Leishmania braziliensis enhances monocyte responses to promote anti-tumor activity.

Innate immune cells can undergo long-term functional reprogramming after certain infections, a process called trained immunity (TI). Here, we focus on antigens of Leishmania braziliensis, which induced anti-tumor effects via trained immunity in human monocytes. We reveal that monocytes exposed to promastigote antigens of L. braziliensis develop an enhanced response to subsequent exposure to Toll-like receptor (TLR)2 or TLR4 ligands. Mechanistically, the induction of TI in monocytes by L. braziliensis is mediated by multiple pattern recognition receptors, changes in metabolism, and increased deposition of H3K4me3 at the promoter regions of immune genes. The administration of L. braziliensis exerts potent anti-tumor capabilities by delaying tumor growth and prolonging survival of mice with non-Hodgkin lymphoma. Our work reveals mechanisms of TI induced by L. braziliensis in vitro and identifies its potential for cancer immunotherapy.

Zika Virus-Infected Monocyte Exosomes Mediate Cell-to-Cell Viral Transmission.

Zika fever is a reemerging arthropod-borne viral disease; however, Zika virus (ZIKV) can be transmitted by other, non-vector means. Severe Zika fever is characterized by neurological disorders, autoimmunity, or congenital Zika syndrome. Monocytes are primary ZIKV targets in humans and, in response to infection, release extracellular vesicles like exosomes. Exosomes mediate intercellular communication and are involved in the virus's ability to circumvent the immune response, promoting pathological processes. This study aimed to evaluate the role of monocyte exosomes in cell-to-cell viral transmission. We isolated exosomes from ZIKV-infected monocytes (Mø exo ZIKV) by differential ultracentrifugation and identified them by nanoparticle tracking analysis; transmission electron microscopy; and CD63, CD81, TSG101, and Alix detection by cytofluorometry. Purified exosome isolates were obtained by uncoupling from paramagnetic beads or by treatment with UV radiation and RNase A. We found that Mø exo ZIKV carry viral RNA and E/NS1 proteins and that their interaction with naïve cells favors viral transmission, infection, and cell differentiation/activation. These data suggest that Mø exo ZIKV are an efficient alternative pathway for ZIKV infection. Knowledge of these mechanisms contributes to understanding the pathogenesis of severe disease and to the development of new vaccines and therapies.

IKAROS expression drives the aberrant metabolic phenotype of macrophages in chronic HIV infection.

The increased risk for acquiring secondary illnesses in people living with HIV (PLWH) has been associated with immune dysfunction. We have previously found that circulating monocytes from PLWH display a trained phenotype. Here, we evaluated the metabolic profile of these cells and found increased mitochondrial respiration and glycolysis of monocyte-derived macrophages (MDMs) from PLWH. We additionally found that cART shifted the energy metabolism of MDMs from controls toward increased utilization of mitochondrial respiration. Importantly, both downregulation of IKAROS expression and inhibition of the mTOR pathway reversed the metabolic profile of MDMs from PLWH and cART-treated control-MDMs. Altogether, this study reveals a very specific metabolic adaptation of MDMs from PLWH, which involves an IKAROS/mTOR-dependent increase of mitochondrial respiration and glycolysis. We propose that this metabolic adaptation decreases the ability of these cells to respond to environmental cues by "locking" PLWH monocytes in a pro-inflammatory and activated phenotype.

Soluble urate-induced effects on cytokine production in vitro - Assessment of methodologies and cell types.

BACKGROUND: Hyperuricemia has been shown to be an inducer of pro-inflammatory mediators by human primary monocytes. To study the deleterious effects of hyperuricemia, a reliable and stable in vitro model using soluble urate is needed. One recent report showed different urate-dissolving methods resulted in either pro-inflammatory or anti-inflammatory properties. The aim of this study was to compare the effect of two methods of dissolving urate on both primary human peripheral blood mononuclear cells (PBMCs) and THP-1 cells. The two methods tested were 'pre-warming' and 'dissolving with NaOH'. METHODS: Primary human PBMCs and THP-1 cells were exposed to urate solutions, prepared using the two methodologies: pre-warming and dissolving with NaOH. Afterwards, cells were stimulated with various stimuli, followed by the measurement of the inflammatory mediators IL-1ß, IL-6, IL-1Ra, TNF, IL-8, and MCP-1. RESULTS: In PBMCs, we observed an overall pro-inflammatory effect of urate, both in the pre-warming and the NaOH dissolving method. A similar pro-inflammatory effect was seen in THP-1 cells for both dissolving methods after restimulation. However, THP-1 cells exhibited pro-inflammatory profile with exposure to urate alone without restimulation. We did not find MSU crystals in our cellular assays. CONCLUSIONS: Overall, the urate dissolving methods do not have critical impact on its inflammatory properties. Soluble urate prepared using either of the two methods showed mostly pro-inflammatory effects on human primary PBMCs and monocytic cell line THP-1. However, human primary PBMCs and the THP-1 differ in their response to soluble urate without restimulation.

Triclosan and its alternatives, especially chlorhexidine, modulate macrophage immune response with distinct modes of action.

Since European regulators restricted the use of bacteriocidic triclosan (TCS), alternatives for TCS are emerging. Recently, TCS has been shown to reprogram immune metabolism, trigger the NLRP3 inflammasome, and subsequently the release of IL-1ß in human macrophages, but data on substitutes is scarce. Hence, we aimed to examine the effects of TCS compared to its alternatives at the molecular level in human macrophages. LPS-stimulated THP-1 macrophages were exposed to TCS or its substitutes, including benzalkonium chloride, benzethonium chloride, chloroxylenol, chlorhexidine (CHX) and cetylpyridinium chloride, with the inhibitory concentration (IC10-value) of cell viability to decipher their mode of action. TCS induced the release of the pro-inflammatory cytokine TNF and high level of IL-1ß, suggesting the activation of the NLRP3-inflammasome, which was confirmed by non-apparent IL-1ß under the NLRP3-inhibitor MCC950 treatment d. While IL-6 release was reduced in all treatments, the alternative CHX completely abolished the release of all investigated cytokines. To unravel the underlying molecular mechanisms, we used untargeted LC-MS/MS-based proteomics. TCS and CHX showed the strongest cellular response at the protein and signalling pathway level, whereby pathways related to metabolism, translation, cellular stress and migration were mainly affected but to different proposed modes of action. TCS inhibited mitochondrial electron transfer and affected phagocytosis. In contrast, in CHX-treated cells, the translation was arrested due to stress conditions, resulting in the formation of stress granules. Mitochondrial (e.g. ATP5F1D, ATP5PB, UQCRQ) and ribosomal (e.g. RPL10, RPL35, RPS23) proteins were revealed as putative key drivers. Furthermore, we have demonstrated the formation of podosomes by CHX, potentially involved in ECM degradation. Our results exhibit modulation of the immune response in macrophages by TCS and its substitutes and illuminated underlying molecular effects. These results illustrate critical processes involved in the modulation of macrophages' immune response by TCS and its alternatives, providing information essential for hazard assessment.

Multiple TLRs elicit alternative NLRP3 inflammasome activation in primary human monocytes independent of RIPK1 kinase activity.

The canonical NOD-like receptor family pyrin domain containing 3 (NLRP3) pathway involves a priming step to induce pro-IL-1ß followed by a secondary signal such as K+ efflux to activate inflammasome formation. This then leads to the maturation of IL-1ß and the formation of gasdermin D (GSDMD) pores that initiate pyroptosis and mediate IL-1ß release. In contrast, primary human monocytes also engage an alternative pathway in response to toll-like receptor (TLR) 4 activation, without the need for a secondary signal. Data from a monocyte-like cell line suggest that the alternative pathway functions via the TLR adaptor protein TIR-domain-containing adapter-inducing interferon-ß (TRIF), receptor-interacting protein kinase 1 (RIPK1), FAS-associated death domain (FADD) and caspase-8 upstream of NLRP3 activation, but in the absence of K+ efflux or pyroptosis. Usage of the alternative pathway by other members of the TLR family that induce IL-1ß but do not signal through TRIF, has yet to be explored in primary human monocytes. Furthermore, the mechanism by which IL-1ß is released from monocytes remains unclear. Therefore, this study investigated if the alternative NLRP3 inflammasome pathway is initiated following activation of TLRs other than TLR4, and if GSDMD was necessary for the release of IL-1ß. Monocytes were stimulated with ligands that activate TLR1/2, TLR2/6, TLR4 and TLR7 and/or TLR8 (using a dual ligand). Similar to TLR4, all of the TLRs investigated induced IL-1ß release in a NLRP3 and caspase-1 dependent manner, indicating that TRIF may not be an essential upstream component of the alternative pathway. Furthermore, inhibition of RIPK1 kinase activity had no effect on IL-1ß release. Although IL-1ß was released independently of K+ efflux and pyroptosis, it was significantly reduced by an inhibitor of GSDMD. Therefore, it is feasible that low level GSDMD pore formation may facilitate the release of IL-1ß from the cell, but not be present in sufficient quantities to initiate pyroptosis. Together these data suggest that the alternative pathway operates independently of RIPK1 kinase activity, downstream of diverse TLRs including TLR4 in primary human monocytes and supports the potential for IL-1ß release via GSDMD pores alongside other unconventional secretory pathways.

Mapping the epigenomic landscape of human monocytes following innate immune activation reveals context-specific mechanisms driving endotoxin tolerance.

BACKGROUND: Monocytes are key mediators of innate immunity to infection, undergoing profound and dynamic changes in epigenetic state and immune function which are broadly protective but may be dysregulated in disease. Here, we aimed to advance understanding of epigenetic regulation following innate immune activation, acutely and in endotoxin tolerant states. METHODS: We exposed human primary monocytes from healthy donors (n = 6) to interferon-γ or differing combinations of endotoxin (lipopolysaccharide), including acute response (2 h) and two models of endotoxin tolerance: repeated stimulations (6 + 6 h) and prolonged exposure to endotoxin (24 h). Another subset of monocytes was left untreated (naïve). We identified context-specific regulatory elements based on epigenetic signatures for chromatin accessibility (ATAC-seq) and regulatory non-coding RNAs from total RNA sequencing. RESULTS: We present an atlas of differential gene expression for endotoxin and interferon response, identifying widespread context specific changes. Across assayed states, only 24-29% of genes showing differential exon usage are also differential at the gene level. Overall, 19.9% (6,884 of 34,616) of repeatedly observed ATAC peaks were differential in at least one condition, the majority upregulated on stimulation and located in distal regions (64.1% vs 45.9% of non-differential peaks) within which sequences were less conserved than non-differential peaks. We identified enhancer-derived RNA signatures specific to different monocyte states that correlated with chromatin accessibility changes. The endotoxin tolerance models showed distinct chromatin accessibility and transcriptomic signatures, with integrated analysis identifying genes and pathways involved in the inflammatory response, detoxification, metabolism and wound healing. We leveraged eQTL mapping for the same monocyte activation states to link potential enhancers with specific genes, identifying 1,946 unique differential ATAC peaks with 1,340 expression associated genes. We further use this to inform understanding of reported GWAS, for example involving FCHO1 and coronary artery disease. CONCLUSION: This study reports context-specific regulatory elements based on transcriptomic profiling and epigenetic signatures for enhancer-derived RNAs and chromatin accessibility in immune tolerant monocyte states, and demonstrates the informativeness of linking such elements and eQTL to inform future mechanistic studies aimed at defining therapeutic targets of immunosuppression and diseases.

Mathematical models disentangle the role of IL-10 feedbacks in human monocytes upon proinflammatory activation.

Inflammation is one of the vital mechanisms through which the immune system responds to harmful stimuli. During inflammation, proinflammatory and anti-inflammatory cytokines interplay to orchestrate fine-tuned and dynamic immune responses. The cytokine interplay governs switches in the inflammatory response and dictates the propagation and development of the inflammatory response. Molecular pathways underlying the interplay are complex, and time-resolved monitoring of mediators and cytokines is necessary as a basis to study them in detail. Our understanding can be advanced by mathematical models that enable to analyze the system of interactions and their dynamical interplay in detail. We, therefore, used a mathematical modeling approach to study the interplay between prominent proinflammatory and anti-inflammatory cytokines with a focus on tumor necrosis factor and interleukin 10 (IL-10) in lipopolysaccharide-primed primary human monocytes. Relevant time-resolved data were generated by experimentally adding or blocking IL-10 at different time points. The model was successfully trained and could predict independent validation data and was further used to perform simulations to disentangle the role of IL-10 feedbacks during an acute inflammatory event. We used the insight to obtain a reduced predictive model including only the necessary IL-10-mediated feedbacks. Finally, the validated reduced model was used to predict early IL-10-tumor necrosis factor switches in the inflammatory response. Overall, we gained detailed insights into fine-tuning of inflammatory responses in human monocytes and present a model for further use in studying the complex and dynamic process of cytokine-regulated acute inflammation.

TLR8 escapes X chromosome inactivation in human monocytes and CD4+ T cells.

BACKGROUND: Human endosomal Toll-like receptors TLR7 and TLR8 recognize self and non-self RNA ligands, and are important mediators of innate immunity and autoimmune pathogenesis. TLR7 and TLR8 are, respectively, encoded by adjacent X-linked genes. We previously established that TLR7 evades X chromosome inactivation (XCI) in female immune cells. Whether TLR8 also evades XCI, however, has not yet been explored. METHOD: In the current study, we used RNA fluorescence in situ hybridization (RNA FISH) to directly visualize, on a single-cell basis, primary transcripts of TLR7 and TLR8 relative to X chromosome territories in CD14+ monocytes and CD4+ T lymphocytes from women, Klinefelter syndrome (KS) men, and euploid men. To assign X chromosome territories in cells lacking robust expression of a XIST compartment, we designed probes specific for X-linked genes that do not escape XCI and therefore robustly label the active X chromosome. We also assessed whether XCI escape of TLR8 was associated with sexual dimorphism in TLR8 protein expression by western blot and flow cytometry. RESULTS: Using RNA FISH, we show that TLR8, like TLR7, evades XCI in immune cells, and that cells harboring simultaneously TLR7 and TLR8 transcript foci are more frequent in women and KS men than in euploid men, resulting in a sevenfold difference in frequency. This transcriptional bias was again observable when comparing the single X of XY males with the active X of cells from females or KS males. Interestingly, TLR8 protein expression was significantly higher in female mononuclear blood cells, including all monocyte subsets, than in male cells. CONCLUSIONS: TLR8, mirroring TLR7, escapes XCI in human monocytes and CD4+ T cells. Co-dependent transcription from the active X chromosome and escape from XCI could both contribute to higher TLR8 protein abundance in female cells, which may have implications for the response to viruses and bacteria, and the risk of developing inflammatory and autoimmune diseases.

Human endosomal Toll-like receptors TLR7 and TLR8, encoded by two adjacent X-linked genes, recognize self and non-self RNA ligands, and are important mediators of innate immunity and autoimmune pathogenesis. We previously reported that TLR7 evades X chromosome inactivation (XCI) in female immune cells, correlating with enhanced functional properties in B cells harboring biallelic expression of this gene. Here, we conducted a comprehensive single-cell resolution analysis of the transcriptional regulation of both TLR7 and TLR8, in CD14⁺ monocytes and CD4⁺ T lymphocytes. We unequivocally demonstrated that TLR8, like TLR7, escapes XCI in immune cells from female and Klinefelter syndrome males. When we analyzed TLR7 and TLR8 transcripts together, cells from women and KS men exhibited higher frequencies of cells co-transcribing the two genes. Surprisingly, these differences were attributable not only to the ability of TLR7 and TLR8 to be expressed on the Xi, but also to the joint transcriptional behavior of the TLR7­TLR8 gene pair on the active X chromosome specifically. This contrasted with a striking pattern of mutually exclusive transcription on the single X of euploid men. Corroborating our RNA FISH results, we found higher TLR8 protein expression in female than in male leukocytes, including all monocyte subpopulations. In summary, our data suggest that sex-biased co-regulation of the Toll-like receptor locus and XCI escape of TLR8 contribute to the sexual dimorphism in TLR8 expression, which may have important consequences for the functional make-up of monocyte and T cell populations.

Posttranslational Modification of Human Cytochrome CYP4F11 by 4-Hydroxynonenal Impairs ω-Hydroxylation in Malaria Pigment Hemozoin-Fed Monocytes: The Role in Malaria Immunosuppression.

Malaria is a frequent parasitic infection becomes life threatening due to the disequilibrated immune responses of the host. Avid phagocytosis of malarial pigment hemozoin (HZ) and HZ-containing Plasmodium parasites incapacitates monocyte functions by bioactive lipoperoxidation products 4-hydroxynonenal (4-HNE) and hydroxyeicosatetraenoic acids (HETEs). CYP4F conjugation with 4-HNE is hypothesised to inhibit ω-hydroxylation of 15-HETE, leading to sustained monocyte dysfunction caused by 15-HETE accumulation. A combined immunochemical and mass-spectrometric approach identified 4-HNE-conjugated CYP4F11 in primary human HZ-laden and 4-HNE-treated monocytes. Six distinct 4-HNE-modified amino acid residues were revealed, of which C260 and H261 are localized in the substrate recognition site of CYP4F11. Functional consequences of enzyme modification were investigated on purified human CYP4F11. Palmitic acid, arachidonic acid, 12-HETE, and 15-HETE bound to unconjugated CYP4F11 with apparent dissociation constants of 52, 98, 38, and 73 µM, respectively, while in vitro conjugation with 4-HNE completely blocked substrate binding and enzymatic activity of CYP4F11. Gas chromatographic product profiles confirmed that unmodified CYP4F11 catalysed the ω-hydroxylation while 4-HNE-conjugated CYP4F11 did not. The 15-HETE dose dependently recapitulated the inhibition of the oxidative burst and dendritic cell differentiation by HZ. The inhibition of CYP4F11 by 4-HNE with consequent accumulation of 15-HETE is supposed to be a crucial step in immune suppression in monocytes and immune imbalance in malaria.

The Enigma of Lymphocyte Apoptosis in the Response to Influenza Virus Infection.

In the pathogenesis of influenza virus infection, lymphocyte apoptosis as a part of the infection and/or the immune response to the virus can be somewhat puzzling. The percentage of human T lymphocytes within the peripheral blood mononuclear cell population that becomes apoptotic greatly exceeds the percentage that are infected after exposure to the virus, consistent with substantial apoptosis of bystander T lymphocytes. Studies reveal an important role of viral neuraminidase expression by co-cultured monocyte/macrophages in induction of apoptosis, including that of uninfected bystander lymphocytes. Despite these observations, it is a reasonable perspective to recognize that the development of lymphocyte apoptosis during the response to infection does not preclude a successful immune response and recovery of the infected host in the great majority of cases. Further investigation is clearly warranted to understand its role in the pathogenesis of influenza virus infection for human subjects.

Concomitant inhibition of PPARγ and mTORC1 induces the differentiation of human monocytes into highly immunogenic dendritic cells.

Monocytes can differentiate into macrophages (Mo-Macs) or dendritic cells (Mo-DCs). The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) induces the differentiation of monocytes into Mo-Macs, while the combination of GM-CSF/interleukin (IL)-4 is widely used to generate Mo-DCs for clinical applications and to study human DC biology. Here, we report that pharmacological inhibition of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) in the presence of GM-CSF and the absence of IL-4 induces monocyte differentiation into Mo-DCs. Remarkably, we find that simultaneous inhibition of PPARγ and the nutrient sensor mammalian target of rapamycin complex 1 (mTORC1) induces the differentiation of Mo-DCs with stronger phenotypic stability, superior immunogenicity, and a transcriptional profile characterized by a strong type I interferon (IFN) signature, a lower expression of a large set of tolerogenic genes, and the differential expression of several transcription factors compared with GM-CSF/IL-4 Mo-DCs. Our findings uncover a pathway that tailors Mo-DC differentiation with potential implications in the fields of DC vaccination and cancer immunotherapy.

Silver nanoparticles exert toxic effects in human monocytes and macrophages associated with the disruption of Δψm and release of pro-inflammatory cytokines.

Silver nanoparticles (AgNP) are the most widely produced type of nanoparticles due to their antimicrobial and preservative properties. However, their systemic bioavailability may be considered a potential hazard. When AgNP reach the bloodstream, they interact with the immune cells, contributing to the onset and development of an inflammatory response. Monocytes and macrophages play a pivotal role in our defense system, but the interaction of AgNP with these cells is still not clear. Therefore, the main objective of this work was to assess the cytotoxic and pro-inflammatory effects induced by 5, 10, and 50 nm AgNP coated with polyvinylpyrrolidone (PVP) and citrate, in concentrations that could be attained in vivo (0-25 µg/mL), in human monocytes isolated from human blood and human macrophages derived from a monocytic cell line (THP-1). The effects of PVP and citrate-coated AgNP on cell viability, mitochondrial membrane potential, and cytokines release were evaluated. The results evidenced that AgNP exert strong harmful effects in both monocytes and macrophages, through the establishment of a strong pro-inflammatory response that culminates in cell death. The observed effects were dependent on the AgNP concentration, size and coating, being observed more pronounced cytotoxic effects with smaller PVP coated AgNP. The results showed that human monocytes seem to be more sensitive to AgNP exposure than human macrophages. Considering the increased daily use of AgNP, it is imperative to further explore the adverse outcomes and mechanistic pathways leading to AgNP-induced pro-inflammatory effects to deep insight into the molecular mechanism involved in this effect.

Upregulation of programmed cell death 1 by interferon gamma and its biological functions in human monocytes.

Programmed cell death 1 (PD-1) is a co-inhibitory checkpoint receptor expressed in various immune cells, especially in activated T cells. Engagement of PD-1 with its ligand leads to the exhausted T cells and impaired antitumor immunity. To date, PD-1 expression and its roles have been widely reported in T cells but not well defined in innate immune cells including monocytes. In this study, expression of PD-1 was investigated in human monocytes. Here we observed that among cytokines tested, IFN-γ significantly upregulated the PD-1 expression in both THP-1 cell line and human primary monocytes in a dose- and time-dependent manner. This effect was reduced by PI3K inhibitor, suggesting that the involvement of PI3K/AKT pathway. Furthermore, enrichment of active histone mark H3K4me3 in the Pdcd1 promotor was also observed in IFN-γ-induced THP-1, indicating that epigenetic regulation also plays a role in IFN-γ-induced PD-1 expression. To investigate the biological functions of PD-1, Pdcd1 was deleted in THP-1 cell line by CRISPR/Cas9 system and the phagocytic ability was investigated. The results showed that the PD-1 deficiency in THP-1 cell line resulted in significantly poor phagocytic potency against carboxylated-modified latex beads. Moreover, the PD-1 deficiency or blocking PD-1/PD-L1 interaction by immune checkpoint inhibitor resulted in an impaired induction of IL-4-induced CD163 expression in THP-1 cell line. Taken together, these results highlighted the importance of PD-1 expression in some of key monocyte functions.

A Pan-RNase Inhibitor Enabling CRISPR-mRNA Platforms for Engineering of Primary Human Monocytes.

Monocytes and their downstream effectors are critical components of the innate immune system. Monocytes are equipped with chemokine receptors, allowing them to migrate to various tissues, where they can differentiate into macrophage and dendritic cell subsets and participate in tissue homeostasis, infection, autoimmune disease, and cancer. Enabling genome engineering in monocytes and their effector cells will facilitate a myriad of applications for basic and translational research. Here, we demonstrate that CRISPR-Cas9 RNPs can be used for efficient gene knockout in primary human monocytes. In addition, we demonstrate that intracellular RNases are likely responsible for poor and heterogenous mRNA expression as incorporation of pan-RNase inhibitor allows efficient genome engineering following mRNA-based delivery of Cas9 and base editor enzymes. Moreover, we demonstrate that CRISPR-Cas9 combined with an rAAV vector DNA donor template mediates site-specific insertion and expression of a transgene in primary human monocytes. Finally, we demonstrate that SIRPa knock-out monocyte-derived macrophages have enhanced activity against cancer cells, highlighting the potential for application in cellular immunotherapies.

Intravenous Immunoglobulins Promote an Expansion of Monocytic Myeloid-Derived Suppressor Cells (MDSC) in CVID Patients.

Common variable immunodeficiency disorders (CVID), the most common primary immune deficiency, includes heterogeneous syndromes characterized by hypogammaglobulinemia and impaired antibody responses. CVID patients frequently suffer from recurrent infections and inflammatory conditions. Currently, immunoglobulin replacement therapy (IgRT) is the first-line treatment to prevent infections and aminorate immune alterations in CVID patients. Intravenous Immunoglobulin (IVIg), a preparation of highly purified poly-specific IgG, is used for treatment of immunodeficiencies as well as for autoimmune and inflammatory disorders, as IVIg exerts immunoregulatory and anti-inflammatory actions on innate and adaptive immune cells. To determine the mechanism of action of IVIg in CVID in vivo, we determined the effect of IVIg infusion on the transcriptome of peripheral blood mononuclear cells from CVID patients, and found that peripheral blood monocytes are primary targets of IVIg in vivo, and that IVIg triggers the acquisition of an anti-inflammatory gene profile in human monocytes. Moreover, IVIg altered the relative proportions of peripheral blood monocyte subsets and enhanced the proportion of CD14+ cells with a transcriptional, phenotypic, and functional profile that resembles that of monocytic myeloid-derived suppressor cells (MDSC). Therefore, our results indicate that CD14 + MDSC-like cells might contribute to the immunoregulatory effects of IVIg in CVID and other inflammatory disorders.

Trained immunity induction by the inactivated mucosal vaccine MV130 protects against experimental viral respiratory infections.

MV130 is an inactivated polybacterial mucosal vaccine that confers protection to patients against recurrent respiratory infections, including those of viral etiology. However, its mechanism of action remains poorly understood. Here, we find that intranasal prophylaxis with MV130 modulates the lung immune landscape and provides long-term heterologous protection against viral respiratory infections in mice. Intranasal administration of MV130 provides protection against systemic candidiasis in wild-type and Rag1-deficient mice lacking functional lymphocytes, indicative of innate immune-mediated protection. Moreover, pharmacological inhibition of trained immunity with metformin abrogates the protection conferred by MV130 against influenza A virus respiratory infection. MV130 induces reprogramming of both mouse bone marrow progenitor cells and in vitro human monocytes, promoting an enhanced cytokine production that relies on a metabolic shift. Our results unveil that the mucosal administration of a fully inactivated bacterial vaccine provides protection against viral infections by a mechanism associated with the induction of trained immunity.

Human monocytes respond to lipopolysaccharide (LPS) stimulation in a sex-dependent manner.

Monocytes play a critical role in inflammation and immune response, their activity being sex-dependent. However, the basis of sex differences is not well understood. Therefore, we investigated the lipopolysaccharide (LPS) effects on tumor necrosis factor-α (TNF-α) release, autophagy, and chemotaxis in freshly isolated monocytes from healthy young men and women. In basal conditions, male and female monocytes had similar TNF-α release, chemotaxis, and estrogen receptors (ER-α) and ER-ß expression, while the LC3II/I ratio was significantly higher in males. LPS treatment induced qualitative and quantitative sex differences. It reduced autophagy and increased TNF-α release only in male monocytes, while, chemotaxis was significantly influenced only in female cells. Moreover, it reduced the expression of ER-α only in female cells, while ER-ß expression was reduced in both sexes, but more markedly in female cells. Finally, the interplay between LPS treatment and 17-ß-estradiol (E2 ) was present only in female cells. Globally, these findings expand the concept that sex plays a role in regulating monocytes' functions, being sex differences cell- and parameter-specific.

Production of IL-6 and Phagocytosis Are the Most Resilient Immune Functions in Metabolically Compromised Human Monocytes.

At sites of inflammation, monocytes carry out specific immune functions while facing challenging metabolic restrictions. Here, we investigated the potential of human monocytes to adapt to conditions of gradually inhibited oxidative phosphorylation (OXPHOS) under glucose free conditions. We used myxothiazol, an inhibitor of mitochondrial respiration, to adjust two different levels of decreased mitochondrial ATP production. At these levels, and compared to uninhibited OXPHOS, we assessed phagocytosis, production of reactive oxygen species (ROS) through NADPH oxidase (NOX), expression of surface activation markers CD16, CD80, CD11b, HLA-DR, and production of the inflammatory cytokines IL-1ß, IL-6 and TNF-α in human monocytes. We found phagocytosis and the production of IL-6 to be least sensitive to metabolic restrictions while surface expression of CD11b, HLA-DR, production of TNF-α, IL-1ß and production of ROS through NOX were most compromised by inhibition of OXPHOS in the absence of glucose. Our data demonstrate a short-term hierarchy of immune functions in human monocytes, which represents novel knowledge potentially leading to the development of new therapeutics in monocyte-mediated inflammatory diseases.

A Two-Stage Flow Cytometry Strategy to Distinguish Single Cells from Doublets in Heterogeneous Cell Mixtures and Improve Cell Cluster Identification: Application to Human Monocyte Subpopulations.

Flow cytometry is a powerful method, widely used to identify cell types present in tissues, to describe their phenotypes, and to purify cells for functional analyses. As a single cell technique, flow cytometry relies on identifying and excluding cell doublets and aggregates present in samples in the initial gating steps. This identification is based on detection of events generating electrical pulses falling outside of linear variations of pulse height, width, and area in a singlet population with increasing cell sizes. In heterogeneous cell mixtures, however, with cell types varying extensively in size and granularity, exclusion of doublets has the risk of removing single cells that co-localize with doublets of another cell type. This is particularly the case when doublets of a smaller cell type overlap with large cells of a distinct, larger cell type. Here, we describe a gating method to reduce this risk. In this protocol, initial gating steps aim to segregate cells according to physical characteristics (such as size and granularity) and gene expression properties in order to obtain more homogeneous cell clusters. Doublet exclusion is then performed separately in each cluster, minimizing the risk of confusion between single cells and doublets. To illustrate this protocol, human blood monocytes are separated and analyzed. By implementing this protocol, we were able to reveal the existence of a population of large monocytes previously unrecognized using conventional gating strategies. In subsequent functional assays, we have shown that this novel population exhibits unique inflammatory responses, highlighting the need and pertinence of this approach to identify and characterize infrequent-yet functionally relevant-cell populations present in complex cell mixtures. © 2021 Wiley Periodicals LLC. Basic Protocol: Distinguishing single cells from doublets in heterogeneous cell mixtures by flow cytometry.