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

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.


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 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.
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 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.
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 structuremediated 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 (m 6 A) modification regulates the circRNA biogenesis, the nuclear export of circRNAs, and the degradation of circRNAs [50][51][52][53][54][55]; YTHDC1, an m 6 A reader, promotes the nuclear export of m 6 A-modified circNSUN2 [50].EIF4A3, another m 6 A 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.

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.

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 circR-NAs 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, circR-NAs 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.

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 proinflammatory 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.

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.CircR-NAs 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 H 2 O 2 .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].

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.

Figure 1 .
Figure1.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.