Caspase-1-driven neutrophil pyroptosis and its role in host susceptibility to Pseudomonas aeruginosa.

Multiple regulated neutrophil cell death programs contribute to host defense against infections. However, despite expressing all necessary inflammasome components, neutrophils are thought to be generally defective in Caspase-1-dependent pyroptosis. By screening different bacterial species, we found that several Pseudomonas aeruginosa (P. aeruginosa) strains trigger Caspase-1-dependent pyroptosis in human and murine neutrophils. Notably, deletion of Exotoxins U or S in P. aeruginosa enhanced neutrophil death to Caspase-1-dependent pyroptosis, suggesting that these exotoxins interfere with this pathway. Mechanistically, P. aeruginosa Flagellin activates the NLRC4 inflammasome, which supports Caspase-1-driven interleukin (IL)-1ß secretion and Gasdermin D (GSDMD)-dependent neutrophil pyroptosis. Furthermore, P. aeruginosa-induced GSDMD activation triggers Calcium-dependent and Peptidyl Arginine Deaminase-4-driven histone citrullination and translocation of neutrophil DNA into the cell cytosol without inducing extracellular Neutrophil Extracellular Traps. Finally, we show that neutrophil Caspase-1 contributes to IL-1ß production and susceptibility to pyroptosis-inducing P. aeruginosa strains in vivo. Overall, we demonstrate that neutrophils are not universally resistant for Caspase-1-dependent pyroptosis.

High Power Electromagnetic Waves Exposure of Healthy and Tumor Bearing Mice: Assessment of Effects on Mice Growth, Behavior, Tumor Growth, and Vessel Permeabilization.

High power radiofrequencies may transiently or permanently disrupt the functioning of electronic devices, but their effect on living systems remains unknown. With the aim to evaluate the safety and biological effects of narrow-band and wide-band high-power electromagnetic (HPEM) waves, we studied their effects upon exposure of healthy and tumor-bearing mice. In field experiments, the exposure to 1.5 GHz narrow-band electromagnetic fields with the incident amplitude peak value level in the range of 40 kV/m and 150 MHz wide-band electric fields with the amplitude peak value in the range of 200 kV/m, did not alter healthy and tumor-bearing animals' growth, nor it had any impact on cutaneous murine tumors' growth. While we did not observe any noticeable behavioral changes in mice during the exposure to narrow-band signals when wide-band HPEM signals were applied, mice could behave in a similar way as they respond to loud noise signals: namely, if a mouse was exploring the cage prior to signal application, it returned to companion mates when wide-band HPEM signals were applied. Moreover, the effect of wide-band signals was assessed on normal blood vessels permeability in real-time in dorsal-chamber-bearing mice exposed in a pilot study using wide-band signal applicators. Our pilot study conducted within the applicator and performed at the laboratory scale suggests that the exposure to wide-band signals with the amplitude of 47.5 kV/m does not result in increased vessel permeability.

L-selectin controls trafficking of chronic lymphocytic leukemia cells in lymph node high endothelial venules in vivo.

B-cell chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. Lymph nodes (LNs) are sites of malignant proliferation and LN enlargement is associated with poor prognosis in the clinics. The LN microenvironment is believed to favor disease progression by promoting CLL cell growth and drug resistance. A better understanding of the mechanisms regulating trafficking of CLL cells to LNs is thus urgently needed. Here, we studied the first step of CLL cell migration to LNs, their interaction with high endothelial venules (HEVs), specialized blood vessels for lymphocyte extravasation in lymphoid organs. We observed that the density of HEV blood vessels was increased in CLL LNs and that CD20(+) CLL cells accumulated within HEV pockets, suggesting intense trafficking. We used intravital imaging to visualize the behavior of human CLL cells within the mouse LN microcirculation, and discovered that CLL cells bind to HEVs in vivo via a multistep adhesion cascade, which involves rolling, sticking, and crawling of the leukemic cells on the endothelium. Functional analyses revealed that the lymphocyte homing receptor L-selectin (CD62L) is the key factor controlling the binding of CLL cells to HEV walls in vivo. Interestingly, L-selectin expression was decreased on CLL cells from patients treated with idelalisib, a phosphoinositide-3-kinase δ inhibitor recently approved for CLL therapy. Interference with L-selectin-mediated trafficking in HEVs could represent a novel strategy to block dissemination of CLL cells to LNs and increase the efficacy of conventional therapy.

Membrane disorder and phospholipid scrambling in electropermeabilized and viable cells.

Membrane electropermeabilization relies on the transient permeabilization of the plasma membrane of cells submitted to electric pulses. This method is widely used in cell biology and medicine due to its efficiency to transfer molecules while limiting loss of cell viability. However, very little is known about the consequences of membrane electropermeabilization at the molecular and cellular levels. Progress in the knowledge of the involved mechanisms is a biophysical challenge. As a transient loss of membrane cohesion is associated with membrane permeabilization, our main objective was to detect and visualize at the single-cell level the incidence of phospholipid scrambling and changes in membrane order. We performed studies using fluorescence microscopy with C6-NBD-PC and FM1-43 to monitor phospholipid scrambling and membrane order of mammalian cells. Millisecond permeabilizing pulses induced membrane disorganization by increasing the translocation of phosphatidylcholines according to an ATP-independent process. The pulses induced the formation of long-lived permeant structures that were present during membrane resealing, but were not associated with phosphatidylcholine internalization. These pulses resulted in a rapid phospholipid flip/flop within less than 1s and were exclusively restricted to the regions of the permeabilized membrane. Under such electrical conditions, phosphatidylserine externalization was not detected. Moreover, this electrically-mediated membrane disorganization was not correlated with loss of cell viability. Our results could support the existence of direct interactions between the movement of membrane zwitterionic phospholipids and the electric field.

Cancer Imaging by Intravital Microscopy: The Dorsal Window Chamber Model.

Intravital microscopy allows a direct visualization of cells' behavior in their environment in a living organism with all its complexity. With appropriated models, longitudinal studies of structural and functional changes can be followed in the same animal on long period. In the field of cancer, the dorsal window chamber model is the model of choice for tumor events such as cells migration, vessels growth, and their permeability or interactions between cells and vessels. Coupled with wide-field, confocal, or multiphoton fluorescence microscopes, high spatial and temporal resolutions of the cellular events can be analyzed in vivo.

Fibroblasts transfection by electroporation in 3D reconstructed human dermal tissue.

The understanding of the mechanisms involved in DNA electrotransfer in human skin remains modest and limits the clinical development of various biomedical applications, such as DNA vaccination. To elucidate some mechanisms of DNA transfer in the skin following electroporation, we created a model of the dermis using a tissue engineering approach. This model allowed us to study the electrotransfection of fibroblasts in a three-dimensional environment that included multiple layers of fibroblasts as well as the self-secreted collagen matrix. With the aim of improving transfection yield, we applied electrical pulses with electric field lines perpendicular to the reconstructed model tissue. Our results indicate that the fibroblasts of the reconstructed skin tissue can be efficiently permeabilized by applied millisecond electrical pulses. However, despite efficient permeabilization, the transfected cells remain localized only on the surface of the microtissue, to which the plasmid was deposited. Second harmonic generation microscopy revealed the extensive extracellular collagen matrix around the fibroblasts, which might have affected the mobility of the plasmid into deeper layers of the skin tissue model. Our results show that the used skin tissue model reproduces the structural barriers that might be responsible for the limited gene electrotransfer in the skin.

TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation.

Epithelium-derived cytokines or alarmins, such as interleukin-33 (IL-33) and thymic stromal lymphopoietin (TSLP), are major players in type 2 immunity and asthma. Here, we demonstrate that TNF-like ligand 1A (TL1A) is an epithelial alarmin, constitutively expressed in alveolar epithelium at steady state in both mice and humans, which cooperates with IL-33 for early induction of IL-9high ILC2s during the initiation of allergic airway inflammation. Upon synergistic activation by IL-33 and TL1A, lung ILC2s acquire a transient IL-9highGATA3low "ILC9" phenotype and produce prodigious amounts of IL-9. A combination of large-scale proteomic analyses, lung intravital microscopy, and adoptive transfer of ILC9 cells revealed that high IL-9 expression distinguishes a multicytokine-producing state-of-activated ILC2s with an increased capacity to initiate IL-5-dependent allergic airway inflammation. Similar to IL-33 and TSLP, TL1A is expressed in airway basal cells in healthy and asthmatic human lungs. Together, these results indicate that TL1A is an epithelium-derived cytokine and an important cofactor of IL-33 in the airways.

LNA-based oligonucleotide electrotransfer for miRNA inhibition.

Micro-RNAs (miRNAs) are small regulatory RNAs that play an important role in disease development and progression and therefore represent a potential new class of therapeutic targets. However, an effective and safe clinical approach for miRNA inhibition remains elusive, primarily due to the lack of effective delivery methods. We proposed to inhibit miRNA by electrotransferring an antisense DNA oligomer containing locked nucleic acids (LNAs) (LNA/DNA oligomer). We observed that electropulsation (EP) led to a strong cellular uptake of LNA/DNA oligomer. The LNA/DNA oligomer electrotransfer mechanism and intracellular localization were visually investigated in real time at the single-cell level. Cyanine 5-labeled oligonucleotide entered exclusively during pulse application on the side of the permeabilized cell membrane facing the cathode, driven by electrophoretic forces. Minutes after the electrotransfer, the LNA/DNA oligomer diffused into the nucleus. EP provided the anti-miRNA oligomer with immediate and direct access to its cytoplasmic mature miRNA target and/or its nuclear precursor miRNA target. We then demonstrated using a LNA/DNA oligomer anti-miR34a that LNA/DNA oligomer electrotransfer decreased the level of the miR34a target and induced its functional inhibition. Our findings show that using the electrotransfer technique for LNA-based oligonucleotide delivery is a promising therapeutic strategy to silence deleterious miRNAs overexpressed in diseases.

Gene Electrotransfer Efficiency in 2D and 3D Cancer Cell Models Using Different Electroporation Protocols: A Comparative Study.

Electroporation, a method relying on a pulsed electric field to induce transient cell membrane permeabilization, can be used as a non-viral method to transfer genes in vitro and in vivo. Such transfer holds great promise for cancer treatment, as it can induce or replace missing or non-functioning genes. Yet, while efficient in vitro, gene-electrotherapy remains challenging in tumors. To assess the differences of gene electrotransfer in respect to applied pulses in multi-dimensional (2D, 3D) cellular organizations, we herein compared pulsed electric field protocols applicable to electrochemotherapy and gene electrotherapy and different "High Voltage-Low Voltage" pulses. Our results show that all protocols can result in efficient permeabilization of 2D- and 3D-grown cells. However, their efficiency for gene delivery varies. The gene-electrotherapy protocol is the most efficient in cell suspensions, with a transfection rate of about 50%. Conversely, despite homogenous permeabilization of the entire 3D structure, none of the tested protocols allowed gene delivery beyond the rims of multicellular spheroids. Taken together, our findings highlight the importance of electric field intensity and the occurrence of cell permeabilization, and underline the significance of pulses' duration, impacting plasmids' electrophoretic drag. The latter is sterically hindered in 3D structures and prevents the delivery of genes into spheroids' core.

In vivo molecular imaging and histological analysis of changes induced by electric pulses used for plasmid DNA electrotransfer to the skin: a study in a dorsal window chamber in mice.

Electropermeabilization/electroporation (EP) is a physical method that by application of electric pulses to cells increases cell membrane permeability and enables the introduction of molecules into the cells. One of the uses of EP in vivo is plasmid DNA electrotransfer to the skin for DNA vaccination. EP of tissues induces reduction of blood flow and, in combination with plasmid DNA, induction of an immune response. One of the EP protocols for plasmid DNA electrotransfer to the skin is a combination of high-voltage (HV) and low-voltage (LV) pulses. However, the effects of this pulse combination on skin-vessel blood flow are not known. Therefore, using intravital microscopy in a dorsal window chamber in mice and fluorescently labeled dextrans, the effects of one HV and eight LV pulses on skin vasculature were investigated. In addition, a detailed histological analysis was performed. Image analysis of fluorescence intensity changes demonstrated that EP induces a transient constriction and increased permeability of blood vessels as well as a "vascular lock." Histological analysis revealed rounding up of endothelial cells and stacking up of erythrocytes at 1 h after EP. In addition, extravasation of erythrocytes and leukocyte infiltration accompanied by edema were determined up to 24 h after EP. In conclusion, our results show that blood flow modifying effects of EP in skin contribute to the infiltration of immune cells in the exposed area. When combined with plasmid DNA for vaccination, this could enable the initial and prolonged contact of immune cells with encoded therapeutic proteins.

Flow cytometry analysis of endothelial cells and subsets of exhausted CD8+ T cells in murine tumor models.

Here, we present a protocol for flow cytometry analysis of endothelial cells (ECs) and CD8+ T cells in murine tumor models, at baseline and after cancer immunotherapy with anti-PD-1/anti-CTLA-4 antibodies. We provide gating strategies for identification of specific cell subsets including ECs from tumor-associated high endothelial venules (TA-HEVs), stem-like, and terminally exhausted CD8+ T cells. This protocol represents a valuable tool for the analysis of rare subsets of tumor ECs and CD8+ T cells with critical roles in antitumor immunity. For complete details on the use and execution of this protocol, please refer to Asrir et al. (2022).

Tumor-associated high endothelial venules mediate lymphocyte entry into tumors and predict response to PD-1 plus CTLA-4 combination immunotherapy.

Recruitment of lymphocytes into tumors is critical for anti-tumor immunity and efficacious immunotherapy. We show in murine models that tumor-associated high endothelial venules (TA-HEVs) are major sites of lymphocyte entry into tumors at baseline and upon treatment with anti-PD-1/anti-CTLA-4 immune checkpoint blockade (ICB). TA-HEV endothelial cells (TA-HECs) derive from post-capillary venules, co-express MECA-79+ HEV sialomucins and E/P-selectins, and are associated with homing and infiltration into tumors of various T cell subsets. Intravital microscopy further shows that TA-HEVs are the main sites of lymphocyte arrest and extravasation into ICB-treated tumors. Increasing TA-HEC frequency and maturation increases the proportion of tumor-infiltrating stem-like CD8+ T cells, and ameliorates ICB efficacy. Analysis of tumor biopsies from 93 patients with metastatic melanoma reveals that TA-HEVs are predictive of better response and survival upon treatment with anti-PD-1/anti-CTLA-4 combination. These studies provide critical insights into the mechanisms governing lymphocyte trafficking in cancer immunity and immunotherapy.

Development of a near infrared protein nanoprobe targeting Thomsen-Friedenreich antigen for intraoperative detection of submillimeter nodules in an ovarian peritoneal carcinomatosis mouse model.

The epithelial ovarian cancer is one of the most lethal gynecological malignancy due to its late diagnostic and many relapses observed after first line of treatment. Once diagnose, the most important prognostic factor is the completeness of cytoreductive surgery. To achieve this goal, surgeons have to pinpoint and remove nodules, especially the smallest nodules. Recent advances in fluorescence-guided surgery led us to develop a recombinant lectin as a nanoprobe for the microscopic detection of nodules in the peritoneal cavity of tumor-bearing mice. This lectin has an intrinsic specificity for a carcinoma-associated glycan biomarker, the Thomsen-Friedenreich antigen. In this study, after its labelling by a near infrared dye, we first demonstrated that this nanoprobe allowed indirect detection of nodules already implanted in the peritoneal cavity, through tumor microenvironment targeting. Secondly, in a protocol mimicking the scattering of cells during surgery, we obtained a direct and long-lasting detection of tumor cells in vivo. This lectin as already been described as a nanocontainer able to do targeted delivery of a therapeutic compound to carcinoma cells. Future developments will focus on the combination of the nanoprobe and nanocontainer aspects in an intraperitoneal nanotheranostic approach.

Electroporation-Induced Stress Response and Its Effect on Gene Electrotransfer Efficacy: In Vivo Imaging and Numerical Modeling.

OBJECTIVE: Skin is an attractive target tissue for gene transfer due to its size, accessibility, and its immune competence. One of the promising delivery methods is gene delivery by means of electroporation (EP), i.e., gene electrotransfer (GET). To assess the importance of different effects of electroporation for successful GET we investigated: stress response and transfection efficacy upon different pulse protocols. Moreover, numerical modeling was used to explain experimental results and to test the agreement of experimental results with current knowledge about GET. METHODS: Double transgenic mice Hspa1b-LucF (+/+) Hspa1b-mPlum (+/+) were used to determine the level of stress sensed by the cell in the tissue in vivo that was exposed to EP. The effect of five different pulse protocols on stress levels sensed by the exposed cells and their efficacy for gene electrotransfer for two plasmids pEGFP-C1 (EGFP) and pCMV-tdTomato was tested. RESULTS: Quantification of the bioluminescence signal intensity shows that EP, regardless of the electric pulse parameters used, increased mean bioluminescence compared to the baseline bioluminescence signal of the non-exposed skin. The results of numerical modeling indicate that thermal stress alone is not sufficient to explain the measured bioluminescence signal. Of the tested pulse protocols, the highest expression of EGFP and tdTomato was achieved with HV-MV (high voltage - medium voltage) protocols, which agrees also with numerical model. SIGNIFICANCE: Although EP is widely used as a method for gene delivery, we show that the field could benefit from the use of mathematical modeling by introducing additional parameters such as EP induced stress and electrophoretic movement of plasmids.

Noninvasive Gene Electrotransfer in Skin.

The skin is considered as well suited for gene therapy and vaccination. DNA vaccines elicit both broad humoral and cellular immune responses when injected in the skin. Physical and chemical methods are needed to boost the expression. Gene electrotransfer (GET) is one of the most effective approaches. This step-by-step protocol describes the procedures to obtain an efficient GET targeted to the skin by using easy-to-use noninvasive electrodes after intradermal plasmid injection (i.d. GET). A specific pulse sequence is reported. Expression is observed by in vivo fluorescence imaging during >2 weeks as the plasmid was coding for tdTomato. The protocol is efficient for the transient expression of clinical proteins.

Evaluations of Acute and Sub-Acute Biological Effects of Narrowband and Moderate-Band High Power Electromagnetic Waves on Cellular Spheroids.

High power electromagnetic signals can disrupt the functioning of electronic devices. As electromagnetism plays a role in cells homeostasis, such electromagnetic signals could potentially also alter some physiological processes. Herein we report on distinct biological parameters assessment after cellular spheroids exposure to high power electromagnetic signals, such as the ones used for defense applications. Signals effects were assessed in tumor cells spheroids and in normal human dermal fibroblasts spheroids, where macroscopic aspect, growth, plasma membrane integrity, induction of apoptosis, ATP content, and mitochondrial potential were investigated after spheroids exposure to high power electromagnetic signals. No significant effects were observed, indicating that 1.5 GHz narrowband electromagnetic fields with incident amplitude level of 40 kV/m, and 150 MHz moderate-band electric fields with an amplitude of 72.5 to approximately 200 kV/m, do not cause any significant alterations of assessed parameters.

Pre-clinical investigation of the synergy effect of interleukin-12 gene-electro-transfer during partially irreversible electropermeabilization against melanoma.

BACKGROUND: Melanoma is a very aggressive skin tumor that can be cured when diagnosed and treated in its early stages. However, at the time of identification, the tumor is frequently in a metastatic stage. Intensive research is currently ongoing to improve the efficacy of the immune system in eliminating cancer cells. One approach is to boost the activation of cytotoxic T cells by IL-12 cytokine that plays a central role in the activation of the immune system. In parallel, physical methods such as electropermeabilization-based treatments are currently under investigation and show promising results. METHODS: In this study, we set electrical parameters to induce a partial-irreversible electropermeabilization (pIRE) of melanoma to induce a sufficient cell death and potential release of tumor antigens able to activate immune cells. This protocol mimics the situation where irreversible electropermeabilization is not fully completed. Then, a peritumoral plasmid IL-12 electrotransfer was combined with pIRE treatment. Evaluation of the tumor growth and survival was performed in mouse strains having a different immunological background (C57Bl/6 (WT), nude and C57Bl6 (TLR9-/-)). RESULTS: pIRE treatment induced apoptotic cell death and a temporary tumor growth delay in all mouse strains. In C57Bl/6 mice, we showed that peritumoral plasmid IL-12 electrotransfer combined with tumor pIRE treatment induced tumor regression correlating with a local secretion of IL-12 and IFN-γ. This combined treatment induced a growth delay of distant tumors and prevented the emergence of a second tumor in 50% of immunocompetent mice. CONCLUSIONS: The combination of pIL-12 GET and pIRE not only enhanced survival but could bring a curative effect in wild type mice. This two-step treatment, named Immune-Gene Electro-Therapy (IGET), led to a systemic activation of the adaptive immune system and the development of an anti-tumor immune memory.

Magnetic Silica-Coated Iron Oxide Nanochains as Photothermal Agents, Disrupting the Extracellular Matrix, and Eradicating Cancer Cells.

Cancerous cells and the tumor microenvironment are among key elements involved in cancer development, progression, and resistance to treatment. In order to tackle the cells and the extracellular matrix, we herein propose the use of a class of silica-coated iron oxide nanochains, which have superior magnetic responsiveness and can act as efficient photothermal agents. When internalized by different cancer cell lines and normal (non-cancerous) cells, the nanochains are not toxic, as assessed on 2D and 3D cell culture models. Yet, upon irradiation with near infrared light, the nanochains become efficient cytotoxic photothermal agents. Besides, not only do they generate hyperthermia, which effectively eradicates tumor cells in vitro, but they also locally melt the collagen matrix, as we evidence in real-time, using engineered cell sheets with self-secreted extracellular matrix. By simultaneously acting as physical (magnetic and photothermal) effectors and chemical delivery systems, the nanochain-based platforms offer original multimodal possibilities for prospective cancer treatment, affecting both the cells and the extracellular matrix.

Biodistribution and Biosafety of a Poly(Phosphorhydrazone) Dendrimer, an Anti-Inflammatory Drug-Candidate.

Dendrimers are nanosized, arborescent polymers of which size and structure are perfectly controlled. This is one reason why they are widely used for biomedical purposes. Previously, we showed that a phosphorus-based dendrimer capped with anionic azabisphosphonate groups (so-called ABP dendrimer) has immuno-modulatory and anti-inflammatory properties towards human immune cells in vitro. Thereafter, we have shown that the ABP dendrimer has a promising therapeutic efficacy to treat models of chronic inflammatory disorders. On the way to clinical translation, the biodistribution and the safety of this drug-candidate has to be thoroughly assessed. In this article, we present preliminary non-clinical data regarding biodistribution, hematological safety, genotoxicity, maximal tolerated doses, and early cardiac safety of the ABP dendrimer. One of the genotoxicity assays reveals a potential mutagen effect of the item at a concentration above 200 µM, i.e., up to 100 times the active dose in vitro on human immune cells. However, as the results obtained for all the other assays show that the ABP dendrimer has promising biodistribution and safety profiles, there is no red flag raised to hamper the regulatory pre-clinical development of the ABP dendrimer.

The Protease-Dependent Mesenchymal Migration of Tumor-Associated Macrophages as a Target in Cancer Immunotherapy.

Macrophage recruitment is essential for tissue homeostasis but detrimental in most cancers. Tumor-associated macrophages (TAMs) play a key role in cancer progression. Controlling their migration is, thus, potentially therapeutic. It is assumed that macrophages use amoeboid motility in vivo like other leukocytes. However, it has not yet been explored. We examined TAM migration using intravital microscopy in mouse tumors and by monitoring ex vivo tissue infiltration in human surgical samples. We demonstrated that TAMs perform protease-dependent and ROCK-independent mesenchymal migration inside mouse fibrosarcoma and breast cancer explants using their own matrix metalloproteases (MMP). In contrast, macrophages use ROCK-dependent and protease-independent amoeboid migration inside inflamed ear derma and in connective tissue at the tumor periphery. We also showed that inhibition of mesenchymal migration correlates with decreased TAM recruitment and tumor growth. In conclusion, this study elucidates how macrophages migrate in vivo, and it reveals that the MMP-dependent migration mode of TAMs provides a rationale for a new strategy in cancer immunotherapy: to target TAMs specifically through their motility. Cancer Immunol Res; 6(11); 1337-51. ©2018 AACR.