Lipopolysaccharide, tumor necrosis factor alpha, or interleukin-1beta triggers reactivation of latent cytomegalovirus in immunocompetent mice

Abstract

We have previously shown that cytomegalovirus (CMV) can reactivate in lungs of nonimmunosuppressed patients during critical illness. Our recent work has shown that polymicrobial bacterial sepsis can trigger reactivation of latent murine CMV (MCMV). We hypothesize that MCMV reactivation following bacterial sepsis may be caused by inflammatory mediators. To test this hypothesis, BALB/c mice latently infected with Smith strain MCMV received sublethal intraperitoneal doses of lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), or saline. Lung tissue homogenates were evaluated for viral reactivation 3 weeks after mediator injection. Because LPS is known to signal via Toll-like receptor 4 (TLR-4) in mice, further studies blocking this signaling mechanism were performed using monoclonal MTS510. Finally, mice were tested with intravenous TNF-alpha to determine whether this would cause reactivation. All mice receiving sublethal intraperitoneal doses of LPS, TNF-alpha, or IL-1beta had pulmonary reactivation of latent MCMV 3 weeks following injection, and LPS caused MCMV reactivation with kinetics similar to those for sepsis. When TLR-4 signaling was blocked, exogenous LPS did not reactivate latent MCMV. Intravenous TNF-alpha administration at near-lethal doses did not reactivate MCMV. Exogenous intraperitoneal LPS, TNF-alpha, and IL-1beta are all capable of reactivating CMV from latency in lungs of previously healthy mice. LPS reactivation of MCMV appears dependent on TLR-4 signaling. Interestingly, intravenous TNF-alpha did not trigger reactivation, suggesting possible mechanistic differences that are discussed. We conclude that inflammatory disease states besides sepsis may be capable of reactivating CMV from latency.

Figures

FIG. 1.

LPS stimulates reactivation of MCMV from latency. RT-PCR of lung homogenates from mice latently infected with MCMV shows that LPS administration stimulates reactivation of viral transcription. In vitro plaque assay confirmed the presence of infectious virus in all LPS-treated mice (4/4), and the more sensitive focused expansion assay showed no reactivation (0/2) in mice receiving saline alone (data not shown). Two dose regimens in comparison to saline (0.2 ml i.p.) are shown: 15 μg/kg/0.2 ml i.p. and 30 μg/kg/0.2 ml i.p. Positive and negative controls refer to technique controls. No-RT reactions were also performed in parallel for MCMV GB and β-actin and were negative (data not shown).

FIG. 2.

Kinetics of LPS-induced MCMV reactivation. Mice latently infected with MCMV received a sublethal dose of LPS (15 μg/kg/0.2 ml i.p.), and cohorts of five were analyzed at 1-, 2-, and 3-week intervals for MCMV GB and β-actin transcription. RT-PCR of lung homogenates from mice latently infected with MCMV shows LPS-induced reactivation of viral transcription is similar to that seen following bacterial peritonitis, becoming detectable by 1 week and persisting for at least 3 weeks after LPS injection. Detection of reactivation required nested RT-PCR at weeks 1 and 2; reactivation became detectable by less sensitive first-round RT-PCR by week 3. Molecular reactivation was confirmed by IVPA with recovery of live virus from five of five week 3 mice receiving LPS (mean ± SEM, 198 ± 27 PFU). Mice receiving saline alone (0.2 ml i.p.) were evaluated at week 3 only and showed no evidence of reactivation by RT-PCR (see above) or by IVPA or focused expansion assay (data not shown). Each lane represents results from one mouse. Positive (Pos) and negative (Neg) controls refer to technique controls. No-RT reactions were also performed in parallel for MCMV GB and β-actin and were all negative (not shown).

FIG. 3.

LPS reactivates MCMV via a TLR-4-mediated mechanism. Mice latently infected with MCMV received MTS510, a monoclonal antibody that blocks TLR-4 signaling (n = 3), or an isotypic rat IgG (n = 2) prior to receiving a sublethal dose of LPS (15 μg/kg/0.2 ml i.p.). Lung homogenates for each mouse were analyzed 3 weeks after LPS injections for reactivation of MCMV. RT-PCR shows that MTS510 prevents reactivation of viral transcription, while isotypic IgG did not prevent reactivation. Each lane represents results from one mouse. Positive (Pos) and negative (Neg) controls refer to technique controls. No-RT reactions were also performed in parallel for MCMV GB and β-actin and were all negative (not shown).

FIG. 4.

Intraperitoneal TNF-α or IL-1β stimulates pulmonary MCMV reactivation. Mice latently infected with MCMV received sublethal doses of either TNF-α (2 μg i.p.) or IL-1β (0.2 μg i.p.). Lung homogenates from each mouse were analyzed by RT-PCR for molecular reactivation of MCMV 3 weeks after inflammatory mediator injections. TNF-α stimulated viral mRNA transcription in 10/10 mice. Similarly, IL-1β caused viral mRNA transcription in 10/10 mice. Molecular reactivation was confirmed by IVPA, with recovery of live virus from five of five week 3 mice receiving TNF-α (mean ± SEM, 170 ± 34 PFU) and from five of five mice receiving IL-1β (mean ± SEM, 156 ± 29 PFU). As in previous experiments, controls receiving saline alone showed no evidence of MCMV reactivation, either by RT-PCR (above) or by IVPA and focused expansion analyses (data not shown). Each experiment was performed in duplicate to give 10 in each group, and representative results from one experiment (5 per group) are shown. Each lane represents results from one mouse. Positive and negative controls refer to technique controls. No-RT reactions were also performed in parallel for MCMV GB and β-actin and were all negative (not shown).

FIG. 5.

Intravenous TNF-α does not stimulate pulmonary MCMV reactivation. Mice latently infected with MCMV received near-lethal doses of TNF-α (1 μg/mouse) intravenously. Lung homogenates from surviving mice were analyzed by RT-PCR for molecular reactivation of MCMV 1, 7, or 21 days after TNF-α injection. Intravenous TNF-α did not stimulate CMV GB mRNA transcription in any mouse. Similarly, in vitro plaque assay and focused expansion analyses showed no productive virus (data not shown). Each lane represents results from one mouse, and positive (Pos) and negative (Neg) controls refer to technique controls. β-Actin results confirm that RNA were recovered for each sample, and no-RT reactions performed in parallel demonstrated no DNA contamination (not shown).

FIG. 6.

Proposed paradigm for MCMV reactivation by intraperitoneal inflammatory mediators. Polymicrobial sepsis, through a variety of pathways, is capable of reactivating latent MCMV. One component released during polymicrobial sepsis, LPS, is capable of reactivating MCMV when introduced exogenously. LPS appears to trigger MCMV reactivation through TLR-4 signaling. TLR-4 signaling is not required for sepsis-triggered reactivation, because other mediators, such as TNF-α and IL-1β, are capable of triggering CMV reactivation.