Immune Cell Migration Research Articles

Immune Cell Migration to Cancer.

Immune cell migration is required for the development of an effective and robust immune response. This elegant process is regulated by both cellular and environmental factors, with variables such as immune cell state, anatomical location, and disease state that govern differences in migration patterns. In all cases, a major factor is the expression of cell surface receptors and their cognate ligands. Rapid adaptation to environmental conditions partly depends on intrinsic cellular immune factors that affect a cell's ability to adjust to new environment. In this review, we discuss both myeloid and lymphoid cells and outline key determinants that govern immune cell migration, including molecules required for immune cell adhesion, modes of migration, chemotaxis, and specific chemokine signaling. Furthermore, we summarize tumor-specific elements that contribute to immune cell trafficking to cancer, while also exploring microenvironment factors that can alter these cellular dynamics within the tumor in both a pro and antitumor fashion. Specifically, we highlight the importance of the secretome in these later aspects. This review considers a myriad of factors that impact immune cell trajectory in cancer. We aim to highlight the immunotherapeutic targets that can be harnessed to achieve controlled immune trafficking to and within tumors.

Enterovirus A71 crosses a human blood-brain barrier model through infected immune cells.

Enterovirus A71 (EV-A71) is associated with neurological conditions such as acute meningitis and encephalitis. The virus is detected in the bloodstream, and high blood viral loads are associated with central nervous system (CNS) manifestations. We used an in vitro blood-brain barrier (BBB) model made up of human brain-like endothelial cells (hBLECs) and brain pericytes grown in transwell systems to investigate whether three genetically distinct EV-A71 strains (subgenogroups C1, C1-like, and C4) can cross the human BBB. EV-A71 poorly replicated in hBLECs, which released moderate amounts of infectious viruses from their luminal side and trace amounts of infectious viruses from their basolateral side. The barrier properties of hBLECs were not impaired by EV-A71 infection. We investigated the passage through hBLECs of EV-A71-infected white blood cells. EV-A71 strains efficiently replicated in immune cells, including monocytes, neutrophils, and NK/T cells. Attachment to hBLECs of immune cells infected with the C1-like virus was higher than attachment of cells infected with C1-06. EV-A71 infection did not impair the transmigration of immune cells through hBLECs. Overall, EV-A71 targets different white blood cell populations that have the potential to be used as a Trojan horse to cross hBLECs more efficiently than cell-free EV-A71 particles.IMPORTANCEEnterovirus A71 (EV-A71) was first reported in the USA, and numerous outbreaks have since occurred in Asia and Europe. EV-A71 re-emerged as a new multirecombinant strain in 2015 in Europe and is now widespread. The virus causes hand-foot-and-mouth disease in young children and is involved in nervous system infections. How the virus spreads to the nervous system is unclear. We investigated whether white blood cells could be infected by EV-A71 and transmit it across human endothelial cells mimicking the blood-brain barrier protecting the brain from adverse effects. We found that endothelial cells provide a strong roadblock to prevent the passage of free virus particles but allow the migration of infected immune cells, including monocytes, neutrophils, and NK/T cells. Our data are consistent with the potential role of immune cells in the pathogenesis of EV-A71 infections by spreading the virus in the blood and across the human blood-brain barrier.

Loss of the TRPM4 channel in humans causes immune dysregulation with defective monocyte migration

BackgroundTransient receptor potential melastatin subfamily member 4 (TRPM4) is a broadly expressed, calcium-activated, monovalent cation channel that regulates immune cell function in mice and cell lines. Clinically however, partial loss or gain of function mutations in TRPM4 lead to arrythmia and heart disease, with no documentation of immunological disorders. ObjectiveTo characterize the functional cellular mechanisms underlying the immune dysregulation phenotype in a proband with a mutated trpm4 gene. MethodsWe employ a combination of biochemical, cell biological, imaging, omics analyses, flow cytometry, and gene editing approaches. ResultsWe report the first human cases with complete loss of the TRPM4 channel leading to immune dysregulation with frequent bacterial and fungal infections. Single cell and bulk RNAseq point to altered expression of genes affecting cell migration, specifically in monocytes. Inhibition of TRPM4 in T cells and the THP1 monocyte cell line reduces migration. More importantly primary T cells and monocytes from the TRPM4 patients migrate poorly. Finally, CRISPR knockout of TRPM4 in THP1 cells greatly reduces their migration potential. ConclusionOur results demonstrate that TRPM4 plays a critical role in regulating immune cell migration, leading to increased susceptibility to infections.

New advances in immune mechanism and treatment during ocular toxoplasmosis.

Ocular toxoplasmosis (OT) is an intraocular infection caused by the parasite Toxoplasma gondii. OT is manifested as retinal choroiditis and is the most common infectious cause of posterior uveitis. Invasion of the retina by T. gondii leads to disruption of the blood-ocular barrier and promotes the migration of immune cells to the ocular tissues. Cytokines such as IFN-γ and IL-1β are effective for controlling parasite growth, but excessive inflammatory responses can cause damage to the host. In this review, we will discuss in detail the latest advances in the immunopathology and treatment of OT.

The Effect of Methyl-Derivatives of Flavanone on MCP-1, MIP-1β, RANTES, and Eotaxin Release by Activated RAW264.7 Macrophages.

Chemokines, also known as chemotactic cytokines, stimulate the migration of immune cells. These molecules play a key role in the pathogenesis of inflammation leading to atherosclerosis, neurodegenerative disorders, rheumatoid arthritis, insulin-resistant diabetes, and cancer. Moreover, they take part in inflammatory bowel disease (IBD). The main objective of our research was to determine the activity of methyl-derivatives of flavanone, namely, 2'-methylflavanone (5B), 3'-methylflavanone (6B), 4'-methylflavanone (7B), and 6-methylflavanone (8B), on the releasing of selected cytokines by RAW264.7 macrophages activated by LPS. We determined the concentration of chemokines belonging to the CC chemokine family, namely, MCP-1, MIP-1β, RANTES, and eotaxin, using the Bio-Plex Magnetic Luminex Assay and the Bio-PlexTM 200 System. Among the tested compounds, only 5B and 6B had the strongest effect on inhibiting the examined chemokines' release by macrophages. Therefore, 5B and 6B appear to be potentially useful in the prevention of diseases associated with the inflammatory process.

Discovery of the Clinical Candidate IDOR-1117-2520: A Potent and Selective Antagonist of CCR6 for Autoimmune Diseases.

Migration of immune cells to sites of inflammation is a critical step in the body's response to infections but also during autoimmune flares. Chemokine receptors, members of the GPCR receptors, are instrumental in directing specific cell types to their target organs. Herein, we describe a highly potent small molecule antagonist of the chemokine receptor CCR6, which came out of fine-tuned structural elaborations from a proprietary HTS hit. Three main issues in the parent chemical series-cytotoxicity, phototoxicity, and hERG, were successfully solved. Biological characterization demonstrated that compound 45 (IDOR-1117-2520) is a selective and insurmountable antagonist of CCR6. In vivo proof-of-mechanism studies in a mouse lung inflammation model using a representative compound from the chemical class of 45 confirmed that the targeted CCR6+ cells were efficiently inhibited from migrating into the bronchoalveoli. Finally, ADMET and physicochemical properties were well balanced and the preclinical package warranted progress in the clinic.

Atypical chemokine receptors in the immune system.

Leukocyte migration is a fundamental component of innate and adaptive immune responses as it governs the recruitment and localization of these motile cells, which is crucial for immune cell priming, effector functions, memory responses and immune regulation. This complex cellular trafficking system is controlled to a large extent via highly regulated production of secreted chemokines and the restricted expression of their membrane-tethered G-protein-coupled receptors. The activity of chemokines and their receptors is also regulated by a subfamily of molecules known as atypical chemokine receptors (ACKRs), which are chemokine receptor-like molecules that do not couple to the classical signalling pathways that promote cell migration in response to chemokine ligation. There has been a great deal of progress in understanding the biology of these receptors and their functions in the immune system in the past decade. Here, we describe the contribution of the various ACKRs to innate and adaptive immune responses, focussing specifically on recent progress. This includes recent findings that have defined the role for ACKRs in sculpting extracellular chemokine gradients, findings that broaden the spectrum of chemokine ligands recognized by these receptors, candidate new additions to ACKR family, and our increasing understanding of the role of these receptors in shaping the migration of innate and adaptive immune cells.

CXCR4-mediated neutrophil dynamics in periodontitis

Background and objectivePeriodontitis is a common oral disease closely related to immune response and this study is aimed to identify the key immune-related pathogenic genes and analyze the infiltration and function of immune cells in the disease using bioinformatics methods. MethodsTranscriptome datasets and single-cell RNA sequencing (scRNA-seq) datasets were downloaded from the GEO database. We utilized weighted correlation network analysis and least absolute selection and shrinkage operator, protein–protein interaction network construction to screen out key pathogenic genes as well as conducted the cell-type identification by estimating relative subsets of RNA transcripts algorithm to analyze and characterize immune cell types in periodontal tissues. In addition to bioinformatics validations, clinical and cell samples were collected and mouse periodontitis models were constructed to validate the important role of key genes in periodontitis. ResultsBioinformatics analysis pointed out the positive correlation between CXCR4 expression and periodontitis, and revealed the increased infiltration of neutrophils in periodontal inflammatory. Similar results were obtained from clinical samples and animal models. In addition, the clustering and functional enrichment results based on CXCR4 expression levels included activation of immune response and cell migration, implying the possible function of CXCR4 on regulating neutrophil dynamics, which might contribute to periodontitis. Subsequent validation experiments confirmed that the increased expression of CXCR4 in neutrophils under periodontitis, where cell migration-related pathways also were activated. ConclusionCXCR4 could be the key pathogenic gene of periodontitis and CXCR4/CXCL12 signal axial might contribute to the development of periodontitis by mediating neutrophil dynamics, suggesting that CXCR4 could be a potential target to help identify novel strategies for the clinical diagnosis and treatment of periodontitis.

(S)-(-)-blebbistatin O-benzoate has the potential to improve atopic dermatitis symptoms in NC/Nga mice by upregulating epidermal barrier function and inhibiting type 2 alarmin cytokine induction.

Atopic dermatitis is a multi-pathogenic disease characterized by chronic skin inflammation and barrier dysfunction. Therefore, improving the skin's ability to form an epidermal barrier and suppressing the production of cytokines that induce type 2 inflammatory responses are important for controlling atopic dermatitis symptoms. (-)-Blebbistatin, a non-muscle myosin II inhibitor, has been suggested to improve pulmonary endothelial barrier function and control inflammation by suppressing immune cell migration; however, its efficacy in atopic dermatitis is unknown. In this study, we investigated whether (S)-(-)-blebbistatin O-benzoate, a derivative of (-)-blebbistatin, improves dermatitis symptoms in a mite antigen-induced atopic dermatitis model using NC/Nga mice. The efficacy of the compound was confirmed using dermatitis scores, ear thickness measurements, serum IgE levels, histological analysis of lesions, and filaggrin expression analysis, which is important for barrier function. (S)-(-)-Blebbistatin O-benzoate treatment significantly reduced the dermatitis score and serum IgE levels compared to those in the vehicle group (p < 0.05). Furthermore, the histological analysis revealed enhanced filaggrin production and a decreased number of mast cells (p < 0.05), indicating that (S)-(-)-blebbistatin O-benzoate improved atopic dermatitis symptoms in a pathological model. In vitro analysis using cultured keratinocytes revealed increased expression of filaggrin, loricrin, involucrin, and ceramide production pathway-related genes, suggesting that (S)-(-)-blebbistatin O-benzoate promotes epidermal barrier formation. Furthermore, the effect of (S)-(-)-blebbistatin O-benzoate on type 2 alarmin cytokines, which are secreted from epidermal cells upon scratching or allergen stimulation and are involved in the pathogenesis of atopic dermatitis, was evaluated using antigens derived from mite feces. The results showed that (S)-(-)-blebbistatin O-benzoate inhibited the upregulation of these cytokines. Based on the above, (S)-(-)-blebbistatin O-benzoate has the potential to be developed as an atopic dermatitis treatment option that controls dermatitis symptoms by suppressing inflammation and improving barrier function by acting on multiple aspects of the pathogenesis of atopic dermatitis.

CCR2-dependent CX3CR1+ colonic macrophages promote Enterococcus faecalis dissemination.

Enterococci are common commensal bacteria that colonize the gastrointestinal tracts of most mammals, including humans. Importantly, these bacteria are one of the leading causes of nosocomial infections. This study examined the role of colonic macrophages in facilitating Enterococcus faecalis infections in mice. We determined that depletion of colonic phagocytes resulted in the reduction of E. faecalis dissemination to the gut-draining mesenteric lymph nodes. Furthermore, we established that trafficking of monocyte-derived CX3CR1-expressing macrophages contributed to E. faecalis dissemination in a manner that was not reliant on CCR7, the conventional receptor involved in lymphatic migration. Finally, we showed that E. faecalis mutants with impaired intracellular survival exhibited reduced dissemination, suggesting that E. faecalis can exploit host immune cell migration to disseminate systemically and cause disease. Our findings indicate that modulation of macrophage trafficking in the context of antibiotic therapy could serve as a novel approach for preventing or treating opportunistic infections by disseminating enteric pathobionts like E. faecalis.

Photoactivated Formation of an Extravascular Dynamic Hydrogel as an Intelligent Blood Flow Regulator to Reprogram the Immunogenic Landscape.

Long-term tumor starvation may be a potential strategy to elevate the antitumor immune response by depriving nutrients. However, combining long-term starvation therapy with immunotherapy often yields limited efficacy due to the blockage of immune cell migration pathways. Herein, an intelligent blood flow regulator (BFR) is first established through photoactivated in situ formation of the extravascular dynamic hydrogel to compress blood vessels, which can induce long-term tumor starvation to elicit metabolic stress in tumor cells without affecting immune cell migration pathways. By leveraging methacrylate-modified nanophotosensitizers (HMMAN) and biodegradable gelatin methacrylate (GelMA), the developed extravascular hydrogel dynamically regulates blood flow via enzymatic degradation. Additionally, aPD-L1 loaded into HMMAN continuously blocks immune checkpoints. Systematic in vivo experiments demonstrate that the combination of immune checkpoint blockade (ICB) and BFR-induced metabolic stress (BIMS) significantly delays the progression of Lewis lung and breast cancers by reshaping the tumor immunogenic landscape and enhancing antitumor immune responses.

Sex Differences in T Cell Migration from the Spleen to the Kidney in Mice at Baseline and in Response to Angiotensin II Infusion

Decades of research has indicated that T cells contribute to the development of hypertension and target organ damage. There is a critical role of spleen-derived T cells in angiotensin II-mediated hypertension and kidney damage in mice. Splenectomy abolished sex differences in the development of hypertension in spontaneously hypertensive rats, suggesting sex differences in the spleen-kidney immune axis. It is well-appreciated that immune cells migrate from different tissues to infiltrate organs. However, whether there are sex differences in T cell migration from the spleen to the kidney remained unexplored. Thus, we hypothesized that male mice would show higher immune cell migration from the spleen to the kidney at baseline and in response to angiotensin II infusion when compared to female mice. To quantify T cell migration in male and female mice, we utilized Kikume Green-Red (KikGR) transgenic mice that ubiquitously express the kikume green protein in all cells throughout the body, and this protein can be stably photoconverted to kikume red with exposure to 405 nm blue light. We specifically photoconverted mouse spleens with 10-minute exposure to blue light and infused angiotensin II via mini-pump implantation (490 ng/kg*min) in half of the photoconverted mice. After 3 days post-photoconversion, all mice were sacrificed, and both kidneys were removed for analysis via spectral flow cytometry to determine spleen-derived infiltrating immune cells (CD45+, KikRed+ cells) as well as spleen-derived T cells (CD45+, KikRed+, CD3+) in the kidney. There were no differences in the immune cell profiles found between left and right kidneys within each sex (p = 0.8 [male kidneys] and 0.8292 [female kidneys], n = 6-10 kidneys per group). At baseline, we found significantly increased spleen-derived immune cells in the kidneys of male compared to female mice (P = 0.0173, n = 3-5 per group). In addition, sex was a significant factor in spleen-derived T cell percentages within the kidney (Two-Way ANOVA, P = 0.029, n = 3-5 mice). In response to 3 days of angiotensin II infusion, spleen-derived immune cells were significantly higher in the kidneys of males compared to females (P = 0.0499, n = 3-5 mice). We conclude that there is reduced immune cell migration to the kidney from the spleen at baseline and in response to angiotensin II infusion in female mice, which may contribute to the lower blood pressure and kidney damage observed in female mice. NIH TL1 DK139566 to LNB, NIH R01 DK134562 to JSP. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Exploring kidney allograft rejection: A proof-of-concept study using spatial transcriptomics

In this proof-of-concept study, spatial transcriptomics combined with public single-cell ribonucleic acid-sequencing data were used to explore the potential of this technology to study kidney allograft rejection. We aimed to map gene expression patterns within diverse pathologic states by examining biopsies classified across nonrejection, T cell–mediated acute rejection, interstitial fibrosis, and tubular atrophy. Our results revealed distinct immune cell signatures, including those of T and B lymphocytes, monocytes, mast cells, and plasma cells, and their spatial organization within the renal interstitium. We also mapped chemokine receptors and ligands to study immune cell migration and recruitment. Finally, our analysis demonstrated differential spatial enrichment of transcription signatures associated with kidney allograft rejection across various biopsy regions. Interstitium regions displayed higher enrichment scores for rejection-associated gene expression patterns than tubular areas, which had negative scores. This implies that these signatures are primarily driven by processes unfolding in the renal interstitium. Overall, this study highlights the value of spatial transcriptomics for revealing cellular heterogeneity and immune signatures in renal transplant biopsies and demonstrates its potential for studying the molecular and cellular mechanisms associated with rejection. However, certain limitations must be borne in mind regarding the development and future applications of this technology.

Biophysical characterization of the CXC chemokine receptor 2 ligands.

The chemokines of the immune system act as first responders by operating as chemoattractants, directing immune cells to specific locations of inflamed tissues. This promiscuous network is comprised of 50 ligands and 18 receptors where the ligands may interact with the receptors in various oligomeric states i.e., monomers, homodimers, and heterodimers. Chemokine receptors are G-protein coupled receptors (GPCRs) present in the membrane of immune cells. The migration of immune cells occurs in response to a concentration gradient of the ligands. Chemotaxis of neutrophils is directed by CXC-ligand (CXCL) activation of the membrane bound CXC chemokine receptor 2 (CXCR2). CXCR2 plays an important role in human health and is linked to disorders such as autoimmune disorders, inflammation, and cancer. Yet, despite their important role, little is known about the biophysical characteristics controlling ligand:ligand and ligand:receptor interaction essential for biological activity. In this work, we study the homodimers of three of the CXCR2 cognate ligands, CXCL1, CXCL5, and CXCL8. The ligands share high structural integrity but a low sequence identity. We show that the sequence diversity has evolved different binding affinities and stabilities for the CXC-ligands resulting in diverse agonist/antagonist behavior. Furthermore, CXC-ligands fold through a three-state mechanism, populating a folded monomeric state before associating into an active dimer.

Dupilumab Therapy Modulates Circulating Inflammatory Mediators in Patients with Prurigo Nodularis

Prurigo nodularis (PN) is a chronic inflammatory skin disease characterized by intense pruritus and skin nodules. Beyond the skin, PN involves circulating blood inflammation that may contribute to systemic disease comorbidities. Dupilumab was recently approved for treatment of PN, but its effects on systemic inflammation are unknown. Thus, we aimed to characterize changes in plasma concentrations of inflammatory proteins following dupilumab treatment. In this exploratory study, plasma samples were collected from three patients with moderate-to-severe PN before and after ≥6 months of dupilumab. All patients exhibited clinically significant improvements post-treatment. Of the 2569 proteins tested, 186 were differentially expressed post-treatment (q<0.1, FC>1.3). Downregulated proteins included cytokines associated with Th1 (IFN-γ, TNF-α), Th2 (IL-4, IL-13), Th17/Th22 (IL-6, IL-22) signaling. Markers of innate immunity (IL-19, TLR1, NOS2), immune cell migration (CCL20, CD177), and fibrosis (IL-11, IL-22) were also decreased (q<0.1). GSVA analysis of Th2, Th17, and Epithelial-Mesenchymal-Transition gene sets showed reduced pathway expression in the post-treatment cohort (p<0.05). Plasma cytokine levels of IL-11, NOS2, IL-13, IL-4, and IFNG (R2>0.75, q<0.10) showed the strongest correlations with pruritus severity. Dupilumab may reduce systemic inflammatory proteins associated with multiple immune and fibrosis pathways in PN patients, potentially modulating the development of systemic disease comorbidities.

A Comparative Study of Human Pluripotent Stem Cell-Derived Macrophages in Modeling Viral Infections.

Macrophages play multiple roles in innate immunity including phagocytosing pathogens, modulating the inflammatory response, presenting antigens, and recruiting other immune cells. Tissue-resident macrophages (TRMs) adapt to the local microenvironment and can exhibit different immune responses upon encountering distinct pathogens. In this study, we generated induced macrophages (iMACs) derived from human pluripotent stem cells (hPSCs) to investigate the interactions between the macrophages and various human pathogens, including the hepatitis C virus (HCV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and Streptococcus pneumoniae. iMACs can engulf all three pathogens. A comparison of the RNA-seq data of the iMACs encountering these pathogens revealed that the pathogens activated distinct gene networks related to viral response and inflammation in iMACs. Interestingly, in the presence of both HCV and host cells, iMACs upregulated different sets of genes involved in immune cell migration and chemotaxis. Finally, we constructed an image-based high-content analysis system consisting of iMACs, recombinant GFP-HCV, and hepatic cells to evaluate the effect of a chemical inhibitor on HCV infection. In summary, we developed a human cell-based in vitro model to study the macrophage response to human viral and bacterial infections; the results of the transcriptome analysis indicated that the iMACs were a useful resource for modeling pathogen-macrophage-tissue microenvironment interactions.

Salvianolic acid B enhances tissue repair and regeneration by regulating immune cell migration and Caveolin-1-mediated blastema formation in zebrafish

IntroductionNon-healing wounds resulting from trauma, surgery, and chronic diseases annually affect millions of individuals globally, with limited therapeutic strategies available due to the incomplete understanding of the molecular processes governing tissue repair and regeneration. Salvianolic acid B (Sal B) has shown promising bioactivities in promoting angiogenesis and inhibiting inflammation. However, its regulatory mechanisms in tissue regeneration remain unclear. PurposeThis study aims to investigate the effects of Sal B on wound healing and regeneration processes, along with its underlying molecular mechanisms, by employing zebrafish as a model organism. MethodsIn this study, we employed a multifaceted approach to evaluate the impact of Sal B on zebrafish tail fin regeneration. We utilized whole-fish immunofluorescence, TUNEL staining, mitochondrial membrane potential (MMP), and Acridine Orange (AO) probes to analyze the tissue repair and regenerative under Sal B treatment. Additionally, we utilized transgenic zebrafish strains to investigate the migration of inflammatory cells during different phases of fin regeneration. To validate the importance of Caveolin-1 (Cav1) in tissue regeneration, we delved into its functional role using molecular docking and Morpholino-based gene knockdown techniques. Additionally, we quantified Cav1 expression levels through the application of in situ hybridization. ResultsOur findings demonstrated that Sal B expedites zebrafish tail fin regeneration through a multifaceted mechanism involving the promotion of cell proliferation, suppression of apoptosis, and enhancement of MMP. Furthermore, Sal B was found to exert regulatory control over the dynamic aggregation and subsequent regression of immune cells during tissue regenerative processes. Importantly, we observed that the knockdown of Cav1 significantly compromised tissue regeneration, leading to an excessive infiltration of immune cells and increased levels of apoptosis. Moreover, the knockdown of Cav1 also affects blastema formation, a critical process influenced by Cav1 in tissue regeneration. ConclusionThe results of this study showed that Sal B facilitated tissue repair and regeneration through regulating of immune cell migration and Cav1-mediated fibroblast activation, promoting blastema formation and development. This study highlighted the potential pharmacological effects of Sal B in promoting tissue regeneration. These findings contributed to the advancement of regenerative medicine research and the development of novel therapeutic approaches for trauma.

Lymphatic vessels in patients withcrescentic glomerulonephritis: association with renal pathology and prognosis.

Various immune cells, including T cells, B cells, macrophages, and neutrophilscontribute to the development of crescentic glomerulonephritis. Previous animal studies have suggested that lymphangiogenesis is involved in the migration of inflammatory cells and the activation of adaptive immunity. However, the extent of the association between lymphatic vessels and crescentic glomerulonephritis severity and prognosis remains unknown. In this study, we assessed lymphatic vessel density in 71 patients withcrescentic glomerulonephritis who underwent renal biopsies between June 2017 and June 2022. By immunohistochemistry and immunofluorescence, we identified increased lymphatic vessel density in thekidneys of patients with crescenticglomerulonephritiscompared to controls. Lymphatic vessels were categorized as total, periglomerular, and interstitial. Spearman's rank correlation analysis showed a positive correlation between total and periglomerular lymphatic vessel density and glomerular crescent proportion. High lymphatic vessel density (total and periglomerular) correlated with declining kidney function, increased proteinuria, and severe glomerular and interstitial pathology. Interstitial lymphatic vessel density had minimal relationship with renal lesions. After a median duration of 13 months of follow-up, higher total and periglomerular lymphatic vessel density was associated with poorer prognosis. Transcriptomic analysis revealed increased immune cell activation and migration in crescentic glomerulonephritis patients compared to healthy controls. Periglomerular lymphatic vessels might play a significant role in immune cell infiltration and renal injury. Elevated lymphatic vessel density in patients withcrescentic glomerulonephritis is associated with poor prognosisand may serve as a predictive factor for adverse outcomes in these patients.

Inflammation-induced TRPV4 channels exacerbate blood–brain barrier dysfunction in multiple sclerosis

BackgroundBlood–brain barrier (BBB) dysfunction and immune cell migration into the central nervous system (CNS) are pathogenic drivers of multiple sclerosis (MS). Ways to reinstate BBB function and subsequently limit neuroinflammation present promising strategies to restrict disease progression. However, to date, the molecular players directing BBB impairment in MS remain poorly understood. One suggested candidate to impact BBB function is the transient receptor potential vanilloid-type 4 ion channel (TRPV4), but its specific role in MS pathogenesis remains unclear. Here, we investigated the role of TRPV4 in BBB dysfunction in MS.Main textIn human post-mortem MS brain tissue, we observed a region-specific increase in endothelial TRPV4 expression around mixed active/inactive lesions, which coincided with perivascular microglia enrichment in the same area. Using in vitro models, we identified that microglia-derived tumor necrosis factor-α (TNFα) induced brain endothelial TRPV4 expression. Also, we found that TRPV4 levels influenced brain endothelial barrier formation via expression of the brain endothelial tight junction molecule claudin-5. In contrast, during an inflammatory insult, TRPV4 promoted a pathological endothelial molecular signature, as evidenced by enhanced expression of inflammatory mediators and cell adhesion molecules. Moreover, TRPV4 activity mediated T cell extravasation across the brain endothelium.ConclusionCollectively, our findings suggest a novel role for endothelial TRPV4 in MS, in which enhanced expression contributes to MS pathogenesis by driving BBB dysfunction and immune cell migration.

Abstract 2319: Deep learning-enabled dynamic infiltration and response to NK therapies in solid tumor organoids

Abstract Patient-derived organoid co-culture models have become a state of the art platform to evaluate the effectiveness of immunotherapeutic agents in preclinical studies. However, preclinical development as well as quantification of therapeutic potency and resistance require differential labeling of cells with fluorescent dyes or transgenic fluorescent proteins, which may dysregulate the effector cell function or produce undesired cytotoxicity. Deep learning-based image analysis allows the use of brightfield images to co-localize tumor organoids (TOs) with effector cells, and measure TO-specific responses to novel candidate immunotherapies in a label-free manner; this can accelerate their evaluation as therapeutic candidates in cancer patients and shed light on tumor-immune interaction mechanisms. Here we record multi-day time-lapse confocal microscopy images of TOs co-cultured with NK cells at increasing concentrations of target to effector cells. Two pre-trained U-Net convolutional neural networks are used to separately segment tumor and immune cells from the brightfield channel, and quantify immune cell infiltration over time. TO segmentation masks are registered with a vital dye (caspase 3/7) channel to selectively quantify TO cell death and correlate it with estimated immune cell infiltration across a large cohort of patient-derived TO models (including breast, colorectal, endometrial, gastric, head and neck, liver, lung and pancreatic cancer), at increasing concentrations of 6 different NK cell types (identified as A through F). We find that the estimated density of infiltrating immune cells is highly correlated with TO death, as quantified by fluorescence intensity of caspase over time (median Pearson’s r=0.89, P&amp;lt;0.001). Higher ratios of effector to target cells lead to higher activation of NK cells as measured by infiltrating cell density. Differential infiltration dynamics are observed across TOs and immune cell lines, as peak infiltration density is affected by co-culture time, ratio of target to effector cells, TO line, and NK cell type. These findings highlight that the paired segmentation models are able to measure varying degrees of infiltration across different types of immune cells and TO models. The approach described here is highly scalable and only requires capturing brightfield images of the assay, therefore eliminating the need to label cells and avoiding phototoxic effects or alteration of the immune cells activity. Time series measurements enable quantification of patterns of immune cell activation, including infiltration, migration and co-localization dynamics, which provide insights into the pharmacokinetics and mechanisms for specific immune therapies. Overall, this methodology enables high throughput screening of many therapeutic candidates across dozens to hundreds of unique TO-models, thereby facilitating targeted precision therapy. Citation Format: Luca Lonini, Sonal Khare, Timothy Don Lopez, Stanislaw Szydlo, Michael Streit, Olga A. Karginova, Mary K. Flaherty, Ameen Salhaudeen, Andre Kunert, Anna Oja, Kate Sasser, Martin C. Stumpe, Chi-Sing Ho. Deep learning-enabled dynamic infiltration and response to NK therapies in solid tumor organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2319.