HLA-DR Expression in Natural Killer Cells Marks Distinct Functional States, Depending on Cell Differentiation Stage.

HLA-DR-positive NK cells, found in both healthy individuals and patients with different inflammatory diseases, are characterized as activated cells. However, data on their capacity for IFNγ production or cytotoxic response vary between studies. Thus, more precise investigation is needed of the mechanisms related to the induction of HLA-DR expression in NK cells, their associations with NK cell differentiation stage, and functional or metabolic state. In this work, HLA-DR-expressing NK cell subsets were investigated using transcriptomic analysis, metabolic activity assays, and analysis of intercellular signaling cascades. We demonstrated that HLA-DR+CD56bright NK cells were characterized by a proliferative phenotype, while HLA-DR+CD56dim NK cells exhibited features of adaptive cells and loss of inhibitory receptors with increased expression of MHC class II trans-activator CIITA. The activated state of HLA-DR-expressing NK cells was confirmed by higher levels of ATP and mitochondrial mass observed in this subset compared to HLA-DR- cells, both ex vivo and after stimulation in culture. We showed that HLA-DR expression in NK cells in vitro can be induced both through stimulation by exogenous IL-2 and IL-21, as well as through auto-stimulation by NK-cell-produced IFNγ. At the intracellular level, HLA-DR expression depended on the activation of STAT3- and ERK1/2-mediated pathways, with subsequent activation of isoform 3 of the transcription factor CIITA. The obtained results broaden the knowledge about HLA-DR-positive NK cell appearance, diversity, and functions, which might be useful in terms of understanding the role of this subset in innate immunity and assessing their possible implications in NK cell-based therapy.

Coordinated Loss and Acquisition of NK Cell Surface Markers Accompanied by Generalized Cytokine Dysregulation in COVID-19.

Coronavirus disease 2019 (COVID-19), caused by the SARS-CoV-2 virus, is accompanied by a dysregulated immune response. In particular, NK cells, involved in the antiviral response, are affected by the infection. This study aimed to investigate circulating NK cells with a focus on their activation, depletion, changes in the surface expression of key receptors, and functional activity during COVID-19, among intensive care unit (ICU) patients, moderately ill patients, and convalescents (CCP). Our data confirmed that NK cell activation in patients with COVID-19 is accompanied by changes in circulating cytokines. The progression of COVID-19 was associated with a coordinated decrease in the proportion of NKG2D+ and CD16+ NK cells, and an increase in PD-1, which indicated their exhaustion. A higher content of NKG2D+ NK cells distinguished surviving patients from non-survivors in the ICU group. NK cell exhaustion in ICU patients was additionally confirmed by a strong negative correlation of PD-1 and natural cytotoxicity levels. In moderately ill patients and convalescents, correlations were found between the levels of CD57, NKG2C, and NKp30, which may indicate the formation of adaptive NK cells. A reduced NKp30 level was observed in patients with a lethal outcome. Altogether, the phenotypic changes in circulating NK cells of COVID-19 patients suggest that the intense activation of NK cells during SARS-CoV-2 infection, most likely induced by cytokines, is accompanied by NK cell exhaustion, the extent of which may be critical for the disease outcome.

Alterations in the CD56- and CD56+ T Cell Subsets during COVID-19.

The effectiveness of the antiviral immune response largely depends on the activation of cytotoxic T cells. The heterogeneous group of functionally active T cells expressing the CD56 molecule (NKT-like cells), that combines the properties of T lymphocytes and NK cells, is poorly studied in COVID-19. This work aimed to analyze the activation and differentiation of both circulating NKT-like cells and CD56- T cells during COVID-19 among intensive care unit (ICU) patients, moderate severity (MS) patients, and convalescents. A decreased proportion of CD56+ T cells was found in ICU patients with fatal outcome. Severe COVID-19 was accompanied by a decrease in the proportion of CD8+ T cells, mainly due to the CD56- cell death, and a redistribution of the NKT-like cell subset composition with a predominance of more differentiated cytotoxic CD8+ T cells. The differentiation process was accompanied by an increase in the proportions of KIR2DL2/3+ and NKp30+ cells in the CD56+ T cell subset of COVID-19 patients and convalescents. Decreased percentages of NKG2D+ and NKG2A+ cells and increased PD-1 and HLA-DR expression levels were found in both CD56- and CD56+ T cells, and can be considered as indicators of COVID-19 progression. In the CD56- T cell fraction, increased CD16 levels were observed in MS patients and in ICU patients with lethal outcome, suggesting a negative role for CD56-CD16+ T cells in COVID-19. Overall, our findings suggest an antiviral role of CD56+ T cells in COVID-19.

Precise tracking of vaccine-responding T cell clones reveals convergent and personalized response in identical twins.

T cell receptor (TCR) repertoire data contain information about infections that could be used in disease diagnostics and vaccine development, but extracting that information remains a major challenge. Here we developed a statistical framework to detect TCR clone proliferation and contraction from longitudinal repertoire data. We applied this framework to data from three pairs of identical twins immunized with the yellow fever vaccine. We identified 600 to 1,700 responding TCRs in each donor and validated them using three independent assays. While the responding TCRs were mostly private, albeit with higher overlap between twins, they could be well-predicted using a classifier based on sequence similarity. Our method can also be applied to samples obtained postinfection, making it suitable for systematic discovery of new infection-specific TCRs in the clinic.

The Biological Role and Therapeutic Potential of NK Cells in Hematological and Solid Tumors.

NK cells are an attractive target for cancer immunotherapy due to their potent antitumor activity. The main advantage of using NK cells as cytotoxic effectors over T cells is a reduced risk of graft versus host disease. At present, several variants of NK-cell-based therapies are undergoing clinical trials and show considerable effectiveness for hematological tumors. In these types of cancers, the immune cells themselves often undergo malignant transformation, which determines the features of the disease. In contrast, the current use of NK cells as therapeutic agents for the treatment of solid tumors is much less promising. Most studies are at the stage of preclinical investigation, but few progress to clinical trials. Low efficiency of NK cell migration and functional activity in the tumor environment are currently considered the major barriers to NK cell anti-tumor therapies. Various therapeutic combinations, genetic engineering methods, alternative sources for obtaining NK cells, and other techniques are aiming at the development of promising NK cell anticancer therapies, regardless of tumorigenesis. In this review, we compare the role of NK cells in the pathogenesis of hematological and solid tumors and discuss current prospects of NK-cell-based therapy for hematological and solid tumors.

Increased Susceptibility of the CD57- NK Cells Expressing KIR2DL2/3 and NKG2C to iCasp9 Gene Retroviral Transduction and the Relationships with Proliferative Potential, Activation Degree, and Death Induction Response.

Nowadays, the use of genetically modified NK cells is a promising strategy for cancer immunotherapy. The additional insertion of genes capable of inducing cell suicide allows for the timely elimination of the modified NK cells. Different subsets of the heterogenic NK cell population may differ in proliferative potential, in susceptibility to genetic viral transduction, and to the subsequent induction of cell death. The CD57-NKG2C+ NK cells are of special interest as potential candidates for therapeutic usage due to their high proliferative potential and certain features of adaptive NK cells. In this study, CD57- NK cell subsets differing in KIR2DL2/3 and NKG2C expression were transduced with the iCasp9 suicide gene. The highest transduction efficacy was observed in the KIR2DL2/3+NKG2C+ NK cell subset, which demonstrated an increased proliferative potential with prolonged cultivation. The increased transduction efficiency of the cell cultures was associated with the higher expression level of the HLA-DR activation marker. Among the iCasp9-transduced subsets, KIR2DL2/3+ cells had the weakest response to the apoptosis induction by the chemical inductor of dimerization (CID). Thus, KIR2DL2/3+NKG2C+ NK cells showed an increased susceptibility to the iCasp9 retroviral transduction, which was associated with higher proliferative potential and activation status. However, the complete elimination of these cells with CID is impeded.

Reduced Immunosenescence of Peripheral Blood T Cells in Parkinson's Disease with CMV Infection Background.

Immunosenescence is a process of remodeling the immune system under the influence of chronic inflammation during aging. Parkinson's disease (PD) is a common age-associated neurodegenerative disorder and is frequently accompanied by neuroinflammation. On the other hand, cytomegalovirus (CMV), one of the most spread infections in humans, may induce chronic inflammation which contributes to immunosenescence, differentiation and the inflation of T cells and NK cells. Currently, there is no clear understanding of immunosenescence severity in PD patients infected with CMV. In this study, we analyzed differentiation stages and immunosenescence characteristics of T cells and NK cells in 31 patients with mild and moderate PD severity, 33 age-matched and 30 young healthy donors. The PD patients were 100% CMV-seropositive compared to 76% age-matched and 73% young CMV-infected healthy donors. The proportion of effector memory T cells re-expressing CD45RA, CD57+CD56- T cells and CD57+CD56+ T cells was significantly reduced in PD patients compared with CMV-seropositive age-matched healthy individuals. The CD57+CD56- T cell proportion in PD patients was similar to that of CMV-seropositive young healthy donors. Thus, PD is characterized by reduced peripheral blood T cell immunosenescence, even against the background of CMV infection.

Recurrent Stimulation of Natural Killer Cell Clones with K562 Expressing Membrane-Bound Interleukin-21 Affects Their Phenotype, Interferon-γ Production, and Lifespan.

A pattern of natural killer cell (NK cell) heterogeneity determines proliferative and functional responses to activating stimuli in individuals. Obtaining the progeny of a single cell by cloning the original population is one of the ways to study NK cell heterogeneity. In this work, we sorted single cells into a plate and stimulated them via interleukin (IL)-2 and gene-modified K562 feeder cells that expressed membrane-bound IL-21 (K562-mbIL21), which led to a generation of phenotypically confirmed and functionally active NK cell clones. Next, we applied two models of clone cultivation, which differently affected their phenotype, lifespan, and functional activity. The first model, which included weekly restimulation of clones with K562-mbIL21 and IL-2, resulted in the generation of relatively short-lived (5⁻7 weeks) clones of highly activated NK cells. Levels of human leukocyte antigen class II molecule-DR isotype (HLA-DR) expression in the expanded NK cells correlated strongly with interferon-γ (IFN-γ) production. The second model, in which NK cells were restimulated weekly with IL-2 alone and once on the sixth week with K562-mbIL21 and IL-2, produced long-lived clones (8⁻14 weeks) that expanded up to 107 cells with a lower ability to produce IFN-γ. Our method is applicable for studying variability in phenotype, proliferative, and functional activity of certain NK cell progeny in response to the stimulation, which may help in selecting NK cells best suited for clinical use.

HLA-DR+ NK cells are mostly characterized by less mature phenotype and high functional activity.

NK cells change their phenotype and functional characteristics during activation. In this work, we searched for a relationship of HLA-DR expression with differentiation stages and functional activity of NK cells ex vivo and stimulated in vitro with IL-2 challenged with gene modified feeder K562 cells expressing membrane-bound IL-21 (K562-mbIL21). This stimulation technique has been described for NK cell expansion in clinical use. We have observed that HLA-DR expression in freshly isolated circulating NK cells was mostly associated with less differentiated CD56bright CD57- cells, although in some individuals it could also be found in terminally differentiated CD57+ cells. Ex vivo HLA-DR+ NK cells possessed better capacity to produce IFN-γ in response to cytokine stimulation compared to their HLA-DR- counterparts. In vitro activation with IL-2 and K562-mbIL21 induces an increase in HLA-DR-positive NK cell proportion, again mostly among CD56bright CD57- NK cells. This happened in particular due to appearance of HLA-DR+ expression de novo in HLA-DR-negative cells. Acquired in vitro HLA-DR expression was associated with NK cell proliferation activity, more intense cytokine-induced IFN-γ production, increased degranulation toward feeder cells, and higher expression of CD86 and NKG2D. Thus, stimulation with IL-2/K562-mbIL21 causes a significant phenotype and functional shift during NK cell activation and expansion.

Ethanol-dependent expression of the NKG2D ligands MICA/B in human cell lines and leukocytes.

Alcohol consumption affects the human immune system, causing a variety of disorders. However, the mechanisms of development of these changes are not fully understood. We hypothesized that ethanol may influence the expression of MICA and MICB, stress-induced molecules capable of regulating the activity of cytotoxic lymphocytes through the interaction with receptor NKG2D, which substantially affects the functionality of cellular immunity. We analyzed the effects of ethanol on MICA/B expression in tumor cell lines and human leukocytes. In the cell line models, ethanol caused different changes in the surface expression of MICA/B; in particular, it induced the translocation of intracellular proteins MICA/B to the cell surface and shedding of MICA (in soluble and microparticle-associated forms) from the plasma membrane. The observed results are not linked with cell death in cultures, taking place only under higher doses of ethanol. Ethanol at physiologically relevant concentrations (and higher) stimulated expression of MICA/B genes in different cell types. The effect of ethanol was more pronounced in hepatocyte line HepG2 compared with hematopoietic cell lines K562, Jurkat, and THP-1. Among the tested leukocytes, the most sensitive to ethanol action were T cells activated ex vivo with IL-2, in which the increase of MICA/B mRNA expression was registered with the smallest dose of ethanol (0.125%). In human monocytes, ethanol may lead to elevations in surface MICA/B levels. Presumably, changes in MICA/B expression caused by ethanol can affect the functions of NKG2D-positive cytotoxic lymphocytes, modulating immune reactions at excessive alcohol consumption.

Similarity and Differences in Inflammation-Related Characteristics of the Peripheral Immune System of Patients with Parkinson's and Alzheimer's Diseases.

Parkinson's disease (PD) and Alzheimer's disease (AD) are the most common age-related neurodegenerative disorders. Both diseases are characterized by chronic inflammation in the brain-neuroinflammation. The first signs of PD and AD are most often manifested in old age, in which the immune system is usually characterized by chronic inflammation, so-called "inflammaging" In recent years, there is growing evidence that pathogenesis of these diseases is connected with both regional and peripheral immune processes. Currently, the association of clinical signs of PD and AD with different characteristics of patient immune status is actively being researched. In this mini-review we compare the association of PD and AD alterations of a number of immune system parameters connected with the process of inflammation.

Towards hybrid biocompatible magnetic rHuman serum albumin-based nanoparticles: use of ultra-small (CeLn)3/4+ cation-doped maghemite nanoparticles as functional shell.

Human serum albumin (HSA) is a protein found in human blood. Over the last decade, HSA has been evaluated as a promising drug carrier. However, not being magnetic, HSA cannot be used for biomedical applications such as magnetic resonance imaging (MRI) and magnetic drug targeting. Therefore, subsequent composites building on iron oxide nanoparticles that are already used clinically as MRI contrast agents are extensively studied. Recently and in this context, innovative fully hydrophilic ultra-small CAN-stabilized maghemite ((CeLn)(3/4+)-γ-Fe2O3) nanoparticles have been readily fabricated. The present study discusses the design, fabrication, and characterization of a dual phase hybrid core (rHSA)-shell ((CeLn)(3/4+)-γ-Fe2O3 NPs) nanosystem. Quite importantly and in contrast to widely used encapsulation strategies, rHSA NP surface-attached (CeLn)(3/4+)-γ-Fe2O3 NPs enabled to exploit both rHSA (protein functionalities) and (CeLn)(3/4+)-γ-Fe2O3 NP surface functionalities (COOH and ligand L coordinative exchange) in addition to very effective MRI contrast capability due to optimal accessibility of H2O molecules with the outer magnetic phase. Resulting hybrid nanoparticles might be used as a platform modular system for therapeutic (drug delivery system) and MR diagnostic purposes.

The hTERT and iCasp9 Transgenes Affect EOMES and T-BET Levels in NK Cells and the Introduction of Both Genes Improves NK Cell Proliferation in Response to IL2 and IL15 Stimulation.

The NK cell exhaustion state evolving during extensive and prolonged cultivation is still one of the limitations of NK cell approaches. In this research, we transduced NK cells with the hTERT and iCasp9 genes. hTERT overexpression can prevent the functional exhaustion of NK cells during long-term cultivation, but, still, the therapeutic use of such cells is unsafe without irradiation. To overcome this obstacle, we additionally transduced NK cells with the iCasp9 transgene that enables the rapid elimination of modified cells. We compared the proliferative and functional activities of the hTERT- and/or iCasp9-modified NK cells, determined their exhaustion state and monitored the levels of EOMES and T-BET, the main NK cell transcription factors. The hTERT and iCasp9 genes were shown to affect the EOMES and T-BET levels differently in the NK cells. The EOMES+T-BET+ phenotype characterized the functionally active NK cells during two months of culture upon stimulation with IL2 and K562-mbIL21 feeder cells, which induced the greatest expansion rates of the NK cells, independently of the transgene type. On the other hand, under cytokine stimulation, the hTERT-iCasp9-NK cells displayed improved proliferation over NK cells modified with iCasp9 alone and showed an increased proliferation rate compared to the untransduced NK cells under stimulation with IL2 and IL15, which was accompanied by reduced immune checkpoint molecule expression. The individual changes in the EOMES and T-BET levels strictly corresponded to the NK cell functional activity, the surface levels of activating and inhibitory receptors along with the expansion rate and expression levels of pro-survival and pro-apoptotic genes.

Autologous T-Cell-Free Antigen Presentation System Unveils hCMV-Specific NK Cell Response.

NK cells play a decisive role in controlling hCMV infection by combining innate and adaptive-like immune reactions. The hCMV-derived VMAPRTLFL (LFL) peptide is a potent activator of NKG2C+ NK cells. Proposed here is an autologous system of LFL stimulation without T lymphocytes and exogenous cytokines that allows us to evaluate NK-cell hCMV-specific responses in more native settings. In this model, we evaluated LFL-induced IFNγ production, focusing on signaling pathways and the degranulation and proliferation of NK cells orchestrated by microenvironment cytokine production and analyzed the transcriptome of expanded NK cells. NK cells of individuals having high anti-hCMV-IgG levels, in contrast to NK cells of hCMV-seronegative and low-positive donors, displayed increased IFNγ production and degranulation and activation levels and enhanced proliferation upon LFL stimulation. Cytokine profiles of these LFL-stimulated cultures demonstrated a proinflammatory shift. LFL-induced NK-cell IFNγ production was dependent on the PI3K and Ras/Raf/Mek signaling pathways, independently of cytokines. In hCMV-seropositive individuals, this model allowed obtaining NK-cell antigen-specific populations proliferating in response to LFL. The transcriptomic profile of these expanded NK cells showed increased adaptive gene expression and metabolic activation. The results complement the existing knowledge about hCMV-specific NK-cell response. This model may be further exploited for the identification and characterization of antigen-specific NK cells.

Three-Dimensional Model Analysis Revealed Differential Cytotoxic Effects of the NK-92 Cell Line and Primary NK Cells on Breast and Ovarian Carcinoma Cell Lines Mediated by Variations in Receptor-Ligand Interactions and Soluble Factor Profiles.

Background/objectives: The functional activity of a certain tumor determines the effectiveness of primary NK cells and NK-92 cell line-based cancer therapy; their therapeutic effectiveness against different tumors can vary. This work provides a direct simultaneous comparison of the cytotoxic effects of in vitro-activated peripheral NK (pNK) cells and NK-92 cells in spheroid models of BT-474, MCF7 and SKOV-3 carcinomas and uncovers the reasons for the differential effectiveness of NK cells against tumors. Methods: Tumor spheroids of similar size and shape, obtained from agarose molds, were incubated with NK-92 or pNK cells for 24 h. Tumor cell death was detected using flow cytometry or confocal microscopy. Cytokine production, granzyme B levels and NK cell degranulation analyses were performed, along with pNK and target-cell phenotypic characterization. Results: While NK-92 and pNK cells lysed BT-474 spheroids with comparably low efficiency, pNK cells were more capable of eliminating MCF7 and SKOV-3 spheroids than NK-92 cells were. The results of the functional and phenotypic analyses strongly support the participation of the NKG2D-NKG2DL pathway in pNK cell activation induced by the most sensitive cytotoxic attack on SKOV-3 spheroids, whereas the CX3CR1-CX3CL1 axis appears to be involved in the pNK reaction against MCF-7 spheroids. Conclusions: We provide a new approach for the preliminary identification of the most promising NK cell receptors that can alter the effectiveness of cancer therapy depending on the specific tumor type. Using this approach, NK-92 cells or pNK subsets can be selected for further accumulation and/or genetic modification to improve specificity and reactivity.

Obtaining Gene-Modified HLA-E-Expressing Feeder Cells for Stimulation of Natural Killer Cells.

Human cytomegalovirus (HCMV)-specific adaptive NK cells are capable of recognizing viral peptides presented by HLA-E on infected cells via the NKG2C receptor. Using retroviral transduction, we have generated a K562-cell-based line expressing HLA-E in the presence of the HLA-E-stabilizing peptide, which has previously shown the capacity to enhance adaptive NK cell response. The obtained K562-21E cell line was employed to investigate proliferative responses of the CD57- NK cell subset of HCMV-seropositive and seronegative donors. Stimulation of CD57- NK cells with K562-21E/peptide resulted in an increased cell expansion during the 12-day culturing period, regardless of the serological HCMV status of the donor. The enhanced proliferation in response to the peptide was associated with a greater proportion of CD56brightHLA-DR+ NK cells. In later stages of cultivation, the greatest proliferative response to K562-21E/peptide was shown for a highly HCMV-seropositive donor. These expanded NK cells were characterized by the accumulation of CD57-KIR2DL2/3+NKG2C+NKG2A- cells, which are hypothesized to represent adaptive NK cell progenitors. The K562-21E feeder cells can be applied both for the accumulation of NK cells as therapeutic effectors, and for the study of NK cell maturation into the adaptive state after the HLA-E peptide presentation.

Methodological Approaches for Increasing the Retroviral Transduction Efficiency of Primary NK Cells.

BACKGROUND: The growing attention to NK cells for cancer cell therapy is associated with the need to establish highly efficient protocols for their genetic modification, particularly by retroviral transduction. OBJECTIVE: In this work, we have optimized several stages of the retroviral-based modification process, and determined the distribution of the amino acid transporter ASCT2 between NK cell subsets. METHODS: Retroviral particles were produced using the Phoenix Ampho cell line transfected with the calcium phosphate method . We used RD114-based retroviral transduction for lymphocyte cell lines and primary NK cells. RESULTS: We have determined the optimal time to collect the RD114-pseudotyped viral supernatants resulting in the titer of viral particles required for efficient NK cell modification to be between 48 and 72 hours. Retroviral modification by retronectin-based method did not alter NK cell functional activity and cell survival. We identified differences in the Multiplicity of Infection (MOI) among cell lines that were partially associated with the ASCT2 surface expression. Cells with higher ASCT2 levels were more susceptible to transduction with RD114-pseudotyped viral particles. Higher ASCT2 expression levels were revealed in activated CD57+ and KIR2DL2DL3+ NK cells compared to their negative counterparts. CONCLUSION: Our findings provide a more nuanced understanding of NK cell transduction, offering valuable insights for improving therapeutic applications involving NK cell modification.

Targeted Delivery of HSP70 to Tumor Cells via Supramolecular Complex Based on HER2-Specific DARPin9_29 and the Barnase:Barstar Pair.

(1) Background: We have previously shown that the use of an artificial supramolecular two-component system based on chimeric recombinant proteins 4D5scFv-barnase and barstar-heat shock protein 70 KDa (HSP70) allows targeted delivery of HSP70 to the surface of tumor cells bearing HER2/neu antigen. In this work, we studied the possibility to using DARPin9_29-barnase as the first targeting module recognizing HER2/neu-antigen in the HSP70 delivery system. (2) Methods: The effect of the developed systems for HSP70 delivery to human carcinomas SK-BR-3 and BT474 cells hyperexpressing HER2/neu on the activation of cytotoxic effectors of the immune cells was studied in vitro. (3) Results: The results obtained by confocal microscopy and cytofluorimetric analysis confirmed the binding of HSP70 or its fragment HSP70-16 on the surface of the treated cells. In response to the delivery of HSP70 to tumor cells, we observed an increase in the cytolytic activity of different cytotoxic effector immune cells from human peripheral blood. (4) Conclusions: Targeted modification of the tumor cell surface with molecular structures recognized by cytotoxic effectors of the immune system is among new promising approaches to antitumor immunotherapy.

Multiple Actions of Telomerase Reverse Transcriptase in Cell Death Regulation.

Telomerase reverse transcriptase (TERT), a core part of telomerase, has been known for a long time only for its telomere lengthening function by reverse transcription of RNA template. Currently, TERT is considered as an intriguing link between multiple signaling pathways. The diverse intracellular localization of TERT corresponds to a wide range of functional activities. In addition to the canonical function of protecting chromosome ends, TERT by itself or as a part of the telomerase complex participates in cell stress responses, gene regulation and mitochondria functioning. Upregulation of TERT expression and increased telomerase activity in cancer and somatic cells relate to improved survival and persistence of such cells. In this review, we summarize the data for a comprehensive understanding of the role of TERT in cell death regulation, with a focus on the interaction of TERT with signaling pathways involved in cell survival and stress response.

Epithelial-Immune Cell Crosstalk Determines the Activation of Immune Cells In Vitro by the Human Cathelicidin LL-37 at Low Physiological Concentrations.

The only human cathelicidin, LL-37, is a host defense antimicrobial peptide with antimicrobial activities against protozoans, fungi, Gram(+) and Gram(-) bacteria, and enveloped viruses. It has been shown in experiments in vitro that LL-37 is able to induce the production of various inflammatory and anti-inflammatory cytokines and chemokines by different human cell types. However, it remains an open question whether such cytokine induction is physiologically relevant, as LL-37 exhibited its immunomodulatory properties at concentrations that are much higher (>20 µg/mL) than those observed in non-inflamed tissues (1-5 µg/mL). In the current study, we assessed the permeability of LL-37 across the Caco-2 polarized monolayer and showed that this peptide could pass through the Caco-2 monolayer with low efficiency, which predetermined its low absorption in the gut. We showed that LL-37 at low physiological concentrations (<5 µg/mL) was not able to directly activate monocytes. However, in the presence of polarized epithelial monolayers, LL-37 is able to activate monocytes through the MAPK/ERK signaling pathway and induce the production of cytokines, as assessed by a multiplex assay at the protein level. We have demonstrated that LL-37 is able to fulfill its immunomodulatory action in vivo in non-inflamed tissues at low physiological concentrations. In the present work, we revealed a key role of epithelial-immune cell crosstalk in the implementation of immunomodulatory functions of the human cathelicidin LL-37, which might shed light on its physiological action in vivo.