Neutrophil microRNAs

Neutrophils are considered ‘first‐line defence’ cells as they can be rapidly recruited to the site of the immune response. As key components of non‐specific immune mechanisms, neutrophils use phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to fight pathogens. Recently, immunoregulatory abilities of neutrophils associated with the secretion of several mediators, including cytokines and extracellular vesicles (EVs) containing, among other components, microRNAs (miRNAs), have also been reported. EVs are small structures released by cells into the extracellular space and are present in all body fluids. Microvesicles show the composition and status of the releasing cell, its physiological state, and pathological changes. Currently, EVs have gained immense scientific interest as they act as transporters of epigenetic information in intercellular communication. This review summarises findings from recent scientific reports that have evaluated the utility of miRNA molecules as biomarkers for effective diagnostics or even as start‐points for new therapeutic strategies in neutrophil‐mediated immune reactions. In addition, this review describes the current state of knowledge on miRNA molecules, which are endogenous regulators of gene expression besides being involved in the regulation of the immune response.


I. INTRODUCTION
The proper functioning of individual human organ systems, conditioned by efficient communication between systems and elements of each of these systems, forms the fundamentals of homeostasis.Communication takes place through endo-, para-, and autocrine pathways via protein messengers, hormones, neurotransmitters, cytokines, and short RNA molecules (Sikora, Ptak & Bryniarski, 2011;Hukowska-Szematowicz, Tokarz-Deptuła & Deptuła, 2012).
MicroRNA molecules (miRNAs, miRs) are single-stranded non-coding structures involved in regulating the expression of about 30-80% of protein-coding genes in humans (Lu & Clark, 2012).Studies on the role of miRNAs have confirmed their involvement in processes crucial to the development and functioning of the organism, such as cell proliferation and differentiation, cell cycle, programmed cell death, blood vessel formation, tumorigenesis, and autoimmunity (Bartel, 2004;Calin et al., 2004;Fujiwara et al., 2022).

II. BIOGENESIS OF miRNAs
Transcription of miRNA genes by RNA polymerase II results in the formation of primary miRNA (pri-miRNA) transcripts of 70 nucleotides in length with a cap and a polyA tail of a 'hairpin' shape (Fig. 1).The Drosha microprocessor enzymatically processes these transcripts to form precursor miRNA (pre-miRNA).The exportin 5 protein, along with the GTP-dependent nuclear protein Ras-related nuclear protein (Ran), transports the pre-miRNA into the cytoplasm of the cell.Then, the endonuclease Dicer trims the pre-miRNA, resulting in miRNA-miRNA* duplexes of 20 nucleotides in length.These duplexes consist of a leading strand and a passenger strand (marked with an asterisk).When the strands of the duplex are separated, the leading strand is usually selected as the functional one, but its passenger strand can also be involved in the regulation of gene expression.A mature miRNA molecule consists of 18-24 nucleotides, which are incorporated into an RNA-induced silencing complex (miRISC) (Finnegan & Pasquinelli, 2013;Rani & Sengar, 2022).These complexes bind to the 3 0 untranslated region (3 0 UTR) of the messenger RNA (mRNA) of the target gene via miRNA.Transcript degradation takes place as a result of complete nucleotide complementarity between miRNA and mRNA, with the Argonaute protein constituting a RISC element with endonucleolytic activity.If complete complementarity is not achieved, translation of the target mRNA is inhibited.The majority of the known miRNA molecules do not show complete complementarity to the 3 0 UTR region (Bartel, 2004;Du & Zamore, 2005;Xiao, Ai & Wilusz, 2020).
Degradation of the target mRNA or repression of translation, or a combination of both, does not damage the miRNA molecule, which can continue to recognise and catalyze the silencing of subsequent genes.A single miRNA molecule can simultaneously regulate the expression of hundreds of target genes, single proteins crucial to a given process, or entire groups of proteins necessary at a given stage of organism development (Hutv agner & Zamore, 2002;Satoh & Tabunoki, 2011).
Genes for miRNAs are often organised in clusters.miRNA can be derived from transcribed regions of other genes (in exons, introns, or UTRs) or be in independent transcriptional units.This arrangement of genes can result in the simultaneous formation of miRNA and mRNA transcripts (Lee, 2002;Shomron & Levy, 2009;Wang, 2010;Isik & Berezikov, 2013).If the miRNA genes are located at or near breakage sites on chromosomes, damage to the genome, due to translocation, deletion, or amplification, affects the expression of not only protein genes but also miRNAs.Abnormal expression of individual miRNA molecules can occur as a result of genomic alterations, impaired biogenesis, or the activity of epigenetic factors that regulate gene expression.As miRNAs regulate gene expression at the post-transcriptional level, they ultimately affect the content of individual proteins in the body (Calin et al., 2004).

III. NOMENCLATURE OF miRNAs
Intensive research on human miRNAs has resulted in the identification of more than 2600 molecules (Natsidis, Kappas & Karlowski, 2018), necessitating the development of a standardised naming system.The name of a miRNA is preceded by a prefix, such as hsa for Homo sapiens and mmu for Mus musculus.The sequences of the mature miRNA molecule are represented by 'miR', and those of the precursor pre-miRNA are represented by 'mir'.In addition, a numerical designation is assigned, which is invariable regardless of species.Differences in the structure of the miRNA molecule are indicated by an additional number or letter.Furthermore, the formation of two different miRNA molecules from a single double-stranded RNA (dsRNA) segment is denoted by adding '-5p' for a miRNA formed on a 5 0 strand or '-3p' on a 3 0 strand (Griffiths-Jones, 2006;Budak et al., 2016).
In the first molecules discovered, the prefix 'lin' (lin-4) or 'let' (let-7) was used in the nomenclature instead of 'miR', which, due to tradition, was not changed after the new nomenclature was introduced (Lagos- Quintana et al., 2001).

IV. TRANSPORT OF miRNAs
Molecules of miRNAs can passively leak into the extracellular space, can undergo secretion in complexes with proteins, or can be transported via extracellular vesicles (EVs), also known as 'physiological liposomes' (van Niel, D'Angelo & Raposo, 2018;Ratajczak & Ratajczak, 2020).EVs are classified as large extracellular vesicles (L-EVs), including apoptotic bodies, large oncosomes and microvesicles, or small extracellular vesicles (S-EVs), including exosomes (Witwer & Théry, 2019;Malkin & Bratman, 2020).Microvesicles are probably secreted by all cells, including stem cells, leukocytes, platelets, and cancer cells, and play an important role in intercellular communication.Microvesicles isolated from human blood plasma are primarily derived from platelets, but also from endothelium, monocytes, lymphocytes, and neutrophils (Bohnsack, Czaplinski & Görlich, 2004).It is hypothesised that the presence of microvesicles in peripheral blood under physiological conditions is related to the intercellular communication of various tissues to which they are directionally transported (He et al., 2021;Dooley et al., 2021).The expression level and profile of miRNAs in vesicles differ from those of miRNAs in leukocytes (Chen, Larregina & Morelli, 2019b).It is likely that the content of microvesicles is determined by the need to alter  In the non-canonical pathway of miRNA formation, intron sequences are transcribed together with exons.The primary transcripts are then spliced into spliceosomes.The removed intronic sequences are separated from the exon nucleotides and trimmed.Both the canonical and non-canonical pathway lead to the formation of a precursor miRNA (pre-miRNA) of about 35 nucleotides long, which is exported from the nucleus to the cytoplasm by Exportin 5 and Ras-related nuclear protein (Ran) in a GTP-dependent manner through the nuclear pores.As a result of the cleavage activity of the Dicer and transactivation response element RNA-binding protein (TRBP) complex, the miRNA/miRNA* duplex is released in the form of two combined miRNA strands.After separation, the miRNA* is degraded while the second miRNA strand is loaded with Argonaute proteins (Ago).The newly formed miRNA-induced silencing complex (RISC) is then ready to interfere with the target messenger RNAs (mRNAs).
the expression of well-defined target genes.The mechanism of formation of microvesicles is not precisely known, and it is presumed that vesicles are randomly detached (shedding vesicles) or that their formation is tightly regulated (exosomes).However, the selection of miRNAs into microvesicles may be mediated by P bodies, which are localised in the cytoplasm of the cell adjacent to miRNAs (Hunter et al., 2008;Vaz et al., 2010).The transfer of miRNAs into the vesicle is regulated by microvesicle proteins.Evidence for the intercellular transport of microvesicles (with miRNAs) suggests that it is probably an active process subject to regulation by receptors on target cells (Yuan et al., 2009;Ismail et al., 2013).

V. METHODS TO EVALUATE miRNA EXPRESSION
The primary technique used to evaluate miRNA expression is the analysis of microarrays containing complementary oligonucleotides.Another method is hybridization in solution with polystyrene grains coated with oligonucleotide probes complementary to the miRNAs under study, in which the resulting hybrids can be analysed using a flow cytometer.The results of microarray analysis can be confirmed using commercial quantitative reverse transcription-precursor polymerase chain reaction (qRT-PCR) assays, which can also be used to evaluate pri-miRNAs and pre-miRNAs (Sassen, Miska & Caldas, 2008).qRT-PCR is the most sensitive and accurate method of miRNA analysis.
The Northern blot technique is the oldest method used to assess miRNAs, and is primarily used to supplement and confirm the results obtained using other techniques (Iorio et al., 2005;De la Rosa & Reyes, 2019).The in situ hybridization technique using high-affinity oligonucleotide probes allows the analysis of expression patterns of miRNAs on formalin-fixed scrapings and paraffin-embedded tissue sections (Sassen et al., 2008;Bugly o et al., 2019).
Recently, miRNA enzyme immunoassay (miREIA) kits for the assessment of miRNAs have become available.This method is based on the hybridization of miRNA isolated from the patient's sample to a complementary probe of biotinylated DNA.The resulting DNA-RNA hybrids are transferred to wells in microplates coated with a monoclonal antibody specific for DNA-RNA hybrids.After washing, the solid phase is incubated with the streptavidin-horseradish peroxidase (HRP) conjugate, and after another washing step, the resulting complexes are visualised by adding a chromogenic substrate (Krepelkova et al., 2019;Dawidowicz et al., 2021;Charkiewicz et al., 2022).

VI. miRNAs IN THE REGULATION OF NEUTROPHIL FUNCTION
Even a small change in the level of a single miRNA molecule can have significant biological effects.miRNAs often form clustersfunctional groups that control multiple components of a single process or single components of various related pathways (Baltimore et al., 2008;O'Connell et al., 2010). In humans, neutrophils express 11 clusters: miR-17-92, miR-106b-25, miR-23a-27a-24, miR-16-1, miR-15b, let-7a-1, let-7a-3, miR-29a, miR-29c, miR-181a, andmiR-181c (Yu et al., 2006;Ward et al., 2011).Distinct miRNAs can coexpress and regulate a process cooperatively.In addition, the pleiotropic nature of miRNA molecules can compensate for the disrupted action of a single miRNA.However, the action of miRNAs is characterised by a 'dose effect', in which defects in more than one molecule of miRNA may have a cumulative effect (Xiao & Rajewsky, 2009).
More than 100 different miRNA molecules that regulate gene expression in immune cells have been described (Fig. 2; Table 1).For instance, human neutrophils express 148 miRNA molecules, but this number may change as new ones are discovered (Ward et al., 2011).It has been confirmed that miRNA molecules control all stages of immune system cell development and function, from stem cells to activated effector cells of both acquired and innate responses (O'Connell et al., 2010;Sun, Wu & Liu, 2021;Wu et al., 2021).
miR-223 controls the development, differentiation, and function of myeloid cells.The presence of miR-223 has been reported in embryonic and progenitor cells of the hematopoietic system.However, increased expression of miR-223 is observed in progenitor cells of the myeloid lineage, and the levels increase with cell differentiation, including in granulocytes (Chen et al., 2004;Johnnidis et al., 2008;Gantier, 2013).miR-223 controls granulopoiesis by suppressing the transcription factor MEF2C (myocyte-specific enhancer factor 2C, also known as MADS box transcription enhancer factor 2, polypeptide C), which promotes the proliferation of myeloid lineage precursors or the insulin-like growth factor receptor.The absence of miR-223 in mice resulted in a twofold increase in hyper-reactive granulocytes, promoting acute inflammation in multiple organs involving myeloperoxidase (MPO) and reactive oxygen species (ROS) (Johnnidis et al., 2008;Kerckhove et al., 2018;Bartneck & Wang, 2019).miR-223 is reported to be essential for the formation of granulocytes (Vian et al., 2014).
The miR-223 molecule is transcribed from an independent promoter located on the X chromosome.Low miR-223 expression is observed in bone marrow stem cells, and its increase is induced as a result of cell differentiation.miR-223 expression is controlled by two transcription factors that compete for binding to the promoter of this molecule.

PMN miRNAs
which is crucial in the subsequent stages of differentiation of the monocytic-granulocytic lineage (Fazi et al., 2005;Johnnidis et al., 2008).
miR-130a is also involved in neutrophil development.It is highly expressed early in the maturation of these cells and regulates target proteins that are important for this process.Transcription factors MYB (myeloblastosis proto-oncogene, transcription factor) and CBF-β (core binding factor beta subunit) are identified as putative targets of miR-130a, which may regulate the expression of MPO and proteinase 3 (Pedersen et al., 2015).miR-130a inhibits the expression of the transcription factor Smad4 (mothers against decapentaplegic homolog 4)a key mediator of transforming growth factor β1 (TGF-β1).Overexpression of miR-130a also inhibits normal cell cycle exit and expression of secondary granule proteins via the C/EBP-ε (transcription factor CCAAT-enhancer Fig. 2. Neutrophil-mediated immune reactions in inflammation and autoimmunity.Dysregulation of non-specific neutrophil defence mechanisms may lead to excessive inflammatory reactions that underlie autoimmunity.Overactivation of neutrophils in terms of secretion of bioactive proteins and ROS, release of NETs or microRNAs (miRNAs) results in the recruitment of leukocytes, activation of immunocompetent cells, activation of the inflammasome, activation of complement and presentation of autoantigens, resulting in increased inflammation and, consequently, autoimmunity.The participation of miRNAs in immune reactions is the subject of extensive study, the results of which place miRNAs in the network of regulatory mechanisms of the immune response (Garley et al., 2017;Fousert et al., 2020;Zhou & Bréchard, 2022).CathG, cathepsin G; CCL, CXCL, chemokines; CCR2, CC chemokine receptor type 2; EV, extracellular vesicle; G-CSF, granulocyte colony-stimulating factor; IL, interleukin; MMPs, metalloproteinases; MPO, myeloperoxidase; NE, neutrophil elastase; NETs, neutrophil extracellular traps; NO, nitric oxide; PAD4, peptidylarginine deiminase 4; ROS; reactive oxygen species; TNF-α, tumour necrosis factor α.
Overexpression of miR-130a reduces the expression of C/EBP-ε and with it the synthesis of the secondary granule proteins lactoferrin, cathelicidin and lipocalin-2, resulting in

PMN miRNAs
differentiating neutrophils with an immature phenotype.In experimental studies, blocking miR-130a allowed granulocytes to achieve cellular maturity, suggesting the temporary involvement of this molecule in granulopoiesis (Larsen et al., 2014).
The miR-146 group was first described while attempting to establish an association between miRNAs and the immune response to infections.Currently, it is acknowledged that miR-146 plays a biological role in the physiological regulation of the response during bacterial infections (Jiang et al., 2022).Activation of non-specific immune response mechanisms results in rapid changes in the expression of miR-146 and miR-155 (Schulte, Westermann & Vogel, 2013).These miRNAs regulate the acute proinflammatory response associated with the release of IL-8 and RANTES (regulated on activation, normal T-cell expressed and secreted, CCL5) either by blocking signal transduction upon activation of Toll-like receptors (TLRs) or by directly affecting the translation of IL-8, RANTES, and TNF-α (Perry et al., 2008).miR-146a directly regulates the expression of TLR4, TNF receptor-associated factor 6, and interleukin 1 receptor-associated kinase 1, which are key molecules involved in the TLR4/nuclear factor kappa B (NF-κB) pathway.Activation of this pathway by lipopolysaccharide (LPS) promotes the transcription of miR-146a (Taganov et al., 2006;Nahid, Satoh & Chan, 2011;Saba, Sorensen & Booth, 2014).
miR-146a-deficient mice have been shown rapidly to develop inflammation, autoimmunity, and even cancer.miR-146a deficiency was accompanied by high miR-155 expression.During the inflammatory response, the NF-κB-miR-155 axis coordinates with the NF-κB-miR-146a axis to regulate the intensity and duration of inflammation.NF-κB signalling during inflammation is self-limiting, and the action of miR-155 and miR-146a molecules is a twostage process.During the first 12 h of inflammation, NF-κB rapidly increases the expression of miR-155, which via SH2 inositol 5-phosphatase 1 (SHIP1) activates the phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt)dependent IkappaB kinase (IKK) signalling complex.This creates a positive feedback loop necessary to amplify the inflammatory signal.In the late phase of inflammation, miR-146a is gradually upregulated, which creates a negative feedback loop through interleukin-1 receptorassociated kinase 1 (IRAK1) and tumour necrosis factor receptor associated factor 6 (TRAF6), ultimately attenuating NF-κB activity and thereby leading to resolution of inflammation.Therefore, the inflammatory phenotype of the response can be attenuated by miR-155 depletion (Mann et al., 2017).
By contrast, in vitro studies in mice showed that overexpression of let-7b inhibits neutrophil TLR4 expression at the post-transcriptional level.Furthermore, let-7b decreases the levels of proinflammatory cytokines, i.e.IL-6, IL-8, and TNF-α, and simultaneously increases IL-10 in freshly isolated human neutrophils involved in the TLR4/NF-κB pathway.Application of a let-7b inhibitor resulted in increased production of IL-8 and TNF-α in neutrophils.Moreover, administration of agomir let-7b (chemically modified double-strand miRNA, which can mimic mature endogenous miRNAs) in septic mice in vivo resulted in the inhibition of neutrophil recruitment, less body mass loss in the animals, and increased survival.These findings are promising in the context of sepsis, suggesting consideration of let-7b as a therapeutic target (Chen et al., 2021).The let-7b molecule is characterised by a high level of evolutionary conservation.In humans, let-7 isoforms share a common sequence called the seed sequence.Because let-7 expression is required for developmental timing and tumour suppressor functions, it must be suppressed for stem cell renewal.Although let-7 biogenesis follows a canonical pathway, its characteristic feature is its strict regulation by RNA-binding proteins.LIN28A/B has been shown to block let-7 biogenesis.Terminal uridyltransferases (TUT4/7) degrade the let-7 precursor (pre-let-7), blocking the production of the mature molecule and 3 0 -5 0exoribonuclease (DIS3L2) has a similar effect (Lee et al., 2016).miR-21 is also a highly conserved molecule, involved in key regulatory pathways, for which the locus is located on chromosome 17.miR-21 transcription is regulated by cytokines, e.g.IFN or hypoxia, and post-translational regulation occurs with the participation of TGF-β.The three main targets of miR-21 are phosphatase and tensin homolog (PTEN), tropomyosin alpha-1 chain (TPM1), and programmed cell death 4 (PDCD4).Downregulation of miR-21 has been shown to increase the rate of cell death (Jenike & Halushka, 2021).Being an important inflammatory regulatory molecule for phagocytes, miR-21 expression is significantly increased in neutrophils during induced sepsis.High miR-21 expression during sepsis or after LPS stimulation has been shown to lead to inflammation and glycolytic metabolism in bone marrow cells (Melo, Alvarez & Serezani, 2019).
The cytokine microenvironment at the site of the neutrophil response is strategically important for the course of the reaction.Additional regulation of cytokine levels with the participation of miRNA molecules guarantees the achievement of locally high cytokine concentrations (Asirvatham, Magner & Tomasi, 2009).High miR-155 expression has been reported in granulocytes and monocytes in response to inflammatory factors.miR-155 enhances the secretion of proinflammatory cytokines and susceptibility to septic shock (Tili et al., 2007).It regulates the expression of genes for IL-4, C-C motif chemokine ligand 5 (CCL5), and the transcription factor c-musculoaponeurotic fibrosarcoma (Maf) (Williams, 2008;Yu et al., 2013).
It has been proved that miR-17 regulates IL-8 levels, which is important in neutrophil recruitment.Together with miR-31, miR-17 controls the process of tight adhesion of these leukocytes to the endothelium (Su arez et al., 2010).Neutrophil migration is suppressed by miR-199 and miR-722, whose high expression inhibits the migration and chemotaxis of human neutrophil-like cells (HL-60) (Hsu et al., 2019a,b).
miR-16 is a constitutive regulator of inflammation.High concentrations of this molecule have been observed in tissues and leukocytes involved in the inflammatory process, including neutrophils.Its action is realised by the rapid degradation of the inflammatory proteins IL-6, IL-8, and TNF-α due to the similarity in the structure of these cytokines, rich in AU sequences at the 3 0 end complementary to the sequence at the 5 0 end of this miRNA.The function of miR-16 is probably related to the inhibition of cell cycle progression during inflammation (Jing et al., 2005;Linsley et al., 2007).

VII. NEUTROPHIL EXTRACELLULAR TRAPS (NETs) AND miRNA
During immune defence, neutrophils form neutrophil extracellular traps (NETs) to eliminate the cause of infection and virulence factors.NETs are made of fibres, primarily composed of DNA from neutrophils, in which highly cytotoxic histones are woven together with antimicrobial proteins.These structures are formed inside the cell and then released into the extracellular space, where they perform their antipathogenic function.The process of releasing NETs from the cell is called NETosis.Depending on the activator, NETosis can be lytic and results in the death of the neutrophil (classical pathway) or non-lytic, in which NETs are released as vesicles and the cell retains its primary functions (Brinkmann et al., 2004;Kenny et al., 2017;Wan et al., 2022).Linhares-Lacerda et al. (2020) reported that NETs are also a carrier of miRNAs.They showed that miR-142-3p is transferred from NETs to macrophages, where it inhibits protein kinase C-α, which regulates TNF-α production.In addition, the miRNA profile depends on the activation pathway, i.e. non-lytic or lytic NETosis (Linhares-Lacerda et al., 2020).These findings show that NETs not only kill pathogens but also are part of important immunoregulatory functions taking part in intercellular communication ( Águila et al., 2021).Hawez et al. (2019) reported that miR-155 also regulates the formation of NETs by affecting peptidylarginine deiminase (PAD4) mRNA, whose protein product PAD4 participates in the regulation of histone citrullination during NETosis.They suggested that NETosis is accompanied by translational activity, which is contrary to previous observations (Hawez et al., 2019).
In a mouse model, miR-146a −/− neutrophils stimulated with phorbol 12-myristate 13-acetate released higher amounts of circulating free DNA (cfDNA) (a NETosis biomarker) and higher expression of citrullinated histone H3 was observed compared with wild-type littermates.These findings show an association between this miRNA molecule and NETosis although the mechanism of this association is unknown (Arroyo et al., 2020).However, some studies have reported that high levels of miR-146a inhibit superoxide dismutase 2 in neutrophils, which in turn contributes to an increase in ROS and thus promotes the formation of NETs.The results of these studies prove that the phenomenon of NET formation is not only enhanced, but can also be inhibited by the participation of miRNA molecules.It can be concluded that the obtained immunological effect depends on the relationship between the miRNA molecules involved in the reaction/process, as well as the ratio of miRNA and mRNA concentrations, complementarity and thermodynamics of their interactions (Zhang et al., 2019b).
miR-505 is a potential regulator of NETs, and exosomes containing miR-505 are shown to enhance the formation of NETs.Researchers have hypothesised that miR-505 inhibits NAD-dependent deacetylase sirtuin-3 (SIRT3), leading to uncontrolled ROS production and, consequently, activation of NETosis (Chen et al., 2019a).
High contents of IL-18, NETs, and miR-223 are observed in patients with autoinflammatory Still's disease.Studies using HL-60 cells have shown that IL-18 induces the formation of

PMN miRNAs
NETs and simultaneously enhances the expression of miR-223.In turn, miR-223 inhibits the formation of NETs by blocking calcium influx into the cell and IL-18-dependent ROS production.In a feedback loop, cfDNA obtained from patients with Still's disease can increase miR-223 expression in human neutrophils, which release secretory vesicles containing these miRNAs (Liao et al., 2021).
Studies in gout patients showed that the high expression of miR-3146 in neutrophils is accompanied by high contents of NETs.Studies in rats showed that miR-3146 antagomir therapy (antisense sequences for miRNAs, specific inhibitors) inhibits ROS and SIRT1-dependent NET formation (Shan et al., 2021).

VIII. miRNAs IN AUTOIMMUNITY
The data presented above prove that miRNAs can regulate the function and activation of neutrophils, by controlling the expression of genes that are involved in processes such as chemotaxis, phagocytosis, and the release of inflammatory mediators.Neutrophils are key players in the initiation and propagation of inflammation.Dysregulation of miRNAs can lead to aberrant neutrophil activity, causing either excessive or inappropriate inflammatory responses, both of which can contribute to autoimmune diseases.miRNAs have been implicated in autoimmune diseases through their ability to influence immune cell differentiation, self-tolerance, and the production of autoantibodies.They can regulate the intensity and duration of the inflammatory response by targeting genes involved in the production of cytokines, chemokines, and other pro-inflammatory molecules (De la Rosa et al., 2020).
miR-155 exhibits highly consistent pro-inflammatory properties.Research has shown that miR-155 is upregulated in neutrophils in response to inflammatory stimuli, and it can modulate the production of ROS, cytokines, and chemotaxis in these cells.Dysregulation of miR-155 expression has been linked to autoimmune diseases, such as rheumatoid arthritis, where increased miR-155 levels can lead to excessive inflammation and tissue damage (Su et al., 2017).miR-146a is another miRNA with a significant role as a negative feedback regulator of the immune system.miR-146a helps control the production of pro-inflammatory cytokines, such as TNF-α and IL-1β, by immune cells, including neutrophils.Dysregulation of miR-146a has been associated with systemic lupus erythematosus (SLE), where a deficiency in miR-146a can contribute to uncontrolled inflammation and tissue damage.miR-146a inhibition in a mouse model of psoriasis promoted earlier psoriasis-like onset, epidermal hyperproliferation, IL-17 skin inflammation, IL-8 secretion, and neutrophil infiltration (Domingo et al., 2020).
Moreover, the formation of NETs, a crucial part of the innate immune response, can have implications for inflammation and autoimmune diseases.NETs can trap and activate immune cells, release pro-inflammatory mediators, and contribute to tissue damage.The presence of NETs can lead to the exposure of autoantigens and trigger the production of autoantibodies, contributing to the autoimmune response.NETs have been implicated in the pathogenesis of SLE, rheumatoid arthritis, and vasculitis, with increased NET formation.miRNAs can regulate the process of NET production.Specific miRNAs can target genes involved in the activation of neutrophils and the formation of NETs.Dysregulated miRNA expression can affect the balance of NET production and clearance.For example, miR-223 has been shown to regulate NET formation negatively by targeting key components involved in this process.miR-21 and miR-155 have been associated with autoimmune diseases, and they may impact NET formation and the subsequent autoantibody response.miR-146a can regulate the TLR pathway by targeting TRAF6 to reduce the expression of IL-8, a NETs inducer, in autoimmune-mediated diffuse alveolar haemorrhage associated with SLE (Hsieh et al., 2022).
Understanding the role of miRNAs in autoimmune diseases and inflammation may lead to more targeted and personalised treatments in the future.It was shown that the administration of miR-155 inhibitors reduced the development of atherosclerotic lesions in mice, the formation of which is mediated by NETs.Therefore, the use of miRNAs as a therapeutic tool in controlling the process of NETs formation represents an interesting future direction for new therapies.The use of exosomes as miRNA carriers that integrate easily with the neutrophil in the regulation of NETosis is highly feasible.Furthermore, transfections with pre-miR-223, pre-miR-126, and pre-miR-148a specifically modulated genes that regulate inflammation, neutrophil survival, and migration in patients with rheumatoid arthritis (De la Rosa et al., 2020).
Advances in miRNA profiling and functional studies are helping to elucidate the complex networks of miRNAs that govern immune responses.Understanding the intricate interplay between miRNAs, NETs, autoimmune diseases, and inflammation is an active area of research with the potential to uncover new therapeutic strategies.

IX. miRNAs AS MARKERS OF ACUTE MYELOID LEUKAEMIA (AML)
Much attention has been paid to profiling the expression of miRNAs in cancer as the miRNA profile has been shown to identify the origin of cells and determine their stage of development and differentiation.miRNA molecules can regulate the expression of many oncogenes and tumour suppressor genes and act as oncogenes or suppressors themselves (Zhang et al., 2007;Xiong et al., 2013;Wu et al., 2020;Guo et al., 2020;Zhou et al., 2021).
Acute myeloid leukaemia (AML) is a malignant neoplasm that originates from a precursor-transformed myeloid cell, which is derived from a bone marrow stem cell.Under normal conditions, divisions from myeloid cells lead to the formation of granulocytes, which are the most numerous neutrophil populations among leukocytes, as well as monocytes.In addition to anemia and thrombocytopenia, one of the hallmarks of AML is an increase in the total number of leukocytes, with significantly low levels of neutrophils (Pelcovits & Niroula, 2020;Newell & Cook, 2021).
Despite numerous limitations of using circulating miRNAs as biomarkers in the diagnostic and clinical practice of haematological malignancies, attempts have been made to use them in AML diagnosis (Wang et al., 2019;Cheng et al., 2020).Studies reported that the expression of miR-92a and the miR92a/miR-638 ratio is significantly reduced in the plasma of acute leukaemia patients compared with healthy subjects (Tanaka et al., 2009).Some studies reported a low expression of let-7d, miR-150, miR-339, and miR-342 in AML patients with concomitant overexpression of let-7b and miR-523.In addition, miR-150 and miR-342 are reported to be of significant value in the detection of AML (Fayyad-Kazan et al., 2013).An inverse correlation between miR-150 expression, and TNF-α, IL-10 and IL-18 levels was observed, indicating potential target genes for this miRNA molecule (Vasilescu et al., 2009).
Other researchers have profiled six miRNA molecules that accurately differentiate AML patients from healthy individuals: miR-10a, miR-93, miR-129, miR-155, miR-181b, and miR-320d.They have confirmed that miR-181b clearly correlates with the overall survival time of these patients and serves as an independent predictor.Physiologically, miR-181b functions as a tumour suppressor, and restoration of its normal expression leads to the promotion of apoptosis, inhibition of tumour cell proliferation, and delay of leukemogenesis (the process of AML formation).The target genes of miR-181b are P27, CREB1, TIMP3, MCL1, and XIAP, which are involved in apoptosis, cell division, resistance to chemotherapy, and cell migration (Zhi et al., 2013).Increased expression of miR-181 family members has been observed in AML cells (Su et al., 2015).
High levels of miR-10a, together with mutations in the NPM1 gene, lead to the decreased expression of the MDM4 target gene, whose product interacts with the TP53 gene protein and induces apoptosis.In AML patients, mutations in NPM1 are associated with the increased expression of miR-196a and miR-196b (Coskun et al., 2011).
High expression of miR-126 in AML cells results in the inhibition of their apoptosis.The increase in miR-126 expression is likely due to hypomethylation of the promoter of the gene encoding this molecule, which leads to the inhibition of the activity of the suppressor gene PLK2.These perturbations block apoptosis and increase the viability of leukaemic cells (Li et al., 2008).
Overexpression of miR-1-3 and miR-133a-1 in AML cells is closely associated with the overexpression of the EVI1 gene and translates into inappropriate cell proliferation and the development of an aggressive form of leukaemia (G omez-Benito et al., 2010).
Since miR-155 stimulates granulocyte proliferation, increased levels of this molecule in bone marrow cells after LPS stimulation can lead to the expansion of myeloid line cells and the development of AML.It is hypothesised that miR-155 may have oncogenic effects (Schneider et al., 2016).However, miR-16, through suppression of oncogenes and proteins associated with cancer cell survival, acts as a suppressor (Gao et al., 2011a).
Molecules in the miR-193 family function as a suppressor of many oncogenes.It has been reported that hypermethylation of promoter gene segments encoding miR-193 leads to the repression of this molecule in AML cells.A low expression of miR-193a is responsible for the increased expression of the c-KIT oncogene and its protein product and consequently the induction of abnormal cell divisions.Unsatisfactory treatment results using miRNAs have also been observed with these disorders.Similar results have been reported for the reduced expression of miR-193b (Gao et al., 2011b,c).
In addition, recent studies show that miRNA molecules are involved in metastasis, acting as activators or inhibitors of this process, including in the course of leukaemia (Wang et al., 2016;Ma, Wang & Jiang, 2020).

X. miRNAs IN CHRONIC MYELOID LEUKAEMIA (CML)
Chronic myeloid leukaemia (CML) results from the translocation of two fragments of chromosomes 9 and 22.The resulting fusion gene (BCR/ABL) product on chromosome 22 (the Philadelphia chromosome) is a tyrosine kinase that alters cell cycle regulation.Thus, proliferation of abnormal cells occurs.As a result of these changes there is overproduction of granulocytes, including neutrophils, many of which have the BCR/ABL translocation [fusion between the Abelson (Abl) tyrosine kinase gene at chromosome 9 and the break point cluster (Bcr) gene at chromosome 22].Both mature and immature cells are present in circulation (Zhou, Medeiros & Hu, 2018;Minciacchi, Kumar & Krause, 2021).
Some studies have shown that the appearance of the BCR/ ABL fusion gene and its kinase leads to decreased miR-451 expression, for which it is a target gene, indicating the existence of a regulatory loop (Lopotov a et al., 2011).Furthermore, low expression of miR-150 and miR-151 is associated with BCR/ABL kinase activity.Moreover, low miR-10a expression in CML is associated with the overexpression of the upstream transcription factor 2 (USF2) gene, encoding the transcription factor USF2 (C-Fos interacting),
Reduced expression of miR-199b and miR-24 in CML cells has also been reported.Since miR-199b controls the expression of genes involved in signal transduction, transcriptional regulation, cell proliferation, and DNA repair, abnormalities in its expression may be crucial to the development of CML.In CML patients, chromosomal alterations involving genes encoding these miRNA molecules are associated with resistance to tyrosine kinase inhibitor therapy and IFN-α therapy (Albano et al., 2009).
By contrast, miR-130a and miR-130b are overexpressed in CML.These oncogenic molecules inhibit the expression of the NOV gene, whose protein product functions as a negative regulator of cell growth.However, the presence of the BCR/ABL fusion gene results in the activation of miR-130a and miR-130b, as well as miR-148a and miR-212.Overexpression of these molecules is associated with NOV activity, resulting in the inhibition of NOV protein regulatory capacity in CML (Suresh et al., 2011).
Overexpression of miR-17-92 has been found to characterise the chronic phase of CML and is dependent on the BCR/ABL and c-MYC gene, indicating the presence of a signalling network (Venturini et al., 2007).

XI. CONCLUSIONS
(1) Recently, analyses of the regulatory functions of miRNA molecules and identification of their expression profiles have increasingly become important clinically, including their involvement in the course of neutrophil-mediated immune reactions.
(2) The high stability of miRNAs in the plasma/serum environment increases their analytical potential for use in routine diagnostics.
(3) High hopes have been placed on research aimed at using miRNAs in new therapeutic applications.It can be presumed that in the future, suppression by synthetic antagomirs or enhancement of expression by transfection of synthetic miRNA or expression of pre-miRNA from plasmids or vectors may find wide-ranging therapeutic applications in the immunoregulation of conditions associated with neutrophil dysfunction.

Fig. 1 .
Fig. 1.Biogenesis of miRNAs.Most miRNAs are transcribed from single DNA sequences or long gene sequences organised in clusters by RNA polymerase II (RNA Pol II) or III.The 5 0 cap and 3 0 polyA tail of the primary miRNA precursor (pri-miRNA) are excised by the microprocessor complex [consisting of Drosha and DiGeorge Syndrome Critical Region 8 (DGCR8)].In the non-canonical pathway of miRNA formation, intron sequences are transcribed together with exons.The primary transcripts are then spliced into spliceosomes.The removed intronic sequences are separated from the exon nucleotides and trimmed.Both the canonical and non-canonical pathway lead to the formation of a precursor miRNA (pre-miRNA) of about 35 nucleotides long, which is exported from the nucleus to the cytoplasm by Exportin 5 and Ras-related nuclear protein (Ran) in a GTP-dependent manner through the nuclear pores.As a result of the cleavage activity of the Dicer and transactivation response element RNA-binding protein (TRBP) complex, the miRNA/miRNA* duplex is released in the form of two combined miRNA strands.After separation, the miRNA* is degraded while the second miRNA strand is loaded with Argonaute proteins (Ago).The newly formed miRNA-induced silencing complex (RISC) is then ready to interfere with the target messenger RNAs (mRNAs).
These are nuclear factor type I (NFI-A) and CCAAT enhancer binding protein alpha (C/EBPα).Under cell resting conditions, NFI-A binds the promoter maintaining low expression of miR-223.During granulocyte differentiation, NFI-A is released and C/EBPα binds to the promoter, which increases the expression of miR-223 in the neutrophil.A positive feedback loop is observed because the target gene for miR-223 is, among others, NFI-A.Therefore, overexpression of miR-223 further suppresses NFI-A expression.Moreover, overexpression of miR-223 induces the CD11b marker, Biological Reviews 99 (2024) 864-877 © 2023 The Authors.Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical Society.

Table 1 .
Key microRNAs (miRNAs) in the development and functions of neutrophils.
Biological Reviews 99 (2024) 864-877 © 2023 The Authors.Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical Society.
Biological Reviews 99 (2024) 864-877 © 2023 The Authors.Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical Society.
Biological Reviews 99 (2024) 864-877 © 2023 The Authors.Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical Society.
Biological Reviews 99 (2024) 864-877 © 2023 The Authors.Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical Society.