Mycobacterium tuberculosis Controls MicroRNA-99b (miR-99b) Expression in Infected Murine Dendritic Cells to Modulate Host Immunity

Mycobacterium tuberculosis resides and replicates within host phagocytes by modulating host microbicidal responses. In addition, it suppresses the production of host protective cytokines to prevent activation of and antigen presentation by M. tuberculosis-infected cells, causing dysregulation of host protective adaptive immune responses. Many cytokines are regulated by microRNAs (miRNAs), a newly discovered class of small noncoding RNAs, which have been implicated in modulating host immune responses in many bacterial and viral diseases. Here, we show that miRNA-99b (miR-99b), an orphan miRNA, plays a key role in the pathogenesis of M. tuberculosis infection. We found that miR-99b expression was highly up-regulated in M. tuberculosis strain H37Rv-infected dendritic cells (DCs) and macrophages. Blockade of miR-99b expression by antagomirs resulted in significantly reduced bacterial growth in DCs. Interestingly, knockdown of miR-99b in DCs significantly up-regulated proinflammatory cytokines such as IL-6, IL-12, and IL-1β. Furthermore, mRNA and membrane-bound protein data indicated that inhibition of miR-99b augments TNF-α and TNFRSF-4 production. Thus, miR-99b targets TNF-α and TNFRSF-4 receptor genes. Treatment of anti-miR-99b-transfected DCs with anti-TNF-α antibody resulted in increased bacterial burden. Thus, our findings unveil a novel host evasion mechanism adopted by M. tuberculosis via miR-99b, which may open up new avenues for designing miRNA-based vaccines and therapies.     

Innate immune recognition of microbial cell wall components and microbial strategies to evade such recognitions

The innate immune system constitutes the first line of defence against invading microbes. The basis of this defence resides in the recognition of defined structural motifs of the microbes called “Microbial associated molecular patterns” that are absent in the host. Cell wall, the outer layer of both bacterial and fungal cells, a unique structure that is absent in the host and is recognized by the germ line encoded host receptors. Nucleotide oligomerization domain proteins, peptidoglycan recognition proteins and C-type lectins are host receptors that are involved in the recognition of bacterial cell wall (usually called peptidoglycan), whereas fungal cell wall components (N- and O-linked mannans, β-glucans etc.) are recognized by host receptors like C-type lectins (Dectin-1, Dectin-2, mannose receptor, DC-SIGN), Toll like receptors-2 and -4 (TLR-2 and TLR-4). These recognitions lead to activation of a variety of host signaling cascades and ultimate production of anti-microbial compounds including phospholipase A2, antimicrobial peptides, lysozyme, reactive oxygen and nitrogen species. These molecules act in cohort against the invading microbes to eradicate infections. Additionally pathogen recognition leads to the production of cytokines, which further activate the adaptive immune system. Both pathogenic and commensal bacteria and fungus use numerous strategies to subvert the host defence. These strategies include bacterial peptidoglycan glycan backbone modifications by O-acetylation, N-deacetylation, N-glycolylation and stem peptide modifications by amidation of meso-Diaminopimelic acid; fungal cell wall modifications by shielding the β-glucan layer with mannoproteins and α-1,3 glucan. This review focuses on the recent advances in understanding the role of bacterial and fungal cell wall in their innate immune recognition and evasion strategies.     

Mycobacterial infection-induced miR-206 inhibits protective neutrophil recruitment via the CXCL12/CXCR4 signalling axis

Pathogenic mycobacteria actively dysregulate protective host immune signalling pathways during infection to drive the formation of permissive granuloma microenvironments. Dynamic regulation of host microRNA (miRNA) expression is a conserved feature of mycobacterial infections across host-pathogen pairings. Here we examine the role of miR-206 in the zebrafish model of Mycobacterium marinum infection, which allows investigation of the early stages of granuloma formation. We find miR-206 is upregulated following infection by pathogenic M. marinum and that antagomir-mediated knockdown of miR-206 is protective against infection. We observed striking upregulation of cxcl12a and cxcr4b in infected miR-206 knockdown zebrafish embryos and live imaging revealed enhanced recruitment of neutrophils to sites of infection. We used CRISPR/Cas9-mediated knockdown of cxcl12a and cxcr4b expression and AMD3100 inhibition of Cxcr4 to show that the enhanced neutrophil response and reduced bacterial burden caused by miR-206 knockdown was dependent on the Cxcl12/Cxcr4 signalling axis. Together, our data illustrate a pathway through which pathogenic mycobacteria induce host miR-206 expression to suppress Cxcl12/Cxcr4 signalling and prevent protective neutrophil recruitment to granulomas.     

Licochalcone A reverses NNK-induced ectopic miRNA expression to elicit in vitro and in vivo chemopreventive effects

BackgroundChemoprevention is the best cost-effective way regarding cancers. MicroRNAs (miRNAs) have been reported to be differentially expressed during the development of lung cancer. However, if lung cancer prevention can be achieved through modulating miRNAs expression so far remains unknown.PurposeTo discover ectopically expressed miRNAs in NNK-induced lung cancer and clarify whether Licochalcone A (lico A) can prevent NNK-induced lung cancer by modulating miRNA expression.Study design and methodsA/J mice were used to construct a lung cancer model by intraperitoneal injection with physiological saline NNK (100 mg/kg). Chemopreventive effects of lico A against lung cancer at 2 mg/kg and 20 mg/kg doses were evaluated in vivo. MicroRNA array and RT-qPCR were used to assess the expression levels of miRNAs. MLE-12 cells were treated with 0.1 mg/ml NNK, stimulating the ectopic expression pattern of miR-144-3p, miR-20a-5p, miR-29c-3p, let-7d-3p, and miR-328-3p. miR-144-3p mimics and inhibitors were used to manipulate miR-144-3p levels. The effects of lico A (10 μM) on cell cycle distribution, apoptosis, and the expression of CK19, RASA1, miR-144-3p, miR-20a-5p, miR-29c-3p, let-7d-3p, and miR-328-3p in NNK-treated MLE-12 cells were studied.ResultsThe expression levels of miR-144-3p, miR-20a-5p, and miR-29c-3p increased, while those of let-7d-3p and miR-328-3p decreased in both NNK-induced A/J mice and MLE-12 cells. Lico A could reverse the NNK-induced ectopic miRNA (miR-144-3p, miR-20a-5p, miR-29c-3p, let-7d-3p, and miR-328-3p) expression both in vivo and in vitro and elicit in vivo lung cancer chemopreventive effect against NNK. In MLE-12 cells, the overexpression of miR-144-3p elicited the same effect as NNK regarding the expression of lung cancer biomarker CK19; the silencing of miR-144-3p reversed the effect of NNK on cell cycle distribution and apoptosis. Lico A could reverse the effect of NNK on the expression of miR-144-3p, CK19, and RASA1 (predicted target of miR-144-3p).ConclusionThe present study suggests that miR-144-3p, miR-20a-5p, miR-29c-3p, let-7d-3p, and miR-328-3p were involved in the in vivo pathogenesis of NNK-induced lung cancer, and lico A could reverse the effect of NNK both in vivo and in vitro to elicit lung cancer chemopreventive effects through, at least partially, these five ectopically expressed miRNAs, especially miR-144-3p.     

The Human Cathelicidin LL-37 Has Antiviral Activity against Respiratory Syncytial Virus

Respiratory syncytial virus is a leading cause of lower respiratory tract illness among infants, the elderly and immunocompromised individuals. Currently, there is no effective vaccine or disease modifying treatment available and novel interventions are urgently required. Cathelicidins are cationic host defence peptides expressed in the inflamed lung, with key roles in innate host defence against infection. We demonstrate that the human cathelicidin LL-37 has effective antiviral activity against RSV in vitro, retained by a truncated central peptide fragment. LL-37 prevented virus-induced cell death in epithelial cultures, significantly inhibited the production of new infectious particles and diminished the spread of infection, with antiviral effects directed both against the viral particles and the epithelial cells. LL-37 may represent an important targetable component of innate host defence against RSV infection. Prophylactic modulation of LL-37 expression and/or use of synthetic analogues post-infection may represent future novel strategies against RSV infection.     

Bacterial Pathogens Activate a Common Inflammatory Pathway through IFNλ Regulation of PDCD4

The type III interferon (IFNλ) receptor IL-28R is abundantly expressed in the respiratory tract and has been shown essential for host defense against some viral pathogens, however no data are available concerning its role in the innate immune response to bacterial pathogens. Staphylococcus aureus and Pseudomonas aeruginosa induced significant production of IFNλ in the lung, and clearance of these bacteria from the lung was significantly increased in IL-28R null mice compared to controls. Improved bacterial clearance correlated with reduced lung pathology and a reduced ratio of pro- vs anti-inflammatory cytokines in the airway. In human epithelial cells IFNλ inhibited miR-21 via STAT3 resulting in upregulation of PDCD4, a protein known to promote inflammatory signaling. In vivo 18 hours following infection with either pathogen, miR-21 was significantly reduced and PDCD4 increased in the lungs of wild type compared to IL-28R null mice. Infection of PDCD4 null mice with USA300 resulted in improved clearance, reduced pathology, and reduced inflammatory cytokine production. These data suggest that during bacterial pneumonia IFNλ promotes inflammation by inhibiting miR-21 regulation of PDCD4.     

Predictive value of circulating miR-328 and miR-134 for acute myocardial infarction

MicroRNA (miRNAs) is demonstrated to be present in the blood of humans and has been increasingly suggested as a novel biomarker for various pathological processes in the heart, including myocardial infarction, myocardial remodeling and progression to heart failure. In this study, we aim to evaluate the diagnostic and prognostic value of circulating miR-328 and miR-134 in patients with acute myocardial infarction (AMI). Circulating levels of miR-328 and miR-134 were detected by quantitative real-time PCR in plasma samples from 359 AMI patients and 30 healthy volunteers. Concentrations of high-sensitivity cardiac troponin T (hs-cTnT) were measured using electrochemiluminescence-based methods. MiRNAs were assessed for discrimination of a clinical diagnosis of AMI and for association with primary clinical endpoint defined as a composite of cardiogenic death and development of heart failure within 6 months after infarction. Results showed that levels of plasma miR-328 and miR-134 were significantly higher in AMI patients than in healthy controls. Receiver operating characteristic curve analyses showed significant diagnostic value of miR-328 and miR-134 for AMI. However, neither of them was superior to hs-cTnT for the diagnosis. Additionally, increased miRNA levels were strongly associated with increased risk of mortality or heart failure within 6 months for miR-328 (OR 7.35, 95 % confidence interval 1.07–17.83, P < 0.001) and miR-134 (OR 2.28, 95 % confidence interval 1.03–11.32 P < 0.001). In conclusion, circulating miR-328 and miR-134 could be potential indicators for AMI, and the miRNA levels are associated with increased risk of mortality or development of heart failure.     

Reactive-Oxygen-Species-Mediated P. aeruginosa Killing Is Functional in Human Cystic Fibrosis Macrophages

Pseudomonas aeruginosa is the most common pathogen for chronic lung infection in cystic fibrosis (CF) patients. About 80% of adult CF patients have chronic P. aeruginosa infection, which accounts for much of the morbidity and most of the mortality. Both bacterial genetic adaptations and defective innate immune responses contribute to the bacteria persistence. It is well accepted that CF transmembrane conductance regulator (CFTR) dysfunction impairs the airways-epithelium-mediated lung defence; however, other innate immune cells also appear to be affected, such as neutrophils and macrophages, which thus contribute to this infectious pathology in the CF lung. In macrophages, the absence of CFTR has been linked to defective P. aeruginosa killing, increased pro-inflammatory cytokine secretion, and reduced reactive oxygen species (ROS) production. To learn more about macrophage dysfunction in CF patients, we investigated the generation of the oxidative burst and its impact on bacterial killing in CF macrophages isolated from peripheral blood or lung parenchyma of CF patients, after P. aeruginosa infection. Our data demonstrate that CF macrophages show an oxidative response of similar intensity to that of non-CF macrophages. Intracellular ROS are recognized as one of the earliest microbicidal mechanisms against engulfed pathogens that are activated by macrophages. Accordingly, NADPH inhibition resulted in a significant increase in the intracellular bacteria survival in CF and non-CF macrophages, both as monocyte-derived macrophages and as lung macrophages. These data strongly suggest that the contribution of ROS to P. aeruginosa killing is not affected by CFTR mutations.     

Detection of Circulating Parasite-Derived MicroRNAs in Filarial Infections

Filarial nematodes cause chronic and profoundly debilitating diseases in both humans and animals. Applications of novel technology are providing unprecedented opportunities to improve diagnosis and our understanding of the molecular basis for host-parasite interactions. As a first step, we investigated the presence of circulating miRNAs released by filarial nematodes into the host bloodstream. miRNA deep-sequencing combined with bioinformatics revealed over 200 mature miRNA sequences of potential nematode origin in Dirofilaria immitis-infected dog plasma in two independent analyses, and 21 in Onchocerca volvulus-infected human serum. Total RNA obtained from D. immitis-infected dog plasma was subjected to stem-loop RT-qPCR assays targeting two detected miRNA candidates, miR-71 and miR-34. Additionally, Brugia pahangi-infected dog samples were included in the analysis, as these miRNAs were previously detected in extracts prepared from this species. The presence of miR-71 and miR-34 discriminated infected samples (both species) from uninfected samples, in which no specific miRNA amplification occurred. However, absolute miRNA copy numbers were not significantly correlated with microfilaraemia for either parasite. This may be due to the imprecision of mf counts to estimate infection intensity or to miRNA contributions from the unknown number of adult worms present. Nonetheless, parasite-derived circulating miRNAs are found in plasma or serum even for those species that do not live in the bloodstream.     

Innate host responses to enteric bacterial pathogens: a balancing act between resistance and tolerance

Infection by enteric bacterial pathogens activates pathogen recognition receptors, leading to innate responses that promote host defence. While responses that promote host 'resistance' to infection, through the release of antimicrobial mediators, or the recruitment of inflammatory cells aimed at clearing the infection are best known, recent studies have begun to identify additional innate driven responses that instead promote intestinal tissue repair and host survival. Described as infection 'tolerance' responses, we and others have primarily studied these responses in the Citrobacter rodentium infection model. In this review we discuss the impact of innate resistance mechanisms on host defence, and describe how 'tolerance' responses act primarily on the intestinal epithelium, triggering epithelial cell proliferation, repair or promoting barrier function. Resistance and tolerance responses appear to work together, with tolerance repairing the tissue injury caused by resistance driven inflammation. Tolerance responses fit a pattern where innate immunity and inflammation are tightly regulated in the gastrointestinal tract. Moreover, tolerance may have developed due to the successful subversion and avoidance of host resistance by enteric bacterial pathogens. Further studies are needed to clarify the contribution of different pathogen recognition receptors to tolerance and resistance responses against bacterial pathogens, in the gut or in other host tissues.     

Bacterial toxins can inhibit host cell autophagy through cAMP generation

Autophagy plays a significant role in innate and adaptive immune responses to microbial infection. Some pathogenic bacteria have developed strategies to evade killing by host autophagy. These include the use of 'camouflage' proteins to block targeting to the autophagy pathway and the use of pore-forming toxins to block autophagosome maturation. However, general inhibition of host autophagy by bacterial pathogens has not been observed to date. Here we demonstrate that bacterial cAMP-elevating toxins from B. anthracis and V. cholera can inhibit host anti-microbial autophagy, including autophagic targeting of S. Typhimurium and latex bead phagosomes. Autophagy inhibition required the cAMP effector protein kinase A. Formation of autophagosomes in response to rapamycin and the endogenous turnover of peroxisomes was also inhibited by cAMP-elevating toxins. These findings demonstrate that cAMP-elevating toxins, representing a large group of bacterial virulence factors, can inhibit host autophagy to suppress immune responses and modulate host cell physiology.     

Bacterial toxins can inhibit host cell autophagy through cAMP generation

Autophagy plays a significant role in innate and adaptive immune responses to microbial infection. Some pathogenic bacteria have developed strategies to evade killing by host autophagy. These include the use of ‘camouflage’ proteins to block targeting to the autophagy pathway and the use of pore-forming toxins to block autophagosome maturation. However, general inhibition of host autophagy by bacterial pathogens has not been observed to date. Here we demonstrate that bacterial cAMP-elevating toxins from B. anthracis and V. cholera can inhibit host anti-microbial autophagy, including autophagic targeting of S. Typhimurium and latex bead phagosomes. Autophagy inhibition required the cAMP effector protein kinase A. Formation of autophagosomes in response to rapamycin and the endogenous turnover of peroxisomes was also inhibited by cAMP-elevating toxins. These findings demonstrate that cAMP-elevating toxins, representing a large group of bacterial virulence factors, can inhibit host autophagy to suppress immune responses and modulate host cell physiology.     

Delineation of the innate and adaptive T-cell immune outcome in the human host in response to Campylobacter jejuni infection

Background

Campylobacter jejuni is the most prevalent cause of bacterial gastroenteritis worldwide. Despite the significant health burden this infection presents, molecular understanding of C. jejuni-mediated disease pathogenesis remains poorly defined. Here, we report the characterisation of the early, innate immune response to C. jejuni using an ex-vivo human gut model of infection. Secondly, impact of bacterial-driven dendritic cell activation on T-cell mediated immunity was also sought.

Methodology

Healthy, control paediatric terminal ileum or colonic biopsy tissue was infected with C. jejuni for 8–12 hours. Bacterial colonisation was followed by confocal microscopy and mucosal innate immune responses measured by ELISA. Marked induction of IFNγ with modest increase in IL-22 and IL-17A was noted. Increased mucosal IL-12, IL-23, IL-1β and IL-6 were indicative of a cytokine milieu that may modulate subsequent T-cell mediated immunity. C. jejuni-driven human monocyte-derived dendritic cell activation was followed by analyses of T cell immune responses utilising flow cytometry and ELISA. Significant increase in Th-17, Th-1 and Th-17/Th-1 double-positive cells and corresponding cytokines was observed. The ability of IFNγ, IL-22 and IL-17 cytokines to exert host defence via modulation of C. jejuni adhesion and invasion to intestinal epithelia was measured by standard gentamicin protection assay.

Conclusions

Both innate and adaptive T cell-immunity to C. jejuni infection led to the release of IFNγ, IL-22 and IL-17A; suggesting a critical role for this cytokine triad in establishing host anti-microbial immunity during the acute and effectors phase of infection. In addition, to their known anti-microbial functions; IL-17A and IL-17F reduced the number of intracellular C. jejuni in intestinal epithelia, highlighting a novel aspect of how IL-17 family members may contribute to protective immunity against C. jejuni.