Natsuo Yamamoto

Dectin-2 Recognition of α-Mannans and Induction of Th17 Cell Differentiation Is Essential for Host Defense against Candida albicans

Dectin-2 (gene symbol Clec4n) is a C-type lectin expressed by dendritic cells (DCs) and macrophages. However, its functional roles and signaling mechanisms remain to be elucidated. Here, we generated Clec4n(-/-) mice and showed that this molecule is important for host defense against Candida albicans (C. albicans). Clec4n(-/-) DCs had virtually no fungal alpha-mannan-induced cytokine production. Dectin-2 signaling induced cytokines through an FcRgamma chain and Syk-CARD9-NF-kappaB-dependent signaling pathway without involvement of MAP kinases. The yeast form of C. albicans induced interleukin-1beta (IL-1beta) and IL-23 secretion in a Dectin-2-dependent manner. In contrast, cytokine production induced by the hyphal form was only partially dependent on this lectin. Both yeast and hyphae induced Th17 cell differentiation, in which Dectin-2, but not Dectin-1, was mainly involved. Because IL-17A-deficient mice were highly susceptible to systemic candida infection, this study suggests that Dectin-2 is important in host defense against C. albicans by inducing Th17 cell differentiation.

Critical role of Vα14+ natural killer T cells in the innate phase of host protection against Streptococcus pneumoniae infection

The present study was designed to elucidate the role of Vα14+ NKT cells in the host defense against pulmonary infection with Streptococcus pneumoniae using Jα281 gene‐disrupted mice (Jα281KO mice) that lacked this lymphocyte subset. In these mice, pneumococcal infection was severely exacerbated, as shown by the shorter survival time and marked increase of live bacteria in the lung compared to wild‐type (WT) mice. The proportion of Vα14+ NKT cells, detected by an α‐galactosylceramide (α‐GalCer)‐loaded CD1d tetramer, increased in thelung after S. pneumoniae infection. This increase was significantly reduced in mice with a genetic disruption of monocyte chemotactic protein (MCP)‐1, which was produced in the early phaseof infection in WT mice. In the lungs of Jα281KO mice, the number of neutrophils was significantly lower at 12 h than that in WT mice. In support of this finding, macrophage inflammatory protein (MIP)‐2 and TNF‐α synthesis in infected lungs was significantly reduced at 3 h and at both 3 and 6 h, respectively, in Jα281KO mice, compared to WT mice. In addition, treatment of mice with α‐GalCer significantly improved the outcome of this infection. Our results demonstrated MCP‐1‐dependent recruitment of Vα14+ NKT cells and their critical role in early host protection against S. pneumoniae by promoting the trafficking of neutrophils to the site of infection.

Toll-Like Receptor 9-Dependent Activation of Myeloid Dendritic Cells by Deoxynucleic Acids from Candida albicans

ABSTRACT The innate immune system of humans recognizes the human pathogenic fungus Candida albicans via sugar polymers present in the cell wall, such as mannan and β-glucan. Here, we examined whether nucleic acids from C. albicans activate dendritic cells. C. albicans DNA induced interleukin-12p40 (IL-12p40) production and CD40 expression by murine bone marrow-derived myeloid dendritic cells (BM-DCs) in a dose-dependent manner. BM-DCs that lacked Toll-like receptor 4 (TLR4), TLR2, and dectin-1, which are pattern recognition receptors for fungal cell wall components, produced IL-12p40 at levels comparable to the levels produced by BM-DCs from wild-type mice, and DNA from a C. albicans pmr1Δ null mutant, which has a gross defect in mannosylation, retained the ability to activate BM-DCs. This stimulatory effect disappeared completely after DNase treatment. In contrast, RNase treatment increased production of the cytokine. A similar reduction in cytokine production was observed when BM-DCs from TLR9−/− and MyD88−/− mice were used. In a luciferase reporter assay, NF-κB activation was detected in TLR9-expressing HEK293T cells stimulated with C. albicans DNA. Confocal microscopic analysis showed similar localization of C. albicans DNA and CpG-oligodeoxynucleotide (CpG-ODN) in BM-DCs. Treatment of C. albicans DNA with methylase did not affect its ability to induce IL-12p40 synthesis, whereas the same treatment completely eliminated the ability of CpG-ODN to induce IL-12p40 synthesis. Finally, impaired clearance of this fungal pathogen was not found in the kidneys of TLR9−/− mice. These results suggested that C. albicans DNA activated BM-DCs through a TLR9-mediated signaling pathway using a mechanism independent of the unmethylated CpG motif.

Efflux pump inhibitors reduce the invasiveness of Pseudomonas aeruginosa.

Efflux systems are thought to contribute to antimicrobial resistance in Pseudomonas aeruginosa. The mexAB-oprM deletion strain of P. aeruginosa PAO1 is compromised in its capacity to invade Madin-Darby canine kidney (MDCK) cells, suggesting that P. aeruginosa exports invasion determinants using a MexAB-OprM system. The influences of efflux pump inhibitors (EPIs), including the broad-spectrum EPI Phe-Arg-beta-naphthylamide (PAbetaN) and MexAB-OprM-specific EPI D13-9001, on the invasion of wild-type (WT) P. aeruginosa PAO1 and its MexAB-OprM-overproducing nalB strain were examined. The invasiveness of PAO1 WT and nalB strains was inhibited in the presence of EPIs in a concentration-dependent manner. Reduction of the invasiveness of both strains was greater for D13-9001 compared with PAbetaN. EPIs are thought to be useful in reducing the invasiveness and antimicrobial resistance of P. aeruginosa and thus may be promising as new anti-infectious agents.

Defect of CARD9 Leads to Impaired Accumulation of Gamma Interferon-Producing Memory Phenotype T Cells in Lungs and Increased Susceptibility to Pulmonary Infection with Cryptococcus neoformans

ABSTRACT Caspase recruitment domain-containing protein 9 (CARD9) is an adaptor molecule signal that is critical for NF-κB activation and is triggered through C-type lectin receptors (CLRs), which are pattern recognition receptors that recognize carbohydrate structures. Previous studies have reported that Cryptococcus neoformans, a fungal pathogen that causes meningoencephalitis in AIDS patients, is recognized through some CLRs, such as mannose receptors or DC-SIGN. However, the role of CARD9 in the host defense against cryptococcal infection remains to be elucidated. In the present study, we analyzed the role of CARD9 in the host defense against pulmonary infection with C. neoformans. CARD9 gene-disrupted (knockout [KO]) mice were highly susceptible to this infection, as shown by the reduced fungal clearance in the infected lungs of CARD9 KO mice, compared to that in wild-type (WT) mice. Gamma interferon (IFN-γ) production was strongly reduced in CARD9 KO mice during the innate-immunity phase of infection. Reduced IFN-γ synthesis was due to impaired accumulation of NK and memory phenotype T cells, which are major sources of IFN-γ innate-immunity-phase production; a reduction in the accumulation of these cells was correlated with reduced CCL4, CCL5, CXCL9, and CXCL10 synthesis. However, differentiation of Th17 cells, but not of Th1 cells, was impaired at the adaptive-immunity phase in CARD9 KO mice compared to WT mice, although there was no significant difference in the infection susceptibility between interleukin 17A (IL-17A) KO and WT mice. These results suggest that CARD9 KO mice are susceptible to C. neoformans infection probably due to the reduced accumulation of IFN-γ-expressing NK and memory phenotype T cells at the early stage of infection.

Toll-like receptor 4-dependent activation of myeloid dendritic cells by leukocidin of Staphylococcus aureus

Leukocidin (Luk), an exotoxin of Staphylococcus aureus consisting of LukF and LukS, is a hetero-oligomeric pore-forming cytolytic toxin toward human and rabbit polymorphonuclear leukocytes. However, it is uncertain how Luk affects the host immune response. In the present study, we investigated whether Luk has the ability to stimulate mouse bone marrow-derived myeloid dendritic cells (BM-DCs). LukF activated BM-DCs to generate IL-12p40 mRNA, induce intracellular expression and extracellular secretion of this cytokine and express CD40 on their surface, whereas LukS showed a much lower or marginal ability in the activation of BM-DCs than its counterpart component. Similarly, TNF-alpha was secreted by BM-DCs upon stimulation with these components. Combined addition of these components did not lead to a further increase in IL-12p40 secretion. IL-12p40 production caused by LukF was completely abrogated in BM-DCs from TLR4-deficient mice similarly to the response to lipopolysaccharide (LPS). Polymixin B did not affect the LukF-induced IL-12p40 production, although the same treatment completely inhibited the LPS-induced response. Boiling significantly inhibited the response caused by LukF, but not by LPS. Finally, in a luciferase reporter assay, LukF induced the activation of NF-kappaB in HEK293T cells transfected with TLR4, MD2 and CD14, whereas LukS did not show such activity. These results demonstrate that LukF caused the activation of BM-DCs by triggering a TLR4-dependent signaling pathway and suggests that Luk may affect the host inflammatory response as well as show a cytolytic effect on leukocytes.

Antimicrobial Activities of Piperacillin-Tazobactam against Haemophilus influenzae Isolates, Including β-Lactamase-Negative Ampicillin-Resistant and β-Lactamase-Positive Amoxicillin- Clavulanate-Resistant Isolates, and Mutations in Their Quinolone Resistance-Determining Regions

ABSTRACT β-Lactamase-negative ampicillin-resistant (BLNAR) isolates of Haemophilus influenzae have been emerging in some countries, including Japan. The Clinical and Laboratory Standards Institute has only a susceptible MIC breakpoint (≤1 μg/ml) for piperacillin-tazobactam and a disclaimer comment that BLNAR H. influenzae should be considered resistant, which was adapted without presentation of data. In addition, fluoroquinolone-resistant H. influenzae isolates have recently been occasionally reported worldwide. To address these problems, we examined susceptibilities to β-lactams, including piperacillin-tazobactam, and ciprofloxacin by microdilution and disk diffusion (only for piperacillin-tazobactam) methods, against a total of 400 recent H. influenzae clinical isolates, including 100 β-lactamase-negative ampicillin-susceptible, β-lactamase-positive ampicillin-resistant, BLNAR, and β-lactamase-positive amoxicillin-clavulanate-resistant (BLPACR) isolates each. BLNAR and BLPACR isolates were tested by PCR using primers that amplify specific regions of the ftsI gene. We also detected mutations in quinolone resistance-determining regions (QRDRs) by direct sequencing of the PCR products of DNA fragments. Among β-lactams, piperacillin-tazobactam exhibited potent activity against all isolates of H. influenzae, with all MICs at ≤0.5 μg/ml (susceptible). A disk diffusion breakpoint for piperacillin-tazobactam of ≥21 mm is proposed. We confirmed that all BLNAR and BLPACR isolates had amino acid substitutions in the ftsI gene and that the major pattern was group III-like (87.5%). One ciprofloxacin-resistant isolate (MIC, 16 μg/ml) and 31 ciprofloxacin-susceptible isolates (MICs, 0.06 to 0.5 μg/ml) had amino acid changes in their QRDRs. Piperacillin-tazobactam was the most potent β-lactam tested against all classes of H. influenzae isolates. It is possible that fluoroquinolone-resistant H. influenzae will emerge since several clinical isolates carried mutations in their QRDRs.

Outbreak of extended-spectrum β-lactamase-producing Escherichia coli transmitted through breast milk sharing in a neonatal intensive care unit

BACKGROUND Routine surveillance in a neonatal intensive care unit (NICU) showed an increased detection of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli (ESBL-E. coli) in August 2012, following nearly a year without detection. AIM To describe the investigation and interventions by a hospital infection control team of an outbreak of ESBL-E. coli in a NICU. METHODS Six neonates with positive cultures of ESBL-E. coli (five with respiratory colonization, one with a urinary tract infection), control infants who were negative for ESBL-E. coli during the study period, and mothers who donated their breast milk were included. A case-control study was performed to identify possible risk factors for positive ESBL-E. coli cultures and molecular typing of isolated strains by pulsed-field gel electrophoresis. FINDINGS The odds ratio for ESBL-E. coli infection after receiving shared unpasteurized breast milk during the study period was 49.17 (95% confidence interval: 6.02-354.68; P < 0.05). The pulsed-field gel electrophoresis pattern showed that all strains were identical, and the same pathogen was detected in freshly expressed milk of a particular donor. After ceasing the breast milk sharing, the outbreak was successfully terminated. CONCLUSION This outbreak indicates that contamination of milk packs can result in transmission of a drug-resistant pathogen to newborn infants. Providers of human breast milk need to be aware of the necessity for low-temperature pasteurization and bacterial cultures, which should be conducted before and after freezing, before prescribing to infants.

Emergence of fluoroquinolone-resistant Streptococcus pyogenes in Japan by a point mutation leading to a new amino acid substitution

OBJECTIVES Streptococcus pyogenes causes various diseases in humans. While the prevalence of fluoroquinolone-resistant S. pyogenes isolates has been increasing since 2000 in the USA and Europe, it has remained very low in Japan. We isolated a fluoroquinolone-resistant S. pyogenes strain and analysed its genetics. METHODS TU-296, a strain of S. pyogenes resistant to levofloxacin (MIC 16 mg/L), was isolated from the throat of a patient in their thirties with pharyngitis in autumn 2007. We carried out susceptibility tests for various antimicrobial agents and PCR analysis of the genes gyrA, gyrB, parC and parE in the quinolone resistance-determining region, followed by sequencing of the PCR products to find mutation(s) and the resulting amino acid substitution(s). We then sequenced the PCR product of the emm gene and determined the emm genotype. RESULTS S. pyogenes TU-296 was found to have the following mutations and amino acid substitutions: adenine 476 to cytosine in gyrA and cytosine 367 to thymine in parC, resulting in Glu-85→Ala in GyrA and Ser-79→Phe in ParC. The genotype of the isolate was emm11. CONCLUSIONS Amino acid substitutions in fluoroquinolone-resistant S. pyogenes have already been reported from Europe and the USA, including Ser-81→Phe or Tyr and Met-99→Leu in GyrA, as well as Ser-79→Phe, Tyr or Ala and others in ParC. Numerous point mutations were found in parC and parE of S. pyogenes TU-296. In addition, a new amino acid substitution was detected (Glu-85→Ala in GyrA). To our knowledge, there have been no previous reports of this substitution in a clinical isolate of S. pyogenes.

Activation of pulmonary invariant NKT cells leads to exacerbation of acute lung injury caused by LPS through local production of IFN-γ and TNF-α by Gr-1+ monocytes

Invariant NK T (iNKT) cells are known to play a critical role in the regulation of inflammatory responses in various clinical settings. In the present study, we assessed the contribution of iNKT cells to the development of acute lung injury (ALI), which was caused by intra-tracheal administration of LPS. Jα18 gene-disrupted mice lacking these cells underwent neutrophilic inflammatory responses in lungs at an equivalent level as control mice. Next, mice were sensitized intra-tracheally with α-galactosylceramide, an activator of iNKT cells, followed by challenge with LPS. In this model, mice showed severe lung injury, and all mice were killed within 72 h after LPS injection. IFN-γ and tumor necrosis factor (TNF)-α were strikingly elevated in the lungs of these mice. Administration of neutralizing mAb against IFN-γ and TNF-α attenuated lung injury in a histopathological analysis and improved their survival rate. Flow cytometric analysis revealed that IFN-γ was expressed in NK cells, iNKT cells and also Gr-1(dull+)Ly-6C(+) monocytes and TNF-α was detected mainly in Gr-1(bright+)Ly-6G(+) neutrophils and Gr-1(dull+)Ly-6C(+) monocytes. Otherwise, in mice treated with LPS alone, IFN-γ was not detected in the lungs and Gr-1(bright+)Ly-6G(+) neutrophil was a main cellular source of TNF-α production. Anti-Gr-1 mAb resulted in the attenuation of ALI and decrease in the level of these cytokines. These results indicated that activation of iNKT cells led to striking exacerbation of ALI caused by LPS and that Gr-1(+) monocytes were recruited in the lungs with expressing IFN-γ and TNF-α and played an important role in the development of these responses.