京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20237号 / 医博第4196号 / 新制||医||1019(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 松原 和夫, 教授 渡邉 大, 教授 伊佐 正 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

ABSTRACT The aim of my thesis is to further investigate the mechanisms underlying inflammatory muscle pain. Despite numerous studies investigating the mechanisms of inflammatory hyperalgesia, little is known of the mechanisms underlying inflammatory muscle hyperalgesia. Using rats as experimental animals, I investigated inflammatory hyperalgesia in muscle and compared it to that of inflamed cutaneous tissue. I injected carrageenan, a plant-origin polysaccharide, into leg muscle and into the hind paw of rats, and measured the behavioural response, as well as cytokine changes, in both plasma and inflamed tissue. Carrageenan induced inflammatory hyperalgesia but the cytokine cascade was not the same in muscle and cutaneous tissue. At no time following carrageenan injection was muscle tumour necrosis factor alpha (TNF-&) concentration elevated above that of muscle injected with saline. TNF-& is a key inflammatory mediator in cutaneous tissue, but apparently not in muscle. Interleukin-1) (IL-1)) and interleukin-6 concentrations also were different during muscle inflammation compared to those of cutaneous inflammation. IL-1) and IL- 6 concentrations, following carrageenan injection, were elevated later in muscle compared to in cutaneous tissue. IL-1) is a potent sensitizer of nociceptors in cutaneous tissue, and also may play an important role in sustaining muscle pain, but it is unlikely to be an initiator of the inflammatory muscle hyperalgesia. In the course of comparing muscle hyperalgesia and cutaneous hyperalgesia, I aimed to identify whether these differences in cytokine concentrations were unique to muscle tissue or if similar differences in cytokine concentrations existed between the hind paw and other cutaneous sites. To explore an alternative cutaneous tissue site, I injected carrageenan into the rat tail and measured the behavioural response, changes in cytokine concentrations and histological changes. Elevations of pro-inflammatory cytokines occurred concurrently with the infiltration of leukocytes into the inflamed tail tissue with the thermal and mechanical hyperalgesia similar to that found in the hind paw. Different mechanisms therefore appear to underlie muscle and cutaneous inflammatory hyperalgesia, regardless of the site used to investigate cutaneous inflammation. One of the consequences of the poor understanding of muscle pain is the lack of a reliable regimen for treating human muscle pain, including delayedonset muscle soreness (DOMS). DOMS, which has a partial inflammatory pathogenesis, is not relieved by non-selective cyclo-oxygenase inhibitors. This phenomenon may be that prostaglandins are not produced peripherally or centrally, when muscle tissue is damaged. I investigated the effect of inhibiting cyclo-oxygenase-2, the isoform released during inflammation, on DOMS in human volunteers. I found that rofecoxib, a cyclo-oxygenase-2 inhibitor, did not attenuate DOMS and nor did tramadol, a central-acting analgesic. The neurochemical pathway underlying DOMS therefore appears to be distinct from the pathways which underlie pain and hyperalgesia in other syndromes. Future research should include investigations into the central mechanisms of muscle pain and blocking the action of IL-1) and CINC-1 both peripherally and centrally may prove a beneficial target for the treatment of clinical muscle pain.

Oxidative stress (OS) and inflammation can be detrimental to exercise performance. Antioxidants such as curcumin are shown to reduce exercise-induced OS, inflammation, muscle damage, and soreness. The purpose of this study was to examine the effect of curcumin on biomarker markers of OS (MDA, TAC), inflammation (TNF-á), muscle damage (CK) and soreness. Participants performed an exercise-induced muscle damage protocol. Before and after supplementation, subjects were randomly assigned to curcumin (1.5 g/day) or placebo for 28 days. Blood was sampled immediately before and after exercise, as well as 60 min, 24, and 48 h after exercise. No significant differences were observed for biomarkers of OS or inflammation. There was a treatment X condition interaction for CK, where CK were significantly lower post supplementation in the curcumin group (p < .0.0001). Curcumin resulted in significantly lower muscle soreness compared to the placebo (p = 0.0120) overall. In conclusion, curcumin may reduce muscle damage, and soreness without affecting the natural OS and inflammatory response to exercise.

Bovine respiratory disease (BRD) is the leading cause of morbidity and mortality in beef cattle. Common management practices in addition to BRD have been shown previously to cause inflammation. The objectives of this study were: (1) characterize the inflammatory profiles as indicated by interleukin-1 beta (IL-1β) and haptoglobin concentrations; (2) evaluate the impact of on-arrival metaphylactic antimicrobial therapy on inflammatory profiles in high risk cattle; and (3) examine the relationship between inflammatory profile and BRD morbidity and mortality. Eighty sale barn heifers were purchased over a two-year period (n=160). At arrival, heifers were randomly assigned to either receive tulathromycin (Draxxin, META, n=40) or not (NO META, n=40). Inflammatory profiles remained increased for all groups through d70 (P = 0.028). Metaphylaxis did not affect haptoglobin concentration (P > 0.10). There was a significant increase in BRD cases from day 0 to 20 (P = 0.002). Morbidity (BRD vs no BRD) did not impact haptoglobin concentrations.

Inflammatory diseases often involve complex and dynamic responses of monocytes, crucial cells of the innate immune system. Understanding these responses, particularly to lipopolysaccharide (LPS), a key inflammatory stimulus, is vital yet remains challenging due to their heterogeneity and plasticity. Upon analyzing available single-cell RNA sequencing data sets, we defined key patterns of monocyte inflammatory responses challenged with varying LPS dosages. We found that high-dose LPS induced the generation of exhausted monocytes with elevated expression of genes associated with pathogenic inflammation and immune suppression.. In contrast, super-low-dose LPS led to a state of low-grade inflammation, characterized by enhanced chemotaxis; immune-enhancement; and adhesion.. Pseudo-time analysis revealed a potential bifurcation of monocytes, starting from a proliferative, less-differentiated and premature state into either the exhausted state (under prolonged high dose LPS challenge) or the low-grade inflammatory state (under the prolonged super-low dose LPS treatment). Complementing our analyses with in vitro cultured murine monocytes, we observed similar exhaustion of monocytes collected from septic murine hearts published in an independent study. Furthermore, we analyzed publicly available scRNAseq datasets regarding monocytes from septic and severe COVID human patients and revealed a similar exhaustion phenotype as we documented in murine exhausted monocytes. In contrast, our analyses of newly published scRNAseq data regarding monocytes from chronic autoimmune patients reveal key distinct low-grade inflammation features. With translational potential, we analyzed the scRNAseq datasets of monocytes trained with 4-PBA, a potent anti-inflammatory compound, and observed that 4-PBA can effectively arrest monocytes in an anti-inflammatory state. Together, our comparative analyses reveal a systems landscape of monocyte memory dynamics with distinct dosage and history of LPS challenges, and offer novel insights for potential therapeutic strategies for modulating both acute sepsis and chronic inflammatory diseases. Our studies also provide a foundation for guiding future mechanistic and translational studies regarding monocyte dynamics and their involvements in health and disease pathogenesis. / Doctor of Philosophy / Inflammation is the body's natural response to injury or infection. A key player in this process is a type of immune cell called monocyte. Monocytes are our body's first line of defense, rushing to the site of injury or infection. However, the way these cells respond can vary greatly, depending on the dosage and duration of external challenges. In our research, we analyzed data collected through an advanced technique called single-cell RNA sequencing, in order to take a detailed look at how an individual monocyte responds to different amounts of LPS, a key substance found in most bacteria. We found that when exposed to a prolonged challenge of higher dose LPS, monocytes become exhausted with pathogenic inflammation and immune suppression, as seen in sepsis. However, when the LPS dose is low, these cells enter a state of low-grade inflammation, responsible for chronic inflammation as seen in autoimmune diseases, atherosclerosis and other chronic diseases. We found that this paradigm of exhaustion and low-grade inflammation can be seen in data analyzed from either patients with severe infections such as sepsis or severe COVID-19, or patients with long-term autoimmune diseases. In simpler terms, our study provides a detailed road map regarding how our body's first responders, namely the monocytes, react under different levels of threat, and how we might be able to guide their responses toward a beneficial direction. Understanding these processes more clearly may lead to new ways to treat a range of infectious or inflammatory diseases.

Recent reports suggest that preeclampsia is the result of an excessive maternal systemic inflammatory response. The role of magnesium and systemic inflammation in normal pregnancy and preeclampsia needs to be defined. The objectives of this study were to compare (1) demographic and (2) anthropometric characteristics of normal and preeclamptic pregnant women; and (3) to determine and compare serum iMg++ and tMg concentration and the association between inflammatory makers such as TNFα, leptin, adiponectin, IL10, ET-1, triacylglycerol, FFA, serum iMg++ and, tMg concentration of normal and preeclamptic pregnant women during the third trimester of pregnancy. Thirtyfive healthy pregnant and thirtyive preeclamptic pregnant women in their third trimester volunteered to participate in the study. Analyses were done by the use of ion-selective electrode, atomic absorption spectrometry, spectrophotometry, and ELISA kits. Pregnant normal and preeclamptic subjects were paired by age, parity, and week of gestation. Gestational age, body weight, age, prepregnancy diastolic BP, intake of birth control pills, supplements, miscarriage, still births, race, and employment status were statistically unchanged between the two groups. Current systolic and diastolic BP, family history of preeclampsia, urine albumin, minimum, maximum temperature and precipitation at the time of presentation were found to be significantly increased in the preeclamptic group. Number of children and pregnancies were significantly higher in the normal pregnant group. In the preeclampsia group, TNFα, ET-1, leptin, FFA, and triacylglycerol blood concentrations increased while tMg, IL10, and adiponectin blood concentrations decreased. Significant positive correlation was recorded between iMg++ and tMg and between iMg++ and tMg with IL10 and adiponectin in the preeclamptic group. An inverse correlation of iMg++ and tMg with TNFα, ET-1, leptin, FFA, and triacylglycerol, was seen in the preeclamptic group. Understanding the role of demographics and anthropometric characteristic during pregnancy and preeclampsia along with high correlation of iMg++ and tMg with each other and with inflammatory markers, increase in blood concentration of TNFα, ET-1, leptin, FFA, and triacylglycerol, and decrease in blood concentration of tMg, IL 10, and adiponectin could be an important step in understanding the pathophysiology of preeclampsia.

No description available.

Given the current obesity epidemic, the prevalence of overweight and obese patients with critical illness is increasing rapidly, however how obesity shapes critical illness and immune response to infection is not entirely understood. We developed a clinically relevant murine model of obesity in the context of sepsis, and examined organ specific inflammatory responses. Male C57BL/6 mice were fed either a high fat Western Diet (WD) (Modified Breslow, 21% Butterfat and 0.15% cholesterol) or normal chow diet (NCD) for 6, 15 or 27 weeks. Sepsis was induced by cecal ligation and perforation (CLP), and six hours post-surgery, plasma and tissue samples were harvested and flash frozen in liquid nitrogen. Septic obese mice at 15 and 27 weeks had significantly (p<0.0001) lower levels of lung myeloperoxidase (26.3±3.8 U/mg tissue) compared to age matched ad libitum (44.1±2.8 U/mg tissue) and diet restricted (63.2±5.60 U/mg tissue) controls, indicative of less lung inflammation. Obese mice (4.23±0.10g) had significantly enlarged livers compared to controls (1.55±0.80g and 1.22±0.031g), with pronounced steatosis, and hepatocyte ballooning, independent of sepsis. These findings are in congruence with clinical observations that obese individuals are protected from sepsis-induced lung injury, however the mechanisms involved are not entirely clear. We also examined effects of housing conditions on susceptibility to developing metabolic syndrome, and inflammatory response in our obesity and sepsis model. For this study, animals were fed either WD or NCD for 15 weeks and were housed in static or ventilated cages. Unlike static cages, ventilated cages have HEPA filtered air supply system and exhaust air ventilation, protecting the animals from air borne particles and preserving the microbiological barrier. Therefore, ventilated cages provide a more sterile environment compared to static cages. After 15 weeks, fecal matter was collected from the cages and mice were subjected to sepsis using the CLP technique. Six hours post surgery, animals were sacrificed and tissues were harvested, snap frozen and stored at -80°C. The animals from the more sterile environment (ventilated cages) had significantly (p<0.0001) less weight gain and did not show signs of overt hyperglycemia, compared to mice housed in a less sterile environment (static cages). In addition, obese mice housed in static cages had less lung injury compared to controls during early sepsis, however this difference was not evident in mice from ventilated cages. There were also significant differences in the fecal microbe composition, where ventilated groups had greater Firmicutes (69% ± 0.06% for WD and 76% ± 0.03%) and less Bacteroidetes population (15% ± 0.04% for WD and 12% ± 0.02% for NCD) compared to static groups (Firmicutes: 42% ± 0.08% for WD and 24% ± 0.02% for NCD, Bacteroidetes: 37% ± 12% for WD and 53% ± 29% for NCD). This study highlighted the impact of environment on the susceptibility to developing metabolic syndrome, and the potential impact on the associated immune responses, in our mouse models of obesity and sepsis. Leptin is an important mediator of immune responses to infection, and the levels are elevated during diet induced obesity in both mice and humans. We found that mice treated with leptin one hour prior to surgery, had significantly less injury (32.62±1.6 U/mg tissue) compared to saline treated animals (46.58±3.48 U/mg tissue), as evident from lung myeloperoxidase levels and histopathology scores. In addition, proprotein convertase subtilisin/kexin type 9 (PCSK9) over expressing mice on a normal diet, had significantly greater lung injury (46.51±4.51 U/mg tissue myeloperoxidase levels) compared to knockouts (31.14±1.75 U/mg tissue), this difference was not observed in WD fed mice with differential PCKS9 expression. In conclusion, WD fed mice had significantly less lung inflammation but greater hepatic injury. Furthermore, both leptin and PCSK9 are important mediators of lung inflammation in early sepsis. / Thesis / Doctor of Science (PhD)

Despite its prevalence, there are currently zero treatments available for traumatic brain injuries (TBI). Neuroinflammation is a key aspect of the secondary injury process, but remains poorly understood. Recent work has shown that Type I Interferons, inflammatory cytokines typically produced in response to viral infection, are present in the post-mortem brains of human TBI patients. The cyclic GMP-AMP Synthase- Stimulator of Interferon Genes (cGAS-STING) pathway is one of the primary methods of producing Type I IFNs; therefore, this work sought to evaluate the role of the cGAS-STING pathway in a murine controlled cortical impact (CCI) model of TBI. Using cGAS knockout (KO) or STING KO mice, we show that global loss of either protein results in substantial neuroprotection. One day after injury, animals have reduced lesion size, cell death, and inflammatory cytokine production, as well as reduced motor deficits several days after injury. We also determined that mitochondrial DNA (mtDNA) is present in the cytosol of injured cortical cells, indicating it is available to bind cGAS, a cytosolic pattern recognition receptor. To determine whether brain-resident or peripheral immune cells are responsible for detrimental cGAS-STING signaling after TBI, we utilized bone marrow chimeric animals lacking STING in either the brain or hematopoietic cells and animals lacking STING specifically in microglia. We found that both microglia and peripheral immune cells contribute to STING signaling after neurotrauma, and that loss of STING in either cell population is beneficial. Taken together, this work demonstrates that canonical, cGAS-dependent STING signaling occurs primarily in microglia and peripheral immune cells, resulting in detrimental neuroinflammatory events after TBI. / Doctor of Philosophy / Traumatic brain injuries (TBI), including concussions and more severe injuries, are a leading cause of death and disability across the globe; yet, there are no Food and Drug Administration (FDA) approved treatments. There are two phases in the injury: primary injury, which is the immediate damage to brain cells upon impact, and secondary injury, which includes a wide range of cellular processes in the minutes to weeks after injury. Because the primary injury is so rapid, we utilize safety measures, such as helmets, to limit the severity of the TBI. The secondary injury, however, occurs over a longer period of time; therefore, this is where most research is focused for developing potential treatments. Inflammation in the brain, termed neuroinflammation, is a key part of this secondary injury. While some inflammation is useful for clearance of damaged cells, too much inflammation can cause additional damage. The goal of this work was to examine how a specific inflammatory pathway, the cGAS-STING (cyclic GMP-AMP synthase- stimulator of interferon genes) pathway, contributes to neuroinflammation after brain injury.

Oxidative stress is a state characterized by an imbalance between the production and elimination of reactive oxygen species (ROS) within cells. ROS, also known as free radicals, are crucial for cellular signaling and are generated through natural processes. The antioxidant defense system typically regulates their concentrations to prevent oxidative stress-related damage. However, when ROS concentrations surpass a certain threshold and overwhelm the antioxidant defense system, it can lead to physiological issues and impairments in athletic performance. Additionally, oxidative stress and inflammation are closely related phenomena that can exacerbate each other, creating a vicious cycle. Both oxidative stress and inflammation play key roles in the pathophysiology of various chronic conditions such as cardiovascular diseases, neurodegenerative diseases, cancer, diabetes mellitus, autoimmune diseases, and accelerated aging. Furthermore, acute oxidative stress and inflammation have been shown to negatively affect performance by reducing skeletal muscle force output and increasing fatigue. Therefore, it is crucial to explore strategies to mitigate uncontrolled elevations of oxidative stress and inflammation. Curcumin, a bioactive compound found in turmeric, has been linked to antioxidant and anti-inflammatory properties. While cell line and animal studies have demonstrated the antioxidant and anti-inflammatory potential of curcumin, its effects in humans remain inconclusive. This dissertation project aimed to evaluate the effect of a four-week turmeric supplementation intervention on biomarkers associated with exercise-induced oxidative stress and inflammation in recreationally active individuals, 18 to 45 years of age. The study investigated curcumin's potential as an antioxidant and anti-inflammatory agent, while contributing to the existing literature on strategies for managing oxidative stress and inflammation. The findings from this research may offer valuable insights for promoting health, well-being, and athletic performance. / Doctor of Philosophy / Oxidative stress arises from an imbalance between the production and elimination of reactive oxygen species (ROS) within cells. ROS, or free radicals, serve essential roles in cell signaling and are naturally generated. While our bodies possess a defense mechanism that typically regulates ROS concentrations to prevent oxidative stress-related damage, an excess of ROS can overwhelm this system, leading to physiological complications and impairing athletic performance. The interplay between oxidative stress and inflammation exacerbates their effects, initiating a detrimental cycle. Both processes are implicated in chronic ailments such as cardiovascular diseases, Alzheimer's disease, cancer, diabetes mellitus, autoimmune disorders, and accelerated aging. Moreover, elevated concentrations of oxidative stress and inflammation can diminish muscle strength and increase fatigue during exercise. Curcumin, a compound found in turmeric, renowned for its antioxidant and anti-inflammatory properties, presents a potential avenue for managing oxidative stress and inflammation. While some studies have demonstrated these benefits in cellular and animal models, the efficacy of curcumin in humans remains uncertain. This study assessed whether a four-week regimen of turmeric supplementation can attenuate markers of oxidative stress and inflammation in physically active individuals, 18 to 45 years of age. By investigating the potential antioxidant and anti-inflammatory properties of curcumin, this research aimed to contribute novel insights into strategies for mitigating oxidative stress and inflammation, thereby promoting health, well-being, and athletic performance.