The results presented here demonstrate that ethanol-treated mice challenged ip with nonpathogenic E. coli
exhibit decreased production of most proinflammatory cytokines and chemokines at early time points, decreased attraction of neutrophils to the peritoneal cavity, decreased clearance of bacteria by macrophages and neutrophils in the peritoneal cavity, and increased mortality. This suggests a scenario in which ethanol inhibits the initial inflammatory response to E. coli
, which decreases the clearance of bacteria in the first few hours after challenge. After ethanol has been cleared (~6 h after dosing) (Carson and Pruett, 1996
), the increased number of bacteria induces an inflammatory response that probably contributes to the lethal outcome observed. Further studies are needed to determine the role of particular inflammatory mediators in lethal outcome in this experimental system.
Some portion of the inhibition of production of these inflammatory cytokines by ethanol could be mediated by stress hormones such as corticosterone that can be induced by ethanol. However, our recent studies indicate that ethanol-induced glucocorticoids and catecholamines do not contribute to the inhibition of cytokine production induced through TLR3 or TLR4, with the possible exception of a partial role in inhibition of IL-6 production (Glover and Pruett, 2006
; Glover et al., 2009
). Thus, it seems unlikely that the stress response is critical to the effects of ethanol on the pathogenesis of sepsis in this model system.
Several investigators have reported that ethanol inhibits TLR signaling (Dai et al., 2005
; Szabo et al., 2007
), and it seemed likely that this was involved in the decreased resistance to infection associated with acute ethanol exposure. However, the results presented here indicate that survival was enhanced in the absence of fully functional TLR4, so inhibition of TLR4 signaling is unlikely to be the major mechanism by which ethanol suppresses resistance to lethality in this experimental system. This is also suggested by the observation that ethanol decreases survival time and/or percentage in TLR4-knockout mice to a similar degree as in wild-type mice, indicating that targets other than TLR4 are involved in lethality. There are cytoplasmic (Cartwright et al., 2007
) as well as membrane-bound receptors that respond to LPS and other TLRs that respond to other components of gram-negative bacteria. It is possible that in the absence of TLR4, these receptors mediate sufficient response to lead to bacterial clearance but not to a lethal overproduction of inflammatory mediators. Our results with regard to cytokine and chemokine production in TLR4-mutant and wild-type mice support this idea (). However, it is also possible that decreased cytokine responses in the first few hours after challenge in mice treated with ethanol allow overgrowth of bacteria which becomes lethal after the ethanol has been eliminated. This lethality may be related to TLR4-induced overproduction of inflammatory mediators, but this cannot be the only mechanism because TLR4-mutant mice treated with ethanol have a similar mortality profile as wild-type mice treated with ethanol. This strongly suggests that lethal effects of sepsis are mediated through other receptors and may not involve well-recognized inflammatory mediators because many of these are decreased in concentration in TLR4-mutant mice. Other receptors that may be involved in the pathogenesis include TLR3 and TLR2. There are reports that TLR3 contributes to lethality in sepsis by sensing the RNA released from necrotic cells during sepsis and amplifies the secondary inflammatory responses (Cavassani et al., 2008
). Similarly, TLR2-dependent signaling has been shown to be critical for host resistance to bacterial infection both in animal studies and humans (Alves-Filho et al., 2009
; Ferwerda et al., 2009
; Mancuso et al., 2004
; Murphey et al., 2008
). Interestingly, ethanol also suppresses TLR3 and TLR2 signaling as reported in our previous study (Pruett et al., 2004a
It remains possible that inhibition of TLR4 signaling by ethanol (or TLR4 mutation) does play an important role in decreased resistance to lower dosages of bacteria, as reported for C3H/HeJ mice (Alves-Filho et al., 2006
), and that clearance of higher dosages of bacteria could be delayed (van Westerloo et al., 2005
) by inhibition or lack of TLR4, even if ultimately effective. Results for enteropathogenic E. coli
indicate that C3H/HeJ mice do not survive as well as wild-type mice when a low dose of bacteria is administered (Cross et al., 1995
). Similar results were obtained by another group when mice were treated with a sublethal challenge dose of bacteria (Alves-Filho et al., 2006
). However, when mice were treated with a greater dose of bacteria (lethal for a major percentage of wild-type mice), a much higher percentage of C3H/HeJ mice survived than wild type, as noted in our study. Another group very recently reported similar results, indicating that TLR4-mutant mice have increased resistance to a lethal outcome in E. coli
sepsis caused by a high dosage of E. coli
(Roger et al., 2009
). Thus, it seems that the role of TLR4 in resistance to sepsis and lethality in sepsis depends on the initial challenge dose of bacteria.
In contrast to the clear protective effect of the absence of functional TLR4 in the C3H/HeJ mice, the lack of MyD88 in MyD88-knockout mice did not significantly improve survival (although there was a tendency in that direction). This leaves open the interesting possibility that the lethal effects of sepsis involve signals transmitted through the alternate adaptor molecule used by TLR4 (and by TLR3 as its only adaptor), TIR-domain-containing adaptor inducing interferon-beta (TRIF). Results indicating that mice lacking TRIF survive sepsis better than wild-type mice are consistent with this possibility (Weighardt and Holzmann, 2007
). Also consistent with this idea is a recent report indicating that IFN-β (the production of which is mediated by the TRIF pathway) is required for release of high-mobility group box 1 protein and lethality in sepsis (Kim et al., 2009
). We previously reported that IL-12 and IL-10 production in response to LPS is decreased almost to the lower limit of detection (Pruett et al., 2005
). The finding that MyD88 is dispensable in survival of E. coli
infection is consistent with findings in humans, in which individuals with a genetic defect in MyD88 were less resistant only to pyogenic bacterial infections, not other to other types of infections (von Bernuth et al., 2008
). In apparent contrast to these findings, Peck-Palmer et al. (2008)
reported that MyD88-knockout mice do not survive as well as wild-type mice in a cecal ligation and puncture model of sepsis. Mortality in that study was 40% in wild-type C57Bl/6 mice, whereas it was 100% in our study (). Thus, the difference in results may simply reflect different outcomes with high versus lower dosages of bacteria, as reported by other investigators (Alves-Filho et al., 2006
; Mancuso et al., 2004
) or differences between polymicrobial sepsis and E. coli
The observation that mice lacking MPO were not significantly more susceptible to sepsis-induced mortality than wild-type mice was not entirely unexpected because similar results have been reported previously (Brovkovych et al., 2008
). However, the finding that wild-type and MPO-knockout mice are similarly and significantly susceptible to ethanol-induced mortality in sepsis indicates that ethanol does not act primarily by inhibiting expression or function of MPO. We had considered this as a potential mechanism because our microarray analysis indicated that early after E. coli
challenge MPO expression was decreased by ethanol (data not shown).
The effects of ethanol on survival were similar for wild-type mice, MyD88-knockout mice, TLR4-mutant mice, and MPO-knockout mice. Significantly decreased survival percentage or survival time was noted in all cases. These results indicate that inhibition of TLR4 signaling through MyD88 (which does occur) is not the major mechanism by which ethanol decreases host resistance. They also demonstrate that inhibition of MPO by ethanol is not a major mechanism for decreased resistance to sepsis in this system.
The cellular targets for decreased antibacterial effectiveness seem to be macrophages and/or neutrophils. Although the results reported here indicate that phagocytosis occurred in ethanol-treated mice (), it was evident that killing and degradation of the bacteria in both macrophages and neutrophils were substantially decreased (). Microarray results using this experimental model indicate inhibition by ethanol of a variety of genes coding for proteins involved in antibacterial effects of phagocytic cells (manuscript in preparation). Others have reported that acute ethanol exposure decreases host resistance by inhibiting upregulation of key antimicrobial effectors (e.g., nitric oxide synthase 2) (Greenberg et al., 1999
). Future studies will be conducted to determine which of these are involved in the inhibition of phagocyte antimicrobial function.
There are at least two obvious potential applications suggested by these results for treatment of sepsis in humans. For E. coli
-mediated sepsis, inhibition of TLR4 signaling may be a useful therapeutic approach because decreased response to TLR4 in a mutant mouse strain improved survival substantially (). This conclusion is consistent with another recently published report (Roger et al., 2009
). This approach has the advantage of decreasing responses of several mediators of inflammation, not just one. Also, greater efficacy of therapy in sepsis might be obtained using treatments that inhibit combinations of the cytokines overproduced in the later stages of sepsis (e.g., IL-1β and IL-6) () and that augment key mediators that are not known to contribute to lethality at intermediate stages of sepsis (e.g., granulocyte-macrophage colony-stimulating-factor) (). It is clear that this animal model is associated with rapid lethality, which is different from the typical course of sepsis in humans, which lasts 15–17 days, possibly due to antibiotic therapy that is not included in the mouse model (Angus et al., 2001
). However, there is evidence that intervention of various types early after the diagnosis of sepsis is more effective than later intervention (Moore et al., 2009
). Thus, the patterns noted in the animal model may be useful, but the results reported here would suggest that the timing of intervention is critical and will need to be determined in studies with human subjects.
In summary, the results presented here demonstrate conclusively that inhibition of TLR4 signaling, MPO expression, and MyD88 expression or function do not represent major mechanisms by which ethanol inhibits resistance to sepsis. The results instead suggest that the primary defect caused by ethanol is in the killing of phagocytosed bacteria by macrophages and neutrophils. Ethanol inhibits cytokine production early, but similar decreases in mice with a defective TLR4 did not decrease resistance to sepsis. These findings highlight how little is known about the quantitative relationships and time dependence of cytokines and chemokines with regard to host resistance to sepsis.