunocompromised hosts. How lung infection starts is unknown. Inhaled murine CMV (MCMV) directly infected alveolar macrophages (AMs) and type 2 alveolar epithelial cells (AEC2s) but not type 1 alveolar epithelial cells (AEC1s). In contrast, herpes simplex virus 1 infected AEC1s and murid herpesvirus 4 (MuHV-4) infected AEC1s via AMs. MCMV-infected AMs prominently expressed viral reporter genes from a human CMV IE1 promoter; but most IE1-positive cells were AEC2s, and CD11c-cre mice, which express cre in AMs, switched the fluorochrome expression of <5% of floxed MCMV in the lungs. In contrast, CD11C-cre mice exhibited fluorochrome switching in >90% of floxed MuHV-4 in the lungs and 50% of floxed MCMV in the blood. AM depletion increased MCMV titers in the lung during the acute phase of infection. Thus, the influence of AMs was more restrictive than permissive. Circulating monocytes entered infected lungs in large numbers and became infected, but not directly; infection occurred mainly via AEC2s. Mice infected with an MCMV mutant lacking its m131/m129 chemokine homolog, which promotes macrophage infection, showed levels of lung infection equivalent to those of wild-type MCMV-infected mice. The level of lung infiltration by Gr-1-positive cells infected with the MCMV m131/m129-null mutant was modestly different from that for wild-type MCMV-infected lungs. These results are consistent with myeloid cells mainly disseminating MCMV from the lungs, whereas AEC2s provide local amplification. IMPORTANCE Cytomegaloviruses (CMVs) chronically and systemically infect most mammals. Human CMV infection is usually asymptomatic but causes lung disease in people with poor immune function. As human infection is hard to analyze, studies with related animal viruses provide important insights. We show that murine CMV has two targets in the lungs: macrophages and surfactant-secreting epithelial cells. Acute virus replication occurred largely in epithelial cells. Macrophages had an important defensive role, as their removal increased the level of infection. These results establish the dual nature of lung infection, with local virus replication occurring in epithelial cells and spread occurring via quiescently infected macrophages. Distinct therapies may be needed to target these contrasting events.

Type 1 Interferons and NK Cells Limit Murine Cytomegalovirus Escape from the Lymph Node Subcapsular Sinus

Cytomegaloviruses (CMVs) establish chronic, systemic infections. Peripheral infection spreads via lymph nodes, which are also a focus of host defence. Thus, this is a point at which systemic infection spread might be restricted. Subcapsular sinus macrophages (SSM) captured murine CMV (MCMV) from the afferent lymph and poorly supported its replication. Blocking the type I interferon (IFN-I) receptor (IFNAR) increased MCMV infection of SSM and of the fibroblastic reticular cells (FRC) lining the subcapsular sinus, and accelerated viral spread to the spleen. Little splenic virus derived from SSM, arguing that they mainly induce an anti-viral state in the otherwise susceptible FRC. NK cells also limited infection, killing infected FRC and causing tissue damage. They acted independently of IFN-I, as IFNAR blockade increased NK cell recruitment, and NK cell depletion increased infection in IFNAR-blocked mice. Thus SSM restricted MCMV infection primarily though IFN-I, with NK cells providing a second line of defence. The capacity of innate immunity to restrict MCMV escape from the subcapsular sinus suggested that enhancing its recruitment might improve infection control.

Murine Cytomegalovirus Spreads by Dendritic Cell Recirculation

ABSTRACT Herpesviruses have coevolved with their hosts over hundreds of millions of years and exploit fundamental features of their biology. Cytomegaloviruses (CMVs) colonize blood-borne myeloid cells, and it has been hypothesized that systemic dissemination arises from infected stem cells in bone marrow. However, poor CMV transfer by stem cell transplantation argues against this being the main reservoir. To identify alternative pathways for CMV spread, we tracked murine CMV (MCMV) colonization after mucosal entry. We show that following intranasal MCMV infection, lung CD11c+ dendritic cells (DC) migrated sequentially to lymph nodes (LN), blood, and then salivary glands. Replication-deficient virus followed the same route, and thus, DC infected peripherally traversed LN to enter the blood. Given that DC are thought to die locally following their arrival and integration into LN, recirculation into blood represents a new pathway. We examined host and viral factors that facilitated this LN traverse. We show that MCMV-infected DC exited LN by a distinct route to lymphocytes, entering high endothelial venules and bypassing the efferent lymph. LN exit required CD44 and the viral M33 chemokine receptor, without which infected DC accumulated in LN and systemic spread was greatly reduced. Taken together, our studies provide the first demonstration of virus-driven DC recirculation. As viruses follow host-defined pathways, high endothelial venules may normally allow DC to pass from LN back into blood. IMPORTANCE Human cytomegalovirus (HCMV) causes devastating disease in the unborn fetus and in the immunocompromised. There is no licensed vaccine, and preventive measures are impeded by our poor understanding of early events in host colonization. HCMV and murine CMV (MCMV) both infect blood-borne myeloid cells. HCMV-infected blood cells are thought to derive from infected bone marrow stem cells. However, infected stem cells have not been visualized in vivo nor shown to produce virus ex vivo, and hematopoietic transplants poorly transfer infection. We show that MCMV-infected dendritic cells in the lungs reach the blood via lymph nodes, surprisingly migrating into high endothelial venules. Dissemination did not require viral replication. It depended on the constitutively active viral chemokine receptor M33 and on the host hyaluronan receptor CD44. Thus, viral chemokine receptors are a possible target to limit systemic CMV infections. IMPORTANCE Human cytomegalovirus (HCMV) causes devastating disease in the unborn fetus and in the immunocompromised. There is no licensed vaccine, and preventive measures are impeded by our poor understanding of early events in host colonization. HCMV and murine CMV (MCMV) both infect blood-borne myeloid cells. HCMV-infected blood cells are thought to derive from infected bone marrow stem cells. However, infected stem cells have not been visualized in vivo nor shown to produce virus ex vivo, and hematopoietic transplants poorly transfer infection. We show that MCMV-infected dendritic cells in the lungs reach the blood via lymph nodes, surprisingly migrating into high endothelial venules. Dissemination did not require viral replication. It depended on the constitutively active viral chemokine receptor M33 and on the host hyaluronan receptor CD44. Thus, viral chemokine receptors are a possible target to limit systemic CMV infections.

Type I Interferons and NK Cells Restrict Gammaherpesvirus Lymph Node Infection

ABSTRACT Gammaherpesviruses establish persistent, systemic infections and cause cancers. Murid herpesvirus 4 (MuHV-4) provides a unique window into the early events of host colonization. It spreads via lymph nodes. While dendritic cells (DC) pass MuHV-4 to lymph node B cells, subcapsular sinus macrophages (SSM), which capture virions from the afferent lymph, restrict its spread. Understanding how this restriction works offers potential clues to a more comprehensive defense. Type I interferon (IFN-I) blocked SSM lytic infection and reduced lytic cycle-independent viral reporter gene expression. Plasmacytoid DC were not required, but neither were SSM the only source of IFN-I, as IFN-I blockade increased infection in both intact and SSM-depleted mice. NK cells restricted lytic SSM infection independently of IFN-I, and SSM-derived virions spread to the spleen only when both IFN-I responses and NK cells were lacking. Thus, multiple innate defenses allowed SSM to adsorb virions from the afferent lymph with relative impunity. Enhancing IFN-I and NK cell recruitment could potentially also restrict DC infection and thus improve infection control. IMPORTANCE Human gammaherpesviruses cause cancers by infecting B cells. However, vaccines designed to block virus binding to B cells have not stopped infection. Using a related gammaherpesvirus of mice, we have shown that B cells are infected not via cell-free virus but via infected myeloid cells. This suggests a different strategy to stop B cell infection: stop virus production by myeloid cells. Not all myeloid infection is productive. We show that subcapsular sinus macrophages, which do not pass infection to B cells, restrict gammaherpesvirus production by recruiting type I interferons and natural killer cells. Therefore, a vaccine that speeds the recruitment of these defenses might stop B cell infection.

Type I Interferons Direct Gammaherpesvirus Host Colonization

Gamma-herpesviruses colonise lymphocytes. Murid Herpesvirus-4 (MuHV-4) infects B cells via epithelial to myeloid to lymphoid transfer. This indirect route entails exposure to host defences, and type I interferons (IFN-I) limit infection while viral evasion promotes it. To understand how IFN-I and its evasion both control infection outcomes, we used Mx1-cre mice to tag floxed viral genomes in IFN-I responding cells. Epithelial-derived MuHV-4 showed low IFN-I exposure, and neither disrupting viral evasion nor blocking IFN-I signalling markedly affected acute viral replication in the lungs. Maximising IFN-I induction with poly(I:C) increased virus tagging in lung macrophages, but the tagged virus spread poorly. Lymphoid-derived MuHV-4 showed contrastingly high IFN-I exposure. This occurred mainly in B cells. IFN-I induction increased tagging without reducing viral loads; disrupting viral evasion caused marked attenuation; and blocking IFN-I signalling opened up new lytic spread between macrophages. Thus, the impact of IFN-I on viral replication was strongly cell type-dependent: epithelial infection induced little response; IFN-I largely suppressed macrophage infection; and viral evasion allowed passage through B cells despite IFN-I responses. As a result, IFN-I and its evasion promoted a switch in infection from acutely lytic in myeloid cells to chronically latent in B cells. Murine cytomegalovirus also showed a capacity to pass through IFN-I-responding cells, arguing that this is a core feature of herpesvirus host colonization.

CD8+ T cell evasion mandates CD4+ T cell control of chronic gamma-herpesvirus infection

Gamma-herpesvirus infections are regulated by both CD4+ and CD8+ T cells. However clinical disease occurs mainly in CD4+ T cell-deficient hosts. In CD4+ T cell-deficient mice, CD8+ T cells control acute but not chronic lung infection by Murid Herpesvirus-4 (MuHV-4). We show that acute and chronic lung infections differ in distribution: most acute infection was epithelial, whereas most chronic infection was in myeloid cells. CD8+ T cells controlled epithelial infection, but CD4+ T cells and IFNγ were required to control myeloid cell infection. Disrupting the MuHV-4 K3, which degrades MHC class I heavy chains, increased viral epitope presentation by infected lung alveolar macrophages and allowed CD8+ T cells to prevent disease. Thus, viral CD8+ T cell evasion led to niche-specific immune control, and an essential role for CD4+ T cells in limiting chronic infection.

Herpes simplex virus type 1 interaction with myeloid cells in vivo

ABSTRACT Herpes simplex virus 1 (HSV-1) enters mice via olfactory epithelial cells and then colonizes the trigeminal ganglia (TG). Most TG nerve endings are subepithelial, so this colonization implies subepithelial viral spread, where myeloid cells provide an important line of defense. The outcome of infection of myeloid cells by HSV-1 in vitro depends on their differentiation state; the outcome in vivo is unknown. Epithelial HSV-1 commonly infected myeloid cells, and Cre-Lox virus marking showed nose and lung infections passing through LysM-positive (LysM+) and CD11c+ cells. In contrast, subcapsular sinus macrophages (SSMs) exposed to lymph-borne HSV-1 were permissive only when type I interferon (IFN-I) signaling was blocked; normally, their infection was suppressed. Thus, the outcome of myeloid cell infection helped to determine the HSV-1 distribution: subepithelial myeloid cells provided a route of spread from the olfactory epithelium to TG neurons, while SSMs blocked systemic spread. IMPORTANCE Herpes simplex virus 1 (HSV-1) infects most people and can cause severe disease. This reflects its persistence in nerve cells that connect to the mouth, nose, eye, and face. Established infection seems impossible to clear. Therefore, we must understand how it starts. This is difficult in humans, but mice show HSV-1 entry via the nose and then spread to its preferred nerve cells. We show that this spread proceeds in part via myeloid cells, which normally function in host defense. Myeloid infection was productive in some settings but was efficiently suppressed by interferon in others. Therefore, interferon acting on myeloid cells can stop HSV-1 spread, and enhancing this defense offers a way to improve infection control.