Leukocyte Infiltration Research Articles

SCUBE2 regulates adherens junction dynamics and vascular barrier function during inflammation.

SCUBE2 (signal peptide-CUB-epidermal growth factor-like domain-containing protein 2) is a secreted or membrane-bound protein originally identified from endothelial cells (ECs). Our previous work showed that SCUBE2 forms a complex with E-cadherin and stabilizes epithelial adherens junctions (AJs) to promote epithelial phenotypes. However, it remains unclear whether SCUBE2 also interacts with vascular endothelial (VE)-cadherin and modulates EC barrier function. In this study, we investigated whether and how SCUBE2 in ECs regulates vascular barrier maintenance. We showed that SCUBE2 colocalized and interacted with VE-cadherin and VE-protein tyrosine phosphatase (VE-PTP) within EC AJs. Furthermore, SCUBE2 knockdown disrupted EC AJs and increased EC permeability. Expression of EC SCUBE2 was suppressed at both mRNA and protein levels via the nuclear factor-κB signalling pathway in response to pro-inflammatory cytokines or permeability-inducing agents. In line with these findings, EC-specific deletion of Scube2 (EC-KO) in mice impaired baseline barrier function and worsened vascular leakiness of peripheral capillaries after local injection of histamine or vascular endothelial growth factor. EC-KO mice were also sensitive to pulmonary vascular hyperpermeability and leucocyte infiltration in response to acute endotoxin- or influenza virus-induced systemic inflammation. Meanwhile, EC-specific SCUBE2-overexpressing mice were protected from these effects. Molecular studies suggested that SCUBE2 acts as a scaffold molecule enabling VE-PTP to dephosphorylate VE-cadherin, which prevents VE-cadherin internalization and stabilizes EC AJs. As such, loss of SCUBE2 resulted in hyperphosphorylation of VE-cadherin at tyrosine 685, which led to its endocytosis, thus destabilizing EC AJs and reducing barrier function. All of these effects were exacerbated by inflammatory insults. We found that SCUBE2 contributes to vascular integrity by recruiting VE-PTP to dephosphorylate VE-cadherin and stabilize AJs, thereby promoting EC barrier function. Moreover, our data suggest that genetic overexpression or pharmacological up-regulation of SCUBE2 may help to prevent vascular leakage and oedema in inflammatory diseases.

Treatment with lipoxin A 4 improves influenza A infection outcome through macrophage reprogramming, anti-inflammatory and pro-resolutive responses.

Here, we evaluated whether a synthetic lipoxin mimetic, designated AT-01-KG, would improve the course of influenza A infection in a murine model. Mice were infected with influenza A/H1N1 and treated with AT-01-KG (1.7 mg/kg/day, i.p.) at day 3 post-infection. Mortality rate was assessed up to day 21 and inflammatory parameters were assessed at days 5 and 7. AT-01-KG attenuated mortality, reducing leukocyte infiltration and lung damage at day 5 and day 7 post-infection. AT-01-KG is a Formyl Peptide Receptor 2 (designated FPR2/3 in mice) agonist, and the protective responses were not observed in FPR2/3 -/- animals. In mice treated with LXA4 (50mg/kg/day, i.p., days 3-6 post-infection), at day 7, macrophage reprogramming was observed, as seen by a decrease in classically activated macrophages and an increase in alternatively activated macrophages in the lungs. Furthermore, the number of apoptotic cells and cells undergoing efferocytosis was increased in the lavage of treated mice. Treatment also modulated the adaptive immune response, increasing the number of anti-inflammatory T cells (Th2) and regulatory T (Tregs) cells in the lungs of the treated mice. Therefore, treatment with a lipoxin A4 analog was beneficial in a model of influenza A infection in mice. The drug decreased inflammation and promoted resolution and beneficial immune responses, suggesting it may be useful in patients with severe influenza.

Skin Manifestations of VEXAS Syndrome and Associated Genotypes

VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome is a newly defined genetic disease with an estimated prevalence of 1 in 4269 men older than 50 years and is marked by systemic inflammation, progressive bone marrow failure, and inflammatory cutaneous manifestations. To define the spectrum of cutaneous manifestations in VEXAS syndrome and the association of these findings with clinical, genetic, and histological features. This observational cohort study included data from 112 patients who were diagnosed with VEXAS-defining genetic variants in UBA1 between 2019 and 2023. Data were collected from medical record review or from patients with VEXAS directly evaluated at the National Institutes of Health in Bethesda, Maryland. To define the spectrum of cutaneous manifestations in VEXAS in association with genetic, histological, and other clinical findings. A secondary outcome was cutaneous response to treatment in VEXAS. Among the 112 patients (median [range] age, 69 [39-79] years; 111 [99%] male), skin involvement was common (93 [83%]), and the most frequent presenting feature of disease (68 [61%]). Of 64 histopathologic reports available from 60 patients, predominant skin histopathologic findings were leukocytoclastic vasculitis (23 [36%]), neutrophilic dermatosis (22 [34%]), and perivascular dermatitis (19 [30%]). Distinct pathogenic genetic variants were associated with specific cutaneous manifestations. The p.Met41Leu variant was most frequently associated with neutrophilic dermal infiltrates (14 of 17 patients [82%]), often resembling histiocytoid Sweet syndrome. In contrast, the p.Met41Val variant was associated with vasculitic lesions (11 of 20 patients [55%]) with a mixed leukocytic infiltrate (17 of 20 patients [85%]). Oral prednisone improved skin manifestations in 67 of 73 patients (92%). Patients with VEXAS treated with anakinra frequently developed severe injection-site reactions (12 of 16 [75%]), including ulceration (2 of 12 [17%]) and abscess formation (1 of 12 [8%]). Results of this cohort study show that skin manifestations are a common and early manifestation of VEXAS syndrome. Genetic evaluation for VEXAS should be considered in older male patients with cutaneous vasculitis, neutrophilic dermatoses, or chondritis. Awareness of VEXAS among dermatologists is critical to facilitate early diagnosis.

A pyroptosis-enhanced leucocyte-hitchhiking liposomal nanoplatform for potentiated immunotherapy of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a highly malignant tumor with unsatisfactory response to immunotherapy. Pyroptosis, a recently discovered form of regulated cell death (RCD), possesses a huge potential to enhance the immunotherapy efficiency against HCC. To achieve efficient drug delivery and ideal activation of antitumor immunity, an E-selectin modified liposomal nanoplatform co-loading gemcitabine elaidate and BMS-202 (a small molecule programmed cell death protein 1 (PD-1)/programmed death ligand 1 (PD-L1) inhibitor) was designed. Following intravenous injection, the liposomal nanoplatform could efficiently bind to sialylated carbohydrates on the surface of peripheral blood leucocytes via E-selectin, subsequently hitchhiking with leucocytes to realize substantial accumulation in the HCC tissue. After cellular uptake by HCC cells, the released gemcitabine could trigger gasdermin E (GSDME)-dependent pyroptosis with the release of danger-associated molecular patterns (DAMPs) and pro-inflammatory cytokines, thus generating antitumor immunity. The released BMS-202 could further relieve immune suppression by blocking the formation of PD-1/PD-L1 complex. More importantly, gemcitabine-triggered tumor pyroptosis enhanced natural orientation of leucocytes to inflammatory tumor site, further increasing the nanoplatform delivery by facilitating tumor leucocyte infiltration through a positive feedback loop. The in vivo efficacy of the fabricated liposomes demonstrated a favorable antitumor immunity by promoting dendritic cell maturation and T cell activation. In summary, this pyroptosis-enhanced leucocyte-hitchhiking liposomal nanoplatform suggests synergistic antitumor activity and unique ability to modulate drug delivery, showing promise as a highly efficient strategy for potentiated tumor immunotherapy, with a potential for clinical translation.

(Homo-)harringtonine prevents endothelial inflammation through IRF-1 dependent downregulation of VCAM1 mRNA expression and inhibition of cell adhesion molecule protein biosynthesis

The plant alkaloid homoharringtonine (HHT) is a Food and Drug Administration (FDA)-approved drug for the treatment of hematologic malignancies. In addition to its well-established antitumor activity, accumulating evidence attributes anti-inflammatory effects to HHT, which have mainly been studied in leukocytes to date. However, a potential influence of HHT on inflammatory activation processes in endothelial cells, which are a key feature of inflammation and a prerequisite for the leukocyte-endothelial cell interaction and leukocyte extravasation, remains poorly understood. In this study, the anti-inflammatory potential of HHT and its derivative harringtonine (HT) on the TNF-induced leukocyte-endothelial cell interaction was assessed, and the underlying mechanistic basis of these effects was elucidated. HHT affected inflammation in vivo in a murine peritonitis model by reducing leukocyte infiltration and proinflammatory cytokine expression as well as ameliorating abdominal pain behavior. In vitro, HT and HHT impaired the leukocyte-endothelial cell interaction by decreasing the expression of the endothelial cell adhesion molecules intracellular adhesion molecule −1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). This effect was mediated by a bipartite mechanism. While HHT did not affect the prominent TNF-induced pro-inflammatory NF-ĸB signaling cascade, the compound downregulated the VCAM1 mRNA expression in an IRF-1-dependent manner and diminished active ICAM1 mRNA translation as determined by polysome profiling. This study highlights HHT as an anti-inflammatory compound that efficiently hampers the leukocyte-endothelial cell interaction by targeting endothelial activation processes.

Chronic endometritis. Local prooxidant, antioxidant and immunometabolic disorders. An open-label, prospective, randomized study

Aim. To determine local features of lipoperoxidation processes, antioxidant defense, state of matrix metalloproteinases in chronic endometritis. Materials and methods. Eighty women aged 29.33±6.71 (min-max [24–36]) with histologically and immunohistochemically confirmed chronic endometritis and a history of reproductive losses were examined (group 1). The control group consisted of 30 patients without chronic endometritis confirmed by the results of a histochemical study and no history of reproductive losses, suffering from infertility of tubal-peritoneal origin at the age of 30.11±6.9 years (min-max [24–37]; group 2). All 130 patients underwent transvaginal echography of the pelvic organs, and histological and immunohistochemical examination of endometrial samples was performed. The activity of lipid peroxidation factors was analyzed: primary, secondary and final products of lipid peroxidation in heptane and isopropanolol fractions of uterine cavity aspirate, enzymes of the antioxidant system: superoxide dismutase, catalase, glutathione peroxidase, deoxyribonuclease I (DNase), by spectrophotometric method, remodeling factors – matrix metalloproteinases MMP-2, -9 by flow fluorimetry. Results. In patients of group 1, according to transvaginal echography of the pelvic organs, M-echo was 6.29±1.22 mm, in group 2 – 9.10±1.15 mm. In chronic endometritis, disruption of pro-oxidant and activity of antioxidant factors in the endometrium in women with chronic endometritis and a history of reproductive losses. In the heptane fraction of the phenolic extract, the concentration of primary products of lipid peroxidation (diene conjugates) was 12.98% higher than in the control, the content of secondary products of lipid peroxidation (ketodienes) was higher by 15.45%, conjugated trienes by 18.67% higher than the values in the control group, which indicates increased lipid peroxidation of endometrial cell membranes during CE. In the isopropanol phase, the products of lipid peroxidation of phospholipid acyl residues are detected. In the isopropanol fraction of the lipid extract, the amount of diene conjugates was higher, ketodienes by 24.32% and conjugated trienes by 26.14%. An increase in the activity of the enzymes superoxide dismutase by 23.98%, catalase by 14.34%, glutathione peroxidase by 12.39%, and DNase by 2.9 times was recorded compared to the indicators of group 2. Conclusion. Chronic endometritis is accompanied by changes in the system of pro-oxidant and antioxidant factors, the content of tissue remodeling products. The pathogenetic changes underlying oxidative stress in the endometrium may be increased leukocyte infiltration, imbalance of primary, secondary and end products of lipid peroxidation, enzymes superoxide dismutase, catalase, glutathione peroxidase, deoxyribonuclease I (DNase), expression of matrix metalloproteinases-2, -9.

Therapeutic Effects of Engineered Exosomes from RAW264.7 Cells Overexpressing hsa-let-7i-5p against Sepsis in Mice-A Comparative Study with Human Placenta-Derived Mesenchymal Stem Cell Exosomes.

This study compared the therapeutic effects of engineered exosomes derived from RAW264.7 cells overexpressing hsa-let-7i-5p (engineered exosomes) to exosomes from human placenta-derived mesenchymal stem cells (hpMSC exosomes) against sepsis-induced acute lung injury. Adult male C57BL/6 mice were divided into lipopolysaccharide (LPS), LPS plus engineered exosome (LEExo), or LPS plus hpMSC exosome (LMExo) groups, alongside control groups. The results showed that lung injury scores (based on pathohistological characteristics) and the levels of lung function alterations, tissue edema, and leukocyte infiltration in LEExo and LMExo groups were comparable and significantly lower than in the LPS group (all p < 0.05). Furthermore, the levels of inflammation (nuclear factor-κB activation, cytokine upregulation), macrophage activation (hypoxia-inducible factor-1α activation, M1 phase polarization), oxidation, and apoptosis were diminished in LEExo and LMExo groups compared to the LPS group (all p < 0.05). Inhibition of hsa-let-7i-5p attenuated the therapeutic effects of both engineered and hpMSC exosomes. These findings underscore the potent therapeutic capacity of engineered exosomes enriched with hsa-let-7i-5p and their potential as an alternative to hpMSC exosomes for sepsis treatment. Continued research into the mechanisms of action and optimization of engineered exosomes could pave the way for their future clinical application.

The analysis of the histomorphometric characteristics and ultrastructural features of the liver in response to environmental cycle desynchronization in the wild desert rodent (Gerbillus tarabuli)

The architecture of the hepatic tissue in the nocturnal wild desert rodent (Gerbillus tarabuli) subjected to a shifted LD (Light/Dark) cycle was investigated using transmission electron microscopy and histomorphometry. Investigation involved two groups: the control group (n = 5), exposed to the regular LD cycle (LD: 12:12), and the shifted group (n = 5), exposed to the LD regimen with a prolonged light phase of 8 h, in a day by day alternation with regular LD cycle (LD: 12:12 / LD: 20:4) during 12 weeks. Histological examination showed in the shifted group disorganization of hepatocyte trabeculae, sinusoidal dilatation, and inflammatory cell infiltration around the portal tracts. Morphometric results revealed hypertrophy of hepatocytes with a size of 29.83 ± 1.69 μm vs. 17.52 ± 0.81 μm and of their nuclei diameter with 8.69 ± 0.25 μm vs. 6.55 ± 0.15 μm. This increase is highly significant (p&lt;0.001) and represents an increase of 70.17% and 32.67%, respectively. We also noticed a significant increase in the number of binucleated cells compared to the control group with an average of 9.73 ± 0.98 vs. 5.53 ± 0.38 binucleated hepatocytes per area unit, respectively. Ultrastructural analysis corroborated the histological findings, confirming the installation of inflammatory foci, characterized by portal and intraparenchymal infiltration of leukocytes, proliferation of Kupffer cells and stellate cells, as well as an expansion of collagen fiber deposits. Electron microscopy also revealed in the shifted group some hepatocytic lesional features represented by vacuolar or liquefied cytoplasm associated with degenerative mitochondria and a rarefaction of organelles in addition to an abundance of lysosomes. Additionally, strong glycogen depletion was observed at the metabolic level. This study showed that 12 weeks of lengthening the light phase of the LD cycle, corresponding to the gerbil's resting period, had a discernible impact on hepatocellular activity, making G. tarabuli an interesting model to study the influence of artificial light at night as a circadian rhythm disruptor.

AN AUTOPSY-BASED STUDY ON THE PATTERNS OF INJURIES OF BURN CASES IN JORHAT DISTRICT, ASSAM

Objective: This autopsy-based study aimed to determine the incidence of burn-related fatalities in Jorhat District, Assam, identify associated factors, investigate injury patterns and severity, explore contributing factors, examine the relationship between injuries and cause of death, differentiate between dermo-epidermal and deep burns, investigate circumstances surrounding burn injuries, and offer recommendations for prevention and forensic investigation improvements. Methods: Data were collected from medico-legal autopsies conducted at the Mortuary of the Department of Forensic Medicine, Jorhat Medical College and Hospital, Jorhat, between May 11, 2020, and May 10, 2021. Demographic information, burn characteristics, autopsy findings, and circumstances of injuries were analyzed. Results: Out of 628 medico-legal autopsies, burn-related deaths comprised 7.32% of cases. Victims with 51–60% total body surface area burns were most common (23.91%). Burns often occurred indoors, notably in kitchens (56.52%) and bedrooms (26.08%), with a majority in closed rooms (54.35%). Synthetic (47.82%) or cotton (41.30%) clothing was prevalent, and kerosene was common in alleged suicidal cases (23.92%). Most victims died within 24 h (43.46%), primarily due to shock (52.17%), and septicemia (45.65%). Dermo-epidermal burns were prevalent (93.47%), and deaths were mainly accidental (65.21%). Histopathological analysis revealed dermis and epidermis separation (82.60%), flattened and elongated epithelial cells (73.91%), congestion (76.09%), leukocyte infiltration (73.91%), capillary dilatation (67.39%), and edema (67.39%). Conclusion: This study provides comprehensive insights into burn injury characteristics, circumstances, and consequences. Recommendations include preventive measures and improvements in forensic investigation practices related to burn injuries, offering valuable implications for health care, forensic medicine, and public awareness initiatives.

Withanicandrin Isolated from Datura Ferox Promotes Antinociception by Modulating the Asics and TRPS Channels and Anti-Inflammation in Adult Zebrafish.

This is the first study to analyze the anti-inflammatory and antinociceptive effect of withanicandrin, isolated from Datura Ferox leaves, and the possible mechanism of action involved in adult zebrafish (ZFa). To this end, the animals were treated intraperitoneally (i. p.) with withanicandrin (4; 20 and 40 mg/kg; 20 μL) and subjected to locomotor activity and acute toxicity. Nociception tests were also carried out with chemical agents, in addition to tests to evaluate inflammatory processes induced by κ-Carrageenan 1.5 % and a Molecular Docking study. As a result, withanicandrin reduced nociceptive behavior by capsaicin at a dose of 40 mg/kg and by acid saline at doses of 4 and 40 mg/kg, through neuromodulation of TRPV1 channels and ASICs, identified through blocking the antinociceptive effect of withanicandrin by the antagonists capsazepine and naloxone. Furthermore, withanicandrin caused an anti-inflammatory effect through the reduction of abdominal edema, absence of leukocyte infiltrate in the liver tissue and reduction of ROS in thel liver tissue and presented better affinity energy compared to control morphine (TRPV1) and ibuprofen (COX-1 and COX-2).

Intratumoral Fc-optimized agonistic CD40 antibody induces tumor rejection and systemic antitumor immunity in patients with metastatic cancer.

While CD40 agonism is an attractive approach for activating antigen-presenting cells and initiating antitumor responses, previous attempts have encountered limited clinical efficacy coupled with toxicity. We previously demonstrated that interactions between the antibody Fc domain and the inhibitory receptor FcγRIIB are critical for enhanced antitumor activity. Here, we present the results of a phase 1 study on intratumoral administration of an anti-CD40 agonistic antibody (2141-V11) Fc-engineered to enhance FcγRIIB binding. Primary endpoints included safety, maximum tolerated dose (MTD), and recommended phase 2 dose. Secondary objectives included preliminary clinical activity and correlative studies from biospecimens. 2141-V11 was well-tolerated without dose-limiting toxicities and MTD was not reached. In ten evaluable patients with metastatic cancer, the overall response rate was 20%, with complete responses in two patients (melanoma and breast carcinoma) and stable disease in six patients. 2141-V11 induced tumor regression in injected and non-injected lesions, with increased leukocyte infiltration and tertiary lymphoid structures (TLS) formation in post-treatment biopsies. In a humanized mouse model for CD40 and FcγRs, 2141-V11 induced TLS formation in mice bearing orthotopic breast carcinoma, correlating with local and abscopal antitumor effects, systemic immune activation, and immune memory. These findings support the safety and efficacy of 2141-V11, warranting phase 2 studies and suggesting a unique mechanism of action for this Fc-enhanced immunotherapy (NCT04059588).

Single-nucleus transcriptome unveils the role of ferroptosis in ischemic stroke

Multiple cell death pathways are involved in neuronal death in ischemic stroke (IS). However, the role of different cell death pathways in different cell types has not been elucidated. By analyzing three single-nucleus RNA sequencing (snRNA-seq) data of IS, we first found that a variety of programmed cell death (PCD) -related genes were significantly changed in different cell types. Based on machine learning and virtual gene knockout, we found that ferroptosis related genes, ferritin heavy chain 1 (Fth1) and ferritin light chain (Ftl1), play a key role in IS. Ftl1 and Fth1 can promote microglia activation, as well as the production of inflammatory factors and chemokines. Cell communication analysis showed that activated microglia could enhance chemotactic peripheral leukocyte infiltration, such as macrophages and neutrophils, through Spp1-Cd44 and App-Cd74 signaling, thereby aggravating brain tissue damage. Furthermore, real‐time quantitative polymerase chain reaction (RT‐qPCR) showed that P2ry12 and Mef2c were significantly decreased in oxygen-glucose deprivation (OGD) group, while Ftl1, Fth1, Apoe, Ctsb, Cd44 and Cd74 were significantly increased in OGD group. Collectively, our findings suggested targeted therapy against microglia Ftl1 and Fth1 might improve the state of microglia, reduce the infiltration of peripheral immune cells and tissue inflammation, and then improve the ischemic brain injury in mouse.

Impact of Cerebrospinal Fluid Leukocyte Infiltration and Neuroimmmune Mediators on Survival with HIV-Associated Cryptococcal Meningitis.

Cryptococcal meningitis remains a prominent cause of death in persons with advanced HIV disease. CSF leukocyte infiltration predicts survival at 18 weeks; however, how CSF immune response relates to CSF leukocyte infiltration is unknown. We enrolled 401 adults with HIV-associated cryptococcal meningitis in Uganda who received amphotericin and fluconazole induction therapy. We assessed the association of CSF leukocytes, chemokine, and cytokine responses with 18-week survival. Participants with CSF leukocytes ≥50/μL, had higher probability 68% (52/77) of 18-week survival compared with 52% (151/292) 18-week survival in those with ≤50 cells/μL (Hazard Ratio=1.63, 95% confidence intervals 1.14-2.23; p=0.008). Survival was also associated with higher expression of T helper (Th)-1, Th17 cytokines, and immune regulatory elements. CSF levels of Programmed Death-1 Ligand, CXCL10, and Interleukin (IL)-2 independently predicted survival. In multivariate analysis, CSF leukocytes were inversely associated with CSF fungal burden and positively associated with CSF protein, interferon-gamma (IFN-γ), IL-17A, tumor necrosis factor (TNF)-α, and peripheral blood CD4+ and CD8+ T cells expression. 18-week survival after diagnosis of cryptococcal meningitis was associated with higher CSF leukocytes at baseline with greater T helper 1 (IFN-γ, IL-2 and TNF-α cytokines), T helper 17 (IL-17A cytokine) and CXCR3+ T cell (CXCL10 chemokine) responses. These results highlight the interdependent contribution of soluble and cellular immune responses in predicting survival with HIV-associated cryptococcal meningitis.

Repeated doses of captopril induce airway hyperresponsiveness by modulating the TRPV1 receptor in rats

Although TRPV1 receptors play an essential role in the adverse effects on the airways following captopril treatment, there is no available evidence of their involvement in treatment regimens involving repeated doses of captopril. Comparing the difference in these two treatment regimens is essential since captopril is a continuous-use medication. Thus, this study explored the role of the transient receptor potential vanilloid 1 (TRPV1) in the effects of captopril on rat airways using two treatment regimens. Airway resistance, bronchoalveolar lavage (BAL), and histological and immunohistochemical analyses were conducted in rats administered with single or repeated doses of captopril. This study showed that the hyperresponsiveness to bradykinin and capsaicin in captopril-treated rats was acute. Treatment with the selective B2 antagonist, HOE140 reduced bradykinin hyperresponsiveness and abolished capsaicin exacerbation in single-dose captopril-treated rats. Likewise, degeneration of TRPV1-positive neurones also reduced hyperresponsiveness to bradykinin. Single-dose captopril treatment increased leukocyte infiltration in the BAL when compared with the vehicle and this increase was reduced by TRPV1-positive neurone degeneration. However, when compared with the vehicle treatment, animals treated with repeated doses of captopril showed an increase in leukocyte influx as early as 1 h after the last captopril treatment, but this effect disappeared after 24 h. Additionally, an increase in TRPV1 expression occurred only in animals who received repeated captopril doses and the degeneration of TRPV1-positive neurones attenuated TRPV1 upregulation. In conclusion, these data strongly indicate that a treatment regimen involving multiple doses of captopril not only enhances sensitisation but also upregulates TRPV1 expression. Consequently, targeting TRPV1 could serve as a promising strategy to reduce the negative impact of captopril on the airways.

Role of ADAR2-mediated innate immune responses in vascular inflammation and atherosclerosis

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft (DFG) Background Adenosine-to-inosine (A-to-I) RNA editing is an epitranscriptional regulator of RNA metabolism, catalyzed by the adenosine deaminases acting on RNA-1/-2 (ADAR1/2). Whether ADAR2 play a role in atherosclerotic processes remains elusive. Aim We examined the role of ADAR2 in vascular endothelial cell (EC) immunomodulatory responses and its biological relevance in murine and human atherosclerosis. Methods The role of ADAR2 in atherosclerosis was determined in a double ADAR2 and ApoE knockout mice fed with Western diet for 20 weeks. Three mouse genetic models (global and inducible vascular endothelial cell (iEC)-restricted ADAR2 and IL6ST knockout) were used to determine the role of ADAR2 in IL-6-related innate immunity. Expression levels of ADAR2 were measured in peripheral blood mononuclear cells derived from 533 individuals at risk for atherosclerotic vascular disease. Structural vascular measurements were used, including intima-media thickness (IMT), carotid wall maximum thickness (maxWT), and the number of atheromatous plaques by carotid and femoral artery ultrasonography as surrogate markers of subclinical arterial disease. Study participants were followed for major adverse cardiovascular events (MACE). Results Genetic ablation of ADAR2 resulted in a reduction of atherosclerotic plaque burden and necrotic core in the aorta and brachiocephalic trunk as well as reduced leukocyte infiltration in atherosclerotic plaques without affecting cholesterol levels in mice. Blood analysis revealed decreased pro-inflammatory cytokine levels including IL-6 after treatment of ADAR2/APOE KO mice compared to littermate control mice. Intravital imaging of IL-6-inflamed vascular endothelium showed that rolling and adhesion of leukocyte subsets to vascular wall were impaired by 2-fold in ADAR2-/- or iEC-ADAR2-/- KO mice. In vivo and ex vivo histological examination revealed that endothelial ADAR2 controls endothelial vascular adhesion molecule expression induced by gp130 signaling. ADAR2-deficient transcriptome revealed a selective upregulation of a conserved group of miRNAs targeting the IL6ST mRNA. Multilayered rescue studies including miR-inhibitors restored IL6ST levels after ADAR2 deficiency. In humans, increased ADAR2 expression was independently associated with the presence of CAD, higher C-reactive protein and mean carotid IMT after adjustment for traditional risk factors. Increased ADAR2 at baseline was prospectively associated with accelerated progression of subclinical atherosclerosis reflected by the increased number of carotid plaques at follow-up (OR=3.74, P&amp;lt;0.05). Finally, high ADAR2 levels were independently associated with higher MACE incidence across a median follow-up period of 54 months. Conclusion Posttranscriptional control of vascular inflammation by ADAR2 is integral for the development and progression of atherosclerosis in mice and humans. Our findings suggest that ADAR2 may serve as a promising therapeutic target for atherosclerosis.Role of ADAR2 in atherosclerosis

Podocyte Pathogenic Bone Morphogenetic Protein-2 Pathway and Immune Cell Behaviors in Primary Membranous Nephropathy.

Primary membranous nephropathy (PMN) is one of the leading causes of end-stage renal disease, and the most frequent cause of massive proteinuria in nondiabetic adults, resulting in fatal complications. However, the underlying pathomechanisms of PMN remain largely unclear. Here, single-cell RNA sequencing is employed to analyze kidney biopsies from eleven PMN patients and seven healthy subjects. Profiling 44060 cells from patients allowed us to characterize the cellular composition and cell-type-specific gene expression in the PMN kidney. The complement-induced BMP2/pSMAD1/COL4 pathway is identified as the pathogenic pathway in podocytes, bridging two key events, i.e., complement system activation and glomerular basement membrane thickening in PMN. Augmented infiltration and activation of myeloid leukocytes and B lymphocytes are found, profiling delicate crosstalk of immune cells in PMN kidneys. Overall, these results provide valuable insights into the roles of podocytes and immune cells in PMN, and comprehensive resources toward the complete understanding of PMN pathophysiology.

#1467 Pegcetacoplan for post-transplant recurrent C3 glomerulopathy or immune complex membranoproliferative glomerulonephritis in NOBLE: 12-week evolution

Abstract Background and Aims C3 glomerulopathy (C3G) and primary immune complex membranoproliferative glomerulonephritis (IC-MPGN) are rare diseases caused by uncontrolled complement activation, resulting in excess deposition of C3 breakdown products in the kidneys, which leads to kidney damage and eventually kidney failure. Kidney transplantation is an option for end-stage kidney disease, but disease recurrence is common (up to 67% of patients), and up to 50% of transplant recipients lose renal allografts due to recurrence. Pegcetacoplan is a targeted C3 and C3b inhibitor that may prevent excess deposition of C3 and C5 breakdown products and subsequent glomerular inflammation and kidney damage in patients with C3G or IC-MPGN, potentially improving clinical outcomes. NOBLE (NCT04572854) was the first prospective randomized controlled trial of pegcetacoplan vs standard of care (SOC) in post-transplant patients with recurrent C3G or IC-MPGN. Method Adult patients were randomized 3:1 to subcutaneous pegcetacoplan 1080 mg twice weekly plus SOC (n = 10) or SOC only (n = 3) for 12 weeks followed by a 40-week non-controlled period in which all patients received pegcetacoplan. Primary and secondary endpoint results for Week-12 data have been reported. We conducted a patient-level post hoc analysis to describe key histology changes from renal biopsy (C3c staining, C3G histologic index activity score), clinical parameters (urine protein-to-creatinine ratio [uPCR; calculated from triplicate first-morning spot urine], estimated glomerular filtration rate [eGFR]), and biomarkers (serum C3, plasma sC5b-9) through Week 12. The C3G histologic index uses a semiquantitative scale of 0–3 for 7 markers of activity (mesangial hypercellularity, endocapillary proliferation, membranoproliferative morphology, leukocyte infiltration, cellular and/or microcellular crescent formation, fibrinoid necrosis, and interstitial inflammation), for a total activity score of 0–21. eGFR stability was defined as a decrease of no more than 25% versus baseline. Results Among the 10 pegcetacoplan-treated patients, 7 (70%) had 1 previous kidney transplant, 2 (20%) had 2, and 1 (10%) had 3. The mean (range) time to disease recurrence after most recent transplant was 10.8 (0.4–31.8) months and from most recent recurrence to randomization was 12.2 (2.1–37.7) months, with 60% randomized &amp;gt;6 months post-disease recurrence. Among the 3 SOC-only patients, 2 (66.7%) had 1 transplant and 1 (33.3%) had 2. Mean (range) time to recurrence was 32.8 (4.6–63.8) months and to randomization was 33.9 (7.0–65.4) months. At Week 12, 8 (80%) pegcetacoplan-treated patients had a reduction in C3c staining of at least 1 order of magnitude. Of these 8, 7 (70%) had a decrease in activity score, and 6 of these 7 (60%) had a decrease in proteinuria. Four (40%) patients had reduced C3c staining, decreased activity score, and cleared electron microscopy deposits at Week 12. eGFR remained stable in 9 (90%) patients. Six of 9 (67%) patients with available Week-12 data (4 of 5 patients with &amp;gt;1 g/g uPCR at baseline) had a reduction in uPCR with 5 of the 6 achieving ≥50% uPCR reduction (Table). All pegcetacoplan-treated patients had increased serum C3, as expected, and 9 (90%) had decreased sC5b-9. At Week 12, no TEAEs led to study discontinuation, treatment withdrawal, or death. No encapsulated bacterial infections or events of rejection/graft loss related to the study drug were reported. Conclusion As early as Week 12, more than half (6/10 [60%]) of post-transplant patients with recurrent C3G or IC-MPGN treated with pegcetacoplan achieved reductions in C3c staining, activity score of C3G histology index, and proteinuria, suggesting that pegcetacoplan targets the underlying pathophysiology of the C3G/IC-MPGN in kidney transplant recipients. The safety and efficacy of pegcetacoplan will be further evaluated at Week 52 of the NOBLE study and as part of the Phase 3 VALIANT (NCT05067127) trial, which enrolled patients with C3G or primary IC-MPGN who have native kidney or post-transplant disease.

#2906 Kidney immune cells and endothelial cells can be modulated by low-salt and high-salt diets

Abstract Background and Aims Kidney immune cells and endothelial cells are involved in pathogenesis of kidney ischemia-reperfusion injury and potential therapeutic targets to improve kidney transplant outcomes. We hypothesized that dietary salt intake can change kidney immune cells and endothelial cells. Method C57BL/6 mice were treated with normal diet, low-salt diet, or high-salt diet. After 6 weeks of allocated diets, mice were euthanized, and their kidneys were collected. Leukocyte infiltration was assessed with CD45 immunohistochemistry. Lymphocytes were isolated from kidneys and were studied by flow cytometry. Human kidney transplant tissues obtained by a protocol biopsy at 2 weeks after kidney transplantation were stained with CD45, CD3, CD20, and CD31. Expression of each marker was quantified using an automated imaging analysis system and analyzed per donors’ pre-donation 24 h urine sodium levels. Induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) from healthy individuals and end-stage kidney disease (ESKD) patients were treated with different sodium concentrations, and their proliferation was assessed. Results Kidneys from mice fed with high-salt diet had higher kidney leukocytes (CD45+) than those fed with normal diet (1.9 ± 0.3% vs 0.9 ± 0.1%, P = .043). Effector-memory CD4+ T cells (normal 44.2 ± 2.6%; low-salt 57.1 ± 3.5%, P = .020; high-salt 62.1 ± 2.5% of CD4+ T cells, P = .002) and mature B cells (70.0 ± 1.5%; 79.1 ± 1.4%, P = .001; 76.7 ± 1.6% of CD19+ cells, P = .015) increased by low-salt and high-salt diets, whereas naïve CD4+ T cells deceased (45.9 ± 2.1%; 32.7 ± 3.2%, P = .016; 27.8 ± 3.1% of CD4+ T cells, P = .001) in kidneys. Human kidney transplants from donors under low-salt diet showed lower total leukocytes (CD45+, 0.3 ± 0.1% vs 0.9 ± 0.3%, P = .019) with lower T and B cells (CD3+, 1.8 ± 0.2% vs 3.5 ± 0.4%, P&amp;lt;.001; CD20+, 0.8 ± 0.1% vs 2.1 ± 0.6%, P = .011) and higher capillary density (CD31+, 29.0 ± 2.2% vs 16.1 ± 1.8%, P = .009) compared to those from donors under high-salt diet. The proliferation of iPSC-ECs from healthy individuals and ESKD patients after hypoxia was reduced under higher sodium concentrations. Conclusion High-salt and low-salt diets affect kidney immune cell compositions and numbers as well as endothelial cells. Reconstitution of kidney microenvironment by modifying dietary salt intake could be a novel approach to improve kidney transplant outcomes.

Cryptococcus neoformans trehalose-6-phosphate synthase (tps1) promotes organ-specific virulence and fungal protection against multiple lines of host defenses.

Trehalose-6-phosphate synthase (TPS1) was identified as a virulence factor for Cryptococcus neoformans and a promising therapeutic target. This study reveals previously unknown roles of TPS1 in evasion of host defenses during pulmonary and disseminated phases of infection. In the pulmonary infection model, TPS1-deleted (tps1Δ) Cryptococci are rapidly cleared by mouse lungs whereas TPS1-sufficent WT (H99) and revertant (tps1Δ:TPS1) strains expand in the lungs and disseminate, causing 100% mortality. Rapid pulmonary clearance of tps1Δ mutant is T-cell independent and relies on its susceptibility to lung resident factors and innate immune factors, exemplified by tps1Δ but not H99 inhibition in a coculture with dispersed lung cells and its rapid clearance coinciding with innate leukocyte infiltration. In the disseminated model of infection, which bypasses initial lung-fungus interactions, tps1Δ strain remains highly attenuated. Specifically, tps1Δ mutant is unable to colonize the lungs from the bloodstream or expand in spleens but is capable of crossing into the brain, where it remains controlled even in the absence of T cells. In contrast, strains H99 and tps1Δ:TPS1 rapidly expand in all studied organs, leading to rapid death of the infected mice. Since the rapid pulmonary clearance of tps1Δ mutant resembles a response to acapsular strains, the effect of tps1 deletion on capsule formation in vitro and in vivo was examined. Tps1Δ cryptococci form capsules but with a substantially reduced size. In conclusion, TPS1 is an important virulence factor, allowing C. neoformans evasion of resident pulmonary and innate defense mechanisms, most likely via its role in cryptococcal capsule formation.

Astrocytic RIPK3 exerts protective anti-inflammatory activity during viral encephalitis via induction of serpin protease inhibitors.

Flaviviruses pose a significant threat to public health due to their ability to infect the central nervous system (CNS) and cause severe neurologic disease. Astrocytes play a crucial role in the pathogenesis of flavivirus encephalitis through their maintenance of blood-brain barrier (BBB) integrity and their modulation of immune cell recruitment and activation within the CNS. We have previously shown that receptor interacting protein kinase-3 (RIPK3) is a central coordinator of neuroinflammation during CNS viral infection, a function that occurs independently of its canonical function in inducing necroptotic cell death. To date, however, roles for necroptosis-independent RIPK3 signaling in astrocytes are poorly understood. Here, we use mouse genetic tools to induce astrocyte-specific deletion, overexpression, and chemogenetic activation of RIPK3 to demonstrate an unexpected anti-inflammatory function for astrocytic RIPK3. RIPK3 activation in astrocytes was required for host survival in multiple models of flavivirus encephalitis, where it restricted neuropathogenesis by limiting immune cell recruitment to the CNS. Transcriptomic analysis revealed that, despite inducing a traditional pro-inflammatory transcriptional program, astrocytic RIPK3 paradoxically promoted neuroprotection through the upregulation of serpins, endogenous protease inhibitors with broad immunomodulatory activity. Notably, intracerebroventricular administration of SerpinA3N in infected mice preserved BBB integrity, reduced leukocyte infiltration, and improved survival outcomes in mice lacking astrocytic RIPK3. These findings highlight a previously unappreciated role for astrocytic RIPK3 in suppressing pathologic neuroinflammation and suggests new therapeutic targets for the treatment of flavivirus encephalitis.