Intrinsic clarithromycin heteroresistance in Mycobacterium avium.

BACKGROUND: Mycobacterium avium is associated with pulmonary disease in otherwise healthy adults. Several clarithromycin-refractory cases have been reported, including some cases caused by clarithromycin-susceptible strains. OBJECTIVES: To characterize the reason for the discrepancy between clinical response and antibiotic susceptibility results. METHODS: We conducted population analysis of clarithromycin-tolerant and heteroresistant subpopulations of M. avium cultured in vitro and in homogenates of infected lungs of mice. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined for 28 M. avium and two M. kansasii strains. Mice were intranasally infected with M. avium and treated with or without clarithromycin (100 mg/kg) thrice weekly. They were sacrificed on day 35 and the bacteria in lung homogenates were tested for clarithromycin resistance. Population analysis assays were performed based on colony growth on plates containing two-fold dilutions of clarithromycin. RESULTS: The MBC/MIC ratios were ≥8 in all 28 strains of M. avium tested. In the population analysis assay, several colonies were observed on the plates containing clarithromycin concentrations above the MIC (2-64 mg/L). No growth of M. kansasii colonies was observed on the plates containing clarithromycin concentrations ≥2 mg/L. M. avium in the homogenates of infected lungs showed clearer clarithromycin-resistant subpopulations than in vitro, regardless of clarithromycin exposure. CONCLUSION: M. avium shows intrinsic heterogeneous resistance (heteroresistance) to clarithromycin. This may explain the observed discrepancies between clarithromycin susceptibility testing results and clinical response to clarithromycin treatment. Further studies are needed to confirm a link between heteroresistance and clinical outcomes.

Apoptosis Inhibitor of Macrophages Contributes to the Chronicity of Mycobacterium avium Infection by Promoting Foamy Macrophage Formation.

In Mycobacterium avium infections, macrophages play a critical role in the host defense response. Apoptosis inhibitor of macrophage (AIM), also known as CD5L, may represent a novel supportive therapy against various diseases, including metabolic syndrome and infectious diseases. The mechanisms of AIM include modulating lipid metabolism in macrophages and other host cells. We investigated the role of AIM in M. avium infections in vitro and in vivo. In a mouse model of M. avium pneumonia, foamy macrophages were induced 6 wk after infection. The bacteria localized in these macrophages. Flow cytometric analysis also confirmed that the percentage of CD11chighMHCclassIIhigh interstitial and alveolar macrophages, a cell surface marker defined as foamy macrophages, increased significantly after infection. AIM in alveolar lavage fluid and serum gradually increased after infection. Administration of recombinant AIM significantly increased the number of bacteria in the lungs of mice, accompanied by the induction of inflammatory cytokine and iNOS expression. In mouse bone marrow-derived macrophages, the mRNA expression of AIM after M. avium infection and the amount of AIM in the supernatant increased prior to the increase in intracellular bacteria. Infected cells treated with anti-AIM Abs had fewer bacteria and a higher percentage of apoptosis-positive cells than infected cells treated with isotype control Abs. Finally, AIM in the sera of patients with M. avium-pulmonary disease was measured and was significantly higher than in healthy volunteers. This suggests that AIM production is enhanced in M. avium-infected macrophages, increasing macrophage resistance to apoptosis and providing a possible site for bacterial growth.

Telomere length and urinary 8-hydroxy-2'-deoxyguanosine and essential trace element concentrations in female Japanese university students.

Telomere length is thought to be a biomarker of biological aging. This study examined whether telomere length was associated with urinary concentrations of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a biomarker of oxidative stress, and antioxidative trace elements in 73 female Japanese university students (age: 19.2 ± 0.7 years). We quantified 8-OHdG and selenium in urine by liquid chromatography-mass spectrometry and inductively coupled plasma-mass spectrometry, respectively. Telomere length and urinary concentrations of other essential trace elements (molybdenum, cobalt, and chromium) that were previously measured in the same study participants, were used in this study. We used multiple linear regression analysis to examine the associations of telomere length with urinary 8-OHdG and essential trace element concentrations (covariates: urinary cotinine concentration, age, BMI, and drinking status). The geometric means (geometric standard deviation) of 8-OHdG and selenium were 3.4 (1.5) and 31 (1.3) µg/g creatinine, respectively. Telomere length was not associated with urinary 8-OHdG concentration, but was negatively associated with urinary selenium concentration. In conclusion, telomere length was not associated with urinary 8-OHdG concentration in the young women in this study. Longitudinal studies should be conducted to clarify the association between telomere shortening rate and oxidative stress level.

Increased Incidence and Plasma-Biofilm Formation Ability of SCCmec Type IV Methicillin-Resistant Staphylococcus aureus (MRSA) Isolated From Patients With Bacteremia.

In Japan, Staphylococcal cassette chromosome mec (SCCmec) type IV methicillin-resistant Staphylococcus aureus (MRSA) is an increasingly prominent cause of bacteremia, but the virulence of most of these strains is unclear. We aimed to investigate the relationship between the molecular characteristics and the ability to form biofilms in the presence of blood plasma (plasma-biofilms) of MRSA strains isolated from bloodstream infections. In this study, the molecular characteristics and biofilms of MRSA strains isolated from blood cultures between 2015 and 2017 were analyzed by PCR-based assays, crystal violet staining, and confocal reflection microscopy methods. Among the 90 MRSA isolates, the detection rate of SCCmec type II clones decreased from 60.7 to 20.6%. The SCCmec type IV clone replaced the SCCmec type II clone as the dominant clone, with a detection rate increasing from 32.1 to 73.5%. The plasma-biofilm formation ability of the SCCmec type IV clone was higher than the SCCmec type II clone and even higher in strains harboring the cna or arcA genes. Plasma-biofilms, mainly composed of proteins, were formed quickly and strongly. Our study demonstrated the increased plasma-biofilm formation ability of SCCmec type IV strains.

Clostridioides difficile toxins enhanced the in vitro production of CXC chemokine ligand 2 and tumor necrosis factor-α via Toll-like receptors in macrophages.

Introduction. Clostridioides difficile infection (CDI) causes toxin-mediated enteropathy, such as antibiotic-associated diarrhoea and pseudomembranous colitis. Rho-glucosylating toxin A (TcdA) and toxin B (TcdB) have been clearly implicated in pathogenesis, whereas the virulence of binary toxin (CDT) is still debated.Hypothesis statement. We hypothesized that CDT is involved in the host immune response and plays a pivotal role in establishing virulence by modulating pro-inflammatory cytokine production; this is achieved through the integral Toll-like receptor (TLR) signalling pathways.Aim. The aim of the present study was to determine whether and how CDT impacts macrophages compared to TcdA or TcdB by examining the induction of CXC chemokine ligand 2 (CXCL2) and tumour necrosis factor-α (TNF-α), both of which are crucial in mediating local and systematic inflammatory responses.Methodology. RAW264.7 cells or transfected human embryonic kidney (HEK) 293 T cells were incubated with TcdA, TcdB, or CDT. In some experiments, a neutralizing antibody against TLR2 or TLR4, or myeloid differentiation 88 inhibitory peptide were added. The amount of CXCL2 and TNF-α secreted was then measured.Results. In RAW264.7 macrophages, CXCL2 and TNF-α were produced via the Toll-like receptor 2 (TLR2) or Toll-like receptor 4 (TLR4) pathway in a TcdA, TcdB, or CDT dose-dependent manner. Interleukin-8 secretion was induced in TLR4/MD2/CD14-transfected, but not in TLR2-transfected, HEK 293 T cells following TcdB or CDT exposure.Conclusion. Our results showed that C. difficile toxins, including CDT, enhanced macrophage-mediated CXCL2 and TNF-α production via TLR2 and TLR4, indicating that CDT affects host immune responses.

Traditional Japanese Herbal Medicine Hochu-Ekki-to Promotes Pneumococcal Colonization Clearance via Macrophage Activation and Interleukin 17A Production in Mice.

Streptococcus pneumoniae may colonize the nasopharynx, and as pneumococcal colonization causes invasive diseases and the subsequent transmission, reducing bacterial burden in the nasal cavity is critical. Hochu-ekki-to (TJ-41) is a traditional Japanese herbal medicine that exerts immunomodulatory effects in host cells. In this study, we investigated the potency of TJ-41 in modulating pneumococcal colonization clearance by activating host immunity. Mice, intranasally inoculated with pneumococci, were treated orally with TJ-41. During colonization, TJ-41 treatment significantly reduced pneumococcal burden and increased macrophage population in the nasopharynx. Furthermore, interleukin 17A production was significantly enhanced after TJ-41 treatment. In vitro experiment using nasal-derived cells revealed that pneumococcal antigen exposure upregulated the transcription of interleukin 17A in the TJ-41-treated group compared with that in the control group. Macrophages activated by killed bacteria were significantly increased in the presence of TJ-41 in an interleukin 17A-dependent manner. Moreover, TJ-41 enhanced phagocytosis, inhibited bacterial growth, and improved the antigen-presenting capacity of macrophages. Our results demonstrate that TJ-41 accelerates the clearance of pneumococcal nasopharyngeal colonization via macrophage activation. Subsequent production of interleukin 17A provides an additional benefit to effector cells.

N-acetyl-cysteine mediates protection against Mycobacterium avium through induction of human ß-defensin-2 in a mouse lung infection model.

Mycobacterium avium complex is a causative organism for refractory diseases. In this study, we examined the effects of N-acetyl-cysteine on M. avium infection in vitro and in vivo. N-acetyl-cysteine treatment suppressed the growth of M. avium in A549 cells in a concentration-dependent manner. This effect was related to the induction of the antibacterial peptide human ß-defensin-2. In a mouse model, N-acetyl-cysteine treatment significantly reduced the number of bacteria in the lungs and induced murine ß-defensin-3. In interleukin-17-deficient mice, the effects of N-acetyl-cysteine disappeared, indicating that these mechanisms may be mediated by interleukin-17. Moreover, an additional reduction in bacterial load was observed in mice administered N-acetyl-cysteine in combination with clarithromycin. Our findings demonstrate the potent antimycobacterial effects of N-acetyl-cysteine against M. avium by inducing antimicrobial peptide, suggesting that N-acetyl-cysteine may have applications as an alternative to classical treatment regimens.

Tissue Damage Caused by Impaired Phagocytosis of Dead Cells: A Previously Unrecognized Adverse Effect Contributing to the Pathogenesis of γδ T Cells in Legionella Pneumonia.

IL-17 plays a critical role in the immunological control of various infectious diseases; its function has been investigated in the removal of both extracellular and intracellular bacteria. Our group previously revealed the importance of IL-17 in neutrophil migration following Legionella infection by using IL-17AF knockout mice; however, aside from neutrophil infiltration, alternative causes for the reduced survival of these mice have not been characterized. In this study, we found that γδ T cells in IL-17AF knockout mice were markedly increased and produced the cytotoxic substances granzyme B and perforin. Moreover, the elimination of γδ T cells from these mice, via an anti-TCRδ Ab, caused a substantial reduction in the level of lactate dehydrogenase in bronchoalveolar lavage fluid, indicating that γδ T cells contribute to lung tissue damage. Moreover, although cells lysed by cytotoxic substances are typically eliminated by phagocytic cells, in IL-17AF knockout mice, lung homeostasis was not maintained because of a decrease in phagocytic cells that impaired the clearance of dead cells. Our results indicate that increased γδ T cells in IL-17AF knockout mice help eliminate Legionella by releasing cytotoxic substances and lysing infected cells; however, this results in tissue damage due to insufficient removal of dead cells by phagocytic cells. This study enhances our understanding of the protective response against Legionella and provides insights into γδ T cell-mediated protective immunity against various infectious diseases.

Pneumococcal conjugate vaccine modulates macrophage-mediated innate immunity in pneumonia caused by Streptococcus pneumoniae following influenza.

Pneumococcal conjugate vaccination (PCV) may prevent influenza-related pneumonia, including Streptococcus pneumoniae pneumonia. To investigate PCV efficacy against secondary pneumococcal pneumonia following influenza, PCV was administered intramuscularly 2 and 5 weeks before S. pneumoniae serotype-3 colonization of murine nasopharynges followed by intranasal challenge with a sublethal dose of influenza A virus. Bacterial and viral loads, including innate immune responses were compared across conditions. PCV vaccination improved the survival of mice with secondary pneumococcal pneumonia and significantly reduced the pulmonary bacterial burden. Increased monocyte/macrophage influx into the lungs, alleviated loss of alveolar macrophages and decreased neutrophil influx into the lungs occurred in PCV-treated mice irrespective of pneumococcal colonization. Higher monocyte chemoattractant protein 1 levels and lower levels of CXCL1, interferon-γ, interleukin-17A, and IL-10, were detected in PCV-treated mice. Additionally, PCV treatment activated the macrophage intracellular killing of S. pneumoniae. Collectively, PCV potentially modulates the host's innate immunity and specific antibodies induction. Macrophage-related innate immunity should be further explored to elucidate the efficacy and mechanisms of PCV versus influenza-related life-threatening diseases.

Analysis of synergy between beta-lactams and anti-methicillin-resistant Staphylococcus aureus agents from the standpoint of strain characteristics and binding action.

In light of the increasing number of clinical cases resistant to traditional monotherapies and the lack of novel antimicrobial agents, combination therapy is an appealing solution for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections. Here, we evaluated the efficacy of anti-MRSA agents, such as vancomycin (VAN), daptomycin (DAP), and linezolid (LZD), in conjunction with 13 beta-lactams and non-beta-lactams. We assessed the in vitro activities of the various combinations against 40 MRSA strains based on the maximum synergistic effect (MSE), an index calculated from the MIC change with a combination agent. Nearly all the anti-MRSA agents, which were combined with beta-lactams as well as VAN and DAP, showed a synergistic effect with arbekacin. VAN also exhibited varying degrees of synergy depending on the type of beta-lactam, whereas DAP and LZD showed similar synergy with different beta-lactams. These effects were confirmed by antibiotic kill curves, except for the apparent interaction between LZD and beta-lactams. The MSE results were analyzed according to strain characteristics including susceptibility to combination agents, staphylococcal cassette chromosome mec type, and presence of the blaZ gene; however, no obvious correlations were observed. In a fluorescence binding assay, the fluorescence intensity of boron-dipyrromethene (BODIPY)-VAN decreased, whereas that of BODIPY-DAP increased in combination with a beta-lactam agent. These findings suggest that beta-lactam combinations are promising treatment options for MRSA infections and that the type of beta-lactam combined with VAN is important for the synergistic effect.

Role of interleukin-17 in a murine community-associated methicillin-resistant Staphylococcus aureus pneumonia model.

Interleukin (IL)-17 is a key member of the Th17 cytokines and has been reported to be involved in the pathomechanisms underlying various diseases, including infectious diseases. Infections with community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) have garnered worldwide attention, and the representative USA300 strain is known to cause pneumonia in healthy people, which can be lethal. However, little is known about the role of IL-17 in CA-MRSA pneumonia. In this study, we investigated the role of IL-17 in a CA-MRSA pneumonia animal model. Mortality was higher and occurred at an earlier stage of infection in the IL-17A-knockout mice than in the wild-type (P < 0.01) and IL-17A/F-knockout mice (P < 0.05); however, no significant difference in the intrapulmonary bacterial counts was observed among the three groups of mice. Moreover, the IL-17A-knockout group showed significantly higher levels of IL-17F and granulocyte-colony stimulating factor (G-CSF) and a significantly higher neutrophil count in the bronchoalveolar lavage fluid than the other groups. These results confirmed that G-CSF expression significantly increased, and significant neutrophilic inflammation occurred under conditions of IL-17A deficiency in the murine CA-MRSA pneumonia model.

Evidence of latent molecular diversity determining the virulence of community-associated MRSA USA300 clones in mice.

INTRODUCTION: The USA300 clone of community-associated MRSA is reported to be hypervirulent and epidemic in the United States. This clone causes a variety of diseases from lethal pneumonia to mild skin infections. We hypothesized that evolutionary diversity may exist among USA300 clones, which may link virulence traits with host responses and mortality rates. METHODS: USA300 isolates from severe pneumonia (IP) and skin infection (IS) were characterized by pulsed-field gel electrophoresis (PFGE) and next-generation sequencing. Their virulence traits and host responses were compared in a lung infection model. RESULTS: The two USA300 isolates were found to be identical in genomic analysis. Robust IL-6 production, aggregation of bacteria, and hemorrhaging were observed in IP-infected lungs, which were associated with a higher rate of mortality than that observed with strain IS. Few neutrophils were detected in the lungs infected with strain IP, even at high bacterial loads. Massive production of α-toxin and coagulase were evident during the early phase of IP infection, and robust gene expression of hla (α-toxin) and lukS-PV (Panton-Valentine leukocidin), but not coa, agrA, or rnaIII, was confirmed in vitro. Strain IP also induced strong hemolysis in red blood cells. CONCLUSIONS: The present data demonstrated latent diversity in the virulence of USA300 clones. Unknown regulatory mechanisms, probably involving a host factor(s) as a trigger, may govern the virulence expression and resultant high mortality in certain sub-clones of USA300.

Potential Role of Gr-1+ CD8+ T Lymphocytes as a Source of Interferon-γ and M1/M2 Polarization during the Acute Phase of Murine Legionella pneumophila Pneumonia.

In this study, we analyzed interferon (IFN)-γ-producing cells and M1/M2 macrophage polarization in Legionella pneumophila pneumonia following anti-Gr-1 antibody treatment. Anti-Gr-1 treatment induced an M1-to-M2 shift of macrophage subtypes in the lungs and weakly in the peripheral blood, which was associated with increased mortality in legionella-infected mice. CD8+ T lymphocytes and natural killer cells were the dominant sources of IFN-γ in the acute phase, and anti-Gr-1 treatment reduced the number of IFN-γ-producing CD8+ T lymphocytes. In the CD3-gated population, most Gr-1-positive cells were CD8+ T lymphocytes in the lungs and lymph nodes (LNs) of infected mice. Additionally, the number of IFN-γ-producing Gr-1+ CD8+ T lymphocytes in the lungs and LNs increased 2 and 4 days after L. pneumophila infection, with anti-Gr-1 treatment attenuating these populations. Antibody staining revealed that Gr-1+ CD8+ T lymphocytes were Ly6C-positive cells rather than Ly6G, a phenotype regarded as memory type cells. Furthermore, the adoptive transfer of Gr-1+ CD8+ T lymphocytes induced increases in IFN-γ, M1 shifting and reduced bacterial number in the Legionella pneumonia model. These data identified Ly6C+ CD8+ T lymphocytes as a source of IFN-γ in innate immunity and partially associated with reduced IFN-γ production, M2 polarization, and high mortality in anti-Gr-1 antibody-treated mice with L. pneumophila pneumonia.

Extracellular and Non-Chaperone Function of Heat Shock Protein-90α Is Required for Skin Wound Healing.

Despite years of effort and investment, there are few topical or systemic medications for skin wounds. Identifying natural drivers of wound healing could facilitate the development of new and effective treatments. When skin is injured, there is a massive increase of heat shock protein (Hsp) 90α inside the wound bed. The precise role for these Hsp90α proteins, however, was unclear. The availability of a unique mouse model that lacked the intracellular ATPase-driven chaperoning, but spared the extracellular fragment-5-supported pro-motility function of Hsp90α allowed us to test specifically the role of the non-chaperone function of Hsp90α in normal wound closure. We found that the chaperone-defective Hsp90α-Δ mutant mice showed similar wound closure rate as the wild-type Hsp90α mice. We generated recombinant proteins from the mouse cDNAs encoding the Hsp90α-Δ and wild-type Hsp90α. Topical application of Hsp90α-Δ mutant protein promoted wound closure as effectively as the full-length wild-type Hsp90α protein. More importantly, selective inhibition of the extracellular Hsp90α-Δ protein function by a monoclonal antibody targeting the fragment-5 region disrupted normal wound closure in both wild-type Hsp90α and Hsp90α-Δ mice. Thus, this study provides direct support for non-chaperone, extracellular Hsp90α as a potential driver for normal wound closure.

Metformin Mediates Protection against Legionella Pneumonia through Activation of AMPK and Mitochondrial Reactive Oxygen Species.

In Legionella pneumophila infection, macrophages play a critical role in the host defense response. Metformin, an oral drug for type 2 diabetes, is attracting attention as a new supportive therapy against a variety of diseases, such as cancer and infectious diseases. The novel mechanisms for metformin actions include modulation of the effector functions of macrophages and other host immune cells. In this study, we have examined the effects of metformin on L. pneumophila infection in vitro and in vivo. Metformin treatment suppressed growth of L. pneumophila in a time- and concentration-dependent fashion in bone marrow-derived macrophages, RAW cells (mouse), and U937 cells (human). Metformin induced phosphorylation of AMP-activated protein kinase (AMPK) in L. pneumophila-infected bone marrow-derived macrophages, and the AMPK inhibitor Compound C negated metformin-mediated growth suppression. Also, metformin induced mitochondrial reactive oxygen species but not phagosomal NADPH oxidase-derived reactive oxygen species. Metformin-mediated growth suppression was mitigated in the presence of the reactive oxygen species scavenger glutathione. In a murine L. pneumophila pneumonia model, metformin treatment improved survival of mice, which was associated with a significant reduction in bacterial number in the lung. Similar to in vitro observations, induction of AMPK phosphorylation and mitochondrial ROS was demonstrated in the infected lungs of mice treated with metformin. Finally, glutathione treatment abolished metformin effects on lung bacterial clearance. Collectively, these data suggest that metformin promotes mitochondrial ROS production and AMPK signaling and enhances the bactericidal activity of macrophages, which may contribute to improved survival in L. pneumophila pneumonia.

Efficacy of ß-Lactam-plus-Macrolide Combination Therapy in a Mouse Model of Lethal Pneumococcal Pneumonia.

Community-acquired pneumonia is a common disease with considerable morbidity and mortality, for which Streptococcus pneumoniae is accepted as a leading cause. Although ß-lactam-plus-macrolide combination therapy for this disease is recommended in several guidelines, the clinical efficacy of this strategy against pneumococcal pneumonia remains controversial. In this study, we examined the effects of ß-lactam-plus-macrolide combination therapy on lethal mouse pneumococcal pneumonia and explored the mechanisms of action in vitro and in vivo We investigated survival, lung bacterial burden, and cellular host responses in bronchoalveolar lavage fluids obtained from mice infected with pneumonia and treated with ceftriaxone, azithromycin, or both in combination. Although in vitro synergy was not observed, significant survival benefits were demonstrated with combination treatment. Lung neutrophil influx was significantly lower in the ceftriaxone-plus-azithromycin-treated group than in the ceftriaxone-treated group, whereas no differences in the lung bacterial burden were observed on day 3 between the ceftriaxone-plus-azithromycin-treated group and the ceftriaxone-treated group. Notably, the analysis of cell surface markers in the ceftriaxone-plus-azithromycin combination group exhibited upregulation of presumed immune checkpoint ligand CD86 and major histocompatibility complex class II in neutrophils and CD11b-positive CD11c-positive (CD11b(+) CD11c(+)) macrophages and dendritic cells, as well as downregulation of immune checkpoint receptors cytotoxic-T lymphocyte-associated antigen 4 and programmed death 1 in T helper and T regulatory cells. Our data demonstrate that the survival benefits of ceftriaxone-plus-azithromycin therapy occur through modulation of immune checkpoints in mouse pneumococcal pneumonia. In addition, immune checkpoint molecules may be a novel target class for future macrolide research.

Endogenous IL-17 as a factor determining the severity of Clostridium difficile infection in mice.

Clostridium difficile infection (CDI) is a toxin-mediated intestinal disease. Toxin A, toxin B and binary toxin are believed to be responsible for the pathogenesis of CDI, which is characterized by massive infiltration of neutrophils at the infected intestinal mucosa. IL-17 is one of the cytokines that play critical roles in several inflammatory and immunological diseases through various actions, including promoting neutrophil recruitment. The aim of this study was to examine the role of this cytokine in CDI by employing IL-17 A and F double knockout (IL-17 KO) mice for the CDI model. We demonstrated that IL-17 KO mice were more resistant to CDI than WT mice using several factors, such as diarrhoea score, weight change and survival rate. Although the bacterial numbers of C. difficile in faeces were not different, the inflammatory mediator levels at the large intestine on day 3 post-infection were attenuated in IL-17 KO mice. Finally, we showed that infiltration of neutrophils, but not macrophages, in the large intestine was significantly decreased in IL-17 KO mice compared to WT mice. In conclusion, the data demonstrate that endogenous IL-17 may be a factor determining the severity of CDI in mice. Although the mechanism is totally unknown, IL-17-mediated inflammatory responses, such as cytokine/chemokine production and neutrophil accumulation, may be plausible targets for future investigations.

Spermatogenesis arrest caused by conditional deletion of Hsp90α in adult mice.

It is controversial whether a functional androgen receptor (AR) on germ cells, including spermatogonia, is essential for their development into sperm and, thus, initiation and maintenance of spermatogenesis. It was recently shown that many spermatocytes underwent apoptosis in the testes of Hsp90α KO mice. We had generated Hsp90α KO mice independently and confirmed this phenotype. However, the important question of whether Hsp90α is required to maintain spermatogenesis in adult mice in which testicular maturation is already completed could not be addressed using these conventional KO mice. To answer this question, we generated a tamoxifen-inducible deletion mutant of Hsp90α and found that conditional deletion of Hsp90α in adult mice caused even more severe apoptosis in germ cells beyond the pachytene stage, leading to complete arrest of spermatogenesis and testicular atrophy. Importantly, immunohistochemical analysis revealed that AR expression in WT testis was more evident in spermatogonia than in spermatocytes, whereas its expression was aberrant and ectopic in Hsp90α KO testis, raising the possibility that an AR abnormality in primordial germ cells is involved in spermatogenesis arrest in the Hsp90α KO mice. Our results suggest that the AR, specifically chaperoned by Hsp90α in spermatogonia, is critical for maintenance of established spermatogenesis and for survival of spermatocytes in adult testis, in addition to setting the first wave of spermatogenesis before puberty.

HSP70 and HSP90 Differentially Regulate Translocation of Extracellular Antigen to the Cytosol for Cross-Presentation.

Antigens (Ag) from cancer or virus-infected cells must be internalized by dendritic cells (DCs) to be presented to CD8(+) T cells, which eventually differentiate into Ag-specific cytotoxic T lymphocytes (CTLs) that destroy cancer cells and infected cells. This pathway is termed cross-presentation and is also implicated as an essential step in triggering autoimmune diseases such as Type I diabetes. Internalized Ag locates within endosomes, followed by translocation through a putative pore structure spanning endosomal membranes into the cytosol, where it is degraded by the proteasome to generate antigen peptides. During translocation, Ag is believed to be unfolded since the pore size is too narrow to accept native Ag structure. Here, we show that paraformaldehyde-fixed, structurally inflexible Ag is less efficient in cross-presentation because of diminished translocation into the cytosol, supporting the "unfolded Ag" theory. We also show that HSP70 inhibitors block both endogenous and cross-presentation. ImageStream analysis revealed that the inhibition in cross-presentation is not due to blocking of Ag translocation because a HSP70 inhibitor rather facilitates the translocation, which is in marked contrast to the effect of an HSP90 inhibitor that blocks Ag translocation. Our results indicate that Ag translocation to the cytosol in cross-presentation is differentially regulated by HSP70 and HSP90.