Approaches for studying human macrophages.

Macrophages are vital tissue components involved in organogenesis, maintaining homeostasis, and responses to disease. Mouse models have significantly improved our understanding of macrophages. Further investigations into the characteristics and development of human macrophages are crucial, considering the substantial anatomical and physiological distinctions between mice and humans. Despite challenges in human macrophage research, recent studies are shedding light on the ontogeny and function of human macrophages. In this opinion, we propose combinations of cutting-edge approaches to examine the diversity, development, niche, and function of human tissue-resident macrophages. These methodologies can facilitate our exploration of human macrophages more efficiently, ideally providing new therapeutic avenues for macrophage-relevant disorders.

Application of a genetically engineered macrophage cell line for evaluating cellular effects of UV/US-treated poly(ethylene terephthalate) microplastics.

Microplastic (MP) pollution is a global environmental problem. To understand the biological effects of MPs on humans, it is essential to evaluate the response of human cells to model plastic particles that mimic environmental MPs in a sensitive and non-invasive manner. In this study, we investigated the preparation of poly(ethylene terephthalate) (PET) fragments with properties similar to those of environmental MPs by combining photo-oxidative degradation via ultraviolet (UV) irradiation with mechanical pulverization and hydrolysis via ultrasound (US) exposure. Combination of UV and US treatments decreased the particle size of PET fragments to 10.2 µm and increased their crystallinity and Young's modulus to 35.7 % and 0.73 GPa, respectively, while untreated PET fragments showed the particle size of 18.9 µm, the crystallinity of 33.7 %, and Young's modulus of 0.48 GPa. In addition, an increase in negative surface potential and O/C ratio were observed for UV/US-treated PET fragments, suggesting surface oxidation via UV/US treatment. Cytokine secretion from human macrophages was evaluated by a highly sensitive inflammation evaluation system using the HiBiT-based chemiluminescence detection method developed by genome editing technology. UV/US-treated PET fragments induced a 1.4 times higher level of inflammatory cytokine secretion on inflammatory macrophages than untreated ones, suggesting that the biological responses of PET fragments could be influenced by changes in material properties via oxidation. In conclusion, UV/US treatment enables efficient preparation of model plastic particles and is expected to provide new insights into the evaluation of biological effects using human cells. (240 words).

Near-Infrared Light Exposure Triggers ROS to Downregulate Inflammatory Cytokines Induced by SARS-CoV-2 Spike Protein in Human Cell Culture.

The leading cause of mortality from SARS-CoV-2 is an exaggerated host immune response, triggering cytokine storms, multiple organ failure and death. Current drug- and vaccine-based therapies are of limited efficacy against novel viral variants. Infrared therapy is a non-invasive and safe method that has proven effective against inflammatory conditions for over 100 years. However, its mechanism of action is poorly understood and has not received widespread acceptance. We herein investigate whether near-infrared (NIR) light exposure in human primary alveolar and macrophage cells could downregulate inflammatory cytokines triggered by the SARS-CoV-2 spike (S) protein or lipopolysaccharide (LPS), and via what underlying mechanism. Our results showed a dramatic reduction in pro-inflammatory cytokines within days of NIR light treatment, while anti-inflammatory cytokines were upregulated. Mechanistically, NIR light stimulated mitochondrial metabolism, induced transient bursts in reactive oxygen species (ROS) and activated antioxidant gene transcription. These, in turn, downregulated ROS and inflammatory cytokines. A causal relationship was shown between the induction of cellular ROS by NIR light exposure and the downregulation of inflammatory cytokines triggered by SARS-CoV-2 S. If confirmed by clinical trials, this method would provide an immediate defense against novel SARS-CoV-2 variants and other inflammatory infectious diseases.

Cross-talk between B cells, microglia and macrophages, and implications to central nervous system compartmentalized inflammation and progressive multiple sclerosis.

BACKGROUND: B cells can be enriched within meningeal immune-cell aggregates of multiple sclerosis (MS) patients, adjacent to subpial cortical demyelinating lesions now recognized as important contributors to progressive disease. This subpial demyelination is notable for a 'surface-in' gradient of neuronal loss and microglial activation, potentially reflecting the effects of soluble factors secreted into the CSF. We previously demonstrated that MS B-cell secreted products are toxic to oligodendrocytes and neurons. The potential for B-cell-myeloid cell interactions to propagate progressive MS is of considerable interest. METHODS: Secreted products of MS-implicated pro-inflammatory effector B cells or IL-10-expressing B cells with regulatory potential were applied to human brain-derived microglia or monocyte-derived macrophages, with subsequent assessment of myeloid phenotype and function through measurement of their expression of pro-inflammatory, anti-inflammatory and homeostatic/quiescent molecules, and phagocytosis (using flow cytometry, ELISA and fluorescently-labeled myelin). Effects of secreted products of differentially activated microglia on B-cell survival and activation were further studied. FINDINGS: Secreted products of MS-implicated pro-inflammatory B cells (but not IL-10 expressing B cells) substantially induce pro-inflammatory cytokine (IL-12, IL-6, TNFα) expression by both human microglia and macrophage (in a GM-CSF dependent manner), while down-regulating their expression of IL-10 and of quiescence-associated molecules, and suppressing their myelin phagocytosis. In contrast, secreted products of IL-10 expressing B cells upregulate both human microglia and macrophage expression of quiescence-associated molecules and enhance their myelin phagocytosis. Secreted factors from pro-inflammatory microglia enhance B-cell activation. INTERPRETATION: Potential cross-talk between disease-relevant human B-cell subsets and both resident CNS microglia and infiltrating macrophages may propagate CNS-compartmentalized inflammation and injury associated with MS disease progression. These interaction represents an attractive therapeutic target for agents such as Bruton's tyrosine kinase inhibitors (BTKi) that modulate responses of both B cells and myeloid cells. FUNDING: Stated in Acknowledgments section of manuscript.

Protective Effect of Glycomacropeptide on the Inflammatory Response of U937 Macrophages.

Macrophages play crucial roles in inflammation and oxidative stress associated with noncommunicable diseases, such as cardiovascular diseases, diabetes, and cancer. Glycomacropeptide (GMP) is a bioactive peptide derived from milk κ-casein that contains abundant sialic acid and has shown anti-inflammatory, antioxidative, anti-obesity, and anti-diabetic properties when is orally administered. The aim of this study was to evaluate the effect of GMP on the regulation of the inflammatory response in human macrophages and the participation of sialic acid in this activity. GMP pretreatment decreased by 35%, 35%, and 49% the production of nitrites, interleukin (IL)-1ß, and tumor necrosis factor (TNF)-α, respectively, in activated human macrophages U937. The same effect was obtained when cells were pretreated with asialo GMP, and no change on the gene expression of the lectins associated with the recognition of sialic acids, SIGLEC5, 7, and 9, was induced by GMP on macrophages, which suggests that sialic acid might not be involved in this immunoregulatory effect. Interestingly, GMP increased 8.9- and 3.5-fold the gene expression of the canonical anti-inflammatory protein SOCS3 and the antioxidant enzyme HMOX1, respectively, in U937 cells. Thus, GMP exerts anti-inflammatory and antioxidative activities on activated macrophages in a sialic acid-independent manner, which might be related to its in vivo reported bioactivity.

Isolation, characterization, and functional study of extracellular vesicles derived from Leishmania tarentolae.

Leishmania (L.) species are protozoan parasites with a complex life cycle consisting of a number of developmental forms that alternate between the sand fly vector and their host. The non-pathogenic species L. tarentolae is not able to induce an active infection in a human host. It has been observed that, in pathogenic species, extracellular vesicles (EVs) could exacerbate the infection. However, so far, there is no report on the identification, isolation, and characterization of L. tarentolae EVs. In this study, we have isolated and characterized EVs from L. tarentolae GFP+ (tEVs) along with L. major GFP+ as a reference and positive control. The EVs secreted by these two species demonstrated similar particle size distribution (approximately 200 nm) in scanning electron microscopy and nanoparticle tracking analysis. Moreover, the said EVs showed similar protein content, and GFP and GP63 proteins were detected in both using dot blot analysis. Furthermore, we could detect Leishmania-derived GP63 protein in THP-1 cells treated with tEVs. Interestingly, we observed a significant increase in the production of IFN-γ, TNF-α, and IL-1ß, while there were no significant differences in IL-6 levels in THP-1 cells treated with tEVs following an infection with L. major compared with another group of macrophages that were treated with L. major EVs prior to the infection. Another exciting observation of this study was a significant decrease in parasite load in tEV-treated Leishmania-infected macrophages. In addition, in comparison with another group of Leishmania-infected macrophages which was not exposed to any EVs, tEV managed to increase IFN-γ and decrease IL-6 and the parasite burden. In conclusion, we report for the first time that L. tarentolae can release EVs and provide evidence that tEVs are able to control the infection in human macrophages, making them a great potential platform for drug delivery, at least for parasitic infections.

Exposure to polystyrene nanoplastics impairs lipid metabolism in human and murine macrophages in vitro.

The use of polystyrene micro and nanoplastics in cosmetics and personal care products continues to grow every day. The harmful effects of their biological accumulation in organisms of all trophic levels including humans have been reported by several studies. While we have accumulating evidence on the impact of nanoplastics on different organ systems in humans, only a handful of reports on the impact of polystyrene nanoplastics upon direct contact with the immune system at the cellular level are avialable. The present study offers significant evidence on the cell-specific harmful impact of sulfate-modified nanoplastics (S-NPs) on human macrophages. Here we report that exposure of human macrophages to S-NPs (100 µg/mL) stimulated the accumulation of lipids droplets (LDs) in the cytoplasm resulting in the differentiation of macrophages into foam cells. The observed effect was specific for human and murine macrophages but not for other cell types, especially human keratinocytes, liver, and lung cell models. Furthermore, we found that S-NPs mediated LDs accumulation in human macrophages was accompanied by acute mitochondrial oxidative stress. The accumulated LDs were further delivered and accumulated into lysosomes leading to impaired lysosomal clearance. In conclusion, our study reveals that exposure to polystyrene nanoplastics stabilized with anionic surfactants can be a potent stimulus for dysregulation of lipid metabolism and macrophage foam cell formation, a characteristic feature observed during atherosclerosis posing a serious threat to human health.

IL-23 production in human macrophages is regulated negatively by tumor necrosis factor α-induced protein 3 and positively by specificity protein 1 after stimulation of the toll-like receptor 7/8 signaling pathway.

The IL-23/IL-17 axis plays an important role in the development of autoimmune diseases, but the mechanism regulating IL-23 production is mainly unknown. We investigated how TNFAIP3 and Sp1 affect IL-23 production by human macrophages after exposure to resiquimod, a TLR7/8 agonist. IL-23 production was significantly upregulated by resiquimod but only slightly by LPS (a TLR4 agonist). Interestingly, IL-23 levels were significantly attenuated after sequential stimulation with LPS and resiquimod, but IL-12p40 and IL-18 levels were not. TLR4-related factors induced by LPS may regulate IL-23 expression via TLR7/8 signaling. LPS significantly enhanced TNFAIP3 and IRAK-M levels but reduced Sp1 levels. After exposure to resiquimod, RNA interference of TNFAIP3 upregulated IL-23 significantly more than siRNA transfection of IRAK-M did. In contrast, knockdown of Sp1 by RNA interference significantly attenuated IL-23 production. Transfection with siRNA for TNFAIP3 enhanced IL-23 expression significantly. After stimulation with resiquimod, GW7647-an agonist for PPARα (an inducer of NADHP oxidase)-and siRNA for UCP2 (a negative regulator of mitochondrial ROS generation) enhanced TNFAIP3 and reduced IL-23. siRNA for p22phox and gp91phox slightly increased Sp1 levels. However, after exposure to resiquimod siRNA-mediated knockout of DUOX1/2 significantly enhanced Sp1 and IL-23 levels, and decreased TNFα-dependent COX-2 expression. Concomitantly, TNFAIP3 levels was attenuated by DUOX1/2 siRNA. TNFAIP3 and Sp1 levels are reciprocally regulated through ROS generation. In conclusion, after stimulation of the TLR7/8 signaling pathway IL-23 production in human macrophages is regulated negatively by TNFAIP3.

Graphene Particles Interfere with Pro-Inflammatory Polarization of Human Macrophages: Functional and Electrophysiological Evidence.

The interaction of two types of fragmented graphene particles (30-160 nm) with human macrophages is studied. Since macrophages have significant phagocytic activity, the incorporation of graphene particles into cells has an effect on the response to functional polarization stimuli, favoring an anti-inflammatory profile. Incubation of macrophages with graphene foam particles, prepared by chemical vapor deposition, and commercially available graphene nanoplatelet particles does not affect cell viability when added at concentrations up to 100 µg mL-1 ; macrophages exhibit differential quantitative responses to each type of graphene particles. Although both materials elicit similar increases in the release of reactive oxygen species, the impact on the transcriptional regulation associated with the polarization profile is different; graphene nanoplatelets significantly modify this transcriptomic profile. Moreover, these graphene particles differentially affect the motility and phagocytosis of macrophages. After the incorporation of both graphene types into the macrophages, they exhibit specific responses in terms of the mitochondrial oxygen consumption and electrophysiological potassium currents at the cell plasma membrane. These data support the view that the physical structure of the graphene particles has an impact on human macrophage responses, paving the way for the development of new mechanisms to modulate the activity of the immune system.

SARS-CoV-2 Isolates Show Impaired Replication in Human Immune Cells but Differential Ability to Replicate and Induce Innate Immunity in Lung Epithelial Cells.

The primary target organ of coronavirus disease 2019 (COVID-19) infection is the respiratory tract. Currently, there is limited information on the ability of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to infect and regulate innate immunity in human immune cells and lung epithelial cells. Here, we compared the ability of four Finnish isolates of SARS-CoV-2 from COVID-19 patients to replicate and induce interferons (IFNs) and other cytokines in different human cells. All isolates failed to replicate in dendritic cells, macrophages, monocytes, and lymphocytes, and no induction of cytokine gene expression was seen. However, most of the isolates replicated in Calu-3 cells, and they readily induced type I and type III IFN gene expression. The hCoV-19/Finland/FIN-25/2020 isolate, originating from a traveler from Milan in March 2020, showed better ability to replicate and induce IFN and inflammatory responses in Calu-3 cells than other isolates of SARS-CoV-2. Our data increase the knowledge on the pathogenesis and antiviral mechanisms of SARS-CoV-2 infection in human cell systems. IMPORTANCE With the rapid spread of the coronavirus disease 2019 (COVID-19) pandemic, information on the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and regulation of innate immunity in human immune cells and lung epithelial cells is needed. In the present study, we show that SARS-CoV-2 failed to productively infect human immune cells, but different isolates of SARS-CoV-2 showed differential ability to replicate and regulate innate interferon responses in human lung epithelial Calu-3 cells. These findings will open up the way for further studies on the mechanisms of pathogenesis of SARS-CoV-2 in human cells.

Innate immune activation by checkpoint inhibition in human patient-derived lung cancer tissues.

Although Pembrolizumab-based immunotherapy has significantly improved lung cancer patient survival, many patients show variable efficacy and resistance development. A better understanding of the drug's action is needed to improve patient outcomes. Functional heterogeneity of the tumor microenvironment (TME) is crucial to modulating drug resistance; understanding of individual patients' TME that impacts drug response is hampered by lack of appropriate models. Lung organotypic tissue slice cultures (OTC) with patients' native TME procured from primary and brain-metastasized (BM) non-small cell lung cancer (NSCLC) patients were treated with Pembrolizumab and/or beta-glucan (WGP, an innate immune activator). Metabolic tracing with 13C6-Glc/13C5,15N2-Gln, multiplex immunofluorescence, and digital spatial profiling (DSP) were employed to interrogate metabolic and functional responses to Pembrolizumab and/or WGP. Primary and BM PD-1+ lung cancer OTC responded to Pembrolizumab and Pembrolizumab + WGP treatments, respectively. Pembrolizumab activated innate immune metabolism and functions in primary OTC, which were accompanied by tissue damage. DSP analysis indicated an overall decrease in immunosuppressive macrophages and T cells but revealed microheterogeneity in immune responses and tissue damage. Two TMEs with altered cancer cell properties showed resistance. Pembrolizumab or WGP alone had negligible effects on BM-lung cancer OTC but Pembrolizumab + WGP blocked central metabolism with increased pro-inflammatory effector release and tissue damage. In-depth metabolic analysis and multiplex TME imaging of lung cancer OTC demonstrated overall innate immune activation by Pembrolizumab but heterogeneous responses in the native TME of a patient with primary NSCLC. Metabolic and functional analysis also revealed synergistic action of Pembrolizumab and WGP in OTC of metastatic NSCLC.

Vitamin D and Beta-Glucans Synergically Stimulate Human Macrophage Activity.

Vitamin D and beta-glucans are both immunostimulants. Vitamin D exerts its beneficial effects on many components of the immune system. In macrophages, the hormone modulates both phagocytic activity and cytokine production; therefore, it plays an important role in mediating the innate immune response to infection. The immunomodulatory properties of beta-glucans are attributed to the ability of these fungal cell wall polysaccharides to bind to different receptors expressed on the cell surface of phagocytic and cytotoxic innate immune cells, including monocytes and macrophages. The intracellular signaling pathways activated by beta-glucans lead to enhanced phagocytosis and cytokine response. In this study we investigated the possible potentiation of immunomodulatory properties of the combined treatment with vitamin D and beta-glucans. The effects of 100 nM 1,25-dihydroxyvitamin D3 or 100 µg/mL beta-glucans were evaluated in human macrophages in terms of cytokine production, intracellular vesicle acidification and changes in energy metabolism, three hallmarks of macrophage antimicrobial activation. We found that all the analyzed parameters were enhanced by the co-treatment compared to the response to single molecules. The results of this study support the validity of a novel therapeutic approach that could boost the immune response, taking advantage of the synergy between two natural compounds.

Biocompatible valproic acid-coupled nanoparticles attenuate lipopolysaccharide-induced inflammation.

Inflammatory diseases like sepsis are associated with dysregulated gene expression, often caused by an imbalance of epigenetic regulators, such as histone acetyltransferases (HATs) and histone deacetylases (HDACs), and consequently, altered epigenetic chromatin signatures or aberrant posttranslational modifications of signalling proteins and transcription factors. Thus, HDAC inhibitors (HDACi) are a promising class of anti-inflammatory drugs. Recently, an efficient drug delivery system carrying the class I/IIa selective HDACi valproic acid (VPA) was developed to circumvent common disadvantages of free drug administration, e.g. short half-life and side effects. The cellulose-based sulphated VPA-coupled (CV-S) nanoparticles (NPs) are rapidly taken up by cells, do not cause any toxic effects and are fully biocompatible. Importantly, VPA is intracellularly cleaved from the NPs and HDACi activity could be proven. Here, we demonstrate that CV-S NPs exhibit overall anti-inflammatory effects in primary human macrophages and are able to attenuate the lipopolysaccharide-induced inflammatory response. CV-S NPs show superior potential to free VPA to suppress the TLR-MyD88-NF-κB signalling axis, leading to decreased TNF-α expression and secretion.

Encephalitozoon intestinalis Infection Impacts the Expression of Apoptosis-Related Genes in U937 Macrophage Cells.

PURPOSE: Encephalitozoon intestinalis affects many physiological processes of host cells to survive, proliferate, and spread to different regions within the body. In this study, the effects of the parasite on host cell apoptosis and proliferation were investigated. METHODS: To determine the impact of the parasite on the host cell apoptosis, changes in the expression profile of genes were investigated with the qPCR array using the Human Apoptosis Panel in infected and non-infected macrophage cells. Also, the rate of apoptosis in the cells was determined by Giemsa staining method. Cell proliferation was determined by measuring the DNA concentration in infected and non-infected cells. RESULTS: The thirty-six of apoptosis-related genes were down-regulated, while 20 of apoptosis-related genes were up-regulated in infected cells compared to uninfected cells. However, there were no significant changes detected in 32 analyzed genes between infected and control groups. E. intestinalis was determined to decrease cell proliferation in U937 macrophage cells. Unexpectedly, Giemsa staining showed an increase in the rate of apoptosis in infected cells. CONCLUSION: Regulated genes after infection are involved in many different biological pathways and various components of the cell. This suggests that the parasite uses highly sophisticated ways to maintain the viability of the cell.

Variations in Antimicrobial Activities of Human Monocyte-Derived Macrophage and Their Associations With Tuberculosis Clinical Manifestations.

Macrophages play a significant role in preventing infection through antimicrobial activities, particularly acidification, and proteolysis. Mycobacterium tuberculosis (Mtb) infection can lead to diverse outcomes, from latent asymptomatic infection to active disease involving multiple organs. Monocyte-derived macrophage is one of the main cell types accumulating in lungs following Mtb infection. The variation of intracellular activities of monocyte-derived macrophages in humans and the influence of these activities on the tuberculosis (TB) spectrum are not well understood. By exploiting ligand-specific bead-based assays, we investigated macrophage antimicrobial activities real-time in healthy volunteers (n = 53) with 35 cases of latent TB (LTB), and those with active TB (ATB), and either pulmonary TB (PTB, n = 70) or TB meningitis (TBM, n = 77). We found wide person-to-person variations in acidification and proteolytic activities in response to both non-immunogenic IgG and pathogenic ligands comprising trehalose 6,6'-dimycolate (TDM) from Mtb or ß-glucan from Saccharamyces cerevisiase. The variation in the macrophage activities remained similar regardless of stimuli; however, IgG induced stronger acidification activity than immunogenic ligands TDM (P = 10-5, 3 × 10-5 and 0.01 at 30, 60, and 90 min) and ß-glucan (P = 10-4, 3 × 10-4 and 0.04 at 30, 60, and 90 min). Variation in proteolysis activity was slightly higher in LTB than in ATB (CV = 40% in LTB vs. 29% in ATB, P = 0.03). There was no difference in measured antimicrobial activities in response to TDM and bacterial killing in macrophages from LTB and ATB, or from PTB and TBM. Our results indicate that antimicrobial activities of monocyte-derived macrophages vary among individuals and show immunological dependence, but suggest these activities cannot be solely responsible for the control of bacterial replication or dissemination in TB.

Exosomes released from Shiga toxin 2a-treated human macrophages modulate inflammatory responses and induce cell death in toxin receptor expressing human cells.

Shiga toxins (Stxs) produced by Stx-producing Escherichia coli are the primarily virulence factors of hemolytic uremic syndrome and central nervous system (CNS) impairment. Although the precise mechanisms of toxin dissemination remain unclear, Stxs bind to extracellular vesicles (EVs). Exosomes, a subset of EVs, may play a key role in Stx-mediated renal injury. To test this hypothesis, we isolated exosomes from monocyte-derived macrophages in the presence of Stx2a or Stx2 toxoids. Macrophage-like differentiated THP-1 cells treated with Stxs secreted Stx-associated exosomes (Stx-Exo) of 90-130 nm in diameter, which induced cytotoxicity in recipient cells in a toxin receptor globotriaosylceramide (Gb3 )-dependent manner. Stx2-Exo engulfed by Gb3 -positive cells were translocated to the endoplasmic reticulum in the human proximal tubule epithelial cell line HK-2. Stx2-Exo contained pro-inflammatory cytokine mRNAs and proteins and induced more severe inflammation than purified Stx2a accompanied by greater death of target cells such as human renal or retinal pigment epithelial cells. Blockade of exosome biogenesis using the pharmacological inhibitor GW4869 reduced Stx2-Exo-mediated human renal cell death. Stx2-Exo isolated from human primary monocyte-derived macrophages activated caspase 3/7 and resulted in significant cell death in primary human renal cortical epithelial cells. Based on these results, we speculate that Stx-containing exosomes derived from macrophages may exacerbate cytotoxicity and inflammation and trigger cell death in toxin-sensitive cells. Therapeutic interventions targeting Stx-containing exosomes may prevent or ameliorate Stx-mediated acute vascular dysfunction.

Generation and characterization of U937-TR: a platform cell line for inducible gene expression in human macrophages.

Monocytes and macrophages are involved in a wide range of biological processes and parasitic diseases. The characterization of the molecular mechanisms governing such processes usually requires precise control of the expression of genes of interest. We implemented a tetracycline-controlled gene expression system in the U937 cell line, one of the most used in vitro models for the research of human monocytes and macrophages. Here we characterized U937-derived cell lines in terms of phenotypic (morphology and marker expression) and functional (capacity for phagocytosis and for Leishmania parasite hosting) changes induced by phorbol-12-myristate-13-acetate (PMA). Finally, we provide evidence of tetracycline-inducible and reversible Lamin-A gene silencing of the PMA-differentiated U937-derived cells.

The rough colony morphotype of Mycobacterium avium exhibits high virulence in human macrophages and mice.

Introduction. The incidence of Mycobacterium avium complex (MAC) pulmonary disease (MAC PD), a refractory chronic respiratory tract infection, is increasing worldwide. MAC has three predominant colony morphotypes: smooth opaque (SmO), smooth transparent (SmT) and rough (Rg).Aim. To determine whether colony morphotypes can predict the prognosis of MAC PD, we evaluated the virulence of SmO, SmT and Rg in mice and in human macrophages.Methodology. We compared the characteristics of mice and human macrophages infected with the SmO, SmT, or Rg morphotypes of M. avium subsp. hominissuis 104. C57BL/6 mice and human macrophages derived from peripheral mononuclear cells were used in these experiments.Results. In comparison to SmO- or SmT-infected mice, Rg-infected mice revealed severe pathologically confirmed pneumonia, increased lung weight and increased lung bacterial burden. Rg-infected macrophages revealed significant cytotoxicity, increased bacterial burden, secretion of proinflammatory cytokines (TNF-α and IL-6) and chemokines (CCL5 and CCL3), and formation of cell clusters. Rg formed larger bacterial aggregates than SmO and SmT. Cytotoxicity, bacterial burden and secretion of IL-6, CCL5 and CCL3 were induced strongly by Rg infection, and were decreased by disaggregation of the bacteria.Conclusion. M. avium Rg, which is associated with bacterial aggregation, has the highest virulence among the predominant colony morphotypes.

The Two Faces of Tumor-Associated Macrophages and Their Clinical Significance in Colorectal Cancer.

Macrophages are one of the immune populations frequently found in colorectal tumors and high macrophage infiltration has been associated with both better and worst prognosis. Importantly, according to microenvironment stimuli, macrophages may adopt different polarization profiles, specifically the pro-inflammatory or M1 and the anti-inflammatory or M2, which display distinct functions. Therefore, concomitantly with the number of tumor-associated macrophages (TAMs), their characterization is fundamental to unravel their relevance in cancer. Here, we profiled macrophages in a series of 150 colorectal cancer (CRC) cases by immunohistochemistry, using CD68 as a macrophage lineage marker, CD80 as a marker of pro-inflammatory macrophages, and CD163 as a marker of anti-inflammatory macrophages. Quantifications were performed by computer-assisted analysis in the intratumoral region, tumor invasive front, and matched tumor adjacent normal mucosa (ANM). Macrophages, specifically the CD163+ ones, were predominantly found at the tumor invasive front, whereas CD80+ macrophages were almost exclusively located in the ANM, which suggests a predominant anti-inflammatory polarization of TAMs. Stratification according to tumor stage revealed that macrophages, specifically the CD163+ ones, are more prevalent in stage II tumors, whereas CD80+ macrophages are predominant in less invasive T1 tumors. Specifically in stage III tumors, higher CD68, and lower CD80/CD163 ratio associated with decreased overall survival. Importantly, despite the low infiltration of CD80+ cells in colorectal tumors, multivariate logistic regression revealed a protective role of these cells regarding the risk for relapse. Overall, this work supports the involvement of distinct microenvironments, present at the intra-tumor, invasive front and ANM regions, on macrophage modulation, and uncovers their prognostic value, further supporting the relevance of including macrophage profiling in clinical settings.

Retention of EsxA in the Capsule-Like Layer of Mycobacterium tuberculosis Is Associated with Cytotoxicity and Is Counteracted by Lung Surfactant.

Mycobacterium tuberculosis, the pathogen that causes tuberculosis, primarily infects macrophages but withstands the host cell's bactericidal effects. EsxA, also called virulence factor 6-kDa early secretory antigenic target (ESAT-6), is involved in phagosomal rupture and cell death. We provide confocal and electron microscopy data showing that M. tuberculosis bacteria grown without detergent retain EsxA on their surface. Lung surfactant has detergent-like properties and effectively strips off this surface-associated EsxA, which advocates a novel mechanism of lung surfactant-mediated defense against pathogens. Upon challenge of human macrophages with these M. tuberculosis bacilli, the amount of surface-associated EsxA rapidly declines in a phagocytosis-independent manner. Furthermore, M. tuberculosis bacteria cultivated under exclusion of detergent exert potent cytotoxic activity associated with bacterial growth. Together, this study suggests that the surface retention of EsxA contributes to the cytotoxicity of M. tuberculosis and highlights how cultivation conditions affect the experimental outcome.