Sepsis is a potentially fatal condition which arises when the body's response to an infection is overly excessive, keeping it in an unbalanced state. Sepsis is often associated with the patients experiencing organ dysfunction resulting in death. Similarly, a majority of those who survive from sepsis suffer long-term sequelae. Identification and understanding of the genes involved in inflammation will help tackle the problem of patient care, sepsis prevention and help decrease the mortality rates. During inflammation certain genes in the body are activated trying to clear out the infection and bring the immune state back into balance. Regulation of specific genes aid the production of signaling proteins which help control the ongoing inflammation. Signaling proteins can be either proinflammatory or anti-inflammatory. Host response to infectious agents trigger immune responses to aid the elimination of pathogens. Immunometabolism encompasses multiple regulators including transcription factors and signaling proteins. This project focuses on a specific gene (IRG1) that is upregulated during inflammation. The inflammatory state is induced using stimulants such as lipopolysaccharide (LPS) to emulate sepsis-like condition. The project utilized in vivo and in vitro methods to investigate expression levels in innate immune system cells. The results showed increased expression of IRG1 gene upon LPS stimulation. The increase in the expression levels was more apparent over time. Under the same conditions cytokine analysis detected increased levels of proinflammatory cytokines in the cells with overexpressed gene compared to the cells with the IRG1 knockout gene. Survival studies in mice were conducted in order to investigate the importance of the gene in animal models. The results showed increased survival rate in mice where the gene was deleted compared to the wild type mice. This study established IRG1 as a potential therapeutic target for bacterial infections.

Background. Small vessel vasculitides (SVVs) are rare, autoimmune, and life-threatening diseases affecting many organs, including the kidney. SVVs trigger chronic thromboinflammation in the renal microvascular network. This process provokes microvascular alterations and rarefaction, promoting renal dysfunction and the development of glomerulosclerosis, all of which are responsible for the development of end stage renal disease (ESRD). We hypothesized that hematopoietic growth factors (HGFs) released by the inflamed kidney may sustain “emergency hematopoiesis” and fuel the thromboinflammatory process. Method. Using a murine model of antibody-mediated chronic kidney disease (AMCKD), mimicking the human pathology, and pharmacological interventions, we comprehensively monitored the response to injury in the circulating blood, urine, bone marrow, and kidney. Results. Experimental AMCKD is characterized by chronic thromboinflammation in glomeruli and the production of HGFs, especially thrombopoietin (TPO), by the injured kidney, which stimulated and skewed hematopoiesis toward megakaryopoiesis. AMCKD was characterized by vascular and kidney dysfunction, TGF-dependent glomerulosclerosis and microvascular rarefaction. In humans, chronic thromboinflammation in glomeruli during SVV-induced glomerulonephritis is associated with TGF-dependent glomerulosclerosis and increased bioavailability of TPO, amongst other HGFs and pro-inflammatory cytokines. The combined analysis of albumin, TPO and pro-inflammatory cytokine levels in sera from patients with SVV-induced glomerulonephritis allowed us to identify treatment responders. Strikingly, TPO neutralization in the experimental AMCKD model normalized hematopoiesis, reduced chronic thromboinflammation, vascular and kidney dysfunction, TGF-dependent glomerulosclerosis and microvascular rarefaction. Conclusion. Our results show that TPO-skewed hematopoiesis exacerbates chronic thromboinflammation in microvessels and worsens AMCKD. TPO is both a relevant biomarker and a promising therapeutic target in humans with CKD and other chronic thrombo-inflammatory diseases.

Endocan was found more than 20 years ago, and this biomarker for inflammation and endothelial dysfunction is still being actively researched. Sometimes referred to as endothelial cell-specific molecule-1 (ESM-1), endocan is a soluble dermatan sulfate proteoglycan produced by endothelial cells. It is expressed in actively proliferating tissues and detected in cultured endothelial cells of the skin, fatty tissue, hepatocytes, pulmonary and coronary arteries, etc. The role of endocan was studied in many diseases associated with inflammation and endothelial dysfunction, such as type 2 diabetes mellitus, arterial hypertension, atherosclerotic cardiovascular diseases, kidney diseases, obesity, polycystic ovary syndrome, metabolic syndrome, non-alcoholic fatty liver disease, sleep apnea syndrome. Given the known association of inflammatory and apoptotic processes of atherogenesis, it would appear relevant to study the relationship between endocan concentration and apoptosis marker levels in patients with coronary artery disease (CAD) within clinical examination and laboratory test context. Objective. Explore the relationship of serum concentration of endocan as a marker of inflammation with the indicators of apoptosis and clinical and laboratory characteristics of patients with CAD. Material and Methods. The study included 264 people (161 males and 103 females), of whom 220 patients had documented CAD. Anthropometric measurements, coronary angiography, echocardiography, duplex ultrasound scanning of extracranial parts of the brachiocephalic arteries were performed for all patients. Concentrations of endocan (ng/ml) and apoptotic markers Bcl-2 (ng/ml), Bax (ng/ml), Bcl-2/Bax, TRAIL (pg/ml) p53 (ng/ml) were measured in blood serum. Patients were divided into groups based on their SYNTAX scores: group 1 with moderate atherosclerotic lesions of the coronary arteries (CA) (score < 22, 124 patients); group 2 with severe CA atherosclerosis (score 23-32, 53 patients); and group 3 with extremely severe CA lesions (score >33, 43 patients). The control group consisted of healthy volunteers (44 subjects). All groups were age- and sex-matched. Differences were considered statistically significant at p<0.05. Results. A correlation was found between endocan concentration and CAD severity (r=0.32, p<0.001). In group 1, the median endocan concentration was 14.40 ng/ml [10.19; 19.91], in group 2, 20.31 ng/ml [12.75; 24.12], in group 3, 32.10 ng/ml [22.12; 38.21] and in the control group, 5.97 ng/ml [4.38; 8.25] (p<0.0001). Correlations of varying strength and significance were observed between the endocan concentration and a number of clinical and instrumental characteristics. Endocan concentrations significantly differed in groups of patients with multivessel disease (p<0.01), angina pectoris (p<0.0001), a history of myocardial infarction (p<0.001), and obesity (p<0.05) from patients without these signs. Also, a correlation was found between serum endocan concentration and apoptotic markers: TRAIL (r= -0.524, p<0.0001); BCL-2 (r= -0.558, p<0.0001), Bax (r= 0.571, p<0.0001), Bcl-2/Bax (r= -0.599, p<0.0001) and p53 (r= 0.560, p <0.0001). Conclusion. The international scientific community has been actively searching for novel laboratory markers of the progression of atherosclerosis processes in recent decades. Attention is paid to the study of the relationship between inflammatory and apoptotic mechanisms of atherosclerotic plaque formation and evolution, including the presence of delay, defective phagocytosis, and active release of pro-inflammatory mediators and signaling molecules in hyperlipidemia. We found that there is a statistically significant increase in serum concentrations of the inflammatory marker endocan in patients with CAD as coronary atherosclerosis becomes more severe. We also revealed correlations of varying strengths between the levels of endocan and some clinical examination and laboratory indicators. The data obtained suggest that endocan can be used as a diagnostic marker of the severity of atherosclerotic processes in patients with CAD.

Collecting lymphatic vessels (CLV) critically transport lymph and immune cells from tissue beds to lymph nodes and back to systemic circulation. Historically, and to perform this function, CLVs have been considered impermeable. However, recent studies have demonstrated increased permeability in situations such as inflammation. We challenged this concept further by assessing the junctional integrity of lymphatic endothelial cells (LECs) in mesenteric CLVs of the TNFΔARE/+mouse, a transgenic model of Crohn’s disease which displays terminal ileal inflammation, dilation of mesenteric CLVs, clustering of immune cells at CLV valve sites leading to the formation of tertiary lymphoid organs, and leakage of inflammatory lymph from these vessels. CLVs isolated from 12- and 28-week-old wild type (WT ) and TNFΔARE/+ mice were fixed pressurised before being assessed for LEC changes to CD31 and VE-Cadherin distribution/expression by fluorescence confocal imaging. While there were no differences between WT and TNFΔARE/+ vessels at 12 weeks, there was a significant loss in junctional protein expression along the length of the 28-week TNFΔARE/+ vessels as well as a distinct loss in junctional protein organization at valve sites. These sites also demonstrated a loss of the CLV-identifying marker FOXC2. To determine whether the overexpressed TNFα altered junctional protein expression and permeability, WT vessels were incubated with 100ng/ml of TNFα for 24 h and compared with untreated vessels. Only CD31 staining was significantly reduced in the post-valve regions of the vessels, which were also more permeable to luminally perfused dextrans (3-5 kDa and 70 kDa) than control vessels. In conclusion, chronic inflammation in TNFΔARE/+ mice increases the permeability of mesenteric CLVs by decreasing junctional protein expression and altering their cell surface distribution. While TNFα contributes to this loss by decreasing CD31 expression in the post-valve region and increasing vessel permeability, the loss of junctional organization may require a longer exposure to TNFα and other inflammatory mediators. We are currently examining whether differences in shear stress experienced in specific regions of the CLVs alter their sensitivity to TNFα and the expression of their junctional proteins.