Eri Ochiai

Interferon-gamma- and perforin-mediated immune responses for resistance against Toxoplasma gondii in the brain

Toxoplasma gondii is an obligate intracellular protozoan parasite that causes various diseases, including lymphadenitis, congenital infection of fetuses and life-threatening toxoplasmic encephalitis in immunocompromised individuals. Interferon-gamma (IFN-γ)-mediated immune responses are essential for controlling tachyzoite proliferation during both acute acquired infection and reactivation of infection in the brain. Both CD4+ and CD8+ T cells produce this cytokine in response to infection, although the latter has more potent protective activity. IFN-γ can activate microglia, astrocytes and macrophages, and these activated cells control the proliferation of tachyzoites using different molecules, depending on cell type and host species. IFN-γ also has a crucial role in the recruitment of T cells into the brain after infection by inducing expression of the adhesion molecule VCAM-1 on cerebrovascular endothelial cells, and chemokines such as CXCL9, CXCL10 and CCL5. A recent study showed that CD8+ T cells are able to remove T. gondii cysts, which represent the stage of the parasite in chronic infection, from the brain through their perforin-mediated activity. Thus, the resistance to cerebral infection with T. gondii requires a coordinated network using both IFN-γ- and perforin-mediated immune responses. Elucidating how these two protective mechanisms function and collaborate in the brain against T. gondii will be crucial in developing a new method to prevent and eradicate this parasitic infection.

Cytotoxic Substances from Aspergillus fumigatus in Oxygenated or Poorly Oxygenated Environment

Aspergillus fumigatus often causes serious health problems. The airway of the human body, the most common initial site of damage, is always exposed to an oxygenated condition, and the oxygen concentration may play a critical role in the virulence of A. fumigatus. In this study, oxygen content, fungal growth, the production of cytotoxic substance(s) in the fungal culture, and their relationship were investigated.Two clinical strains of A. fumigatus were cultured under certain oxygen contents (10, 14 and 20%), and cytotoxicity of their culture filtrates on murine macrophages and their fungal growth were evaluated. The components of these filtrates were analyzed by gas chromatography-mass spectrometry.All culture filtrates contained gliotoxin and showedpotent cytotoxicity on macrophages at very low concentration. The amount of gliotoxin in the culture filtrate prepared at 10% oxygen was markedly less, but diminutions in fungal growth and cytotoxicity of this culture filtrate were negligible.These results suggest that a well-oxygenated condition is suitable for the production of gliotoxin by A. fumigatus. A significant role of cytotoxic substances(s) other than gliotoxin is also suggested.

Secondary metabolite profiles and antifungal drug susceptibility of Aspergillus fumigatus and closely related species, Aspergillus lentulus, Aspergillus udagawae, and Aspergillus viridinutans.

The incidence of Aspergillus infection has been increasing in the past few years. Also, new Aspergillus fumigatus-related species, namely Aspergillus lentulus, Aspergillus udagawae, and Aspergillus viridinutans, were shown to infect humans. These fungi exhibit marked morphological similarities to A. fumigatus, albeit with different clinical courses and antifungal drug susceptibilities. The present study used liquid chromatography/time-of-flight mass spectrometry to identify the secondary metabolites secreted as virulence factors by these Aspergillus species and compared their antifungal susceptibility. The metabolite profiles varied widely among A. fumigatus, A. lentulus, A. udagawae, and A. viridinutans, producing 27, 13, 8, and 11 substances, respectively. Among the mycotoxins, fumifungin, fumiquinazoline A/B and D, fumitremorgin B, gliotoxin, sphingofungins, pseurotins, and verruculogen were only found in A. fumigatus, whereas auranthine was only found in A. lentulus. The amount of gliotoxin, one of the most abundant mycotoxins in A. fumigatus, was negligible in these related species. In addition, they had decreased susceptibility to antifungal agents such as itraconazole and voriconazole, even though metabolites that were shared in the isolates showing higher minimum inhibitory concentrations than epidemiological cutoff values were not detected. These strikingly different secondary metabolite profiles may lead to the development of more discriminative identification protocols for such closely related Aspergillus species as well as improved treatment outcomes.

VCAM-1/α4β1 Integrin Interaction Is Crucial for Prompt Recruitment of Immune T Cells into the Brain during the Early Stage of Reactivation of Chronic Infection with Toxoplasma gondii to Prevent Toxoplasmic Encephalitis

ABSTRACT Reactivation of chronic infection with Toxoplasma gondii can cause life-threatening toxoplasmic encephalitis in immunocompromised individuals. We examined the role of VCAM-1/α4β1 integrin interaction in T cell recruitment to prevent reactivation of the infection in the brain. SCID mice were infected and treated with sulfadiazine to establish a chronic infection. VCAM-1 and ICAM-1 were the endothelial adhesion molecules detected on cerebral vessels of the infected SCID and wild-type animals. Immune T cells from infected wild-type mice were treated with anti-α4 integrin or control antibodies and transferred into infected SCID or nude mice, and the animals received the same antibody every other day. Three days later, sulfadiazine was discontinued to initiate reactivation of infection. Expression of mRNAs for CD3δ, CD4, CD8β, gamma interferon (IFN-γ), and inducible nitric oxide synthase (NOS2) (an effector molecule to inhibit T. gondii growth) and the numbers of CD4+ and CD8+ T cells in the brain were significantly less in mice treated with anti-α4 integrin antibody than in those treated with control antibody at 3 days after sulfadiazine discontinuation. At 6 days after sulfadiazine discontinuation, cerebral tachyzoite-specific SAG1 mRNA levels and numbers of inflammatory foci associated with tachyzoites were markedly greater in anti-α4 integrin antibody-treated than in control antibody-treated animals, even though IFN-γ and NOS2 mRNA levels were higher in the former than in the latter. These results indicate that VCAM-1/α4β1 integrin interaction is crucial for prompt recruitment of immune T cells and induction of IFN-γ-mediated protective immune responses during the early stage of reactivation of chronic T. gondii infection to control tachyzoite growth.

Inhalation of Stachybotrys chartarum causes pulmonary arterial hypertension in mice

Inhalation of Stachybotrys chartarum, a ubiquitous fungus in our living environment, has been suspected as a cause of acute idiopathic pulmonary haemorrhage in infants, but its relation to human diseases is not yet known. The aim of present study was to investigate the effect of repeated intratracheal injection of the fungus into mice, paying special attention to the pulmonary vascular system. Spores of S. chartarum were injected into the trachea of mice from 6 to 18 times over 4–12 weeks, and the lungs were examined by histopathology, morphometrics and haemodynamics. When 1 × 104 spores/mouse were injected, histopathological examination showed the development of pulmonary arterial hypertension (PAH). Symmetrical thickening of the intima and media of the pulmonary arterial walls was seen after six injections over 4 weeks. Right ventricular hypertrophy was also evident after 12 injections. PAH was confirmed by the elevation of right ventricular systolic pressure (20.1 ± 5.7 mmHg in the injected group vs. 12.0 ± 2.4 mmHg in the control group, P < 0.01). This study showed that the inhalation of S. chartarum caused PAH in mice, suggesting a potential of S. chartarum as a cause of human health problem such as PAH.

CXCL9 Is Important for Recruiting Immune T Cells into the Brain and Inducing an Accumulation of the T Cells to the Areas of Tachyzoite Proliferation to Prevent Reactivation of Chronic Cerebral Infection with Toxoplasma gondii

T cells are required to maintain the latency of chronic infection with Toxoplasma gondii in the brain. Here, we examined the role of non-glutamic acid-leucine-arginine CXC chemokine CXCL9 for T-cell recruitment to prevent reactivation of infection with T. gondii. Severe combined immunodeficient (SCID) mice were infected and treated with sulfadiazine to establish a chronic infection. Immune T cells from infected wild-type mice were transferred into the SCID mice in combination with treatment with anti-CXCL9 or control sera. Three days later, sulfadiazine was discontinued to initiate reactivation of infection. Numbers of CD4(+) and CD8(+) T cells isolated from the brains were markedly less in mice treated with anti-CXCL9 serum than in mice treated with control serum at 3 days after sulfadiazine discontinuation. Amounts of tachyzoite (acute stage form of T. gondii)-specific SAG1 mRNA and numbers of foci associated with tachyzoites were significantly greater in the former than the latter at 5 days after sulfadiazine discontinuation. An accumulation of CD3(+) T cells into the areas of tachyzoite growth was significantly less frequent in the SCID mice treated with anti-CXCL9 serum than in mice treated with control serum. These results indicate that CXCL9 is crucial for recruiting immune T cells into the brain and inducing an accumulation of the T cells into the areas where tachyzoites proliferate to prevent reactivation of chronic T. gondii infection.

Cutting Edge: IFN-γ Produced by Brain-Resident Cells Is Crucial To Control Cerebral Infection with Toxoplasma gondii

In vitro studies demonstrated that microglia and astrocytes produce IFN-γ in response to various stimulations, including LPS. However, the physiological role of IFN-γ production by brain-resident cells, including glial cells, in resistance against cerebral infections remains unknown. We analyzed the role of IFN-γ production by brain-resident cells in resistance to reactivation of cerebral infection with Toxoplasma gondii using a murine model. Our study using bone marrow chimeric mice revealed that IFN-γ production by brain-resident cells is essential for upregulating IFN-γ–mediated protective innate immune responses to restrict cerebral T. gondii growth. Studies using a transgenic strain that expresses IFN-γ only in CD11b+ cells suggested that IFN-γ production by microglia, which is the only CD11b+ cell population among brain-resident cells, is able to suppress the parasite growth. Furthermore, IFN-γ produced by brain-resident cells is pivotal for recruiting T cells into the brain to control the infection. These results indicate that IFN-γ produced by brain-resident cells is crucial for facilitating both the protective innate and T cell–mediated immune responses to control cerebral infection with T. gondii.

A simple method for confirming correct endotracheal intubation in mice

Endotracheal intubation in mice is both a common and important technique. However, it is a difficult procedure because of the small orotracheal size and the success rate is variable. There have been many reports of refined techniques that facilitate intubation but only a few reports have proposed how to verify the proper placement of the endotracheal tube. We describe a novel, safe and reliable method to confirm endotracheal intubation in mice using an extension tube for intravenous infusion. One drop of water was instilled in the extension tube and connected to the end of the catheter used as an endotracheal tube. When the catheter was inserted correctly into the trachea, the water in the extension tube oscillated in synchrony with the movement of the mouses thorax, indicating correct placement of the catheter. This method was simple, reliable and use materials that are routinely available. This method is helpful for experimental mouse models that require airway access.

Gene expression analysis of a murine model with pulmonary vascular remodeling compared to end-stage IPAH lungs

BackgroundIdiopathic pulmonary arterial hypertension (IPAH) continues to be one of the most serious intractable diseases that might start with activation of several triggers representing the genetic susceptibility of a patient. To elucidate what essentially contributes to the onset and progression of IPAH, we investigated factors playing an important role in IPAH by searching discrepant or controversial expression patterns between our murine model and those previously published for human IPAH. We employed the mouse model, which induced muscularization of pulmonary artery leading to hypertension by repeated intratracheal injection of Stachybotrys chartarum, a member of nonpathogenic and ubiquitous fungus in our envelopment.MethodsMicroarray assays with ontology and pathway analyses were performed with the lungs of mice. A comparison was made of the expression patterns of biological pathways between our model and those published for IPAH.ResultsSome pathways in our model showed the same expression patterns in IPAH, which included bone morphogenetic protein (BMP) signaling with down-regulation of BMP receptor type 2, activin-like kinase type 1, and endoglin. On the other hand, both Wnt/planar cell polarity (PCP) signaling and its downstream Rho/ROCK signaling were found alone to be activated in IPAH and not in our model.ConclusionsActivation of Wnt/PCP signaling, in upstream positions of the pathway, found alone in lungs from end stage IPAH may play essential roles in the pathogenesis of the disease.

CD8(+) T cells remove cysts of Toxoplasma gondii from the brain mostly by recognizing epitopes commonly expressed by or cross-reactive between type II and type III strains of the parasite.

Our previous study demonstrated that CD8(+) T cells remove cysts of Toxoplasma gondii from the brain through perforin-mediated mechanisms. We here show that a transfer of CD8(+) immune T cells primed with a type II or a type III strain of T. gondii both efficiently removed cysts of a type II strain from infected SCID mice, although the former tended to be slightly more efficient than the latter. Similarly, a transfer of type II-primed CD8(+) T cells removed cysts of a type III strain. Therefore, CD8(+) T cells are capable of removing T. gondii cysts by recognizing epitopes commonly expressed in types II and III strains or cross-reactive between these two genotypes.