Optimized detection of acute MHV68 infection with a reporter system identifies large peritoneal macrophages as a dominant target of primary infection

Abstract

Investigating the dynamics of virus-host interactions in vivo remains an important challenge, often limited by the ability to directly identify virally-infected cells. Here, we combine detection of a beta-lactamase activated fluorescent substrate with full spectrum flow cytometry to identify primary targets of murine gammaherpesvirus 68 (MHV68) infection in the peritoneal cavity. By optimizing substrate and detection conditions, we were able to achieve multiparameter characterization of infected cells and the ensuing host response. MHV68 infection leads to a pronounced increase in immune cells, with CD8+ T cells increasing by 3 days, and total infiltrate peaking around 8 days post-infection. MHV68 infection results in near elimination of large peritoneal macrophages by 8 days post-infection, and a concordant increase in small peritoneal macrophages and monocytes. Infection is associated with prolonged changes to myeloid cells, with a distinct population of MHC IIhigh large peritoneal macrophages emerging by 14 days. Targets of MHV68 infection could be readily detected. Between 1 to 3 days post-infection, MHV68 infects ~5-10% of peritoneal cells, with >75% being large peritoneal macrophages. By 8 days post-infection, the frequency of MHV68 infection is reduced at least 10-fold, with infection primarily in small peritoneal macrophages, with few infected dendritic cells and B cells. MHV68 infection at 3 days post-infection contains both lytic and latent infection, consistent with the identification of cells with active reporter gene expression. Our findings demonstrate the utility of the beta-lactamase MHV68 reporter system for high throughput single-cell analysis and identify dynamic changes during primary gammaherpesvirus infection.ImportanceIdentifying virally-infected cells in vivo is key to tracking viral infection and understanding host-pathogen interactions. The ability to further characterize and phenotype virally-infected cells is technically challenging. We use a mouse gammaherpesvirus, MHV68, expressing a reporter gene to identify infected cells during primary infection via flow cytometry. Optimization using this reporter system allowed us to further characterize infected cells via multiparameter full spectrum flow cytometry. Our study provides a technical model for high throughput single-cell immunophenotyping methods in the context of gammaherpesvirus infection. Furthermore, we show that acute MHV68 infection in the peritoneal cavity dramatically changes the immune landscape of this tissue, results in a high number of infected macrophages at early times, and is characterized by both lytic and latent infection within immune cells.

Coinfection with Intestinal Parasite Expands Resident Macrophages and Impairs Control of Chronic Herpesvirus Infection

“…When compared to the infection data with the marker virus, this suggests that even though there was increased MHV68-infected cells in the coinfected mice, that these infected cells were not replicating more virus and instead harbor latent virus. This observation supports the idea that some MHV68 genomes become latent immediately (16,27). Overall, our data shows that coinfected mice have increased numbers of infected LPMs.…”

“…In particular, the impact of LPM expansion on peritoneal viral infection has never been reported, even though we routinely model viral infection in mice by peritoneal injection. Murine gammaherpsvirus-68 (MHV68) infects and establishes latent, chronic infection in B cells, dendritic cells, macrophages, and in particular, large peritoneal macrophages( 16, 24, 25 ). We asked whether HP infection altered acute infection with MHV68.…”

“…Certain signals, such as HDAC inhibitors (12), parasite infection (13), or IL-4 complexed with IFN-γ blockade (14), promote virus reactivation from latency, which is important for herpesvirus-induced lymphomas (15). Interestingly, MHV68 was recently shown to infect LPMs (16), leading us to question what the impact of parasite-mediated expansion of LPMs would be on peritoneal viral infection. Studies examining the outcomes of parasite and gammaherpesvirus coinfection reveal the complex nature of coinfection studies.…”

“…Interestingly, MHV68 was recently shown to infect LPMs ( 16 ), leading us to question what the impact of parasite-mediated expansion of LPMs would be on peritoneal viral infection. Studies examining the outcomes of parasite and gammaherpesvirus coinfection reveal the complex nature of coinfection studies.…”

IKKalpha-Mediated Non-canonical NF-kappaB Signaling is Required to Support Murine Gammaherpesvirus 68 LatencyIn Vivo

“…MEFs were stimulated for 18 h with antibody against the LTβR and tested for p100 cleavage in the whole cell, cytoplasmic, and nuclear fractions. We also stimulated MEFs with the canonical NF-κB pathway activator TNFα for Macrophages of the peritoneal compartment comprise another reservoir of MHV68 latency (33). Peritoneal exudate cells isolated 16 days post-intraperitoneal infection revealed nearly identical levels of reactivation from latency in mice infected with IKKα-SA1 (1/2,218) or IKKα- 4E).…”