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Women acquire HIV through sexual transmission, with increasing incidence in women >50 years old. Identifying protective mechanisms in the female genital tract (FGT) is important to prevent HIV-acquisition in women as they age. Human genital and blood neutrophils inactivate HIV by releasing neutrophil extracellular traps (NETs), an innate protective mechanism against HIV-infection. However, how NET formation is triggered by HIV in different tissues and whether this mechanism is affected by aging remain unknown. We demonstrate that the mechanisms that trigger NET release in response to HIV are different in blood and genital tissues, and that NET release decreases with aging. In blood neutrophils, HIV stimulation independently activated calcium pathways and endosomal TLR8, but aging reduced calcium responses, resulting in delayed NET release. In contrast, calcium responses were absent in genital neutrophils and NET release was triggered preferentially through TLR8 activation, but aging impaired this pathway. HIV induced NET formation through non-lytic pathways in blood and FGT neutrophils, except for a small subset of NETs that incorporated annexin V and lactoferrin predominantly in blood, suggesting proinflammatory and lytic NET release. Our findings demonstrate that blood neutrophils cannot model genital neutrophil responses which has important implications to understanding protection against HIV acquisition., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Moreno de Lara, Werner, Borchers, Carrillo-Salinas, Marmol, Parthasarathy, Iyer, Vogell, Illanes, Abadía-Molina, Ochsenbauer, Wira and Rodriguez-Garcia.)

Background: Approximately 13.8% and 6.1% of coronavirus disease 2019 (COVID-19) patients require hospitalization and sometimes intensive care unit (ICU) admission, respectively. There is no biomarker to predict which of these patients will develop an aggressive stage that we could improve their quality of life and healthcare management. Our main goal is to include new markers for the classification of COVID-19 patients., Methods: Two tubes of peripheral blood were collected from a total of 66 (n = 34 mild and n = 32 severe) samples (mean age 52 years). Cytometry analysis was performed using a 15-parameter panel included in the Maxpar ® Human Monocyte/Macrophage Phenotyping Panel Kit. Cytometry by time-of-flight mass spectrometry (CyTOF) panel was performed in combination with genetic analysis using TaqMan ® probes for ACE2 (rs2285666), MX1 (rs469390), and TMPRSS2 (rs2070788) variants. GemStone™ and OMIQ software were used for cytometry analysis., Results: The frequency of CD163 + /CD206 - population of transitional monocytes (T-Mo) was decreased in the mild group compared to that of the severe one, while T-Mo CD163 - /CD206 - were increased in the mild group compared to that of the severe one. In addition, we also found differences in CD11b expression in CD14 dim monocytes in the severe group, with decreased levels in the female group (p = 0.0412). When comparing mild and severe disease, we also found that CD45 - [p = 0.014; odds ratio (OR) = 0.286, 95% CI 0.104-0.787] and CD14 dim /CD33 + (p = 0.014; OR = 0.286, 95% CI 0.104-0.787) monocytes were the best options as biomarkers to discriminate between these patient groups. CD33 was also indicated as a good biomarker for patient stratification by the analysis of GemStone™ software. Among genetic markers, we found that G carriers of TMPRSS2 (rs2070788) have an increased risk (p = 0.02; OR = 3.37, 95% CI 1.18-9.60) of severe COVID-19 compared to those with A/A genotype. This strength is further increased when combined with CD45 - , T-Mo CD163 + /CD206 - , and C14 dim /CD33 + ., Conclusions: Here, we report the interesting role of TMPRSS2 , CD45 - , CD163/CD206, and CD33 in COVID-19 aggressiveness. This strength is reinforced for aggressiveness biomarkers when TMPRSS2 and CD45 - , TMPRSS2 and CD163/CD206, and TMPRSS2 and CD14 dim /CD33 + are combined., Competing Interests: Author CE-B is employed by LORGEN G.P. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Martínez-Diz, Marín-Benesiu, López-Torres, Santiago, Díaz-Cuéllar, Martín-Esteban, Cortés-Valverde, Arenas-Rodríguez, Cuenca-López, Porras-Quesada, Ruiz-Ruiz, Abadía-Molina, Entrala-Bernal, Martínez-González and Álvarez-Cubero.)

Ly108 (SLAMF6) is a homophilic cell surface molecule that binds SLAM-associated protein (SAP), an intracellular adapter protein that modulates humoral immune responses. Furthermore, Ly108 is crucial for the development of natural killer T (NKT) cells and CTL cytotoxicity. Significant attention has been paid towards expression and function of Ly108 since multiple isoforms were identified, i.e., Ly108-1, Ly108-2, Ly108-3, and Ly108-H1, some of which are differentially expressed in several mouse strains. Surprisingly, Ly108-H1 appeared to protect against disease in a congenic mouse model of Lupus. Here, we use cell lines to further define Ly108-H1 function in comparison with other isoforms. We show that Ly108-H1 inhibits IL-2 production while having little effect upon cell death. With a refined method, we could detect phosphorylation of Ly108-H1 and show that SAP binding is retained. We propose that Ly108-H1 may regulate signaling at two levels by retaining the capability to bind its extracellular as well as intracellular ligands, possibly inhibiting downstream pathways. In addition, we detected Ly108-3 in primary cells and show that this isoform is also differentially expressed between mouse strains. The presence of additional binding motifs and a non-synonymous SNP in Ly108-3 further extends the diversity between murine strains. This work highlights the importance of isoform awareness, as inherent homology can present a challenge when interpreting mRNA and protein expression data, especially as alternatively splicing potentially affects function.

Research Question: Are the alterations observed in the endometriotic cells, such as progesterone resistance, already present in the eutopic endometrium or acquired in the ectopic location?, Design: The response to decidualization with progesterone and cyclic AMP for up to 28 days was compared in different endometrial stromal cell (EnSC) lines established from samples of endometriomas (eEnSC), eutopic endometrium from women with endometriosis (eBEnSC), endometrial tissue from healthy women (BEnSC) and menstrual blood from healthy donors (mEnSC)., Results: Usual features of decidualized cells, such as changes in cell morphology and expression of prolactin, were similarly observed in the three types of eutopic EnSC studied, but not in the ectopic cells upon decidualization. Among the phenotypic markers analysed, CD105 was down-regulated under decidualization in all cell types (mEnSC, P = 0.005; BEnSC, P = 0.029; eBEnSC, P = 0.022) except eEnSC. mEnSC and BEnSC underwent apoptosis during decidualization, whereas eBEnSC and eEnSC were resistant to the induction of cell death. Lastly, migration studies revealed that mEnSC secreted undetermined factors during decidualization that inhibited cell motility, whereas eEnSC showed a significantly lower ability to produce those migration-regulating factors (P < 0.0001, P  < 0.001 and P = 0.0013 for the migration of mEnSC at 24, 48 and 72 h, respectively; P  < 0.0001 for the migration of eEnSC at all times studied)., Conclusions: This study provides novel insights into the differences between endometriotic and eutopic endometrial cells and reinforces the idea that the microenvironment in the ectopic location plays additional roles in the acquisition of the alterations that characterize the cells of the endometriotic foci., (Copyright © 2022 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Background & Aims: Barrett's esophagus is considered to be a metaplastic lesion that predisposes for esophageal adenocarcinoma. Development of Barrett's esophagus is considered to be driven by sonic hedgehog mediated bone morphogenetic protein (BMP) signaling. We aimed to investigate in preclinical in vivo models whether targeting canonical BMP signaling could be an effective treatment for Barrett's esophagus., Methods and Results: Selective inhibition of BMP2 and BMP4 within an in vivo organoid model of Barrett's esophagus inhibited development of columnar Barrett's cells, while favoring expansion of squamous cells. Silencing of noggin, a natural antagonist of BMP2, BMP4, and BMP7, in a conditional knockout mouse model induced expansion of a Barrett's-like neo-columnar epithelium from multi-lineage glands. Conversely, in this model specific inhibition of BMP2 and BMP4 led to the development of a neo-squamous lineage. In an ablation model, inhibition of BMP2 and BMP4 resulted in the regeneration of neo-squamous epithelium after the cryoablation of columnar epithelium at the squamocolumnar junction. Through lineage tracing the generation of the neo-squamous mucosa was found to originate from K5+ progenitor squamous cells., Conclusions: Here we demonstrate that specific inhibitors of BMP2 and BMP4 attenuate the development of Barrett's columnar epithelium, providing a novel potential strategy for the treatment of Barrett's esophagus and the prevention of esophageal adenocarcinoma., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Signaling lymphocytic activation molecule family 8 (SLAMF8) is involved in the negative modulation of NADPH oxidase activation. However, the impact of SLAMF8 downregulation on macrophage functionality and the microbicide mechanism remains elusive. To study this in depth, we first analyzed NADPH oxidase activation pathways in wild-type and SLAMF8-deficient macrophages upon different stimulus. Herein, we describe increased phosphorylation of the Erk1/2 and p38 MAP kinases, as well as increased phosphorylation of NADPH oxidase subunits in SLAMF8-deficient macrophages. Furthermore, using specific inhibitors, we observed that specific PI3K inhibition decreased the differences observed between wild-type and SLAMF8-deficient macrophages, stimulated with either PMA, LPS, or Salmonella typhimurium infection. Consequently, SLAMF8-deficient macrophages also showed increased recruitment of small GTPases such as Rab5 and Rab7, and the p47 phox subunit to cytoplasmic Salmonella , suggesting an impairment of Salmonella- containing vacuole (SCV) progression in SLAMF8-deficient macrophages. Enhanced iNOS activation, NO production, and IL-6 expression were also observed in the absence of SLAMF8 upon Salmonella infection, either in vivo or in vitro , while overexpression of SLAMF8 in RAW264.7 macrophages showed the opposite phenotype. In addition, SLAMF8-deficient macrophages showed increased activation of Src kinases and reduced SHP-1 phosphate levels upon IFNγ and Salmonella stimuli in comparison to wild-type macrophages. In agreement with in vitro results, Salmonella clearance was augmented in SLAMF8-deficient mice compared to that in wild-type mice. Therefore, in conclusion, SLAMF8 intervention upon bacterial infection downregulates mouse macrophage activation, and confirmed that SLAMF8 receptor could be a potential therapeutic target for the treatment of severe or unresolved inflammatory conditions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Romero-Pinedo, Barros, Ruiz-Magaña, Maganto-García, Moreno de Lara, Abadía-Molina, Terhorst and Abadía-Molina.)

Progress in the understanding of the biology of perinatal tissues has contributed to the breakthrough revelation of the therapeutic effects of perinatal derivatives (PnD), namely birth-associated tissues, cells, and secreted factors. The significant knowledge acquired in the past two decades, along with the increasing interest in perinatal derivatives, fuels an urgent need for the precise identification of PnD and the establishment of updated consensus criteria policies for their characterization. The aim of this review is not to go into detail on preclinical or clinical trials, but rather we address specific issues that are relevant for the definition/characterization of perinatal cells, starting from an understanding of the development of the human placenta, its structure, and the different cell populations that can be isolated from the different perinatal tissues. We describe where the cells are located within the placenta and their cell morphology and phenotype. We also propose nomenclature for the cell populations and derivatives discussed herein. This review is a joint effort from the COST SPRINT Action (CA17116), which broadly aims at approaching consensus for different aspects of PnD research, such as providing inputs for future standards for the processing and in vitro characterization and clinical application of PnD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Silini, Di Pietro, Lang-Olip, Alviano, Banerjee, Basile, Borutinskaite, Eissner, Gellhaus, Giebel, Huang, Janev, Kreft, Kupper, Abadía-Molina, Olivares, Pandolfi, Papait, Pozzobon, Ruiz-Ruiz, Soritau, Susman, Szukiewicz, Weidinger, Wolbank, Huppertz and Parolini.)

Menstrual blood-derived stromal cells (MenSCs) are emerging as a strong candidate for cell-based therapies due to their immunomodulatory properties. However, their direct impact on innate immune populations remains elusive. Since macrophages play a key role in the onset and development of inflammation, understanding MenSCs implication in the functional properties of these cells is required to refine their clinical effects during the treatment of inflammatory disorders. In this study, we assessed the effects that MenSCs had on the recruitment of macrophages and other innate immune cells in two mouse models of acute inflammation, a thioglycollate (TGC)-elicited peritonitis model and a monobacterial sepsis model. We found that, in the TGC model, MenSCs injection reduced the percentage of macrophages recruited to the peritoneum and promoted the generation of peritoneal immune cell aggregates. In the sepsis model, MenSCs exacerbated infection by diminishing the recruitment of macrophages and neutrophils to the site of infection and inducing defective bacterial clearance. Additional in vitro studies confirmed that co-culture with MenSCs impaired macrophage bactericidal properties, affecting bacterial killing and the production of reactive oxygen intermediates. Our findings suggest that MenSCs modulate the macrophage population and that this modulation must be taken into consideration when it comes to future clinical applications.

Monocytes and macrophages constitute the first line of defense of the immune system against external pathogens. Macrophages have a highly plastic phenotype depending on environmental conditions; the extremes of this phenotypic spectrum are a pro-inflammatory defensive role (M1 phenotype) and an anti-inflammatory tissue-repair one (M2 phenotype). The Inhibitor of Apoptosis (IAP) proteins have important roles in the regulation of several cellular processes, including innate and adaptive immunity. In this study we have analyzed the differential expression of the IAPs, NAIP, cIAP1 and cIAP2, during macrophage differentiation and polarization into M1 or M2. In polarized THP-1 cells and primary human macrophages, NAIP is abundantly expressed in M2 macrophages, while cIAP1 and cIAP2 show an inverse pattern of expression in polarized macrophages, with elevated expression levels of cIAP1 in M2 and cIAP2 preferentially expressed in M1. Interestingly, treatment with the IAP antagonist SMC-LCL161, induced the upregulation of NAIP in M2, the downregulation of cIAP1 in M1 and M2 and an induction of cIAP2 in M1 macrophages.

Introduction: Human decidual stromal cells (DSCs) play a key role in maternal-fetal interactions. Precursors of DSCs (preDSCs) localize around vessels in both the endometrium and decidua. Previous studies suggested a relationship between preDSCs and pericytes because these cells share a perivascular location, alpha smooth muscle actin (α-SM actin) expression and the ability to contract under the effects of cytokines., Methods: To further study this relationship, we established 15 human preDSC lines and 3 preDSC clones. The preDSC lines and clones were tested by flow cytometry with a panel of 29 monoclonal antibodies, 14 of which are pericyte markers. The expression of angiogenic factors was determined by RT-PCR, chemotactic activity was studied with the migration assay, and cell contractility was evaluated with the collagen cell contraction assay. Confocal microscopy was used to study decidual sections., Results: Under the effect of progesterone and cAMP, these lines decidualized in vitro: the cells became rounder and secreted prolactin, a marker of physiological DSC differentiation (decidualization). The antigen phenotype of these preDSC lines and clones was fully compatible with that reported for pericytes. PreDSC lines displayed pericyte characteristics: they expressed angiogenic factors and showed chemotactic and cytokine-induced contractile activity. Confocal microscopic examination of decidual sections revealed the expression of antigens detected in preDSC lines: α-SM actin colocalized with CD146, CD140b, MFG-E8, nestin, and STRO-1 (all of which are pericyte markers) in cells located around the vessels, a distinctive location of preDSCs and pericytes., Discussion: Taken together, our results show that preDSCs are pericyte-like cells., (Copyright © 2017 Elsevier Ltd. All rights reserved.)