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1.
Mol Oral Microbiol ; 2024 Mar 21.
Article in English | MEDLINE | ID: mdl-38511906

ABSTRACT

BACKGROUND: Considered the second largest and most diverse microbiome after the gut, the human oral ecosystem is complex with diverse and niche-specific microorganisms. Although evidence is growing for the importance of oral microbiome in supporting a healthy immune system and preventing local and systemic infections, the influence of craniomaxillofacial (CMF) trauma and routine reconstructive surgical treatments on community structure and function of oral resident microbes remains unknown. CMF injuries affect a large number of people, needing extensive rehabilitation with lasting morbidity and loss of human productivity. Treatment efficacy can be complicated by the overgrowth of opportunistic commensals or multidrug-resistant pathogens in the oral ecosystem due to weakened host immune function and reduced colonization resistance in a dysbiotic oral microbiome. AIMS: To understand the dynamics of microbiota's community structure during CMF injury and subsequent treatments, we induced supra-alveolar mandibular defect in Hanford miniature swine (n = 3) and compared therapeutic approaches of immediate mandibullar reconstructive (IMR) versus delayed mandibullar reconstructive (DMR) surgeries. METHODS: Using bacterial 16S ribosomal RNA gene marker sequencing, the composition and abundance of the bacterial community of the uninjured maxilla (control) and the injured left mandibula (lingual and buccal) treated by DMR were surveyed up to 70-day post-wounding. For the injured right mandibula receiving IMR treatment, the microbial composition and abundance were surveyed up to 14-day post-wounding. Moreover, we measured sera level of biochemical markers (e.g., osteocalcin) associated with bone regeneration and healing. Computed tomography was used to measure and compare mandibular bone characteristics such as trabecular thickness between sites receiving DMR and IMR therapeutic approaches until day 140, the end of study period. RESULTS: Independent of IMR versus DMR therapy, we observed similar dysbiosis and shifts of the mucosal bacteria residents after CMF injury and/or following treatment. There was an enrichment of Fusobacterium, Porphyromonadaceae, and Bacteroidales accompanied by a decline in Pasteurellaceae, Moraxella, and Neisseria relative abundance in days allotted for healing. We also observed a decline in species richness and abundance driven by reduction in temporal instability and inter-animal heterogeneity on days 0 and 56, with day 0 corresponding to injury in DMR group and day 56 corresponding to delayed treatment for DMR or injury and immediate treatment for the IMR group. Analysis of bone healing features showed comparable bone-healing profiles for IMR vs. DMR therapeutic approach.

2.
Infect Drug Resist ; 14: 1-10, 2021.
Article in English | MEDLINE | ID: mdl-33442271

ABSTRACT

PURPOSE: The biology of chronic wounds is complex and many factors act concurrently to impede healing progress. In this study, the dynamics of microflora changes and their antibiotic susceptibility patterns were evaluated longitudinally over 30 days using data from 28 patients with a total of 47 chronic lower extremity wounds. MATERIALS AND METHODS: In this study, colonized wound isolates were characterized using cultural, biochemical, and VITEK 2 methods. Antibiotic susceptibility patterns of the wound isolates were analyzed using various phenotypic assays. Furthermore, antimicrobial resistance patterns and the presence of mutations were evaluated by a genotypic assay, whole-genome sequencing (WGS). RESULTS: Staphylococcus aureus and Pseudomonas aeruginosa were found to be the most common strains at early time points, while members of Enterobacteriaceae were prevalent at later stages of infection. Antimicrobial resistance testing and whole-genome sequencing revealed that the molecular and phenotypic characteristics of the identified wound pathogens remained relatively stable throughout the study period. It was also noted that Enterobacter and Klebsiella species may serve as reservoirs for quinolone resistance in the Pacific region. CONCLUSION: Our observations showed that wounds were colonized with diverse bacteria and interestingly their numbers and/or types were changed over the course of infection. The rapid genetic changes that accompanied the first 4 weeks after presentation did not directly contribute to the development of antibiotic resistance. In addition, standard wound care procedures did not appear to select for resistant bacterial strains. Future efforts should focus on defining those genetic changes associated with the wound colonizing microorganisms that occur beyond 4 weeks.

3.
J Microbiol Methods ; 169: 105833, 2020 02.
Article in English | MEDLINE | ID: mdl-31904440

ABSTRACT

Virulence is the relative capacity of a pathogenic microorganism to cause damage in susceptible host cells such as those found in airway passages and the gut. In this study, the effect of clinical bacterial isolates on the monolayer integrity of cultured human alveolar basal epithelial cells (A549) was evaluated using the Electric Cell-Substrate Impedance Sensing (ECIS) system. ECIS is a morphological biosensor which records electrical properties of cell-covered microelectrodes in an AC circuit including impedance (ohm), resistance (ohm), and capacitance (µFarad). In the current study, fluctuations in the electrical properties of cell-covered microelectrodes reflect dynamic changes in cell morphology resulting from disrupted cell monolayers following exposure to bacteria. Using the ECIS system, real-time changes of cell morphology and disruption of monolayer integrity of cell-cultures in vitro were revealed for A549 cells infected with either Pseudomonas aeruginosa, ESBL Escherichia coli, Staphylococcus aureus (MRSA), or Enterococcus (VRE). We determined empirically that the optimal signal response was obtained for resistance (ohm) measurements at 4000 hertz. Following infection of A549 cells, the data revealed that Pseudomonas aeruginosa resulted in little change in microelectrode resistance (ohm @4 kHz) as compared to pathogen-free controls within the first 12 h. In contrast, E. coli, MRSA, and VRE caused significant changes in electrode resistance (ohm @4 kHz) values in the infected cells compared to controls over the first 5 h. Resistance (ohm @4 kHz) changes were also observed in cell monolayers infected with different bacterial concentrations for all isolates over 24 h. The highest concentration of bacteria caused the measured resistance (ohm @4 kHz) to drop faster than its' immediate lower concentration, suggesting a dose-dependent effect. Compared to live bacteria, cells exposed to heat-killed bacteria did not show significant changes in resistance (ohm @4 kHz) over 48 h post-exposure. Functionally, cytokine responses were different between cells treated with live and heat-killed bacteria. Of note, live bacteria induced IFNγ, IL-13, and IL-1ß production in A549 cells, whereas heat-killed bacteria induced IL-8 production suggesting a differential interaction with cells that could reveal the underlying causes of resistance (ohm @4 kHz) changes. Our findings indicate that ECIS provides a means to quantify, automate, and measure bacterial virulence, which may have broader implications governing the course of treatment compared to traditional methods alone.


Subject(s)
Bacteria/metabolism , Cell Physiological Phenomena/physiology , Electric Impedance , Epithelial Cells/microbiology , A549 Cells , Biosensing Techniques/methods , Cell Line , Cytokines/metabolism , Enterococcus/isolation & purification , Enterococcus/metabolism , Enterococcus/pathogenicity , Escherichia coli/isolation & purification , Escherichia coli/metabolism , Escherichia coli/pathogenicity , Humans , Microelectrodes , Pseudomonas aeruginosa/isolation & purification , Pseudomonas aeruginosa/metabolism , Pseudomonas aeruginosa/pathogenicity , Staphylococcus aureus/isolation & purification , Staphylococcus aureus/metabolism , Staphylococcus aureus/pathogenicity , Tight Junctions/microbiology
4.
J Emerg Nurs ; 45(2): 169-177.e1, 2019 Mar.
Article in English | MEDLINE | ID: mdl-30573161

ABSTRACT

INTRODUCTION: The emergency department is a fast-paced, high-volume environment, serving patients with diverse and evolving acuities. Personnel providing direct care are continually exposed to pathogenic microorganisms from patients and everyday surfaces, to which the organisms may spread. Indeed, hospital items-such as electronic devices, stethoscopes, and staff clothing-have demonstrated high rates of contamination. Despite this, policies governing the use, disinfection, and wear of various environmental surfaces remain relaxed, vague, and/or difficult to enforce. This study aimed to examine the bacterial contamination on 2 hospital uniform types in a large military hospital within the emergency department. METHODS: Environmental sampling of military and civilian nursing staff uniforms was performed on 2 separate occasions. Emergency nurses wore hospital-provided freshly laundered scrubs on the first sampling day and home-laundered personally owned uniforms complicit with ED policy on the second sampling day. Samples were collected by impressing of contact blood agar growth medium at arrival (0 hour), 4 hours, and 8 hours of wear. Microbiological methods were used to enumerate and identify bacterial colonies. RESULTS: Bacterial contamination of personally owned uniforms was significantly higher than freshly laundered hospital-provided scrubs on 4 different sampling sites and across the span of an 8-hour workday. No significant differences were observed between military and civilian personally owned uniforms. However, several risk factors for nosocomial infection were increased in the military subgroup. DISCUSSION: Re-evaluating organizational factors (such as uniform policies) that increase the propensity for pathogenic contamination are critical for mitigating the spread and acquisition of multidrug-resistant organisms in the emergency department.


Subject(s)
Clothing , Cross Infection/microbiology , Emergency Service, Hospital , Equipment Contamination/statistics & numerical data , Hospitals, Military , Nursing Staff, Hospital , Adult , Cross-Over Studies , Female , Humans , Infection Control , Male , Middle Aged , Military Personnel
5.
Sci Rep ; 8(1): 11736, 2018 08 06.
Article in English | MEDLINE | ID: mdl-30082843

ABSTRACT

Hawaii has one of the highest incidences of Campylobacteriosis in the United States, but there remains little published data on circulating strains or antimicrobial resistance. We characterized 110 clinical Campylobacter isolates (106 C. jejuni, 4 C. coli) processed at Tripler Army Medical Center in Honolulu, HI from 2012-2016. Twenty-five percent of C. jejuni isolates exhibited fluoroquinolone (FQ) resistance, compared with 16% for tetracycline (TET), and 0% for macrolides. Two of the four C. coli isolates were resistant to FQ, TET, and macrolides. C. jejuni isolates further underwent multilocus sequence typing, pulsed-field gel electrophoresis, and molecular capsular typing. Nineteen capsule types were observed, with two capsule types (HS2 and HS9) being associated with FQ resistance (p < 0.001 and p = 0.006, respectively). HS2 FQ-resistant isolates associated with clonal complex 21, possibly indicating clonal spread in FQ resistance. Macrolides should be considered for treatment of suspect cases due to lack of observed resistance.


Subject(s)
Campylobacter/drug effects , Adult , Anti-Bacterial Agents/pharmacology , Campylobacter/genetics , Campylobacter Infections/prevention & control , Drug Resistance, Bacterial/genetics , Electrophoresis, Gel, Pulsed-Field , Female , Fluoroquinolones/pharmacology , Hawaii , Humans , Macrolides/pharmacology , Male , Multilocus Sequence Typing , Retrospective Studies , Tetracycline/pharmacology , Young Adult
6.
Infect Control Hosp Epidemiol ; 39(11): 1316-1321, 2018 11.
Article in English | MEDLINE | ID: mdl-30156175

ABSTRACT

OBJECTIVE: To compare bacterial contamination of military-approved uniforms and hospital-provided scrubs donned by nursing staff in an inpatient setting. DESIGN: Randomized experimental crossover study. SETTING: Large academic military medical center. METHODS: Inpatient units were randomized to predetermine the order of uniform sampling. Participants included nursing staff who provided direct patient care across 7 eligible inpatient units. Sampling of 6 designated sites on the uniform was completed on arrival to work, at ~4 hours into their shift, and at the 8-hour time point, for a total of 18 samples. Sampling of each participant occurred on 2 separate occasions, once in a military-approved uniform, and once in hospital-provided scrubs. After 24 hours of incubation, a colony-counting machine was used to calculate the total colony-forming units (CFU) of the sample. RESULTS: Across all time points, military-approved uniforms demonstrated a 2-fold bacterial increase at the abdominal site and 3-fold increases at the sleeve cuff and waist pocket regions compared to the same regions on hospital-provided scrubs. CONCLUSION: Nurses should be aware that bacteria are present at much higher levels on their personal military uniforms compared to hospital-provided scrubs. Additional research is needed to determine whether these findings are a function of wear, laundering, or environmental factors. Nurses should adhere to daily uniform washing to reduce bacterial load and minimize risk of nosocomial infections to the patients they care for.


Subject(s)
Cross Infection/microbiology , Equipment Contamination/statistics & numerical data , Hospitals, Military , Nursing Staff, Hospital , Protective Clothing/microbiology , Adult , Bacterial Load/methods , Colony Count, Microbial/methods , Cross-Over Studies , Female , Humans , Infection Control , Male , Military Personnel , Patient Care , Young Adult
7.
J Innate Immun ; 7(4): 428-40, 2015.
Article in English | MEDLINE | ID: mdl-25896300

ABSTRACT

Unwarranted overproduction of cytokines, such as interleukin (IL)-1ß, can cause moderate to severe pathological complications, and thus elaborate mechanisms are needed to regulate its onset and termination. One such, well-known, mechanism is endotoxin tolerance, generally described as controlling lipopolysaccharide Toll-like receptor 4 (LPS-TLR4) signaling. Similarly, cytokine-induced tolerance plays an important role in regulating an overactive cytokine response. In this report, the capability of IL-1ß to induce tolerance and cross-tolerance to various inflammatory ligands was investigated. IL-1ß-stimulated THP-1 monocytes showed a gradual increase of microRNA (miR)-146a, reaching 15-fold expression by 24 h. miR-146a upregulation induced tolerance toward subsequent challenges of IL-1ß, LPS, peptidoglycan, Pam and flagellin in THP-1 cells. The induction of tolerance was dependent on the IL-1ß priming dose and associated increase of miR-146a expression. Moreover, IL-1ß-treated THP-1 cells showed sustained miR-146a upregulation that repressed IRAK1 and TRAF6 adaptor molecules. Transfection of miR-146a alone mimicked IL-1ß-induced tolerance in monocytes, while cells transfected with miR-146a inhibitor increased chemokine production. A comparable cytokine response regulated by miR-146a was also detected in lung epithelial A549 cells, purified human monocytes and mouse peritoneal macrophages. Thus, our studies showed that miR-146a was crucial for monocytic cell-based IL-1ß tolerance and cross-tolerance, and thus opens the way for future research in the development of therapeutics for inflammatory diseases.


Subject(s)
Immune Tolerance , Interleukin-1beta/immunology , Macrophages, Peritoneal/immunology , MicroRNAs/immunology , Toll-Like Receptors/immunology , Animals , Cell Line, Tumor , Humans , Interleukin-1 Receptor-Associated Kinases/genetics , Interleukin-1 Receptor-Associated Kinases/immunology , Interleukin-1beta/genetics , Mice , MicroRNAs/genetics , TNF Receptor-Associated Factor 6/genetics , TNF Receptor-Associated Factor 6/immunology , Toll-Like Receptors/genetics
8.
J Immunol ; 190(3): 1250-63, 2013 Feb 01.
Article in English | MEDLINE | ID: mdl-23264652

ABSTRACT

Innate immune response is the first defense against pathogens via recognition by various conserved pattern recognition receptors, such as TLRs, to initiate a rapid and strong cytokine alarm. TLR signaling-mediated cytokine production must be properly regulated to prevent pathological conditions deriving from overproduction of cytokines. In this study, the role of specific microRNAs in TLR-signaling pathway was investigated to reveal the cross-interaction and -regulation in the MyD88 pathway. In peptidoglycan (PGN)/TLR2-stimulated THP-1 monocytes, PBMCs, and primary macrophages showed rapid and dramatic miR-132 and miR-212 (miR-132/-212) upregulation. This newly identified response appeared earlier in time than the characteristic miR-146a response in LPS-TLR4 stimulation. The rapid induction of miR-132/-212 was transcription factor CREB dependent, and the sustained expression of miR-132/-212 was responsible for inducing tolerance to subsequent PGN challenge. Cross-tolerance was observed by TLR5 ligand flagellin and heat-killed or live bacteria resulting from miR-132/-212 upregulation. Mechanistically, IRAK4 was identified and validated as a target of miR-132/-212 by luciferase reporter assay and seed-sequence mutagenesis of the reporter. Transfection of miR-132 or miR-212 alone mimicked PGN tolerance in monocytes, whereas transfected specific miRNA inhibitors tampered the tolerance effect. During bacterial infection, PGN-mediated TLR2 signaling induces miR-132/-212 to downregulate IRAK4, an early component in the MyD88-dependent pathway, whereas LPS/TLR4-induced miR-146a downregulates downstream components of the same MyD88-dependent pathway. The identification of miR-132/-212 and miR-146a together to prevent damaging consequences from the overproduction of proinflammatory cytokines by targeting a common signaling pathway is significant and will provide insights into future design and development of therapeutics.


Subject(s)
Immune Tolerance/immunology , Interleukin-1 Receptor-Associated Kinases/immunology , Macrophages, Peritoneal/immunology , MicroRNAs/immunology , Monocytes/immunology , Peptidoglycan/immunology , Signal Transduction/immunology , Toll-Like Receptor 2/immunology , Animals , Bacterial Proteins/immunology , Cells, Cultured/immunology , Cyclic AMP Response Element-Binding Protein/immunology , Female , Flagellin/immunology , Flagellin/pharmacology , Gene Expression Regulation , Genes, Reporter , Lipopolysaccharides/immunology , Lipoproteins/pharmacology , Macrophage Activation , Mice , Mice, Inbred C57BL , MicroRNAs/antagonists & inhibitors , MicroRNAs/biosynthesis , MicroRNAs/genetics , Myeloid Differentiation Factor 88/immunology , RNA, Small Interfering/pharmacology , Tumor Necrosis Factor-alpha/metabolism
9.
Vet Immunol Immunopathol ; 144(3-4): 255-8, 2011 Dec 15.
Article in English | MEDLINE | ID: mdl-22041600

ABSTRACT

The reactivity of different lectins with crude chromogranin A (CgA) obtained from different animals, namely, cow, horse, dog, pig, and dolphin, was examined to identify lectin(s) that would be useful as coating reagent(s) in a sandwich enzyme-linked immunosorbent assay (ELISA). Of the different lectins studied, the Amaranthus caudatus lectin (ACA), which is specific for the Thomsen-Friedenreich (T)-antigen (Galß1-3GalNAc), was found to react with the CgA from different animals by western blotting. Purified rabbit anti-bovine CgA antibody was also found to cross-react with the crude CgA preparations. On the basis of these findings, a sandwich ELISA was developed with ACA as the coating reagent and anti-bovine CgA antibody as the probing antibody. Using this method, concentration-dependent curves ranging from 0.003 µg/mL to 25 µg/mL and from 0.02 µg/mL to 25 µg/mL were obtained for bovine CgA and canine CgA, respectively. Similarly, concentration-dependent curves were obtained for the equine, swine, and dolphin crude CgA extracts. Thus, ACA is concluded to be a valuable reagent for CgA detection in crude extracts from different animal species, and for CgA isolation/purification.


Subject(s)
Adrenal Glands/chemistry , Amaranthus/immunology , Antigens, Tumor-Associated, Carbohydrate/immunology , Chromogranin A/analysis , Enzyme-Linked Immunosorbent Assay/methods , Plant Lectins/immunology , Animals , Blotting, Western , Cattle , Chromogranin A/immunology , Dogs , Dolphins , Electrophoresis, Polyacrylamide Gel , Horses , Rabbits/immunology , Swine , Tissue Extracts/chemistry
10.
Cell Mol Immunol ; 8(5): 388-403, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21822296

ABSTRACT

Toll-like receptors (TLRs) in innate immune cells are the prime cellular sensors for microbial components. TLR activation leads to the production of proinflammatory mediators and thus TLR signaling must be properly regulated by various mechanisms to maintain homeostasis. TLR4-ligand lipopolysaccharide (LPS)-induced tolerance or cross-tolerance is one such mechanism, and it plays an important role in innate immunity. Tolerance is established and sustained by the activity of the microRNA miR-146a, which is known to target key elements of the myeloid differentiation factor 88 (MyD88) signaling pathway, including IL-1 receptor-associated kinase (IRAK1), IRAK2 and tumor-necrosis factor (TNF) receptor-associated factor 6 (TRAF6). In this review, we comprehensively examine the TLR signaling involved in innate immunity, with special focus on LPS-induced tolerance. The function of TLR ligand-induced microRNAs, including miR-146a, miR-155 and miR-132, in regulating inflammatory mediators, and their impact on the immune system and human diseases, are discussed. Modulation of these microRNAs may affect TLR pathway activation and help to develop therapeutics against inflammatory diseases.


Subject(s)
Endotoxins/pharmacology , Gene Expression Regulation/immunology , Immune Tolerance/genetics , Immunity, Innate , MicroRNAs/immunology , Signal Transduction/immunology , Animals , Bacteria/growth & development , Bacterial Infections/immunology , Bacterial Infections/microbiology , Cytokines/genetics , Cytokines/immunology , Cytokines/metabolism , Endotoxins/immunology , Humans , Immune System Diseases/genetics , Interleukin-1 Receptor-Associated Kinases/genetics , Interleukin-1 Receptor-Associated Kinases/immunology , Interleukin-1 Receptor-Associated Kinases/metabolism , Mice , MicroRNAs/genetics , MicroRNAs/metabolism , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/immunology , Myeloid Differentiation Factor 88/metabolism , Rats , Signal Transduction/genetics , TNF Receptor-Associated Factor 6/genetics , TNF Receptor-Associated Factor 6/immunology , TNF Receptor-Associated Factor 6/metabolism , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/immunology , Toll-Like Receptor 4/metabolism
11.
Arthritis Res Ther ; 13(4): 229, 2011 Jul 13.
Article in English | MEDLINE | ID: mdl-21787439

ABSTRACT

MicroRNAs (miRNAs) are endogenous, non-coding, single-stranded RNAs about 21 nucleotides in length. miRNAs have been shown to regulate gene expression and thus influence a wide range of physiological and pathological processes. Moreover, they are detected in a variety of sources, including tissues, serum, and other body fluids, such as saliva. The role of miRNAs is evident in various malignant and nonmalignant diseases, and there is accumulating evidence also for an important role of miRNAs in systemic rheumatic diseases. Abnormal expression of miRNAs has been reported in autoimmune diseases, mainly in systemic lupus erythematosus and rheumatoid arthritis. miRNAs can be aberrantly expressed even in the different stages of disease progression, allowing miRNAs to be important biomarkers, to help understand the pathogenesis of the disease, and to monitor disease activity and effects of treatment. Different groups have demonstrated a link between miRNA expression and disease activity, as in the case of renal flares in lupus patients. Moreover, miRNAs are emerging as potential targets for new therapeutic strategies of autoimmune disorders. Taken together, recent data demonstrate that miRNAs can influence mechanisms involved in the pathogenesis, relapse, and specific organ involvement of autoimmune diseases. The ultimate goal is the identification of a miRNA target or targets that could be manipulated through specific therapies, aiming at activation or inhibition of specific miRNAs responsible for the development of disease.


Subject(s)
MicroRNAs , Rheumatic Diseases/genetics , Humans , Rheumatic Diseases/immunology
12.
FEBS Lett ; 585(23): 3667-74, 2011 Dec 01.
Article in English | MEDLINE | ID: mdl-21600203

ABSTRACT

Rheumatoid arthritis (RA) is a chronic and severe autoimmune disease that affects joint tissues, bone, and cartilage. However, the pathogenesis of RA is still unclear. Autoantibodies such as rheumatoid factor and anti-cyclic citrullinated peptide are useful tools for early diagnosis, monitoring disease activity, and predicting prognosis. Recently, many groups have focused their attention on the role of microRNAs in the pathogenesis of RA, as well as a potential biomarker to monitor RA. In fact, the expression of some microRNAs, such as miR-146a, is upregulated in different cell types and tissues in RA patients. MicroRNAs in RA could also be considered as possible future targets for new therapeutic approaches.


Subject(s)
Arthritis, Rheumatoid/genetics , MicroRNAs/metabolism , Animals , Arthritis, Rheumatoid/immunology , Humans , Immune System/metabolism , MicroRNAs/genetics , Models, Immunological
13.
Infect Immun ; 79(4): 1597-605, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21263019

ABSTRACT

Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia are periodontal pathogens associated with the etiology of adult periodontitis as polymicrobial infections. Recent studies demonstrated that oral infection with P. gingivalis induces both periodontal disease and atherosclerosis in hyperlipidemic and proatherogenic ApoE(-/-) mice. In this study, we explored the expression of microRNAs (miRNAs) in maxillas (periodontium) and spleens isolated from ApoE(-/-) mice infected with P. gingivalis, T. denticola, and T. forsythia as a polymicrobial infection. miRNA expression levels, including miRNA miR-146a, and associated mRNA expression levels of the inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1ß (IL-1ß) were measured in the maxillas and spleens from mice infected with periodontal pathogens and compared to those in the maxillas and spleens from sham-infected controls. Furthermore, in response to these periodontal pathogens (as mono- and polymicrobial heat-killed and live bacteria), human THP-1 monocytes demonstrated similar miRNA expression patterns, including that of miR-146a, in vitro. Strikingly, miR-146a had a negative correlation with TNF-α secretion in vitro, reducing levels of the adaptor kinases IL-1 receptor-associated kinase 1 (IRAK-1) and TNF receptor-associated factor 6 (TRAF6). Thus, our studies revealed a persistent association of miR-146a expression with these periodontal pathogens, suggesting that miR-146a may directly or indirectly modulate or alter the chronic periodontal pathology induced by these microorganisms.


Subject(s)
Gram-Negative Bacterial Infections/genetics , Gram-Negative Bacterial Infections/microbiology , MicroRNAs/biosynthesis , Periodontitis/genetics , Periodontitis/microbiology , Animals , Apolipoproteins E/deficiency , Apolipoproteins E/immunology , Bacteroides/immunology , Blotting, Western , Disease Models, Animal , Gram-Negative Bacterial Infections/immunology , Humans , Interleukin-1beta/biosynthesis , Interleukin-1beta/immunology , Male , Mice , Mice, Knockout , MicroRNAs/genetics , MicroRNAs/immunology , Monocytes/immunology , Monocytes/metabolism , Periodontitis/immunology , Porphyromonas gingivalis/immunology , Reverse Transcriptase Polymerase Chain Reaction , Spleen/immunology , Spleen/metabolism , Treponema denticola/immunology , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/immunology
14.
J Immunol ; 186(3): 1723-34, 2011 Feb 01.
Article in English | MEDLINE | ID: mdl-21178010

ABSTRACT

Human TLRs are critical sensors for microbial components leading to the production of proinflammatory cytokines that are controlled by various mechanisms. Monocytes pretreated with LPS exhibit a state of hyporesponsiveness, referred to as cross-tolerance, to both homologous and heterologous ligands, which play a broader role in innate immunity. To date, LPS-induced cross-tolerance has not been examined regarding microRNA expression kinetics. In this study, THP-1 monocytes treated with various inflammatory ligands showed a continuous amplification of microRNA (miR)-146a over 24 h that is inversely correlated to TNF-α production. In contrast, inhibition of miR-146a showed a reciprocal effect. Thus, the characteristic upregulation of miR-146a in LPS-exposed THP-1 monocytes was studied for cross-tolerance. Strikingly, in LPS-tolerized THP-1 monocytes, only miR-146a showed a continuous overexpression, suggesting its crucial role in cross-tolerance. Similarly, peptidoglycan-primed THP-1 cells showed homologous tolerance associated with miR-146a upregulation. Subsequently, interchangeable differential cross-regulation was observed among non-LPS ligands. TLR2 and TLR5 ligands showed both homologous and heterologous tolerance correlated to miR-146a overexpression. More importantly, inflammatory responses to TLR4, TLR2, and TLR5 ligands were reduced due to knockdown of miR-146a targets IL-1R-associated kinase 1 or TNFR-associated factor 6, suggesting the regulatory effect of miR-146a on these TLRs signaling. Transfection of miR-146a into THP-1 cells caused reduction of TNF-α production, mimicking LPS-induced cross-tolerance. Aside from individual ligands, a whole bacterial challenge in LPS-primed THP-1 monocytes was accompanied by less TNF-α production, which is conversely correlated to miR-146a expression. Our studies have thus demonstrated that miR-146a plays a crucial role for in vitro monocytic cell-based endotoxin-induced cross-tolerance.


Subject(s)
Immune Tolerance , Lipopolysaccharides/toxicity , MicroRNAs/physiology , Signal Transduction/immunology , Toll-Like Receptors/metabolism , Animals , Bacteroidaceae Infections/immunology , Bacteroidaceae Infections/metabolism , Cell Line, Tumor , Cytokines/antagonists & inhibitors , Cytokines/biosynthesis , Escherichia coli Infections/immunology , Escherichia coli Infections/metabolism , Female , Humans , Immunity, Innate , Inflammation Mediators/antagonists & inhibitors , Inflammation Mediators/metabolism , Ligands , Mice , Mice, Inbred C57BL , MicroRNAs/antagonists & inhibitors , MicroRNAs/biosynthesis , Monocytes/immunology , Monocytes/metabolism , Staphylococcal Infections/immunology , Staphylococcal Infections/metabolism , Toll-Like Receptors/physiology
15.
J Biol Chem ; 284(50): 34590-9, 2009 Dec 11.
Article in English | MEDLINE | ID: mdl-19840932

ABSTRACT

The human toll-like receptor 4 (TLR4) pathway is activated in response to lipopolysaccharide (LPS), and subsequent signal transductions lead to the production of cytokines such as tumor necrosis factor-alpha (TNF-alpha) by innate immune cells. Defects in innate immune response may contribute to the overproduction of TNF-alpha leading to systemic inflammation and diseases. Thus, the innate immune response needs to be tightly regulated by elaborate mechanisms to control its onset and termination. LPS tolerance is a state of hyporesponsiveness to subsequent LPS challenge and is achieved by monocytic cells after prolonged exposure to LPS. In this report, kinetics of endotoxin-responsive microRNAs expression analysis revealed a unique pattern of gradual increase for miR-146a starting 4 h after LPS stimulation in THP-1 cells and continued up to 35-fold over 24 h. Conversely, TNF-alpha increased up to 4 h and then decreased gradually implicating a negative correlation with miR-146a progression. The characteristic up-regulation of miR-146a toward subsequent LPS challenge in THP-1 cells was studied. Strikingly, microRNA expression analysis during the tolerized state of THP-1 cells showed only miR-146a overexpression suggesting its important role in LPS tolerance. In addition, LPS tolerance was dependent on a LPS-priming dose and associated miR-146a up-regulation. LPS-tolerized cells were observed to regain responsiveness in TNF-alpha production 22 h after LPS removal correlating with a decrease in miR-146a level. Transfection of miR-146a into THP-1 cells mimicked LPS priming, whereas transfection of miR-146a inhibitor largely abolished LPS tolerance. Thus our studies demonstrated that miR-146a is critical for the in vitro monocytic cell-based endotoxin tolerance.


Subject(s)
Endotoxins , Immune Tolerance , Immunity, Innate , MicroRNAs/immunology , Monocytes/drug effects , Monocytes/immunology , Animals , Cell Line , Endotoxins/immunology , Endotoxins/pharmacology , Gene Knockdown Techniques , Humans , Immune Tolerance/drug effects , Immune Tolerance/immunology , Interleukin-1 Receptor-Associated Kinases/genetics , Interleukin-1 Receptor-Associated Kinases/metabolism , Lipopolysaccharides/immunology , Lipopolysaccharides/pharmacology , MicroRNAs/genetics , Monocytes/cytology , Signal Transduction/immunology , TNF Receptor-Associated Factor 6/genetics , TNF Receptor-Associated Factor 6/metabolism , Toll-Like Receptor 4/immunology , Tumor Necrosis Factor-alpha/immunology
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