An extracellular microRNA can rescue lives in sepsis

Sepsis, or blood poisoning, is a savage response of the body to infection. It can lead to organ failure, blood pressure decline, heart failure, and coma. Between 20 and 30 million people suffer from sepsis each year, leading to 8 million deaths. Although certain people are more at risk than others (young children, elderly), anyone can develop sepsis. Patients are resuscitated and treated with antibiotics, and their organ functions are supported. Despite the investment in sepsis research during the previous decades, successful clinical trials are scarce and sepsis remains one of the most difficult and deadly unmet medical needs of today. A study in this issue now provides new insight into sepsis and points to a therapeutic future [¹].

During sepsis, host proteins (Damage Associated Molecular Patterns) are released into the blood. A study in this issues shows that an extracellular microRNA inhibits one of these, thereby reducing pro‐inflammatory cytokine production.

Since sepsis starts with (usually bacterial) infections, inflammation is induced, as well as innate and adaptive immune pathways. Because of their importance in these processes, white blood cells (WBCs) have been studied in sepsis and a profound “genomic storm” has been described in these cells, followed by the expression of inflammatory genes, metabolic reprogramming, and activation of microbe‐killing mechanisms among other effects 2. Obviously, genomic regulation goes beyond mRNA expression of protein‐coding genes, but also includes epigenetic mechanisms, such as DNA methylation, histone acetylation, and the regulation of long non‐coding RNAs (lncRNAs) and microRNAs (miRs). The latter have attracted attention in several diseases, since they are often released into the blood and can regulate gene expression in distant target cells. miRs are short single‐stranded RNA molecules of 20–22 bases, which can bind mRNAs by base‐pairing, leading to the breakdown of the target or repression of protein translation from this mRNA. One miR can target many mRNAs, and one mRNA can be subject of regulation by several miRs 3. This simple mechanism suffices to explain most actions of miRs, but a number of studies, including one of Gurien et al 1, have reported on other biological activities of miRs.

In diseases, miRs that are often appearing in the blood may indicate underlying mechanisms, they may also serve as biomarkers, and they may provide opportunities for therapeutic intervention. In sepsis models, as well as in patients, it has been reported frequently that miRs are increased in the blood 4. In the paper by Gurien et al, the authors did miR profiling and found numerous miRs increased in the well‐validated sepsis model known as the cecal ligation and puncture (CLP). In this model, mice undergo an operation where the cecum is isolated, ligated by a surgical wire, and punctured. Following surgery, bacteria leak from the cecum and colonize the peritoneum, leading to a septic peritonitis, which can be lethal and which resembles human septic peritonitis quite well 5. In CLP mice, compared with sham‐operated mice, several miRs attracted attention by their upregulation in the blood. miR‐130b‐3p (miR‐130) was found as most strongly induced by CLP and moreover was also found induced in human sepsis patients 1. The question about the function of a specific miR is usually approached by studying its sequence and interrogating prediction programs about which mRNA sequences are complementary, which usually leads to a long list of potential mRNA targets. The great novelty of the study by Gurien et al is that they focused their attention elsewhere (Fig 1).

Figure 1. The non‐genomic inhibitory effect of microRNA‐130b‐3p in sepsis.

Bacterial sepsis (here gram‐negative bacteria) leads to the activation of TLRs on many cell types, including macrophages via PAMPs. LPS is such a PAMP, binding to and activating TLR4. This will cause inflammation and other systemic events, such as blood coagulation and complement activation, leading to reduced blood flow, hypoxia, and tissue damage. This will trigger cell death and the release of DAMPs, some of which will activate macrophages via TLR4 even further. eCIRP, an RNA‐binding molecule, will detect and bind circulating microRNAs, released during infection. How and where these microRNAs are released is unclear. miR‐130b‐3p binds eCIRP and reduces its binding to TLR4. This limits ongoing inflammation and has protective effects. Macrophages are also involved in eliminating bacteria (green line), but the impact of reduced eCIRP on this activity is not known.

As mentioned above, in sepsis and other inflammatory diseases, WBCs are essential in the propagation of the disease as well as its termination. In sepsis, macrophages, a subset of WBCs, have an additional role, namely phagocytosis of infectious agents such as bacteria. Macrophages can be stimulated by a variety of ways, but the membrane‐bound Toll‐like receptors (TLRs) are best known. Each mammal has about a dozen different TLRs. TLR4 detects lipopolysaccharides (LPSs), which are cell‐wall components of gram‐negative bacteria (e.g., Escherichia coli), many of which can cause sepsis. LPS belongs to a group of immunogenic molecules known as pathogen‐associated molecular patterns (PAMPs). In the early hours of sepsis, also several host proteins released from the nucleus, cytoplasm, or even mitochondria of damaged cells are present in the blood. They are called damage‐associated molecular patterns (DAMPs). The list of DAMPs is long and contains several highly inflammatory molecules, e.g., high mobility group box 1 protein (HMGB1) and heat shock protein 70 (HSP70) 6.

In previous papers, the research group of Gurien and co‐workers has focused on a novel DAMP, namely an RNA‐binding protein known as cold‐inducible RNA‐binding protein (CIRP) 7. Like other DAMPs, CIRP leaks from damaged cells and binds TLR4 on macrophages, inducing inflammation. In the CLP model, miRs are released into the blood, as well as CIRP, now referred to as extracellular CIRP (eCIRP). In a key experiment, the authors found that miR‐130 co‐purified with eCIRP from the blood of CLP mice 1. Molecular docking experiments found specific binding of the miR on eCIRP, and the interaction was confirmed using recombinant CIRP in a BIAcore experiment. Finally, and most importantly, the binding of miR‐130 led to a reduced biological activity of CIRP, since it induced less inflammation in macrophages 1. Again using BIAcore, the authors confirmed that CIRP, once bound to miR‐130, showed less binding to TLR4. Whether miR‐130 causes a small conformational change in CIRP, or competes for binding to TLR4, is not yet known.

The impact of miR‐130 on the biological activity of CIRP was further confirmed in mice by two experiments. Firstly, by injecting recombinant CIRP, inflammation and organ damage are induced, but less so when the miR was co‐injected. Secondly, it was found that the injection of miR‐130 caused strong protection in numerous inflammation and organ damage readouts in the CLP model 1. Whether miR‐130 causes more survival in this aggressive sepsis model was not recorded, though.

It is fair to say that this paper is a game‐changer. It makes us wonder whether the release of DAMPs, miRs, and other molecules, which are considered as collateral damage, reflects maybe another level of regulation, yet to be discovered. By realizing that RNA‐binding proteins as well as miRs end up in the blood, the epigenetic factors that regulate gene expression in cells may simply be transported to the extracellular milieu to exert their functions, or even other functions fitting to the needs. We may start wondering whether other DAMPs once released into the blood also have biological functions (distantly) linked to their intracellular functions. Does HSP70 acts as a chaperone in the blood in sepsis? Are there other DAMPs with RNA‐binding protein function that bind miRs and undergo changes by this? Is this regulation specific for TLR4, or are there miR‐based systems for other TLRs or other PAMP receptors?

The paper also raises questions that should be addressed in future research. Knowing that there are different forms of macrophages (pro‐inflammatory, anti‐inflammatory), we wonder whether the miR‐130‐eCIRP interplay is equally strong and important in all of these? Also, does eCIRP modulate the bacterial clearing mechanism of macrophages, and what is the impact of miR‐130 on this function? As shown by Gurien et al, in sepsis, many dozens of miRs are released into the blood. Does miR‐130 undergo competition to bind to eCIRP? Are there other miRs binding to eCIRP, and maybe rather stimulate than inhibit its function? Where do the miRs come from? Does binding a protein in the blood undermine other functions of the miR, e.g., mRNA binding after cell penetration? And most importantly, since TLR4 and LPS inhibitors have been quite disappointing drugs in human sepsis clinical trials 8, is it conceivable that miR‐130 (mimics) as a drug will make a real difference at the bedside?

In any case, the paper by Gurien et al is of high interest, because it makes people consider that behind each molecule or biomarker, several layers and stories may be hidden, and we must uncover them all to get a complete picture of a complex setting like sepsis.

EMBO Reports (2020) 21: e49193