Macrophage Infiltration Research Articles

Subcutaneously transplanted xenogeneic protein recruits treg cells and M2 macrophages to induce browning of inguinal white adipose tissue.

To study whether subcutaneously embedding xenogeneic protein threads or synthetic polymer absorbable threads can improve obesity phenotypes and metabolic conditions, and to further explore its underlying mechanism. Thirty-six 8-week-old ob/ob mice were randomly allocated to three groups, respectively, receiving catgut embedding, PGA thread embedding or sham treatment bilaterally to the groin. Individual parameters including weight, food intake, and core temperature are recorded and metabolism assessment, energy expenditure analysis, and PET/CT scanning are also performed at fixed timepoints. After surgical incision, the inguinal white adipose tissue was histologically examined and its expression profile was tested and compared among groups 4 weeks and 12 weeks after operation. Catgut embedding reduced weight gain and improved metabolic status in ob/ob mice. Browning of bilateral inguinal WAT (white adipose tissue) was induced after catgut embedding, with massive infiltration of Treg cells and M2 macrophages in the tissue slices of fat pads. IL-10 and TGF-β released by Treg cells targeted macrophages and the induced M2 macrophages increased the expression of thermogenic and anti-inflammatory genes in fat. The secretion of catecholamines by polarized M2 macrophages led to the activation of β3-AR-related pathways in adipocytes and the browning of adipose tissue. Abdominal subcutaneous catgut embedding has the potential to combat obesity through the induction of WAT browning mediated by infiltrated Treg cells and macrophages.

Synthesis and preclinical evaluation of [18F]AlF-NODA-MP-C6-CTHRSSVVC as a PET tracer for CD163-positive tumor-infiltrating macrophages

Positron emission tomography (PET) can provide information about tumor-associated macrophage (TAM) infiltration, as long as a suitable tracer is available. This study aimed to evaluate the radiolabeled peptide [18F]AlF-NODA-MP-C6-CTHRSSVVC as a potential PET tracer for imaging of the CD163 receptor, which is expressed on M2-type tumor-associated macrophages. The conjugated peptide NODA-MP-C6-CTHRSSVVC was labeled with aluminum [18F]fluoride. Tracer binding and its biodistribution were evaluated in an in vitro binding assay and in healthy BALB/c mice, respectively. In addition, different treatments with cyclophosphamide in tumor-bearing mice were used to assess whether the tracer could detect differences in CD163 expression caused by differential TAM infiltration. After 7 days of treatment, animals were injected with [18F]AlF-NODA-MP-C6-CTHRSSVVC, and a 60-min dynamic PET scan was performed, followed by an ex vivo biodistribution study. [18F]AlF-NODA-MP-C6-CTHRSSVVC was prepared in 23 ± 6 % radiochemical yield and showed approximately 50 % of specific receptor-mediated binding in an in vitro binding assay on human CD163-expressing tissue homogenates. No CD163-mediated binding of [18F]AlF-NODA-MP-C6-CTHRSSVVC was detected by PET under normal physiological conditions in healthy BALB/c mice. On the other hand, CD163-positive xenograft tumors were clearly visualized with PET and a positive correlation was found between CD163 levels and the [18F]AlF-NODA-MP-C6-CTHRSSVVC tumor-to-muscle ratio (TMR) obtained from the PET images (Pearson r = 0.76, p = 0.002). No significant differences in the CD163 protein level and in the tracer uptake between treatment groups were found in the tumors. Taken together, [18F]AlF-NODA-MP-C6-CTHRSSVVC appears a promising candidate PET tracer for M2-type TAM, as it binds specifically to CD163 in vitro and its tumor uptake correlates well with CD163 expression in vivo.

Diallyl trisulfide alleviates dextran sulphate sodium-induced colitis in mice by inhibiting NLRP3 inflammasome activation via ROS/Trx-1 pathway.

Diallyl trisulfide (DATS), a sulphur-containing compound isolated from the medicinal food plant garlic, has been previously reported to attenuate experimental colitis induced by either dextran sodium sulphate (DSS) or 2,4,6-trinitrobenzenesulfonic acid (TNBS) in mice; however, the underlying mechanism remains to be identified. In this study, we deciphered the key mechanism by which DATS alleviates ulcerative colitis (UC). We showed that oral administration of DATS for 10 consecutive days greatly restrained the infiltration of macrophages and the pathological changes in colonic tissues of mice with DSS-induced colitis. DATS treatment notably dampened the content of IL-1β and IL-18 and suppressed NLRP3 inflammasome activation in colon. Mechanistically, DATS effectively diminished the generation of ROS in macrophages. The suppressive effect of DATS on the activation of NLRP3 inflammasome and downregulation of IL-18 and IL-1β levels was blunted by xanthine oxidase. Further studies revealed that DATS inhibited NF-κB pathway activation by suppressing the expression of Trx-1, thereby inhibiting NLRP3 inflammasome activation. Trx-1 overexpression and interference in macrophages promoted and diminished NLRP3 inflammasome activation, respectively. In summary, garlic and its main active ingredient DATS have potentials to prevent and treat UC, and DATS functions by inhibiting NLRP3 inflammasome activation via Trx-1/ROS pathway.

Au/Ag@ZnS yolk-shell photocatalysts enhanced with noble metals and hyaluronic acid for efficient hydrogen production in rheumatoid arthritis therapy

Rheumatoid arthritis, characterized by the abnormal proliferation of synovial cells and extensive macrophage infiltration, is a chronic inflammatory disease. Molecular hydrogen, known for its antioxidant properties, has shown promise in eliminating reactive oxygen species. However, the low solubility and bioavailability of hydrogen limit the effectiveness of this therapy. To overcome these issues, we developed a novel yolk-shell heterostructure, H-AAZS (Au/Ag@ZnS modified hyaluronic acid), utilizing a hydrothermal cation exchange process. Through ion doping, semiconductor hybridization, and Schottky barriers in H-AAZS, photocatalysis for hydrogen generation has been successfully implemented using 660 nm laser irradiation. Additionally, the H-AAZS demonstrate the capacity for mild photothermal therapy, inducing apoptosis in synovial cells with Au's hot electrons with 660 nm laser irradiation. This strategy not only improves the abnormal proliferation of synovial cells but also avoids the exacerbation of inflammation caused by thermal stimulation. Both in vitro and in vivo experiments validate the synergistic effects of hydrogen production mediated anti-inflammatory responses, macrophage polarization and photothermal therapy. Therefore, this work represents a significant advancement as it ingeniously harnesses photocatalysis to modulate the synovial microenvironment while mitigating the side effects associated with photothermal therapy. This nanocrystal provides new and valuable insights into the potential treatment of Rheumatoid arthritis.

Sexual Dimorphism in the Immunometabolic Role of Gpr183 in Mice

Abstract Aim Excessive eating and intake of a Western diet negatively affect the intestinal immune system, resulting in compromised glucose homeostasis and lower gut bacterial diversity. The G protein-coupled receptor GPR183 regulates immune cell migration and intestinal immune response and has been associated with tuberculosis, type 1 diabetes, and inflammatory bowel diseases. We hypothesized that with these implications, GPR183 has an important immunometabolic role and investigated this using a global Gpr183 knock-out mouse model. Material and methods Wild-type (WT) and Gpr183 deficient (Gpr183-/-) mice were fed a high-fat, high-sucrose diet (HFSD) for 15 weeks. We investigated changes in weight, body composition, fecal IgA levels, fecal microbiome, and glucose tolerance before and after the diet. Macrophage infiltration into visceral fat was determined by flow cytometry, and hepatic gene expression was measured. Results and conclusions A sexual dimorphism was discovered, where female Gpr183-/- mice showed adverse metabolic outcomes compared to WT counterparts with inferior glucose tolerance, lower fecal IgA levels, and increased macrophage infiltration in visceral fat. In contrast, male Gpr183-/- mice had significantly lower fasting blood glucose after diet than male WT mice. Liver gene expression showed reduced inflammation and macrophage markers in Gpr183-/- livers, regardless of sex, while the pancreatic islet area did not differ between the groups. No conclusive differences were found after microbiome sequencing. To conclude, Gpr183 maintains metabolic homeostasis in female but not in male mice independent of diet. If confirmed in humans, future therapy targeting GPR183 should consider this sexual dimorphism.

Historical Control Background Incidence of Spontaneous Nonneoplastic Lesions of Sprague Dawley Rats in 104-Week Carcinogenicity Studies.

Microscopic observation data collected from approximately 1800 male and female Sprague Dawley (SD) control rats used on 104-week carcinogenicity studies performed at North American Labcorp Early Development, Inc, Madison, WI, were retrospectively evaluated for spontaneous nonneoplastic findings. This study provides incidence of the most common spontaneous nonneoplastic microscopic findings in each organ system of SD rats encountered during 104-week carcinogenicity studies. Some of the most common spontaneous background findings were cardiomyopathy; chronic progressive nephropathy; uterine cystic endometrial hyperplasia; prostate inflammation; pulmonary alveolar macrophage infiltrates; hepatocyte vacuolation, bile duct hyperplasia, and basophilic foci in the liver; pancreatic fibrosis; splenic extramedullary hematopoiesis and pigment; decreased lymphocytes and epithelial hyperplasia in the thymus; ventral brain compression; cystic degeneration and hyperplasia of the adrenal cortex; and mammary gland hyperplasia. The most common nonneoplastic findings in male SD rats were chronic progressive nephropathy (80.9%) and rodent progressive cardiomyopathy (73.2%). The most common nonnenoplastic findings in female SD rats were cystic degeneration of the adrenal cortex (64.7%) and ventral compression of the brain due to pituitary neoplasms (62.7%).

Pnn deficiency alters expressions of calcium handling proteins and impairs nuclear envelope structure in cardiomyocytes

Abstract Pnn is a multi-functional protein playing roles in the regulation of gene transcription, mRNA alternative splicing, as well as cell-cell connection. Previous studies suggested an association between Pnn genetic defects and arrhythmogenic right ventricular cardiomyopathy (ARVC) in human. In this study, we applied an inducible cardiomyocyte-specific Pnn depletion mouse model (Myh6-CreERT2, Pnnflox/flox) to determine the impact of loss of Pnn on cardiac structure and function in mice. Six weeks after inducing Pnn gene disruption, electrocardiographic abnormalities, ventricular dilatation, and reduced left ventricular ejection fraction (LVEF) were observed in mice with loss of Pnn in cardiomyocytes. Histopathological examinations showed that the proliferation of cardiac fibroblasts and expression of α-smooth muscle actin were increased with Pnn depletion, while the cell-cell connection between cardiomyocytes were impaired. In addition to impaired intercalated discs, loss of Pnn also altered the distribution and expression of nuclear envelope proteins and the linker of nucleoskeleton and cytoskeleton (LINC) complex proteins, such as nuclear lamins, Emerin, and SUNs, in cardiomyocytes. Microarray analysis as well as subsequent RT-PCR and Western blots also indicated that loss of Pnn regulated myocardial expressions of genes responsible for calcium handling, such as RYR2, Atp2a2, Slc8a1, and Kcnj3. Furthermore, infiltrations of macrophages and neutrophiles were observed in the myocardium with Pnn depletion, while expression levels of oxidative stress-associated proteins were also significantly regulated by Pnn depletion. In conclusion, Pnn plays an essential role in the maintenance of intercalated disc integrity and nuclear envelope structure of cardiomyocytes, while cardiomyocyte-specific loss of Pnn impairs the calcium handling and leads to arrhythmogenic dilated cardiomyopathy in mice.Cardiac MRI of mice with Pnn depletionMyocardial expression of SUN2 and Emerin

Inhibiting chondroitin sulfate synthase 1 ameliorates hyperplastic arterial remodelling

Abstract Introduction Hyperplastic arterial remodelling is characterized by intima-media thickening, lumen narrowing, inflammation and increased extracellular matrix (ECM) deposition. Chondroitin sulfate (CS) glycosaminoglycans, the unbranched polysaccharide chains of proteoglycans, are important components of ECM. We previously found CS accumulated in adversely remodelled heart and artery, aggravating cardiac deterioration. Objective We aim to explore a therapeutic strategy to interfere with CS synthesis and investigate its potential in ameliorating hyperplastic arterial remodelling. Methods Through single-cell RNA sequencing of non-cardiomyocytes cardiac cells from mouse myocardial infarction model, chondroitin sulfate synthase 1 (CHSY1) was identified as the primary CS synthase and was increased during disease progression. A Chsy1 knockout (KO) mouse strain was generated. Fibrosis and arterial remodelling were induced using: (i) 4 weeks of angiotensin II/phenylephrine (AngII/PE) infusion through subcutaneous osmotic minipumps; and (ii) transverse aortic constriction surgery. To assess the therapeutic potential of CHSY1 inhibition in ameliorating hyperplastic arterial remodelling, Chsy1 was knockdown by dsiRNA post disease onset. Mechanism of action was investigated using primary vascular smooth muscle cells (VSMCs). Results Chsy1 KO reduced CS and its stereoisomer dermatan sulfate in the heart to 42 ± 22% and 36 ± 7% respectively, whereas other types of glycosaminoglycans remain intact. KO mice were protected against phenotypes of hyperplastic arterial remodelling induced by the two disease models in both the coronary and carotid arteries. For example, 90.1% of coronary arterial fibrosis and 79.7% of macrophage infiltration induced by AngII/PE was declined in KO mice compared to WT mice, while cardiac function as stroke volume was augmented by 41.3%. Concomitantly, carotid arterial wall thickening was lower by 36.5% and distensibility was higher by 36.7%, validating Chsy1 as a potent target. As a therapeutic approach, weekly intravenous injection of dsiRNA achieved Chsy1 reduced to 30.2 ± 5.5% in the carotid artery and 72.5 ± 4.7% in the heart. Post disease onset, Chsy1 KD therapy reduced fibrosis by 59.9% in the coronary artery (P = 0.006) and improved stroke volume by 25.6% (P = 0.036). The structural integrity and function of carotid arteries were well-reserved, manifesting as improved distensibility (29%, P = 0.016), increased elastin/perimeter ratio (15%, P = 0.002), and reduced macrophage infiltration (38%, P = 0.029). In vitro, CHSY1 inhibition suppressed migration and cytokine production of macrophages, fibroblast-to-myofibroblast transition, and VSMC hyperproliferation. NOTCH3 signalling was found to be positively correlated with CHSY1 modulation. Conclusions We demonstrated that inhibiting CHSY1 is necessary and sufficient to block CS synthesis. Chsy1 KD by dsiRNA is an effective therapy to ameliorate hyperplastic arterial remodelling.

Combined administration of atorvastatin and mesenchymal stem cells synergizes to attenuate inflammation and promote efferocytosis in atherosclerotic mice

Abstract Background Atherosclerosis (AS), characterized by chronic systemic and lesional inflammation, is by far the most frequent underlying cause of cardio-cerebrovascular diseases. Recently, impaired efferocytosis, referring to defective removal of dead cells, was identified as another essential factor contributing to the development of AS, which is partly attributed to upregulation of CD47 and explain the benefits of atorvastatin (ATV). Mesenchymal stem cells (MSCs) were reported to protect against AS by inhibiting inflammation; while whether ATV and MSCs could synergize to attenuate inflammation and promote efferocytosis and consequently, reduce AS burden, remains unclear. Methods To establish the AS model, 6-week-old male ApoE-/- mice fed with Western diet for 12 weeks were used and grouped randomly (n=15 per group). The scheme of administration of ATV and human umbilical cord MSCs was shown in Figure 1A. After 6-week treatment, mice were sacrificed, and hearts and aortas were harvested. Oil Red O staining was used to evaluate the lesional burden. Immunofluorescence staining was used to assess the inflammation and efferocytosis in the plaque. A high level of CD11b+ cells (macrophages) infiltration was considered an indicator of inflammation; a high level of cleaved caspase 3 puncta unmerged with macrophages (Mac3+ area) was considered an indicator of impaired efferocytosis. Results Human umbilical cord MSCs were identified as CD73+CD90+CD105+CD45-CD14- cells. Evoked inflammation and defective clearance of apoptotic cells in the plaque were observed (Figure 1B, C). ATV and MSC treatment inhibited macrophage infiltration and activated efferocytosis. Given the mechanistic idea that upregulated CD47 blocks the Signal regulatory protein α (SIRPα) / src homology-2 (SH2)-domain-containing protein tyrosine phosphatases (SHP) signal and leads to efferocytosis impairment, we evaluated the expression levels of CD47 in plaque and phosphorylated SHP (p-SHP) in lesional macrophages (Figure 2A, B). The elevation of CD47 expression and consequent downregulation of p-SHP was reversed by both ATV and MSCs, with a lower lesion burden observed in NS+MSC, ATV+PBS and ATV+MSC groups. All benefits were more pronounced in the group received combined treatment (ATV+MSC). Conclusions The results indicated ATV and MSC administration could synergizes to attenuate inflammation and promote efferocytosis in AS plaque, and the combined treatment led to the lowest lesion burden. CD47/p-SHP signal plays an essential role in the activation of efferocytosis. The mechanisms underlying the inhibition of CD47 are currently under investigation.

Gradual hypertensive response in aortic alterations and myocardial inflammatory activation in progressing HFpEF in mice

Abstract Introduction Hypertension is the most common comorbidity in patients affected by heart failure with preserved ejection fraction (HFpEF). Early effects of pressure overload and its contribution to the gradual development of cardiac and vascular pathology leading to HFpEF are not well recognized, as patients are diagnosed when the disease has already advanced and show symptoms. Several hypertensive HFpEF mouse models possessing the hallmarks of the clinical-like phenotype are available. Nonetheless, initial and gradual pathological manifestations of hypertensive HFpEF have not been comprehensively described. Purpose This study aimed to investigate the gradual progression of HFpEF phenotype development in mice from the early manifestation of cardiac and vascular pathology to global cardiac dysfunction to multiorgan syndrome in response to hypertension. Methods Hypertensive HFpEF was induced by angiotensin II infusion in C57BL/6JOlaHsd 18-20-week-old male mice that were given low, moderate and high doses (0.5mg/kg/day - 1.5 mg/kg/day) subcutaneously for 4 weeks. Cardiac physiology and alterations in haemodynamics were assessed with echocardiography of heart and aorta, weekly ECG monitoring, and blood pressure measurements from the mouse tail. Extensive histology and immunohistochemistry analyses of cardiac tissue, microvasculature, lungs and kidneys were performed. Results In the low dose group, loss of aortic wall flexibility and presence of aortic root dilatation were detected. This group also demonstrated initiation of concentric remodelling process with the upsurge in macrophage infiltration in myocardium, but the signs of diastolic dysfunction were not yet present. The high dose group exhibited characteristic HFpEF features with diastolic dysfunction, left ventricular hypertrophy and fibrosis. Pathology in cardiac conduction system was noticed in all groups, with prominent ECG waveform abnormalities already on week one. However, only the high dose group experienced non-recovery in impulse propagation, accompanied by significant prolongation of ventricular depolarization. Conclusion This study demonstrates that change in aortic root dimensions and aortic wall dynamics are the first hypertension-sensitive alterations together with myocardial inflammatory cell infiltration that may contribute to myocardial remodelling and subsequent development of HFpEF and systemic effects. Hypertensive angiotensin II-induced HFpEF is a relevant mouse model, recapitulating not only hallmarks of cardiac pathology but also an extensive range of vascular and multiorgan alterations, demonstrating similarities with the clinical phenotype.

CD14 blockade preserves left ventricular structure and systolic function in a murine model of ST-elevation myocardial infarction

Abstract Background Cluster of differentiation-14 (CD14) facilitates the excessive pro-inflammatory monocyte-macrophage response to myocardial injury. Both acute and chronic infiltration of pro-inflammatory macrophages are implicated in the immunological progression of ischemic heart failure. However, investigation of clinically practicable strategies of CD14 blockade are yet to be reported. Purpose This fully blinded and randomised preclinical study evaluated the efficacy of a neutralising anti-CD14 antibody given at reperfusion in a murine model of reperfused ST-elevation myocardial infarction (STEMI) with key clinical features. Methods Large anterior STEMI was induced in 48 adult mice by left anterior descending coronary artery occlusion for 1 h prior to reperfusion and randomisation to receive anti-CD14 antibody, isotype control (IgG2a) or saline weekly for four weeks (independently blinded). Outcome measures included functional assessments by 9.4T cardiac magnetic resonance (CMR) imaging and ultra-high-resolution echocardiography at 21- and 28-days post-STEMI, respectively. Results Multi-plane short axis CMR at 21-days post-STEMI revealed greater left ventricular (LV) ejection fraction in the anti-CD14 antibody-treated group compared with saline- and isotype-treated controls (30±1 vs 19±1 and 20±1 %, respectively, p<0.001; Figure 1A). CMR also revealed reduced LV dilatation with anti-CD14-treatment compared to control groups (76±5 vs 102±8 and 107±7 µl EDV in saline- and isotype-treated controls, respectively, p<0.05). Parasternal long axis echocardiography at 28 days post-STEMI confirmed that anti-CD14 antibody treatment was associated with greater LV systolic function compared to saline and isotype controls (30±1 vs 24±1 and 25±2 % EF, respectively, p<0.05; Figure 1B). Preservation of LV end-diastolic (86±4 vs 111±6 and 101±5 µl, p<0.01) and end-systolic (61±3 vs 84±5 and 77±5 µl, p<0.01) volumes was also observed with anti-CD14 treatment. Global longitudinal strain (GLS) of the LV was also significantly greater at 28 days post-STEMI with anti-CD14 treatment relative to saline and isotype controls (-8.5±0.4 vs -6.8±0.3 and -6.6±0.4 %, respectively, p<0.05). Conclusion(s) The findings of this fully blinded and randomised preclinical study provide evidence of a clinically practicable strategy of CD14 blockade initiated at reperfusion to mitigate progressive LV systolic dysfunction and dilatation post-STEMI.Figure 1.Functional outcome measures

Human epicardial adipose tissue-derived factors promote atrial endothelial dysfunction: role of pro-inflammatory cytokines and AT1R/NADPH oxidases/SGLT2 pro-oxidant pathway

Abstract Introduction Epicardial adipose tissue (EAT) has been suggested to contribute to left atrium dysfunction via paracrine mechanisms to induce endothelial dysfunction, pro-arrhythmogenic and pro-remodeling responses thereby favoring atrial fibrillation (AF) onset. Recently, sodium-glucose co-transporter2 inhibitors (SGLT2i) have been shown to reduce AF incidence, EAT volume, differentiation and inflammation, however, the underlying mechanisms are unknown. Purpose This study investigates the impact of human EAT-derived mediators on atrial endothelial cell function and, if so, to characterize the role of SGLT2 using the inhibitor empagliflozin (EMPA). Methods Epicardial and subcutaneous adipose tissues (SAT) were collected from 70 cardiac patients at a university hospital. Conditioned medium (CM, 24 h) from fats were applied to porcine atrial endothelial cells (AECs, first passage) for 24 h. Histology of tissues was assessed by haematoxylin and eosin staining, protein expression by Western blot analysis or immunofluorescence staining, mRNA levels by RT-qPCR, formation of reactive oxygen species (ROS) and nitric oxide (NO) by fluorescent probes, and pro-inflammatory cytokine levels by ELISA. Results Compared to SAT, EATs were more vascularized with increased infiltration of M1-like macrophages, expression of proinflammatory cytokines (IL-1ß, IL-6, TNF-α), markers of ECs VCAM-1 and eNOS, fibrosis TGF-β, NADPH oxidases NOX-1 and SGLT1/2. These effects were associated with a pro-oxidant response that was inhibited by neutralizing Abs directed against IL-1ß or IL-6, and by inhibition of either NOS, NADPH oxidases, ACE, AT1R or SGLT2. Levels of SGLT2 and ROS in EATs were higher in AF compared to non-AF patients and progressively increasing with age. CM of EATs showed higher concentrations of IL-1ß, IL-6, TNF-α and MCP-1 than that of SAT. Exposure of AECs to CM of EATs increased the level of oxidative stress that was positively correlated to the level of pro-inflammatory cytokines, and reduced basal and bradykinin-induced NO formation that was prevented by EMPA. It also resulted in upregulation of p53 and SGLT2 expression, and nuclear translocation of NF-kB by CM of the top quartile inflamed EATs but not by the lowest quartile of EATs or SATs, prevented by EMPA. Conclusion The findings indicate that compared to SATs, the EATs showed higher in situ expression and release of pro-inflammatory cytokines that contributed to oxidative stress involving the AT1R/NADPH oxidases/SGLT2 pro-oxidant pathway. CMs of inflamed EATs induced AECs dysfunction, oxidative stress and activation of NF-kB involving pro-inflammatory cytokines and SGLT2. Thus, SGLT2i appear as an interesting strategy to decrease EAT inflammation and improve endothelial function contributing to prevent cardiac remodeling and fibrotic responses.

A novel murine wire injury based infectious endocarditis model aggravates aortic valve stenosis

Abstract Background With mortality rates up to 40% under optimal medical treatment, infectious endocarditis (IE) remains one of the deadliest infectious diseases worldwide. In the early stages of IE, diagnosis and treatment initiation are often difficult and research is limited due to unreliable models. Most murine IE models rely on permanent prosthetic wire placement and i.v. bacterial injection, thus making it inadequate to study IE pathophysiology with the aim to reflect the course of disease in humans. Purpose To develop a reproducible murine IE model, based on aortic valve injury preceding intravenous S. aureus bacterial challenge. Methods Using a wire-injury model we induced endothelial lesions. 72h after wire injury (WI), we performed intravenous bacterial challenge using S. aureus. Echocardiography was performed to confirm bacterial vegetations and assess valvular function. Cross sections of valvular leaflets were prepared for scanning electron microscopy (SEM). Macrophage, neutrophil and S. aureus-specific immunofluorescence staining was performed and visualized by immunofluorescence microscopy. Bacterial cultivation was performed from peripheral blood and valve tissue. Systemic immune response was analyzed using flow cytometry. Results WI induced endothelial damage in all mice verified using scanning electron microscopy imaging. The combination of wire injury and injection of 10^5 and 10^6 colonies-forming-units (CFU) S. aureus reliably caused IE validated via in vivo echocardiography as well as S.aureus immunofluorescence staining and SEM imaging. Mice undergoing bacterial challenge responded with significant neutrophilia in the blood. Using 10^6 CFU S. aureus was associated with an increased mortality, liver and kidney abscess formation due to systemic bacterial spreading compared to 10^5 CFU. Peak velocity across the aortic valve and aortic valve leaflet thickness as markers for aortic valve stenosis were increased in IE compared to WI only mice. Aortic regurgitation was more prevalent after bacterial challenge mediating increased left ventricular volumes, meanwhile ejection fraction was not altered. Immunofluorescence staining revealed a pro-inflammatory milieu including increased macrophage and neutrophil infiltration in IE compared to WI only mice. Conclusion In vivo echocardiography and ex vivo histological staining revealed reliable IE induction using our new model with S. aureus concentrations of 10^5 CFU. The additional bacterial challenge causes a more severe aortic valve stenosis compared to WI only mice, potentially due to an observed increase in valvular inflammation.Fig 1 Scanning electron microscopy after

Sinomenine hydrochloride alleviates autoimmune myocarditis via suppressing Th1 cell induced-M1 macrophage pyroptosis

Abstract Background Myocarditis is typical of the complex inflammatory response in the heart with high morbidity and mortality, accompanied by tissue damage and cardiac fibrosis. Previous studies have demonstrated that CD4+ T cells are crucial to the cardiac autoimmunity of myocarditis, but the role of different CD4+ T cell subsets remains unclear. Recently, sinomenine hydrochloride, a natural compound from the traditional Chinese herb Sinomenium acutum, is reported to inhibit T cell proliferation in some systemic autoimmune diseases such as rheumatoid arthritis. Given that autoimmune myocarditis is an organ-specific autoimmune disease, the effect of sinomenine hydrochloride on autoimmune myocarditis demands further study. Purpose This study aims to elucidate the role of CD4+ T-helper 1 (Th1) cells in regulating inflammatory cell death in autoimmune myocarditis and investigate the pharmacological effect of sinomenine hydrochloride on CD4+ Th1 cells and autoimmune myocarditis. Methods Male Balb/c mice were immunized with myosin heavy chain-α peptides to establish the experimental autoimmune myocarditis (EAM) model, followed by the treatments with sinomenine hydrochloride by gavage. Tohoku Hospital Pediatrics-1 (THP-1) cell line and murine T cells were used for in vitro experiments. Results In the acute phase of EAM, we observed plenty of CD4+ T cells infiltrating the heart tissue. RNA sequencing revealed that among the genes encoding typical transcription factors of CD4+ T cell subset, only Tbx21 and Stat4, both representing Th1 cells, were upregulated, which consisted with immunofluorescence staining. Further studies indicated that Th1 cells not only mediated classical activated macrophage (M1 macrophage) polarization by secreting interferon-γ (IFN-γ) but also associated with pro-inflammatory macrophage pyroptosis in the EAM, with the cleavage of gasdermin D (GSDMD), caspase-1 and IL-1β. Subsequent in vitro experiments confirmed that IFN-γ could dramatically promote macrophage pyroptosis combined with ATP, leading to the release of IL-1β and IL-18(Figure 1). To find an effective method for Th1 cell suppression and myocarditis remission, we identified that sinomenine hydrochloride impeded Th1 cell activation, differentiation and proliferation in vitro. Furthermore, sinomenine hydrochloride inhibited Th1 cell infiltration and M1 macrophage pyroptosis in the EAM heart, In the chronic phase, cardiac remodeling and fibrosis were suppressed, and cardiac systolic function was significantly improved in the treatment group (Figure 2). Conclusion Our study demonstrates that Th1 cells-induced M1 macrophage pyroptosis is an essential pathogenic mechanism of EAM. Sinomenine hydrochloride can alleviate the severity and progression of EAM, which may become a novel therapeutic strategy for treating myocarditis.Figure 1Figure 2

Association of polarimetric signatures with coronary plaque composition in patients with acute and chronic coronary syndrome: an intravascular polarization-sensitive optical coherence tomography study

Abstract Background Intravascular polarimetry with polarization-sensitive (PS-) optical coherence tomography (OCT) measures polarization properties, birefringence and depolarization, in parallel with structural features available with conventional OCT (Figure 1). Birefringence is elevated in collagen and collagen-synthesizing smooth muscle cells, which imparts mechanical integrity to fibrous caps. Depolarization is elevated in the presence of macrophages and lipid/necrotic core. Purpose This study aimed to investigate polarimetric signatures of coronary lesions in patients with acute coronary syndrome (ACS) and chronic coronary syndrome (CCS). Furthermore, we aimed to investigate diagnostic value for birefringence and depolarization of ACS culprit lesions discriminating from CCS target lesions. Methods This was the first-in-human study of intracoronary PS-OCT in Japan, consisting of 17 patients with ACS (n = 5) or CCS (n = 12) who underwent intravascular PS-OCT. ACS culprit lesions (ACS-lesions) and CCS target lesions (CCS-lesions) were included in the analysis, resulting in a total of 862 cross-sections. In the cross-sectional images, lumen segmentation and guidewire artifact were automatically segmented. The median value of birefringence and depolarization were automatically analyzed using depolarization cut-off value (cross-sectional level). Birefringence and depolarization of ACS- and CCS-lesions were compared by using a generalized estimating equation model. Receiver operating characteristic (ROC) analysis was used to investigate diagnostic performance of polarimetric signatures for the identification of ACS-lesions. Results Compared to CCS-lesions, ACS-lesions featured higher lipid-burden index and maximum lipid arc (both p < 0.05). Compared to the CCS-lesions, ACS-lesions exhibited significantly lower birefringence (p < 0.05) and higher depolarization (p < 0.05) (Figure 2). In the ROC analysis for differentiating ACS-lesions from CCS-lesions, area under the curves (AUC) for birefringence and depolarization were 0.712 and 0.672, respectively. In the multivariable ROC analysis in diagnosing ACS lesions, combination of birefringence with depolarization improved the AUC to 0.755 (p ＝ 0.025). Conclusions Intravascular PS-OCT provides quantitative compositional assessment of coronary lesions. Decreased birefringence and pronounced depolarization within the ACS-lesions indicate increased collagenolytic activity and the presence of macrophage infiltration, respectively. These results suggest that polarization properties may serve as quantitative imaging markers for assessing natural history of coronary atherosclerosis.

Analysis of risk factors in high-risk plaque in acute coronary syndrome based on LASSO regression

Abstract Background The CLIMA Study (Relationship between OCT Coronary Plaque Morphology and Clinical Outcome) put forward the concept of High-risk plaque (HRP) and demonstrated that HRP were correlated with the high risk of major coronary events[1, 2]. HRP were defined as the presence of four simultaneous characteristics: minimum lumen area (MLA) <3.5mm2, fibrous cap thickness (FCT) <75 μm, lipid arc circumferential extension >180°, and the presence of macrophage infiltration. Early prediction of HRP formation are of great significance for the prevention and treatment of Acute Coronary Syndrome (ACS), but there are no relevant studies. Purpose To identify the risk factors related to OCT HRP in ACS and establish risk prediction model for HRPs in ACS. Methods A prospective observational cohort study was carried out on the patients with ACS from September 2019 to August 2022. A total of 169 patients were divided into OCT HRP (n=55) and OCT non-HRP (n=114) groups. Clinical information, laboratory examination and the OCT characteristics of the patients were collected. LASSO regression was used to screen variables, and multivariate logistic regression was used to establish risk prediction model. A nomogram was formulated, use the ROC curve to evaluate the discrimination of the model and the Bootstrap method to internally validate the model. Results The most frequently observed HRP characteristic was Lipid plague>180°(147 patients），followed by MLA<3.5mm2(141 patients), macrophages(127 patients), FCT<75μm(64 patients). The LASSO regression model was used to screen variables and construct an HRP risk factor model. This final model included age, BMI≥25, TC, LDL-C and Log NT-proBNP. The model is well differentiated (AUC 0.780, 95%CI 0.705-0.855) and calibrated. Decision Curve Analysis shows that the model has good net benefits. Conclusion This prediction model can accurately predict the risk of OCT HRP in patients with ACS, and is expected to assist clinicians in diagnosis and prevention of plaque stability.

Hypertension is associated with the reduction in epidermal small fibres independently of sural nerve inflammation in type 2 diabetic subjects.

Diabetic polyneuropathy (DPN) is a multifactorial disease associated not only with hyperglycaemia but also with circulatory disturbances such as hypertension. A close interaction between the immune system and hypertension is known. It remains unclear whether the inflammatory response is associated with hypertension in the pathology of human DPN. Autopsied patients were evaluated: 7 non-diabetic patients (nDM), 11 non-diabetic patients with hypertension (nDMHT), 6 patients with diabetes (DM) and 9 patients with hypertension and diabetes (DMHT). Intraepidermal nerve fibre density (IENFD) was examined by immunofluorescent staining. Dissected sural nerve (SNs) were morphometrically quantified. Dermal and endoneurial macrophage infiltration was evaluated by double immunostaining using anti-CD68 and anti-CD206 antibodies. IENFD was significantly decreased in DM compared to nDM (p < 0.05) and was further decreased in DMHT (p < 0.05). Myelinated nerve fibre density (MNFD) in the SN was significantly decreased in DM compared with nDM (p < 0.05) and further decreased in DMHT (p < 0.01 vs. DM). The infiltration of CD206-/CD68+ proinflammatory macrophages in the SN was significantly increased in DM compared to nDM (p < 0.05), whilst the number of CD206+/CD68+ anti-inflammatory macrophages was decreased in DM (p < 0.05). Hypertension had no impact on macrophage infiltration. The ratio of CD206- and CD206+ macrophage was negatively correlated with MNFD (r = 0.42, p < 0.05) but not IENFD (r = 0.30, p = 0.09). Dermal CD206+ macrophage infiltration was similar amongst all groups. Diabetes complicated by hypertension significantly increased the total diffusion barrier thickness (p < 0.01 vs. DM). Total diffusion barrier thickness was inversely correlated with both IENFD (r = -0.59, p < 0.01) and MNFD (r =-0.62, p < 0.01). Our results suggest that vascular factors and inflammation might be synergistically involved in pathological changes in human diabetic patients through different mechanisms.

Identification and validation of M2 macrophage-related gene signature as a novel prognostic model for head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is a highly heterogeneous tumor. Commonly used tumor staging don’t sufficiently and accurately assess the prognosis of HNSCC patients, resulting in a lack of guidance for clinical treatment decisions. M2 macrophage infiltration has been shown to be strongly associated with the tumor prognosis. In this study, we used the Cancer Genome Atlas (TCGA) data to screen for genes co-expressed with M2 macrophages in HNSCC. We used univariate Cox regression to screen out the genes associated with HNSCC prognosis, and constructed a HNSCC prognosis model by Lasso regression analysis. The results confirmed that the model had good predictive value and accuracy for the prognosis of HNSCC patients by survival analysis, ROC curve and nomogram. We divided the HNSCC samples into high-risk and low-risk groups according to the risk score, and the results showed that patients in the high-risk group were more prone to genetic mutations and had a higher tumor mutational burden. In addition, there were significant differences between risk groups in terms of immune cell infiltration and drug sensitivity. The HNSCC prognostic model established in this study may provide guidance for clinical therapeutic decision-making and provide a theoretical foundation for the development of new immunotherapy methods.

Osteopontin is a therapeutic target that drives breast cancer recurrence

Recurrent breast cancers often develop resistance to standard-of-care therapies. Identifying targetable factors contributing to cancer recurrence remains the rate-limiting step in improving long-term outcomes. In this study, we identify tumor cell-derived osteopontin as an autocrine and paracrine driver of tumor recurrence. Osteopontin promotes tumor cell proliferation, recruits macrophages, and synergizes with IL-4 to further polarize them into a pro-tumorigenic state. Macrophage depletion and osteopontin inhibition decrease recurrent tumor growth. Furthermore, targeting osteopontin in primary tumor-bearing female mice prevents metastasis, permits T cell infiltration and activation, and improves anti-PD-1 immunotherapy response. Clinically, osteopontin expression is higher in recurrent metastatic tumors versus female patient-matched primary breast tumors. Osteopontin positively correlates with macrophage infiltration, increases with higher tumor grade, and its elevated pathway activity is associated with poor prognosis and long-term recurrence. Our findings suggest clinical implications and an alternative therapeutic strategy based on osteopontin’s multiaxial role in breast cancer progression and recurrence.

Apigenin's Influence on Inflammatory and Epigenetic Responses in Rat Lungs After Radiotherapy.

The lung is a moderately radio-sensitive organ. When cells are damaged due to accidental radiation exposure or treatment, they release molecules that lead to the recruitment of immune cells, accumulating inflammatory cytokines at the site of damage. Apigenin (Api) is a natural flavonoid known for its anti-inflammatory properties. In this study, we investigated the radioprotective properties of Api in the lung. Thirty-six Wistar rats were randomly assigned to nine groups: control, radiation (Rad), CMC+Rad, Api10+Rad, and Api20+Rad. Api was administered with an intraperitoneal injection for 7 days, after which the rats were irradiated with 6 Gy whole-body X-ray. At 6 and 72 hours post-irradiation, the rats were euthanized, and their lung tissue was extracted. Radiation led to increased alveolar wall thickness and the infiltration of macrophages and lymphocytes. Furthermore, the expression levels of inflammatory factors such as a nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-ĸB), Glycogen synthase kinase-3 beta (GSK-3β), transforming growth factor-beta1 (TGF-β1), and epigenetic factors including DNA methyltransferase 3a (DNMT3a) and Histone deacetylase 2 (HDAC2) were elevated in the lung tissue following radiation. Meanwhile, the expression level of IκB-α decreased. However, administration of Api (at both 10&20 mg/kg) reversed the adverse effects of radiation. Api administration mitigated radiation-induced lung damage by reversing inflammatory and epigenetic changes.