Regulatory Role of Non-Coding RNAs on Immune Responses During Sepsis

Sepsis is resulted from a systemic inflammatory response to bacterial, viral, or fungal agents. The induced inflammatory response by these microorganisms can lead to multiple organ system failure with devastating consequences. Recent studies have shown altered expressions of several non-coding RNAs such as long non-coding RNAs (lncRNAs), microRNAs (miRNAs) and circular RNAs (circRNAs) during sepsis. These transcripts have also been found to participate in the pathogenesis of multiple organ system failure through different mechanisms. NEAT1, MALAT1, THRIL, XIST, MIAT and TUG1 are among lncRNAs that participate in the pathoetiology of sepsis-related complications. miR-21, miR-155, miR-15a-5p, miR-494-3p, miR-218, miR-122, miR-208a-5p, miR-328 and miR-218 are examples of miRNAs participating in these complications. Finally, tens of circRNAs such as circC3P1, hsa_circRNA_104484, hsa_circRNA_104670 and circVMA21 and circ-PRKCI have been found to affect pathogenesis of sepsis. In the current review, we describe the role of these three classes of noncoding RNAs in the pathoetiology of sepsis-related complications.


INTRODUCTION
Sepsis is a systemic inflammatory response to different infections, namely bacterial, viral, or fungal agents. This condition is the principal source of mortality in intensive care units (1). These infectious microorganisms can stimulate inflammatory reactions through induction of cytokines release. These reactions lead to multiple organ system failure. Other factors that contribute in this devastating condition during sepsis are systemic hypotension and abnormal perfusion of the microcirculatory system (2). No specific treatment modality has been suggested for prevention of multiple organ system failure during sepsis (2). Thus, identification of sepsis-related changes at cellular and biochemical levels is important. Currently, there is no effective pharmacological therapy for sepsis. Thus, early diagnosis, resuscitation and instant administration of suitable antibiotics are essential steps in decreasing the burden of this condition {Thompson, 2019 #562}.
Lipopolysaccharide (LPS) as the main constituent of the cell wall of Gram-negative bacteria has been found to stimulate apoptotic pathways in tubular epithelial cells of kidney (3). Moreover, it can prompt acute inflammatory responses through activation of NF-kB during the course of acute kidney injury (4). This molecular pathway is an important axis in mediation of immune-related organ damage.
Recent studies have shown altered expressions of several non-coding RNAs such as long non-coding RNAs (lncRNAs), microRNAs (miRNAs) and circular RNAs (circRNAs) during sepsis. These transcripts have also been found to participate in the pathogenesis of multiple organ system failure through different mechanisms. In the current review, we describe the role of these three classes of noncoding RNAs in the pathoetiology of sepsis-related complications.

LNCRNAS AND SEPSIS
LncRNAs are transcripts with sizes larger than 200 nucleotides. These transcripts regulate gene expression through modulation of chromatin configuration, regulation of splicing events, serving as decoys for other transcripts and making structures for recruitment of regulatory proteins (5). These transcripts participate in the regulation of immune reactions and pathoetiology of several immune-related disorders (6). Experiments in animal model of acute lung injury have shown down-regulation of TUG1 and induction of apoptosis and inflammation. Up-regulation of TUG1 in these animals could ameliorate sepsis-associated lung injury, apoptosis and inflammatory reactions. TUG1 could also protect lung microvascular endothelial cells from deteriorative effects of LPS. In fact, TUG1 inhibits cell apoptosis and inflammatory reactions in LPS-stimulated microvascular endothelial cells through sponging miR-34b-5p and releasing GAB1 from its inhibitory effects. Cumulatively, TUG1 ameliorates sepsisassociated inflammation and apoptosis through miR-34b-5p/ GAB1 axis (7). Another study has demonstrated downregulation of TUG1 while up-regulation of miR-223 in the plasma samples of sepsis patients. They have also reported a negative correlation between expressions of TUG1 and miR-223 in sepsis patients. Besides, expression levels of TUG1 have been negatively correlated with respiratory infection, serum creatinine, white blood cell, C-reactive protein, APACHE II score, and SOFA score. Based on these results, TUG1 has been suggested as a biomarker for prediction of course and prognosis of sepsis (8). TUG1 has also been shown to interact with miR-27a. Over-expression of TUG1 has resulted in down-regulation of TNF-a, while up-regulation of miR-27a has enhanced expression of TNF-a in cardiomyocytes. TNF-a and miR-27a up-regulation could enhance LPS-induced apoptosis of cardiomyocytes. On the other hand, TUG1 up-regulation has exerted opposite effects (9).
MALAT1 is another lncRNA that affects immune responses of rats with LPS-induced sepsis through influencing the miR-146a/NF-kB P65 axis (10). Moreover, MALAT1 could increase apoptosis skeletal muscle cells and sepsis-associated immune responses through down-regulating BRCA1 levels via recruitment of EZH2 (11). The miR-150-5p/NF-kB axis is another axis that mediates the effects of MALAT1 in sepsisassociated cardiac inflammation (12). In addition, the protective effects of Ulinastatin against LPS-associated dysfunction of heart microvascular endothelial cells have been shown to be exerted through down-regulation of MALAT1 (13). Most notably, MALAT1/miR-125a axis has been shown to discriminate sepsis patients based on their severity of diseases, organ damage, levels of inflammatory responses and mortality (14). Figure 1 depicts function of MALAT1 in sepsis-related events.
NEAT1 is another lncRNA whose participation in the pathophysiology of sepsis has been vastly investigated. This lncRNA could promote inflammatory responses and aggravate sepsis-associated hepatic damage through the Let-7a/TLR4 axis (15). Moreover, NEAT1 can accelerate progression of sepsis via miR-370-3p/TSP-1 axis (16). This lncRNA could also promote LPS-induced inflammatory responses in macrophages through regulation of miR-17-5p/TLR4 axis (17). NEAT1 silencing could suppress immune responses during sepsis through miR-125/ MCEMP1 axis (18). Figure 2 shows the function of NEAT1 in sepsis-related events. Several other lncRNAs have also been found to influence course of sepsis through modulation of immune responses ( Table 1).

miRNAs AND SEPSIS
miRNAs have sizes about 22 nucleotides and regulate expression of genes through binding with different regions of target mRNAs, particularly their 3' UTR. They can either degrade target mRNA or suppress its translation. Several miRNAs have been found to influence course of sepsis. Altered expression of these small-sized transcripts has been reported in sepsis by numerous research groups. For instance, plasma levels of miR-494-3p have been shown to be decreased in sepsis patients compared with healthy controls in correlation with up-regulation of TLR6. Expression level of miR-494-3p has been decreased in LPS-induced RAW264.7 cells, parallel with up-regulation of TLR6 and TNF-a. Forced over-expression of miR-494-3p in RAW264.7 cells could reduce TNF-a level and suppress translocation of NF-kB p65 to the nucleus.  Table 1).  TLR6 has been shown to be targeted by miR-494-3p. Taken together, miR-494-3p could attenuate sepsis-associated inflammatory responses through influencing expression of TLR6 (132). miR-218 is another miRNA which participates in the pathoetiology of sepsis. This miRNA could reduce inflammatory responses in the sepsis through decreasing expression of VOPP1 via JAK/STAT axis (133). miR-122 is another important miRNA in the sepsis which has superior diagnostic power compared with CRP and total leucocytes count for distinguishing sepsis from wound infection. miR-122 has also been found to be a prognostic marker for sepsis, albeit with poor specificity and accuracy values (134).
In the mice model of sepsis, decreased levels of miR-208a-5p and increased levels of SOCS2 has been associated with enhanced activity of SOD, while reduction in LDH and MDA activities. Moreover, down-regulation of miR-208a-5p has been associated with low levels TNF-a, IL-6, NF-kB p65 and HIF-1a in this animal model. miR-208a-5p silencing could decrease the extent of mitochondria swelling, and inhibit apoptosis of cardiomyocytes in animal model of sepsis. Taken together, miR-208a-5p suppression has been suggested as a modality to attenuate sepsis-related myocardial damage. This function is mediated through NF-kB/HIF-1a axis (135). miR-21 is another miRNA whose role in sepsis has been investigated by several groups. Down-regulation of miR-21 has been shown to inhibit inflammasome activation, ASC pyroptosome, LPS-induced pyroptosis and septic shock in one study (136). On the other hand, another study in animal models of sepsis has shown that up-regulation of miR-21 reduced inflammation and apoptosis (137). Similarly, bMSCs-derived exosomes have been shown to reduce symptoms in septic mice and improve their survival rate through up-regulation of miR-21 (138). miR-328 is another miRNA which is dysregulated in sepsis patients as well as animal models of sepsis. Serum levels of this miRNA could properly differentiate sepsis from normal conditions. Thus, miR-328 has been suggested as a diagnostic biomarker for sepsis. Moreover, down-regulation of miR-328 could amend sepsis-related heart dysfunction and inflammatory responses in this tissue (139). miR-452 is another miRNA with diagnostic applications in sepsis. Notably, serum and urinary levels of this miRNA have been suggested as possible markers for early diagnosis of sepsis-associated acute kidney injury, since expression of this miRNA has been higher in sepsis patients with acute kidney injury compared with those without this condition (140) ( Table 2). Figure 3 depicts miRNAs that are downregulated in sepsis.

CircRNAs AND SEPSIS
CircRNAs are a recently appreciated group of non-coding RNAs with enclosed circular configuration formed by covalent bonds between two ends of linear transcripts. However, some of these transcripts have been shown to produce proteins. They mostly exert regulatory functions in the transcriptome. Impact of circRNAs in the sepsis has been assessed by several groups (303). For instance, circC3P1 has been shown to attenuate production of inflammatory cytokines and decrease cell apoptosis in sepsis-associated acute lung injury via influencing expression of miR-21 (304).
A microarray-based has shown differential expression of 132 circRNAs between sepsis patients and healthy controls am ong them have been hsa_circRNA_10 448 4 a nd hsa_circRNA_104670 whose up-regulation in sepsis serum exosomes has been verified been RT-PCR. Expression levels of these two circRNAs have been suggested as diagnostic biomarkers for sepsis (305).
CircVMA21 is another circRNA that has been shown to ameliorate sepsis-related acute kidney injury through modulation of oxidative stress and inflammatory responses via miR-9-3p/SMG1 axis (306). Circ_0114428/miR-495-3p/CRBN axis is another molecular axis which is involved in the pathoetiology of sepsis-related acute kidney injury (307). Moreover, expression levels of circPRKCI have been correlated with sepsis risk, severity of sepsis and mortality during a period of 28 days (308). Table 3 summarizes the role of circRNAs in sepsis.
Downregulation of CRNDE reduced edema, necrosis and apoptosis in sepsis-induced AKI.
Up-regulation of CRNDE enhanced cell injuries, inflammatory responses and apoptosis in sepsisinduced AKI.
Downregulation of CRNDE increased the urea nitrogen and serum creatinine, and reduced proliferation and promoted apoptosis.
_ Up-regulation of CRNDE increased viability and repressed apoptosis in sepsis-induced liver injury.   NF-kB, PI3K/AKT, JAK/STAT and Wnt/b-catenin pathways are the most important pathways being regulated by lncRNAs, circRNAs and miRNAs in the context of sepsis. These transcripts, particularly miRNAs can be used as diagnostic or prognostic markers in sepsis. Expression levels of these regulatory transcripts might be used for diagnosis of organ specific damages during the course of sepsis.
In general, the pathophysiology of sepsis is considered as an initial hyperinflammatory phase ("cytokine storm") followed by a protracted immunosuppressive phase. Since no data is available about the differential expression of non-coding RNAs during these two distinct phases, future studies are needed to evaluate expression patterns of non-coding RNAs in these two phases. It is possible that some of the non-coding RNAs that suppress the immune response could be used as biomarkers to indicate the immunoparalysis in sepsis.
From a therapeutic point of view, several in vitro and in vivo studies have shown that up-regulation/silencing of circRNAs, HCs, healthy controls; AKI, acute kidney injury; ARDS, acute respiratory distress syndrome.
lncRNAs and miRNAs can ameliorate the pathologic events in the target organs, particularly heart and kidney during sepsis. Yet, this field is still in its infancy needing verification in additional animal models and cell lines. Moreover, since sepsis is an emergency situation, any therapeutic option should be verified in terms of bioavailability, efficiency and instant amelioration of pathological events.
Since the pathoetiology of sepsis-related complications is not completely understood, high throughput sequencing strategies focusing on different classes of non-coding as well coding RNAs are necessary to find the complicated networks between these transcripts in the context of sepsis.

AUTHOR CONTRIBUTIONS
SG-F wrote the draft and revised it. MT designed and supervised the study. NA, BH, and TK collected the data and designed the figures and tables. All authors contributed to the article and approved the submitted version.