Cultures Of Human Macrophages Research Articles

Some new insights into the biological activities of carboxymethylated polysaccharides from Lasiodiplodia theobromae

BackgroundCarboxymethylated Lasiodiplodan (LaEPS-C), Lasiodiplodia theobromae β-glucan exopolysaccharide derivative, has a well-known range of biological activities. Compared to LaEPS-C, its fractions, Linear (LLaEPS-C) and Branched (BLaEPS-C), have biological potentialities scarcely described in the literature. So, in this study, we investigate the immunomodulatory, antiviral, antiproliferative, and anticoagulant activities of LLaEPS-C and BLaEPS-C and compare them to the LaEPS-C.MethodsLaEPS was obtained from L. theobromae MMBJ. After carboxymethylation, LaEPS-C structural characteristics were confirmed by Elementary Composition Analysis by Energy Dispersive X-Ray Detector (EDS), Fourier Transform Infrared (FTIR), and Nuclear Magnetic Resonance (NMR). The immunomodulatory activity on cytokine secretion was evaluated in human monocyte-derived macrophage cultures. The antiviral activity was evaluated by Hep-2 cell viability in the presence or absence of hRSV (human respiratory syncytial virus). In vitro antiproliferative activity was tested by sulforhodamine B assay. The anticoagulant activity was determined by APTT (Activated Partial Thromboplastin Time) and PT (Prothrombin Time).ResultsLaEPS-C showed low macrophage cell viability only at 100 µg/mL (52.84 ± 24.06, 48 h), and LLaEPS-C presented no effect. Conversely, BLaEPS-C showed cytotoxicity from 25 to 100 µg/mL (44.36 ± 20.16, 40.64 ± 25.55, 33.87 ± 25.16; 48 h). LaEPS-C and LLaEPS-C showed anti-inflammatory activity. LaEPS-C presented this at 100 µg/mL (36.75 ± 5.53, 48 h) for IL-10, and LLaEPS-C reduces TNF-α cytokine productions at 100 µg/mL (18.27 ± 5.80, 48 h). LLaEPS-C showed an anti-hRSV activity (0.7 µg/ml) plus a low cytotoxic activity for Hep-2 cells (1.4 µg/ml). LaEPS-C presented an antiproliferative activity for NCI-ADR/RES (GI50 65.3 µg/mL). A better PT was achieved for LLaEPS-C at 5.0 µg/mL (11.85 ± 0.87s).ConclusionsThese findings demonstrated that carboxymethylation effectively improves the biological potential of the LaEPS-C and their fractions. From those polysaccharides tested, LLaEPS provided the best results with low toxicity for anti-inflammatory, antiviral, and anticoagulant activities.

An in vitro model for osteoarthritis using long-cultured inflammatory human macrophages repeatedly stimulated with TLR agonists.

Osteoarthritis (OA) is characterized by an abundance of inflammatory M1-like macrophages damaging local tissues. The search for new potential drugs for OA suffers from the lack of appropriate methods of long-lasting inflammation. Here we developed and characterized an in vitro protocol of long-lasting culture of primary human monocyte-derived macrophages differentiated with a combination of M-CSF+GM-CSF that optimally supported long-cultured macrophages (LC-Mϕs) for up to 15 days, unlike their single use. Macrophages repeatedly stimulated for 15 days with the TLR2 ligand Pam3CSK4 (LCS-Mϕs), showed sustained levels over time of IL-6, CCL2, and CXCL8, inflammatory mediators that were also detected in the synovial fluids of OA patients. Furthermore, macrophages isolated from the synovia of two OA patients showed an expression profile of inflammation-related genes similar to that of LCS-Mϕs, validating our protocol as a model of chronically activated inflammatory macrophages. Next, to confirm that these LCS-Mϕs could be modulated by anti-inflammatory compounds, we employed dexamethasone and/or celecoxib, two drugs widely used in OA treatment, that significantly inhibited the production of inflammatory mediators. This easy-to-use in vitro protocol of long-lasting inflammation with primary human macrophages could be useful for the screening of new compounds to improve the therapy of inflammatory disorders.

Immunomodulatory effects of new phytotherapy on human macrophages and TLR4- and TLR7/8-mediated viral-like inflammation in mice.

BackgroundWhile all efforts have been undertaken to propagate the vaccination and develop remedies against SARS-CoV-2, no satisfactory management of this infection is available yet. Moreover, poor availability of any preventive and treatment measures of SARS-CoV-2 in economically disadvantageous communities aggravates the course of the pandemic. Here, we studied a new immunomodulatory phytotherapy (IP), an extract of blackberry, chamomile, garlic, cloves, and elderberry as a potential low-cost solution for these problems given the reported efficacy of herbal medicine during the previous SARS virus outbreak.MethodsThe key feature of SARS-CoV-2 infection, excessive inflammation, was studied in in vitro and in vivo assays under the application of the IP. First, changes in tumor-necrosis factor (TNF) and lnteurleukin-1 beta (IL-1β) concentrations were measured in a culture of human macrophages following the lipopolysaccharide (LPS) challenge and treatment with IP or prednisolone. Second, chronically IP-pre-treated CD-1 mice received an agonist of Toll-like receptors (TLR)-7/8 resiquimod and were examined for lung and spleen expression of pro-inflammatory cytokines and blood formula. Finally, chronically IP-pre-treated mice challenged with LPS injection were studied for “sickness” behavior. Additionally, the IP was analyzed using high-potency-liquid chromatography (HPLC)-high-resolution-mass-spectrometry (HRMS).ResultsLPS-induced in vitro release of TNF and IL-1β was reduced by both treatments. The IP-treated mice displayed blunted over-expression of SAA-2, ACE-2, CXCL1, and CXCL10 and decreased changes in blood formula in response to an injection with resiquimod. The IP-treated mice injected with LPS showed normalized locomotion, anxiety, and exploration behaviors but not abnormal forced swimming. Isoquercitrin, choline, leucine, chlorogenic acid, and other constituents were identified by HPLC-HRMS and likely underlie the IP immunomodulatory effects.ConclusionsHerbal IP-therapy decreases inflammation and, partly, “sickness behavior,” suggesting its potency to combat SARS-CoV-2 infection first of all via its preventive effects.

Oleamide-Mediated Polarization of M1 Macrophages and IL-1β Production by Regulating NLRP3-Inflammasome Activation in Primary Human Monocyte-Derived Macrophages.

Macrophages are a type of innate immune cell that activates the NLRP3 inflammasome, causing the release of the cytokine IL-1β, which is a crucial mediator of the inflammatory response. NLRP3 activation that is dysregulated worsens a variety of inflammatory and autoimmune diseases, as well as neurodegenerative diseases. Oleamide is an endogenous fatty acid amide that was first determined as a sleep-inducing molecule and later shown to have wide-ranging beneficial effects on the central nervous system. How oleamide influences human macrophage polarization and NLRP3-inflammasome activation remains unclear. The effect of oleamide on macrophage polarization was explored using an in vitro culture of primary human monocyte-derived macrophages (MDMs) supplemented with human serum-containing media. Cellular and molecular mechanisms of oleamide-regulated MDMs polarization were also investigated. Results showed that oleamide promoted naïve macrophages (M0) toward the M1 phenotype by upregulating M1-associated genes (IL-1β, iNOS, CXCL10), along with downregulation of M2-associated genes (Arg-1, CD206, CCL22). Cell surface expression indicated that oleamide enhanced CD80 expression in M0 naïve macrophages and hider CD206 and CD163 expression in M2 macrophages. Higher production of IL-1β cytokine was observed but with no alteration in IL-6 and TNF-α levels by MDMs and differentiated THP-1 models. Whether oleamide functioned as a second signal that activated the NLRP3 inflammasome and mediated IL-1β production was further investigated using LPS-primed MDMs followed by oleamide treatment that induced activation of inflammasome-related proteins including NLRP3, ASC, cleaved casp-1, and cleaved IL-1β. These findings suggested that oleamide promoted M1 macrophage polarization and increased IL-1β production by activating the NLRP3 inflammasome in primary MDMs. This research reveals a new function for oleamide as well as prospective targets for treating NLRP3-related inflammatory disorders.

Multiscale Invasion Assay for Probing Macrophage Response to Gram-Negative Bacteria.

The immune system is a complex network of various cellular components that must differentiate between pathogenic bacteria and the commensal bacteria of the human microbiome, where misrecognition is linked to inflammatory disorders. Fragments of bacterial cell wall peptidoglycan bind to pattern recognition receptors within macrophages, leading to immune activation. To study this complex process, a methodology to remodel and label the bacterial cell wall of two different species of bacteria was established using copper (I) catalyzed azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne cycloaddition (SPAAC). Additionally, an approach for three-dimensional (3D) culture of human macrophages and their invasion with relevant bacteria in a well-defined hydrogel-based synthetic matrix inspired by the microenvironment of the gut was established. Workflows were developed for human monocyte encapsulation and differentiation into macrophages in 3D culture with high viability. Bacteria invaded into macrophages permitted in situ peptidoglycan labeling. Macrophages exhibited biologically-relevant cytokine release in response to bacteria. This molecularly engineered, multi-dimensional bacteria-macrophage co-culture system will prove useful in future studies to observe immunostimulatory, bacterial fragment production and localization in the cell at the carbohydrate level for insights into how the immune system properly senses bacteria.

Abstract 99: Single Cell RNA-Sequencing Reveals Array Of Immune Activation States In Intracerebral Hemorrhage

Introduction: Secondary inflammation is a well-established cause of injury in intracerebral hemorrhage (ICH). One potential treatment for ICH limits inflammation through targeted metabolic modulation of immune cells, but leukocyte activation states in ICH are poorly defined. Using single-cell RNA sequencing (scRNA-seq) of patient hematoma evacuates, we characterized the inflammatory and metabolic profile of over a dozen identifiable leukocyte populations. This research provides insight into the dynamics of immune activation in ICH. Methods: Evacuated hematoma and peripheral blood samples from 8 ICH patients (62 ± 2.1 years) were collected via minimally invasive surgery (n=5) or craniotomy (n=3) (Fig. 1a). Collection time ranged from 0 to 4 days after ICH onset (1.5 ± 0.5 days). scRNA-seq was done on the 10X Genomics platform and analyzed using Seurat. Human monocyte-derived macrophage cultures were stimulated with S100A9 and IL-1β, two ICH-relevant inflammatory molecules, or LPS. Echinomycin and 2-deoxy-D-glucose were used to inhibit HIF signaling and glycolysis respectively. Results: Clustering revealed over a dozen transcriptionally distinct CNS-resident and immune cell types, many of which have little prior study in ICH (Fig. 1b). 3,395 differentially expressed genes between hematoma and peripheral blood (p<0.05) were found in CD14 + monocytes and 932 in CD4 + effector T cells. Pathway analysis found upregulation of HIF signaling, glycolysis, and correlated networks of cytokine expression in the hematoma. The findings were significant across all immune populations but most prominent in myeloid cells (Fig. 1c). Inhibition of HIF and glycolysis in human macrophages in-vitro abrogated inflammatory and reparative cytokine production following inflammatory stimulation. In conclusion, this work highlights the interplay of inflammation and metabolism at a single cell level in ICH, contributing to knowledge of the immune response to brain injury.

798 CDX-585, A bispecific antibody with dual targeting of ILT4 and PD-1 checkpoint pathways

BackgroundActivation of the ITIM-bearing ILT4/LILRB2 receptor by its cognate ligands (HLA-G and HLA Class I) has been postulated as a resistance mechanism for checkpoint blockade of PD-1 and CTLA-4. Dual inhibition of receptors that suppress myeloid and T cell compartments through the generation of bispecific antibodies (bsAbs) is a promising strategy to improve outcomes for patients whose tumors are resistant to checkpoint inhibition.MethodsWe describe the discovery and characterization of CDX-585 a bsAb developed from novel ILT4 and PD-1 antagonist mAbs that revert myeloid cell suppression by antagonizing ILT4 and activating T-cell responses through PD-1 inhibition. The bsAb was engineered as a tetravalent molecule using the PD-1 IgG1 mAb linked to scFv of the ILT4 mAb at the C-terminus of the heavy chain. A series of mutations were introduced in the Fc domain to eliminate Fcy receptor binding and increase affinity to the neonatal Fc receptor. CDX-585 has good biophysical characteristics and retains functional properties similar to, or better, than the parental mAbs.ResultsCDX-585 has sub-nanomolar affinity binding to ILT4 and PD-1 and is a potent competitor of their respective ligands. Primary cultures of human macrophages and dendritic cells treated with CDX-585 enhanced production of inflammatory cytokines/chemokines, which was further potentiated in the presence of toll like receptor activation with lipopolysaccharide (LPS). CDX-585 was particularly effective in promoting T cell activation as measured by mixed lymphocyte reactions, and in polarizing macrophages towards M1 based on their cytokine profile. Pilot studies in mice and cynomolgus macaques confirmed a favorable pharmacokinetic profile without adverse effects of treatment noted in clinical observations or clinical chemistry.ConclusionsCDX-585 effectively combines ILT4 and PD-1 blockade into one molecule with favorable biophysical and functional characteristics supporting the initiation of development activities including manufacturing and IND-enabling studies.

Enhanced Skin Incisional Wound Healing With Intracellular ATP Delivery via Macrophage Proliferation and Direct Collagen Production.

This study sought to use a newly developed intracellular ATP delivery to enhance incisional wound healing to reduce surgical wound dehiscence and to explore possible mechanism for this effect. Thirty-five adult New Zealand white rabbits were used. Skin incisions were made on the back and closed. ATP-vesicles were mixed with a neutral cream for one side of the wounds while the neutral cream alone was used on the other side of the wounds. Laser speckle contrast imaging (LSCI), biomechanical, histological, and immunohistochemical analyses were performed 7 and 14 days after surgery, and macrophage culture was used to test the enhanced collagen production ability. Among them, 10 were used for wound perfusion study and 25 were used for wound biomechanical and histological/immunohistochemical studies. Wound tissue perfusion was reduced after surgery especially in early days. Wound tissue tensile strength, breaking stress, and elasticity were all much higher in the ATP-vesicle treated group than in the cream treated group at days 7 and 14. The healing was complemented by earlier macrophage accumulation, in situ proliferation, followed by direct collagen production. The results were further confirmed by human macrophage culture. It was concluded that intracellular ATP delivery enhanced healing strength of incisional wounds via multiple mechanisms.

Monoclonal antibodies capable of binding SARS-CoV-2 spike protein receptor-binding motif specifically prevent GM-CSF induction.

A severe acute respiratory syndrome (SARS)‐like coronavirus 2 (SARS‐CoV‐2) has recently caused a pandemic COVID‐19 disease that infected approximately 94 million and killed more than 2,000,000 people worldwide. Like the SARS‐CoV, SARS‐CoV‐2 also employs a receptor‐binding motif (RBM) of its envelope spike protein for binding the host angiotensin‐converting enzyme 2 (ACE2) to gain viral entry. Currently, extensive efforts are being made to produce vaccines against a surface fragment of a SARS‐CoV‐2, such as the spike protein, in order to boost protective antibodies that can inhibit virus‐ACE2 interaction to prevent viral entry. It was previously unknown how spike protein‐targeting antibodies would affect innate inflammatory responses to SARS‐CoV‐2 infections. Here we generated a highly purified recombinant protein corresponding to the RBM of SARS‐CoV‐2, and used it to screen for cross‐reactive monoclonal antibodies (mAbs). We found two RBM‐binding mAbs that competitively inhibited its interaction with human ACE2, and specifically blocked the RBM‐induced GM‐CSF secretion in both human peripheral blood mononuclear cells and murine macrophage cultures. Our findings have suggested a possible strategy to prevent SARS‐CoV‐2‐elicited “cytokine storm,” and revealed a potentially anti‐inflammatory and protective mechanism for SARS‐CoV‐2 spike‐based vaccines.

Small Noncoding RNAs MTS0997 and MTS1338 Affect the Adaptation and Virulence of Mycobacterium tuberculosis

Tuberculosis (TB) is currently the leading cause of death among bacterial infectious diseases. The spectrum of disease manifestations depends on both host immune responses and the ability of Mycobacterium tuberculosis to resist it. Small non-coding RNAs are known to regulate gene expression; however, their functional role in the relationship of M. tuberculosis with the host is poorly understood. Here, we investigated the effect of small non-coding sRNAs MTS1338 and MTS0997 on M. tuberculosis properties by creating knockout strains. We also assessed the effect of small non-coding RNAs on the survival of wild type and mutant mycobacteria in primary cultures of human alveolar macrophages and the virulence of these strains in a mouse infection model. Wild-type and mutants survived differentially in human alveolar macrophages. Infection of I/St mice with KO M. tuberculosis H37RV strains provided beneficial effects onto major TB phenotypes. We observed attenuated tuberculosis-specific inflammatory responses, including reduced cellular infiltration and decreased granuloma formation in the lungs. Infections caused by KO strains were characterized by significantly lower inflammation of mouse lung tissue and increased survival time of infected animals. Thus, the deletion of MTS0997 and MTS1338 lead to a significant decrease in the virulence of M. tuberculosis.

Sintered S53P4 bioactive glass scaffolds have anti-inflammatory properties and stimulate osteogenesis in vitro

Bioactive glasses (BAG) are used as bone-graft substitutes in orthopaedic surgery. A specific BAG scaffold was developed by sintering BAG-S53P4 granules. It is hypothesised that this scaffold can be used as a bone substitute to fill bone defects and induce a bioactive membrane (IM) around the defect site. Beyond providing the scaffold increased mechanical strength, that the initial inflammatory reaction and subsequent IM formation can be enhanced by coating the scaffolds with poly(DL-lactide-co-glycolide) (PLGA) is also hypothesised. To study the immunomodulatory effects, BAG-S53P4 (± PLGA) scaffolds were placed on monolayers of primary human macrophage cultures and the production of various pro- and anti-inflammatory cytokines was assessed using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and ELISA. To study the osteogenic effects, BAG-S53P4 (± PLGA) scaffolds were cultured with rabbit mesenchymal stem cells and osteogenic differentiation was evaluated by RT-qPCR and matrix mineralisation assays. The scaffold ion release was quantified and the BAG surface reactivity visualised. Furthermore, the pH of culture media was measured. BAG-S53P4 scaffolds had both anti-inflammatory and osteogenic properties that were likely attributable to alkalinisation of the media and ion release from the scaffold. pH change, ion release, and immunomodulatory properties of the scaffold could be modulated by the PLGA coating. Contrary to the hypothesis, the coating functioned by attenuating the BAG surface reactions and subsequent anti-inflammatory properties, rather than inducing an elevated inflammatory response compared to BAG-S53P4 alone. These results further validated the use of BAG-S53P4 (± PLGA) scaffolds as bone substitutes and indicate that scaffold properties can be tailored to a specific clinical need.

Designing 3D Synthetic Matrices for Studying Interactions Between Bacterial Peptidoglycan and Macrophages in the Context of Immune Misrecognition Diseases

The immune system is a complex network comprised of various cellular components that work together to protect the body from damage, with a key function of differentiating between pathogenic bacteria and the 39 trillion commensal bacteria that constitute our microbiome. Misrecognition of commensal bacteria has been linked to many inflammatory bowel disorders in humans yet remains not fully understood and difficult to study. Enabled by recent advances in molecular tools, fragments of the bacterial cell wall peptidoglycan have been identified that bind to pattern recognition receptors within macrophages, which activate macrophages to start a signaling cascade that turns on the immune response. Typically, these interactions are studied using two‐dimensional invasion assays on tissue culture plastic plates; however, the dimensionality and ‘stiffness’ of these traditional culture systems may not be representative of the cell‐microenvironment interactions that exist in vivo. In order to better simulate the native cellular environment, three‐dimensional (3D) cell culture systems can be used to control key biochemical and mechanical properties of the cell microenvironment. Here, we established an approach for the encapsulation and culture of human macrophages in 3D within well‐defined, biomimetic hydrogels decorated with integrin binding peptides that mimic the natural in vivo environment and linked with cell‐degradable sites for cell‐driven migration and remodeling to probe macrophage response to bacterial invasion. With this approach, we have shown invasion with bacteria whose glycans are remodeled and their engulfment through immunostaining and assessed activation of macrophages using RT‐qPCR and ELISA. These studies provide new insights into the interactions between bacteria and immune cells and this new method affords unique opportunities for examining how our immune system recognizes and misrecognizes bacteria toward a better understanding of the mechanisms of immune misrecognition diseases.Support or Funding InformationNIH CBI Training Grant‐5T32GM008550NIH U01 Common Fund program‐U01CA221230‐01

Abstract A39: Inflammatory macrophage derived TNF alpha induces downregulation of estrogen receptor alpha in breast cancer cells by inactivation of Foxo3A

Abstract Breast cancers are divided into different subtypes based on receptor expression status: estrogen receptor (ER), progesterone receptor (PR) and Her2. The luminal A subtype (ER+PR+Her2-) is most often associated with a good prognosis while the triple-negative (TN) cancers (ER-PR-Her2-) have a poor prognosis. ER+ cancer cells depend on estrogen for their growth and can be treated with drugs to block its effects. While most human breast cancers express ER alpha, some tumors are hormone independent despite the presence of ER and others become ER negative over time. Myeloid cells are known to affect tumor development, and infiltration of tumor-associated macrophages is associated with a worse prognosis. Theoretically, however, macrophages can be both beneficial and bad for the patient. Proinflammatory M1 macrophages (CD68+ CD163-) can eliminate cancer cells and promote antitumor immune responses while anti-inflammatory M2 macrophages (CD163+CD68+) or anti-inflammatory myeloid cells (CD68-CD163+) can promote tumor progression by inhibiting proinflammatory immune responses and induce wound-healing processes. This includes angiogenesis and matrix degradation, both of which promote metastatic spread and possibly even EMT. Macrophages have also been proposed to downregulate ER in breast cancer cells through an unknown mechanism. Due to the recent reports suggesting that macrophages can induce EMT and downregulate ER, we evaluated the effect of human primary macrophages on ER expression in breast cancer cells. Immunohistochemistry analyses of breast cancer tumor xenografts, established with co-transplantation of primary human myeloid cells together with ER+ breast cancer cells in NSG mice, showed association of monocytes with downregulation of ER-receptor expression. Using primary human monocyte-derived macrophage cultures In vitro, we show that the different subtypes of macrophages have unique cytokine secretion profiles with M1 macrophages secreting significantly higher levels of TNF alpha compared to M2 macrophages. ER alpha positive MCF-7 and ER alpha negative MDA-MB-231 breast cancer cells were cultured in macrophage condition media, and both Western blot and qPCR analysis showed downregulation of ER was caused by M1 macrophage-derived TNF alpha that inhibits FoxO3A transcription of ER alpha. We propose that proinflammatory macrophages (M1), despite being tumoricidal, may have direct effects on endocrine resistance in breast cancer patients. Our findings suggest that different macrophage subtypes have various and unique impacts on breast cancer progression, and that proinflammatory macrophages, despite being tumoricidal, may have unwanted and direct effects on endocrine resistance mechanisms in breast cancer patients. Citation Format: Frida Björk Gunnarsdottir, Catharina Hagerling, Caroline Bergenfelz, Camilla M. Rydberg, Meliha Mehmeti, Roni Allaoui, Sofie Mohlin, Sven Påhlman, Daniel Bexell, Karin Leandersson. Inflammatory macrophage derived TNF alpha induces downregulation of estrogen receptor alpha in breast cancer cells by inactivation of Foxo3A [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A39.

Tropism of the Novel Coronavirus SARS-CoV-2 in Human Respiratory Tract: An Analysis in <i>Ex Vivo</i> and <i>In Vitro</i> Cultures

Background: A novel human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in December 2019, to cause a respiratory disease (COVID-19) of varying severity in Wuhan China, subsequently spreading to other parts of China and beyond. Methods: We infected ex vivo explant cultures of the human conjunctiva, bronchus and lung, and in vitro cultures of primary human alveolar epithelial cells and macrophages with SARS-CoV-2, and assessed viral tropism, replication competence and innate immune responses, in comparison with SARS-CoV, MERS-CoV, and the 2009 pandemic influenza H1N1 (pdmH1N1) virus.Findings: SARS-CoV-2 infected ciliated, mucus secreting and club cells of bronchial epithelium, spindled morphologically type I pneumocytes in the lung, and the conjunctival mucosa. Virus replication competence of SARS-CoV-2 in the bronchus was higher than that of SARS-CoV but lower than pdmH1N1. SARS-CoV-2 replication was comparable with SARS-CoV and pdmH1N1 in the lung but was lower than MERS-CoV. SARS-CoV-2 virus was a less potent inducer of pro-inflammatory cytokines compared with H5N1 and MERS-CoV. Influenza virus infection of alveolar epithelial cells increased ACE2 expression.Interpretation: The conjunctival epithelium and the conducting airways appear to be potential portals of infection of SARS-CoV-2. Both SARS-CoV and SARS-CoV-2 replicated comparably in the alveolar epithelium explaining the progression of infection to a primary viral pneumonia.Funding Statement: US National Institute of Allergy and Infectious Diseases (NIAID) under Centers of Excellence for Influenza Research and Surveillance (CEIRS) contract no. HHSN272201400006C and the Theme Based Research Scheme (Ref: T11-705/14N), Hong Kong Special Administrative Region.Declaration of Interests: There is no conflict of interest for all authors.Ethics Approval Statement: All experiments were carried out in a Bio-safety level 3 (BSL-3) facility. Informed consent was obtained from all subjects and approval was granted by the Institutional Review Board (IRB) of the University of Hong Kong and the Hospital Authority (Hong Kong West) (approval no: UW 20-167).

Transition of Macrophages to Fibroblast-Like Cells in Healing Myocardial Infarction

BackgroundMacrophages and fibroblasts are 2 major cell types involved in healing after myocardial infarction (MI), contributing to myocardial remodeling and fibrosis. Post-MI fibrosis progression is characterized by a decrease in cardiac macrophage content. ObjectivesThis study explores the potential of macrophages to express fibroblast genes and the direct role of these cells in post-MI cardiac fibrosis. MethodsProlonged in vitro culture of human macrophages was used to evaluate the capacity to express fibroblast markers. Infiltrating cardiac macrophages was tracked in vivo after experimental MI of LysM(Cre/+);ROSA26(EYFP/+) transgenic mice. The expression of Yellow Fluorescent Protein (YFP) in these animals is restricted to myeloid lineage allowing the identification of macrophage-derived fibroblasts. The expression in YFP-positive cells of fibroblast markers was determined in myocardial tissue sections of hearts from these mice after MI. ResultsExpression of the fibroblast markers type I collagen, prolyl-4-hydroxylase, fibroblast specific protein-1, and fibroblast activation protein was evidenced in YFP-positive cells in the heart after MI. The presence of fibroblasts after MI was evaluated in the hearts of animals after depletion of macrophages with clodronate liposomes. This macrophage depletion significantly reduced the number of Mac3+Col1A1+ cells in the heart after MI. ConclusionsThe data provide both in vitro and in vivo evidence for the ability of macrophages to transition and adopt a fibroblast-like phenotype. Therapeutic manipulation of this macrophage-fibroblast transition may hold promise for favorably modulating the fibrotic response after MI and after other cardiovascular pathological processes.

Junin Virus Triggers Macrophage Activation and Modulates Polarization According to Viral Strain Pathogenicity.

The New World arenavirus Junin (JUNV) is the etiological agent of Argentine hemorrhagic fever (AHF). Previous studies of human macrophage infection by the Old-World arenaviruses Mopeia and Lassa showed that while the non-pathogenic Mopeia virus replicates and activates human macrophages, the pathogenic Lassa virus replicates but fails to activate human macrophages. Less is known in regard to the impact of New World arenavirus infection on the human macrophage immune response. Macrophage activation is critical for controlling infections but could also be usurped favoring immune evasion. Therefore, it is crucial to understand how the JUNV infection modulates macrophage plasticity to clarify its role in AHF pathogenesis. With this aim in mind, we compared infection with the attenuated Candid 1 (C#1) or the pathogenic P strains of the JUNV virus in human macrophage cultures. The results showed that both JUNV strains similarly replicated and induced morphological changes as early as 1 day post-infection. However, both strains differentially induced the expression of CD71, the receptor for cell entry, the activation and maturation molecules CD80, CD86, and HLA-DR and selectively modulated cytokine production. Higher levels of TNF-α, IL-10, and IL-12 were detected with C#1 strain, while the P strain induced only higher levels of IL-6. We also found that C#1 strain infection skewed macrophage polarization to M1, whereas the P strain shifted the response to an M2 phenotype. Interestingly, the MERTK receptor, that negatively regulates the immune response, was down-regulated by C#1 strain and up-regulated by P strain infection. Similarly, the target genes of MERTK activation, the cytokine suppressors SOCS1 and SOCS3, were also increased after P strain infection, in addition to IRF-1, that regulates type I IFN levels, which were higher with C#1 compared with P strain infection. Together, this differential activation/polarization pattern of macrophages elicited by P strain suggests a more evasive immune response and may have important implications in the pathogenesis of AHF and underpinning the development of new potential therapeutic strategies.

Mitochondrial Calcium Uptake Is Instrumental to Alternative Macrophage Polarization and Phagocytic Activity.

Macrophages are highly plastic and dynamic cells that exert much of their function through phagocytosis. Phagocytosis depends on a coordinated, finely tuned, and compartmentalized regulation of calcium concentrations. We examined the role of mitochondrial calcium uptake and mitochondrial calcium uniporter (MCU) in macrophage polarization and function. In primary cultures of human monocyte-derived macrophages, calcium uptake in mitochondria was instrumental for alternative (M2) macrophage polarization. Mitochondrial calcium uniporter inhibition with KB-R7943 or MCU knockdown, which prevented mitochondrial calcium uptake, reduced M2 polarization, while not affecting classical (M1) polarization. Challenging macrophages with E. coli fragments induced spikes of mitochondrial calcium concentrations, which were prevented by MCU inhibition or silencing. In addition, mitochondria remodelled in M2 macrophages during phagocytosis, especially close to sites of E. coli internalization. Remarkably, inhibition or knockdown of MCU significantly reduced the phagocytic capacity of M2 macrophages. KB-R7943, which also inhibits the membrane sodium/calcium exchanger and Complex I, reduced mitochondria energization and cellular ATP levels, but such effects were not observed with MCU silencing. Therefore, phagocytosis inhibition by MCU knockdown depended on the impaired mitochondrial calcium buffering rather than changes in mitochondrial and cellular energy status. These data uncover a new role for MCU in alternative macrophage polarization and phagocytic activity.

Effect of Apoptotic Neutrophils on the Production of Erythropoietin, MMP-9, and TIMP-1 in Cultures of Human Macrophages.

We studied the effect of apoptotic neutrophils on the production of erythropoietin, MMP-9, and TIMP-1 by GM-CSF-induced human macrophages. GM-CSF-induced macrophages spontaneously produce erythropoietin and secrete MMP-9 and TIMP-1. Polarization of these macrophages towards the M2-like phenotype after exposure to apoptotic neutrophils considerably increased the production of erythropoietin; the MMP-9/TIMP-1 ratio tended to increase under these conditions due to a decrease in TIMP-1.

Pathogenic Mechanisms of Endothelial Damage in Severe Scrub Typhus

Abstract Our newly established lethal and sublethal Orientia tsutsugamushi infection mouse models, which mimic clinical/pathologic features of human scrub typhus, are valuable for mechanistic studies. A hallmark of lethal Orientia infection is Th1-skewed, but Th2-impared, immune responses, accompanied with severe endothelial damage and acute tissue injury. Importantly, while tissue bacteria peak at disease onset (D6) in mice, neutrophil and CD8 T cell influx/activation reach peaks in the lungs around D10 (prior to host death). We hypothesize a two-stage-dysfunction loop for lethal infection: 1) Orientia replication in phagocytes and endothelial cells (EC) triggers inflammatory responses and host defense machinery; and 2) infection-mediated release of DAMP molecules exacerbates vascular dysfunction. This hypothesis was supported by immunofluorescent co-staining of bacteria with leukocyte/EC/platelet-specific markers, multi-color FACS, Western blot, and qRT-PCR. The progressive loss of vasculature and junction proteins (VE-cad, occludin) and reduction in vascular function (angiopoietin 1, Tie2, pTie2) and CD41+ platelets in the lungs were positively linked to neutrophil and CD8 T cell influx and activation, rather than to body bacterial loads. The mechanisms of immune dysregulation were further examined by using infected human endothelial cells and mouse primary macrophage and neutrophil cultures, as well as knockout mice. While Orientia infection directly altered the Ang-Tie2 axis in EC, activated IL-33/IL-36-like DAMP molecules further contributed to vascular/tissue injury. Our studies, for the first time, have revealed immune dysregulation and prognostic biomarkers in the context of scrub typhus pathogenesis.

Immunotoxicological Evaluation of Schinus molle L. (Anacardiaceae) Essential Oil in Lymphocytes and Macrophages.

Schinus molle L. is used to treat various diseases; however, the literature lacks information regarding its possible immunotoxic effects. The aim of the study was to investigate the immunotoxic effects of essential oil from leaves of Schinus molle L. in cultures of human lymphocytes and macrophages. The cultures were treated with essential oil (EO) of Schinus molle L. and subsequently subjected to genotoxic analysis (comet assay), mutagenic analysis (micronucleus frequency and chromosomal aberration), and cytotoxic (cell viability) and functional parameters (interleukins secretions). Our analyses have determined that the essential oil from leaves of Schinus molle L. presents several compounds with α-pinene being the major compound; in addition, the compound verbenene was firstly identified; genotoxic effects were detected only in macrophages and only at the two highest concentrations tested. An important finding is that Schinus molle L. oil causes an activation of the immune system. This action has its mechanism centered by the cascade nitric oxide-interleukin-10-tumor necrosis factor alpha.