Kyosuke Yamanishi

All Publications

• IL-18 primes T cells with an antigen-inexperienced memory phenotype for proliferation and differentiation into effector cells through Notch signaling. Journal of leukocyte biology Li, W., Jin, D., Takai, S., Inoue, N., Yamanishi, K., Tanaka, Y., Okamura, H. 2024; 117 (1)

  Abstract

  Recent studies have revealed that a subset of CD8+ T cells exhibit innate features and can be activated by cytokines. However, the precise mechanisms underlying the proliferation and differentiation of these cells remain unclear. Here, we demonstrated that CD44highCD8+ T cells in the mouse spleen express functional interleukin-18 (IL-18) receptors, whereas CD44lowCD8+ T cells do not. In response to IL-18 stimulation, these cells activated various metabolic pathways, upregulated the expression of surface molecules, such as c-Kit (CD117), CD25, and PD-1, and induced progression through the G1/S phase in the cell cycle. IL-18-primed cells, expressing a high-affinity receptor for IL-2, exhibited robust proliferation in response to IL-2 and underwent differentiation into effector cells. The splenic CD44highCD8+ T cells exhibited high expression levels of CD122, CD62L, CCR7, and CXCR3, along with CD5, indicating their potential for migration to the lymph nodes, where they could undergo expansion and terminal differentiation into effector cells. Additionally, in a tumor model, administration of IL-18 increased the accumulation of CD8+ T cells in both the lymph nodes and tumors. It is noteworthy that stimulation of CD44highCD8+ T cells with IL-18 upregulated the Notch-1 receptor and c-Myc. Moreover, inclusion of γ-secretase inhibitors attenuated the effect of IL-18 on both proliferation and interferon-γ production in the cells. These results demonstrate that IL-18 primes CD44highCD122highCXCR3highCD62LhighCD8+ T cells for expansion and differentiation into effector cells in a Notch signaling-dependent manner.

  View details for DOI 10.1093/jleuko/qiae172

  View details for PubMedID 39213165

• LPS-induced delirium-like behavior and microglial activation in mice correlate with bispectral electroencephalography (BSEEG). The journals of gerontology. Series A, Biological sciences and medical sciences Nishiguchi, T., Yamanishi, K., Gorantla, N., Shimura, A., Seki, T., Ishii, T., Aoyama, B., Malicoat, J. R., Phuong, N. J., Dye, N. J., Yamanashi, T., Iwata, M., Shinozaki, G. 2024

  Abstract

  Delirium is a multifactorial medical condition characterized by impairment across various mental functions and is one of the greatest risk factors for prolonged hospitalization, morbidity, and mortality. Research focused on delirium has proven to be challenging due to a lack of objective measures for diagnosing patients, and few laboratory models have been validated. Our recent studies report the efficacy of bispectral electroencephalography (BSEEG) in diagnosing delirium in patients and predicting patient outcomes. We applied BSEEG to validate a lipopolysaccharide (LPS)-induced mouse model of delirium. Moreover, we investigated the relationship between BSEEG score, delirium-like behaviors, and microglia activation in hippocampal dentate gyrus and cortex regions in young and aged mice. There was a significant correlation between BSEEG score and impairment of attention in young mice. Additionally, there was a significant correlation between BSEEG score and microglial activation in hippocampal dentate gyrus and cortex regions in young and aged mice. We have successfully validated the BSEEG method by showing its associations with a level of behavioral change and microglial activation in an LPS-induced mouse model of delirium. In addition, the BSEEG method was able to sensitively capture an LPS-induced delirium-like condition that behavioral tests could not capture because of a hypoactive state.

  View details for DOI 10.1093/gerona/glae261

  View details for PubMedID 39492697

• Discovery of novel protective agents for infection-related delirium through bispectral electroencephalography. Translational psychiatry Nishiguchi, T., Yamanishi, K., Patel, S., Malicoat, J. R., Phuong, N. J., Seki, T., Ishii, T., Aoyama, B., Shimura, A., Gorantla, N., Yamanashi, T., Iwata, M., Pieper, A. A., Shinozaki, G. 2024; 14 (1): 413

  Abstract

  Delirium is a multifactorial medical condition of waxing and waning impairment across various domains of mental functioning over time. Importantly, delirium is also one of the greatest risk factors for prolonged hospitalization, morbidity, and mortality. Studying this important condition is challenging due to the difficulty in both objective diagnosis in patients and validation of laboratory models. As a result, there is a lack of protective treatments for delirium. Our recent studies report the efficacy of bispectral electroencephalography (BSEEG) in diagnosing delirium in patients and predicting patient outcomes, advancing the concept that this simple measure could represent an additional vital sign for patients. Here, we applied BSEEG to characterize and validate a novel lipopolysaccharide (LPS) mouse model of infection-related delirium. We then applied this model to evaluate the protective efficacy of three putative therapeutic agents: the conventional antipsychotic medication haloperidol, the neuroprotective compound P7C3-A20, and the antibiotic minocycline. Aged mice were more susceptible than young mice to LPS-induced aberration in BSEEG, reminiscent of the greater vulnerability of older adults to delirium. In both young and old mice, P7C3-A20 and minocycline administration prevented LPS-induced BSEEG abnormality. By contrast, haloperidol did not. P7C3-A20 and minocycline have been shown to limit different aspects of LPS toxicity, and our data offers proof of principle that these agents might help protect patients from developing infection-related delirium. Thus, utilization of BSEEG in a mouse model for infection-related delirium can identify putative therapeutic agents for applications in patient clinical trials.

  View details for DOI 10.1038/s41398-024-03130-4

  View details for PubMedID 39358319

• The Genome-wide DNA methylation changes in gastrointestinal surgery patients with and without postoperative delirium: Evidence of immune process in its pathophysiology. Journal of psychiatric research Nishizawa, Y., Yamanashi, T., Nishiguchi, T., Kajitani, N., Miura, A., Matsuo, R., Tanio, A., Yamamoto, M., Sakamoto, T., Fujiwara, Y., Thompson, K., Malicoat, J., Yamanishi, K., Seki, T., Kanazawa, T., Iwata, M., Shinozaki, G. 2024; 177: 249-255

  Abstract

  The pathophysiological mechanisms of postoperative delirium (POD) are still unclear, and there is no reliable biomarker to distinguish between those with and without POD. Our aim was to discover DNAm markers associated with POD in blood collected from patients before and after gastrointestinal surgery.We collected blood samples from 16 patients including 7 POD patients at three timepoints; before surgery (pre), the first and third postoperative days (day1 and day3). We measured differences in DNA methylation between POD and control groups between pre and day1 as well as between pre and day3 using the Illumina EPIC array method. Besides, enrichment analysis with Gene Ontology and Kyoto Encyclopedia of Genes and Genomes terms were also performed after excluding influence of common factors related to surgery and anesthesia.The results showed that pre and day1 comparisons showed that immune and inflammatory signals such as 'T-cell activation' were significantly different, consistent with our previous studies with non-Hispanic White subjects. In contrast, we found that these signals were not significant any more when pre was compared with day3.These results provide strong evidence for the involvement of inflammatory and immune-related epigenetic signals in the pathogenesis of delirium, including POD, regardless of ethnic background. These findings also suggest that DNAm, which is involved in inflammation and immunity, is dynamically altered in patients with POD. In summary, the present results indicate that these signals may serve as a new diagnostic tool for POD.

  View details for DOI 10.1016/j.jpsychires.2024.07.013

  View details for PubMedID 39043004

• A web-based delirium detection application using bispectral electroencephalography (BSEEG). General hospital psychiatry Shimura, A., Seki, T., Nishiguchi, T., Yamanishi, K., Ishii, T., Aoyama, B., Nguyen, H. D., Gorantla, N., Inoue, T., Shinozaki, G. 2024

  View details for DOI 10.1016/j.genhosppsych.2024.07.005

  View details for PubMedID 39060191

• Epigenetic signals associated with delirium replicated across four independent cohorts. Translational psychiatry Nishizawa, Y., Thompson, K. C., Yamanashi, T., Wahba, N. E., Saito, T., Marra, P. S., Nagao, T., Nishiguchi, T., Shibata, K., Yamanishi, K., Hughes, C. G., Pandharipande, P., Cho, H., Howard, M. A., Kawasaki, H., Toda, H., Kanazawa, T., Iwata, M., Shinozaki, G. 2024; 14 (1): 275

  Abstract

  Delirium is risky and indicates poor outcomes for patients. Therefore, it is crucial to create an effective delirium detection method. However, the epigenetic pathophysiology of delirium remains largely unknown. We aimed to discover reliable and replicable epigenetic (DNA methylation: DNAm) markers that are associated with delirium including post-operative delirium (POD) in blood obtained from patients among four independent cohorts. Blood DNA from four independent cohorts (two inpatient cohorts and two surgery cohorts; 16 to 88 patients each) were analyzed using the Illumina EPIC array platform for genome-wide DNAm analysis. We examined DNAm differences in blood between patients with and without delirium including POD. When we compared top CpG sites previously identified from the initial inpatient cohort with three additional cohorts (one inpatient and two surgery cohorts), 11 of the top 13 CpG sites showed statistically significant differences in DNAm values between the delirium group and non-delirium group in the same directions as found in the initial cohort. This study demonstrated the potential value of epigenetic biomarkers as future diagnostic tools. Furthermore, our findings provide additional evidence of the potential role of epigenetics in the pathophysiology of delirium including POD.

  View details for DOI 10.1038/s41398-024-02986-w

  View details for PubMedID 38965205

  View details for PubMedCentralID 2698979

• The bispectral electroencephalography (BSEEG) method quantifies post-operative delirium-like states in young and aged male mice after head mount implantation surgery. The journals of gerontology. Series A, Biological sciences and medical sciences Nishiguchi, T., Shibata, K., Yamanishi, K., Dittrich, M. N., Islam, N. Y., Patel, S., Phuong, N. J., Marra, P. S., Malicoat, J. R., Seki, T., Nishizawa, Y., Yamanashi, T., Iwata, M., Shinozaki, G. 2024

  Abstract

  Delirium, a syndrome characterized by an acute change in attention, awareness, and cognition, is commonly observed in older adults, although there are few quantitative monitoring methods in the clinical setting. We developed a bispectral electroencephalography (BSEEG) method capable of detecting delirium and can quantify the severity of delirium using a novel algorithm. Pre-clinical application of this novel BSEEG method can capture a delirium-like state in mice following LPS administration. However, its application to postoperative delirium (POD) has not yet been validated in animal experiments. This study aimed to create a POD model in mice with the BSEEG method by monitoring BSEEG scores following EEG head-mount implantation surgery and throughout the recovery. We compared the BSEEG scores of C57BL/6J young (2-3 months old) with aged (18-19 months old) male mice for quantitative evaluation of POD-like states. Postoperatively, both groups displayed increased BSEEG scores and a loss of regular diurnal changes in BSEEG scores. In young mice, BSEEG scores and regular diurnal changes recovered relatively quickly to baseline by postoperative day 3. Conversely, aged mice exhibited prolonged increases in postoperative BSEEG scores and it reached steady states only after postoperative day 8. This study suggests that the BSEEG method can be utilized as a quantitative measure of POD and assess the effect of aging on recovery from POD in the pre-clinical model.

  View details for DOI 10.1093/gerona/glae158

  View details for PubMedID 38877811

• Large-scale animal model study uncovers altered brain pH and lactate levels as a transdiagnostic endophenotype of neuropsychiatric disorders involving cognitive impairment. eLife Hagihara, H., Shoji, H., Hattori, S., Sala, G., Takamiya, Y., Tanaka, M., Ihara, M., Shibutani, M., Hatada, I., Hori, K., Hoshino, M., Nakao, A., Mori, Y., Okabe, S., Matsushita, M., Urbach, A., Katayama, Y., Matsumoto, A., Nakayama, K. I., Katori, S., Sato, T., Iwasato, T., Nakamura, H., Goshima, Y., Raveau, M., Tatsukawa, T., Yamakawa, K., Takahashi, N., Kasai, H., Inazawa, J., Nobuhisa, I., Kagawa, T., Taga, T., Darwish, M., Nishizono, H., Takao, K., Sapkota, K., Nakazawa, K., Takagi, T., Fujisawa, H., Sugimura, Y., Yamanishi, K., Rajagopal, L., Hannah, N. D., Meltzer, H. Y., Yamamoto, T., Wakatsuki, S., Araki, T., Tabuchi, K., Numakawa, T., Kunugi, H., Huang, F. L., Hayata-Takano, A., Hashimoto, H., Tamada, K., Takumi, T., Kasahara, T., Kato, T., Graef, I. A., Crabtree, G. R., Asaoka, N., Hatakama, H., Kaneko, S., Kohno, T., Hattori, M., Hoshiba, Y., Miyake, R., Obi-Nagata, K., Hayashi-Takagi, A., Becker, L. J., Yalcin, I., Hagino, Y., Kotajima-Murakami, H., Moriya, Y., Ikeda, K., Kim, H., Kaang, B., Otabi, H., Yoshida, Y., Toyoda, A., Komiyama, N. H., Grant, S. G., Ida-Eto, M., Narita, M., Matsumoto, K., Okuda-Ashitaka, E., Ohmori, I., Shimada, T., Yamagata, K., Ageta, H., Tsuchida, K., Inokuchi, K., Sassa, T., Kihara, A., Fukasawa, M., Usuda, N., Katano, T., Tanaka, T., Yoshihara, Y., Igarashi, M., Hayashi, T., Ishikawa, K., Yamamoto, S., Nishimura, N., Nakada, K., Hirotsune, S., Egawa, K., Higashisaka, K., Tsutsumi, Y., Nishihara, S., Sugo, N., Yagi, T., Ueno, N., Yamamoto, T., Kubo, Y., Ohashi, R., Shiina, N., Shimizu, K., Higo-Yamamoto, S., Oishi, K., Mori, H., Furuse, T., Tamura, M., Shirakawa, H., Sato, D. X., Inoue, Y. U., Inoue, T., Komine, Y., Yamamori, T., Sakimura, K., Miyakawa, T. 2024; 12

  Abstract

  Increased levels of lactate, an end-product of glycolysis, have been proposed as a potential surrogate marker for metabolic changes during neuronal excitation. These changes in lactate levels can result in decreased brain pH, which has been implicated in patients with various neuropsychiatric disorders. We previously demonstrated that such alterations are commonly observed in five mouse models of schizophrenia, bipolar disorder, and autism, suggesting a shared endophenotype among these disorders rather than mere artifacts due to medications or agonal state. However, there is still limited research on this phenomenon in animal models, leaving its generality across other disease animal models uncertain. Moreover, the association between changes in brain lactate levels and specific behavioral abnormalities remains unclear. To address these gaps, the International Brain pH Project Consortium investigated brain pH and lactate levels in 109 strains/conditions of 2294 animals with genetic and other experimental manipulations relevant to neuropsychiatric disorders. Systematic analysis revealed that decreased brain pH and increased lactate levels were common features observed in multiple models of depression, epilepsy, Alzheimer's disease, and some additional schizophrenia models. While certain autism models also exhibited decreased pH and increased lactate levels, others showed the opposite pattern, potentially reflecting subpopulations within the autism spectrum. Furthermore, utilizing large-scale behavioral test battery, a multivariate cross-validated prediction analysis demonstrated that poor working memory performance was predominantly associated with increased brain lactate levels. Importantly, this association was confirmed in an independent cohort of animal models. Collectively, these findings suggest that altered brain pH and lactate levels, which could be attributed to dysregulated excitation/inhibition balance, may serve as transdiagnostic endophenotypes of debilitating neuropsychiatric disorders characterized by cognitive impairment, irrespective of their beneficial or detrimental nature.

  View details for DOI 10.7554/eLife.89376

  View details for PubMedID 38529532

• Genome-wide DNA methylation analysis in female veterans with military sexual trauma and comorbid PTSD/MDD. Journal of affective disorders Marra, P., Seki, T., Nishizawa, Y., Chang, G., Yamanishi, K., Nishiguchi, T., Shibata, K., Braun, P., Shinozaki, G. 2024

  Abstract

  Military sexual trauma (MST) is a prevalent issue within the U.S. military. Victims are more likely to develop comorbid diseases such as posttraumatic stress disorder (PTSD) and major depressive disorder (MDD). Nonetheless, not everyone who suffers from MST develops PTSD and/or MDD. DNA methylation, which can regulate gene expression, might give us insight into the molecular mechanisms behind this discrepancy. Therefore, we sought to identify genomic loci and enriched biological pathways that differ between patients with and without MST, PTSD, and MDD.Saliva samples were collected from 113 female veterans. Following DNA extraction and processing, DNA methylation levels were measured through the Infinium HumanMethylationEPIC BeadChip array. We used limma and bump hunting methods to generate the differentially methylated positions and differentially methylated regions (DMRs), respectively. Concurrently, we used Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genome to find enriched pathways.A DMR close to the transcription start site of ZFP57 was differentially methylated between subjects with and without PTSD, replicating previous findings and emphasizing the potential role of ZFP57 in PTSD susceptibility. In the pathway analyses, none survived multiple correction, although top GO terms included some potentially relevant to MST, PTSD, and MDD etiology.We conducted one of the first DNA methylation analyses investigating MST along with PTSD and MDD. In addition, we found one DMR near ZFP57 to be associated with PTSD. The replication of this finding indicates further investigation of ZFP57 in PTSD may be warranted.

  View details for DOI 10.1016/j.jad.2024.01.241

  View details for PubMedID 38309478

• A prospective investigation of impacts of comorbid attention deficit hyperactivity disorder (ADHD) on clinical features and long-term treatment response in adult patients with obsessive-compulsive disorder (OCD). Comprehensive psychiatry Miyauchi, M., Matsuura, N., Mukai, K., Hashimoto, T., Ogino, S., Yamanishi, K., Yamada, H., Hayashida, K., Matsunaga, H. 2023; 125: 152401

  Abstract

  A close association between obsessive-compulsive disorder (OCD) and attention-deficit/hyperactivity disorder (ADHD) in children and adolescents has been investigated in previous studies. However, few studies examined the relationship between lifetime comorbidity of ADHD and OCD in adults. Therefore, we sought to investigate the clinical and psychopathological features related to comorbid ADHD in Japanese adult patients with OCD.We assessed lifetime comorbidity of ADHD in 93 adult Japanese patients with OCD. Additionally, we used the Japanese version of Conners' Adult ADHD Rating Scales to assess the characteristics and severity of ADHD in each participant. According to the results, we excluded OCD patients that did not have ADHD but who exhibited elevated levels of ADHD traits. We compared OCD patients with ADHD (ADHD+ group) and those without ADHD or its trait (ADHD- group) in terms of background profiles and clinical features, such as OCD symptomatology and psychometric test results. Additionally, the 6-month treatment outcome was compared prospectively between groups.Of the 93 OCD participants, the prevalence of lifetime comorbidity of ADHD was estimated as 16.1%. Compared with the ADHD- group, participants in the ADHD+ group had an earlier age of onset of OCD, higher frequencies of hoarding symptoms, higher levels of depressive and anxiety symptoms and lower quality of life, more elevated levels of impulsivity, and higher rates of substance or behavioral addiction and major depression. Finally, the mean improvement rate on the Yale-Brown Obsessive Compulsive Scale after 6 months of standardized OCD treatment in the ADHD+ group (16.1%) was significantly lower than that in the ADHD- group (44.6%).The lifetime comorbidity of ADHD is likely to exert a significant effect on clinical features and treatment outcome in adult patients with OCD. It is important to consider that underlying ADHD pathology may function as a facilitator for increased severity of global clinical features and treatment refractory conditions in OCD patients. Further studies are required to examine treatment strategies for such patients.

  View details for DOI 10.1016/j.comppsych.2023.152401

  View details for PubMedID 37454485

• NSAIDs use history: impact on the genome-wide DNA methylation profile and possible mechanisms of action. Clinical and experimental medicine Marra, P. S., Nishizawa, Y., Yamanashi, T., Sullivan, E. J., Comp, K. R., Crutchley, K. J., Wahba, N. E., Shibata, K., Nishiguchi, T., Yamanishi, K., Noiseux, N. O., Karam, M. D., Shinozaki, G. 2023

  Abstract

  NSAIDs inhibit cyclooxygenase, but their role in aging and other diseases is not well understood. Our group previously showed the potential benefit of NSAIDs in decreasing the risk of delirium and mortality. Concurrently, epigenetics signals have also been associated with delirium. Therefore, we sought to find differentially methylated genes and biological pathways related to exposure with NSAIDs by comparing the genome-wide DNA methylation profiles of patients with and without a history of NSAIDs use.Whole blood samples were collected from 171 patients at the University of Iowa Hospital and Clinics from November 2017 to March 2020. History of NSAIDs use was assessed through a word-search function in the subjects' electronic medical records. DNA was extracted from the blood samples, processed with bisulfite conversion, and analyzed using Illumina's EPIC array. The analysis of top differentially methylated CpG sites and subsequent enrichment analysis were conducted using an established pipeline using R statistical software.Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) showed several biological pathways relevant to NSAIDs' function. The identified GO terms included "arachidonic acid metabolic process," while KEGG results included "linoleic acid metabolism," "cellular senescence," and "circadian rhythm." Nonetheless, none of the top GO and KEGG pathways and the top differentially methylated CpG sites reached statistical significance.Our results suggest a potential role of epigenetics in the mechanisms of the action of NSAIDs. However, the results should be viewed with caution as exploratory and hypothesis-generating given the lack of statistically significant findings.

  View details for DOI 10.1007/s10238-023-01119-9

  View details for PubMedID 37341931

  View details for PubMedCentralID 7486988

• Elevated levels of interleukin-18 are associated with several indices of general and visceral adiposity and insulin resistance in women with polycystic ovary syndrome. Archives of endocrinology and metabolism Kabakchieva, P., Gateva, A., Velikova, T., Georgiev, T., Yamanishi, K., Okamura, H., Kamenov, Z. 2022; 66 (1): 3-11

  Abstract

  Our aim was to analyze levels of proinflammatory biomarker interleukin-18 (IL-18) in healthy controls and patients with polycystic ovary syndrome (PCOS) focusing on its association with obesity, clinical, hormonal, and metabolic characteristics.Fifty-eight patients with PCOS were enrolled in the study fulfilling the Rotterdam criteria and were matched for age, body mass index (BMI), and ethnicity with 30 healthy controls. Detailed anthropometric measurements, clinical investigations, hormonal and biochemical tests were obtained between the 3rd and 5th day of a menstrual cycle. A subanalysis of the PCOS group was performed separating patients into several groups according to a waist-to-height ratio (WHtR), insulin resistance (IR), and free androgen index (FAI). Serum IL-18 levels were measured using the ELISA method.Levels of IL-18 were similar between PCOS patients and controls. IL-18 was higher in overweight/obese women compared to normal-weight women when analyzing all participants together and separately PCOS or controls group (p < 0.001, p < 0.001, p = 0.01, respectively). Additionally, IL-18 levels were higher in high-WHtR and IR subgroups compared to low-WHtR (p < 0.001) and non-IR PCOS women (p < 0.001). PCOS women with high FAI had greater serum IL-18 levels than normal-FAI patients (p = 0.002). Levels of IL-18 correlated positively with most of the anthropometric and metabolic parameters. In multiple linear regression, age, waist circumference, and fasting insulin were independently related factors with IL-18.Elevated levels of IL-18 were related to several indices of general and visceral adiposity and insulin resistance in PCOS.

  View details for DOI 10.20945/2359-3997000000442

  View details for PubMedID 35263047

  View details for PubMedCentralID PMC9991028

• Exploring Molecular Mechanisms Involved in the Development of the Depression-Like Phenotype in Interleukin-18-Deficient Mice. BioMed research international Yamanishi, K., Miyauchi, M., Mukai, K., Hashimoto, T., Uwa, N., Seino, H., Li, W., Gamachi, N., Hata, M., Kuwahara-Otani, S., Maeda, S., Watanabe, Y., Yamanishi, H., Yagi, H., Okamura, H., Matsunaga, H. 2021; 2021: 9975865

  Abstract

  Interleukin-18 (IL-18) is an inflammatory cytokine that has been linked to energy homeostasis and psychiatric symptoms such as depression and cognitive impairment. We previously revealed that deficiency in IL-18 led to hippocampal abnormalities and resulted in depression-like symptoms. However, the impact of IL-18 deficiency on other brain regions remains to be clarified. In this study, we first sought to confirm that IL-18 expression in neural cells can be found in human brain tissue. Subsequently, we examined the expression of genes in the prefrontal cortex of Il18 -/- mice and compared it with gene expression in mice subjected to a chronic mild stress model of depression. Extracted genes were further analyzed using Ingenuity® Pathway Analysis, in which 18 genes common to both the chronic mild stressed model and Il18 -/- mice were identified. Of those, 16 were significantly differentially expressed between Il18+/+ and Il18 -/- mice. We additionally measured protein expression of α-2-HS-glycoprotein (AHSG) and transthyretin (TTR) in serum and the brain. In the prefrontal cortex of Il18 -/- mice, TTR but not AHSG was significantly decreased. Conversely, in the serum of Il18 -/- mice, AHSG was significantly increased but not TTR. Therefore, our results suggest that in IL-18-deficit conditions, TTR in the brain is one of the mediators causally related to depression, and AHSG in peripheral organs is one of the regulators inducing energy imbalance. Moreover, this study suggests a possible "signpost" to clarify the molecular mechanisms commonly underlying the immune system, energy metabolism, neural function, and depressive disorders.

  View details for DOI 10.1155/2021/9975865

  View details for PubMedID 34708129

  View details for PubMedCentralID PMC8545524

• Evaluation of hemodynamic changes using near-infrared spectroscopy in patients with tic-related obsessive-compulsive disorder. Psychiatry and clinical neurosciences Mukai, K., Matsuura, N., Miyauchi, M., Hashimoto, T., Yamanishi, K., Maebayashi, K., Hayashida, K., Matsunaga, H. 2021; 75 (6): 191-199

  Abstract

  A tic-related specifier is included in the DSM-5 diagnostic criteria to identify a clinically specific obsessive-compulsive disorder (OCD) subtype. The current study sought to evaluate hemodynamic changes during executive function tasks among OCD patients with and without a lifetime history of tic disorder (TD) and healthy controls, and to investigate the relation between brain activation and clinical variables in each group using structured equation modeling.Twenty-nine OCD patients diagnosed according to the DSM-IV-TR and 15 healthy controls were recruited. Patients were divided into two groups according to the presence or absence of a lifetime history of TD (TD+, n = 11; TD-, n = 18). Prefrontal hemodynamic changes were measured using multi-channel near-infrared spectroscopy during the Verbal Fluency Task, Trail-Making Task, and Tower of London (ToL) Task.There were significant brain activation differences in the frontopolar cortex between OCD patients with and without TD during Verbal Fluency Task and ToL performance. Brain activation in the dorsolateral prefrontal cortex (DLPFC) during the ToL Task in OCD patients with TD exerted a direct causal effect on the severity of compulsions. In addition, we detected a direct causal effect of the severity of obsessions in OCD patients without TD on brain activation in the DLPFC during the ToL Task.Brain activation in the frontopolar cortex exhibits different hemodynamics depending on the task, and DLPFC function may play a different role in the neural basis of developing OCD symptoms between OCD patients with and without TD.

  View details for DOI 10.1111/pcn.13207

  View details for PubMedID 33543818

• Acute impact of COVID-19 pandemic on phenomenological features in fully or partially remitted patients with obsessive-compulsive disorder. Psychiatry and clinical neurosciences Matsunaga, H., Mukai, K., Yamanishi, K. 2020; 74 (10): 565-566

  View details for DOI 10.1111/pcn.13119

  View details for PubMedID 32697002

  View details for PubMedCentralID PMC7404884

• Analysis of genes linked to depressive-like behaviors in interleukin-18-deficient mice: Gene expression profiles in the brain. Biomedical reports Yamanishi, K., Hashimoto, T., Miyauchi, M., Mukai, K., Ikubo, K., Uwa, N., Watanabe, Y., Ikawa, T., Okuzaki, D., Okamura, H., Yamanishi, H., Matsunaga, H. 2020; 12 (1): 3-10

  Abstract

  Interleukin (IL)-18 is an interferon γ-inducing inflammatory cytokine associated with function of the immune system and other physiological functions. IL-18-deficient (Il18 -/-) mice exhibit obesity, dyslipidemia, non-alcoholic steatohepatitis and depressive-like behavioral changes. Therefore, IL-18 has a number of important roles associated with immunity, energy homeostasis and psychiatric conditions. In the present study, gene expression in the brains of Il18 -/- mice was analyzed to identify genes associated with the depressive-like behaviors and other impairments displayed by Il18 -/- mice. Using whole genome microarray analysis, gene expression patterns in the brains of Il18 +/+ and Il18 -/- mice at 6 and 12 weeks of age were examined and compared. Subsequently, genes were categorized using Ingenuity® Pathway Analysis (IPA). At 12 weeks of age, 2,805 genes were identified using microarray analysis. Genes related to 'Major depression' and 'Depressive disorders' were identified by IPA core analysis, and 13 genes associated with depression were isolated. Among these genes, fibroblast growth factor receptor 1 (Fgfr1); protein tyrosine phosphatase, non-receptor type 1 (Ptpn1); and urocortin 3 (Ucn3) were classed as depression-inducing and the other genes were considered depression-suppressing genes. Subsequently, the interactions between the microarray results at 6 weeks of age and the above three depression-inducing genes were analyzed to search for effector genes of depression at 12 weeks of age. This analysis identified cyclin D1 (Ccnd1) and NADPH oxidase 4 (Nox4). The microarray analysis results were correlated with the results of reverse transcription-quantitative PCR (RT-qPCR). Overall, the results suggest that Fgfr1, Ptpn1 and Ucn3 may be involved in depression-like changes and Ccnd1 and Nox4 regulate these three genes in IL-18-deficient mice.

  View details for DOI 10.3892/br.2019.1259

  View details for PubMedID 31839943

  View details for PubMedCentralID PMC6906543

• Impaired function of aorta and perivascular adipose tissue in IL-18-deficient mice. American journal of physiology. Heart and circulatory physiology Li, W., Jin, D., Takai, S., Hayakawa, T., Ogata, J., Yamanishi, K., Yamanishi, H., Okamura, H. 2019; 317 (5): H1142-H1156

  Abstract

  IL-18 is ubiquitously produced by both hematopoietic and non-hematopoietic cells. The present study examined the thoracic aorta, including the surrounding perivascular adipose tissue (PVAT), of IL-18KO mice from functional and histological perspectives. IL-18KO mice exhibited raised blood pressure compared with wild-type mice. Echocardiographic examination showed a thickened vascular wall and narrowed vascular diameter of the aorta. Examination by the Magnus test demonstrated dysfunction of endothelial cells (ECs) in the IL-18KO thoracic aorta and impairment of the anticontractile function of IL-18KO PVAT. Histological examination showed no inflammatory lesions in the aorta but indicated progressive fibrosis in the vessel and conversion of PVAT from brown adipose tissue-like features to white adipose tissue-like features. Electron microscopic observation suggested several deformed mitochondria in the aorta and vacuole-like structures in ECs from IL-18KO mice. In addition, activity of complex IV was lower and production of reactive oxygen species was augmented in the mitochondria of IL-18KO aorta. Although expression of LC3 B was higher, rapamycin-induced autophagy flux was impaired in the IL-18KO PVAT. Moreover, Western blot analysis revealed that LAMP 1/2 was increased in IL-18KO PVAT, and measurement of cathepsin-D activity indicated decreased levels in IL-18KO PVAT. The IL-18KO thoracic aorta thus showed defects in physiological functions related to histological alterations, and the inflammasome/IL-18 system was suggested to play a protective role in cardiovascular cells, probably through quality control of mitochondria via promotion of autophagosome/autophagolysosome formation.NEW & NOTEWORTHY IL-18 deficiency caused aortic abnormalities in terms of morphology and functions in parallel with an accumulation of damaged mitochondria and anomalous turnover of protein complexes, such as PGC-1 and LAMP1 and -2 in PVAT. These findings suggested that IL-18 plays roles in maintaining the homeostasis of vessels and PVAT around the aorta, possibly by promoting autophagy.

  View details for DOI 10.1152/ajpheart.00813.2018

  View details for PubMedID 31518161

• A new approach to identifying hypertension-associated genes in the mesenteric artery of spontaneously hypertensive rats and stroke-prone spontaneously hypertensive rats. Journal of hypertension Ikawa, T., Watanabe, Y., Okuzaki, D., Goto, N., Okamura, N., Yamanishi, K., Higashino, T., Yamanishi, H., Okamura, H., Higashino, H. 2019; 37 (8): 1644-1656

  Abstract

  Hypertension is one of the most prevalent diseases in humans who live a modern lifestyle. Alongside more effective care, clarification of the genetic background of hypertension is urgently required. Gene expression in mesenteric resistance arteries of spontaneously hypertensive rats (SHR), stroke-prone SHR (SHRSP) and two types of renal hypertensive Wistar Kyoto rats (WKY), two kidneys and one clip renal hypertensive rat (2K1C) and one kidney and one clip renal hypertensive rat (1K1C), was compared using DNA microarrays.We used a simultaneous equation and comparative selection method to identify genes associated with hypertension using the Reactome analysis tool and GenBank database.The expression of 298 genes was altered between SHR and WKY (44 upregulated and 254 downregulated), while the expression of 290 genes was altered between SHRSP and WKY (83 upregulated and 207 downregulated). For SHRSP versus SHR, the expression of 60 genes was altered (36 upregulated and 24 downregulated). Several genes expressed in SHR and SHRSP were also expressed in the renovascular hypertensive 2K1C and 1K1C rats, indicative of the existence of hyper-renin and/or hypervolemic pathophysiological changes in SHR and SHRSP.The overexpression of Kcnq1, Crlf1, Alb and Xirp1 and the inhibition of Galr2, Kcnh1, Ache, Chrm2 and Slc5a7 expression may indicate that a relationship exists between these genes and the cause and/or worsening of hypertension in SHR and SHRSP.

  View details for DOI 10.1097/HJH.0000000000002083

  View details for PubMedID 30882592

  View details for PubMedCentralID PMC6615961

• Interleukin-18-deficient mice develop hippocampal abnormalities related to possible depressive-like behaviors. Neuroscience Yamanishi, K., Doe, N., Mukai, K., Ikubo, K., Hashimoto, T., Uwa, N., Sumida, M., El-Darawish, Y., Gamachi, N., Li, W., Kuwahara-Otani, S., Maeda, S., Watanabe, Y., Hayakawa, T., Yamanishi, H., Matsuyama, T., Yagi, H., Okamura, H., Matsunaga, H. 2019; 408: 147-160

  Abstract

  Interleukin-18 (IL-18) is an inflammatory cytokine linked to major depressive disorder (MDD). MDD is closely related to metabolic disorders, such as diabetes mellitus (DM) and obesity. Moreover, DM is associated with cognitive impairment and promotes apoptosis of hippocampal cells by activating pro-apoptotic and inhibiting anti-apoptotic factors. IL-18-deficient (Il18-/-) mice are obese and have DM. Therefore, we hypothesized a close relationship between IL-18 and death of hippocampal cells, affecting neurogenesis related to behavioral changes such as MDD. Il18-/- male mice were generated on the C57Bl/6 background and Il18+/+ mice were used as controls. Behavioral, histopathological, and molecular responses, as well as responses to intracerebral recombinant IL-18 administration, were examined. Compared with Il18+/+ mice, Il18-/- mice had impaired learning and memory and exhibited lower motivation. In the Il18-/- mice, degenerated mitochondria were detected in synaptic terminals in the molecular layer, the polymorphic layer, and in mossy fibers in the dentate gyrus, suggesting mitochondrial abnormalities. Because of the degeneration of mitochondria in the dentate gyrus, in which pro-apoptotic molecules were upregulated and anti-apoptotic factors were decreased, apoptosis inducers were not cleaved, indicating inhibition of apoptosis. In addition, neurogenesis in the dentate gyrus and the maturity of neuronal cells were decreased in the Il18-/- mice, while intracerebral administration of recombinant IL-18 promoted significant recovery of neurogenesis. Our findings suggested that IL-18 was indispensable for mitochondrial homeostasis, sustaining clearance of degenerative neural cells, and supporting neurogenesis, normal neuronal maturation and hippocampal function.

  View details for DOI 10.1016/j.neuroscience.2019.04.003

  View details for PubMedID 30981863

• Deficiency in interleukin-18 promotes differentiation of brown adipose tissue resulting in fat accumulation despite dyslipidemia. Journal of translational medicine Yamanishi, K., Maeda, S., Kuwahara-Otani, S., Hashimoto, T., Ikubo, K., Mukai, K., Nakasho, K., Gamachi, N., El-Darawish, Y., Li, W., Okuzaki, D., Watanabe, Y., Yamanishi, H., Okamura, H., Matsunaga, H. 2018; 16 (1): 314

  Abstract

  The cytokine, interleukin-18 (IL-18), was originally identified as an interferon-γ-inducing proinflammatory factor; however, there is increasing evidence suggesting that it has non-immunological effects on physiological functions. We have previously investigated the potential pathophysiological relationship between IL-18 and dyslipidemia, non-alcoholic fatty liver disease and non-alcoholic steatohepatitis, which were mediated by lipid energy imbalance. Therefore, herein we focused on brown adipocytes (BAs) and brown adipose tissue (BAT) related to energy consumption as non-shivering thermogenesis.Il18-/- male mice were generated on the C57Bl/6 background, and littermate C57Bl/6 Il18+/+ male mice were used as controls. To reveal the direct effect of IL-18, primary cell cultures derived from both mice were established. Moreover, for molecular analysis, microarray, quantitative reverse transcription PCR and western blotting were performed using 6 and 12 weeks old mice. To evaluate the short- and long-term effects of IL-18 on BAT, recombinant IL-18 was administered for 2 and 12 weeks, respectively.Compared with Il18+/+ mice, BAT of Il18-/- mice showed earlier differentiation and lipid accumulation. To examine the direct effect of IL-18 on BAT, BA cell cultures were established. Myogenic factor 5-expressing adipose precursor cells were extracted from Il18+/+ and Il18-/- mice. PR domain containing 16 (PRDM16), a differentiation inducer, was strongly expressed in Il18-/- BAs, and uncoupling protein 1, a thermogenic and differentiation marker, was upregulated, resulting in the promotion of BA differentiation. Moreover, PRDM16-dependent and independent molecules related to BAT function, such as fibroblast growth factor 21, were activated. These findings were confirmed by comparing Il18+/+ and Il18-/- mice at 6 and 12 weeks of age. Additional analyses of the molecular mechanisms influencing the 'Quantity of adipocytes' identified three associated genes, apolipoprotein C3 (Apoc3), insulin-induced gene 1 (Insig1) and vitamin D (1,25-dihydroxyvitamin D3) receptor (Vdr). Intravenous administration of IL-18 not only significantly improved the expression of some of these genes, but it also significantly decreased the adipocytes' size.This study demonstrated the critical function of IL-18 in differentiation and lipid metabolism in BAs. Furthermore, IL-18 may contribute to novel treatments by improving the energy imbalance.

  View details for DOI 10.1186/s12967-018-1684-3

  View details for PubMedID 30453990

  View details for PubMedCentralID PMC6245626

• Frontline Science: IL-18 primes murine NK cells for proliferation by promoting protein synthesis, survival, and autophagy. Journal of leukocyte biology El-Darawish, Y., Li, W., Yamanishi, K., Pencheva, M., Oka, N., Yamanishi, H., Matsuyama, T., Tanaka, Y., Minato, N., Okamura, H. 2018; 104 (2): 253-264

  Abstract

  Combined stimulation by IL-2 and IL-18 effectively promotes proliferation of NK cells, whereas singular stimulation does not. In this study, synergistic effects of these cytokines on NK cells proliferation was analyzed, focusing on the roles of IL-18. In splenic resting NK cells from IL-18KO mice, IL-18 rapidly activated NF-κB independently of IL-2, and activated or up-regulated various molecules downstream of PI3K/AKT and mTOR, including S6, Bcl-XL, ATG5, and LC3II, accompanying increases in cell growth and survival. Thus, IL-18 alone was revealed to augment various cellular processes (gene transcription, protein synthesis, survival) in the absence or presence of IL-2. Notably, combined IL-18 and IL-2 promoted autophagosome formation. In addition, priming NK cells with IL-18 augmented IL-2R, especially CD25, and enabled cells to respond to IL-2, resulting in activation of STAT3 and STAT5, followed by increase of cyclin B1 leading to proliferation. However, IL-2 alone failed to activate STAT3 or STAT5 in resting IL18KO NK cells. These results clarify the distinct roles of IL-2 and IL-18 in NK cell proliferation, and the intrinsic roles of IL-18 in various cellular processes, suggesting a range of functions of IL-18 expressed in an array of nonhematopoietic cells.

  View details for DOI 10.1002/JLB.1HI1017-396RR

  View details for PubMedID 29603367

• Ten-year follow-up study of Japanese patients with obsessive-compulsive disorder. Psychiatry and clinical neurosciences Nakajima, A., Matsuura, N., Mukai, K., Yamanishi, K., Yamada, H., Maebayashi, K., Hayashida, K., Matsunaga, H. 2018; 72 (7): 502-512

  Abstract

  Obsessive-compulsive disorder (OCD) is a well-known chronic illness. This study retrospectively investigated 10-year outcomes and associated clinical factors in Japanese OCD patients. We focused on the impact of several sociocultural factors, including medical expenses and insurance systems specific to each country, on the differences or biases in follow-up procedures of OCD.Seventy-nine patients diagnosed with OCD who received a standardized combination of treatments for 10 continuous years were divided into three groups according to their improvement rates on the Yale-Brown Obsessive-Compulsive Scale after 10 years of treatment.A survival analysis revealed that the rate of patients achieving full remission increased every year. Following 10 years of treatment, 56% of OCD patients experienced 'full remission' for at least 1 year. Consequently, 48% exhibited full remission, and 37% exhibited partial remission at the end-point of this study. We identified several factors that were predictive of poorer outcomes, including lower Global Assessment of Functioning Scale scores and the presence of hoarding symptoms or involvement behaviors. In addition, improvement rates after 1 year significantly predicted better 10-year outcomes.Our findings highlight the transcultural nature of long-term outcomes of OCD treatment, which appear to be independent of sociocultural differences.

  View details for DOI 10.1111/pcn.12661

  View details for PubMedID 29652103

• Physiological and molecular effects of interleukin-18 administration on the mouse kidney. Journal of translational medicine Yamanishi, K., Mukai, K., Hashimoto, T., Ikubo, K., Nakasho, K., El-Darawish, Y., Li, W., Okuzaki, D., Watanabe, Y., Hayakawa, T., Nojima, H., Yamanishi, H., Okamura, H., Matsunaga, H. 2018; 16 (1): 51

  Abstract

  The cytokine interleukin-18 was originally identified as an interferon-γ-inducing proinflammatory factor; however, there is increasing evidence to suggest that it has non-immunological effects on physiological functions. We previously investigated the potential pathophysiological relationship between interleukin-18 and dyslipidemia, non-alcoholic fatty liver disease, and non-alcoholic steatohepatitis, and suggested interleukin-18 as a possible novel treatment for not only these diseases but also for cancer immunotherapy. Before clinical application, the effects of interleukin-18 on the kidney need to be determined. In the current study, we examined the kidney of interleukin-18 knockout (Il18-/-) mice and the effects of interleukin-18 on the kidney following intravenous administration of recombinant interleukin-18.Il18-/- male mice were generated on the C57Bl/6 background and littermate C57Bl/6 Il18+/+ male mice were used as controls. To assess kidney damage, serum creatinine and blood urea nitrogen levels were measured and histopathological analysis was performed. For molecular analysis, microarray and quantitative reverse transcription PCR was performed using mice 6 and 12 weeks old. To evaluate the short- and long-term effects of interleukin-18 on the kidney, recombinant interleukin-18 was administered for 2 and 12 weeks, respectively.Compared with Il18+/+ mice, Il18-/- mice developed kidney failure in their youth-6 weeks of age, but the condition was observed to improve as the mice aged, even though dyslipidemia, arteriosclerosis, and higher insulin resistance occurred. Analyses of potential molecular mechanisms involved in the onset of early kidney failure in Il18-/- mice identified a number of associated genes, such as Itgam, Nov, and Ppard. Intravenous administration of recombinant interleukin-18 over both the short and long term showed no effects on the kidney despite significant improvement in metabolic diseases.Short- and long-term administration of interleukin-18 appeared to have no adverse effects on the kidney in these mice, suggesting that administration may be a safe and novel treatment for metabolic diseases and cancer.

  View details for DOI 10.1186/s12967-018-1426-6

  View details for PubMedID 29514661

  View details for PubMedCentralID PMC5842592

• Molecular analysis of the mouse brain exposed to chronic mild stress: The influence of hepatocyte nuclear factor 4α on physiological homeostasis. Molecular medicine reports Ikubo, K., Yamanishi, K., Doe, N., Hashimoto, T., Sumida, M., Watanabe, Y., El-Darawish, Y., Li, W., Okamura, H., Yamanishi, H., Matsunaga, H. 2017; 16 (1): 301-309

  Abstract

  Major depressive disorder (MDD) is a prevalent disorder that causes considerable disability in social functioning and is a risk factor for physical diseases. Recent clinical reports have demonstrated a marked association between MDD and physiological dyshomeostasis induced by metabolic disorders, including diabetes, hormone abnormalities and autoimmune diseases. The authors of the present study have previously analyzed comparative gene expression profiles in the prefrontal cortex (PFC) of a chronic mild stress (CMS) animal model of MDD. Hepatocyte nuclear factor 4α (Hnf4α) was identified as a central regulator that exerted significant influence on genes associated with physiological homeostasis. The aim of the present study was to investigate: i) the molecular mechanism of the depressive state in the PFC, and ii) the involvement of genes extracted from the comparative gene expression profiles, particularly those applicable to MDD in clinical practice. Core analysis of the previous PFC microarray results was performed using Ingenuity Pathway Analysis (IPA). Subsequently, IPA was used to search for molecules that are regulated by Hnf4α, and exist in the PFC and serum. From the core analysis, 5 genes that are associated with cell death and are expressed in the cortex were selected. Four of the extracted genes, insulin‑like growth factor 1, transthyretin, serpin family A member 3 and plasminogen, were markedly affected by Hnf4α. S100 calcium‑binding protein A9 (S100a9) and α2-HS-glycoprotein (Ahsg) were also chosen as they exist in serum and are also affected by Hnf4α. A significant group difference in the expression of these two genes was detected in the PFC, thalamus and hippocampus. The protein levels of AHSG and S100A9 in the PFC and hippocampus of the CMS group increased significantly when compared with the control group. These findings support the close association of Hnf4α (through genes such as S100a9 and Ahsg) with the development of various diseases induced by deregulation of physiological homeostasis during the progression of MDD.

  View details for DOI 10.3892/mmr.2017.6577

  View details for PubMedID 28498421

• Interleukin-18 and its receptor are expressed in gonadotropin-releasing hormone neurons of mouse and rat forebrain. Neuroscience letters Kuwahara-Otani, S., Maeda, S., Kobayashi, K., Minato, Y., Tanaka, K., Yamanishi, K., Hata, M., Li, W., Hayakawa, T., Noguchi, K., Okamura, H., Yagi, H. 2017; 650: 33-37

  Abstract

  Interleukin-18 (IL-18) is a pro-inflammatory cytokine and an important mediator of peripheral inflammation and host immune response. IL-18 functions through its binding with the IL-18 receptor (IL-18R), which consists of two chains, an IL-18-binding α chain (IL-18Rα) and a signaling β chain. IL-18 and IL-18R are expressed in the brain; however, limited information is available on IL-18R expression and the role of IL-18 in neurosecretory cells. In the present study, we used immunohistochemical techniques to investigate the distribution of IL-18Rα and IL-18 in the hypothalamus of male mice and rats. IL-18Rα-positive and IL-18-positive perikarya and fibers were found scattered throughout the medial septal nucleus, the nuclei of the vertical and horizontal limbs of the diagonal band, the organum vasculosum of the laminae terminalis, the preoptic area, and the anterior hypothalamic area. It is well known that gonadotropin-releasing hormone (GnRH) neuronal somata and/or fibers are found in these regions. Therefore, we performed double-label immunofluorescence for IL-18Rα/IL-18 and GnRH. IL-18Rα was expressed in approximately 60% of GnRH-immunopositive perikarya, and IL-18 was distributed in all GnRH-immunopositive perikarya. These observations suggest that IL-18 exerts direct effects upon the GnRH neuron via IL-18Rα and acts on GnRH neurons through an autocrine or paracrine pathway.

  View details for DOI 10.1016/j.neulet.2017.03.051

  View details for PubMedID 28373090

• Fine structure of interleukin 18 (IL-18) receptor-immunoreactive neurons in the retrosplenial cortex and its changes in IL18 knockout mice. Journal of chemical neuroanatomy Hayakawa, T., Hata, M., Kuwahara-Otani, S., Yamanishi, K., Yagi, H., Okamura, H. 2016; 78: 96-101

  Abstract

  Interleukin 18 (IL-18) participates in the inflammatory immune response of lymphocytes. Delay in learning or memory are common in the IL-18 knockout mouse. Many IL-18-immunoreactive neurons are found in the retrosplenial cortex (RSC) and the subiculum. These neurons also contain the IL-18 receptor. We determined the location and the ultrastructure of the IL-18 receptor-immunoreactive neurons in the RSC and observed changes in the IL-18 receptor-immunoreactive neurons of the IL-18 knockout mouse. The IL-18 receptor-immunoreactive neurons were found specifically in layer V of the granular RSC. They were medium-sized neurons with a light oval nucleus and had little cytoplasm with many free ribosomes, rough endoplasmic reticulum and many mitochondria, but no Nissl bodies. The number of axosomatic terminals was about six per section. The IL-18 receptor-immunoreactive neurons were not found in the RSC in the IL-18 knockout mouse at 5 or 9 weeks of age. However, many small electron-dense neurons were found in layer V. Both the nucleus and cytoplasm were electron-dense, but not necrotic. The mitochondria and rough endoplasmic reticulum were swollen. The IL-18 receptor-immunoreactive neurons were presumed to be degenerating. The degeneration of the IL18-receptor-immunoreactive neurons in the RSC may cause the abnormal behaviors of the IL-18 knockout mice.

  View details for DOI 10.1016/j.jchemneu.2016.08.009

  View details for PubMedID 27593389

• Dysfunction of mitochondria and deformed gap junctions in the heart of IL-18-deficient mice. American journal of physiology. Heart and circulatory physiology Li, W., Jin, D., Hata, M., Takai, S., Yamanishi, K., Shen, W., El-Darawish, Y., Yamanishi, H., Okamura, H. 2016; 311 (2): H313-25

  Abstract

  Interleukin-18 (IL-18) was discovered as an interferon-γ-inducing factor and has been regarded as a proinflammatory cytokine. However, IL-18 is ubiquitously expressed both in immune/inflammatory cells and in nonimmune cells, and its biological roles have not been sufficiently elucidated. Here, we demonstrate that IL-18-deficient [IL-18 knockout (KO)] mice have heart abnormalities that may be related to impaired autophagy. In endurance running tests, IL-18KO mice ran significantly shorter distances compared with wild-type (WT) mice. Echocardiographs indicated disability in the systolic and diastolic functions of the IL-18KO mouse heart. Immunostaining of connexin 43 showed heterogeneous localization of gap junctions in the lateral membranes of the IL-18KO cardiac myocytes. Western blotting analysis revealed decreased phosphorylated connexin 43 in the IL-18KO heart. Electron microscopy revealed unusual localization of intercalated disks, swollen or damaged mitochondria, and broad, indistinct Z-lines in the IL-18KO heart. In accordance with the morphological observation, mitochondrial respiratory function, including that of complexes I and IV, was impaired, and production of reactive oxygen species was augmented in IL-18KO hearts. Notably, levels of LC3-II were markedly lower in the IL-18KO hearts than in WT hearts. In the culture of cardiac myocytes of IL-18KO neonates, exogenous IL-18 upregulated LC3-II and increased the number of intact mitochondria with high mitochondrial membrane potential. These results indicated that IL-18 has roles apart from those as a proinflammatory cytokine in cardiac myocytes and suggested that IL-18 contributes to the homeostatic maintenance of mitochondrial function and gap-junction turnover in cardiac myocytes, possibly by upregulating autophagy.

  View details for DOI 10.1152/ajpheart.00927.2015

  View details for PubMedID 27288439

• Interleukin-18-deficient mice develop dyslipidemia resulting in nonalcoholic fatty liver disease and steatohepatitis. Translational research : the journal of laboratory and clinical medicine Yamanishi, K., Maeda, S., Kuwahara-Otani, S., Watanabe, Y., Yoshida, M., Ikubo, K., Okuzaki, D., El-Darawish, Y., Li, W., Nakasho, K., Nojima, H., Yamanishi, H., Hayakawa, T., Okamura, H., Matsunaga, H. 2016; 173: 101-114.e7

  Abstract

  We investigated potential pathophysiological relationships between interleukin 18 (IL-18) and dyslipidemia, nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis (NASH). Compared with Il18(+/+) mice, IL-18 knockout (Il18(-/-)) mice developed hypercholesterolemia and hyper-high-density-lipoprotein-cholesterolemia as well as hypertriglyceridemia as they aged, and these disorders occurred before the manifestation of obesity and might cause secondary NASH. The analyses of molecular mechanisms involved in the onset of dyslipidemia, NAFLD, and NASH in Il18(-/-) mice identified a number of genes associated with these metabolic diseases. In addition, molecules related to circadian rhythm might affect these extracted genes. The intravenous administration of recombinant IL-18 significantly improved dyslipidemia, inhibited the body weight gain of Il18(+/+) mice, and prevented the onset of NASH. The expression of genes related to these dysfunctions was also affected by recombinant IL-18 administration. In conclusion, this study demonstrated the critical function of IL-18 in lipid metabolism and these findings might contribute to the progress of novel treatments for NAFLD or NASH.

  View details for DOI 10.1016/j.trsl.2016.03.010

  View details for PubMedID 27063959

• Augmentation of Immune Checkpoint Cancer Immunotherapy with IL18. Clinical cancer research : an official journal of the American Association for Cancer Research Ma, Z., Li, W., Yoshiya, S., Xu, Y., Hata, M., El-Darawish, Y., Markova, T., Yamanishi, K., Yamanishi, H., Tahara, H., Tanaka, Y., Okamura, H. 2016; 22 (12): 2969-80

  Abstract

  Recent clinical trials and animal models demonstrated that immune checkpoint blockade enhanced effector cell responses and tumor rejection; however, further development and improvement of cancer immunotherapy is necessary for more favorable objective responses. In this study, we examined the effect of IL18 on the antitumor effect of immune checkpoint inhibitors.We examined the effect of IL18 on the peritoneal dissemination of CT-26 cells or tail vein injection metastasis of B16/F10 cells using antiprogrammed death-1 ligand-1 (αPD-L1) and/or anti-CTL-associated antigen-4 (αCTLA-4) mAbs.Massive ascites developed after intraperitoneal inoculation of CT-26, resulting in animal death within 30 days. Treatment of mice with αPD-L1 and/or αCTLA-4 significantly prolonged their survival, and a combination of the antibodies and IL18 provided a much greater therapeutic benefit. The combination modality led to the accumulation of precursor of mature natural killer (pre-mNK) cells in the peritoneal cavity together with increased CD8(+) T and decreased CD4(+)CD25(+)Foxp3(+) T cells. Depletion of the pre-mNK cells abrogated the therapeutic effects and increased the number of CD4(+)CD25(+)Foxp3(+) T cells. The combination treatment also suppressed tail vein injection metastasis of B16/F10 cells.The results demonstrated that IL18 enhanced therapeutic effects of immune checkpoint blockade against peritoneal dissemination of carcinoma or tail vein injection metastasis of melanoma through accumulation of pre-mNK cells, memory-type CD8(+) T cells, and suppression of CD4(+)CD25(+)Foxp3(+) T cells. A combination of immune checkpoint inhibitors with IL18 may give a suggestion to the development of next-generation cancer immunotherapy. Clin Cancer Res; 22(12); 2969-80. ©2016 AACR.

  View details for DOI 10.1158/1078-0432.CCR-15-1655

  View details for PubMedID 26755531

• Genetic analysis of genes causing hypertension and stroke in spontaneously hypertensive rats: Gene expression profiles in the kidneys. International journal of molecular medicine Watanabe, Y., Yoshida, M., Yamanishi, K., Yamamoto, H., Okuzaki, D., Nojima, H., Yasunaga, T., Okamura, H., Matsunaga, H., Yamanishi, H. 2015; 36 (3): 712-24

  Abstract

  Spontaneously hypertensive rats (SHRs) and stroke-prone SHRs (SHRSP) are frequently used as models not only of essential hypertension and stroke, but also of attention-deficit hyperactivity disorder (ADHD). Normotensive Wistar-Kyoto (WKY) rats are normally used as controls in these studies. In the present study, we aimed to identify the genes causing hypertension and stroke, as well as the genes involved in ADHD using these rats. We previously analyzed gene expression profiles in the adrenal glands and brain. Since the kidneys can directly influence the functions of the cardiovascular, endocrine and sympathetic nervous systems, gene expression profiles in the kidneys of the 3 rat strains were examined using genome-wide microarray technology when the rats were 3 and 6 weeks old, a period in which rats are considered to be in a pre-hypertensive state. Gene expression profiles were compared between the SHRs and WKY rats and also between the SHRSP and SHRs. A total of 232 unique genes showing more than a 4-fold increase or less than a 4-fold decrease in expression were isolated as SHR- and SHRSP-specific genes. Candidate genes were then selected using two different web tools: the 1st tool was the Database for Annotation, Visualization and Integrated Discovery (DAVID), which was used to search for significantly enriched genes and categorized them using Gene Ontology (GO) terms, and the 2nd was Ingenuity Pathway Analysis (IPA), which was used to search for interactions among SHR- and also SHRSP‑specific genes. The analyses of SHR-specific genes using IPA revealed that B-cell CLL/lymphoma 6 (Bcl6) and SRY (sex determining region Y)-box 2 (Sox2) were possible candidate genes responsible for causing hypertension in SHRs. Similar analyses of SHRSP-specific genes revealed that angiotensinogen (Agt), angiotensin II receptor-associated protein (Agtrap) and apolipoprotein H (Apoh) were possible candidate genes responsible for triggering strokes. Since our results revealed that SHRSP-specific genes isolated from the kidneys of rats at 6 weeks of age, included 6 genes related to Huntington's disease, we discussed the genetic association between ADHD and Huntington's disease.

  View details for DOI 10.3892/ijmm.2015.2281

  View details for PubMedID 26165378

  View details for PubMedCentralID PMC4533772

• Clinically related or predictive factors and impacts on long-term treatment outcomes of involvement behaviors in patients with obsessive-compulsive disorder. Comprehensive psychiatry Yanagisawa, Y., Matsuura, N., Mukai, K., Nakajima, A., Motoyama, M., Yamanishi, K., Yamada, H., Hayashida, K., Matsunaga, H. 2015; 60: 105-13

  Abstract

  Patients with obsessive-compulsive disorder (OCD) frequently exhibit involvement behaviors (IBs) in which they accommodate their caregivers to their OCD symptoms by 1) asking them for reassurance, 2) forcing them to participate in their rituals, or 3) forcing them to assist in avoidance according to the patients' rules or demands. Such behaviors correspond to those of their caregivers, and are referred to as family accommodation (FA).We performed multifactorial comparisons between 256 OCD patients with and without IBs in order to examine the clinical characteristics and long-term treatment outcomes of OCD patients with IBs. A multiple logistic regression analysis was also performed to identify the related and predictive factors of IBs.A total of 108 out of the 256 OCD patients examined (42%) were determined to exhibit IBs. OCD patients with IBs were differentially characterized by demographic and phenomenological characteristics (e.g. female predominance and poorer insight), more severe psychopathological features (e.g. lower GAFS, higher anxiety, or a depressive status), and poorer treatment outcomes. Furthermore, the predictive factors of IBs such as being female, having a higher compulsive score, and lower GAFS were identified by logistic analyses and structural equation modeling.The presence of IBs correlated with the severe clinical features, treatment refractoriness, and poorer long-term outcome of OCD. The severity of compulsions, being female, and lower GAFS were identified as predictive factors for the presence of IBs. Similar to FA, these findings appear to support the effectiveness of early identification and family-focused intervention in the treatment of OCD patients with IBs.

  View details for DOI 10.1016/j.comppsych.2015.03.002

  View details for PubMedID 25861961

• Hepatocyte nuclear factor 4 alpha is a key factor related to depression and physiological homeostasis in the mouse brain. PloS one Yamanishi, K., Doe, N., Sumida, M., Watanabe, Y., Yoshida, M., Yamamoto, H., Xu, Y., Li, W., Yamanishi, H., Okamura, H., Matsunaga, H. 2015; 10 (3): e0119021

  Abstract

  Major depressive disorder (MDD) is a common psychiatric disorder that involves marked disabilities in global functioning, anorexia, and severe medical comorbidities. MDD is associated with not only psychological and sociocultural problems, but also pervasive physical dysfunctions such as metabolic, neurobiological and immunological abnormalities. Nevertheless, the mechanisms underlying the interactions between these factors have yet to be determined in detail. The aim of the present study was to identify the molecular mechanisms responsible for the interactions between MDD and dysregulation of physiological homeostasis, including immunological function as well as lipid metabolism, coagulation, and hormonal activity in the brain. We generated depression-like behavior in mice using chronic mild stress (CMS) as a model of depression. We compared the gene expression profiles in the prefrontal cortex (PFC) of CMS and control mice using microarrays. We subsequently categorized genes using two web-based bioinformatics applications: Ingenuity Pathway Analysis and The Database for Annotation, Visualization, and Integrated Discovery. We then confirmed significant group-differences by analyzing mRNA and protein expression levels not only in the PFC, but also in the thalamus and hippocampus. These web tools revealed that hepatocyte nuclear factor 4 alpha (Hnf4a) may exert direct effects on various genes specifically associated with amine synthesis, such as genes involved in serotonin metabolism and related immunological functions. Moreover, these genes may influence lipid metabolism, coagulation, and hormonal activity. We also confirmed the significant effects of Hnf4a on both mRNA and protein expression levels in the brain. These results suggest that Hnf4a may have a critical influence on physiological homeostasis under depressive states, and may be associated with the mechanisms responsible for the interactions between MDD and the dysregulation of physiological homeostasis in humans.

  View details for DOI 10.1371/journal.pone.0119021

  View details for PubMedID 25774879

  View details for PubMedCentralID PMC4361552

• The impacts of elevated autism spectrum disorder traits on clinical and psychosocial features and long-term treatment outcome in adult patients with obsessive-compulsive disorder. Comprehensive psychiatry Mito, H., Matsuura, N., Mukai, K., Yanagisawa, Y., Nakajima, A., Motoyama, M., Arikawa, A., Yamanishi, K., Matsunaga, H. 2014; 55 (7): 1526-33

  Abstract

  While a close relation between obsessive-compulsive disorder (OCD) and autism spectrum disorder (ASD) has been pointed out, there are few studies that have investigated whether highly elevated ASD traits may have significant impacts on clinical and psychosocial features as well as long-term treatment outcome in adult OCD patients.We assessed ASD traits using the Autism Spectrum Quotient (AQ) in 81 Japanese patients with OCD. The relation between degree of ASD traits and clinical and psychosocial variables and the 48-week treatment outcomes was analyzed in the subjects.A substantial proportion of the subjects showed higher ASD traits (35%) with more severe depressive or pervasive anxiety status, and social impairments and lower QOL compared to other OCD individuals. However, elevated ASD traits may exert rather smaller impact on the OCD phenomenology along with on the long-term treatment outcome than expected.Elevated ASD traits may further emphasize the general psychopathological and socio-dysfunctional features rather than clinical aspects associated with OCD. Co-existing depressive or anxious symptom severity may further exacerbate the core-deficits related to ASD pathology. Thus the assessment of ASD traits should be important for understanding the clinical and psychosocial features and treatment responses in OCD patients.

  View details for DOI 10.1016/j.comppsych.2014.05.005

  View details for PubMedID 24957957

• Chymase inhibition improves vascular dysfunction and survival in stroke-prone spontaneously hypertensive rats. Journal of hypertension Takai, S., Jin, D., Chen, H., Li, W., Yamamoto, H., Yamanishi, K., Miyazaki, M., Higashino, H., Yamanishi, H., Okamura, H. 2014; 32 (8): 1637-48; discussion 1649

  Abstract

  To clarify the role of chymase in hypertension, we evaluated the effect of a chymase inhibitor, TY-51469, on vascular dysfunction and survival in stroke-prone spontaneously hypertensive rats (SHR-SP).SHR-SP were treated with TY-51469 (1 mg/kg per day) or placebo from 4 to 12 weeks old or until death. Wistar-Kyoto rats were used as a normal group.SBP was significantly higher in both the placebo and TY-51469 groups than in the normal group, but there was no significant difference between the two treatment groups. Plasma renin, angiotensin-converting enzyme activity and angiotensin II levels were not different between the placebo and TY-51469 groups. In contrast, vascular chymase-like activity was significantly higher in the placebo than in the normal group, but it was reduced by TY-51469. Acetylcholine-induced vascular relaxation was significantly higher in the TY-51469 group than in the placebo group. There was significant augmentation of the number of monocytes/macrophages and matrix metalloproteinase-9 activity in aortic tissue from the placebo group compared with the normal group, and these changes were attenuated by TY-51469. There were also significant increases in mRNA levels of monocyte chemoattractant protein-1 and tumor necrosis factor-α in the placebo group that were attenuated by TY-51469. Cumulative survival was significantly prolonged in the TY-51469 group compared with the placebo group.Chymase might play an important role in vascular dysfunction via augmentation both of matrix metalloproteinase-9 activity and monocyte/macrophage accumulation in SHR-SP, and its inhibition may be useful for preventing vascular remodeling and prolonging survival.

  View details for DOI 10.1097/HJH.0000000000000231

  View details for PubMedID 24886822

• Analysis of genes causing hypertension and stroke in spontaneously hypertensive rats: gene expression profiles in the brain. International journal of molecular medicine Yoshida, M., Watanabe, Y., Yamanishi, K., Yamashita, A., Yamamoto, H., Okuzaki, D., Shimada, K., Nojima, H., Yasunaga, T., Okamura, H., Matsunaga, H., Yamanishi, H. 2014; 33 (4): 887-96

  Abstract

  Spontaneously hypertensive rats (SHR) and stroke-prone SHR (SHRSP) are frequently used as rat models not only of essential hypertension and stroke, but also of attention-deficit hyperactivity disorder (ADHD). Normotensive Wistar-Kyoto rats (WKY) are used as the control rats in these cases. An increasing number of studies has demonstrated the critical role of the central nervous system in the development and maintenance of hypertension. In a previous study, we analyzed the gene expression profiles in the adrenal glands of SHR. Thus, in this study, we analyzed gene expression profiles in the brains of SHR in order to identify the genes responsible for causing hypertension and stroke, as well as those involved in ADHD. Using genome-wide microarray technology, we examined the gene expression profiles in the brains of 3 rat strains (SHR, SHRSP and WKY) when the rats were 3 and 6 weeks of age, a period in which the rats are considered to be in a pre-hypertensive state. Gene expression profiles in the brain were compared between SHR and WKY, and between SHRSP and SHR. A total of 179 genes showing a >4- or <-4-fold change in expression were isolated, and candidate genes were selected using two different web tools: the first tool was the Database for Annotation, Visualization and Integrated Discovery (DAVID), which was used to search for significantly enriched genes, and categorized them using Gene Ontology (GO) terms, and the second was the network explorer of Ingenuity Pathway Analysis (IPA), which was used to search for interaction networks among SHR- and SHRSP-specific genes. The IPA of SHR-specific genes revealed that prostaglandin E receptor 4 (Ptger4) is one of the candidate genes responsible for causing hypertension in SHR, and that albumin (Alb) and chymase 1 (Cma1) are also responsible for causing hypertension in SHR in the presence of angiotensinogen (Agt). Similar analyses of SHRSP-specific genes revealed that the angiotensin II receptor-associated gene (Agtrap) interacts with the FBJ osteosarcoma oncogene (Fos), and with the angiotensin II receptor type-1b (Agtr1b). As Agtrap and Agtr1b not only participate in the 'uptake of norepinephrine' and 'blood pressure', but also in the 'behavior' of SHRSP at 6 weeks of age, our data demonstrate a close association between hypertension and ADHD.

  View details for DOI 10.3892/ijmm.2014.1631

  View details for PubMedID 24452243

• Regulation of development of CD56 bright CD11c + NK-like cells with helper function by IL-18. PloS one Li, W., Okuda, A., Yamamoto, H., Yamanishi, K., Terada, N., Yamanishi, H., Tanaka, Y., Okamura, H. 2013; 8 (12): e82586

  Abstract

  Human γδ T cells augment host defense against tumors and infections, and might have a therapeutic potential in immunotherapy. However, mechanism of γδ T cell proliferation is unclear, and therefore it is difficult to prepare sufficient numbers of γδ T cells for clinical immunotherapy. Recently, natural killer (NK)-like CD56(bright)CD11c(+) cells were shown to promote the proliferation of γδ T cells in an IL-18-dependent manner. In this study, we demonstrated that the NK-like CD56(bright)CD11c(+) cells could directly interact with γδ T cells to promote their sustained expansion, while conventional dendritic cells (DCs), IFN-α-induced DCs, plasmacytoid DCs or monocytes did not. We also examined the cellular mechanism underlying the regulation of CD56(bright)CD11c(+) cells. CD14(+) monocytes pre-incubated with IL-2/IL-18 formed intensive interactions with CD56(int)CD11c(+) cells to promote their differentiation to CD56(bright)CD11c(+) cells with helper function. The development of CD56(bright)CD11c(+) cells was suppressed in an IFN-α dependent manner. These results indicate that CD14(+) monocytes pretreated with IL-2/IL-18, but neither DCs nor monocytes, play a determining role on the development and proliferation of CD56(bright)CD11c(+) cells, which in turn modulate the expansion of γδ T cells. CD56(bright)CD11c(+) NK-like cells may be a novel target for immunotherapy utilizing γδ T cells, by overcoming the limitation of γδ T cells proliferation.

  View details for DOI 10.1371/journal.pone.0082586

  View details for PubMedID 24376549

  View details for PubMedCentralID PMC3869690

• Genetic analysis of genes causing hypertension and stroke in spontaneously hypertensive rats. International journal of molecular medicine Yamamoto, H., Okuzaki, D., Yamanishi, K., Xu, Y., Watanabe, Y., Yoshida, M., Yamashita, A., Goto, N., Nishiguchi, S., Shimada, K., Nojima, H., Yasunaga, T., Okamura, H., Matsunaga, H., Yamanishi, H. 2013; 31 (5): 1057-65

  Abstract

  Spontaneously hypertensive rats (SHR) and stroke-prone SHR (SHRSP) are frequently used as model rats not only in studies of essential hypertension and stroke, but also in studies of attention deficit hyperactivity disorder (ADHD). Normotensive Wistar-Kyoto rats (WKY) are normally used as controls in these studies. In this study, using these rats, we aimed to identify the genes causing hypertension and stroke, as well as the genes involved in ADHD. Since adrenal gland products can directly influence cardiovascular, endocrine and sympathetic nervous system functions, gene expression profiles in the adrenal glands of the 3 rat strains were examined using genome-wide microarray technology when the rats were 3 and 6 weeks of age, a period in which the rats are considered to be in a pre-hypertensive state. Gene expression profiles were compared between SHR and WKY and between SHRSP and SHR. A total of 353 genes showing more than a 4-fold increase or less than a 4-fold decrease in expression were isolated and candidate genes were selected as significantly enriched genes. SHR-specific genes isolated when the rats were 3 weeks of age contained 12 enriched genes related to transcriptional regulatory activity and those isolated when the rats were 6 weeks of age contained 6 enriched genes related to the regulation of blood pressure. SHRSP-specific genes isolated when the rats were 3 weeks of age contained 4 enriched genes related to the regulation of blood pressure and those isolated when the rats were 6 weeks of age contained 4 enriched genes related to the response to steroid hormone stimulus. Ingenuity pathway analysis of enriched SHR-specific genes revealed that 2 transcriptional regulators, cAMP responsive element modulator (Crem) and Fos-like antigen 1 (Fosl1), interact with blood pressure-regulating genes, such as neurotensin (Nts), apelin (Apln) and epoxide hydrolase 2, cytoplasmic (Ephx2). Similar analyses of SHRSP-specific genes revealed that angiotensinogen (Agt), one of the blood pressure-regulating genes, plays pivotal roles among SHRSP-specific genes. Moreover, genes associated with ADHD, such as low density lipoprotein receptor (Ldlr) and Crem, are discussed.

  View details for DOI 10.3892/ijmm.2013.1304

  View details for PubMedID 23525202