Studies of streptococcal infections (16
), adenoviral infections, and viral hepatitis (20
) indicate that scavenger receptors participate in the uptake and clearance of these pathogens from the lung and other sites. In contrast, IAV infection in MARCO-deficient mice resulted in better survival and earlier recovery from weight loss and morbidity symptoms than the WT control mice in the three trials reported in this manuscript. Similar findings of increased survival with influenza in MARCO−/−
mice have been observed by another laboratory (Z. Chroneos, personal communication).
The course of infection was marked by a transient increase in inflammation early in the innate defense phase in the absence of obvious differences in the adaptive response measured by the lymphocyte counts (see
). The molecular mechanism that links the transient inflammatory up-regulation to an enhanced recovery later is unclear, although it is evident that inflammatory cells suppress viral replication and may result in effectively lower viral levels during the peak period (). The total lymphocyte influx in the lungs remained comparable between the groups. Kai and colleagues reported that IL-10–deficient mice showed increased survival via increased TH17 response in the absence of alteration in the magnitude of cellular response (35
). In addition, lower virus load does not necessarily correlate with survival advantages (35
). Our results also show that LPS administration 12 hours after influenza infection could effectively lower the viral mRNA levels, but the excessive inflammation that LPS caused resulted in increased death rates of mice, as expected (). Our findings support the interpretation that a critical window for a beneficial inflammatory response exists for influenza immunity, where there is a mild but early inflammatory response within the first 2 to 3 days followed by effective down-regulation and prevention of further inflammatory amplification sequelae. MARCO−/−
mice exhibited this pattern.
In an earlier study, therapeutic blockage of MIP-2 blocked PMN influx in the lungs and reduced the severity of lung pathology to IAV (36
). Fujisawa (9
) showed that depletion of neutrophils in rats by the use of monoclonal antibody RB6 was associated with significant elevations in the pulmonary viral titers on Days 3 through 5 and that it took longer for the viral titer to decrease than in control rats. Although these reports appear to contradict regarding the role of inflammatory cellular influx in influenza, they illustrate the importance of timing and of the level of neutrophil response in influenza in leading to a beneficial versus a deleterious role. Recently, Tate and colleagues (10
) reported the use of anti-Ly6G (1A8) antibody with increased specificity to neutrophils to block their influx before influenza. They reported that viral titer was greatly increased in the experimentally neutropenic mice and was associated with a worse pathological outcome and edema. We show here in our MARCO-deficient model that a more subtle differential neutrophil response, without exaggerated peak levels or duration of neutrophil influx, resulted in lower peak RNA levels of the virus and was associated with early recovery and better survival.
We inquired if MARCO mediated the uptake of IAV. MARCO deficiency did not result in quantitative differences in IAV entry in macrophages. However, MARCO−/− macrophages showed higher levels of IL1b (data not shown), CCL5, and CCL7 mRNA upon infection in vitro (). This suggested that differential intracellular responses exist as a consequence of MARCO, probably in the mode of viral entry and intracellular processing, and we are currently investigating this. The role of IL-17 during the initial stages before 2 days after infection is not clear. Our initial attempts to neutralize IL-17 in vivo using antibody did not result in abrogation of early neutrophil influx in the WT or the MARCO−/− lungs at Day 2 after infection (data not shown), but additional studies are needed to fully address this question. It is possible that the release of IL-17 was delayed further from our messenger RNA studies or that neutrophils expressed higher IL-17, which may have implications later in the disease process.
We considered the most likely initial cause of enhanced acute inflammation in MARCO−/−
mice to be the compromised scavenging of oxidized phospholipid and postcellular debris, which trigger proinflammatory gene expression. As initially shown in studies related to atherosclerosis, oxidized phospholipids and LDLs are scavenged by SRA and CD36 (37
). Macrophage class A scavenger receptors readily take up oxidized liposomes (40
). In prior studies of acute inflammation in ozone-damaged lungs, we found that the absence of MARCO leads to diminished clearance of proinflammatory oxidized phospholipids, with a resultant robust acute inflammatory effect. We have shown here () that an inflammatory component existed in the BALF of MARCO−/−
mice as early as 8 hours after infection, when neutrophil influx or reactive oxygen species were not detectable. We also noted that even in the uninfected state, BALF of MARCO−/−
mice contained greater oxidized phospholipid than the WT mice but did not have detectable increased neutrophil levels or proinflammatory gene messenger levels at basal state. We believe that a threshold value is reached with additional release and accumulation of ox-PL upon infection and initiation of cell death, which then triggers increased inflammation. Fitting with our observation of an early yet transient rise in inflammation in the MARCO−/−
mice's response to influenza, the enhanced levels of ox-PL were not evident at 4 days after infection (), although cell death must have increased at this time. One possibility is that the function of MARCO in this case is significant in the early stages, mostly in the resident macrophages encountering the earliest wave of oxidized lipids and cell debris from the first cellular victims of infection. Due to functional redundancy of these scavenger receptors, it is possible that activated neutrophils and macrophages recruited into the lungs have compensatory scavenger receptors, which may nullify the effect of the SR deficiency at a later period. Further study is needed to evaluate a time-dependent role of MARCO versus other scavenger receptors during lung infection. In summary, this study identifies an unexpected harmful role for the macrophage scavenger receptor MARCO in early innate immune responses to influenza. We speculate that possible variations in expression or functional efficacy of this receptor in the human population could contribute to individual variation in influenza disease severity.