Multifaceted Roles of microRNAs in Host-Bacterial Pathogen Interaction

ABSTRACT

MicroRNAs (miRNAs) are a well-characterized class of small noncoding RNAs that act as major posttranscriptional regulators of gene expression. Accordingly, miRNAs have been associated with a wide range of fundamental biological processes and implicated in human diseases. During the past decade, miRNAs have also been recognized for their role in the complex interplay between the host and bacterial pathogens, either as part of the host response to counteract infection or as a molecular strategy employed by bacteria to subvert host pathways for their own benefit. Importantly, the characterization of downstream miRNA targets and their underlying mechanisms of action has uncovered novel molecular factors and pathways relevant to infection. In this article, we review the current knowledge of the miRNA response to bacterial infection, focusing on different bacterial pathogens, including Salmonella enterica, Listeria monocytogenes, Mycobacterium spp., and Helicobacter pylori, among others.

INTRODUCTION

MicroRNAs (miRNAs) are a class of small noncoding RNAs (typically 20 to 22 nucleotides long) that posttranscriptionally regulate the expression of target mRNAs exhibiting partially complementary binding sites (1). miRNAs are found in a wide range of organisms, including animals, plants, and viruses. According to the latest release of miRBase (http://www.mirbase.org/; release 22 March 2018), a total of 48,885 mature miRNAs are currently annotated in 271 species; 2,694 mature miRNAs are annotated in the human genome.

miRNA biogenesis is a well-described multistep process (reviewed in reference 2) (Fig. 1). Typically, the primary miRNA transcripts (pri-miRNAs) are long, capped, and polyadenylated RNA molecules containing hairpin structures transcribed by RNA polymerase II. The pri-miRNA is then processed in the nucleus by the microprocessor complex (comprising the RNase III enzyme Drosha and the double-stranded RNA binding protein DGCR8) into a 60- to 100-nucleotide hairpin precursor miRNA. Exportin-5 mediates the transport of the precursor miRNA from the nucleus to the cytoplasm, where it is further processed into a miRNA duplex of approximately 20 bp by Dicer, an enzyme that also belongs to the RNase III family, in complex with the RNA binding protein TRBP. One of the strands of the miRNA is then loaded into the miRNA-induced silencing complex, which contains, among multiple components, an Argonaute (AGO) protein that binds to the mature miRNA. Target mRNAs are identified by base-pairing between the miRNA and mRNA, usually involving nucleotides 2 to 7/8 of the 5′ end of the miRNA (known as the seed-region). miRNAs repress target gene expression by a combination of mechanisms involving translation repression and target mRNA degradation following deadenylation and decapping (reviewed in reference 3).

FIGURE 1.

Overview of the canonical miRNA biogenesis pathway. miRNA genes are transcribed as pri-miRNAs by RNA polymerase II. The main proteins involved in the multistep miRNA processing are indicated. Repression of target gene expression occurs through inhibition of translation and mRNA degradation.

Based on the high number of miRNAs identified in the human genome, bioinformatic predictions, and experimental identification of hundreds of target mRNAs for a number of individual miRNAs, it has been suggested that approximately 60% of the human genome is under the regulation of miRNAs (4), though this may be an underestimation. Consistent with a pervasive role of miRNAs in the control of gene expression, miRNAs have been shown to regulate countless fundamental biological processes (e.g., cellular proliferation, differentiation, apoptosis [5–7]), and miRNA dysregulation has been implicated in a wide spectrum of human diseases (e.g., cancer, cardiovascular disorders [8, 9]).

In addition to these functions, it is now clear that miRNAs have a preponderant role during infections caused by viruses, parasites, fungi, and bacterial pathogens. Initial work addressing miRNA function in the context of infection focused on viruses (reviewed in reference 10). In these seminal studies, the expression of miRNAs encoded by DNA viruses (herpesvirus, polyomavirus, and adenovirus) was revealed; this was first shown for Epstein-Barr virus (11). Equally important, the repertoire of host miRNAs is vastly changed in response to viral infections. Indeed, viral and host miRNAs modulate multiple processes relevant to infection, ranging from virus replication and propagation to host antiviral responses and/or promotion of the viral life cycle through complex regulatory pathways.

During the past decade, miRNAs have also emerged as powerful players in the interaction of bacterial pathogens with the host. It has become clear that bacteria have evolved sophisticated mechanisms of harnessing host miRNAs to create an immune-tolerant environment and/or to modulate host pathways for their own benefit, promoting pathogen survival, replication, and latency/persistence. From the host perspective, miRNAs are an integral part of an effective immunological response, relevant for the control and clearance of infection.

In this article, we summarize a growing body of literature on the modulation and function of host miRNAs in the context of infection by bacterial pathogens.

miRNA RESPONSE TO BACTERIAL INFECTION

The first evidence of the regulatory role of miRNAs in response to bacterial infections was obtained in 2006 from studies performed with plants. In this seminal work, Navarro and colleagues described the increased expression of miR-393a as an important mediator of the resistance of Arabidopsis thaliana to infection by the extracellular pathogen Pseudomonas syringae (12). Specifically, the authors showed that recognition of a flagellin-derived peptide from P. syringae by the FLS2 receptor of A. thaliana induces the transcription of miR-393a, which in turn represses the expression of three F-box auxin receptors. This blunts signaling by auxin, a plant hormone that negatively regulates the plant immune system, ultimately restraining bacterial spreading and enhancing plant resistance to P. syringae infection (12). Interestingly, the authors later demonstrated that P. syringae is able to counteract this miRNA-mediated antibacterial response through the secretion into host cells of effector proteins that suppress transcription, biogenesis, stability, and activity of pathogen-associated molecular pattern-responsive miRNAs (13).

In mammalian cells, the first report implicating miRNAs in the innate immune response to bacterial components resulted from the pioneering study of Taganov and colleagues (14). These authors investigated the expression of a panel of 200 miRNAs in human monocytes stimulated with lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria that is sensed by the Toll-like receptor 4 (TLR4). This work led to the identification of the first three endotoxin-responsive miRNAs (miR-146a/b, miR-132, and miR-155). Interestingly, the characterization of miR-146a/b targets uncovered several components of the TLR4 signaling cascade, suggesting the existence of a negative-feedback loop that might protect host cells from an excessive inflammatory response.

Subsequent studies established miRNA regulation upon infection as a common phenomenon (summarized in Table 1), with implications for multiple host cell functions ranging from the control of the immune response and autophagy to cell cycle and cell death, among others (Fig. 2). The main findings obtained in this context are described below, grouped by bacterial pathogen.

TABLE 1.

Host cell miRNAs regulated upon infection by bacterial pathogens^a

Bacterium	miRNA	Regulation	Cell/tissue	Reference
Salmonella enterica	let-7 family	Down	HeLa, RAW264.7	17
	miR-1, miR-125a/b, miR-130, miR-148	Down	Piglet MLN	28
	miR-15 family	Down	HeLa	22
	miR-21, miR-29a/b, miR-146a/b	Up	Zebrafish embryos	25
	miR-21, miR-146a/b, miR-155	Up	RAW264.7	17
	miR-26, miR-143	Up	Pig whole blood	30
	miR-29a	Up	Piglet ileal tissue	27
	miR-30c/e	Up	HeLa, J774	24
	miR-34a-5p, miR-215-5p, miR-1662	Down	Chicken cecum	32
	miR-101-3p	Down	Chicken spleen tissue	19
	miR-125b-5p, miR-1416-5p	Up	Chicken cecum	32
	miR-128	Up	HT-29, murine small intestine and colon tissue	31
	miR-146a/b	Up	THP-1	18
	miR-155	Up	Chicken spleen tissue	19
	miR-193a-5p, miR-3525	Up	Chicken cecum	26
	miR-214	Down	Piglet whole blood	29
	miR-331-3p	Up	Piglet whole blood	29
	miR-1308	Up	HeLa	17
Helicobacter pylori	let-7 family, miR-103, miR-125a, miR-130a, miR-491-5p, miR-500, miR-532	Down	AZ-521, human gastric mucosa	58
	let-7b	Down	AGS, GES-1, human gastric mucosa	57
	miR-21	Up	AGS, human gastric mucosa	35
	miR-30b	Up	AGS, human gastric mucosa	62
	miR-30d	Up	AGS, GES-1	63
	miR-101	Down	GES-1, MKN-45, SGC-7901, primary gastric cells	38
	miR-143-3p	Up	Human gastric mucosa	47
	miR-146a	Up	MNK-45, GES-1, HGC-27, AGS, human gastric mucosa	56
	miR-146a	Up	GES-1	55
	miR-146a	Up	HGC-27	54
	miR-146a, miR-16	Up	GES-1	52
	miR-146a, miR-155	Up	Human gastric mucosa	50
	miR-152, miR-200b	Down	AGS, human gastric mucosa	59
	miR-155	Up	AGS, GES-1, MKN-45, human gastric mucosa	52
	miR-155	Up	Jurkat, CCRF-CEM, AGS, MKN-74, J774A, murine BMDMs, human gastric mucosa	48
	miR-155	Up	Human gastric mucosa	56
	miR-155	Up	Murine gastric mucosa	51
	miR-155	Up	AZ-521	58
	miR-155	Up	J774A.1, murine BMDMs	49
	miR-210	Down	Mongolian gerbil and human gastric mucosa	39
	miR-212-3p, miR-361-3p	Down	Human esophagus tissue, HET-1A, OE33	45
	miR-222	Up	HGC-27, AGS, BGC-823, SGC-7901, GES-1	36
	miR-222	Up	Human gastric mucosa	37
	miR-223	Up	Human gastric mucosa	58
	miR-223	Up	THP-1, AGS	53
	miR-320	Down	MKN-28, AGS	40
	miR-370	Down	AGS, human and murine gastric mucosa	41
	miR-371, miR-372, miR-373	Down	AGS	46
	miR-584, miR-1290	Up	AGS	42
	miR-1289	Up	AGS, human gastric mucosa	64
	miR-4270, miR-4459	Down	Human MDMs	61
Mycobacterium spp.	let-7e, miR-29a, miR-146a, miR-155, miR-886-5p	Up	Human MDMs	75
	let-7f	Down	RAW264.7; human MDMs; murine BMDMs; lung, spleen, and lymph node tissue	79
	miR-15a, miR-21-3p, miR-22-3p, miR-23a, miR-30b-5p, miR-142-5p, miR-146a/b	Up	Bovine alveolar macrophages	100
	miR-17-5p	Down	RAW264.7; murine BMDMs; lung, spleen, and lymph node tissue	90
	miR-17-5p	Up	RAW264.7	91
	miR-20a	Up	RAW264.7	98
	miR-20b	Down	Human MDMs, murine lung tissue	88
	miR-21	Up	RAW264.7	86
	miR-21	Up	Human PBMCs, skin samples	82
	miR-21, miR-26a, miR-29a, miR-142-3p	Down	Human T-cells, peripheral blood	84
	miR-26a	Down	RAW264.7; THP1; human MDMs; murine BMDMs; lung, spleen, and lymph nodes	101
	miR-27a	Up	Human PBMCs, murine lung tissue, peritoneal macrophages	95
	miR-27b	Up	RAW264.7, murine BMDMs, lung and spleen tissue	78
	miR-29a	Up	Human serum, sputum	83
	miR-29a/b	Down	Murine splenocytes, T-cells	85
	miR-30a	Up	THP1, human alveolar macrophages	92
	miR-30a/e, miR-155, miR-1275, miR-3178, miR-3665, miR-4484, miR-4497, miR-4668-5p	Up	THP1	68
	miR-33, miR-33*	Up	Murine peritoneal and alveolar macrophages, THP1, BMDMs	94
	miR-106b-5p	Up	Human MDMs	102
	miR-125a-3p	Up	RAW264.7, murine BMDMs	93
	miR-125b, miR-146a, miR-155	Up	RAW264.7, THP1, murine peritoneal macrophages	72
	miR-125b, miR-155	Up	Human MDMs	73
	miR-142-3p	Up	J774A.1, human MDMs	81
	miR-144-3p	Up	RAW264.7	97
	miR-144-5p	Up	THP1, human MDMs, lung and lymph nodes tissue	96
	miR-155	Up	Human PBMCs, murine spleen, lymph node and peritoneal macrophages	66
	miR-155	Up	RAW264.7, murine BMDMs	67
	miR-155	Up	RAW264.7, murine lung tissue and BMDMs	76
	miR-155	Up	THP1, human PMBCs	71
	miR-155	Up	Human dendritic cells	70
	miR-155	Up	Murine BMDMs	74
	miR-155	Up	RAW264.7	77
	miR-199a	Up	J774A.1, murine BMDMs, lung and spleen tissue	99
	miR-223	Up	Murine whole blood and lung tissue, human peripheral blood and lung tissue	80
	miR-582-5p	Up	Human PBMCs	87
Listeria monocytogenes	miR-16, miR-146b, miR-155	Up	Caco-2	107
	miR-21	Up	Murine BMDMs	106
	miR-29a/b	Down	Murine splenocytes and NK cells	85
	miR-125a-3p/-5p, miR-146a, miR-155, miR-149	Up	Murine BMDMs	104
	miR-143, miR-148a, miR-194, miR-200b/c, miR-378	Down	Murine ileal tissue	113
	miR-145, let-7a1	Down	Caco-2	107
	miR-192, miR-200b, miR-215	Down	Murine ileal tissue	112
Francisella spp.	miR-133a, miR-146a, miR-150, miR-155, miR-886-5p	Up	Human MDMs	114
	miR-155	Up	Human PBMCs, murine lung, liver and spleen tissue	115
Citrobacter rodentium	miR-7a, miR-17, miR-20a, miR-21, miR-142-3p, miR-203	Up	Murine colonic crypts	117
Staphylococcus aureus	miR-15b-5p	Up	Human and porcine skin tissue	121
	miR-20b, miR-31, miR-155, miR-182, miR-222	Up	Murine lung monocytes	118
	miR-24	Down	U937, RAW264.7	119
Pseudomonas aeruginosa	miR-26a-5p, miR-155, miR-182-5p, miR-200c-3p, miR-294-3p, miR-302b-3p, miR-495-3p, miR-669k-3p	Up	MH-S	124
	miR-302b	Up	MLE-12, MH-S, murine lung tissue	124
Chlamydia spp.	miR-16, miR-23b, miR-30c/e, miR-125b-5p, miR-135a, miR-182, miR-183, miR-214	Down	Murine genital tract tissue	130
	miR-30c-5p	Up	HUVEC, hFIMB, HFF	127
	miR-100-5p, miR-200a-3p, miR-200b-3p/-5p, miR-411-5p	Down	Murine genital tract tissue	131
	miR-132, miR-142-3p, miR-147-3p, miR-149-3p, miR-212-3p	Up	Murine genital tract tissue	131
	miR-146, miR-451	Up	Murine genital tract tissue	130
	miR-155	Up	Murine dendritic cells	129
	miR-182, miR-183	Up	Murine splenic T-cells	129
	miR-214	Down	Murine genital tract tissue	128
Brucella spp.	let-7b, miR-92a, miR-99a, miR-142-5p, miR-181b, miR-1981	Up	RAW264.7	132
	miR-93, miR-151-3p	Down	RAW264.7	132
	miR-125b-5p	Down	RAW264.7	133
	miR-130a-3p	Up	PAM	134
	miR-146a, miR-181a/b, miR-301a-3p	Up	RAW264.7, PAM	134
	miR-351-5p	Up	RAW264.7	134
Shigella flexneri	miR-29b-2-5p	Down	HeLa	135

^a Abbreviations: AGS, •••; BMDM, bone marrow-derived macrophages; CCRF-CEM, •••; HFF, primary human foreskin fibroblasts; hFIMB, human fallopian tube fimbriae cells; HUVEC, •••; MDM, monocyte-derived macrophages; MH-S, •••; MLN, mesenteric lymph node; PAM, •••; PBMC, peripheral blood mononuclear cells.

FIGURE 2.

Regulation of miRNAs upon infection by bacterial pathogens impacts multiple crucial host cell functions. miRNA modulation upon infection has been shown to be an integral part of the host response or a mechanism exploited by bacteria to promote infection.

Salmonella enterica

S. enterica remains a leading cause of gastroenteritis worldwide, with S. enterica subspecies enterica serovar Typhimurium (S. Typhimurium) being one of the most frequent serovars causing foodborne disease (15). Salmonella spp. can infect phagocytic and nonphagocytic cells in humans as well as in a wide range of domestic and wild animals (16).

Host miRNA regulation of S. Typhimurium infection was first shown in mouse macrophages, where miR-155, miR-146a/b, and miR-21 were strongly induced (17). Interestingly, regulation of these miRNAs was also observed with S. Typhimurium mutant strains defective in cell invasion (ΔSPI-1) and replication (ΔSPI-2), as well as upon treatment with purified Salmonella LPS, showing that sensing of extracellular stimuli triggers the regulation of these miRNAs. Similar results were obtained in human monocytes (18). These studies and additional findings have singled miR-155 as a key immune miRNA supporting the proinflammatory host response against infection (17, 19). However, miR-155 also participates in negative-feedback regulation of the proinflammatory signaling, ultimately protecting the host from a potentially damaging inflammation overreaction (20). Moreover, this miRNA was shown to be essential for normal immune function, particularly of dendritic cells and B and T lymphocytes, and as part of immunization with attenuated S. Typhimurium (21).

By comparing miRNome (the full spectrum of expressed miRNAs) changes in macrophages and epithelial cells, Schulte et al. showed that members of the let-7 miRNA family are downregulated in both cell types, revealing a common denominator of the phagocytic and nonphagocytic cell response to S. Typhimurium infection (17). Interestingly, the let-7 family was shown to target two major cytokines—the anti-inflammatory cytokine interleukin 10 (IL-10) and the proinflammatory cytokine IL-6. The decreased let-7 expression upon infection and consequent derepression of cytokines with opposing effects likely contributes to a balanced inflammatory response to S. Typhimurium infection.

In addition to participating in the host response to S. Typhimurium infection, miRNA regulation can be harnessed to modulate host physiology, ultimately rendering host cells more permissive to bacterial infection. By employing a high-content screening approach in which the individual effect of ca. 1,000 miRNAs was tested on S. Typhimurium replication in epithelial cells, our group has identified multiple miRNAs controlling bacterial interaction with host cells (22). Among the strongest inhibitors of S. Typhimurium infection, we identified the miR-15 family, which blocks host G₁/S cell cycle transition through the repression of cyclin D1. Interestingly, expression of the miR-15 miRNA family is downregulated upon S. Typhimurium infection, thus favoring cell cycle progression and, ultimately, bacterial replication.

SUMOylation, a posttranslational modification pathway central for cell homeostasis (23), is essential for Salmonella intracellular survival, and it has also been shown to be subverted by modulation of host miRNAs. Indeed, S. Typhimurium survival inside host cells relies on the depletion of Ubc-9, a crucial enzyme of the SUMO pathway, which is achieved by the increased levels of miR-30c and miR-30e upon infection (24).

The effect of Salmonella infection on miRNAs has been also studied in vivo in different models of infection, including zebrafish, chicken, and especially pig. In zebrafish embryos, several miRNAs were shown to be upregulated upon S. Typhimurium infection, including the two miR-146 family members, miR-146a/b (25). Interestingly, combined knockdown of these miRNAs in zebrafish embryos infected with S. Typhimurium had a minor effect on the proinflammatory response, but it induced apolipoprotein-mediated lipid transport genes, suggesting a possible function of these miRNAs in regulating lipid metabolism during inflammation (25). In the chicken model, 14 miRNAs were shown to be differentially expressed upon S. Typhimurium infection. From these, the most strongly induced miRNAs were miR-3525 and miR-193a-5p, which might impact the immune response against S. Typhimurium by targeting IL-6 signal transducer and interferon-γ (IFN-γ), respectively (26). In the ileum of piglets, S. Typhimurium upregulated miR-29a expression (27). This miRNA was shown to target Caveolin-2, an inhibitor of the small Rho GTPase Cdc42, thus favoring S. Typhimurium invasion of epithelial cells. In a recent study, Herrera-Uribe et al. identified 110 dysregulated miRNAs in mesenteric lymph nodes of S. Typhimurium-infected piglets (28). Among these, the authors highlighted several miRNAs with predicted and validated targets among major histocompatibility complex class I and II antigen presentation pathways. The miRNA expression profile of whole-blood samples of pigs challenged with S. Typhimurium has also been analyzed, revealing 62 differentially expressed miRNAs (29). From these, the decrease of miR-214 was shown to increase expression of SLC11A1 and LILR-like expression, potentially regulating bacterial replication by actively removing iron from the phagosomal space and by negatively regulating TLR-mediated responses to maintain a balanced inflammatory response, respectively. Induction of miR-331-3p was also validated and suggested to contribute to blocking S. Typhimurium uptake by suppressing the activity of Rho GTPase family members (e.g., RhoA, Cdc42, and Rac1) through targeting of VAV2 (29). Similarly, Yao et al. demonstrated that miR-143, miR-26, and miR-4335 are differentially expressed in whole-blood samples of S. Typhimurium-infected pigs (30).

Although to a lesser extent, the miRNA regulation by other Salmonella serovars, specifically S. enterica subsp. enterica serovar Enteritidis (S. Enteritidis), has also been investigated. Analysis of the miRNome of colon epithelial cells infected with two strains of S. Enteritidis revealed 22 differentially regulated miRNAs (31). miR-128 was shown to be consistently upregulated in S. Enteritidis-infected samples in vitro and in vivo, through a mechanism likely involving activation of the p53 signaling pathway by bacterial secreted proteins. miR-128 was shown to target macrophage colony-stimulating factor (M-CSF), leading to impaired M-CSF-mediated macrophage recruitment and thus benefiting bacterial survival. In chicken, 37 and 32 miRNAs were shown to be differentially regulated between noninfected and S. Enteritidis-infected cecum (32) and spleen (19) samples, respectively. Through integrated analysis of miRNA and transcriptome datasets obtained from spleen samples, two miRNAs were identified in hub positions of the regulatory network—miR-155 and miR-101-3p—likely with important consequences to the immune response upon infection (19).

Helicobacter pylori

H. pylori is a Gram-negative bacterium estimated to chronically colonize the gastric mucosa of more than half of the human population. Unless treated, colonization persists lifelong and represents a key factor in the etiology of gastritis, peptic ulcer, and gastric cancer (33). Similar to other pathogens, H. pylori pathogenesis depends on its bacterial virulence factors, such as the cytotoxin-associated gene A (CagA) and vacuolating cytotoxin A (VacA), which affect a multitude of host cell pathways (34).

The first evidence that bacterial pathogens can affect the expression of mammalian host miRNAs was obtained in a pioneering study by Zhang and colleagues, who used H. pylori as a model pathogen (35). This study demonstrated that miR-21 is upregulated in gastric epithelium tissue samples of H. pylori-infected patients compared to noninfected samples; similar results were reported in vitro in the human gastric cancer cell line AGS. Interestingly, miR-21 overexpression was shown to promote cellular proliferation and invasion by targeting RECK, a known tumor suppressor. These results suggested a strong link between miR-21 regulation upon H. pylori infection and the development of gastric cancers (35). Other studies have further explored the connection between regulation of host miRNA expression upon H. pylori infection and the ability of this pathogen to induce tumorigenesis. In one of these studies, H. pylori infection was shown to induce the expression of miR-222, another oncomiR (oncogenic miRNA) that also targets RECK (36). Recently, miR-222 was also shown to directly target the homeodomain-interacting protein kinase-2, promoting cell proliferation and invasion and inhibiting apoptosis of gastric cancer cells (37). Along the same line, miR-101 was shown to be downregulated in H. pylori-infected samples and cultured cells, with similar consequences for cell growth and tumorigenesis, elicited by the derepression of its direct target, the oncogene SOCS2 (38). In addition, DNA methylation of the miR-210 gene promoter was shown to be increased in H. pylori-infected human gastric mucosa samples compared to negative controls, promoting cell proliferation through derepression of STMN1 (involved in the initiation of tumor development) and DIMT1 (a member of the S-adenosylmethionine-dependent methyltransferase superfamily) (39).

Mechanistically, the H. pylori virulence factor CagA was shown to be an important player in H. pylori-induced miRNA expression changes, with 61 miRNAs differentially expressed in a CagA-dependent manner (40). CagA-dependent downregulation of miR-320 upon H. pylori infection results in increased expression of its target MCL1, an antiapoptotic gene, likely contributing to an increased risk of tumorigenesis (40). Similarly, the downregulation of miR-370, in a CagA-dependent manner, leads to increased expression of FoxM1, a transcription factor involved in promoting cell cycle progression (41). CagA was also shown to stimulate expression of miRNAs, specifically of miR-584 and miR-1290. These miRNAs have as a common target FOXA1, which is an important negative regulator of epithelial-mesenchymal transition, critical for the development of cancer metastasis (42).

Although H. pylori primarily affects the stomach mucosa, its presence has also been associated with other pathological conditions, such as Barrett’s esophageal disease and esophagus adenocarcinoma (43, 44). In the context of esophageal epithelial cells, Teng et al. showed that H. pylori decreases miR-212-3p and miR-361-3p expression, leading to derepression of their targets, COX2 and CDX2, respectively, two oncoproteins associated with transformation of esophageal epithelial cells (45).

Although a large body of literature coherently supports the involvement of miRNA regulation during H. pylori infection to promote cell proliferation and, ultimately, favor tumorigenesis, H. pylori has also been shown to block cell cycle progression and cell proliferation in gastric epithelial cells through the regulation of miRNAs (46, 47). Indeed, CagA-dependent downregulation of miR-372 and miR-373 in H. pylori-infected cells increases expression of their target LATS2, a tumor suppressor, leading to G₁ cell cycle arrest (46). Recently, Wang et al. analyzed miRNA expression in gastric cancer patients, comparing H. pylori-positive and -negative subgroups. Among the 53 miRNAs differentially expressed in these samples, the authors focused on miR-143-3p, which was the most upregulated miRNA in H. pylori-positive gastric cancer tissues. The authors demonstrated that high levels of miR-143-3p dampen cell growth, migration, and invasion through the direct targeting of AKT2 (47), a pro-survival protein that is frequently overexpressed in cancer. Although these studies reveal an unexpected facet of H. pylori, i.e., bacterial-induced host cell cycle arrest and inhibition of cell proliferation, it is conceivable that this can be relevant to inhibit gastric epithelium renewal and therefore constitute a host defense mechanism against infection.

H. pylori infection elicits a strong immune response, triggering the expression of diverse cytokines and chemokines by gastric epithelial cells. Along the same line, several studies have reported the deregulation of immune-related miRNAs in response to H. pylori infection. For example, miR-155 is strongly upregulated upon H. pylori infection, both in vitro (epithelial cells, macrophages, and lymphocytes) and in vivo (human biopsies from infected gastric mucosa) (48–52). Upregulation of miR-155 was shown to be dependent on sensing of H. pylori LPS by TLR4 and subsequent NF-κB pathway activation (49, 52), as well as on the secretion of bacterial proteins by the type-IV secretion system, particularly VacA and γ-glutamyl transpeptidase (48). miR-155 inhibits the release of the proinflammatory cytokines IL-8 and GRO-α, resulting in attenuated NF-κB activity and weakened inflammatory response against H. pylori (52). In macrophages, upregulation of miR-155 was also shown to inhibit DNA damage-induced apoptosis, a mechanism that might increase cell survival upon DNA damage induced by H. pylori (49). Interestingly, miR-155 knockout mice are unable to control H. pylori infection, owing to a deficient pathogen-specific Th1 and Th17 response (51). Pachathundikandi et al. showed that in infected THP-1 monocytes, the inflammasome-forming NLRP3 protein is downregulated upon H. pylori infection through the upregulation of miR-223-3p (53). The immune-related miRNA miR-146a is also upregulated by H. pylori infection, both in vitro and in vivo (52, 54–56). miR-146a negatively regulates TRAF6, PTGS2, and IRAK1 and, consequently, blunts the inflammatory response against H. pylori (54–56). Similar to Salmonella spp., H. pylori infection downregulates the let-7 miRNA family both in vitro and in vivo through its major virulence factor CagA (57, 58). Decreased expression of let-7 family members leads to the derepression of their target TLR4 and to the consequent activation of the NF-κB-dependent inflammatory response. In addition to classical immune-related miRNAs, other miRNAs have been implicated in the regulation of immune function in the context of H. pylori infection. In a recent study, Xie and colleagues showed that H. pylori induces the expression of B7-H1 by decreasing the levels of miR-152 and miR-200b (59). Indeed, B7-H1 functions as a negative regulator of cell-mediated immune response by inhibiting proliferation and inducing apoptosis of activated T-cells (60). In a recent study, Pagliari et al. described that H. pylori can alter miR-4270 expression as a strategy to persist in macrophages and thus evade the immune system (61). miR-4270 controls the expression of the plasma membrane receptor CD300E, compromising the ability of macrophages to expose major histocompatibility complex class II molecules, ultimately compromising recognition by T-cells, and providing a survival niche for the bacterium.

Beyond immune-related functions, H. pylori subverts other cellular processes for its own benefit through miRNA regulation. Upregulation of miR-30b was shown to occur in AGS cells and gastric tissues infected with H. pylori (62). miR-30b negatively regulates the autophagy pathway by targeting Beclin-1 and ATG12, two proteins involved in the formation and maturation of autophagosomes, likely contributing to preventing H. pylori clearance by autophagy. The expression of these two autophagy proteins (along with ATG2B, ATG5, and BNIP3L) was also shown to be repressed by the upregulation of miR-30d induced during H. pylori infection (63). H. pylori can also decrease the gastric acidity by inducing the expression of miR-1289 in a CagA- and soluble lytic transglycolase-dependent manner. miR-1289 represses HKα, a subunit of the gastric H⁺/K⁺ ATPase (64), leading to a transient hypochlorhydria that favors H. pylori colonization of the gastric mucosa.

Mycobacterium Species

The genus Mycobacterium includes highly pathogenic species responsible for diseases that remain major public health challenges, such as tuberculosis (caused by Mycobacterium tuberculosis) and leprosy (caused by Mycobacterium leprae). In addition, it also encompasses opportunistic pathogens, such as Mycobacterium avium, that can infect immunocompromised patients. Not surprisingly, miRNAs play relevant roles in the modulation of the host response to mycobacterial infection (reviewed in reference 65).

Several studies have examined the regulation and role of miR-155 in mycobacterial infection using different cellular models, experimental conditions, and Mycobacterium species (66–77). These studies have generated divergent results, particularly regarding the function of miR-155 during infection, though the majority underlined the recurrent function of miR-155 in the regulation of the innate immune response. Adding to the body of knowledge on the role of miR-155 in infection, a recent study by Rothchild and colleagues applied an integrative approach to analyze the miRNA network in infected macrophages, revealing a critical and dual role for miR-155 in M. tuberculosis infection (74): on the one hand, miR-155 promotes the survival of infected macrophages, providing a bacterial niche during the early stages of infection, while on the other hand, it promotes the survival and function of specific T-cells, enabling an effective adaptive immune response. miR-27b, another miRNA with a dual role in mycobacterial infection, has recently been shown to be induced upon infection. miR-27b targets the Bcl-2-associated athanogene 2 protein (Bag2)—which inhibits the production of proinflammatory factors by blunting NF-κB activity but also increases p53-dependent apoptosis and reactive oxygen species production—thus increasing bacterial clearance (78). Other miRNAs have been shown to blunt the proinflammatory response against Mycobacterium to favor bacterial survival. For example, downregulation of let-7f in macrophages infected with M. tuberculosis was shown to derepress the expression of A20 deubiquitinase, a negative regulator of the NF-κB pathway (79). Along the same line, induction of miR-223 reduces the expression of the cytokine IL6 and chemokines CXCL2 and CCL3 upon infection, inhibiting proper recruitment of neutrophils to the infection site and increasing bacterial survival (80).

The miRNA response against Mycobacterium spp. has been shown to be highly dependent on the cell context and bacterial species. For example, while miR-21, miR-29a, and miR-142-3p levels were shown to increase in infected macrophages and serum of tuberculosis patients (75, 81–83), they are downregulated in CD4⁺ T-cells and samples of peripheral blood of tuberculosis patients (84). Similar to the observations in Listeria (see “Listeria monocytogenes” below), miR-29 is downregulated in natural killer (NK) cells upon M. bovis infection (85), which was shown to enhance bacterial clearance by derepressing IFN-γ expression. In contrast, in macrophages infected with M. avium, miR-29a expression, along with let-7e, is upregulated, resulting in inhibition of apoptosis through a decrease of caspase-3 and caspase-7 (75). Regulation of host cell death is emerging as a recurrent target of miRNAs regulated during mycobacterial infection. Along this line, upon M. tuberculosis infection, upregulation of miR-582-5p, miR-155, and miR-21 was also shown to inhibit apoptosis, by targeting FOX1, FOX3, and NF-κB/Bcl-2, respectively (71, 86, 87). On the other hand, a decrease of miR-20b in M. tuberculosis-infected macrophages derepresses NLRP3, leading to the activation of the NRLP3/caspase-1/IL-1β pathway and, consequently, enhancing inflammation and pyroptosis (88).

Autophagy is a process by which intracellular components are targeted for degradation, and it plays a crucial role in the host defense against intracellular pathogens, including mycobacteria (89). Mycobacterium spp. have evolved strategies based on host miRNA modulation to subvert the autophagy pathway and ensure intracellular survival. Figure 3 illustrates the impact of mycobacteria-regulated miRNAs on autophagy and lysosomal trafficking. The role of miR-155 as a positive or negative regulator of autophagy in the context of mycobacterial infection is still controversial, and it likely depends on cell context and bacterial strain. A proautophagic function of miR-155 through the repression of the negative autophagy regulator RHEB was shown in macrophages (76), compromising M. tuberculosis survival. Conversely, upon M. tuberculosis infection of dendritic cells, upregulated miR-155 was shown to directly target ATG3 and reduce the lipidated form of LC3 in the autophagosomes, indicating negative regulation of autophagy (70). In addition to miR-155, other miRNAs were shown to impinge on autophagic regulation during M. tuberculosis infection. Downregulation of miR-17 leads to an increase of STAT3, a transcriptional activator of the autophagy regulator MCL-1 (90), impairing autophagy and contributing to mycobacterial survival. In the context of M. bovis bacillus Calmette-Guérin (BCG) infection, upregulation of miR-17 has been reported to repress ULK1, an essential autophagy initiation protein, ultimately favoring BCG growth (91). This apparently contradictory result on miR-17 expression might be linked to the different Mycobacterium spp. used. Mycobacterium was also shown to block autophagy by increasing the levels of miR-30a and miR-125a-3p, which target Beclin-1 and UVRAG, respectively, two essential players in autophagy induction (92, 93). M. tuberculosis also induces the expression of miR-33 and miR-33*, which target several players in the autophagy and lysosomal pathways (ATG5, ATG12, LC3B, LAMP1, AMPK, FOXO3, and TFEB) (94). Recently, Liu et al. showed that induction of miR-27a upon infection represses the expression of the endoplasmic reticulum-located calcium transporter CAC-NA2D3, impairing the calcium signaling required for autophagosome formation (95). miR-144-5p and miR-144-3p have also been implicated in the control of autophagy: miR-144-5p is upregulated during M. tuberculosis infection, suppressing autophagy by targeting the autophagy regulator DRAM2 (96), while an increase of miR-144-3p upon BCG infection inhibits autophagy by targeting ATG4a (97). miR-20a and miR-199a are also upregulated upon M. bovis infection and inhibit autophagy through downregulation of ATG7, ATG16L1 (98), and TBK1 (99), respectively. Overall, the majority of the studies reported above support a model in which mycobacteria-modulated host miRNAs have a predominant effect on dampening the autophagic process, with positive consequences for bacterial survival.

FIGURE 3.

Mycobacterium spp.-induced miRNA changes have a strong impact on autophagy. The autophagic flux is controlled by multiple miRNAs that are regulated as a consequence of mycobacterial infection. Most studies report that miRNA modulation inhibits specific steps of the autophagy pathway, thus impairing bacterial degradation.

Mycobacterium spp. can also modulate phagosomal trafficking for their own benefit by modulating several miRNAs. Vegh et al. found a set of miRNAs regulated in bovine macrophages upon infection with M. bovis (6 miRNAs at 24 h postinfection and 40 miRNAs at 48 h postinfection); analysis of the predicted targets of the upregulated miRNAs showed an enrichment for genes related to the lysosome and endocytosis, including members of the membrane trafficking Rab family (100). Downregulation of miR-26a during M. tuberculosis infection was shown to derepress KLF4, preventing bacterial trafficking to lysosomes and reducing inducible nitric oxide synthase production (101). Recently, it was also shown that M. tuberculosis upregulates miR-106b-5p expression, which directly represses cathepsin-S, decreasing the lysosomal enzymatic activity and consequently increasing bacterial intracellular survival (102). miRNAs have also been shown to negatively modulate bacterial uptake, likely as part of the host response to counteract infection. Specifically, an increase of miR-142-3p in response to M. tuberculosis infection represses N-WASP, an actin binding protein essential for phagocytosis (81).

Listeria monocytogenes

L. monocytogenes is a Gram-positive facultative intracellular bacterium that is the causative agent of human listeriosis, a foodborne disease. Listeria can cause gastroenteritis in healthy adults and severe illness in children, the elderly, and other immunocompromised individuals (103).

Genome-wide miRNA profiling in murine bone marrow-derived macrophages infected with L. monocytogenes identified 13 miRNAs that were significantly upregulated, including miRNAs known to regulate the inflammatory response (e.g., miR-155 and miR-146a) (104). In addition, miR-155 has been shown to be essential for the CD8⁺ T-cell response to L. monocytogenes (105). Recently, the immune-related miRNA miR-21 was also shown to be upregulated following L. monocytogenes infection of bone marrow-derived macrophages (106). Interestingly, miR-21 knockout macrophages showed an increased bacterial burden at early times postinfection (30 min). The identification of the actin-modulating proteins RHOB and MARCKS as putative miR-21 targets suggests a possible role for this miRNA in the negative regulation of phagocytosis during infection.

The effect of L. monocytogenes infection on host miRNA expression has also been addressed in human intestinal epithelial Caco-2 cells (107); in this study, five miRNAs (miR-146b, miR-16, let-7a1, miR-145, and miR-155) were found to be dysregulated upon L. monocytogenes infection. Similar to what has been described in macrophages (104), in Caco-2 cells the expression of miR-155 was induced to a comparable extent by infection with wild-type L. monocytogenes or with a mutant deficient for listeriolysin, a secreted toxin that is essential for bacterial vacuolar escape, among other functions, in L. monocytogenes virulence. Strikingly, upregulation of miR-155 also occurs following incubation of cells with purified listeriolysin (107). Among the miRNAs dysregulated by L. monocytogenes in Caco-2 cells, Izar and colleagues showed the downregulation of miR-145 (107). This miRNA exerts a proinflammatory effect by potentiating the production of the proinflammatory cytokines IL-5 and IL-13 (108) and by decreasing the levels of the anti-inflammatory cytokine IFN-β (109), a cytokine shown to be beneficial to L. monocytogenes infection (reviewed in 110). However, a recent study by Li and colleagues revealed an anti-inflammatory effect of miR-145 by reducing ARF6 expression and downstream signal transduction via NF-κB (111). Taken together, these studies suggest that downregulation of miR-145 could play an essential role in balancing the inflammatory response during Listeria infection.

In vivo, challenge with L. monocytogenes has also been shown to induce significant host miRNome changes. Systemic infection of mice with L. monocytogenes downregulates miR-29 expression in NK cells, CD4⁺ T-cells, and CD8⁺ T-cells (85). Ma et al. (85) suggested that decreased miR-29 expression upon infection facilitates IFN-γ production, promoting bacterial clearance and host resistance to L. monocytogenes infection. miR-192, miR-215, and miR-200b were shown to be downregulated in the ileum of orally infected gnotobiotic humanized mice (112). Interestingly, pretreatment with Lactobacillus casei prevented the decrease of expression of these miRNAs upon L. monocytogenes infection. In another study, Archambaud et al. reported the decreased expression of six miRNAs (miR-143, miR-148a, miR-194, miR-200b, miR-200c, and miR-378) in the ileum of conventional mice infected with L. monocytogenes (113). Interestingly, regulation of four of these miRNAs (miR-143, miR-148a, miR-200b, and miR-200c) occurs only in the presence of a normal microbiota, whereas miR-194 is decreased in both the presence and absence of gut microbiota and miR-378 expression is increased in germ-free mice. Overall, these studies reveal an interesting microbiota-dependent regulation of miRNA expression during Listeria infection, suggesting that the microbiota plays an important role in the miRNA host cell response, with relevant implications for infection with bacterial pathogens.

Other Bacterial Pathogens

As described above, analysis of miRNA expression and the characterization of the role of miRNAs are well under way for certain bacterial pathogens. For most bacterial pathogens, however, this analysis has not yet been performed or is just at its inception. We provide below a nonexhaustive review of what is known concerning the role of miRNAs in infection by other bacterial pathogens. Of note, most of these studies have focused on immune response-related miRNAs, most prominently miR-155. For example, miR-155 was shown to be differentially induced upon infection with Francisella tularensis (114, 115), a Gram-negative facultative intracellular bacterium that causes turalemia. Interestingly, miR-155 is strongly induced in an LPS-dependent manner upon infection with F. tularensis subsp. novicida, a low-virulence F. tularensis subspecies, while infection with the highly virulent F. tularensis subsp. tularensis leads to significantly lower miR-155 induction, probably due to the low bioactivity of its LPS. This might contribute to explaining the higher bacterial dissemination and more severe disease caused by F. tularensis subsp. tularensis (114, 115). Indeed, the increase of miR-155 upon F. tularensis subsp. novicida infection represses SHIP (a key negative regulator of the PI3K/Akt pathway), similarly to what was described for Mycobacterium infection (67), thus enhancing the proinflammatory cytokine response and contributing to infection clearance (115). Clare and colleagues demonstrated that miR-155 knockout mice are unable to clear infection by Citrobacter rodentium (116), a murine pathogen that is used as a model for understanding enteropathogenic and enterohemorrhagic Escherichia coli in vivo since it shares several pathogenic mechanisms with these two important human gastrointestinal pathogens. In addition to miR-155, the increased expression of miR-203 in colonic crypts of C. rodentium-infected mice was detected and shown to deregulate Wnt/β-catenin signaling through the direct repression of the Wnt antagonist WIF1 (117). This may contribute to the observed Wnt/β-catenin-dependent crypt hyperplasia in response to C. rodentium infection.

Staphylococcus aureus is another example of a pathogen for which the role of miRNAs during infection is starting to be addressed. Initial reports investigated the impact of staphylococcal enterotoxin B (SEB), an exotoxin responsible for food poisoning and toxic shock, on miRNA expression in lung-infiltrating mononuclear cells isolated from mice exposed to SEB (118). miR-155, the most upregulated miRNA, was shown to enhance the accumulation of IFN-γ by targeting SOCS1, leading to an exacerbated inflammatory response. Interestingly, miR-155 knockout mice are protected against the overinflammation and lung injury elicited by SEB (118), further supporting the relevance of this miRNA on SEB toxicity and, arguably, on S. aureus pathogenicity. Decreased expression of miR-24 has been described in S. aureus-infected macrophages (119). In these cells, reduced miR-24 levels were shown to contribute to a macrophage M1 polarization phenotype via increased CHI3L1 expression. Colonization by S. aureus is frequently linked to chronic skin wounds, particularly in diabetic patients and obese or immunosuppressed individuals (120). The presence of S. aureus in wounds increases miR-15b-5p expression, impairing DNA repair and the inflammatory response, with likely negative consequences to wound healing (121). Using an miR-142 knockout mouse, miR-142-3p and miR-142-5p were also shown to contribute to healing of S. aureus-infected skin wounds (122). Indeed, this phenotype was related to impaired neutrophil chemotactic and phagocytic behavior due to abnormal expression of Rac and Rho GTPases. Recently, the therapeutic potential of miRNA modulation was shown in a model of S. aureus-infected wounds (123). Mice with a specific deletion of miR-223 showed enhanced repair of S. aureus-infected wounds compared to wild-type mice. Similar improvement of wound healing was observed in wild-type mice transplanted with neutrophils lacking miR-223 or treated with inhibitors of miR-223. Interestingly, miR-223 was identified as one of the most highly expressed miRNAs at wound sites during the inflammatory phase.

More systematic studies of miRNA expression have also been performed for a number of pathogens. For example, infection of mouse alveolar macrophages with the opportunistic pathogen Pseudomonas aeruginosa was shown to upregulate eight miRNAs, miR-302b being the most prominently increased (124). Upregulation of this miRNA results in a dampened inflammatory response via targeting of IRAK4, a component of the MyD88 signaling complex critical for NF-κB activation.

The effect of infection with Chlamydia trachomatis, an obligate intracellular pathogen that is the causative agent of trachoma and sexually transmitted infections, on host miRNA expression has been analyzed (reviewed in reference 125). Derrick et al. analyzed miRNA expression profiles of trachoma disease patients, including conjunctival scarring with and without inflammation, revealing 82 differentially expressed miRNAs (126). Pathway analysis revealed an enrichment of genes related to fibrosis and epithelial cell differentiation among the predicted targets of these miRNAs. A recent study by Chowdhury and colleagues analyzed differentially expressed miRNAs in primary human umbilical vein endothelial cells infected with C. trachomatis, revealing miR-30c-5p as one of the most upregulated miRNAs (127). miR-30c-5p upregulation leads to p53-mediated downregulation of Drp1, a mitochondrial fission regulator, thereby inhibiting stress-induced mitochondrial fission and promoting cell survival during infection. In addition, several groups used the mouse pathogen Chlamydia muridarum, a model organism to study human C. trachomatis urogenital tract infections, to investigate miRNA expression changes (128–131). Interestingly, Yeruva et al. showed that C. muridarum infection decreases the protein levels of two components of the miRNA machinery, Dicer and Ago2 (131). Although the consequence of these changes to infection is not yet clear, this suggests a broad effect of C. muridarum in blunting host miRNA responses.

In macrophages, infection by the Gram-negative bacterium Brucella melitensis, the causative agent of brucellosis, was shown to regulate the expression of 57 miRNAs (132). Putative targets of the six most abundant differentially expressed miRNAs (miR-92a, miR-93, miR-151-3p, miR-181b, miR-1981, and let7-b) revealed enrichment for apoptosis, autophagy, and immune response-related pathways. Recent studies have demonstrated that the related pathogen Brucella abortus is also able to modulate host miRNA expression to increase its intracellular survival in macrophages. Indeed, downregulation of miR-125b-5p by B. abortus was shown to diminish the proinflammatory host response via derepression of the inhibitor of NF-κB activation A20 (133). miRNA expression upon Brucella suis infection of porcine and murine macrophages revealed that a common set of five miRNAs (miR-130a-3p, miR-146a, miR-181a, miR-301a-3p, and miR-351-5p) are upregulated. By controlling these miRNAs, B. suis negatively regulates TNFα to promote bacterial intracellular survival (134).

High-throughput functional screenings have recently started to be applied to identify miRNAs controlling different biological processes. We have pioneered the application of this approach to infections by bacterial pathogens, specifically S. Typhimurium (cf. section above [22]) and Shigella flexneri (135). S. flexneri is a major causative agent of shigellosis in humans (136). Using an unbiased functional screening approach, we identified miR-29b-2-5p as a miRNA with a dual regulatory effect on S. flexneri infection, enhancing both bacterial binding to host cells and intracellular bacterial replication (135). miR-29b-2-5p leads to increased filopodia formation through the direct regulation of UNC5C, ultimately enhancing S. flexneri binding to host cells. Interestingly, S. flexneri intracellular replication decreases miR-29b-2-5p expression at late times postinfection by specific degradation of the mature miRNA by the exonuclease PNPT1. A decrease of miR-29b-2-5p may constitute a bacterial strategy to promote balanced intracellular replication, avoiding premature host cell death and favoring spreading to neighboring cells or, alternatively, may be part of the host response to counteract Shigella infection.

PERSPECTIVES

Advances in RNA sequencing have contributed to the extensive analysis of the host miRNome and contributed to demonstrating that infection by various bacterial pathogens induces major miRNome changes. Of note, these studies highlighted that even closely related species (e.g., M. tuberculosis versus M. bovis versus M. avium, or F. tularensis subsp. tularensis versus F. tularensis subsp. novicida) lead to widely different host miRNA profiles. However, a common set of miRNAs regulated as part of the host immune response (e.g., miR-155, miR-146, and let-7) has been described for a wide spectrum of pathogens. Along this line, a recent analysis of miRNA response to infection of dendritic cells by six bacteria revealed a core of 49 miRNAs with consistently altered expression (137). Interestingly, this study also showed that the relative abundance of miRNA duplex arms (–3p and –5p miRNAs) and the expression dynamics of miRNA isoforms (isomiRs) are strongly impacted by bacterial infection, although the consequences of these phenomena to infection are yet to be determined.

In addition to the identity of the pathogen, the regulation of host miRNAs upon infection is dependent on the cellular context (e.g., epithelial cells versus macrophages). Added to this, the majority of the miRNA expression analysis in the context of infection has been performed in bulk cell populations comprising both cells with internalized bacteria and bystander cells. The heterogeneity of the miRNA response of individual cells to infection and its dependency on the extent of bacterial internalization and/or replication still require further investigation. With the improvement of single-cell RNA sequencing, reliable analysis of miRNA profiles with sufficient coverage will be attainable (138). One additional interesting point concerns differences in the response of cells with internalized bacteria and bystander cells. Initial reports in sorted populations of infected and bystander cells suggest that although miRNA changes in the bystander population generally echo those observed in infected cells, a portion of the changes appear to be specific to bystander cells (22). The relevance of the miRNA reprogramming in bystander cells and their consequences to infection clearly deserve further investigation. Importantly, the molecular mechanisms underlying most of the observed miRNA regulations, irrespective of whether these occur in infected or bystander cells, remain poorly understood. In this regard, the direct manipulation of the miRNA pathway by secreted bacterial factors remains a fascinating hypothesis prompted by the discovery of RNA silencing suppressor proteins in viruses and plant bacterial pathogens.

The impacts of microbiota on host miRNA expression and their implications for organ/tissue homeostasis and/or infections are also an exciting subject. In this regard, the resident gut microbiota has been shown to modulate host miRNA expression (139, 140), and a recent report suggests that the host might influence the gut microbiome through miRNAs (141). Moreover, analysis of the host miRNA response to L. monocytogenes infection in conventional versus germ-free mice identified five miRNAs that were downregulated upon infection in a microbiota-dependent manner (113).

Overall, miRNAs are now considered major players in the infection process. As summarized in this article, a growing number of studies are uncovering host miRNAs as part of the defense strategies mounted by the host to fight infection but also as pathogen strategies to subvert host functions and promote virulence. The study of miRNAs in the context of infection and, equally importantly, the identification and characterization of downstream targets and underlying mechanisms of action will continue to provide important insights into the intricate interplay between pathogens and host.