Biology subjects

Paschalis, E. I.

Publications and source records attributed to Paschalis, E. I..

10 recordsLinked to original sources

bioRxiv2024

A TROP2/Claudin Program Mediates Immune Exclusion to Impede Checkpoint Blockade in Breast Cancer

BackgroundImmune exclusion inhibits anti-tumor immunity and response to immunotherapy, but its mechanisms remain poorly defined. In triple-negative breast cancer (TNBC), an aggressive and generally immune-rich subtype, an immune-cold microenvironment predicts poor prognosis due to a limited response to chemotherapy and immune checkpoint inhibitors. This study aimed to identify mechanisms regulating immune infiltration in TNBC. MethodsWe performed spatial transcriptomic analysis comparing immune-enriched versus immune-cold treatment-naive TNBCs. Functional analyses, including loss-of-function and reconstitution experiments, were conducted to investigate the role of Trophoblast Cell-Surface Antigen 2 (TROP2), a key target of anti-cancer Antibody Drug Conjugates (ADCs), in promoting TNBC progression. A humanized TROP2 syngeneic TNBC model was used to assess the effects of TROP2-targeting in combination with anti-PD1 therapy. Additionally, data from patients treated with immune checkpoint blockade were used to test hypotheses from the preclinical findings. ResultsWe reveal that TROP2 controls barrier-mediated immune exclusion in TNBC through Claudin 7 association and tight junction regulation. TROP2 expression is inversely correlated with T cell infiltration and predicts poor outcomes in TNBC. We demonstrate that TROP2 is sufficient to drive tumor progression in vivo in a CD8 T cell-dependent manner, while its loss deregulates expression and localization of multiple tight junction proteins, enabling T cell infiltration. We show that TROP2 targeting via hRS7, the antibody component of the ADC Sacituzumab govitecan (SG), enhances the anti-PD1 response and improves T cell accessibility and effector function. Correspondingly, TROP2 expression is highly associated with lack of response to anti-PD1 therapy in human breast cancer. ConclusionsThis study defines a new mechanism of barrier-mediated immune exclusion in cancer controlled by TROP2-dependent tight junctions. This mechanism drives tumor progression but can be targeted via TROP2-directed therapy to activate anti-tumor immunity and enhance immunotherapy response. What is already known on this topicBarrier-mediated immune exclusion is emerging as a mechanism of immune evasion in cancer such as triple-negative breast cancer (TNBC), but its molecular underpinnings remain poorly understood. TROP2 is a surface glycoprotein targeted by antibody-drug conjugates such as Sacituzumab govitecan, yet its functional role beyond drug delivery has been unclear. What this study addsThis study identifies TROP2 as a key regulator of tight junction-mediated immune exclusion in TNBC, independent of its intracellular signaling function. TROP2 promotes an immune-cold tumor microenvironment by enforcing mechanical barriers that limit T cell infiltration. How this study might affect research, practice or policyThese findings establish TROP2 as a functional driver of immune evasion and provide a mechanistic rationale for combining TROP2-targeting therapies with immune checkpoint inhibitors to overcome resistance in immune-excluded tumors.

cancer biology↗

bioRxiv2024

Epigenetic adaptation drives monocyte differentiation into microglia-like cells upon engraftment into the retina

The identification of specific markers to distinguish resident microglia from infiltrating monocytes has been a long-standing challenge in neuroscience. Recently, proteins such as P2RY12, TMEM119, and FCRLS have been proposed as microglia-specific and are now widely used to define microglial populations in health and disease. The specificity of these markers was predicated on the assumption that circulating monocytes retain their distinct signatures after entering the central nervous system (CNS). Here, we challenge this paradigm. Using a combination of bone marrow chimeras, single-cell RNA sequencing, ATAC-seq, flow cytometry, and immunohistochemistry, we demonstrate that monocytes engrafting into the CNS acquire de novo expression of these established microglia markers. This phenotypic conversion is driven by profound epigenetic reprogramming, characterized by dynamic changes in chromatin accessibility at key gene loci, including P2ry12, Tmem119, and Aif1 (Iba1), and a shift in transcription factor binding motifs toward a microglial profile. We show this process occurs in the retina following injury and, remarkably, under physiological conditions in the brain and spinal cord, where blood-derived monocytes progressively contribute to the resident myeloid pool. Furthermore, engrafted monocytes downregulate canonical monocyte markers (Ly6C, CD45), eventually becoming indistinguishable from embryonic microglia based on conventional phenotyping. Our findings reveal that infiltrating monocytes undergo extensive epigenetic and transcriptional remodeling to adopt a microglia-like fate, challenging the specificity of current markers and necessitating a re-evaluation of the distinct roles of these two cell populations in CNS pathology. Significance StatementDistinguishing resident CNS microglia from infiltrating monocytes is fundamental to understanding neuro-inflammation. This study reveals that widely used "microglia-specific" markers are not exclusive, as monocytes entering the central nervous system are epigenetically reprogrammed to express them. This mimicry invalidates long-held assumptions about microglial identity and demonstrates that many cells previously identified as microglia may have a peripheral origin. Our work underscores the critical need for more reliable methods to differentiate these populations to accurately define their respective contributions to CNS health and disease.

immunology↗

bioRxiv2024

Deletion of the endothelial glycocalyx component endomucin leads to impaired glomerular structure and function

BackgroundEndomucin (EMCN), an endothelial-specific glycocalyx component, was found to be highly expressed by the endothelium of the renal glomerulus. We reported an anti-inflammatory role of EMCN and its involvement in the regulation of vascular endothelial growth factor (VEGF) activity through modulating VEGF receptor 2 (VEGFR2) endocytosis. The goal of this study is to investigate the phenotypic and functional effects of EMCN deficiency using the first global EMCN knockout mouse model. MethodsGlobal EMCN knockout mice were generated by crossing EMCN-floxed mice with ROSA26-Cre mice. Flow cytometry was employed to analyze infiltrating myeloid cells in the kidneys. The ultrastructure of the glomerular filtration barrier was examined by transmission electron microscopy, while urinary albumin, creatinine, and total protein levels were analyzed from freshly collected urine samples. Expression and localization of EMCN, EGFP, CD45, CD31, CD34, podocin, albumin, and -smooth muscle actin were examined by immunohistochemistry. Mice were weighed regularly, and their systemic blood pressure was measured using a non-invasive tail-cuff system. Glomerular endothelial cells and podocytes were isolated by fluorescence-activated cell sorting for RNA-seq. Transcriptional profiles were analyzed to identify differentially expressed genes in both endothelium and podocytes, followed by gene ontology analysis of up- and down-regulated genes. Protein levels of EMCN, albumin, and podocin were quantified by Western blot. ResultsEMCN-/- mice were viable with no gross anatomical defects in kidneys. The EMCN-/- mice exhibited increased infiltration of CD45+ cells, with an increased proportion of Ly6GhighLy6Chigh myeloid cells and higher VCAM-1 expression. EMCN-/- mice displayed albuminuria with increased albumin in the Bowmans space compared to the EMCN+/+ littermates. Glomeruli in EMCN-/- mice revealed fused and effaced podocyte foot processes and disorganized endothelial fenestrations. We found no significant difference in blood pressure between EMCN knockout mice and their wild-type littermates. RNA-seq of glomerular endothelial cells revealed downregulation of cell-cell adhesion and MAPK/ERK pathways, along with glycocalyx and extracellular matrix remodeling. In podocytes, we observed reduced VEGF signaling and alterations in cytoskeletal organization. Notably, there was a significant decrease in both mRNA and protein levels of podocin, a key component of the slit diaphragm. ConclusionOur study demonstrates a critical role of the endothelial marker EMCN in supporting normal glomerular filtration barrier structure and function by maintaining glomerular endothelial tight junction and homeostasis and podocyte function through endothelial-podocyte crosstalk.

pathology↗

bioRxiv2023

A novel sustained release therapy of combined VEGF and TNF-α inhibitors leads to pan-ocular protection for months after severe ocular trauma.

PurposeTo develop a clinically feasible and practical therapy for multi-ocular protection following ocular injury by using a thermosensitive drug delivery system (DDS) for sustained delivery of TNF- and VEGF inhibitors to the eye. MethodsA thermosensitive, biodegradable hydrogel DDS (PLGA-PEG-PLGA triblock polymer) loaded with 0.7mg of adalimumab and 1.4 mg of aflibercept was injected subconjunctivally in Dutch-belted pigmented rabbits after corneal alkali injury. The polymer was tuned to transition from liquid to gel upon contact with body temperature without need of a catalyst. Control rabbits received 2mg of IgG loaded DDS or 1.4mg aflibercept loaded DDS. Animals were followed for 3 months and assessed for tolerability and prevention of corneal neovascularization (NV), improvement of corneal re-epithelialization, inhibition of retinal ganglion cell (RGC) and optic nerve axon loss, and inhibition of immune cell infiltration into the cornea. Drug release kinetics was assessed in vivo using aqueous humor protein analysis. ResultsA single subconjunctival administration of dual anti-TNF/anti-VEGF DDS achieved sustained 3-month delivery of antibodies to the anterior chamber, iris, ciliary body, and retina. Administration after corneal alkali burn suppressed CD45+ immune cell infiltration into the cornea, completely inhibited cornea NV for 3 months, accelerated corneal re-epithelialization and wound healing, and prevented RGC and optic nerve axon loss at 3 months. In contrast, anti-VEGF alone or IgG DDS treatment led to persistent corneal epithelial defect, increased infiltration of CD45+ immune cells into the cornea, and significant loss of RGCs and optic nerve axons at 3 months. Aqueous humor protein analysis showed first-order release kinetics without adverse effects at the injection site. ConclusionSustained concomitant inhibition of TNF- and VEGF using a biodegradable, slow-release thermosensitive DDS provides significant ocular protection and prevents corneal neovascularization and irreversible damage to retina and optic nerve after corneal alkali injury. This therapeutic approach has the potential to dramatically improve the outcomes of severe ocular injuries in patients.

pathology↗

bioRxiv2022

The effect of subconjunctival TNF-α inhibitors on retinal ganglion cell apoptosis and optic nerve degeneration after corneal surgery or trauma--of possible prophylactic value against secondary glaucoma.

AbstractO_ST_ABSBackground and PurposeC_ST_ABSLate secondary glaucoma is an often-severe complication after acute events like anterior segment surgery, trauma, infection, etc. TNF- is a major mediator that is rapidly upregulated, diffusing also to the retina and causes apoptosis of the ganglion cells and degeneration of their optic nerve axons (mediating steps to glaucomatous damage). Anti-TNF- antibodies are in animals very effective in protecting the retinal cells and the optic nerve--and might therefore be useful prophylactically against secondary glaucoma in future such patients.Here we evaluate 1) toxicity and 2) efficacy of two TNF- inhibitors (adalimumab and infliximab), in rabbits by subconjunctival administration. MethodsFor drug toxicity, animals with normal, unburned corneas were injected with adalimumab (0.4, 4, or 40 mg), or infliximab (1, 10, or 100 mg). For drug efficacy, other animals were subjected to alkali burn before such injection, or steroids (for control). The rabbits were evaluated clinically with slit lamp and photography, electroretinography, optical coherence tomography, and intraocular pressure manometry. A sub-set of eyes were stained ex vivo after 3 days for retinal cell apoptosis (TUNEL). In other experiments the optic nerves were evaluated by paraphenylenediamine staining after 50 or 90 days. Loss of retinal cells and optic nerve degeneration were quantified. ResultsSubconjunctival administration of 0.4 mg or 4.0 mg adalimumab were well tolerated, whereas 40.0 mg was toxic to the retina. 1, 10, or 100 mg infliximab were also well tolerated.Analysis of the optic nerve axons after 50 days confirmed the safety of 4.0 mg adalimumab and of 100 mg infliximab. For efficacy, 4.0 mg adalimumab subconjunctivally in 0.08 mL provided practically full protection against retinal cell apoptosis 3 days following alkali burn, and infliximab 100 mg only slightly less. At 90 days following burn injury, control optic nerves showed about 50% axon loss as compared to 8% in the adalimumab treatment group. ConclusionsSubconjunctival injection of 4.0 mg adalimumab in rabbits shows no eye toxicity and provides excellent neuroprotection, both short (3 days) and long-term (90 days). Ourtotal accumulated data from several of our studies, combined with the present paper, suggest that corneal injuries, including surgery, might benefit from routine administration of anti-TNF- biologics to reduce inflammation and future secondary glaucoma.

pathology↗

bioRxiv2021

CSF1R inhibition by small molecule affects T-helper cell differentiation independently of microglia depletion

Colony-stimulating factor 1 receptor (CSF1R) inhibition has been proposed as a specific method for microglia depletion. However, recent work revealed that in addition to microglia, CSF1R inhibition also affects other innate immune cells, such as peripheral monocytes and tissueresident macrophages of the lung, liver, spleen, and peritoneum. Here, we show that this effect is not restricted to innate immune cells only, but extends to the adaptive immune compartment. CSF1R inhibition alters the transcriptional profile of bone marrow cells that control T helper cell activation. In vivo or ex vivo inhibition of CSF1R profoundly changes the transcriptional profile of CD4+ cells and suppresses Th1 and Th2 differentiation in directionally stimulated and unstimulated cells and independently of microglia depletion. Given that T cells also contribute in CNS pathology, these effects may have practical implications in the interpretation of relevant experimental data. Significance statementHere we show that CSF1R inhibition affects not only innate immune cells and microglia, but also the adaptive immune compartment by suppressing Th1/Th2 differentiation of CD4+ cells, independently of microglia depletion.

immunology↗

bioRxiv2021

Opposing roles of blood-borne monocytes and tissue-resident macrophages in limbal stem cell damage after ocular injury

Limbal stem cell (LSC) deficiency is a frequent and severe complication after chemical injury to the eye. Previous studies have assumed that this is mediated directly by the caustic agent. Here we show that LSC damage occurs through immune cell mediators, even without direct injury to LSCs. In particular, pH elevation in the anterior chamber (AC) causes acute uveal stress, release of inflammatory cytokines at the basal limbal tissue, and subsequent LSC loss. Peripheral CCR2+ CX3CR1- monocytes are the key mediators of LSC damage, through the upregulation of tumor necrosis factor alpha (TNF-) at the limbus. In contrast to peripherally-derived monocytes, CX3CR1+ CCR2- tissue-resident macrophages have a protective role and their depletion prior to injury exacerbates LSC loss and increases LSC vulnerability to TNF--mediated apoptosis independently of CCR2+ cell infiltration into the tissue. Consistently, repopulation of the tissue by new resident macrophages not only restores the protective M2-like phenotype of macrophages but also suppresses LSC loss after exposure to inflammatory signals. These findings may have clinical implication in patient with chemical burns. SignificanceLimbal stem cell (LSC) loss after chemical burn is a significant clinical problem that has long been thought to be the result of direct chemical injury. This study demonstrates the existence of competing inflammatory mechanism responsible for LSC death, mediated by infiltrating peripheral monocytes that antagonize and overcome the protective role of tissue-resident macrophages. Thus, enhancing the role of tissue-resident macrophages while suppressing that of infiltrating monocytes could be a novel therapeutic strategy for LSC survival.

immunology↗

bioRxiv2019

CSF1R inhibition by small molecule inhibitor affects hematopoiesis and the function of macrophages.

Colony-stimulating factor 1 receptor (CSF1R) inhibition has been proposed as a method for microglia depletion, with the assumption that it does not affect peripheral immune cells. Here, we show that CSF1R inhibition by PLX5622 indeed affects the myeloid and lymphoid compartments, causes long-term changes in bone marrow-derived macrophages by suppressing their IL-1{beta}, CD68 and phagocytosis, but not CD208, following exposure to endotoxin, and also reduces the population of tissue resident macrophages of peritoneum, lung, liver, but not spleen. Thus, small molecule CSF1R inhibition is not restricted to microglia only, but rather causes strong effects on circulating and tissue macrophages that perdure long after cessation of the treatment. Given that peripheral monocytes repopulate the CNS after CSF1R inhibition, these changes may have practical implications on relevant experimental data.

neuroscience↗

bioRxiv2018

Neuroglial Remodeling After Various Ocular Injuries is Regulated by Microglia

Reactive microglia and infiltrating peripheral monocytes have been implicated in many neurodegenerative diseases of the retina and central nervous system (CNS). However, their specific contribution in retinal degeneration remains unclear. We recently showed that peripheral monocytes that infiltrate the retina after ocular injury in mice become permanently engrafted into the tissue, establishing a pro-inflammatory phenotype that promotes neurodegeneration. Here, we show in mice that microglia regulate the process of neuroglia remodeling during ocular injury, and their depletion results in marked upregulation of inflammatory markers, such as Il17f, Tnfsf11, Ccl4, Il1a, Ccr2, Il4, Il5, and Csf2 in the retina, abnormal engraftment of peripheral CCR2+ CX3CR1+ monocytes into the retina and is associated with increased retinal ganglion cell (RGC) loss, retinal nerve fiber layer thinning, and RPE65+ cell migration onto the retinal surface. Furthermore, we show that other types of ocular injuries, such as penetrating corneal trauma and ocular hypertension, also cause similar changes. However, optic nerve crush injury mediated RGC loss evokes neither peripheral monocyte response in the retina, nor RPE65+ cell migration, although peripheral CX3CR1+ and CCR2+ monocytes infiltrate the optic nerve injury site and remain present for months. Our study suggests that microglia are key regulators of peripheral monocyte infiltration and RPE migration and their depletion results in abnormal neuroglia remodeling that exacerbates neuroretinal tissue damage. This mechanism of retinal damage through neuroglia remodeling may be clinically important for the treatment of patients with ocular injuries, including surgical traumas.

immunology↗

bioRxiv2018

Permanent neuroglial remodeling of the retina following infiltration of CSF1R-inhibition resistant peripheral monocytes.

Previous studies have demonstrated that ocular injury can lead to prompt infiltration of bone marrow-derived peripheral monocytes into the retina. However, the ability of these cells to integrate into the tissue and become microglia has not been studied. Here we show that such peripheral monocytes not only infiltrate into the retina after ocular injury, but that they engraft permanently, migrate to the three distinct microglia strata, and adopt a microglia-like morphology. However, contrary to the original microglia, after injury the engrafted peripheral monocytes are resistant to depletion by colony stimulating factor 1 receptor (CSF1R) inhibitor and remain pro-inflammatory, expressing high levels of major histocompatibility complex II (MHC-II) for long-term. In the absence of ocular injury, on the other hand, the peripheral monocytes that repopulate the retina after CSF1R inhibition remain sensitive to CSF1R inhibition and can be re-depleted. The observed permanent neuroglia remodeling after injury constitutes a major immunological deviation that may contribute to progressive retinal degeneration. These findings may be relevant also to other degenerative conditions of the retina and central nervous system.

immunology↗