The systemic inflammatory response syndrome [SIRS (1)] can be triggered by diverse forms of injury including burns, ischemia, autoimmune diseases, injuries including surgery and infection. The severity of illness seen in SIRS varies widely but severe SIRS accompanied by multiple organ dysfunction syndrome (MODS), especially in the setting of sepsis (a condition which shares common clinical manifestations with SIRS), may be lethal (2). Even with optimal medical care, mortality rates in severe sepsis increase to around 50% (3). In this respect, clinical scores, such as the sequential organ failure assessment [SOFA (4)] and the acute physiology and chronic health evaluation II [APACHE II (5)], are useful to evaluate SIRS severity and patient mortality risk.

During SIRS, immune cell activation spreads to the whole body driving severe immunopathology (1, 6, 7). Damage- and/or pathogen-associated molecular pattern molecules (respectively, DAMPs and PAMPs) released after injury (or infection) initiate, via toll-like receptor (TLR) signals (6, 8), an activation cascade in immune and endothelial cells leading to inflammatory cytokine production [e.g., tumor necrosis factor (TNF) α, IL-1, IL-6, and IL-8], in a so-called “cytokine storm” (9, 10). Activated inflammatory cells migrate to affected organs and release secondary inflammatory mediators (1), including reactive oxygen species (ROS) and nitrogen species (11–15), resulting in oxidative stress (16, 17). Inflammatory cell migration to tissues and endothelial cell activation (18) lead to disseminated intravascular coagulation (19) aggravating organ damage. Increased levels of TNFα, IL-1, and IL-6 have been reported to occur more often in sepsis than SIRS (1, 20–22). Anti-inflammatory mediators (e.g., TGFβ and cytokine antagonists) are also present in plasma during SIRS, with soluble cytokine receptors often found at concentration higher than the respective cytokines (20, 23), hence compensatory anti-inflammatory responses [CAR (24)] may be activated during SIRS. Understanding the responses which occur in non-infective inflammation and sepsis and how they are regulated will aid development of diagnostics and therapeutics in these major inflammatory diseases.

MicroRNAs (miRNAs) are small (~23 nt) RNAs that function as post-transcriptional gene regulators (25, 26). By annealing to complementary sequences in the 3′ untranslated region (3′ UTR) of target mRNAs, they reduce expression of specific proteins by promoting target degradation or inhibition of translation (26). The role of miRNAs in SIRS remains unclear, although many inflammatory cytokines, mediators, and their regulators are miRNA targets (27). Approximately 100–200 miRNAs are found in human blood in health and disease (28–30). Previous studies (31, 32) suggest that exogenous miRNAs may be detected by TLRs (33, 34), particularly TLR7 (32), as potential DAMPs leading to inflammatory signals. We previously identified a pool of circulating inflammatory-relevant miRNA (CIR-miRNAs) that robustly distinguish sepsis from non-infective SIRS (35). We were the first to find that, in these conditions, CIR-miRNA levels inversely correlate with those of inflammatory cytokines (35), suggesting that they may be part of the CAR.

To extend these observations, we set out to determine how CIR-miRNAs reflect SIRS severity and whether they are likely to arise directly from immune activation. In this study, we investigated the correlation of plasma levels of CIR-miRNAs with disease severity and other inflammatory parameters. We found that SIRS severity significantly impacts on the levels of miR-378a-3p, miR-30a-5p, miR-30d-5p, and miR-192-5p. These CIR-miRNAs are unlikely to derive from red blood cell (RBC) damage consequent to hemolysis or coagulopathy. Instead their levels positively correlate with levels of the redox biomarker, Prdx-1, which is released by immune cells during inflammation (36, 37). Finally, we show that activated immune cells produce SIRS-associated miR-378a-3p, miR-30a-5p, miR-30d-5p, and miR-192-5p, in vitro. Our study has implications for furthering the understanding of the origin and role and trafficking of CIR-miRNAs in SIRS, sepsis, and potentially other inflammatory disease.

We report here that, during non-infective SIRS, the blood levels of CIR-miRNAs increase in parallel with the severity of inflammation. Levels of CIR-miRNAs do not correlate with those of free Hb, indicating that they do not derive from RBCs during SIRS. Instead, CIR-miRNAs positively correlate with levels of the inflammatory mediator and redox enzyme, Prdx-1 which is released by immune cells in inflammation. Consistently miR-378a-3p, miR-30a-5p, miR-30d-5p, and miR-192-5p affected by severity of SIRS are produced by immune cells upon activation. As CIR-miRNAs are increasingly proposed as biomarkers for sepsis, cancer, and other disease (35, 53–55), their inflammatory cell origin should be considered in future research.

In our study, CIR-miRNAs distinguish non-severe from severe SIRS better than inflammatory cytokines and mediators. Inflammatory cytokines are thought to be released during SIRS as part of the cytokine storm, during MODS (1). As in our current study, plasma levels of cytokines have not previously been found to reliably increase in severe SIRS (1), perhaps due to low cytokine concentration and short half-life in samples (56, 57). By contrast, regulators of inflammatory mediators are abundant in the blood of SIRS patients (20), as we found to be the case for the CIR-miRNAs affected by SIRS severity (Figure 3; Figure S3 in Supplementary Material). In addition, it should be noted that blood levels of miR-378a-3p, miR-30a-5p, miR-30d-5p, and miR-192-5p were regulated by disease severity, irrespectively from whether patients were taking anti-inflammatory drugs (at the time of admission) (Figure S8 and Table S8 in Supplementary Material).

CIR-miRNAs may act as regulators of inflammation (35) by targeting the 3′UTR of mRNAs encoding pro-inflammatory cytokines/mediators (58–61). Many miRNAs are involved in inflammation/immune function (27), including miR-378 (62, 63), miR-30a/d (64, 65), miR-192 (64–68), and others found in this study (69). Interestingly, the more severe SIRS is, the more CIR-miRNAs are released, potentially counteracting systemic inflammation as part of the CAR (24). Significantly, direct or indirect targets of miR-378, miR-30a/d, and miR-192 include, among other genes, IL-1A (70), IL-1R (71), IL-17 (72), IL-17RA, and IL-17RE (73). In contrast, CIR-miRNA levels did not correlate with sepsis severity (compare Tables 1 and 2; Tables S1 and S2 in Supplementary Material), suggesting that inflammatory pathways leading to CIR-miRNA accumulation in blood are dysregulated in sepsis. Consequently, upon sepsis progression, the negative regulation exerted by CIR-miRNAs may be restrained, thus boosting immunopathology. Inflammatory cytokine (IL-1, IL-6, etc.) levels increased consistently much more in sepsis than in SIRS (35), therefore inflammatory mRNAs may act as a “sponge” to sequestrate miRNAs in sepsis, but not in other trauma including SIRS (74). Although miR-30a was recently associated with induction of myeloid derived suppressor cells in cancer (75), the question of whether CIR-miRNAs are anti-inflammatory and ameliorate sepsis outcome remains to be further investigated.

Alternatively, CIR-miRNAs may act as modulators of inflammation via indirect effects (i.e., by not targeting directly inflammatory cytokine expression). For instance, at least in mice, miR-378 is known to target phosphoinositide 3 kinase (PI3K) expression in liver cells, affecting liver metabolism with systemic effects which may be relevant during systemic inflammatory disease and MODS (76). Furthermore, PI3K pathway is also crucially regulated during immune cell proliferation, differentiation, and apoptosis (77), all of which may affect immune cell function during SIRS. Similarly, miR-30a has been shown to target the expression of Blimp-1 (78) (an important differentiation factor in immune cells) and various members of the calcineurin signaling pathway, including NFATc3 (79) at least in podocytes and cardiomyocytes, which might have implications for MODS and inflammation. Finally, miR-192 has been shown to suppress cell-proliferation pathways in myeloma (80) and other leukemic cells (81). However for all abovementioned cases, whether the same targets are also regulated in normal immune cells remains so far elusive.

Altogether, our results have implications for clinical practice and future therapeutic interventions. In particular in ICU/HDU, using miRNAs to distinguish non-infective SIRS from sepsis and severe from non-severe SIRS could help guide patient management, for example, triaging patients based on severity, informing decisions based on prognosis, and helping target therapy including the need for and choice of antibiotics. In future, miRNAs may become markers and/or targets for novel immunotherapy for acute inflammatory conditions, potentially providing a non-antibiotic alternative intervention.

The origin of circulating miRNAs is unclear. RBCs contain miRNAs that they release upon hemolysis (39, 40). To exclude artifacts from sample processing, we removed hemolytic samples and used plasma rather than serum, as the latter contains more Hb (and RBC miRNAs) due to coagulation damage (82, 83). Coagulopathy may drive pathophysiological levels of hemolysis and release of CIR-miRNAs, as seen for miR-486-5p and miR-451a [abundant in RBCs (29)]. Although free Hb levels did not significantly increase with SIRS severity, levels of multiple CIR-miRNAs inversely correlate with free Hb, indicating that these do not derive from RBCs in SIRS as in sepsis.

Our data suggest that CIR-miRNAs affected in SIRS may derive from inflammatory cells activated during disease. In particular, CIR-miRNAs may be released in the blood in association with inflammation-induced oxidative stress, as suggested by the positive correlation between Prdx-1 and multiple CIR-miRNAs. Prdx-1 is an anti-oxidant enzyme that mediates the elimination of H₂O₂ and can be secreted as a dimer by macrophages, upon TLR triggering (36, 37). Its expression is regulated mainly by the transcription factor Nrf2, activated by ROS and various other reactive, electrophilic species (84). Its induction may be an indicator of a protective response to oxidative stress (85, 86). Because Prdx-1 is associated with immune cells activation (37), we asked whether SIRS-relevant CIR-miRNAs were derived from circulating immune cells upon activation. We found that miRNAs affected by SIRS severity were indeed increased >10-fold in supernatants of PBMC cultures after activation with bacterial SAg that drove ~1.5-fold cell-expansion. Thus, sustained immune cell activation may explain the massive release of miRNAs regulated during severe SIRS. Macrophages/monocytes, T and NK cells could be possible candidates for the release of CIR-miRNAs.

Of note, activated PBMCs did not produce significant increase in the supernatant levels of miR-10b-5p, which was not affected by SIRS severity (Figure S3 in Supplementary Material). Unlike other miRNAs, the modest (<2-fold) increase of miR-122-5p upon SAg stimulation may be explained simply by cell proliferation, suggesting that PBMCs contribute little to miR-122-5p blood levels. As hepatocytes highly express miR-122 and increased blood levels of miR-122 were previously associated with liver pathology (87), epithelial cell release of miR-122 and potentially other CIR-miRNAs during SIRS should be further investigated in future.

In conclusion, our work opens up a number of questions about CIR-miRNAs for future investigation. Currently, the exact function of circulating miRNAs in inflammatory conditions remains unknown. CIR-miRNAs released by cells in the body after inflammatory damage may act as DAMPs that bind to TLR ligands (33), a property that is shared by Prdx-1 (86). For instance, small RNAs contained in exosomes can be phagocytosed in myeloid-origin, antigen-presenting like cells and trigger TLR7 signaling, in vitro (31). In this context, miRNAs may act as pro-inflammatory DAMPs to drive inflammatory cytokines (including IL-6) production downstream of intracellular TLRs (31). However, we previously found that pro-inflammatory cytokine levels inversely correlate with blood levels of CIR-miRNAs, suggesting that CIR-miRNAs may negatively regulate inflammation in vivo (35). In mice, regulatory T cells (Tregs) have been shown to dampen the activity of conventional T cells by miRNA transfer (88). Yet, it is unclear whether miRNAs from Tregs can exert suppression at distance. Furthermore, beyond targeting cytokine and inflammatory mediator expression, CIR-miRNAs may regulate inflammation also via indirect mechanisms, or a complex combination of direct and indirect mechanisms, by acting in concert on distinct targets, in multiple cell types, and in different organs. The dynamic contribution of immune cells to blood miRNAs has been frequently overlooked, despite inflammation clearly affecting them. Thus, while differences in miRNA transfer may exist in vivo compared to in vitro, future research is needed to clarify the role of CIR-miRNAs in immunopathological and homeostatic conditions. In particular, more research is needed to address which immune cells specifically produce CIR-miRNAs to be released in the blood, including when and how. Also the mode of trafficking and the cellular and molecular targets of CIR-miRNAs need to be identified yet. In future, such research may allow to harness CIR-miRNAs as immunomodulatory drugs useful for therapeutic purposes in inflammatory conditions as in other diseases.

Written informed consent or consultee approval to enroll was secured for all study participants (patients and healthy donors). This study was approved by the North Wales Research Ethics Committee (Central and East, reference 10/WNo03/19) and the BSMS Research Governance and Ethics Committee (reference: 13/182/LLE) and carried out in accordance with the approved guidelines. All data were anonymized.

SC and MM performed experiments and analyzed the data; SC, PG, SN, FK, and ML conceived and designed experiments; SC, SN, and ML wrote the first manuscript draft. All authors critically contributed to the final version of the manuscript.

The authors have no conflict of interest directly related to the current manuscript. However, SC, FK, SN, and ML are inventors in a patent submitted by the University of Sussex for the use of CIR-miRNAs as biomarkers of sepsis.