Next Article in Journal
Comparing Viral Vectors and Fate Mapping Approaches for Astrocyte-to-Neuron Reprogramming in the Injured Mouse Cerebral Cortex
Previous Article in Journal
Correlation between Periostin Expression and Pro-Angiogenic Factors in Non-Small-Cell Lung Carcinoma
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Cells
Volume 13
Issue 17
10.3390/cells13171407
cells-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Review

Emerging Roles of Circular RNA in Macrophage Activation and Inflammatory Lung Responses

by
Chang Jun Son
1,†,
Jonathan M. Carnino
1,†,
Heedoo Lee
1,2 and
Yang Jin
1,*
1
Division of Pulmonary and Critical Care Medicine, Department of Medicine, Boston University, Boston, MA 02118, USA
2
Department of Biology and Chemistry, Changwon National University, Changwon 51140, Republic of Korea
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Cells 2024, 13(17), 1407; https://doi.org/10.3390/cells13171407
Submission received: 16 July 2024 / Revised: 20 August 2024 / Accepted: 20 August 2024 / Published: 23 August 2024
Browse Figure
Versions Notes

Abstract

:
Circular RNA (circRNA) is a type of single-stranded RNA that forms a covalently closed continuous loop, unlike linear RNA. The expression of circRNAs in mammals is often conserved across species and shows tissue and cell specificity. Some circRNA serve as gene regulators. However, the biological function of most circRNAs is unclear. CircRNA does not have 5′ or 3′ ends. The unique structure of circRNAs provides them with a much longer half-life and more resistance to RNase R than linear RNAs. Inflammatory lung responses occur in the pathogenesis and recovery of many lung diseases. Macrophages form the first line of host defense/innate immune responses and initiate/mediate lung inflammation. For example, in bacterial pneumonia, upon pro-inflammatory activation, they release early response cytokines/chemokines that recruit neutrophils, macrophages, and lymphocytes to sites of infection and clear pathogens. The functional effects and mechanisms by which circRNAs exert physiological or pathological roles in macrophage activation and lung inflammation remain poorly understood. In this article, we will review the current understanding and progress of circRNA biogenesis, regulation, secretion, and degradation. Furthermore, we will review the current reports on the role of circRNAs in macrophage activation and polarization, as well as in the process of inflammatory lung responses.

1. Introduction

Non-coding RNAs are emerging targets for developing novel therapeutic and diagnostic strategies. Among all the types of non-coding RNAs, unlike long non-coding RNAs (lncRNAs) and microRNAs (miRNAs), circular RNAs (circRNAs) carry unique features that have attracted emerging attention recently. Circular RNAs were initially reported in 1976 as RNA viruses [1]. Several years later, the circular form of RNA was identified under electron microscopy in the cytoplasm of eukaryotic cells [2] and mitochondria of yeast [3]. Initially, most circRNAs were thought to be generated by mis-splicing and had no meaningful biological functions [4]. With the development of deep RNA sequencing and bioinformatics, circRNAs are found widely expressed in different species including mammalian cells [5,6,7]. Recently, emerging evidence has shown that circRNAs carry an essential role in various physiological and pathological processes. The involvement of circRNAs in human diseases has been reported, including but not limited to cancer [8,9], cardiovascular disease [10], and neuronal diseases [11]. In the setting of host defense, innate immunity, and inflammatory responses in response to noxious stimuli, circRNAs have been reported to exert antiviral effects [12]. Furthermore, circRNAs are differentially detected in patients with systemic lupus erythematosus (SLE), suggesting their potential to serve as novel biomarkers [13] in the process of inflammation. Inflammatory responses are common features in the lungs in the process of host defense against noxious stimuli, including both infectious and sterile stimuli, such as bacterial, and viral infections, acid inhalational injury, and hyperoxic injury [14,15]. Macrophages including alveolar macrophages (AMs) are the main immune cell population in the lungs and alveolar sacs. They are responsible for maintaining homeostasis and serve as the first line of defense against external insults [16,17]. In this review, we will discuss the current updates on the potential role of circular RNAs in lung inflammation and macrophages.

2. Circular RNA Characteristics, Biogenesis, Degradation, and Removal from Intracellular Compartments

Circular RNAs (circRNAs) represent a distinct category of noncoding RNAs characterized by their covalently closed loop structure. This design provides them with a notable resistance to enzymatic breakdown because they do not have the 5′ caps and 3′ tails that are usually targeted by exonucleases. Consequently, circRNAs have at least a 2.5-fold longer half-life than linear RNAs, enhancing their ability to fulfill various roles, such as serving as biomarkers and therapeutic targets for human diseases. [18,19,20,21] The sequences of circRNAs are conservative to a certain extent [21]. Most circRNAs are located in the cytoplasm and some are in the nucleus such as the exon-intro circRNAs (EIcirRNAs) [22,23]. Most circRNAs belong to ncRNAs and are produced by exons [24] despite that certain single gene loci can be cycled into multiple circRNAs and translated into peptides/proteins [24].
CircRNA biogenesis is a specific process unique to each cell type. Most circRNAs are produced utilizing a spliceosome-dependent process known as back-splicing. During this process, a splice donor site downstream connects to an upstream splice acceptor site, crafting a circRNA molecule devoid of the linear RNA’s conventional 5′ cap and 3′ poly-A tail. This circular formation is facilitated by RNA-binding proteins and the intron sequences flanking the exons that will circularize, promoting the close alignment necessary for circRNA creation [25,26,27,28,29,30]. As shown in Schema 1, various types of circRNA formation have been identified. Besides the most recognized spliceosome-mediated back-splicing, other types of circRNAs exist, such as intron-derived nuclear circRNAs. Unfortunately, RNA-seq reads that span back splicing junctions often do not reveal the internal structure of the circular RNA. The most formed circRNAs are produced by exons sharing the same sequences in the cognate linear RNA. CircRNA and its host linear RNA are normally coexpressed. Therefore, the back-splice junction (BSJ) is the only sequence that allows unambiguous identification of the circular form [25,26,27,28,29,30]. To date, circRNA biogenesis is not completely understood. Under the assistance of specific RNA-binding proteins (RBPs), base pairing between inverted repeats in the flanking introns potentially induces back-splicing (Figure 1) [25,26,27]. Based on their sequences, circRNAs are divided into four classes. In addition to the intronic circRNAs (ciRNAs) mentioned above, exonic circRNAs contain two or more exons or their partial sequences. The third type of circRNA refers to the exon-intron circRNAs (EIciRNAs), which retain introns between exons. The fourth type of circRNAs has recently been reported, referring to the tRNA intronic circular RNAs, which are spliced from the endonuclease complex. The differential mechanisms involved in the biogenesis of circRNAs dictate the specific locations of these circRNAs. For example, ciRNAs are mainly located in the nucleus [28]; exonic circRNAs are often distributed in the cytoplasm and nucleus [29]; and EIciRNAs are located mainly in the nucleus, like ciRNAs [30].
As mentioned above, due to the circular form and the absence of a free 5′ cap and 3′ poly A ends, circRNAs are more resistant to exonucleases [5,19], resulting in the accumulation of circRNAs intracellularly. In certain scenarios, the intracellular level of circRNAs exceeds their counterpart linear form RNAs. This raises important questions, e.g., what are the consequences of accumulated circRNAs in the cells? Are cells capable of removing the unwanted circRNAs? Are any human diseases associated with the accumulation of circRNAs? [5,19,31]. Can the circRNAs potentially be diagnostic markers or, more importantly, therapeutic targets?
Although we do not have all the answers to the above questions, a partial understanding of the fates of circRNAs has revealed that cells remove intracellular circRNAs via active secretion. BSJ-containing circRNAs are exported to the cytoplasm. Numerous reports have further demonstrated the presence of abundant circRNAs in extracellular vesicles (EVs) and exosomes. The higher amount of circRNAs were enriched in exosomes compared to the producer cells [32,33], suggesting that EV (exosome)-mediated secretion is one of the mechanisms by which cells remove the accumulated intracellular circRNAs.
There are several hypotheses regarding the encapsulation of circRNAs into EVs. In 2015, circRNAs were first reported to be stably enriched in exosomes [23]. The abundance of circRNAs in exosomes is increased by at least 2- to 6-fold compared with their intracellular levels, suggesting that circRNAs are transported into EVs via an active process. More than 1000 different circRNA candidates have been identified in human serum exosomes [34]. One of the studies has further shown that KRAS (Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) mutations lead to a differential abundance of each specific exosomal-cargo circRNA. Ref. [35], suggesting that circRNAs are selectively packaged into EVs. Interestingly, circRNAs are more pronounced in exosomes, the smaller size EVs, which usually have a different mechanism of generation compared with larger size EVs, which usually are generated via direct plasma membrane budding. EVs are a general term describing lipid bilayer vesicles that are released from cells ranging from 20 to 5000 nm [36]. Based on the current guidelines issued by the International Society of Extracellular Vesicles (ISEV) in 2024 [36], small EVs formerly called exosomes refer to the EVs with a diameter of ~ 30–200 nm that are produced by the fusion of multivesicular bodies (MVBs) with the cell plasma membrane [37]. The molecular mechanisms of exosome biogenesis remain incompletely explored. Early endosomes, the intraluminal vesicles (ILVs), and MVBs are all involved in exosome generation and dynamically communicate with other organelles, such as the trans-Golgi network (TGN) and the endoplasmic reticulum (ER), mitochondrion, or phagosome [38,39,40,41]. MVBs eventually fuse with lysosomes to be degraded or fuse with the plasma membrane to generate exosomes (Schema 1). Larger EVs including formerly called microvesicles (MVs) are often generated by directly budding from the cell membrane [36]. Based on the different mechanisms of EV biogenesis, it is not surprising to us that exosomes consist of the majority of circRNA cargos, given that exosomes experience a long journey from ILV formation along with various cargo incorporations to the fusion of MVBs with the cell membrane. CircRNA-containing mature MVBs can fuse with lysosome or autophagosome to be degraded. That said, circRNAs are much more resistant to nuclease-mediated degradation. It remains unclear what percentage of the MVB-transported circRNAs are degraded in the lysosomes and what percentage of the circRNAs are recycled into the intracellular vesicle systems or secreted via exosomes.
Scattered reports involving circular RNA degradation show that specific endonucleases participate in circRNA degradation. RNase H1 cleaves the R-loops formed between the circular intronic RNAs and the DNA at their expression sites, such as ci-ankrd52. Therefore, ci-ankrd52 can be degraded via RNase H1 [42]. The circRNAs with high GC content are more likely to form R-loops, and cells can recruit RNase H1 to degrade circRNAs in such R-loops [42]. RNase L has been reported to restrict circPTPN22 [43]. Upon inflammation or virus infection, circRNAs have been reported globally degraded by activated RNase L [44]. The second key components involved in circRNA degradation are RNA binding proteins. Argonaute protein-2 (Ago2) recognizes and cleaves the complex formed by miRNA-1224 and pre-circRNA-Filip1l, leading to a decreased mature circRNA-Filip1l in the spinal nucleus [45]. The miRNA-671-circRNA-CDR1 complex is cleaved by Ago2 and degraded afterward [46]. GW182 deletion results in elevated circdati and circlaccase2 levels [47]. GW182 may regulate circRNA degradations in an Ago-slicer- or P-body-independent manner via the ABD and/or the UBA [47]. CircRNAs can also be degraded via structure-mediated RNA decay (SRD). SRD is a specific mechanism by which the highly folded and structured RNA is degraded. RNA-binding proteins, up-frameshift protein 1 (UPF1), and Ras-Gap-SH3 domain-binding protein 1 (G3BP1) facilitate the RNAs to form the highly structured RNA [48]. Recent studies suggest that UPF1 and G3BP1 bind to highly structured base-pair regions of circRNAs and promote circRNA degradation [48].
Thus far, we do not have a complete understanding of the selection of specific circRNAs that are transported from the nucleus to cytoplasm and/or secreted from the cells to exosomes. However, the lengths of mature circRNAs potentially dictate nuclear exportation. Two closely related RNA helicases, URH49 and UAP56, control the transportation of short and long circRNAs from the nucleus, respectively [49]. Next, the post-transcriptional modifications (PTMs) play a role in the nuclear export and/or secretion of the circRNAs. N6-methyladenosine (m6A) modification regulates the circRNA biogenesis, the nuclear export of circRNAs, and the degradation of circRNAs [50,51,52,53,54,55]; YTHDC1, an m6A reader, promotes the nuclear export of m6A-modified circNSUN2 [50]. EIF4A3, another m6A reader, increases the nuclear export of circPRKCI [51,52]. The deletion of Exportin 4 (XPO4) results in the nuclear accumulation of exonic circRNAs, subsequently leading to the formation of harmful R-loops [53].
There are several hypothetic mechanisms of the circRNAs sorting into exosomes. First, the size of the circRNAs potentially dictates their encapsulation into the exosomes. The average size of the circRNAs that were not secreted from cells was 459 nucleotides (nts), while the average size of the circRNAs released by exosomes was 435 nts [54]. Next, RNA binding proteins (RBPs), such as hnRNPs, have been shown to mediate microRNA(miRNA) encapsulations into the exosomes [55]. MiRNAs and other long-non-coding RNAs potentially competitively regulate circRNA sorting into exosomes. One example is that the deletion of lncRNA UCA1 in serum exosomes leads to an elevated circHIPK3 expression, indicating a competitive mechanism of lncRNA UCA1 and exosomal circHIPK3 [56]. The sponging effects of miR-7 against circCDR1 raised intracellular levels of miR-7, diminished the circCDR1 in exosomes, and up-regulated the circCDR1 level in the cells [23]. That said, the regulations and mechanisms involved in the exosomal secretion of circRNAs remain largely unexplored.

3. Circular RNA and Its Potential Roles in Macrophage Activation and Inflammatory Lung Responses

CircRNA has been reported to regulate a variety of human diseases including but not limited to aging-related diseases, cancer, cardiovascular diseases, diabetes, osteoarthritis, stress, and viral diseases [57,58,59,60,61,62,63,64]. CircRNAs have also been reported to be involved in pulmonary fibrosis, acute respiratory distress syndrome (ARDS), cystic fibrosis, pulmonary hypertension, pulmonary tuberculosis, asthma, and silicosis [65,66,67,68,69,70,71,72,73,74]. Inflammatory lung responses often play an important role in the pathogenesis of these pulmonary disorders. Macrophages are the first-line cells in host defense/innate immunity and regulate the initiation, maintenance, and resolution of the inflammatory response. Here, we will review the current understanding of the role of circRNAs in inflammatory lung responses and macrophage activations.

4. CircRNAs and Macrophage Activation, Differentiation, and Polarization

CircRNAs are reported to regulate macrophage polarization and activation in response to LPS. CircRNAs can also inhibit macrophage biogenesis [75,76,77]. In one study, approximately 2000 circular RNAs were altered after TLR4 stimulation. In response to LPS stimulation, circRNA circRasGEF1b is stably expressed and required for NF-kB and macrophage activations [75,76,77]. Furthermore, Gonzalez et al. demonstrated the effects of circRNAs on macrophage polarization using cirRNA cdr1as as an example [78]. In this report, the authors performed circRNA microarray analyses in bone marrow-derived macrophages (BMDM) which were differentiated into the pro-and anti-inflammatory phenotypes. Using circ-Cdr1as as an example, the authors investigated the macrophage polarizations using the “gain or loss of function” approaches by deletion or overexpression circ-Cdr1as in BMDMs. The author showed that Cdr1as is one of the most downregulated circRNAs in pro-inflammatory macrophages and robustly upregulated in anti-inflammatory macrophages. Circ-Cdr1as potentially promotes anti-inflammatory markers and steers the cell toward M2 polarization. Circ17725 has also been demonstrated to be downregulated in peripheral blood mononuclear cells (PBMCs) obtained from RA patients. Circ17725 is found to interact with miR-4668-5p-FAM46C. Functionally, circ17725 inhibits the proliferation and enhances the apoptosis of macrophages. Additionally, overexpression of circ17725 decreases the expression of TNF-α, IL-1β, and MMP-9 in Raw264.7 macrophages, suggesting that it promotes macrophage polarization towards M2 by targeting miR-4668-5p/FAM46C as a miRNA sponge. CircANKRD36 sponges miR-330, resulting in increased ROCK1 in LPS-treated Raw264.7 macrophages [79].
One recent report analyzed the circRNA expression in polarized human macrophages. The authors demonstrated that 9720 circular RNAs are detected using RNA sequencing in human THP-1 macrophages. A total of 71 up-regulated circRNAs and 69 down-regulated circRNAs are detected in the M1-geared THP-1 cells, confirmed by Quantitative real-time PCR (qRT-PCR) [80]. Using circRNA-RNF19B (circRNF19B) as an example, it is significantly up-regulated in M1 macrophages. On the other hand, circRNF19B expression is increased when the M2 phenotype is converted to M1 and vice versa. Additionally, circRNAs are differentially expressed in human macrophages after Mycobacterium tuberculosis infection; 32 circRNAs were up-regulated and 110 were down-regulated [81]. The author hypothesized that circ_0043497 and circ_0001204 are potentially effective diagnostic biomarkers for TB. In septic patients, circular RNA ASPH (circASPH) is highly expressed in peripheral blood mononuclear cells. CircASPH levels peaked after 24 h of M1 polarization and after 12 h of M2 polarization in THP1 cells [82]. Deletion of circASPH leads to the downregulation of M1 gene expression and cytokine secretion.
CircRNAs can be secreted into extracellular space via exosomes. Therefore, not only do the endogenous circRNAs play a role in macrophage activation, but the exogenous circRNAs, after entering the target cells, potentially regulate the cellular events of the recipients. Recent reports show that exogenous circRNAs interact with RNA sensors in macrophages [83]. Delivery of exogenous circRNAs potentially stimulates a greater innate immune response than that stimulated by their linear counterparts [83]. The transfection of purified in vitro-produced circRNAs into the recipient cells results in significant innate immune responses against viral infection [83]. Furthermore, Retinoic acid-inducible gene I (RIG-I) has been demonstrated to sense foreign circRNA and co-aggregate in cytoplasmic foci. The introns involved in circRNAs generation play an essential role in the recognition of foreign circRNAs and initiate immune responses. The summary of the recently discovered circRNAs that play a role in regulating macrophage activation, apoptosis, and polarization are listed in Table 1.

5. circRNAs in Inflammatory Lung Responses

Although macrophage forms the first line of innate immunity, inflammatory lung responses involve many more cell types and pathways. Therefore, many more studies have reported the potential roles of circRNAs in the initiation, development, and resolution of lung inflammation and, in the end, lung injury. These reports revealed that not only macrophages but also epithelial cells, endothelial cells, dendritic cells, and fibroblasts all potentially are regulated by circRNAs in the process of inflammatory lung responses. For example, the deletion of circ0038467 reduces LPS-induced inflammatory injury [100]. CircHECTD1 inhibits the apoptosis of AECs [101]. CircPALM2 is increased in LPS-caused MLE-12 cell damage [102]. Circ0054633 is over-expressed in LPS-induced rats and murine pulmonary microvascular endothelial cells, through activating the NF-κB pathways [103]. Deletion of circANKRD36 suppressed cell viability and migration and alleviated inflammation of LPS-treated macrophages, probably serving as a sponge for miR-330 and ROCK1 [79]. Circ_0001679 is upregulated in LPS-induced MLE-12 cells and leads to increased apoptosis [104]. Furthermore, LPS induces circTMOD3 in WI-38 lung fibroblasts, and circTMOD3 sponges miR-146b-3p to induce CXCR1 expression [105]. Circ_VMA21 was downregulated in LPS-treated WI-38 cells after pneumonia, and circ_VMA21 sponges miR-409-3p to induce the expression of Kruppel-like transcription factor 4 (KLF4), an inflammatory palliative in sepsis [106].
However, many more studies did not focus on specific cells; rather, they used in vivo studies to demonstrate the overall picture. Circ42341, circ44122, and circ44123 were robustly upregulated, whereas circ010498, circ25030, and circ010498 were significantly downregulated through microchip analysis in total lung tissue from the LPS-mouse lung injury model [107]. CircC3P1 reduces pro-inflammatory cytokine production and cell apoptosis [108]. CircVMA21 decreases oxidative stress, apoptosis, and inflammation in sepsis rats [109]. Circ0001679 and circ0001212 are increased in septic mice and regulate Nprl3 inflammasome expression [110]. CircNCLN sponge and antagonize miR-291a-3p to alleviate LPS-induced acute lung injury [111]. In the plasma of influenza A-induced lung injury patients, circRNAs Slco3a1 and Wdr33 were aberrantly expressed [84]. CircVPS33A and circ_0000455 are highly expressed in a murine asthma model. CircVPS33A sponges miR-192-5p to increase the level of high-mobility group box 1 (HMGB1), a strong pro-inflammatory mediator [112,113] Many current studies in lung inflammation focus on specific lung cells and lung tissue. Our newly published study showed that the profiles of circRNAs in BALF exosomes are also significantly altered in the setting of bacterial infections [114]. The exosome-cargo circRNAs potentially can travel to adjacent and distant target cells and mediate signal exchanges among cells.

6. circRNAs Derived from Microbes Participate in the Pathogenesis of Lung Inflammation and Injury

In addition to host cell-derived circRNAs, circRNAs derived from microbes contribute significantly to human diseases, including but not limited to respiratory disorders. CircRNAs can be derived from both DNA and RNA viruses. A repertoire of latent and lytic viral circular RNAs has been well studied and reported in the Epstein Barr virus (EBV) transcriptome [115]. Its oncogenic role in liver disease has been studied. EBV circRNAs have also been detected in patient samples [116]. In brief, the mechanisms through which circRNAs regulate pathogenesis include but are not limited to miRNA sponges [117], protein sponges, regulating parental gene expression, and peptide translating [118,119].
Besides the well-studied circRNAs in liver diseases, virus-derived circRNAs have also been reported in respiratory disorders. For example, circRNAs are reported to be generated from MERS-CoV, SARS-CoV-1, and SARS-CoV-2, the RNA coronaviridae that cause acute lung injury and have caused several endemic/pandemic episodes [120]. In 2022, Yang et al. predicted that 351, 224, and 2764 circRNAs derived from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, and Middle East respiratory syndrome coronavirus, respectively [121]. They further confirmed that 75 SARS-CoV-2 circRNAs are potentially derived from SARS-CoV-2. Computational analysis indicated that SARS-CoV-2–circRNAs–miRNAs potentially promote the host genes involved in the response to the virus and muscular skeletal development [122]. Further biomedical informatic analysis suggests that the viral circular RNAs target the host genes regulating, “cytokine receptor binding” and “growth factor activity”. For example, SARS-CoV-2 circRNA 29122|28295 contains the binding site of hsa-miR-3194-5p, and functions as a sponge for host cellular miRNAs, subsequently manipulating the downstream host gene GPR115.
In addition to virus-derived circRNAs, circular RNAs derived from bacteria have been reported lately. He et al. demonstrated that a regulatory noncoding RNA (DucS) carries both linear and circular conformation derived from Bacillus altitudinis [123]. The circular form of DucS is likely regulated by the 3′ end sequences of the linear forms. Interestingly, the linear forms of DucS enhance the tolerance of oxidative stress by B. altitudinis via increased translation of htrA stress-responsive gene. The formation of circular DucS potentially decreases the levels of the regulatory linear counterpart, leading to intolerance to H2O2. Moreover, via bioinformatic analysis, 30 bacterial species have been identified to potentially produce circular forms of RNAs. The authors validated four and seven circular RNAs from Bacillus paralicheniformis and B. altitudinis, respectively. However, the predicted circular RNAs from Bacillus subtilis and Escherichia coli were not confirmed, suggesting the circulation of specific RNAs may not be conserved among different bacterial species.
Trichophyton rubrum (T. rubrum) belongs to human pathogenic filamentous fungi. Cao et al. identified 4254 circRNAs in T. rubrum and found 940 differentially expressed circRNAs between the conidial and mycelial stages [124]. Interestingly, they found that unlike the circRNAs discovered in plants and animals, most circRNAs in fungi originated from intergenic regions. Functional analysis suggested that these circRNAs may regulate posttranscriptional processes and protein synthesis. Numerous other reports confirmed that circRNAs exist in the world of fungi that cause both human and plant disorders [125].

7. Remaining Questions and Future Directions

One of the most studied mechanisms is circRNA-miRNA sponges. Most of the studies mentioned above have revealed these findings. However, as part of non-coding RNAs, circRNAs are likely to also modify DNA structure and modulate RNA transcription and protein translation. EcircRNAs have been reported to be translated into proteins. Without a 5′cap, circRNAs adopt a cap-independent translation mechanism. They potentially use IRES or MIRES to bind to the initiation factor eIF4G2 complex and anchor the 43S complex for protein translation [85,126]. The next direction regarding the circRNAs and lung inflammation will be to explore the circRNA secretion and reuptake. To date, it remains unclear on which regulators control circRNA secretion, degradation, and uptake. Additionally, most works study the ecircRNAs generated via back-splicing. Intronic circRNAs and EIcircRNAs, as well as tRNA-facilitated circRNAs, require further investigation.
In summary, circRNAs have become a newly targeted non-coding RNA category and potentially have essential roles in the process of inflammatory lung responses and macrophage polarization.

Funding

This research was funded by National Institutes of Health R01HL142758.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Sanger, H.L.; Klotz, G.; Riesner, D.; Gross, H.J.; Kleinschmidt, A.K. Viroids are single-stranded covalently closed circular RNA molecules existing as highly base-paired rod-like structures. Proc. Natl. Acad. Sci. USA 1976, 73, 3852–3856. [Google Scholar] [CrossRef] [PubMed]
  2. Hsu, M.-T.; Coca-Prados, M. Electron microscopic evidence for the circular form of RNA in the cytoplasm of eukaryotic cells. Nature 1979, 280, 339–340. [Google Scholar] [CrossRef] [PubMed]
  3. Arnberg, A.C.; Van Ommen, G.J.; Grivell, L.A.; Van Bruggen, E.F.J.; Borst, P. Some yeast mitochondrial RNAs are circular. Cell 1980, 19, 313–319. [Google Scholar] [CrossRef]
  4. Cocquerelle, C.; Mascrez, B.; Hétuin, D.; Bailleul, B. Mis-splicing yields circular RNA molecules. FASEB J. 1993, 7, 155–160. [Google Scholar] [CrossRef]
  5. Jeck, W.R.; Sorrentino, J.A.; Wang, K.; Slevin, M.K.; Burd, C.E.; Liu, J.; Marzluff, W.F.; Sharpless, N.E. Circular RNAs are abundant, conserved, and associated with ALU repeats. RNA 2012, 19, 141–157. [Google Scholar] [CrossRef]
  6. Guo, J.U.; Agarwal, V.; Guo, H.; Bartel, D.P. Expanded identification and characterization of mammalian circular RNAs. Genome Biol. 2014, 15, 409. [Google Scholar] [CrossRef] [PubMed]
  7. Jeck, W.R.; Sharpless, N.E. Detecting and characterizing circular RNAs. Nat. Biotechnol. 2014, 32, 453–461. [Google Scholar] [CrossRef]
  8. Guarnerio, J.; Bezzi, M.; Jeong, J.; Paffenholz Stella, V.; Berry, K.; Naldini Matteo, M.; Lo-Coco, F.; Tay, Y.; Beck Andrew, H.; Pandolfi, P. Oncogenic Role of Fusion-circRNAs Derived from Cancer-Associated Chromosomal Translocations. Cell 2016, 165, 289–302. [Google Scholar] [CrossRef]
  9. Wang, K.; Sun, Y.; Tao, W.; Fei, X.; Chang, C. Androgen receptor (AR) promotes clear cell renal cell carcinoma (ccRCC) migration and invasion via altering the circHIAT1/miR-195-5p/29a-3p/29c-3p/CDC42 signals. Cancer Lett. 2017, 394, 1–12. [Google Scholar] [CrossRef]
  10. Baulina, N.M.; Kiselev, I.S.; Chumakova, O.S.; Favorova, O.O. Circular RNAs: Biogenesis, Functions, and Role in Myocardial Hypertrophy. Biochemistry 2024, 89 (Suppl. 1), S1–S13. [Google Scholar] [CrossRef] [PubMed]
  11. Kumar, L.; Shamsuzzama; Jadiya, P.; Haque, R.; Shukla, S.; Nazir, A. Functional Characterization of Novel Circular RNA Molecule, circzip-2 and Its Synthesizing Gene zip-2 in C. elegans Model of Parkinson’s Disease. Mol. Neurobiol. 2018, 55, 6914–6926. [Google Scholar] [CrossRef] [PubMed]
  12. Wang, M.; Yu, F.; Wu, W.; Zhang, Y.; Chang, W.; Ponnusamy, M.; Wang, K.; Li, P. Circular RNAs: A novel type of non-coding RNA and their potential implications in antiviral immunity. Int. J. Biol. Sci. 2017, 13, 1497–1506. [Google Scholar] [CrossRef] [PubMed]
  13. Li, H.; Li, K.; Lai, W.; Li, X.; Wang, H.; Yang, J.; Chu, S.; Wang, H.; Kang, C.-M.; Qiu, Y.-R. Comprehensive circular RNA profiles in plasma reveals that circular RNAs can be used as novel biomarkers for systemic lupus erythematosus. Clin. Chim. Acta 2018, 480, 17–25. [Google Scholar] [CrossRef] [PubMed]
  14. Moldoveanu, B.; Otmishi, P.; Jani, P.; Walker, J.; Sarmiento, X.; Guardiola, J.; Saad, M.; Yu, J. Inflammatory mechanisms in the lung. J. Inflamm. Res. 2009, 2, 1–11. [Google Scholar] [PubMed]
  15. Keskinidou, C.; Vassiliou, A.; Dimopoulou, I.; Kotanidou, A.; Orfanos, S. Mechanistic Understanding of Lung Inflammation: Recent Advances and Emerging Techniques. J. Inflamm. Res. 2022, 15, 3501–3546. [Google Scholar] [CrossRef]
  16. Bain, C.C.; MacDonald, A.S. The impact of the lung environment on macrophage development, activation and function: Diversity in the face of adversity. Mucosal Immunol. 2022, 15, 223–234. [Google Scholar] [CrossRef]
  17. Hou, F.; Xiao, K.; Tang, L.; Xie, L. Diversity of Macrophages in Lung Homeostasis and Diseases. Front. Immunol. 2021, 12, 753940. [Google Scholar] [CrossRef]
  18. Song, H.; Yang, Y.; Sun, Y.; Wei, G.; Zheng, H.; Chen, Y.; Cai, D.; Li, C.; Ma, Y.; Lin, Z.; et al. Circular RNA Cdyl promotes abdominal aortic aneurysm formation by inducing M1 macrophage polarization and M1-type inflammation. Mol. Ther. 2022, 30, 915–931. [Google Scholar] [CrossRef] [PubMed] [PubMed Central]
  19. Enuka, Y.; Lauriola, M.; Feldman, M.E.; Sas-Chen, A.; Ulitsky, I.; Yarden, Y. Circular RNAs are long-lived and display only minimal early alterations in response to a growth factor. Nucleic Acids Res. 2015, 44, 1370–1383. [Google Scholar] [CrossRef]
  20. Salzman, J.; Gawad, C.; Wang, P.L.; Lacayo, N.; Brown, P.O. Circular RNAs Are the Predominant Transcript Isoform from Hundreds of Human Genes in Diverse Cell Types. PLoS ONE 2012, 7, e30733. [Google Scholar] [CrossRef]
  21. Wang, P.L.; Bao, Y.; Yee, M.-C.; Barrett, S.P.; Hogan, G.J.; Olsen, M.N.; Dinneny, J.R.; Brown, P.O.; Salzman, J. Circular RNA Is Expressed across the Eukaryotic Tree of Life. PLoS ONE 2014, 9, e90859. [Google Scholar] [CrossRef] [PubMed]
  22. Danan, M.; Schwartz, S.; Edelheit, S.; Sorek, R. Transcriptome-wide discovery of circular RNAs in Archaea. Nucleic Acids Res. 2011, 40, 3131–3142. [Google Scholar] [CrossRef] [PubMed]
  23. Li, Y.; Zheng, Q.; Bao, C.; Li, S.; Guo, W.; Zhao, J.; Chen, D.; Gu, J.; He, X.; Huang, S. Circular RNA is enriched and stable in exosomes: A promising biomarker for cancer diagnosis. Cell Res. 2015, 25, 981–984. [Google Scholar] [CrossRef] [PubMed]
  24. Zhang, X.-O.; Wang, H.-B.; Zhang, Y.; Lu, X.; Chen, L.-L.; Yang, L. Complementary Sequence-Mediated Exon Circularization. Cell 2014, 159, 134–147. [Google Scholar] [CrossRef]
  25. Patop, I.L.; Wüst, S.; Kadener, S. Past, present, and future of circ RNAs. EMBO J. 2019, 38, e100836. [Google Scholar] [CrossRef]
  26. Kristensen, L.S.; Andersen, M.S.; Stagsted, L.V.W.; Ebbesen, K.K.; Hansen, T.B.; Kjems, J. The biogenesis, biology and characterization of circular RNAs. Nat. Rev. Genet. 2019, 20, 675–691. [Google Scholar] [CrossRef]
  27. Li, X.; Yang, L.; Chen, L.-L. The Biogenesis, Functions, and Challenges of Circular RNAs. Mol. Cell 2018, 71, 428–442. [Google Scholar] [CrossRef]
  28. Zhang, Y.; Zhang, X.-O.; Chen, T.; Xiang, J.-F.; Yin, Q.-F.; Xing, Y.-H.; Zhu, S.; Yang, L.; Chen, L.-L. Circular Intronic Long Noncoding RNAs. Mol. Cell 2013, 51, 792–806. [Google Scholar] [CrossRef]
  29. Toptan, T.; Abere, B.; Nalesnik, M.A.; Swerdlow, S.H.; Ranganathan, S.; Lee, N.; Kathy Moore, P.S.; Chang, Y. Circular DNA tumor viruses make circular RNAs. Proc. Natl. Acad. Sci. USA 2018, 115, E8737–E8745. [Google Scholar] [CrossRef]
  30. Li, Z.; Huang, C.; Bao, C.; Chen, L.; Lin, M.; Wang, X.; Zhong, G.; Yu, B.; Hu, W.; Dai, L.; et al. Exon-intron circular RNAs regulate transcription in the nucleus. Nat. Struct. Mol. Biol. 2015, 22, 256–264. [Google Scholar] [CrossRef]
  31. Memczak, S.; Jens, M.; Elefsinioti, A.; Torti, F.; Krueger, J.; Rybak, A.; Maier, L.; Mackowiak, S.D.; Gregersen, L.H.; Munschauer, M.; et al. Circular RNAs are a large class of animal RNAs with regulatory potency. Nature 2013, 495, 333–338. [Google Scholar] [CrossRef] [PubMed]
  32. Hansen, T.B.; Wiklund, E.D.; Bramsen, J.B.; Villadsen, S.B.; Statham, A.L.; Clark, S.J.; Kjems, J. miRNA-dependent gene silencing involving Ago2-mediated cleavage of a circular antisense, R.N.A. EMBO J. 2011, 30, 4414–4422. [Google Scholar] [CrossRef]
  33. Han, Z.; Chen, H.; Guo, Z.; Shen, J.; Luo, W.; Xie, F.; Wan, Y.; Wang, S.; Li, J.; He, J. Circular RNAs and Their Role in Exosomes. Front. Oncol. 2022, 12, 848341. [Google Scholar] [CrossRef]
  34. Bao, C.; Lyu, D.; Huang, S. Circular RNA expands its territory. Mol. Cell. Oncol. 2015, 3, e1084443. [Google Scholar] [CrossRef] [PubMed]
  35. Dou, Y.; Diana, J.-S.; Franklin, J.L.; Higginbotham, J.N.; Jeppesen, D.K.; Weaver, A.M.; Prasad, N.; Levy, S.; Coffey, R.J.; Patton, J.G.; et al. Circular RNAs are down-regulated in KRAS mutant colon cancer cells and can be transferred to exosomes. Sci. Rep. 2016, 6, 37982. [Google Scholar] [CrossRef] [PubMed]
  36. Welsh, J.A.; Goberdhan, D.C.I.; O’Driscoll, L.; Buzas, E.I.; Blenkiron, C.; Bussolati, B.; Cai, H.; Di Vizio, D.; Driedonks, T.A.P.; Erdbrügger, U.; et al. Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches. J. Extracell. Vesicles 2024, 13, e12404. [Google Scholar] [CrossRef] [PubMed] [PubMed Central]
  37. Crescitelli, R.; Lasser, C.; Lotvall, J. Isolation and characterization of extracellular vesicle subpopulations from tissues. Nat. Protoc. 2021, 16, 1548–1580. [Google Scholar] [CrossRef]
  38. Di Mattia, T.; Tomasetto, C.; Alpy, F. Faraway, so close! Functions of Endoplasmic reticulum–Endosome contacts. Biochim. Biophys. Acta (BBA) Mol. Cell Biol. Lipids 2020, 1865, 158490. [Google Scholar] [CrossRef]
  39. Kwon, S.-H.; Oh, S.; Nacke, M.; Mostov, K.E.; Lipschutz, J.H. Adaptor Protein CD2AP and L-type Lectin LMAN2 Regulate Exosome Cargo Protein Trafficking through the Golgi Complex. J. Biol. Chem. 2016, 291, 25462–25475. [Google Scholar] [CrossRef]
  40. Rabas, N.; Palmer, S.; Mitchell, L.; Ismail, S.; Gohlke, A.; Riley, J.S.; Tait, S.W.G.; Gammage, P.; Soares, L.L.; Macpherson, I.R.; et al. PINK1 drives production of mtDNA-containing extracellular vesicles to promote invasiveness. J. Cell Biol. 2021, 220, e202006049. [Google Scholar] [CrossRef]
  41. Zhao, Y.G.; Codogno, P.; Zhang, H. Machinery, regulation and pathophysiological implications of autophagosome maturation. Nat. Rev. Mol. Cell Biol. 2021, 22, 733–750. [Google Scholar] [CrossRef] [PubMed]
  42. Li, X.; Zhang, J.-L.; Lei, Y.-N.; Liu, X.-Q.; Xue, W.; Zhang, Y.; Nan, F.; Gao, X.; Zhang, J.; Wei, J.; et al. Linking circular intronic RNA degradation and function in transcription by RNase H1. Sci. China Life Sci. 2021, 64, 1795–1809. [Google Scholar] [CrossRef] [PubMed]
  43. Miao, Q.; Zhong, Z.; Jiang, Z.; Lin, Y.; Ni, B.; Yang, W.; Tang, J. RNA-seq of circular RNAs identified circPTPN22 as a potential new activity indicator in systemic lupus erythematosus. Lupus 2019, 28, 520–528. [Google Scholar] [CrossRef] [PubMed]
  44. Zheng, Z.-M. Circular RNAs and RNase L in PKR activation and virus infection. Cell Biosci. 2019, 9, 43. [Google Scholar] [CrossRef]
  45. Pan, Z.; Li, G.-F.; Sun, M.-L.; Xie, L.; Liu, D.; Zhang, Q.; Yang, X.-X.; Xia, S.; Liu, X.; Zhou, H.; et al. MicroRNA-1224 Splicing CircularRNA-Filip1l in an Ago2-Dependent Manner Regulates Chronic Inflammatory Pain via Targeting Ubr5. J. Neurosci. 2019, 39, 2125–2143. [Google Scholar] [CrossRef]
  46. Piwecka, M.; Glažar, P.; Hernandez-Miranda, L.R.; Memczak, S.; Wolf, S.A.; Rybak-Wolf, A.; Filipchyk, A.; Klironomos, F.; Cerda Jara, C.A.; Fenske, P.; et al. Loss of a mammalian circular RNA locus causes miRNA deregulation and affects brain function. Science 2017, 357, eaam8526. [Google Scholar] [CrossRef]
  47. Jia, R.; Xiao, M.-S.; Li, Z.; Shan, G.; Huang, C. Defining an evolutionarily conserved role of GW182 in circular RNA degradation. Cell Discov. 2019, 5, 45. [Google Scholar] [CrossRef]
  48. Fischer, J.W.; Busa, V.F.; Shao, Y.; Leung, A.K. Structure-mediated RNA decay by UPF1 and G3BP1. Mol. Cell 2020, 78, 70–84. [Google Scholar] [CrossRef]
  49. Huang, C.; Liang, D.; Tatomer, D.C.; Wilusz, J.E. A length-dependent evolutionarily conserved pathway controls nuclear export of circular RNAs. Genes. Dev. 2018, 32, 639–644. [Google Scholar] [CrossRef]
  50. Chen, R.; Chen, X.; Xia, L.; Zhang, J.X.; Pan, Z.; Ma, X.; Han, K.; Chen, J.; Judde, J.-G.; Déas, O.; et al. N6-methyladenosine modification of circNSUN2 facilitates cytoplasmic export and stabilizes HMGA2 to promote colorectal liver metastasis. Nat. Commun. 2019, 10, 4695. [Google Scholar] [CrossRef]
  51. Wang, X.; Song, H.; Fang, L.; Wu, T. EIF4A3-mediated circPRKCI expression promotes triple-negative breast cancer progression by regulating WBP2 and PI3K/AKT signaling pathway. Cell Death Discov. 2022, 8, 92. [Google Scholar] [CrossRef] [PubMed]
  52. Ron, M.; Ulitsky, I. Context-specific effects of sequence elements on subcellular localization of linear and circular RNAs. Nat. Commun. 2022, 13, 2481. [Google Scholar] [CrossRef]
  53. Chen, L.; Wang, Y.; Lin, J.; Song, Z.; Wang, Q.; Zhao, W.; Wang, Y.; Xiu, X.; Deng, Y.; Li, X.; et al. Exportin 4 depletion leads to nuclear accumulation of a subset of circular RNAs. Nat. Commun. 2022, 13, 5769. [Google Scholar] [CrossRef]
  54. Preußer, C.; Hung, L.-H.; Schneider, T.; Schreiner, S.; Hardt, M.; Moebus, A.; Santoso, S.; Bindereif, A. Selective release of circRNAs in platelet-derived extracellular vesicles. J. Extracell. Vesicles 2018, 7, 1424473. [Google Scholar] [CrossRef]
  55. Lee, H.; Li, C.; Zhang, Y.; Zhang, D.; Otterbein, L.E.; Jin, Y. Caveolin-1 selectively regulates microRNA sorting into microvesicles after noxious stimuli. J. Exp. Med. 2019, 216, 2202–2220. [Google Scholar] [CrossRef] [PubMed] [PubMed Central]
  56. Barbagallo, C.; Duilia Brex Caponnetto, A.; Cirnigliaro, M.; Scalia, M.; Magnano, A.; Caltabiano, R.; Barbagallo, D.; Biondi, A.; Alessandro Cappellani Basile, F.; Cinzia Di Pietro Purrello, M.; et al. LncRNA UCA1, Upregulated in CRC Biopsies and Downregulated in Serum Exosomes, Controls mRNA Expression by RNA-RNA Interactions. Mol. Ther. Nucleic Acids 2018, 12, 229–241. [Google Scholar] [CrossRef] [PubMed]
  57. Yao, Y.; Chen, C.; Wang, J.; Xuan, H.; Chen, X.; Li, Z.; Yang, F.; Wang, B.; Lin, S.; Li, S.; et al. Circular RNA circATP9A promotes non-small cell lung cancer progression by interacting with HuR and by promoting extracellular vesicles-mediated macrophage M2 polarization. J. Exp. Clin. Cancer Res. 2023, 42, 330, Erratum in: J. Exp. Clin. Cancer Res. 2023, 42, 345. [Google Scholar] [CrossRef] [PubMed] [PubMed Central]
  58. Yin, W.; Zhang, Z.; Xiao, Z.; Li, X.; Luo, S.; Zhou, Z. Circular RNAs in diabetes and its complications: Current knowledge and future prospects. Front. Genet. 2022, 13, 1006307. [Google Scholar] [CrossRef]
  59. Long, Q.; Lv, B.; Jiang, S.; Lin, J. The Landscape of Circular RNAs in Cardiovascular Diseases. Int. J. Mol. Sci. 2023, 24, 4571. [Google Scholar] [CrossRef]
  60. Dong, X.; Bai, Y.; Liao, Z.; Gritsch, D.; Liu, X.; Wang, T.; Borges-Monroy, R.; Ehrlich, A.; Serrano, G.E.; Feany, M.B.; et al. Circular RNAs in the human brain are tailored to neuron identity and neuropsychiatric disease. Nat. Commun. 2023, 14, 5327. [Google Scholar] [CrossRef]
  61. Li, H.-Z.; Lin, Z.; Xu, X.-H.; Lin, N.; Lu, H.-D. The potential roles of circRNAs in osteoarthritis: A coming journey to find a treasure. Biosci. Rep. 2018, 38, BSR20180542. [Google Scholar] [CrossRef]
  62. Zhang, S.; Wang, X.; Chen, G.; Tong, L.; Dai, T.; Wang, L.; Zhu, L.; Zhang, H.; Du, D. CircRNA Galntl6 sponges miR-335 to ameliorate stress-induced hypertension through upregulating Lig3 in rostral ventrolateral medulla. Redox Biol. 2023, 64, 102782. [Google Scholar] [CrossRef] [PubMed]
  63. Ziegler, C.G.K.; Miao, V.N.; Owings, A.H.; Navia, A.W.; Tang, Y.; Bromley, J.D.; Lotfy, P.; Sloan, M.; Laird, H.; Williams, H.B.; et al. Impaired local intrinsic immunity to SARS-CoV-2 infection in severe COVID-19. Cell 2021, 184, 4713–4733.e22. [Google Scholar] [CrossRef] [PubMed]
  64. Gao, X.; Fang, D.; Liang, Y.; Deng, X.; Chen, N.; Zeng, M.; Luo, M. Circular RNAs as emerging regulators in COVID-19 pathogenesis and progression. Front. Immunol. 2022, 13, 980231. [Google Scholar] [CrossRef] [PubMed]
  65. Qi, F.; Li, Y.; Yang, X.; Wu, Y.; Lin, L.; Liu, X. Hsa_circ_0044226 knockdown attenuates progression of pulmonary fibrosis by inhibiting CDC27. Aging 2020, 12, 14808–14818. [Google Scholar] [CrossRef]
  66. Cheng, Z.; Zhang, Y.; Wu, S.; Zhao, R.; Yu, Y.; Zhou, Y.; Zhou, Z.; Dong, Y.; Qiu, A.; Xu, H.; et al. Peripheral blood circular RNA hsa_circ_0058493 as a potential novel biomarker for silicosis and idiopathic pulmonary fibrosis. Ecotoxicol. Environ. Saf. 2022, 236, 113451. [Google Scholar] [CrossRef]
  67. Li, R.; Wang, Y.; Song, X.; Sun, W.; Zhang, J.; Liu, Y.; Li, H.; Meng, C.; Zhang, J.; Zheng, Q. Changjun Lv Potential regulatory role of circular RNA in idiopathic pulmonary fibrosis. Int. J. Mol. Med. 2018, 42, 3256–3268. [Google Scholar] [CrossRef]
  68. Xu, P.; Zhang, J.; Wang, M.; Liu, B.; Li, R.; Li, H.; Zhai, N.; Liu, W.; Lv, C.; Song, X. hnRNPL-activated circANKRD42 back-splicing and circANKRD42-mediated crosstalk of mechanical stiffness and biochemical signal in lung fibrosis. Mol. Ther. 2022, 30, 2370–2387. [Google Scholar] [CrossRef]
  69. Yang, L.; Liu, X.; Zhang, N.; Chen, L.; Xu, J.; Tang, W. Investigation of circular RNAs and related genes in pulmonary fibrosis based on bioinformatics analysis. J. Cell. Biochem. 2019, 120, 11022–11032. [Google Scholar] [CrossRef]
  70. Liu, X.; Liu, H.; Jia, X.; He, R.; Zhang, X.; Zhang, W. Changing Expression Profiles of Messenger RNA, MicroRNA, Long Non-coding RNA, and Circular RNA Reveal the Key Regulators and Interaction Networks of Competing Endogenous RNA in Pulmonary Fibrosis. Front. Genet. 2020, 11, 558095. [Google Scholar] [CrossRef]
  71. Fang, S.; Guo, H.; Cheng, Y.; Zhou, Z.; Zhang, W.; Han, B.; Luo, W.; Wang, J.; Xie, W.; Chao, J. circHECTD1 promotes the silica-induced pulmonary endothelial–mesenchymal transition via HECTD1. Cell Death Dis. 2018, 9, 396. [Google Scholar] [CrossRef]
  72. Jiang, R.; Zhou, Z.; Liao, Y.; Yang, F.; Cheng, Y.; Huang, J.; Wang, J.; Chen, H.; Zhu, T.; Chao, J. The emerging roles of a novel CCCH-type zinc finger protein, ZC3H4, in silica-induced epithelial to mesenchymal transition. Toxicol. Lett. 2019, 307, 26–40. [Google Scholar] [CrossRef] [PubMed]
  73. Zeng, H.; Gao, H.; Zhang, M.; Wang, J.; Gu, Y.; Wang, Y.; Zhang, H.; Liu, P.; Zhang, X.; Zhao, L. Atractylon Treatment Attenuates Pulmonary Fibrosis via Regulation of the mmu_circ_0000981/miR-211-5p/TGFBR2 Axis in an Ovalbumin-Induced Asthma Mouse Model. Inflammation 2021, 44, 1856–1864. [Google Scholar] [CrossRef]
  74. Yao, W.; Li, Y.; Han, L.; Ji, X.; Pan, H.; Liu, Y.; Yuan, J.; Yan, W.; Ni, C. The CDR1as/miR-7/TGFBR2 Axis Modulates EMT in Silica-Induced Pulmonary Fibrosis. Toxicol. Sci. 2018, 166, 465–478. [Google Scholar] [CrossRef] [PubMed]
  75. Holdt, L.M.; Stahringer, A.; Sass, K.; Pichler, G.; Kulak, N.A.; Wilfert, W.; Kohlmaier, A.; Herbst, A.; Northoff, B.H.; Nicolaou, A.; et al. Circular non-coding RNA ANRIL modulates ribosomal RNA maturation and atherosclerosis in humans. Nat. Commun. 2016, 7, 12429. [Google Scholar] [CrossRef] [PubMed]
  76. Ng, W.L.; Marinov, G.K.; Chin, Y.-M.; Lim, Y.-Y.; Ea, C.-K. Transcriptomic analysis of the role of RasGEF1B circular RNA in the TLR4/LPS pathway. Sci. Rep. 2017, 7, 12227. [Google Scholar] [CrossRef] [PubMed]
  77. Ng, W.L.; Marinov, G.K.; Liau, E.S.; Lam, Y.L.; Lim, Y.-Y.; Ea, C.-K. Inducible RasGEF1B circular RNA is a positive regulator of ICAM-1 in the TLR4/LPS pathway. RNA Biol. 2016, 13, 861–871. [Google Scholar] [CrossRef]
  78. Gonzalez, C.; Cimini, M.; Cheng, Z.; Benedict, C.; Wang, C.; Trungcao, M.; Mallaredy, V.; Rajan, S.; Garikipati, V.N.S.; Kishore, R. Role of circular RNA cdr1as in modulation of macrophage phenotype. Life Sci. 2022, 309, 121003. [Google Scholar] [CrossRef] [PubMed] [PubMed Central]
  79. Lin, Q.; Liang, Q.; Qin, C.; Li, Y. CircANKRD36 Knockdown Suppressed Cell Viability and Migration of LPS-Stimulated RAW264.7 Cells by Sponging MiR-330. Inflammation 2021, 44, 2044–2053. [Google Scholar] [CrossRef]
  80. Zhou, R.M.; Shi, Z.H.; Shan, K.; Zhang, S.J.; Zhang, Y.H.; Liang, Y.; Yan, B.; Zhao, C. Comparative Analysis of Differentially Expressed Circular RNAs in Polarized Macrophages. Front. Genet. 2022, 13, 823517. [Google Scholar] [CrossRef] [PubMed] [PubMed Central]
  81. Huang, Z.; Su, R.; Deng, Z.; Xu, J.; Peng, Y.; Luo, Q.; Li, J. Identification of differentially expressed circular RNAs in human monocyte derived macrophages response to Mycobacterium tuberculosis infection. Sci. Rep. 2017, 7, 13673. [Google Scholar] [CrossRef] [PubMed] [PubMed Central]
  82. Wang, T.Z. Role of Circular RNA ASPH in Macrophage Polarization and Response in Sepsis. Master’s Thesis. Electronic Thesis and Dissertation Repository. 2023. Available online: https://ir.lib.uwo.ca/etd/9528 (accessed on 19 August 2024).
  83. Chen, Y.G.; Kim, M.V.; Chen, X.; Batista, P.J.; Aoyama, S.; Wilusz, J.E.; Iwasaki, A.; Chang, H.Y. Sensing Self and Foreign Circular RNAs by Intron Identity. Mol. Cell 2017, 67, 228–238.e5. [Google Scholar] [CrossRef] [PubMed]
  84. Yang, C.; Ni, B.; Li, C.; Sun, W.; Wang, Z.; Wang, H.; Hou, X.; Yan, S.; Wang, X.; Xu, D. circRNA_17725 Promotes Macrophage Polarization towards M2 by Targeting FAM46C to Alleviate Arthritis. Mediators Inflamm. 2023, 2023, 6818524. [Google Scholar] [CrossRef] [PubMed] [PubMed Central]
  85. Zhang, C.; Han, X.; Yang, L.; Fu, J.; Sun, C.; Huang, S.; Xiao, W.; Gao, Y.; Liang, Q.; Wang, X.; et al. Circular RNA circPPM1F modulates M1 macrophage activation and pancreatic islet inflammation in type 1 diabetes mellitus. Theranostics 2020, 10, 10908–10924. [Google Scholar] [CrossRef] [PubMed] [PubMed Central]
  86. Fan, L.; Yao, L.; Li, Z.; Wan, Z.; Sun, W.; Qiu, S.; Zhang, W.; Xiao, D.; Song, L.; Yang, G.; et al. Exosome-Based Mitochondrial Delivery of circRNA mSCAR Alleviates Sepsis by Orchestrating Macrophage Activation. Adv. Sci. (Weinh). 2023, 10, e2205692. [Google Scholar] [CrossRef] [PubMed]
  87. Yang, X.; Wang, J.; Zhou, Z.; Jiang, R.; Huang, J.; Chen, L.; Cao, Z.; Chu, H.; Han, B.; Cheng, Y.; et al. Silica-induced initiation of circular ZC3H4 RNA/ZC3H4 pathway promotes the pulmonary macrophage activation. FASEB J. 2018, 32, 3264–3277. [Google Scholar] [CrossRef] [PubMed]
  88. Zhang, Y.; Zhang, Y.; Li, X.; Zhang, M.; Lv, K. Microarray analysis of circular RNA expression patterns in polarized macrophages. Int. J. Mol. Med. 2017, 39, 373–379. [Google Scholar] [CrossRef] [PubMed]
  89. Bao, X.; Zhang, Q.; Liu, N.; Zhuang, S.; Li, Z.; Meng, Q.; Sun, H.; Bai, J.; Zhou, X.; Tang, L. Characteristics of circular RNA expression of pulmonary macrophages in mice with sepsis-induced acute lung injury. J. Cell Mol. Med. 2019, 23, 7111–7115. [Google Scholar] [CrossRef] [PubMed]
  90. Yi, B.; Dai, K.; Yan, Z.; Yin, Z. Circular RNA PLCE1 promotes epithelial mesenchymal transformation, glycolysis in colorectal cancer and M2 polarization of tumor-associated macrophages. Bioengineered 2022, 13, 6243–6256. [Google Scholar] [CrossRef] [PubMed]
  91. Yang, D.; Zhao, D.; Ji, J.; Wang, C.; Liu, N.; Bao, X.; Liu, X.; Jiang, S.; Zhang, Q.; Tang, L. CircRNA_0075723 protects against pneumonia-induced sepsis through inhibiting macrophage pyroptosis by sponging miR-155-5p and regulating SHIP1 expression. Front. Immunol. 2023, 14, 1095457. [Google Scholar] [CrossRef] [PubMed]
  92. Zhu, Y.; Kong, D.; Wang, Z.; Li, T.; Tang, T.; Peng, Y.; Hu, C.; Chao, J.; Chen, H.; Chen, Y.; et al. Identification of Differential Circular RNA Expression Profiles and Functional Networks in Human Macrophages Induced by Virulent and Avirulent Mycobacterium tuberculosis Strains. Int. J. Mol. Sci. 2023, 24, 17561. [Google Scholar] [CrossRef] [PubMed]
  93. Li, H.; Luo, F.; Jiang, X.; Zhang, W.; Xiang, T.; Pan, Q.; Cai, L.; Zhao, J.; Weng, D.; Li, Y.; et al. CircITGB6 promotes ovarian cancer cisplatin resistance by resetting tumor-associated macrophage polarization toward the M2 phenotype. J. Immunother Cancer 2022, 10, e004029. [Google Scholar] [CrossRef] [PubMed] [PubMed Central]
  94. Yin, J.; Hu, T.; Xu, L.; Li, P.; Li, M.; Ye, Y.; Pang, Z. Circular RNA expression profile in peripheral blood mononuclear cells from Crohn disease patients. Medicine 2019, 98, e16072. [Google Scholar] [CrossRef] [PubMed] [PubMed Central]
  95. Li, H.; Guo, H.; Huang, Q.; Wang, S.; Li, X.; Qiu, M. Circular RNA P4HB promotes glycolysis and tumor progression by binding with PKM2 in lung adenocarcinoma. Respir. Res. 2023, 24, 252. [Google Scholar] [CrossRef] [PubMed] [PubMed Central]
  96. Lian, C.; Sun, J.; Guan, W.; Zhang, L.; Zhang, X.; Yang, L.; Hu, W. Circular RNA circHIPK3 Activates Macrophage NLRP3 Inflammasome and TLR4 Pathway in Gouty Arthritis via Sponging miR-561 and miR-192. Inflammation 2021, 44, 2065–2077. [Google Scholar] [CrossRef] [PubMed]
  97. Huang, Z.; Yao, F.; Liu, J.; Xu, J.; Guo, Y.; Su, R.; Luo, Q.; Li, J. Up-regulation of circRNA-0003528 promotes mycobacterium tuberculosis associated macrophage polarization via down-regulating miR-224-5p, miR-324-5p and miR-488-5p and up-regulating CTLA4. Aging 2020, 12, 25658–25672. [Google Scholar] [CrossRef] [PubMed]
  98. Wang, X.; Sheng, W.; Xu, T.; Xu, J.; Gao, R.; Zhang, Z. CircRNA hsa_circ_0110102 inhibited macrophage activation and hepatocellular carcinoma progression via miR-580-5p/PPARα/CCL2 pathway. Aging 2021, 13, 11969–11987. [Google Scholar] [CrossRef] [PubMed]
  99. Gu, J.; Wu, J.; Wang, C.; Xu, Z.; Jin, Z.; Yan, D.; Chen, S. BMSCs-derived exosomes inhibit macrophage/microglia pyroptosis by increasing autophagy through the miR-21a-5p/PELI1 axis in spinal cord injury. Aging 2024, 16, 5184–5206. [Google Scholar] [CrossRef] [PubMed]
  100. Liu, G.; Wan, Q.; Li, J.; Hu, X.; Gu, X.; Xu, S. Circ_0038467 regulates lipopolysaccharide-induced inflammatory injury in human bronchial epithelial cells through sponging miR-338-3p. Thorac. Cancer 2020, 11, 1297–1308. [Google Scholar] [CrossRef]
  101. Li, H.; Niu, X.; Shi, H.; Feng, M.; Du, Y.; Sun, R.; Ma, N.; Wang, H.; Wei, D.; Gao, M. circHECTD1 attenuates apoptosis of alveolar epithelial cells in acute lung injury. Lab. Investig. J. Tech. Methods Pathol. 2022, 102, 945–956. [Google Scholar] [CrossRef]
  102. Ren, Y.; Li, L.; Wang, M.; Yang, Z.; Sun, Z.; Zhang, W.; Cao, L.; Nie, S. Knockdown of circRNA Paralemmin 2 Ameliorates Lipopolysaccharide-induced Murine Lung Epithelial Cell Injury by Sponging miR-330-5p to Reduce ROCK2 Expression. Immunol. Investig. 2022, 51, 1707–1724. [Google Scholar] [CrossRef]
  103. Yang, C.-L.; Yang, W.-K.; He, Z.-H.; Guo, J.-H.; Yang, X.-G.; Li, H.-B. Quietness of circular RNA circ_0054633 alleviates the inflammation and proliferation in lipopolysaccharides-induced acute lung injury model through NF-κB signaling pathway. Gene 2021, 766, 145153. [Google Scholar] [CrossRef]
  104. Lu, S.; Wu, X.; Xin, S.; Zhang, J.; Lin, H.; Miao, Y.; Li, Y. Knockdown of circ_0001679 alleviates lipopolysaccharide-induced MLE-12 lung cell injury by regulating the miR-338-3p/mitogen-activated protein kinase 1 axis. Bioengineered 2022, 13, 5803–5817. [Google Scholar] [CrossRef] [PubMed]
  105. Ma, K.; Wang, W.; Gao, C.; He, J. The role of circTMOD3 in regulating LPS-induced acute inflammation and injury in human lung fibroblast WI-38 cells. Exp. Lung Res. 2021, 47, 311–322. [Google Scholar] [CrossRef] [PubMed]
  106. Wang, Q.; Zhang, X.; Chen, D. circ_VMA21 protects WI-38 cells against LPS-induced apoptotic and inflammatory injury by acting on the miR-409-3p/KLF4 axis. Gen. Physiol. Biophys. 2021, 40, 275–287. [Google Scholar] [CrossRef] [PubMed]
  107. Li, X.; Yuan, Z.; Chen, J.; Wang, T.; Shen, Y.; Chen, L.; Wen, F. Microarray analysis reveals the changes of circular RNA expression and molecular mechanism in acute lung injury mouse model. J. Cell. Biochem. 2019, 120, 16658–16667. [Google Scholar] [CrossRef]
  108. Jiang, W.-Y.; Ren, J.; Zhang, X.-H.; Lu, Z.-L.; Feng, H.-J.; Yao, X.-L.; Li, D.-H.; Xiong, R.; Fan, T.; Geng, Q. CircC3P1 attenuated pro-inflammatory cytokine production and cell apoptosis in acute lung injury induced by sepsis through modulating miR-21. J. Cell. Mol. Med. 2020, 24, 11221–11229. [Google Scholar] [CrossRef]
  109. Ke, J.; Chen, M.; Ma, S.; Zhang, L.; Zhang, L. Circular RNA VMA21 ameliorates lung injury in septic rat via targeting microRNA-497-5p/CD2-associated protein axis. Bioengineered 2022, 13, 5453–5466. [Google Scholar] [CrossRef]
  110. Zou, Z.; Wang, Q.; Zhou, M.; Li, W.; Zheng, Y.; Li, F.; Zheng, S.; He, Z. Protective effects of P2X7R antagonist in sepsis-induced acute lung injury in mice via regulation of circ_0001679 and circ_0001212 and downstream Pln, Cdh2, and Nprl3 expression. J. Gene Med. 2020, 22, e3261. [Google Scholar] [CrossRef]
  111. Cao, J.; Kuang, D.; Luo, M.; Wang, S.; Fu, C. Targeting circNCLN/miR-291a-3p/TSLP signaling axis alleviates lipopolysaccharide-induced acute lung injury. Biochem. Biophys. Res. Commun. 2022, 617 Pt 1, 60–67. [Google Scholar] [CrossRef]
  112. Imbalzano, E.; Quartuccio, S.; Di Salvo, E.; Crea, T.; Casciaro, M.; Gangemi, S. Association between HMGB1 and asthma: A literature review. Clin. Mol. Allergy CMA 2017, 15, 12. [Google Scholar] [CrossRef]
  113. Su, Y.; Geng, L.; Ma, Y.; Yu, X.; Kang, Z.; Kang, Z. Identification of circular RNA circVPS33A as a modulator in house dust mite-induced injury in human bronchial epithelial cells. Exp. Lung Res. 2021, 47, 368–381. [Google Scholar] [CrossRef] [PubMed]
  114. Lee, H.; Hong, R.; Jin, Y. Altered circular RNA expressions in extracellular vesicles from bronchoalveolar lavage fluids in mice after bacterial infections. Front. Immunol. 2024, 15, 2024. [Google Scholar]
  115. Ungerleider, N.; Concha, M.; Lin, Z.; Roberts, C.; Wang, X.; Cao, S.; Baddoo, M.; Moss, W.N.; Yu, Y.; Seddon, M.; et al. The Epstein Barr virus circRNAome. PLoS Pathog. 2018, 14, e1007206. [Google Scholar] [CrossRef] [PubMed]
  116. Lee, N. The many ways Epstein-Barr virus takes advantage of the RNA tool kit. RNA Biol. 2021, 18, 759–766. [Google Scholar] [CrossRef]
  117. Thomson, D.W.; Dinger, M.E. Endogenous microRNA sponges: Evidence and controversy. Nat. Rev. Genet. 2016, 17, 272–283. [Google Scholar] [CrossRef]
  118. Yao, T.; Chen, Q.; Fu, L.; Guo, J. Circular RNAs: Biogenesis, properties, roles, and their relationships with liver diseases. Hepatol. Res. 2017, 47, 497–504. [Google Scholar] [CrossRef]
  119. Haque, S.; Harries, L.W. Circular RNAs (circRNAs) in Health and Disease. Genes 2017, 8, 353. [Google Scholar] [CrossRef]
  120. Abdelrahman, Z.; Li, M.; Wang, X. Comparative Review of SARS-CoV-2, SARS-CoV, MERS-CoV, and Influenza A Respiratory Viruses. Front. Immunol. 2020, 11, 552909. [Google Scholar] [CrossRef]
  121. Yang, S.; Zhou, H.; Liu, M.; Jaijyan, D.; Cruz-Cosme, R.; Ramasamy, S.; Subbian, S.; Liu, D.; Xu, J.; Niu, X.; et al. SARS-CoV-2, SARS-CoV, and MERS-CoV encode circular RNAs of spliceosome-independent origin. J. Med. Virol. 2022, 94, 3203–3222. [Google Scholar] [CrossRef]
  122. Saçar Demirci, M.D.; Adan, A. Computational analysis of microRNA-mediated interactions in SARS-CoV-2 infection. PeerJ 2020, 8, e9369. [Google Scholar] [CrossRef]
  123. He, T.T.; Xu, Y.F.; Li, X.; Wang, X.; Li, J.-Y.; Ou-Yang, D.; Cheng, H.-S.; Li, H.-Y.; Qin, J.; Huang, Y.; et al. A linear and circular dual-conformation noncoding RNA involved in oxidative stress tolerance in Bacillus altitudinis. Nat. Commun. 2023, 14, 5722. [Google Scholar] [CrossRef] [PubMed]
  124. Cao, X.; Xu, X.; Dong, J.; Xue, Y.; Sun, L.; Zhu, Y.; Liu, T.; Jin, Q. Genome-wide identification and functional analysis of circRNAs in Trichophyton rubrum conidial and mycelial stages. BMC Genom. 2022, 23, 21. [Google Scholar] [CrossRef] [PubMed]
  125. Gu, A.; Jaijyan, D.K.; Yang, S.; Zeng, M.; Pei, S.; Zhu, H. Functions of Circular RNA in Human Diseases and Illnesses. Noncoding RNA. 2023, 9, 38. [Google Scholar] [CrossRef] [PubMed]
  126. Godet, A.C.; David, F.; Hantelys, F.; Tatin, F.; Lacazette, E.; Garmy-Susini, B.; Prats, A.-C. IRES Trans-Acting Factors, Key Actors of the Stress Response. Int. J. Mol. Sci. 2019, 20, 924. [Google Scholar] [CrossRef]
Cells 13 01407 g001
Figure 1. circRNA formation, transportation, and secretion. CircRNAs can be generated by back splicing in four different manners. Here, three common categories of circRNAs are illustrated: intronic circRNAs (ciRNAs); exonic circRNAs, which contain two or more exons or their partial sequences; and exon-intron circRNAs (EIciRNAs), which retain introns between exons. The fourth type of circRNAs refers to the tRNA intronic circular RNAs spliced from the endonuclease complex. CiRNAs are mainly located in the nucleus; exonic circRNAs are often distributed in the cytoplasm and nucleus; and EIciRNAs are located mainly in the nucleus, like ciRNAs. Cytoplasmic circRNAs can be enwrapped into the MVBs and, subsequently, transported to autophagosomes and lysosomes, and/or merged with cytoplasmic membrane and, subsequently, secreted into the extracellular spaces.
Figure 1. circRNA formation, transportation, and secretion. CircRNAs can be generated by back splicing in four different manners. Here, three common categories of circRNAs are illustrated: intronic circRNAs (ciRNAs); exonic circRNAs, which contain two or more exons or their partial sequences; and exon-intron circRNAs (EIciRNAs), which retain introns between exons. The fourth type of circRNAs refers to the tRNA intronic circular RNAs spliced from the endonuclease complex. CiRNAs are mainly located in the nucleus; exonic circRNAs are often distributed in the cytoplasm and nucleus; and EIciRNAs are located mainly in the nucleus, like ciRNAs. Cytoplasmic circRNAs can be enwrapped into the MVBs and, subsequently, transported to autophagosomes and lysosomes, and/or merged with cytoplasmic membrane and, subsequently, secreted into the extracellular spaces.
Cells 13 01407 g001
Table 1. CircRNAs in Macrophage Activations.
Table 1. CircRNAs in Macrophage Activations.
YearConclusions/FindingsCitation
2022circRNF19B increased when M2 converted to M1.PMID 35368656 [80]
2022circ-Cdr1as serves as an anti-inflammatory regulator in BMDM. PMID 36181865 [78]
2022circCdyl promotes M1 polarization.PMID 34547461 [18]
2023circATP9A promotes macrophage M2 polarization PMID 38049814 [57]
2023circ17725 Promotes Macrophage Polarization towards M2 PMID 37035757 [84]
2020circPPM1F modulates M1 macrophage activation PMID 33042261 [85]
2023circmSCAR in septic mice is closely related to M1 macrophage polarization. PMID 36965082 [86]
2018circZC3H4 RNA/ZC3H4 promotes pulmonary macrophage activation. PMID 29401612 [87]
2016circRasGEF1B upregulates LPS-induced ICAM-1 expression in macrophages PMID 27362560 [77]
2016circ-ANRIL is involved in the apoptosis of macrophages PMID 27539542 [75]
2017circ003780, circ010056, and circ010231 are upregulated and circ003424, circ013630, circ001489 and circ018127 are downregulated in M1.PMID 28075448 [88]
201911 and 126 circRNAs are upregulated and downregulated in lung macrophages in CLP/sepsis mice PMID 31411002 [89]
2022CircRNA PLCE1 promotes M2 polarization of tumor-associated macrophages PMID 35349390 [90]
2023Circ0075723 inhibits macrophage pyroptosis via miR-155 sponging PMID 36923408 [91]
2023circSOD2, circCHSY1, circTNFRSF21, and circDHTKD1 are differentially expressed in TB-infected macrophages PMID 38139387 [92]
2023circASPH upregulates M1 gene expression in macrophage in sepsis. Western Univ. Thesis [82]
2022 circITGB6- mediated tumor- associated macrophage (TAM) polarization into M2 phenotype PMID 35277458 [93]
2019 circRNAs (092520, 102610, 004662, or 103124) in PBMCs increase in CD, PMID 31261517 [94]
2023 circP4HB promoted M2 macrophage phenotype PMID 37880717 [95]
2021 circHIPK3 sponges miR-192 and miR-561 to promote NLRP3 in macrophages PMID 34085163 [96]
2020 circRNA-0003528 promoted tuberculosis associated macrophage polarization PMID 33318319 [97]
2021 Circ0110102 inhibits macrophage activation PMID 33891564 [98]
2024 circ0000381 promotes microglial/macrophage pyroptosis PMID 38466640 [99]
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Son, C.J.; Carnino, J.M.; Lee, H.; Jin, Y. Emerging Roles of Circular RNA in Macrophage Activation and Inflammatory Lung Responses. Cells 2024, 13, 1407. https://doi.org/10.3390/cells13171407

AMA Style

Son CJ, Carnino JM, Lee H, Jin Y. Emerging Roles of Circular RNA in Macrophage Activation and Inflammatory Lung Responses. Cells. 2024; 13(17):1407. https://doi.org/10.3390/cells13171407

Chicago/Turabian Style

Son, Chang Jun, Jonathan M. Carnino, Heedoo Lee, and Yang Jin. 2024. "Emerging Roles of Circular RNA in Macrophage Activation and Inflammatory Lung Responses" Cells 13, no. 17: 1407. https://doi.org/10.3390/cells13171407

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop