Effect of sp3/sp2 carbon ratio and hydrodynamic size on the biodistribution kinetics of nanodiamonds in mice via intravenous injection.

BACKGROUND: Nanodiamonds (NDs) have gained a rapidly growing interest in biomedical applications; however, little is known regarding their biokinetics owing to difficulties in measurements and limited synthesis/purification technologies. In this study, we investigated the distribution kinetics of detonation-synthesized NDs in mice via intravenous injection to evaluate the parameters that determine the behavior of the particles. We prepared two distinctive NDs that controlled the sp3/sp2 carbon ratio and particle size by coating them with serum proteins. The four control samples were intravenously injected into mice, and tissue distribution and clearance were evaluated at 30 min and 1, 7, and 28 days post-injection. RESULTS: The sp3/sp2 carbon ratio showed no correlation with the organ distribution of the NDs. However, hydrodynamic size showed an excellent correlation with organ distribution levels: a negative correlation in the liver and positive correlations in the spleen and lungs. Furthermore, the deposition levels of NDs in the lung suggest that particles smaller than 300 nm could avoid lung deposition. Finally, a similar organ distribution pattern was observed in mice injected with carbon black nanoparticles controlled hydrodynamic size. CONCLUSIONS: In conclusion, the tissue distribution of NDs is modulated not by the sp3/sp2 carbon ratio but by the hydrodynamic size, which can provide helpful information for targeting the tissue of NDs. Furthermore, the organ distribution pattern of the NDs may not be specific to NDs but also can apply to other nanoparticles, such as carbon black.

Understanding Mental Health Issues in Different Subdomains of Social Networking Services: Computational Analysis of Text-Based Reddit Posts.

BACKGROUND: Users increasingly use social networking services (SNSs) to share their feelings and emotions. For those with mental disorders, SNSs can also be used to seek advice on mental health issues. One available SNS is Reddit, in which users can freely discuss such matters on relevant health diagnostic subreddits. OBJECTIVE: In this study, we analyzed the distinctive linguistic characteristics in users' posts on specific mental disorder subreddits (depression, anxiety, bipolar disorder, borderline personality disorder, schizophrenia, autism, and mental health) and further validated their distinctiveness externally by comparing them with posts of subreddits not related to mental illness. We also confirmed that these differences in linguistic formulations can be learned through a machine learning process. METHODS: Reddit posts uploaded by users were collected for our research. We used various statistical analysis methods in Linguistic Inquiry and Word Count (LIWC) software, including 1-way ANOVA and subsequent post hoc tests, to see sentiment differences in various lexical features within mental health-related subreddits and against unrelated ones. We also applied 3 supervised and unsupervised clustering methods for both cases after extracting textual features from posts on each subreddit using bidirectional encoder representations from transformers (BERT) to ensure that our data set is suitable for further machine learning or deep learning tasks. RESULTS: We collected 3,133,509 posts of 919,722 Reddit users. The results using the data indicated that there are notable linguistic differences among the subreddits, consistent with the findings of prior research. The findings from LIWC analyses revealed that patients with each mental health issue show significantly different lexical and semantic patterns, such as word count or emotion, throughout their online social networking activities, with P<.001 for all cases. Furthermore, distinctive features of each subreddit group were successfully identified through supervised and unsupervised clustering methods, using the BERT embeddings extracted from textual posts. This distinctiveness was reflected in the Davies-Bouldin scores ranging from 0.222 to 0.397 and the silhouette scores ranging from 0.639 to 0.803 in the former case, with scores of 1.638 and 0.729, respectively, in the latter case. CONCLUSIONS: By taking a multifaceted approach, analyzing textual posts related to mental health issues using statistical, natural language processing, and machine learning techniques, our approach provides insights into aspects of recent lexical usage and information about the linguistic characteristics of patients with specific mental health issues, which can inform clinicians about patients' mental health in diagnostic terms to aid online intervention. Our findings can further promote research areas involving linguistic analysis and machine learning approaches for patients with mental health issues by identifying and detecting mentally vulnerable groups of people online.

Spatiotemporal Clusters and Trend of Trichomonas vaginalis Infection in Korea.

This study was done to provide an overview of the latest trichomoniasis status in Korea by finding disease clusters and analyzing temporal trends during 2012-2020. Data were obtained from the Health Insurance Review & Assessment Service (HIRA) of Korea. SaTScan and Joinpoint programs were used for statistical analyses. Gyeonggi-do had the highest average population and highest number of cases. The high incidence of T. vaginalis infections were observed among women aged 40-49 and 30-39 years (33,830/year and 33,179/year, respectively). Similarly, the 40-49 and 30-39 age group in men showed the highest average cases (1,319/year and 1,282/year, respectively). Jeollabuk-do was the most likely cluster, followed by Busan/Gyeongsangnam-do/Ulsan/Daegu and Jeju-do and Gwangju. Urban and rural differences were prominent. Trichomoniasis has decreased significantly in most clusters, except for Incheon. Trichomoniasis was decreasing in women recently after peaking around 2014. Men showed different trends according to age. Trichomoniasis was increasing in the 10-39 age groups, but decreasing in the 40-59 age groups. This study might provide an analytic basis for future health measures, policy-makers, and health authorities in developing effective system for prevention of trichomoniasis.

Relationship between Clonorchis sinensis Infection and Cholangiocarcinoma in Korea.

This study provides an overview of the current status of clonorchiasis and cholangiocarcinoma (CCA), and their relationship in Korea during 2012-2020. Data were obtained from the Health Insurance Review & Assessment Service of Korea. Cluster, trend, and correlation analyses were performed. Gyeongsangnam-do and Seoul had the highest average number of cases (1,026 and 4,208) and adjusted rate (306 and 424) for clonorchiasis and CCA, respectively. The most likely clusters (MLC) for clonorchiasis and CCA were Busan/Gyeongsangnam-do/Ulsan/Daegu/Gyeongsangbuk-do (Relative Risk; RR = 4.55, Likelihood Ratio; LLR = 9,131.115) joint cluster and Seoul (RR = 2.29, LLR = 7,602.472), respectively. The MLC for clonorchiasis was in the southeastern part of Korea, while that for CCA was in the southern part. Clonorchiasis showed a decreasing trend in the southeastern districts, while increased in the southwestern districts. Cities in the central region had a decreasing trend, while the western districts had an increasing trend. In most adults (30-59), infection rate of clonorchiasis showed a significant decrease until 2018, while thereafter increased, although not significant. CCA showed a sharply decreasing tendency. The incidence of clonorchiasis and CCA were positively correlated. In general, the correlation was weak (r = 0.39, P < 0.001), but it was strongly positive around the 4 river basins (r = 0.74, P < 0.001). This study might provide an analytic basis for developing an effective system against clonorchiasis and CCA.

TLR2 and the NLRP3 inflammasome mediate IL-1ß production in Prevotella nigrescens-infected dendritic cells.

Prevotella nigrescens is an oral pathogen that is frequently observed in the subgingival plaque of periodontitis patients. Interleukin-1ß (IL-1ß) is known to be involved in the immunopathology of periodontal diseases and has been implicated in the destruction of bone. In this study, we investigated the mechanism of IL-1ß production by P. nigrescens in murine bone marrow-derived dendritic cells (BMDCs). Our results showed that a host receptor, Toll-like receptor 2 (TLR2), but not TLR4 is required for pro-IL-1ß induction and nucleotide-binding oligomerization domain like receptor pyrin domain containing 3 (NLRP3) priming in BMDCs in response to P. nigrescens and activation of the NLRP3 inflammasome is necessary for processing of pro-IL-1ß into mature IL-1ß. In addition, an inhibitor assay revealed that production of reactive oxygen species, P2X7R activity, and release of cathepsin B are involved in IL-1ß production in BMDCs in response to P. nigrescens.

Clusters of Toxoplasmosis in Ganghwa-gun, Cheorwon-gun, and Goseong-gun, Korea.

We find out the clusters with high toxoplasmosis risk to discuss the geographical pattern in Gyodong-myeon and Samsan-myeon of Ganghwa-gun, Cheorwon-gun, and Goseong-gun, Korea. Seroepidemiological data of toxoplasmosis surveyed using rapid diagnostic tests for the residents in the areas in 2019 were analyzed to detect clusters of the infection. The cluster was investigated using the SaTScan program which is based on Kulldorff's scan statistic. The clusters were found with P-values in each region analyzed in the program, and the risk and patient incidence of specific areas can be examined by the values such as relative risk and log likelihood ratio. Jiseok-ri and Insa-ri were found to be a cluster in Gyodong-myeon and Seokmo-ri was the cluster in Samsan-myeon. Yangji-ri and Igil-ri were found to be a cluster in Cheorwon-gun and Madal-ri and Baebong-ri were the cluster in Goseong-gun. This findings can be used to monitor and prevent toxoplasmosis infections occurring in vulnerable areas.

Recent Spatial and Temporal Trends of Malaria in Korea.

This study was done to provide an analytical overview on the latest malaria infection clusters by evaluating temporal trends during 2010-2019 in Korea. Incheon was the most likely cluster (MLC) for all cases of malaria during the total period. MLCs for P. falciparum, vivax, malariae, ovale, and clinically diagnosed malaria without parasitological confirmation were Jeollanam-do, Incheon, Gangwon-do, Gyeongsangnam-do, and Jeollabuk-do, respectively. Malaria was decreasing in most significant clusters, but Gwangju showed an increase for all cases of malaria, P. vivax and clinically diagnosed cases. Malaria overall, P. falciparum and P. vivax seem to be under control thanks to aggressive health measures. This study might provide a sound scientific basis for future control measures against malaria in Korea.

Seroprevalence of Toxoplasma gondii assayed using Rapid Diagnostic Tests among Residents in Three Counties Adjacent to The Demilitarized Zone, Korea.

Toxoplasma gondii seroprevalence have been rapidly increasing in some parts of Korea. We analyzed prevalence of anti-Toxoplasma gondii antibodies, using a rapid diagnostic test (RDT), in the sera of 552 residents in Ganghwa-gun, 661 ones in Cheorwon-gun, and 305 ones in Goseong-gun, Korea in 2019. IgG/IgM RDT mounted with recombinant fragment of major surface antigen (SAG1), glutathione-S-transferase-linker-SAG1A, were applied to the sera. IgG seroprevalence was 28.1% in Ganghwa-gun, 19.5% in Cheorwon-gun and 35.7% in Goseong-gun. Odds ratios comparing Cheorwon vs Ganghwa was 0.63 (P=0.001) and Goesong versus Ganghwa was 1.47 (P=0.01) adjusting age and sex. Goseong had highest seroprevalence among the 3 counties both in crude rates and logistic regression. Although Cheorwon and Goseong are adjacent to the demilitarized zone (DMZ) in Korea, seroprevalence rate was much higher in Goseong. Further investigation on other DMZ-closed areas is necessary whether they have high prevalence rates compared to the other areas. T. gondii prevalence in Korea is still persists; proper health policy should be established.

Transcriptional profiling of single fiber cells in a transgenic paradigm of an inherited childhood cataract reveals absence of molecular heterogeneity.

Our recent single-cell transcriptomic analysis has demonstrated that heterogeneous transcriptional activity attends molecular transition from the nascent to terminally differentiated fiber cells in the developing mouse lens. To understand the role of transcriptional heterogeneity in terminal differentiation and the functional phenotype (transparency) of this tissue, here we present a single-cell analysis of the developing lens, in a transgenic paradigm of an inherited pathology, known as the lamellar cataract. Cataracts hinder transmission of light into the eye. Lamellar cataract is the most prevalent bilateral childhood cataract. In this disease of early infancy, initially, the opacities remain confined to a few fiber cells, thus presenting an opportunity to investigate early molecular events that lead to cataractogenesis. We used a previously established paradigm that faithfully recapitulates this disease in transgenic mice. About 500 single fiber cells, manually isolated from a 2-day-old transgenic lens were interrogated individually for the expression of all known 17 crystallins and 78 other relevant genes using a Biomark HD (Fluidigm). We find that fiber cells from spatially and developmentally discrete regions of the transgenic (cataract) lens show remarkable absence of the heterogeneity of gene expression. Importantly, the molecular variability of cortical fiber cells, the hallmark of the WT lens, is absent in the transgenic cataract, suggesting absence of specific cell-type(s). Interestingly, we find a repetitive pattern of gene activity in progressive states of differentiation in the transgenic lens. This molecular dysfunction portends pathology much before the physical manifestations of the disease.

Cluster of Parasite Infections by the Spatial Scan Analysis in Korea.

This study was performed to find out the clusters with high parasite infection risk to discuss the geographical pattern. Clusters were detected using SatScan software, which is a statistical spatial scan program using Kulldorff's scan statistic. Information on the parasitic infection cases in Korea 2011-2019 were collected from the Korea Centers for Disease Control and Prevention. Clusters of Ascaris lumbricoides infection were detected in Jeollabuk-do, and T. trichiura in Ulsan, Busan, and Gyeongsangnam-do. C. sinensis clusters were detected in Ulsan, Daegu, Busan, Gyeongsangnam-do, and Gyeongsangbuk-do. Clusters of intestinal trematodes were detected in Ulsan, Busan, and Gyeongsangnam-do. P. westermani cluster was found in Jeollabuk-do. E. vermicularis clusters were distributed in Gangwon-do, Jeju-do, Daegu, Daejeon, and Gwangju. This clustering information can be referred for surveillance and control on the parasitic infection outbreak in the infection-prone areas.

Clusters of Toxoplasmosis in Gyodong-Myeon and Samsan-Myeon, Ganghwa-Gun, Korea.

The purpose of this study is to find out the clusters with high toxoplasmosis risk to discuss the geographical pattern in 2 islands of Gyodong-myeon and Samsan-myeon in Ganghwa-gun, Korea. Seroepidemiological data of toxoplasmosis surveyed using rapid diagnostic tests for the residents in 2 islands from 2010 to 2013 were analyzed to detect clusters of the infection. The cluster was investigated using the SatScan program which is based on Kulldorff's scan statistic. The clusters were found with P-values in each region analyzed in the program, and the risk and patient incidence of specific areas can be examined by the values such as relative risk and log likelyhood ratio. Jiseok-ri was found to be a cluster in Gyodong-myeon and Ha-ri was the cluster in Samsan-myeon. This findings can be used to monitor and prevent toxoplasmosis infections occurring in vulnerable areas.

Image Segmentation for FIB-SEM Serial Sectioning of a Si/C-Graphite Composite Anode Microstructure Based on Preprocessing and Global Thresholding.

The choice of materials that constitute electrodes and the way they are interconnected, i.e., the microstructure, influences the performance of lithium-ion batteries. For batteries with high energy and power densities, the microstructure of the electrodes must be controlled during their manufacturing process. Moreover, understanding the microstructure helps in designing a high-performance, yet low-cost battery. In this study, we propose a systematic algorithm workflow for the images of the microstructure of anodes obtained from a focused ion beam scanning electron microscope (FIB-SEM). Here, we discuss the typical issues that arise in the raw FIB-SEM images and the corresponding preprocessing methods that resolve them. Next, we propose a Fourier transform-based filter that effectively reduces curtain artifacts. Also, we propose a simple, yet an effective, global-thresholding method to identify active materials and pores in the microstructure. Finally, we reconstruct the three-dimensional structures by concatenating the segmented images. The whole algorithm workflow used in this study is not fully automated and requires user interactions such as choosing the values of parameters and removing shine-through artifacts manually. However, it should be emphasized that the proposed global-thresholding method is deterministic and stable, which results in high segmentation performance for all sectioning images.

NLRP3 inflammasome mediates interleukin-1ß production in immune cells in response to Acinetobacter baumannii and contributes to pulmonary inflammation in mice.

Acinetobacter baumannii is a multi-drug resistant, Gram-negative bacteria and infection with this organism is one of the major causes of mortality in intensive care units. Inflammasomes are multiprotein oligomers that include caspase-1, and their activation is required for maturation of interleukin-1ß (IL-1ß). Inflammasome signalling is involved in host defences against various microbial infections, but the precise mechanism by which A. baumannii activates inflammasomes and the roles of relevant signals in host defence against pulmonary A. baumannii infection are unknown. Our results showed that NLRP3, ASC and caspase-1, but not NLRC4, are required for A. baumannii-induced production of IL-1ß in macrophages. An inhibitor assay revealed that various pathways, including P2X7R, K+ efflux, reactive oxygen species production and release of cathepsins, are involved in IL-1ß production in macrophages in response to A. baumannii. Interleukin-1ß production in bronchoalveolar lavage (BAL) fluid was impaired in NLRP3-deficient and caspase-1/11-deficient mice infected with A. baumannii, compared with that in wild-type (WT) mice. However, the bacterial loads in BAL fluid and lungs were comparable between WT and NLRP3-deficient or caspase-1/11-deficient mice. The severity of lung pathology was reduced in NLRP3- deficient, caspase-1/11- deficient and IL-1-receptor-deficient mice, although the recruitment of immune cells and production of inflammatory cytokines and chemokines were not altered in these mice. These findings indicate that A. baumannii leads to the activation of NLRP3 inflammasome, which mediates IL-1ß production and lung pathology.

Employment of gold-coated silver nanowires as transparent conductive electrode for organic light emitting diodes.

This study proposes a simple method of Au coating on silver nanowires (Ag NWs) transparent conductive films as the anode of organic light emitting diodes (OLED) to increase the work function of the film and thus enhance hole transport. We carefully engineer the process conditions (pretreatment, solution concentrations, and coating number) of the coating using a diluted HAuCl4 solution on the Ag NWs film to minimize etching damage on Ag NWs accompanying the galvanic replacement reaction. Ultraviolet photoelectron spectroscopy and Kelvin probe force microscopy show work function increase of Ag NWs upon Au coating. OLED devices based on Au-coated Ag NWs show a lower turn-on voltage and higher luminance, compared with pristine Ag NWs device. Although the Ag NWs device displays poor efficiencies in the low luminance range due to a high leakage, some of the Au-coated Ag NWs devices showed efficiencies higher than those of the ITO device in a high luminance.

Shiga Toxins Activate the NLRP3 Inflammasome Pathway To Promote Both Production of the Proinflammatory Cytokine Interleukin-1ß and Apoptotic Cell Death.

Shiga toxin (Stx)-mediated immune responses, including the production of the proinflammatory cytokines tumor necrosis-α (TNF-α) and interleukin-1ß (IL-1ß), may exacerbate vascular damage and accelerate lethality. However, the immune signaling pathway activated in response to Stx is not well understood. Here, we demonstrate that enzymatically active Stx, which leads to ribotoxic stress, triggers NLRP3 inflammasome-dependent caspase-1 activation and IL-1ß secretion in differentiated macrophage-like THP-1 (D-THP-1) cells. The treatment of cells with a chemical inhibitor of glycosphingolipid biosynthesis, which suppresses the expression of the Stx receptor globotriaosylceramide and subsequent endocytosis of the toxin, substantially blocked activation of the NLRP3 inflammasome and processing of caspase-1 and IL-1ß. Processing and release of both caspase-1 and IL-1ß were significantly reduced or abolished in Stx-intoxicated D-THP-1 cells in which the expression of NLRP3 or ASC was stably knocked down. Furthermore, Stx mediated the activation of caspases involved in apoptosis in an NLRP3- or ASC-dependent manner. In Stx-intoxicated cells, the NLRP3 inflammasome triggered the activation of caspase-8/3, leading to the initiation of apoptosis, in addition to caspase-1-dependent pyroptotic cell death. Taken together, these results suggest that Stxs trigger the NLRP3 inflammasome pathway to release proinflammatory IL-1ß as well as to promote apoptotic cell death.

MetaPathways v2.5: quantitative functional, taxonomic and usability improvements.

UNLABELLED: Next-generation sequencing is producing vast amounts of sequence information from natural and engineered ecosystems. Although this data deluge has an enormous potential to transform our lives, knowledge creation and translation need software applications that scale with increasing data processing and analysis requirements. Here, we present improvements to MetaPathways, an annotation and analysis pipeline for environmental sequence information that expedites this transformation. We specifically address pathway prediction hazards through integration of a weighted taxonomic distance and enable quantitative comparison of assembled annotations through a normalized read-mapping measure. Additionally, we improve LAST homology searches through BLAST-equivalent E-values and output formats that are natively compatible with prevailing software applications. Finally, an updated graphical user interface allows for keyword annotation query and projection onto user-defined functional gene hierarchies, including the Carbohydrate-Active Enzyme database. AVAILABILITY AND IMPLEMENTATION: MetaPathways v2.5 is available on GitHub: http://github.com/hallamlab/metapathways2. CONTACT: shallam@mail.ubc.ca SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Nucleotide-binding oligomerization domain 2 (NOD2) activation induces apoptosis of human oral squamous cell carcinoma cells.

OBJECTIVES: Microbial Pattern-recognition receptors (PRRs), such as nucleotide-binding oligomerization domains (NODs), are essential for mammalian innate immune response. This study was designed to determine the effect of NOD1 and NOD2 agonist on innate immune responses and antitumor activity in oral squamous cell carcinoma (OSCC) cells. MATERIALS AND METHODS: NODs expression was examined by RT-PCR, and IL-8 production by NODs agonist was examined by ELISA. Western blot analysis was performed to determine the MAPK activation in response to their agonist. Cell proliferation was determined by MTT assay. Flow cytometry and Western blot analysis were performed to determine the MDP-induced cell death. RESULTS: The levels of NODs were apparently expressed in OSCC cells. NODs agonist, Tri-DAP and MDP, led to the production of IL-8 and MAPK activation. NOD2 agonist, MDP, inhibited the proliferation of YD-10B cells in a dose-dependent manner. Also, the ratio of Annexin V-positive cells and cleaved PARP was increased by MDP treatment in YD-10B cells, suggesting that MDP-induced cell death in YD-10B cells may be owing to apoptosis. CONCLUSIONS: Our results indicate that NODs are functionally expressed in OSCC cells and can trigger innate immune responses. In addition, NOD2 agonist inhibited cell proliferation and induced apoptosis. These findings provide the potential value of MDP as novel candidates for antitumor agents of OSCC.

Establishment of a mouse model for pulmonary inflammation and fibrosis by intratracheal instillation of polyhexamethyleneguanidine phosphate.

Although several animal models have been developed to study human pulmonary fibrosis, lack of a perfect model has raised the need for various animal models of pulmonary fibrosis. In this study, we evaluated the pulmonary effect of polyhexamethyleneguanidine phosphate instillation into the lungs of mice to determine the potential of these mice as a murine model of pulmonary fibrosis. Intratracheal instillation of polyhexamethyleneguanidine phosphate induced severe lung inflammation manifested by the infiltration of mononuclear cells and neutrophils and increased production of IL-6, TNF-α, CCL2 and CXCL1. The lung inflammation gradually increased until 28 days after polyhexamethyleneguanidine phosphate exposure, and increases of collagen deposition and TGF-ß production, which are indicators of pulmonary fibrosis, were seen. Our study showed that intratracheal instillation of polyhexamethyleneguanidine phosphate induces pulmonary inflammation and fibrosis in mice.

Critical role of TRIF and MyD88 in Mycobacterium tuberculosis Hsp70-mediated activation of dendritic cells.

As a potent immune regulator, heat shock protein 70 derived from Mycobacterium tuberculosis (Mtb Hsp70) has adjuvant effect and activates immune cells such as macrophages and dendritic cells (DCs). Although Toll-like receptors (TLRs) are known to involve in DCs activation by Mtb Hsp70, there is still a controversy and the underlying mechanism is not well understood. In this study, we examined whether TRIF and MyD88, the core adaptor molecules for TLRs signaling, regulate Mtb Hsp70-induced DCs activation. Although Mtb Hsp70 produced substantial level of cytokines (IL-6, IL-12p40, and TNF-α) in TRIF-deficient DCs in a dose-dependent manner, each level was significantly lower than that in WT cells. The cytokines production was almost abolished in MyD88-deficient DCs. Consistent with cytokine results, Mtb Hsp70-induced activation of NF-κB and MAPKs was also impaired in both TRIF- and MyD88-deficient DCs, as compared with WT cells. Inhibitor assay revealed that NF-κB, ERK, and JNK, but not p38, regulate Mtb Hsp70-induced production of cytokines. In addition, the up-regulation of co-stimulatory molecules and MHC class II was mostly TRIF-dependent in DCs in response Mtb Hsp70, whereas MyD88 was only partially involved. Finally, mixed leukocytes reaction (MLR) assay revealed that both TRIF and MyD88 are critical for DCs ability promoted by Mtb Hsp70 to differentiate naïve T cells into effector T cells of producing IFN-γ. Our findings suggest that both TRIF and MyD88 are essential for the activation and maturation of DCs in response to Mtb Hsp70.

Diverse Toll-like receptors mediate cytokine production by Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans in macrophages.

Toll-like receptors (TLRs) orchestrate a repertoire of immune responses in macrophages against various pathogens. Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans are two important periodontal pathogens. In the present study, we investigated TLR signaling regulating cytokine production of macrophages in response to F. nucleatum and A. actinomycetemcomitans. TLR2 and TLR4 are redundant in the production of cytokines (interleukin-6 [IL-6] and tumor necrosis factor alpha [TNF-α]) in F. nucleatum- and A. actinomycetemcomitans-infected macrophages. The production of cytokines by macrophages in response to F. nucleatum and A. actinomycetemcomitans infection was impaired in MyD88-deficient macrophages. Moreover, cytokine concentrations were lower in MyD88-deficient macrophages than in TLR2/TLR4 (TLR2/4) double-deficient cells. An endosomal TLR inhibitor, chloroquine, reduced cytokine production in TLR2/4-deficient macrophages in response to F. nucleatum and A. actinomycetemcomitans, and DNA from F. nucleatum or A. actinomycetemcomitans induced IL-6 production in bone marrow-derived macrophages (BMDMs), which was abolished by chloroquine. Western blot analysis revealed that TLR2/4 and MyD88 were required for optimal activation of NF-κB and mitogen-activated protein kinases (MAPKs) in macrophages in response to F. nucleatum and A. actinomycetemcomitans, with different kinetics. An inhibitor assay showed that NF-κB and all MAPKs (p38, extracellular signal-regulated kinase [ERK], and Jun N-terminal protein kinase [JNK]) mediate F. nucleatum-induced production of cytokines in macrophages, whereas NF-κB and p38, but not ERK and JNK, are involved in A. actinomycetemcomitans-mediated cytokine production. These findings suggest that multiple TLRs may participate in the cytokine production of macrophages against periodontal bacteria.