miRNAs as regulators of extracellular TLRs in bacterial infections

miRNAs as regulators of extracellular TLRs in bacterial infections | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 12 September 2025 V1 Latest version Share on miRNAs as regulators of extracellular TLRs in bacterial infections Authors : Paulina Puente Mancera , Erandi Pérez Figueroa 0009-0005-0605-0177 , Mariana López Mejía , and Antonia Castillo-Rodal I [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.175769486.69748216/v1 205 views 129 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract The detection of pathogens by the immune system is facilitated through various cellular interactions and molecules, including pattern recognition receptors that activate signaling pathways in response to the pathogen. Among the most studied receptors are Toll-like receptors, which are activated upon ligand recognition and are considered the first event in innate immunity. The transcriptional response of TLRs in infected cells is modified by pathogens, where miRNAs, which are post-transcriptional regulators of gene expression, play a role. We review some bacteria that alter the signaling cascade of extracellular TLRs and the co-participation of miRNAs, most of which are analyzed downstream, and a very low percentage act as ligands of the TLRs. There is an increase in knowledge of the regulation of miRNAs by bacteria; however, the functional complexity of miRNAs has hindered progress in utilizing them as biomarkers or for therapeutic purposes. Introduction The immune system is an evolutionarily and phylogenetically conserved system composed of different cells and molecules that become activated when homeostasis is altered. The immune system is classified in a coordinated manner into two stages: innate immunity, in which cells respond immediately to foci of inflammation with the production of cytokines and chemokines, and adaptive immunity. The adaptive immunity helps innate immunity, but its response takes several days to generate and leaves a memory response. Innate immunity is made up of cells with different functions, both hematopoietic and non-hematopoietic cells, including macrophages, dendritic cells, neutrophils, innate lymphocytes, natural killer cells, and epithelial cells. Cells of innate immunity respond to pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) through pattern recognition receptors (PRRs). This response is nonspecific, immediate, and long considered to be a memoryless response. Activation of the innate immune response immediately incorporates cells at the required site that produce an inflammatory response mediated by the cytokines IL-6, IL-1β, and TNFα and chemokines. This action stimulates antigen presentation through major histocompatibility complexes I and II, which activates the adaptive immune response (Kaur and Secord, 2021; Marshall et al., 2018). However, the imprint left by micro-organisms on immune innate cells, following their first encounter, induces a more intense memory response to subsequent exposures. These changes include epigenetic modifications that induce the reprogramming of innate cells, resulting in a memory immune response. Recently, it has been shown that immune progenitor cells also acquire a memory immune response. Considered a defense mechanism that occurs with greater intensity in secondary events. Different reports suggest that trained immunity is linked to metabolic pathways and epigenetic remodeling, which involve diverse cell types and receptor recognition pathways. Of the main stimuli identified that induce trained immunity are LPS or β-glucan inducers of chromatin modulation. Trained immunity or memory immunity induced by PAMPs and DAMPs is responsible for eliminating any foreign agent because long-term epigenetic reprogramming directly at the promoters of genes responsible for the induction of inflammatory molecules by reconfiguring memory metabolism in cells. This gene reconfiguration also involves small messenger RNAs (miRNAs). (Arneth, n.d.) A frequent event occurs when pathogens are able to control the cell by producing a tolerogenic immune response, which is associated with the loss of the memory response. However, this tolerogenic immune response can be reversed by trained immunity. The training immunity is associated with gene activation of H3K4me monomethylation and H3K4me3 trimethylation, as well as the presence of H3K27ac in trained macrophages and metabolic reconfiguration related to metabolic changes, such as the overregulation of glycolysis in trained monocytes. A frequently mentioned example is the immunity trained by the application of the responsible M. bovis BCG vaccine, which sensitizes immune cells, and they respond immediately and efficaciously to other pathogen stimuli. It is suggested that BCG induces epigenetic modifications in bone marrow myeloid precursors and that the information is maintained after migration of the cells to the site of infection. (Ochando et al., 2023) Specialized molecules mediate microorganism detection by the innate immune system, called patterns recognition receptors (PRRs), within which Toll-like receptors (TLRs), retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), C-type lectin receptors (CLRs), absent in melanoma-2 (AIM2) like receptors (ALRs), and the nucleotide oligomerization domain-like receptors (NLR). TLRs play a critical role in the defense of the system against various infections. TLRs are the best characterized PPRs and are classified into two subfamilies based on their localization: cell surface TLRs and intracellular TLRs. TLRs recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). PAMPs are highly conserved and specific molecular structures, such as lipopolysaccharide (LPS), lipoteichoic acids (LTA), proteins, and deoxyribonucleic acid (DNA), typical of microorganisms. TLRs perform two essential functions: specific binding to their ligand and signal transmission that induces the activation of intracellular signaling transduction that promotes inflammatory responses, producing growth factors, cytokines, and chemokines, as well as linking innate immunity to adaptive immunity. Innate immune cells, such as macrophages, neutrophils, dendritic cells (DCs), mast cells, NK cells, as well as epithelial and endothelial cells, express various TLRs for the recognition of PAMPs in the extracellular and intracellular environment. The recognition of PAMPs by TLRs is key to the initiation of the innate immune response and is recognized within different molecules, three main molecules involved in signal transduction: transcription factors, adaptor proteins, and protein kinases. These molecules often converge into common signaling pathways. (Duan et al., 2022; Li and Wu, 2021) TLRs are highly conserved PRRs, and they are involved in the recognition of pathogens like bacteria, viruses, fungi, and parasites. Ten TLRs have been identified in humans: TLR-1, TLR-2, TLR-4, TLR-5, TLR-6, and TLR-10 are found on the cell surface and recognize mainly components of the bacterial cell membrane, inducing the inflammatory response. While TLRs 3, 7, 8, and 9 recognize DNA from both bacteria and viruses, they induce an inflammatory process accompanied by IFN-I production. Following ligand binding to the receptor, the carboxyl (C)-terminal globular cytoplasmic Toll/interleukin-1 (IL-1) receptor (TIR domain) in the cytosol dimerizes, leading to the stimulation of various downstream molecules and finally to the translation of the cytosolic signal. TLRs are synthesized in the endoplasmic reticulum (ER), where several proteins play a key role in this function. The proper functioning of TLRs depends on a controlled regulation involving various regulatory molecules, including co-receptors, soluble receptors, and intracellular regulators, among others, which maintain a balance between activation and inhibition of TLR signaling to multiple stimuli. (Duan et al., 2022) Innate memory immunity is exerted by both immune and non-immune cells. Immune cells exposed to various PAMPs, which in turn are recognized by different PRRs such as LPS by TLR4 or peptidoglycan by TLR2, induce resistance to infection and trained immunity. The binding of LPS or monophosphoryl lipid A (MPLA) to TLR4 induces resistance to both Gram-positive, Gram-negative and fungal pathogen while gram positive peptidoglycan binding to TLR2 produces resistance to infection of both Gram-positive and Gram-negative bacteria. Gene expression in memory immunity occurs through the modulation of chromatin structure and the control of epigenetic modifications, such as acetylation and methylation. Similarly, in metabolic reprogramming, methyl and acyl groups play a key role in mediating the process.(Sherwood et al., 2022) TLRs are synthesized and transported to the Golgi apparatus to be taken to endosomes or to the cell surface by the Multi-pass transmembrane proteins (UNC93B1), PRAT4A, and the gp96 protein. The functional maturation of TLRs to recognize their ligands requires proteolytic cleavage of nucleic acid-sensing by cathepsin B, S, L, H, and K as well as asparaginyl endopeptidase. The immune response of the TLRs after ligand recognition is expressed differently among the various cells of the immune system. One example is the release of TNF-α and IL-1β at high concentrations by monocytes as opposed to human cord cells that secrete low concentrations of both cytokines. (Vijay, 2018) Another critical component of the immune response is microRNAs (miRNAs), which regulate gene expression in cells across various biological processes and in diseases such as cancer. miRNA formation initiates in the nucleus with destination in the cytoplasm. The presence of a database of miRNAs gives us about 250,000 miRNAs in more than 168 species; however, the function of many of them is not known(Jorge et al., 2021). miRNAs play an essential role in infection control, or to the benefit of the pathogen. In an infection, pathogens utilize cellular miRNAs, altering their function in their favor, regulating the inflammatory response, highlighting in this response the expression of miRNA 155, miRNA 146, and miRNA 223. (Chandan et al., 2020)Recently, some authors point out that miRNAs not only act by controlling the gene expression of TLRs, but also act as ligands of TLRs by inducing or altering the cellular signaling pathway. LPS-activated DC shows decreased expression in the TLR/IL-1 signaling cascade by the action of miRNA-155. (Bayraktar et al., 2019) This review also contributes to a deeper understanding of the role of host-derived miRNAs in bacterial infections, highlighting their involvement in the activation, signaling, and regulation of TLR pathways, as well as the modulation of gene networks. TLRs TLRs are collectively referred to as the “interleukin-1 receptor/Toll-like receptor superfamily.” TLRs are type I transmembrane domain proteins. Their amino (N)-terminal extracellular domain contains leucine-rich repeats (LRRs) that fold into a characteristic horseshoe-like structure due to the distinctive modular arrangement of the LRR-containing proteins. The extracellular domain mediates the recognition of specific ligands like proteins, sugars, or lipids. TLRs also contain a single transmembrane spanning region required for downstream signaling pathways and a carboxyl (C)-terminal globular cytoplasmic Toll/interleukin-1 (IL-1) receptor (TIR) domain that is fundamental for downstream signalling. TIR domain has five-β stranded sheet region and five alpha-helices, which together form interleukin-1 receptor/Toll-like receptor family. TLRs are involved in innate and adaptive immune response through inflammatory reaction to fight infectious diseases and cancer. (Asami and Shimizu, 2021; Duan et al., 2022; Sameer and Nissar, 2021) All mammalian TLRs use their ectodomain of leucine-rich repeats to bind to microbial ligands by interacting with a TLR dimer. The affinity of this binding varies from low to very high ligand concentrations and depends on cell type. There are significant advances in the knowledge of signaling through TLRs and the molecules involved; however, we don’t know some events of the host-pathogen interaction. TLRs activate the immune system after bacterial recognition, to perform their function, TLRs form homodimers or heterodimers to recognize different PAMPs or DAMPs molecules. It is established in several reports that TLR4 binds to LPS and fibronectin, TLR2, one of the most versatile extracellular TLRs, forms heterodimers with other TLRs, binds to zymosan, peptidoglycans, diacylated, porin, and triacylated lipopeptides (TLR2/TLR1 and TLR2/TLR6). In contrast, homodimer TLR2 binds lipoarabinomannan, polysaccharides, and diacyls and triacylglycerols. On the other hand, TLR5 recognize flagellin. (Fitzgerald and Kagan, 2020) Assembly of a TLR dimer is required upon ligand recognition; the dimerized TLR ectodomain results in dimerization of the cytosolic TIR domains, producing various cellular changes, resulting in the formation of a protein scaffold known as a supramolecular organizing center (SMOC). Dimerized TIR domains are recognized by five proteins: TRIF, TIRAP, MyD88, MAL, and MAL, which are responsible for the expression of several cellular cytokines and chemokines. (El-Zayat et al., 2019; Wicherska‐pawłowska et al., 2021) Signaling through TLRs is divided into two pathways: the MyD88-dependent pathway and the TRIF-dependent pathway. MyD88 recruitment is facilitated by two adaptor molecules: MAL, which is used by all receptors, and TIRAP, which is required by TLR1, 2, 4, and 6 receptors. TLR3 and TLR4 utilize the TRIF-dependent pathway by stimulating IFN-I expression. TLR4 requires TRAM, an adaptor protein to activate TRIF. The downstream kinases that are directly recruited to the TLR-MyD88 complex are interleukin-1 receptor-associated kinase 4 (IRAK4), IRAK1/IRAK2, and IRAK-M. (Fig 1) The MyD88–IRAK4–IRAK1 complex is a single-stranded left-handed helix of DDs composed of six molecules of MyD88, followed by four molecules of IRAK4 and ending with four molecules of IRAK2, and is called “the Myddosome.”A subsequent series of phosphorylation and ubiquitination steps further activate transcription factors such as nuclear factor-κB (NF-κB), activator protein 1 (AP-1), and interferon regulatory factors (IRFs) to induce the transcription of immune effectors of antipathogen mechanisms and inflammation. TLRs activate different transcriptional responses depending on the adaptor involved. MyD88 is an adapter utilized by all TLRs. TLR4 is unique because it utilizes both MyD88 and TRIF pathways following ligand binding and engages TRAM/TRIF adaptors from the endosomes. (Wicherska‐pawłowska et al., 2021) microRNAs MicroRNAs (miRNAs) are part of the regulatory networks of gene expression with negative or positive feedback mechanisms. The first miRNA, lin-4, was discovered in Caenorhabditis elegans in 1993, responsible for regulating the developmental timing of larval C. elegans , the same gene was discovered in humans 7 years later. We know that most of the human genome is transcribed into RNAs, and only 1 to 2 % code for proteins. RNA can be divided into that which codes for proteins and that which does not code for proteins, called ”non-coding RNAs.” The location of most miRNAs’ sequences is within introns or exons of noncoding RNAs and introns of pre-miRNAs. Noncoding RNAs are also classified by their size into small noncoding RNAs with less than 200 nucleotides and long noncoding RNAs greater than 200. Most known miRNAs are small RNA molecules of 20 to 22 nucleotides of noncoding RNA strands that negatively regulate expression by directly binding to their target mRNA’s 3’ untranslated region (3’ UTR) through sequence complementarity, which changes the translation pattern of mRNA proteins. They use the seed sequence (nucleotides 2-7) to recognize and suppress diverse target mRNAs. The application of miRNAs as a treatment for various diseases is being used secondary to the knowledge that they participate in intercellular communication. One of the most studied is the regulation of the immune response. (Chandan et al., 2020; Saliminejad et al., 2019) A database has been constructed by various researchers using methods for predicting and validating micro-ribonucleic acid (miRNA)-target mRNA interactions, which contains approximately 2700 genes. Additionally, the application of computational kinetic modeling techniques to elucidate the function of miRNAs in physiological states and diseases. miRNA represses or degrades the critical region of the mRNA and controls its translational function. Currently, we do not fully understand all their functions, so it is essential to validate the analyzed interactions and ensure accurate identification. Every year, the number of known miRNAs related to different diseases increases, with emphasis on the response of the immune system. For miRNAs to act on mRNA, about 20 nucleotides are needed, increasing the prediction of false positives, so experimental verification is essential. Validated miRNAs give outstanding information on such interactions. It should be considered that most genes are targets of miRNAs, many miRNAs can regulate only one mRNA target, and a single miRNA can regulate many mRNA targets. (Kariuki et al., 2023; Zhao et al., 2019) miRNAs biogenesis The biogenesis of miRNAs occurs through canonical and non-canonical pathways. Multiple steps mediate it: transcription of primary miRNA transcripts, nuclear processing by Drosha, nucleocytoplasmic export, cytoplasmic processing by Dicer, and formation of RNA-induced silencing complex (RISC) with Argonaute (Ago) proteins. (Jorge et al., 2021) The biogenesis of canonical miRNAs begins with the generation of transcripts by RNA polymerase (Pol) II or RNA Pol III, giving rise to pri-miRNAs. The RNase III endonuclease Drosha and (DiGeorge syndrome critical region 8) DGCR8 endonuclease complex processes the pri-miRNA into precursors pre-miRNAs in the nucleus, the pre-miRNAs are exported to the cytoplasm and processed by exportin 5 (XPO5) and (Ras related Nuclear protein- guanosine triphosphate) RAN-GTP, become mature under the influence of RNAse III Dicer interacting with several proteins like TAR RNA binding protein (TRBP), Protein ACTivator (PACT), and (adenosine deaminase RNA-specific binding protein) ADAR1, liberating a 21-24 nt miRNA duplex. (de Sousa et al., 2019; Jorge et al., 2021; Komatsu et al., 2023) Only one strand of the miRNA duplex, called the guide strand, is retained in the Ago proteins to form the AGO-miRNA complex by means of the interaction of the 5’ nucleotide with the MID domain of the AGO proteins. A miRNA strand binds to complementary miRNA recognition elements encoded within the mRNA and forms an active complex with effector proteins for complex formation (RISC). The Ago-miRNA complex binds to the untranslated region (3’ UTR) that leads to the repression, silencing, or degradation of mRNA. (de Sousa et al., 2019; Komatsu et al., 2023) The generation of miRNAs is controlled, and we see it in some pri-miRNAs that determine the cutting site of DROSHA in a poorly matched GHG motif. In fact, it was reported that from a database of almost 2000 pri-miRNAs, only 758 were processed by DROSHA. This showed that some endogenous pri-miRNAs are not optimal to be processed by DROSHA, and that, thanks to a group of pri-miRNA forks present, they help them to be optimal. On the other hand, the processing of pre-miRNAs requires DICER to have a PAZ domain in the middle region and three tandem RNA helical domains (DExD/H domain) in the N-terminal region associated with the binding and cutting of pre-miRNAs. The generation of miRNAs and their processing is carried out at a faster rate than mRNAs, with an average life span of days in which they remain stable; however, the half-life of each miRNA is different depending on the cell type and function performed. The complementary target mRNA promotes the decay of miRNAs through a process called miRNA decay by degradation of target miRNAs (TDMD). It is worth mentioning that the fine regulation of miRNA biogenesis pathways integrally controls the abundance of miRNAs. The seed sequence of miRNAs is significant for their recognition by mRNA whether elaborated by the canonical or non-canonical pathway. (Komatsu et al., 2023) Modulation of miRNAs and TLRs in bacterial infections Various investigations on the responsibility of miRNAs in altering cell function in bacterial infections are reported. However, the diversity of PAMPs from different bacteria, as well as the type of cell or sample analyzed, and heterogeneity in studies, make it difficult to understand the function of different cellular miRNAs fully. Specific studies of the regulation of TLRs by miRNAs indicate that TLR cell signaling pathways are modulated by direct transcriptional regulation of miRNAs or act directly as a ligand for TLRs. (Banerjee et al., 2021) In addition, it is also noted that TLR regulatory pathways induce the expression of miRNAs. There is diverse research where miR-21, miR-146, miR-155, and the let-7 family are involved in various biological processes. Indeed, miRNAs regulate the immune response at the extracellular and intracellular level by regulating the function of PRRs and transcription factors respectively, as well as signaling proteins. In bacterial infection, both miRNAs and TLRs, which are responsible for both the regulation of pathogenic infection and tolerance in the innate immune system, are expressed. An example is Francisella tularensis , recognized by TLR2 that stimulates the MyD88-MAPKs signaling pathway and do not induce miR-155 expression, so the inflammatory response is diminished. In L. monocytogenes infection miR-155, miR146a, miR-125-3p/5p, and miR-149 are overregulated. miR-155 is upregulated through NF-kB while miR-125a3p/5p is sensed by TLR2 and leads to IL-1 and IL-6 secretion (Riahi Rad et al., 2021) miRNAs are essential molecules related to different cellular functions. However, their functions are complex, and their expression and activity depend on several factors, such as DNA methylation or acetylation, and their interaction with RNA-binding proteins. The survival and multiplication of bacteria depend on several factors, including control in the regulation of host cell miRNAs. M. tuberculosis is sensed by TLR4, decreasing the inflammatory response via NF-kB involving several miRNAs, including let-7f, miR-223. miR-21 and miR-146, among others. (Riahi Rad et al., 2021) Recent studies of the miRNA/TLR relationship in bacterial infections, reported that the analysis of miRNAs in human cell line culture as well as biopsies patients infected with H. pylori showed deregulation significant of miR-7 and miR-153 and increase in IL-1β related to the virulence factor CagA. They also observed apoptosis induction as well as decreased expression of TLR4, regulating the NF-kB signaling pathway (Song et al., 2023). H. pylori, through miRNA-155, produces negative feedback by decreasing the inflammatory response via NF-kB. miRNA-146 stimulates the dysregulation of inflammation by a negative feedback mechanism using the TRAF6 and IRAK1 pathway. It also decreases the expression of IL-8 growth-related oncogene (GRO) and macrophage inflammatory protein (MIP) using the NF-kB pathway (Săsăran et al., 2021). Figure 1. TLR2 recognize a wide range of pathogens following ligand recognition by forming heterodimers with TLR1, TLR4, TLR6 and TLR10 with high specificity. TLR1 dimerizes with TLR2 to recognize bacterial lipoproteins from Gram-positive bacteria, mycoplasma, and mycobacteria that contain conserved triacylated lipopeptides and trigger the production of TNF-α and IL-6 by macrophages. TLR2 and TLR6 recognize diacylated LPS like lipoteichoic acid (LTA) from mycoplasma and gram-positive bacteria, as well as acid-lactic bacteria, TLR2/TLR10 recognize Helicobacter pylori LPS. However, TLR2 has a dual function; activation TLR2 produces pro-inflammatory response or tissue damage by to excessive TLR2 activation. (Colleselli et al., 2023) Patients infected with Vibrio cholerae O1 exhibited increased expression of miR-146 and miR-155 in duodenal tissue during the acute phase of the disease; this expression decreased during the patient’s recovery. Similarly, the mRNA concentration of IL-8, IL-1β, and CCLX9 increased in the acute phase of intestinal mucosal infection. (Bitar et al., 2017) TLR1 and TLR4 expression in the Caco-2 cell line and PBMC showed a concentration-dependent increase following exposure to recombinant Vibrio cholerae coregulator pili toxin (rTcpA) protein. (Ghasemi et al., 2020) In an interesting review, Kazemi et al. showed the presence of a great variety of miRNAs related to the acute and chronic immune response by Brucella genus that eludes the human immune system. Brucella strategies to evade the immune response include interference with the complement system and TLR signaling pathways. The miRNAs: miR-146a and miR-155 are well defined in the modulation of immunity and inflammation during brucellosis. The signaling cascades starting with TLR4 induction include targets of miR-146: IRAK1 and TRAF6 and miR-155. Brucella signals via TLR4, sensing through IRAK1 and TRAF6 to translocate NF-kB to the nucleus, while miR-155 does the same through this pathway. (Kazemi et al., 2021) The effect of the Bacillus Calmette-Guérin vaccine on three miRNAs in peripheral blood monocyte-derived macrophages (MDM) was studied. miR-1224 expressed in MDM challenged with M. bovis BCG increased concentration 72h after infection. miR-1224 negatively regulates TNF-α, an important cytokine in mycobacterial infections, because it induces phagosome-lysosome maturation as well as apoptosis, whereas miR-484 and miR-425 did not show significant changes (Alipoor et al., 2018). Data analyzed both in vitro and in vivo in leptospirosis infection, the authors report that TLR2 induces the production of proinflammatory mediators, including tumor necrosis factor-alpha (TNF-α), IL-6, IL-12p40, and monocyte chemoattractant protein-1 (MCP-1), by activating downstream events. miR-21-5p, miR-144-3p, and miR-let-7b-5p play essential roles in disease signal transduction, signaling by interleukins, the MAPK signaling pathway, and many other functions, and small-RNA loading onto the RNA-induced silencing complex. Leptospiral LPS upregulates miR-21-5p, miR-144-3p, and miR-let-7b-5p through the TLR2 immune axis, and these miRNAs can be significant signature molecules to differentiate leptospiral infection from other bacterial infections with which it is often confused. Because other spirochetes, like Borrelia and Treponema, do not possess LPS, identification of leptospiral LPS-stimulated miRNAs could differentiate leptospiral infection from other spirochetal infections. This recognition is through the activation of NF-kB and MAPK, stimulating the production of pro-inflammatory cytokines. Novel research has shown that miR-101-3p levels are significantly higher in peripheral blood mononuclear cells of patients with primary syphilis and those in the Sero fast state, whereas TLR2 receptor levels were higher in patients with syphilis than in healthy controls. In vitro, stimulation of THP-1 cells with T. pallidum increased miR-101-3p expression. Moreover, miR-101-3p reduced expression levels of TLR2 mRNA and protein in THP-1 cells via binding to the 3’ untranslated region of TLR2. Likewise, miR-101-3p inhibited production of inflammatory cytokines, including IL-1b, IL-6, TNF-α, and IL-12, in T. pallidum -stimulated macrophages. IL-1b and IL-6 mRNA expression levels were reduced by transfection of macrophages with a TLR2-specific small interfering RNA. Conversely, overexpression of TLR2 upregulated cytokine expression. Patients with secondary syphilis exhibited the highest levels of plasma IL-6, which were negatively correlated with miR-101-3p. In conclusion, T. pallidum infection upregulates miR-101-3p expression, which in turn inhibits the TLR2 signaling pathway, leading to reduced cytokine production. (Akino Mercy et al., 2020; Huang et al., 2020; Xu et al., 2017) miR-146a found in Cultibacterium acnes is elevated in the sebaceous glands of human acne tissue samples. Regarding dermatological diseases, increased levels of miR-146a were already confirmed in keratinocytes of acne, just as in psoriasis and atopic dermatitis samples, with a suggested role in regulating inflammation. Characterizing sebocytes with altered levels of miR-146a; miR-146a is not only a marker for activation but could have a regulatory role on cell proliferation and on the immune competence of sebocytes. Moreover, in psoriasis, genetic alterations have even been shown to be associated with disease severity. Higher levels of miR-146a were also detected in keratinocytes treated with LTA (TLR2 activator), where it may down-regulate C. acnes induced production of IL-6, -8, and TNF-α by inhibiting the TLR2/IRAK1/TRAF6/NF-κB and MAPK pathways. The fact that miR-146a is highly expressed in sebaceous glands of acne samples confirms that miR-146a may be involved in acne also at the level of sebocytes and adds further important details on the immune competence of this cell type. Therefore, the induction of the TLR-miR-146a axis in sebocytes may result in a decreased production of IL-8, a cytokine characteristic of acne-related inflammation, and in a reduced chemoattractant potential of sebocytes (Dull et al., 2021) A series of rescue experiments were carried out to clarify whether microRNA-494-3p attenuated the inflammatory response by inhibiting TLR6. Cells were co-overexpressed with microRNA-494-3p and TLR6 or only overexpressed with microRNA-494-3p. The mRNA and protein level of TNF-α increased in co-overexpressed cells more than those only overexpressing microRNA-494-3p. It is suggested that the effect of microRNA-494-3p on inhibiting TNF-α level was reversed by TLR6 overexpression. Moreover, the inhibitory role of microRNA-494-3p in the nuclear translocation of NF-κB p65 was partially reversed by TLR6 overexpression. To sum up, microRNA-494-3p exerted its anti-inflammation function in sepsis through target-degrading TLR6. Lactobacillus acidophilus increases the expresión of miR-146, accompanied by decreased expression of IRAK1 and TRAF6. Moreover, the up-regulation of miR-146a in L. acidophilus - treated was positively correlated with reduced mRNA expression of TLR4 and NF-κB, as well as TNF-α, IL-6, and IL-1β. Studies have shown that blocking the TLR4 signaling might thus reduce the allergic inflammation caused by beta-lactoglobulin. In conclusion, miR-146a is a crucial mediator of L. acidophilus strains to reduce β-Lg-induced inflammation in macrophages through the TLR4 pathway. (Li et al., 2021) Several studies show the expression of let-7 in various cellular events, mainly in carcinogenesis. let-7b signals through TLR4, binds to the 3’-UTR region of the mRNA, and at the transcriptional level suppresses its activity. let-7b expression decreased and TLR4 expression and NF-kB activation secondary to H. pylori infection were upregulated, resulting in an inflammatory process (Săsăran et al., 2021). Figure 1. The infection by Legionella pneumophila in human macrophages target 85 miRNAs in a human macrophage model infection. The host miRNAs miR-125b, miR-221, and miR-579 are decisive factors for intracellular bacterial replication by downregulation of dynamin-like GTPase 1 (MX1). On the other hand, the galectin-8 protein (LGALS8) is known to be involved in antibacterial defense and displayed decreased levels upon miRNA-579 transfection. A new miRNA-regulated cell-autonomous immune network containing LGALS8 and the antiviral factor MX1, which restricts L. pneumophila replication in human macrophages, was found. In this research miR-146a, miR-155, miR-27a, and miR-125a were upregulated in a time-dependent manner, whereas miR-221, miR-222, miR-125b, miR-26a, and miR-29b were downregulated in response to L. pneumophila infection. miR-146a can facilitate Legionella replication inside the host cell via degradation of IRAK1 mRNA. In the present study, miR-125b, miR-221, and miR-579 were of particular interest, while miR-579, unlike miR-125b and miR-221, was only weakly detected. (Herkt et al., 2020) The virulence protein Streptococcus pneumoniae endopeptidase O (PepO) induces miR-155 upregulation, relying on TLR2/NF-kB, and enhances phagocytosis by macrophages, while macrophages TLR-2 deficient are incapable of enhancing phagocytosis in the presence of miR-155. (Yao et al., 2017) There are several reports of miRNA expression analysis in tuberculosis. miR-206, miR-147, miR-148 are deregulated in M. tuberculosis infection and the virulence factors EsxA and EsxB are responsible for this regulation, preventing phagosome maturation as well as the release of inflammatory cytokines. In the same research, they demonstrated that miR-20a-3p is overexpressed, and this produces the reduction of pro-inflammatory cytokines through the IKK/NF-kB pathway, while miR-99b overexpression is induced via MyD88, resulting in a decrease in cytokines, and miR-1178 overexpression allows pathogen survival. (Mourenza et al., 2022) Analyses of miR-21, miR-31, miR-146, and miR-155 in peripheral blood mononuclear cells of patients with active tuberculosis, latent tuberculosis, and healthy patients showed over-regulation of miR-21 and miR-31 after stimulation of cells with PPD in patients with active tuberculosis. Expression of miR-146 increased in patients with active TB and latent TB, while miR-155 only increased in response to the challenge with active TB PPD (Alijani et al., 2023) Chen YC and coworkers demonstrated the importance of miR-23-3a-3p in tuberculosis patients by decreasing reactive oxygen species (ROS) generation, blocking apoptosis, and decreasing inflammatory response via TNF-alpha/TLR4/IL10/TGF-beta 1/SP1/IRF1 signaling pathway. While miR-146a was decreased in patients with active tuberculosis with high bacillary load. (Chen et al., 2020) An interesting review of miRNAs and tuberculosis highlights the various reports on the analysis of the immune response and miRNAs. miR-21-5p decreases the expression of IL-1 beta, IL-6, and TNF-alpha while miR-27a via IRAK4 decreases the production of IFN- gamma, IL-6, and TNF-alpha. M. tuberculosis represses NF-kB target miR-125a while miR-32-5p diminishes IL-1 beta, IL-6, and TNF-alfa by TLR4/FSTL1 (follistin-like protein 1) signaling pathway (Kundu and Basu, 2021). Figure 2. Cystic Fibrosis is an autosomal recessive genetic disease highly sensitive to bacterial infections, and among the most frequent is Pseudomona aeruginosa . Analysis in miRNAs expression during the infection, show us miRNAs differential expression in Cystic Fibrosis patients infected with P. aeruginosa . The over-expression of 6 miRNAs: hsa-miR-1247, hsa-miR-1276, hsa-miR-449c, hsa-miRNA-3170, hsa- miRNA-432-5p and hsa-miR-548 was observed in the PA positive group when compared to the control group. miR-449 and miR-532 are involved in Wnt/-Beta -catenin and NF-kB-TNF alpha signaling, as well as in ciliogenesis. In PA infection, these signaling pathways are defective. (Fesen et al., 2019) Concluding remarks Most of the studies describing the structure and function of TLRs have been performed using synthetic or single commercial molecules. However, the ability to mount tissue- and cell-specific responses depends on the microbial ligand, and only a few studies have characterized specific ligand-driven responses. Several challenges need to be addressed in this field, including ligand identification for specific bacterial infections and the modulation and shaping of innate responses by pathogen-derived ligands. Understanding the host-pathogen interaction in infectious processes involving TLRs and miRNAs is a key point for controlling infections in the near future. Although there is an essential advance in the knowledge of this interaction, there are still gaps to be uncovered. Progress in the analysis of the expression and effect of miRNAs on TLRs and vice versa is arduous, as it requires the use of various modern technological tools to understand better the mechanisms that regulate miRNAs and TLRs. This involves the use of multiple platforms, data normalization, and separation of different physiological and biological factors such as sample type, source, and origin, among others. The analysis should be based on the over-expression or deregulation of both miRNAs and TLRs evaluated under homeostasis conditions, as well as in knockout models of infectious processes for each ligand and bacterium analyzed, as well as various molecules and signaling pathways of interest. All changes must be examined at both the molecular and protein levels, including normalizing strategies, the type of sample used, and the research method of analysis, among others. Knowing the function of miRNAs as ligands for TLRs or as a conduit for changes in TLRs signaling pathways in bacterial infections, their use in diagnosis, prognosis, and treatment would be of significant contribution to humanity. This requires verification and validation of their function as biomarkers before clinical use. Figure 1: TLRs and miRNA in infection by Helicobacter pylori Helicobacter pylori (H. pylori) infection induces dysregulation of several microRNAs, notably the downregulation of miR-153 and miR-7. This downregulation is associated with enhanced autophagy, increased apoptosis, and elevated IL-1β production through NF-κB activation. Both miR-153 and miR-7 participate in the regulation of TLR4 expression and transcriptional modulators such as KLF5. In contrast, miR-155 exerts a negative feedback effect by attenuating the inflammatory response via NF-κB signaling. Similarly, miR-146 regulates inflammation through a negative feedback mechanism involving the TRAF6 and IRAK1 pathway. Figure 2: TLR-mediated infection with Mycobacterium tuberculosis Multiple microRNAs (miRNAs) have been associated with M. tuberculosis infection. Among them, miR-21, miR-31, miR-146, and miR-155 have been detected in serum or plasma samples during both active and latent infection. Several of these miRNAs modulate the TLR4 signaling pathway, attenuating the inflammatory response by downregulating the expression of proinflammatory cytokines such as TNF-α, IL-1β, IL-6, and IFN-γ, thereby promoting M. tuberculosis survival and replication. Overexpression of miR-99b and miR-20a-3p has been shown to target MyD88 and NF-κB (indicated by brown and pink arrows), leading to reduced production of inflammatory cytokines. 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Int J Mol Sci. https://doi.org/10.3390/ijms20020421 Information & Authors Information Version history V1 Version 1 12 September 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords bacterial infections innate immunity recognition mirnas toll-like receptors Authors Affiliations Paulina Puente Mancera Universidad Nacional Autonoma de Mexico Facultad de Medicina View all articles by this author Erandi Pérez Figueroa 0009-0005-0605-0177 Universidad Nacional Autonoma de Mexico Facultad de Medicina View all articles by this author Mariana López Mejía Universidad Nacional Autonoma de Mexico Facultad de Medicina View all articles by this author Antonia Castillo-Rodal I [email protected] Universidad Nacional Autonoma de Mexico Facultad de Medicina View all articles by this author Metrics & Citations Metrics Article Usage 205 views 129 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Paulina Puente Mancera, Erandi Pérez Figueroa, Mariana López Mejía, et al. miRNAs as regulators of extracellular TLRs in bacterial infections. Authorea . 12 September 2025. 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