In the previous two posts (here and here) we laid out some new results that dissect what might be happening at the molecular level when a patient infected with SARS or bird flu descends into Acute Respiratory Distress Syndrome (ARDS) from Acute Lung Injury (ALI) in a just published paper in the journal Cell (Imai et al., "Identification of Oxidative Stress and Toll-like Receptor 4 Signaling as a Key Pathway of Acute Lung Injury", Cell, Vol 133, 235-249, 18 April 2008). We have already discussed their experiments showing that TLR4, a receptor that is part of the innate immune system, was needed for ALI caused by an acid mist; and that a locally released cytokine, IL-6, was connected with whatever was going on. We left off at the point where we were presenting the evidence connecting activation of TLR4, with oxidative damage to the phospholipids making up the lung cell membrane and the bath of surfactant in which they sit. Their results show that oxidized phospholipids can both cause ALI and induce expression of the cytokine IL-6 from pulmonary macrophages, the wandering scavenger/killer cells that patrol the lung looking for pathogens. The next step is perhaps the most dramatic:
Patients who died of H5N1 avian influenza or Spanish influenza developed ARDS, and extremely high levels of cytokines have been observed in patients and animals infected with these viruses (Beigel et al., 2005, Tumpey et al., 2005). Our data so far showed that acid aspiration triggers production of OxPLs [oxidized phospholipids] that can augment the severity of ALI and cytokine production via TLR4. We wanted to extend these findings to H5N1-mediated ALI. (Imai et al.)
They instilled inactivated H5N1 (i.e., virus that retained hemagglutin (HA) that could bind to host cell receptor but the virus could not infect or replicate) into mouse lung and showed that it acted like acid mist, i.e., it induced ALI and generated cytokine IL-6 by a pathway that was dependent on TLR4 through the TRIF branch (not the MyD88 branch). There was another interesting effect on the host cells. Inactivated H5N1 caused them to express more TLR4 on their surface. They also showed that H5N1 induced oxidative stress that led to oxidized phospholipids. Was the production of oxidized phospholipids caused by the lung injury or the other way around? By using mice that couldn't produce a respiratory burst (see previous post) they determined that the oxidative stress came first. Somehow H5N1 produced oxidized phospholipids which then stimulated TLR4 and consequent ALI and IL-6 production.
To cap things off, examination of tissues from two actual H5N1 patients showed "massive formation" of oxidized phospholipids, but patients who died of non lung diseases showed no such changes. It gets better. The same oxidized phospholipids were found in nine patients who developed ARDS after coming down with SARS and similarly in monkeys with pulmonary anthrax, Monkeypox and Yersinia pneumonitis (plague in the lung). The experiments with mice deficient in oxidative stress mechanisms seem to rule out the explanation that the oxidized phospholipids were the result, not the cause of the lung injury.
The bottom line here seems to be that Acute Lung Injury (ALI) with resulting ARDS is the result of oxidative stress coupled to the machinery of the innate immune system through one pathway leading out of TLR4. Does it matter that the path goes through TRIF and not MyD88? The authors make this intriguing suggestion. Since TLR4 utilizes both the classical MyD88 pathway and the TRIF pathway but the ALI is associated only with the TRIF path, by finding a way to suppress TRIF or some of its downstream proteins we might be able to treat ALI or ARDS without disturbing the functions of the innate immune system that work through MyD88. Thus many of the usual functions of ATLR4 would not be affected.
Assuming this story holds together we need to know more about what the TLR4-TRIF pathway is doing and what triggers the oxidation of phospholipids (and what phospholipids are involved and where). We know from recent work that blocking IL-6 doesn't stop damage from flu, so whatever TLR4 sets in motion might include IL-6 but clearly involves more, possibly other cytokines or maybe the cytokines are just effects, not causes of what is going on. The participation of TLR4 is somewhat surprising since it has been associated with defense against bacteria, not viruses. Moreover oxidized phospholipids have been shown to be protective against damage by bacterial LPS. Here's a possible explanation. The oxidized phospholipids are also produced by bacterial infection but they compete with LPS in someway. This might be some kind of brake on a runaway inflammatory response to LPS. In a viral infection there is no LPS to compete with and the oxidized phospholipids have nothing to counterbalance them. They thus make things worse, not better.
Of course I am just speculating. There's still a lot to learn. But this elegant series of experiments makes me feel as if we have just taken a big step forward and just as importantly, suggested a direction to go. All we need is enough time and the will and resources to use what we find out.
Thank you for your lucid and detailed explanation. I'm intrigued by the revelations on the role of oxidative stress as mechanism for ALI. Forgive my ignorance, but is this widely accepted or a fairly new line of investigation? Also, can you explain how this may relate to the mechanisms of action of statins and related drugs? Especially in relation to the various studies that suggest improved survival in sepsis and pneumonia for patients taking statins.
Again, much thanks for your work!!
Susan: I think the oxidized phospholipid connection is new or almost new. The statin connection is interesting. As far as I understand it, these two lines of evidence are working at opposite ends of the causal chain. The TLR4 is what gets the process going, but the statins are working on the outcome, perhaps activation of NFkappaB. Others may know more details. This paper is largely about dissecting the chain of events that sets ALI in motion. We'll have to see what its therapeutic implications are.
I think the oxidized phospholipid connection is new or almost new.
Well, this makes it all the more exciting then. Thanks!
Most intriguing. If only we had a compound that would anchor the pro and anti-inflammatory response together, so one side was unable to run amok. Not supress one side or the other but, link the two so neither side of the equation could overwhelm the other.
Shannon: But we don't want to knock off all inflammatory and anti-inflammatory effects. They protect us. We want to be very selective and prevent them just for runaway flu infection. And remember that at least a third of flu infections are asymptomatic. Perhaps this is because the mechanism works well a lot of the time. It's always a balancing act.
I understand it is a balancing act. You must have inflammation to kill the invading virus. Yet, there is something within the virus which stimulates a deregulation of the pro and anti-inflammatory response balance. However, this phenomenon isn't exclusive to H5N1 infection. Catastrophic injury from whatever source can do the same thing and elicit the same catastrophic response. So too can other disease causing agents. In these cases, it would then be beneficial to have a drug on hand that would forge a link that would have some elasticity so pro-inflammation can occur but, it couldn't ever become so predominant it overwhelms the anti-inflammatory response. A chemical bonding between the two that acts rather like a bungee cord. The two halves are not able to ever pull so far apart that one side can start the cytokine cascade leading to death. Rather than attempt to specifically reduce IL-6 or IL-10 (or whatever), it would not suppress but balance the two halves.
[From my paper published last year]:
The mechanism by which influenza viruses can effect
hypercytokinaemia is poorly understood. Cells sense the
presence of viral material through a variety of pattern recognition receptors, such as the Toll-like receptors (TLR). Both TLR7 and TLR8 are involved in the recognition of
single-stranded RNA, and TLR3 in the recognition of
double-stranded RNA (Garcia-Sastre, 2006; Matikainen
et al., 2006). Induction of IFN-a/b follows recognition,
although influenza A viruses are able to prevent this via
the antagonistic action of the NS1 protein (Garcia-Sastre,
2006), which also inhibits adaptive immunity by attenuating
human dendritic cell maturation (Fernandez-Sesma
et al., 2006).