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Background: The African swine fever (ASF) virus (ASFV) and ASF-like viral sequences were identified in human samples and sewage as well as in different water environments. Pigs regularly experience infections by the ASFV. The considerable stability of the virus in the environment suggests that there is ongoing and long-term contact between humans and the ASFV. However, humans exhibit resistance to the ASFV, and the decisive factor in developing infection in the body is most likely the reaction of target macrophages to the virus. Therefore, this study aimed to characterize the responses of human macrophages to the virus and explore the distinct features of the viral replication cycle within human macrophages., Methods: The ASFV Armenia/07 strain was used in all experiments. In this study, quantitative real-time polymerase chain reaction (qRT-PCR) was used to determine the ASFV gene expression; flow cytometry analysis was performed to evaluate the effects of the inactive and active ASFV (inASFV and aASFV) treatments on the phenotype of THP-1-derived macrophages (Mφ0) and inflammatory markers. Moreover, other methods such as cell viability and apoptosis assays, staining techniques, phagocytosis assay, lysosome-associated membrane protein (LAMP-1) cytometry, and cytokine detection were used during experiments., Results: Our findings showed that the virus initiated replication by entering human macrophages. Subsequently, the virus shed its capsid and initiated the transcription of numerous viral genes, and at least some of these genes executed their functions. In THP-1-derived macrophages (Mφ0), the ASFV implemented several functions to suppress cell activity, although the timing of their implementation was slower compared with virus-sensitive porcine alveolar macrophages (PAMs). Additionally, the virus could not complete the entire replication cycle in human Mφ0, as indicated by the absence of viral factories and a decrease in infectious titers of the virus with each subsequent passage. Overall, the infection of Mφ0 with the ASFV caused significant alterations in their phenotype and functions, such as increased TLR2, TLR3, CD80, CD36, CD163, CXCR2, and surface LAMP-1 expression. Increased production of the tumor necrosis factor (TNF) and interleukin (IL)-10 and decreased production of interferon (IFN)-α were also observed. Taken together, the virus enters human THP-1-derived macrophages, starts transcription, and causes immunological responses by target cells but cannot complete the replicative cycle., Conclusion: These findings suggest that there may be molecular limitations within human macrophages that at least partially restrict the complete replication of the ASFV. Understanding the factors that hinder viral replication in Mφ0 can provide valuable insights into the host-virus interactions and the mechanisms underlying the resistance of human macrophages to the ASFV., Competing Interests: The authors declare no conflict of interest., (© 2024 The Author(s). Published by IMR Press.)

Fatty liver disease, characterized by excessive inflammation and lipid deposition, is becoming one of the most prevalent liver metabolic diseases worldwide owing to the increasing global incidence of obesity. However, the underlying mechanisms of fatty liver disease are poorly understood. Accumulating evidence suggests that hepatic macrophages, specifically Kupffer cells (KCs), act as key players in the progression of fatty liver disease. Thus, it is essential to examine the current evidence of the roles of hepatic macrophages (both KCs and monocyte-derived macrophages). In this review, we primarily address the heterogeneities and multiple patterns of hepatic macrophages participating in the pathogenesis of fatty liver disease, including Toll-like receptors (TLRs), NLRP3 inflammasome, lipotoxicity, glucotoxicity, metabolic reprogramming, interaction with surrounding cells in the liver, and iron poisoning. A better understanding of the diverse roles of hepatic macrophages in the development of fatty liver disease may provide a more specific and promising macrophage-targeting therapeutic strategy for inflammatory liver diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Xu, Liu, Bai, Xu, Liang, Ma and Gao.)

The activation of proinflammatory cellular processes and signals such as those linked to NF-kB in macrophages are involved in the control of infection by Leishmania ssp. However, little is known about the influence of the drugs used in the treatment on the host cellular inflammatory signaling pathways. This study aimed to evaluate the effects of different drugs used in the treatment of leishmaniasis on inflammatory profile related to Toll-like receptors (TLRs) from L. amazonensis-infected macrophages. J774 macrophage-like cells were infected with the promastigote forms (5:1) and 24 hs incubated with Amphotericin B (AmB), Glucantime® (GLU) or Pentamidine (Pent). The following inflammatory pathways were evaluated: NF-κB p65, NF-κB p65 phosphorylated (Ser536), stress-activated protein kinase/c-Jun NH(2)-terminal kinase (SAPK/JNK) phosphorylated (Thr183/Tyr185), p38 mitogen activated protein kinase (MAPK p38) phosphorylated (Thr180/Tyr182), signal transducer and activator of transcription-3 (Stat3) phosphorylated (Tyr705) and inhibitor kappa B-α (IκB-α) phosphorylated (Ser32). In silico tests were performed to evaluate the molecular affinity between TLRs and antileishmanial drugs. Molecular docking showed that affinities varied significantly among the binders evaluated. The lowest affinity (-8.6 Kcal/Mol) was calculated for AmB in complex with TLR4. Pent showed higher values for TLR1, TLR2 and TLR3, while for TLR4 the affinity value was lower (5.5 Kcal/Mol). The values obtained for GLU were the highest for the set of binders tested. From the infected macrophages, treatments inhibited NF-kB p65 for GLU (65.44%), for Pent (46.43%) and for AmB (54.07%) compared to untreated infected macrophages. The activation of the signaling pathway of NF-kB, SAPK/JNK and IκB-α caused by AmB and Pent may potentiate the microbicidal mechanisms of the infected macrophages., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objectives: Macrophages are conventionally classified as pro-inflammatory (M1) and anti-inflammatory (M2) functional types. There is evidence for a predominance of macrophages with an inflammatory phenotype (M1) in the rheumatoid arthritis (RA) synovium. MicroRNAs (miRs) play a pivotal role in regulating the inflammatory response in innate immune cells and are found at dysregulated levels in RA patients. Here we explored miRs that tune the inflammatory function of M2-macrophages. Methods: Expression profiles of miR-221-3p and miR-155-5p were analyzed in clinical samples from RA, other inflammatory arthritis (OIA), osteoarthritis (OA), and healthy donors (HD) by qPCR. In vitro generated macrophages were transfected with miR-mimics and inhibitors. Transcriptome profiling through RNA-sequencing was performed on M2-macrophages overexpressing miR-221-3p mimic with or without LPS treatment. Secretion of IL-6, IL-10, IL-12, IL-8, and CXCL13 was measured in M1- and M2-macrophages upon TLR2/TLR3/TLR4-stimulation using ELISA. Inflammatory pathways including NF-κB, IRF3, MAPKs, and JAK3/STAT3 were evaluated by immunoblotting. Direct target interaction of miR-221-3p and predicted target sites in 3'UTR of JAK3 were examined by luciferase reporter gene assay. Results: miR-221-3p in synovial tissue and fluid was increased in RA vs. OA or OIA. Endogenous expression levels of miR-221-3p and miR-155-5p were higher in M1- than M2-macrophages derived from RA patients or HD. TLR4-stimulation of M1- and M2-macrophages resulted in downregulation of miR-221-3p, but upregulation of miR-155-5p. M2-macrophages transfected with miR-221-3p mimics secreted less IL-10 and CXCL13 but more IL-6 and IL-8, exhibited downregulation of JAK3 protein and decreased pSTAT3 activation. JAK3 was identified as new direct target of miR-221-3p in macrophages. Co-transfection of miR-221-3p/miR-155-5p mimics in M2-macrophages increased M1-specific IL-12 secretion. Conclusions: miR-221-3p acts as a regulator of TLR4-induced inflammatory M2-macrophage function by directly targeting JAK3. Dysregulated miR-221-3p expression, as seen in synovium of RA patients, leads to a diminished anti-inflammatory response and drives M2-macrophages to exhibit a M1-cytokine profile., (Copyright © 2020 Quero, Tiaden, Hanser, Roux, Laski, Hall and Kyburz.)

Seven non-toxigenic C. diphtheriae strains and one toxigenic strain were analyzed with regard to their interaction with murine macrophages (BMM) and human THP-1 macrophage-like cells. Proliferation assays with BMM and THP-1 revealed similar intracellular CFUs for C. diphtheriae strains independent of the host cell. Strain ISS4060 showed highest intracellular CFUs, while the toxigenic DSM43989 was almost not detectable. This result was confirmed by TLR 9 reporter assays, showing a low signal for DSM43989, indicating that the bacteria are not endocytosed. In contrast, the non-pathogenic C. glutamicum showed almost no intracellular CFUs independent of the host cell, but was recognized by TLR9, indicating that the bacteria were degraded immediately after endocytosis. In terms of G-CSF and IL-6 production, no significant differences between BMM and THP-1 were observed. G-CSF production was considerably higher than IL-6 for all C. diphtheriae strains and the C. glutamicum did not induce high cytokine secretion in general. Furthermore, all corynebacteria investigated in this study were able to induce NFκB signaling but only viable C. diphtheriae strains were able to cause host cell damage, whereas C. glutamicum did not. The absence of Mincle resulted in reduced G-CSF production, while no influence on the uptake of the bacteria was observed. In contrast, when MyD88 was absent, both the uptake of the bacteria and cytokine production were blocked. Consequently, phagocytosis only occurs when the TLR/MyD88 pathway is functional, which was also supported by showing that all corynebacteria used in this study interact with human TLR2.

Brown seaweeds are well-known source of bioactive compounds, which are producing a variety of secondary metabolites with promising bioactive properties. Traditionally, seaweeds used as ingredients in medicine for many centuries in Asian countries. However, the protective mechanisms of many metabolites found in seaweeds are remains to be determined. Thus, applications of seaweeds are limited because of poor understanding of their structural features and mechanisms responsible for their bioactive properties. In the present study, anti-inflammatory properties of fucoidan isolated from the brown seaweed Padina commersonii (PCF) was evaluated against LPS-activated RAW 264.7 macrophages. PCF was characterized using NMR, FT-IR, and HPAE-PAD spectrum (for mono sugar composition). It was observed that PCF is rich in fucose and sulfate as well as a similar structure to the commercial fucoidan. Western blots and RT-qPCR analysis were used to determine the protective effects of PCF after LPS challenge using RAW 264.7 macrophages. According to the results, PCF significantly down-regulated LPS-activated mRNA and protein expression levels of TLR2, TLR4, and MyD88 which are known inducers/activators of NF-κB transcriptional factors. The results, obtained from this study demonstrated PCF has a potential to inhibit LPS-induced inflammatory responses via blocking TLR/MyD88/ NF-κB signal transduction., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The tetraspanin CD82 is a potent suppressor of tumor metastasis and regulates several processes including signal transduction, cell adhesion, motility, and aggregation. However, the mechanisms by which CD82 participates in innate immunity are unknown. We report that CD82 is a key regulator of TLR9 trafficking and signaling. TLR9 recognizes unmethylated cytosine-phosphate-guanine (CpG) motifs present in viral, bacterial, and fungal DNA. We demonstrate that TLR9 and CD82 associate in macrophages, which occurs in the endoplasmic reticulum (ER) and post-ER. Moreover, CD82 is essential for TLR9-dependent myddosome formation in response to CpG stimulation. Finally, CD82 modulates TLR9-dependent NF-κB nuclear translocation, which is critical for inflammatory cytokine production. To our knowledge, this is the first time a tetraspanin has been implicated as a key regulator of TLR signaling. Collectively, our study demonstrates that CD82 is a specific regulator of TLR9 signaling, which may be critical in cancer immunotherapy approaches and coordinating the innate immune response to pathogens.-Khan, N. S., Lukason, D. P., Feliu, M., Ward, R. A., Lord, A. K., Reedy, J. L., Ramirez-Ortiz, Z. G., Tam, J. M., Kasperkovitz, P. V., Negoro, P. E., Vyas, T. D., Xu, S., Brinkmann, M. M., Acharaya, M., Artavanis-Tsakonas, K., Frickel, E.-M., Becker, C. E., Dagher, Z., Kim, Y.-M., Latz, E., Ploegh, H. L., Mansour, M. K., Miranti, C. K., Levitz, S. M., Vyas, J. M. CD82 controls CpG-dependent TLR9 signaling.

Long noncoding RNAs (lncRNAs) can regulate target gene expression by acting in cis (locally) or in trans (non-locally). Here, we performed genome-wide expression analysis of Toll-like receptor (TLR)-stimulated human macrophages to identify pairs of cis -acting lncRNAs and protein-coding genes involved in innate immunity. A total of 229 gene pairs were identified, many of which were commonly regulated by signaling through multiple TLRs and were involved in the cytokine responses to infection by group B Streptococcus We focused on elucidating the function of one lncRNA, named lnc-MARCKS or ROCKI (Regulator of Cytokines and Inflammation), which was induced by multiple TLR stimuli and acted as a master regulator of inflammatory responses. ROCKI interacted with APEX1 (apurinic/apyrimidinic endodeoxyribonuclease 1) to form a ribonucleoprotein complex at the MARCKS promoter. In turn, ROCKI -APEX1 recruited the histone deacetylase HDAC1, which removed the H3K27ac modification from the promoter, thus reducing MARCKS transcription and subsequent Ca 2+ signaling and inflammatory gene expression. Finally, genetic variants affecting ROCKI expression were linked to a reduced risk of certain inflammatory and infectious disease in humans, including inflammatory bowel disease and tuberculosis. Collectively, these data highlight the importance of cis -acting lncRNAs in TLR signaling, innate immunity, and pathophysiological inflammation., (© 2019 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Exposure to mold-contaminated indoor air has been associated with various respiratory diseases, and there is a need for experimental data to confirm these associations. The pro-inflammatory properties of well-characterized aerosolized spores and hyphal fragments from Aspergillus fumigatus and Aspergillus versicolor were examined and compared using various human macrophage cell models including phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 macrophages (THP-1 Ma), primary peripheral blood monocyte-derived macrophages (MDM), and primary airway macrophages (AM) from induced sputum. X-ray treated samples of the two mold species induced different responses with A. fumigatus displaying the most potent induction of pro-inflammatory responses. While hyphal fragments from A. fumigatus were more potent than spores, similar responses were produced by the two growth stages of A. versicolor . THP-1 Ma was the most sensitive model releasing a broad range of cytokines/chemokines. MDM exhibited a similar cytokine/chemokine profile as THP-1 Ma, except for a low-quantity release of interleukin-1β (IL-1β). In contrast, AM appeared to be nonresponsive and yielded a different pattern of pro-inflammatory markers. Toll-like receptor (TLR)4, but also to a certain degree TLR2, was involved in several responses induced by spores and aerosolized hyphal fragments of A. fumigatus in MDM. Taken together, MDM seems to be the most promising experimental macrophage model. Abbreviations : AF: A. fumigatus, Aspergillus fumigatus ; AV: A. versicolor, Aspergillus versicolor ; AM: Airway Macrophage; CBA: Cytometric Bead Array; CD: Cluster of Differentiation; DTT: dithiothreitol; ELISA: Enzyme Linked Immunosorbent Assay; FBS: fetal bovine serum; GM-CSF: Granulocyte macrophage colony-stimulating factor; IL-1β: Interleukin-1beta; MDM: Monocyte-Derived Macrophages; NF-κB: Nuclear Factor kappa light chain enhancer of activated B cells; NLR: NOD-like Receptor; PAMP: Pathogen Associated Molecular Pattern; PMA: Phorbol 12-myristate 13-acetate; PRR: Pattern Recognition Receptor; THP-1: Human leukemia monocyte cell line; TLR: Toll-like Receptor; TNF-α: Tumor Necrosis Factor- alpha.

Mycobacterium tuberculosis is a highly virulent bacterium that causes tuberculosis. It infects about one third of the world's population. Type I interferons (IFNs) play a detrimental role in host defense against M. tuberculosis infection. Proteins secreted by M. tuberculosis through ESX-1 secretion system contribute to type I IFNs production. However, the precise mechanism by which 6-kDa early secretory antigen target (ESAT6), one of ESX-1-mediated secretory proteins, induces type I IFNs production in host cells is currently unclear. Therefore, the objective of the present study was to determine the underlying molecular mechanism regulating ESAT6-mediated gene expression of IFN-β in macrophages. Recombinant ESAT6 produced from E. coli expression system induced IFN-β gene expression in various types of macrophages such as mouse bone marrow-derived macrophages (BMDMs), peritoneal macrophages, and MH-S cells (murine alveolar macrophage cell line). Deficiency of TLR4 and TRIF absolutely abrogated ESAT6-induced IFN-β gene expression. TLR2 and MyD88 were partially involved in IFN-β gene expression in response to low dose of ESAT6. Another recombinant ESAT6 produced from baculovirus system also upregulated IFN-β gene expression via TLR4-dependent pathway. Polymyxin B (PMB) treatment impaired LPS-induced IFN-β expression. However, IFN-β expression induced by ESAT6 was not influenced by PMB. This suggests that ESAT6-mediated IFN-β expression is not due to LPS contamination. Treatment with ESAT6 resulted in activation of TBK1 and IRF3 in macrophages. Such activation was abolished in TLR4- and TRIF-deficient cells. Moreover, inhibition of IRF3 and TBK1 suppressed IFN-β gene expression in response to ESAT6. Our results suggest that ESAT6 might contribute to virulence of M. tuberculosis by regulating type I IFNs production through TLR4-TRIF signaling pathway., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Dengue, one the most important public health problems in tropical and subtropical areas, is the most important mosquito-borne viral infection in humans. In the absence of effective treatment and vaccine against dengue, the active form of vitamin D could play a central role in protection against dengue virus (DENV), the causal agent of dengue. Recently we reported that monocyte-derived macrophages (MDMs) differentiated in the presence of vitamin D, in addition to expressing lower levels of mannose receptor, are less susceptible to DENV infection and produce low levels of pro-inflammatory cytokines, compared to MDMs differentiated in the absence of vitamin D., Objective: The aim of this study was to determine that oral vitamin D supplementation exerts an effect on DENV susceptibility and pro-inflammatory cytokine production in MDMs., Methods: Healthy individuals were supplemented with 1000 or 4000 international units (IU)/day of vitamin D during 10days. Before and after vitamin D supplementation, a peripheral blood (PB) sample was taken and the monocytes recovered were used to obtain MDMs and were challenged with DENV-2. Furthermore, the expression of genes encoding vitamin D receptor (VDR), CYP24A1 and CAMP were analyzed using real-time quantitative PCR., Results: The data indicate that macrophages differentiated from monocytes obtained from healthy donors who received higher doses of vitamin D (4000IU/day), exhibited higher resistance to DENV-2 infection and produced a significant decrease of pro-inflammatory cytokines and high production of interleukin-10 (IL-10). Furthermore, a significant decrease in intracellular toll-like receptor (TLR) and CAMP mRNA was observed., Conclusion: A supplement of 4000IU/day of vitamin D may represent an adequate dose to control dengue progression and DENV replication. Although the results of our study suggest that the vitamin D status can influence the immune response, further studies are needed to determine the feasibility of vitamin D as anti-DENV agent and immune modulator., (Copyright © 2017. Published by Elsevier B.V.)

Macrophages are linchpins of innate immunity, responding to invading microorganisms by initiating coordinated inflammatory and antimicrobial programs. Immediate antimicrobial responses, such as NADPH-dependent reactive oxygen species (ROS), are triggered upon phagocytic receptor engagement. Macrophages also detect and respond to microbial products through pattern recognition receptors (PRRs), such as TLRs. TLR signaling influences multiple biological processes including antigen presentation, cell survival, inflammation, and direct antimicrobial responses. The latter enables macrophages to combat infectious agents that persist within the intracellular environment. In this review, we summarize our current understanding of TLR-inducible direct antimicrobial responses that macrophages employ against bacterial pathogens, with a focus on emerging evidence linking TLR signaling to reprogramming of mitochondrial functions to enable the production of direct antimicrobial agents such as ROS and itaconic acid. In addition, we describe other TLR-inducible antimicrobial pathways, including autophagy/mitophagy, modulation of nutrient availability, metal ion toxicity, reactive nitrogen species, immune GTPases (immunity-related GTPases and guanylate-binding proteins), and antimicrobial peptides. We also describe examples of mechanisms of evasion of such pathways by professional intramacrophage pathogens, with a focus on Salmonella, Mycobacteria, and Listeria. An understanding of how TLR-inducible direct antimicrobial responses are regulated, as well as how bacterial pathogens subvert such pathways, may provide new opportunities for manipulating host defence to combat infectious diseases., (©2018 Society for Leukocyte Biology.)

Background: Toll-like receptors (TLRs) and macrophages play an important role in rheumatoid arthritis (RA). Currently, it is not clear whether inflammatory M1 or anti-inflammatory M2 predominate among the resident macrophages in the synovium. In the present study, we set out to investigate the impact of TLR stimulation on monocyte-derived M1 and M2 macrophage function and phenotype by mimicking the exposure to abundant TLR agonists as occurs in the context of RA. The response of macrophage subsets to TLR2 and TLR4 activation was evaluated on cluster of differentiation (CD) marker profile; cytokine secretion; gene expression; and NF-κB, interferon regulatory factors 3 and 7 (IRF3/7), and mitogen-activated protein kinase (MAPK) activation., Methods: Human monocytes were isolated from peripheral blood of healthy individuals and patients with RA and differentiated into M1-like and M2-like macrophages by granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF), respectively. Cells were either (1) stimulated with TLR ligands Pam3 or lipopolysaccharide (LPS) or (2) classically activated via interferon (IFN)-γ/LPS. Cytokine production was measured by enzyme-linked immunosorbent assay, and gene expression was measured by qPCR. Cells were stained for CD markers and analyzed by fluorescence-activated cell sorting. NF-κB, IRF3/7, and MAPKs were detected by Western blotting., Results: Monocyte-derived macrophages of healthy donors (HD) or patients with RA displayed comparable subset-specific phenotypes upon exposure to TLR agonists. CD14 and CD163 marker expression on M2 macrophages did not change upon TLR2 and TLR4 engagement. By contrast, M2 gene markers HMOX1, FOLR2, and SLC40A1 were decreased. Importantly, M2 macrophages derived from HD or patients with RA showed both a decreased ratio of interleukin (IL)-10/IL-6 and IL-10/IL-8 upon stimulation with TLR2 ligand Pam3 compared with TLR4 ligand LPS. Gene expression of TLR2 was increased, whereas TLR4 expression was decreased, by TLR ligand stimulation. MAPKs p38, extracellular signal-regulated kinase 1/2, and c-Jun N-terminal kinase were activated more strongly in M2 than in M1 macrophages by Pam3 or LPS., Conclusions: We show that the anti-inflammatory activity of M2 macrophages is reduced in the presence of abundant TLR2 ligands without significant changes in cell surface markers. Thus, the classical M1/M2 paradigm based on cellular markers does not apply to macrophage functions in inflammatory conditions such as RA.

Toll-like receptor (TLR) agonists drive hematopoietic stem and progenitor cells (HSPCs) to differentiate along the myeloid lineage in vitro and also in vivo following infection. In this study, we used an in vitro model of HSPC differentiation to investigate the functional consequences (cytokine production) that exposing HSPCs to various pathogen-associated molecular patterns (PAMPs) and Candida albicans cells have on the subsequently derived macrophages. Mouse HSPCs (Lin - cells) were cultured with GM-CSF to induce macrophage differentiation in the presence or absence of the following pattern recognition receptor (PRR) agonists: Pam 3 CSK 4 (TLR2 ligand), LPS (TLR4 ligand), depleted zymosan (which only activates Dectin-1), or inactivated C. albicans yeasts (which activate several PRRs, mainly TLR2 and Dectin-1). Our data show that only pure TLR2 ligand exposure (transient and continuous) impacts the inflammatory function of GM-CSF-derived macrophages, because Pam 3 CSK 4 -exposed HSPCs generate macrophages with a diminished ability to produce inflammatory cytokines. Interestingly, the Pam 3 CSK 4 -induced tolerance of macrophages (by transient exposure of HSPCs) is reinforced by subsequent exposure to C. albicans cells in GM-CSF-derived macrophages; however, the induced tolerance is partially reversed in M-CSF-derived macrophages. Therefore, the ability of macrophages to produce inflammatory cytokines is extremely dependent on how the HSPCs from which they are derived receive and integrate multiple microenvironmental signals (PRR ligands and/or CSFs).

Leukemia is often accompanied by enhanced susceptibility to infection due to compromised B cell and T cell functions. The alterations in macrophage functions in leukemia remain less investigated. Herein, we examined macrophages for their functions and responsiveness to IL-3 and GM-CSF in ENU-induced leukemia in BALB/c mice. We observed that the macrophages from the leukemic mice had less phagocytic activity, reduced chemotactic activity in response to monocyte chemoattractant protein-1(MCP-1), and decreased expression of iNOS, GM-CSF, TLR2, TLR4, IFN-γ and TNF-α. These impaired macrophage functions in leukemic mice were significantly corrected by IL-3 and GM-CSF treatment indicating the therapeutic benefit of these two cytokines in leukemia., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Herpes simplex virus type 1 (HSV-1) cause not only mild symptoms but also blindness and encephalitis. It was previously shown that the immune response against HSV-1 occurs mainly in the trigeminal ganglia (TG) and that Toll-like receptors 2 and 9 (TLR2/9) are important in mediating this response. It was also demonstrated that iNOS (nitric oxide synthase) and interleukin 1 beta (IL-1β) play an essential role in the defense against HSV-1 infection. Importantly, the present work aimed to identify the primary cells responsible for iNOS and IL-1β production and search for other important molecules and cells that might or might not depend on TLR2/9 receptors to mediate the immune response against HSV-1., Methods: C57BL/6 (wild type, WT) and TLR2/9 -/- mice were infected by the intranasal route with HSV-1 (1 × 10 6 p.f.u.). Cells were obtained from the TG and spleen tissues and the profile of immune cells was determined by flow cytometry in infected and mock infected WT and knockout mice. The percentage of cells producing iNOS, IL-1β, granzyme B and perforin was also determined by flow cytometry. Chemokine monocyte chemoattractant protein-1 (MCP1) was measured by Cytometric Bead Array (CBA) in the TG, spleen and lung. Expression of type I interferons (IFNs), interleukins (IL) 5 and 10, IL-1β and granzyme B were quantified by real time PCR., Results: The results indicate that dendritic cells (DCs) and monocytes/macrophages (Mo/Mϕ) were the main sources of IL-1β and iNOS, respectively, which, together with type I IFNs, were essential for the immune response against HSV-1. Additionally, we showed that granzyme B produced by CD8 + T and NK lymphocytes and MCP-1 were also important for this immune response. Moreover, our data indicate that the robust production of MCP-1 and granzyme B is either TLR-independent or down regulated by TLRs and occurs in the TG of TLR2/9 -/- infected mice., Conclusion: Taken together, our data provide strong evidence that the responses mediated by DCs, Mo/Mϕ, NK and CD8 + T lymphocytes through IL-1β, iNOS and granzyme B production, respectively, together with the production of type I IFN early in the infection, are crucial to host defense against HSV-1.

The dermis, a major reservoir of immune cells in immediate vicinity to the colonizing skin microflora, serves as an important site of host-pathogen interactions. Macrophages (Mϕ) are the most frequent resident immune cell type in the dermis. They protect the host from invasive infections by highly adapted bacteria, such as staphylococci via pattern recognition of bacterial effectors, phagocytosis, and recruitment of other myeloid cells from the blood. Already under homeostatic conditions, the dermal Mϕ population receives a dynamic input of monocytes invading from the bloodstream. This quantitative renewal is promoted further at the beginning of life, when prenatally seeded cells are rapidly replaced and in healing phases after injuries or infections. Here, we discuss the potential implications of the dynamic dermal Mϕ biology on the establishment and maintenance of immunity against Staphylococcus aureus, which can either be a harmless colonizer or an invasive pathogen. The understanding of the heterogeneity of the "mature" dermal Mϕ compartment driven both by the influx of differentiating monocytes and by a bone marrow-independent Mϕ persistence and expansion may help to explain failing immunity and immunopathology originating from the skin, the important interface between host and environment., (© Society for Leukocyte Biology.)

Skeletal muscle regeneration requires coordination between dynamic cellular populations and tissue microenvironments. Macrophages, recruited via CCR2, are essential for regeneration; however, the contribution of macrophages and the role of CCR2 on nonhematopoietic cells has not been defined. In addition, aging and sex interactions in regeneration and sarcopenia are unclear. Muscle regeneration was measured in young (3-6 mo), middle (11-15 mo), old (24-32 mo) male and female CCR2 -/- mice. Whereas age-related muscle atrophy/sarcopenia was present, regenerated myofiber cross-sectional area (CSA) in CCR2 -/- mice was comparably impaired across all ages and sexes, with increased adipocyte area compared with wild-type (WT) mice. CCR2 -/- mice myofibers achieved approximately one third of baseline CSA even 84 d after injury. Regenerated CSA and clearance of necrotic tissue were dependent on bone marrow-derived cellular expression of CCR2. Myogenic progenitor cells isolated from WT and CCR2 -/- mice exhibited comparable proliferation and differentiation capacity. The most striking cellular anomaly in injured muscle of CCR2 -/- mice was markedly decreased macrophages, with a predominance of Ly6C - anti-inflammatory monocytes/macrophages. Ablation of proinflammatory TLR signaling did not affect muscle regeneration or resolution of necrosis. Of interest, many proinflammatory, proangiogenic, and chemotactic cytokines were markedly elevated in injured muscle of CCR2 -/- relative to WT mice despite impairments in macrophage recruitment. Collectively, these results suggest that CCR2 on bone marrow-derived cells, likely macrophages, were essential to muscle regeneration independent of TLR signaling, aging, and sex. Decreased proinflammatory monocytes/macrophages actually promoted a proinflammatory microenvironment, which suggests that inflammaging was present in young CCR2 -/- mice., (© Society for Leukocyte Biology.)

Triggering receptor expressed on myeloid cells 1(TREM-1) is a newly identified member of the immunoglobulin superfamily and is extensively involved in the regulation of innate immunity. To determine the role of TREM-1 in innate antiviral immunity, we investigated TREM-1 expression and its downstream signaling effect in the murine bone marrow-derived macrophages or RAW264.7 macrophage-like mouse cell line by double-stranded RNA (dsRNA) stimulation. The level of TREM-1 expression was low at the baseline and could up-regulate markedly in dose- and time-dependent manners upon stimulation by dsRNA/poly IC. Inhibitor studies disclosed mitogen-activated protein kinase (MAPK) p38 and PI3K pathways were involved in dsRNA-induced up-regulation of TREM-1. Compared with lipopolysaccharide (LPS), the peak response of poly IC-induced TREM-1 expression is delayed, and cells pretreated with scrambled RNA presented higher expression of TREM-1 upon LPS challenge. After ligation with the agonist antibody, TREM-1 can potentiate type I interferon (IFN) production and antiviral inflammation induced by dsRNA, which is ralated to the enhanced phosphorylation of MAPKs and expression of RLRs and TLRs by TREM-1 ligation. This study is the first to show the regulatory role of TREM-1 in RLRs and TLRs expression, and these findings might enrich the understanding of the up-regulation mechanism and the function of TREM-1., (Copyright © 2016 Elsevier Inc. All rights reserved.)

TLRs represent an attractive target for the stimulation of myeloid cell production by HSPCs. We have previously demonstrated that HSPCs use TLR2 to sense Candida albicans in vivo and induce the production of macrophages. In this work, we used an in vitro model of HSPCs differentiation to investigate the functional consequences for macrophages of exposure of HSPCs to various PAMPs and C. albicans cells. Mouse HSPCs (Lin(-) cells) were cultured with M-CSF to induce macrophage differentiation, in the presence or absence of the following PRR agonists: Pam3CSK4 (TLR2 ligand), LPS (TLR4 ligand), depleted zymosan (which only activates Dectin-1), or C. albicans yeasts (which activate several PRRs, but principally TLR2 and Dectin-1). Our data show that these PAMPs differentially impact the anti-microbial function of the macrophages produced by the exposed HSPCs. Pure TLR2 and TLR4 ligands generate macrophages with a diminished ability to produce inflammatory cytokines. In contrast, HSPCs activation in response to C. albicans leads to the generation of macrophages that are better prepared to deal with the infection, as they produce higher amounts of inflammatory cytokines and have higher fungicidal capacity than control macrophages. Therefore, the tailored manipulation of the differentiation process may help to boost the innate immune response to infection., (Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Our previous studies have showed that tumor-derived autophagosomes (termed "DRibbles") induce B cell activation, resulting in antibody production and cytokine secretion. Unexpectedly, we found that unfractionated splenocytes produced a higher level of antibody and cytokine than that of purified B cells. In the current study, we investigated the role of accessory cells in DRibbles-induced B cell activation. We found that cognate macrophages, but not T cells, significantly enhanced the B cell activities. Such an enhancement required cell-cell contact. Furthermore, DRibbles stimulation up-regulated CD40L expression on macrophages, resulting in increased level of CD40 expressed on B cells. The accessory role of macrophages in DRibbles-activated B cells is critically dependent on the CD40/CD40L interaction. In addition, the effects of macrophages were found to be largely dependent on TLR4 and MyD88 signaling pathway. Finally, our results showed that macrophages were able to enhance the antigen presentation function of B cells for specific T cell stimulation. Thus, these results suggest that macrophages play an important accessory role for DRibbles-induced B cell immune function., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Toll-like receptors (TLR) 7 and 9 transduce a cellular signal through the MyD88-dependent pathway and induce the production of inflammatory mediators against microbial nucleotide components. The repeated stimulation of TLR4 leads to endotoxin tolerance, but the molecular mechanisms of tolerance induced through the costimulation of individual TLR has not yet been established, although endosomal TLRs share signaling pathways with TLR4. In the present study, mouse macrophages were simultaneously stimulated with the TLR7 agonist, gardiquimod (GDQ), and the TLR9 agonist, CpG ODN 1826, to examine the mechanism and effector functions of macrophage tolerance. Compared with individual stimulation, the costimulation of both TLRs reduced the secretion of TNF-α and IL-6 through the delayed activation of the NF-κB pathway; notably, IL-10 remained unchanged in costimulated macrophages. This tolerance reflected the early induction of suppressor of cytokine signaling-1 (SOCS-1), according to the detection of elevated TNF-α secretion and restored NF-κB signaling in response to the siRNA-mediated abrogation of SOCS-1 signaling. In addition, the restimulation of each TLRs using the same ligand significantly reduced the expression of both TLRs in endosomes. These findings revealed that the costimulation of TLR7 and TLR9 induced macrophage tolerance via SOCS-1, and the restimulation of each receptor or both TLR7 and TLR9 downregulated TLR expression through a negative feedback mechanisms that protects the host from excessive inflammatory responses. Moreover, the insufficient and impaired immune response in chronic viral infection might also reflect the repeated and simultaneous stimulation of those endosomal TLRs.

Problem: Microbial-driven responses in placenta are linked with adverse pregnancy outcomes. The role of Toll-like receptor (TLR) function in Hofbauer cells (HBCs) and fetal macrophages of the placental villous core remains understudied., Method of Study: Flow cytometry and immunohistochemistry (IHC) were used to establish the phenotype of HBCs. Regulation of cytokine secretion in response to treatment with TLR agonists and expression levels of TLRs and co-activators were compared in HBCs, placental fibroblasts (FIBs), and human umbilical vein endothelial cells (HUVECs) using ELISA and qPCR., Results: Although flow cytometry and IHC revealed HBCs to be M2 (anti-inflammatory) macrophages, LPS and polyinosinic: polycytidylic acid [poly (I:C)] treatments markedly enhanced IL-6 secretion by HBCs, and expression of TLR-2, TLR-3, TLR-4, CD14, and MD-2 was the highest in HBCs., Conclusion: These results indicate that although HBCs are M2 macrophages, inflammatory responses are induced through TLR-3 and TLR-4 in this cell type, suggesting a role in microbial-driven placental/fetal inflammation., (© 2014 The Authors. American Journal of Reproductive Immunology Published by John Wiley & Sons Ltd.)

This study compares the human immune systems of neonates and adults. Flow cytometric analysis was used to study the cellular phenotypes of cord blood (CB) and adult peripheral blood (APB). Luminex analysis was used to determine the levels of cytokines in cell culture supernatants. Our findings indicate that T cells in CB were mainly naïve and thus less responsive to PMA/ionomycin with the synthesis of cytokines. The percentages of CD3(+)CD4(+)CD25(high) and of CD3(+)CD4(+)CD25(dim) cells expressing chemokine receptors were different between CB and APB. TLR1, TLR6 and TLR9 expressions on NK and NKT cells also differed between CB and APB. CB monocyte-derived macrophages responded better than APB macrophages to TLR ligands with increased secretion of inflammatory cytokines, especially IL-6. The high levels of the inflammatory cytokines in cell culture supernatants of CB were mainly due to higher numbers of responsive macrophages, since dendritic cell numbers were lower in CB than APB., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

TLR22 is a fish-specific TLR that recognizes dsRNAs. In the present study, a TLR22 homologue gene from gilthead seabream (sbTLR22) was identified and characterized. The full coding sequence contained a single open-reading frame of 2895 nucleotides encoding a predicted protein of 964 amino acids in length. Its 3'-UTR was relatively long, 1380 nucleotides, and contained three AU-rich sequences frequently associated with mRNA instability. Functional studies showed that the sbTLR22 transcript had a short half-life, although the three AU-rich sequences in its 3'-UTR did not seem to be related with this fact. The sbTLR22 was highly expressed in the spleen, thymus and gills of healthy fish. After Vibrio anguillarum infection, the mRNA levels of sbTLR22 increased greatly in head kidney, blood and peritoneal exudate, but were only moderately induced in spleen and liver, suggesting the involvement of sbTLR22 in the immune response against bacterial infections. In addition, acidophilic granulocytes and macrophages, both considered professional phagocytes in seabream, displayed cell-type-specific sbTLR22 expression profiles when stimulated with different pathogen-associated molecular patterns (PAMPs). Although acidophilic granulocytes expressed sbTLR22, polyinosinic:polycytidylic acid (poly I:C) was unable to up-regulate the expression of this receptor. In contrast, poly I:C induced the expression of sbTLR22 in macrophages, in a process that was partially endosome-dependent. Taken together, our results suggest that sbTLR22 is involved in bacterial infection and might sense bacterial PAMPs., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The activation of TLRs by microbial molecules triggers intracellular-signaling cascades and the expression of cytokines such as IL-10. Il10 expression is tightly controlled to ensure effective immune responses, while preventing pathology. Maximal TLR-induction of Il10 transcription in macrophages requires signaling through the MAPKs, ERK, and p38. Signals via p38 downstream of TLR4 activation also regulate IL-10 at the post-transcriptional level, but whether this mechanism operates downstream of other TLRs is not clear. We compared the regulation of IL-10 production in TLR2 and TLR4-stimulated BM-derived macrophages and found different stability profiles for the Il10 mRNA. TLR2 signals promoted a rapid induction and degradation of Il10 mRNA, whereas TLR4 signals protected Il10 mRNA from rapid degradation, due to the activation of Toll/IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF) and enhanced p38 signaling. This differential post-transcriptional mechanism contributes to a stronger induction of IL-10 secretion via TLR4. Our study provides a molecular mechanism for the differential IL-10 production by TLR2- or TLR4-stimulated BMMs, showing that p38-induced stability is not common to all TLR-signaling pathways. This mechanism is also observed upon bacterial activation of TLR2 or TLR4 in BMMs, contributing to IL-10 modulation in these cells in an infection setting., (© 2013 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA Weinheim.)

Dectin-2, a C-type lectin receptor (CLR), plays an essential role in mediating nuclear factor-kappa B (NF-κB) activation and anti-fungal immunity in response to Candida albicans infection. However, the molecular mechanisms and function of Dectin-2 signaling in response to infection by the pathogenic fungus Aspergillus fumigatus have not been characterized. In order to characterize Dectin-2 signaling in response to A. fumigatus infection, activation of Dectin-2 was analyzed at both transcriptional and translational levels. Spleen tyrosine kinase (Syk) phosphorylation, NF-κB activation and cytokine production downstream of Dectin-2 activation were also investigated. In addition, Dectin-2-Syk function and its ability to mediate reactive oxygen species (ROS) production and elimination of A. fumigatus conidia was examined. We demonstrate that Syk is involved in Dectin-2-induced IκBα (inhibitor of kappa B protein) phosphorylation and NF-κB activation following A. fumigatus stimulation in a time dependent manner. Silencing of Dectin-2 and Syk as well as Syk inhibition blocks NF-κB activation and cytokine secretion. Furthermore, the killing of A. fumigatus conidia and ROS production are significantly affected by Dectin-2 or Syk silencing as well as Syk inhibition. Swelling and germination of the fungus followed by hyphae formation and not the resting and heat-inactivated form of A. fumigatus mediate the activation of Dectin-2 signaling. In conclusion, Syk plays an essential role in IκBα kinase phosphorylation, NF-κB activation, and ROS production mediated by Dectin-2 activation in response to A. fumigatus infection., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Activations of endosomal TLRs include TLR3, TLR7/8, and TLR9 stimulates the production of cytokines, such as type I interferons (IFNs), and therefore involves in virus-host interactions. In the present study, two equine anemia virus (EIAV) strains EIAVFDDV13 and EIAVFDDV3-8, which showed different induction on protective immunity, were compared regarding their ability to regulate the expression of endosomal TLRs, as well as type I IFNs, after infection of equine monocyte-derived macrophages (eMDMs). Our results showed that EIAVFDDV13 dramatically up-regulated the expression of TLR3 and IFNβ and less robustly up-regulated the expression of TRL9 and IFNα1, whereas EIAVFDDV3-8 induced significantly lower expression of type I IFN mRNA and protein and more strongly down-regulated the expression of TLR7 and TLR8. In addition, no significant differences in cell apoptosis were observed between these two strains. Given that the genomic variation of EIAVFDDV13 is considerably higher than that of molecular clone EIAVFDDV3-8, our results suggest that stronger TLR3 activation and increased INFβ production induced by the multi-species strain are associated with an effective vaccine-elicited protective immune response., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Several groups have shown that detection of microbial components by TLRs on hematopoietic stem and progenitor cells (HSPCs) instructs myeloid cell generation, raising interest in the possibility of targeting TLRs on HSPCs to boost myelopoiesis. However, although "TLR-derived" cells exhibit myeloid cell characteristics (phagocytosis, cytokine production, antigen presentation), it is not clear whether they are functionally equivalent to macrophages derived in the absence of TLR activation. Our in vitro and in vivo studies show that macrophages derived from mouse and human HSPC subsets (including stem cells) exposed to a TLR2 agonist prior to or during macrophage differentiation produce lower levels of inflammatory cytokines (TNF-α, IL-6, and IL-1β) and reactive oxygen species. This is in contrast to prior exposure of differentiated macrophages to the TLR2 agonist ("tolerance"), which suppresses inflammatory cytokine production, but elevates reactive oxygen species. Soluble factors produced following exposure of HSPCs to a TLR2 agonist can also act in a paracrine manner to influence the function of macrophages derived from unexposed HSPCs. Our data demonstrate that macrophage function can be influenced by TLR signaling in the HSPCs from which they are derived, and that this may impact the clinical utility of targeting TLRs on HSPCs to boost myelopoiesis., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

As a highly organized system, endo‐lysosomes play a crucial role in maintaining immune homeostasis. However, the mechanisms involved in regulating endo‐lysosome progression and subsequent inflammatory responses are not fully understood. By screening 103 E3 ubiquitin ligases in regulating endo‐lysosomal acidification, it is discovered that lysosomal RNF13 inhibits lysosome maturation and promotes inflammatory responses mediated by endosomal Toll‐like receptors (TLRs) in macrophages. Mechanistically, RNF13 mediates K48‐linked polyubiquitination of LAMP‐1 at residue K128 for proteasomal degradation. Upon TLRs activation, LAMP‐1 promotes lysosomes maturation, which accelerates lysosomal degradation of TLRs and reduces TLR signaling in macrophages. Furthermore, peripheral blood mononuclear cells (PBMCs) from patients with rheumatoid arthritis (RA) show increased RNF13 levels and decreased LAMP‐1 expression. Accordingly, the immunosuppressive agent hydroxychloroquine (HCQ) can increase the polyubiquitination of RNF13. Taken together, the study establishes a linkage between proteasomal and lysosomal degradation mechanisms for the induction of appropriate innate immune response, and offers a promising approach for the treatment of inflammatory diseases by targeting intracellular TLRs. [ABSTRACT FROM AUTHOR]

Abstract As a highly organized system, endo‐lysosomes play a crucial role in maintaining immune homeostasis. However, the mechanisms involved in regulating endo‐lysosome progression and subsequent inflammatory responses are not fully understood. By screening 103 E3 ubiquitin ligases in regulating endo‐lysosomal acidification, it is discovered that lysosomal RNF13 inhibits lysosome maturation and promotes inflammatory responses mediated by endosomal Toll‐like receptors (TLRs) in macrophages. Mechanistically, RNF13 mediates K48‐linked polyubiquitination of LAMP‐1 at residue K128 for proteasomal degradation. Upon TLRs activation, LAMP‐1 promotes lysosomes maturation, which accelerates lysosomal degradation of TLRs and reduces TLR signaling in macrophages. Furthermore, peripheral blood mononuclear cells (PBMCs) from patients with rheumatoid arthritis (RA) show increased RNF13 levels and decreased LAMP‐1 expression. Accordingly, the immunosuppressive agent hydroxychloroquine (HCQ) can increase the polyubiquitination of RNF13. Taken together, the study establishes a linkage between proteasomal and lysosomal degradation mechanisms for the induction of appropriate innate immune response, and offers a promising approach for the treatment of inflammatory diseases by targeting intracellular TLRs.

Probiotics have been reported to have immunomodulatory properties in the context of infectious disease and inflammation, although the underlying mechanisms are not fully understood. Here, we aimed to determine how different probiotic bacterial strains modulated macrophage function during TLR3 stimulation mimicking viral infection. We screened 14 different strains for their ability to modulate TNF‐α, IL‐6 IL‐10, IFN‐α, IFN‐β and IFN‐γ secretion in RAW 264.7 macrophages with or without poly(I:C) stimulation. Seven strains were selected for further analysis using primary porcine alveolar macrophages. In‐depth transcriptomic analysis on alveolar macrophages was conducted for two strains. Most strains induced a synergistic effect when co‐incubated with poly(I:C) resulting in increased levels of IL‐6 and TNF‐α secretion from RAW 264.7 cells. This synergistic effect was found to be TLR2 independent. Only strains of Bacillus spp. could induce this effect in alveolar macrophages. Transcriptomic analysis indicated that the increased TNF‐α secretion in alveolar macrophages after co‐incubation with poly(I:C) correlated with significant upregulation of TNF and IL23A‐related pathways. Collectively, our data show that probiotic bacteria possess strain‐dependent immunomodulatory properties that may be harnessed to enhance innate immune responses to pathogens. [ABSTRACT FROM AUTHOR]

To the current data, there have been 6,955,141 COVID-19-related deaths worldwide, reported to WHO. Toll-like receptors (TLRs) implicated in bacterial and virus sensing could be a crosstalk between activation of persistent innate-immune inflammation, and macrophage's sub-population alterations, implicated in cytokine storm, macrophage over-activation syndrome, unresolved Acute Respiratory Disease Syndrome (ARDS), and death. The aim of this study is to demonstrate the association between Toll-like-receptor-4 (TLR-4)-induced inflammation and macrophage imbalance in the lung inflammatory infiltrate of lethal COVID-19 disease. Twenty-five cases of autopsy lung tissues were studied by digital pathology-based immunohistochemistry to evaluate expression levels of TLR-4 (CD 284), pan-macrophage marker CD68 (clone KP1), sub-population marker related to alveolar macrophage Galectin-3 (GAL-3) (clone 9C4), and myeloid derived CD163 (clone MRQ-26), respectively. SARS-CoV-2 viral persistence has been evaluated by in situ hybridation (ISH) method. This study showed TLR-4 up-regulation in a subgroup of patients, increased macrophage infiltration in both Spike-1(+) and Spike-1(−) lungs (p < 0.0001), and a macrophage shift with important down-regulation of GAL-3(+) alveolar macrophages associated with Spike-1 persistence (p < 0.05), in favor of CD163(+) myeloid derived monocyte-macrophages. Data show that TLR-4 expression induces a persistent activation of the inflammation, with inefficient resolution, and pathological macrophage shift, thus explaining one of the mechanisms of lethal COVID-19. [ABSTRACT FROM AUTHOR]

Helicobacter pylori is one of the most successful gastric pathogens that has co-existed with human for centuries. H. pylori is recognized by the host immune system through human pattern recognition receptors (PRRs), such as toll-like receptors (TLRs), C-type lectin like receptors (CLRs), NOD-like receptors (NLRs), and RIG-I-like receptors (RLRs), which activate downstream signaling pathways. Following bacterial recognition, the first responders of the innate immune system, including neutrophils, macrophages, and dendritic cells, eradicate the bacteria through phagocytic and inflammatory reaction. This review provides current understanding of the interaction between the innate arm of host immunity and H. pylori, by summarizing H. pylori recognition by PRRs, and the subsequent signaling pathway activation in host innate immune cells. [ABSTRACT FROM AUTHOR]

Leptospira interrogans are pathogenic bacteria responsible for leptospirosis, a zoonosis impacting 1 million people per year worldwide. Leptospires can infect all vertebrates, but not all hosts develop similar symptoms. Human and cattle may suffer from mild to acute illnesses and are therefore considered as sensitive to leptospirosis. In contrast, mice and rats remain asymptomatic upon infection, although they get chronically colonized in their kidneys. Upon infection, leptospires are stealth pathogens that partially escape the recognition by the host innate immune system. Although leptospires are mainly extracellular bacteria, it was suggested that they could also replicate within macrophages. However, contradictory data in the current literature led us to reevaluate these findings. Using a gentamicin-protection assay coupled to high-content (HC) microscopy, we observed that leptospires were internalized in vivo upon peritoneal infection of C57BL/6J mice. Additionally, three different serotypes of pathogenic L. interrogans and the saprophytic L. biflexa actively infected both human (PMA differentiated) THP1 and mouse RAW264.7 macrophage cell lines. Next, we assessed the intracellular fate of leptospires using bioluminescent strains, and we observed a drastic reduction in the leptospiral intracellular load between 3 h and 6 h post-infection, suggesting that leptospires do not replicate within these cells. Surprisingly, the classical macrophage microbicidal mechanisms (phagocytosis, autophagy, TLR-mediated ROS, and RNS production) were not responsible for the observed decrease. Finally, we demonstrated that the reduction in the intracellular load was associated with an increase of the bacteria in the supernatant, suggesting that leptospires exit both human and murine macrophages. Overall, our study reevaluated the intracellular fate of leptospires and favors an active entrance followed by a rapid exit, suggesting that leptospires do not have an intracellular lifestyle in macrophages. [ABSTRACT FROM AUTHOR]

Leptospira interrogans are pathogenic bacteria responsible for leptospirosis, a zoonosis impacting 1 million people per year worldwide. Leptospires can infect all vertebrates, but not all hosts develop similar symptoms. Human and cattle may suffer from mild to acute illnesses and are therefore considered as sensitive to leptospirosis. In contrast, mice and rats remain asymptomatic upon infection, although they get chronically colonized in their kidneys. Upon infection, leptospires are stealth pathogens that partially escape the recognition by the host innate immune system. Although leptospires are mainly extracellular bacteria, it was suggested that they could also replicate within macrophages. However, contradictory data in the current literature led us to reevaluate these findings. Using a gentamicin–protection assay coupled to high-content (HC) microscopy, we observed that leptospires were internalized in vivo upon peritoneal infection of C57BL/6J mice. Additionally, three different serotypes of pathogenic L. interrogans and the saprophytic L. biflexa actively infected both human (PMA differentiated) THP1 and mouse RAW264.7 macrophage cell lines. Next, we assessed the intracellular fate of leptospires using bioluminescent strains, and we observed a drastic reduction in the leptospiral intracellular load between 3 h and 6 h post-infection, suggesting that leptospires do not replicate within these cells. Surprisingly, the classical macrophage microbicidal mechanisms (phagocytosis, autophagy, TLR–mediated ROS, and RNS production) were not responsible for the observed decrease. Finally, we demonstrated that the reduction in the intracellular load was associated with an increase of the bacteria in the supernatant, suggesting that leptospires exit both human and murine macrophages. Overall, our study reevaluated the intracellular fate of leptospires and favors an active entrance followed by a rapid exit, suggesting that leptospires do not have an intracellular lifestyle in macrophages.

Escalated innate immunity plays a critical role in SARS-CoV-2 pathology; however, the molecular mechanism is incompletely understood. Thus, we aim to characterize the molecular mechanism by which SARS-CoV-2 Spike protein advances human macrophage (Mϴ) inflammatory and glycolytic phenotypes and uncover novel therapeutic strategies. We found that human Mϴs exposed to Spike protein activate IRAK4 phosphorylation. Blockade of IRAK4 in Spike protein-stimulated Mϴs nullifies signaling of IRAK4, AKT, and baseline p38 without affecting ERK and NF-κB activation. Intriguingly, IRAK4 inhibitor (IRAK4i) rescues the SARS-CoV-2-induced cytotoxic effect in ACE2+HEK 293 cells. Moreover, the inflammatory reprogramming of Mϴs by Spike protein was blunted by IRAK4i through IRF5 and IRF7, along with the reduction of monokines, IL-6, IL-8, TNFα, and CCL2. Notably, in Spike protein-stimulated Mϴs, suppression of the inflammatory markers by IRAK4i was coupled with the rebalancing of oxidative phosphorylation over metabolic activity. This metabolic adaptation promoted by IRAK4i in Spike protein-activated Mϴs was shown to be in part through constraining PFKBF3, HIF1α, cMYC, LDHA, lactate expression, and reversal of citrate and succinate buildup. IRAK4 knockdown could comparably impair Spike protein-enhanced inflammatory and metabolic imprints in human Mϴs as those treated with ACE2, TLR2, and TLR7 siRNA. Extending these results, in murine models, where human SARS-CoV-2 Spike protein was not recognized by mouse ACE2, TLRs were responsible for the inflammatory and glycolytic responses instigated by Spike protein and were dysregulated by IRAK4i therapy. In conclusion, IRAK4i may be a promising strategy for severe COVID-19 patients by counter-regulating ACE2 and TLR-mediated Mϴ hyperactivation. IRAK4i therapy counteracts Mϴ inflammatory and glycolytic reprogramming triggered by Spike protein. This study illustrates that SARS-CoV-2 Spike protein activates IRAK4 signaling via ACE2 as well as TLR2 and TLR7 sensing in human Mϴs. Remarkably, IRAK4i treatment can dysregulate both ACE-dependent and independent (via TLR sensing) SARS-CoV-2 Spike protein-activated inflammatory and metabolic imprints. [ABSTRACT FROM AUTHOR]

Fatty liver disease, characterized by excessive inflammation and lipid deposition, is becoming one of the most prevalent liver metabolic diseases worldwide owing to the increasing global incidence of obesity. However, the underlying mechanisms of fatty liver disease are poorly understood. Accumulating evidence suggests that hepatic macrophages, specifically Kupffer cells (KCs), act as key players in the progression of fatty liver disease. Thus, it is essential to examine the current evidence of the roles of hepatic macrophages (both KCs and monocyte-derived macrophages). In this review, we primarily address the heterogeneities and multiple patterns of hepatic macrophages participating in the pathogenesis of fatty liver disease, including Toll-like receptors (TLRs), NLRP3 inflammasome, lipotoxicity, glucotoxicity, metabolic reprogramming, interaction with surrounding cells in the liver, and iron poisoning. A better understanding of the diverse roles of hepatic macrophages in the development of fatty liver disease may provide a more specific and promising macrophage-targeting therapeutic strategy for inflammatory liver diseases.

Seven non-toxigenic C. diphtheriae strains and one toxigenic strain were analyzed with regard to their interaction with murine macrophages (BMM) and human THP-1 macrophage-like cells. Proliferation assays with BMM and THP-1 revealed similar intracellular CFUs for C. diphtheriae strains independent of the host cell. Strain ISS4060 showed highest intracellular CFUs, while the toxigenic DSM43989 was almost not detectable. This result was confirmed by TLR 9 reporter assays, showing a low signal for DSM43989, indicating that the bacteria are not endocytosed. In contrast, the non-pathogenic C. glutamicum showed almost no intracellular CFUs independent of the host cell, but was recognized by TLR9, indicating that the bacteria were degraded immediately after endocytosis. In terms of G-CSF and IL-6 production, no significant differences between BMM and THP-1 were observed. G-CSF production was considerably higher than IL-6 for all C. diphtheriae strains and the C. glutamicum did not induce high cytokine secretion in general. Furthermore, all corynebacteria investigated in this study were able to induce NFκB signaling but only viable C. diphtheriae strains were able to cause host cell damage, whereas C. glutamicum did not. The absence of Mincle resulted in reduced G-CSF production, while no influence on the uptake of the bacteria was observed. In contrast, when MyD88 was absent, both the uptake of the bacteria and cytokine production were blocked. Consequently, phagocytosis only occurs when the TLR/MyD88 pathway is functional, which was also supported by showing that all corynebacteria used in this study interact with human TLR2.

Leptospira interrogans are pathogenic bacteria responsible for leptospirosis, a worldwide zoonosis. All vertebrates can be infected, and some species like humans are susceptible to the disease whereas rodents such as mice are resistant and become asymptomatic renal carriers. Leptospires are stealth bacteria that are known to escape several immune recognition pathways and resist killing mechanisms. We recently published that leptospires may survive intracellularly in and exit macrophages, avoiding xenophagy, a pathogen-targeting form of autophagy. Interestingly, the latter is one of the antimicrobial mechanisms often highjacked by bacteria to evade the host immune response. In this study we explored whether leptospires subvert the key molecular players of autophagy to facilitate infection. We showed in macrophages that leptospires triggered a specific accumulation of autophagy-adaptor p62 in puncta-like structures, without altering autophagic flux. We demonstrated that Leptospira -induced p62 accumulation is a passive mechanism depending on the leptospiral virulence factor LPS signaling via TLR4/TLR2. p62 is a central pleiotropic protein, also mediating cell stress and death, via the translocation of transcription factors. We demonstrated that Leptospira -driven accumulation of p62 induced the translocation of transcription factor NRF2, a key player in the anti-oxidant response. However, NRF2 translocation upon Leptospira infection did not result as expected in antioxydant response, but dampened the production of inflammatory mediators such as iNOS/NO, TNF and IL6. Overall, these findings highlight a novel passive bacterial mechanism linked to LPS and p62/NRF2 signaling that decreases inflammation and contributes to the stealthiness of leptospires. [ABSTRACT FROM AUTHOR]

Abstract Background Toll-like receptors (TLRs) and macrophages play an important role in rheumatoid arthritis (RA). Currently, it is not clear whether inflammatory M1 or anti-inflammatory M2 predominate among the resident macrophages in the synovium. In the present study, we set out to investigate the impact of TLR stimulation on monocyte-derived M1 and M2 macrophage function and phenotype by mimicking the exposure to abundant TLR agonists as occurs in the context of RA. The response of macrophage subsets to TLR2 and TLR4 activation was evaluated on cluster of differentiation (CD) marker profile; cytokine secretion; gene expression; and NF-κB, interferon regulatory factors 3 and 7 (IRF3/7), and mitogen-activated protein kinase (MAPK) activation. Methods Human monocytes were isolated from peripheral blood of healthy individuals and patients with RA and differentiated into M1-like and M2-like macrophages by granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF), respectively. Cells were either (1) stimulated with TLR ligands Pam3 or lipopolysaccharide (LPS) or (2) classically activated via interferon (IFN)-γ/LPS. Cytokine production was measured by enzyme-linked immunosorbent assay, and gene expression was measured by qPCR. Cells were stained for CD markers and analyzed by fluorescence-activated cell sorting. NF-κB, IRF3/7, and MAPKs were detected by Western blotting. Results Monocyte-derived macrophages of healthy donors (HD) or patients with RA displayed comparable subset-specific phenotypes upon exposure to TLR agonists. CD14 and CD163 marker expression on M2 macrophages did not change upon TLR2 and TLR4 engagement. By contrast, M2 gene markers HMOX1, FOLR2, and SLC40A1 were decreased. Importantly, M2 macrophages derived from HD or patients with RA showed both a decreased ratio of interleukin (IL)-10/IL-6 and IL-10/IL-8 upon stimulation with TLR2 ligand Pam3 compared with TLR4 ligand LPS. Gene expression of TLR2 was increased, whereas TLR4 expression was decreased, by TLR ligand stimulation. MAPKs p38, extracellular signal-regulated kinase 1/2, and c-Jun N-terminal kinase were activated more strongly in M2 than in M1 macrophages by Pam3 or LPS. Conclusions We show that the anti-inflammatory activity of M2 macrophages is reduced in the presence of abundant TLR2 ligands without significant changes in cell surface markers. Thus, the classical M1/M2 paradigm based on cellular markers does not apply to macrophage functions in inflammatory conditions such as RA.

A zoonotic pathogen, Brucella spp. is the causative agent of brucellosis, which results in abortion and loss in milk production in domestic animals, and undulant fever, osteoarticular pain and splenomegaly in humans. Due to the capability of the bacteria to modulate the host cell functions and survive in macrophages, early detection and eradication of the intracellular bacteria has received significant attention. Moreover, understanding the immunological alterations in Brucella infection is crucial to help develop control measures. Cytokines and toll-like receptors (TLRs) are some of the major compounds that play important roles in modulating the innate immunity and acquired immunity in host after infection. In this study, therefore, human leukemic monocyte cells (THP-1 cells) were stimulated with five Brucella abortus cellular components: outer membrane protein 10 (OMP10), outer membrane protein 19 (OMP19), thiamine transporter substrate-binding protein (TbpA), arginase (RocF), and malate dehydrogenase (Mdh). Post stimulation, the cytokine productions and TLR expressions in the cells were evaluated at different time points (12 h and 24 h), and analyzed using ELISA and real time RT-PCR, respectively. In the production of cytokines, it was observed that the production of TNF-α and IL-6 was highly induced in THP-1 cells stimulated with five recombinant protein antigens. Also, TLR8 was induced in a time-dependent manner after stimulation with two recombinant proteins, rOMP19 and rMdh, until 24 h. These results suggest that the two B. abortus antigens, rOMP19 and rMdh, might be involved in TLR8 signaling pathway in THP-1 cells in a time-dependent manner. These two proteins are therefore potentially effective antigen candidates which would help to provide better understandings of the immune responses after Brucella infection. [ABSTRACT FROM AUTHOR]

Background: Herpes simplex virus type 1 (HSV-1) cause not only mild symptoms but also blindness and encephalitis. It was previously shown that the immune response against HSV-1 occurs mainly in the trigeminal ganglia (TG) and that Toll-like receptors 2 and 9 (TLR2/9) are important in mediating this response. It was also demonstrated that iNOS (nitric oxide synthase) and interleukin 1 beta (IL-1β) play an essential role in the defense against HSV-1 infection. Importantly, the present work aimed to identify the primary cells responsible for iNOS and IL-1β production and search for other important molecules and cells that might or might not depend on TLR2/9 receptors to mediate the immune response against HSV-1. Methods: C57BL/6 (wild type, WT) and TLR2/9-/- mice were infected by the intranasal route with HSV-1 (1 × 106 p.f.u.). Cells were obtained from the TG and spleen tissues and the profile of immune cells was determined by flow cytometry in infected and mock infected WT and knockout mice. The percentage of cells producing iNOS, IL-1β, granzyme B and perforin was also determined by flow cytometry. Chemokine monocyte chemoattractant protein-1 (MCP1) was measured by Cytometric Bead Array (CBA) in the TG, spleen and lung. Expression of type I interferons (IFNs), interleukins (IL) 5 and 10, IL-1β and granzyme B were quantified by real time PCR. Results: The results indicate that dendritic cells (DCs) and monocytes/macrophages (Mo/Mϕ) were the main sources of IL-1β and iNOS, respectively, which, together with type I IFNs, were essential for the immune response against HSV-1. Additionally, we showed that granzyme B produced by CD8+ T and NK lymphocytes and MCP-1 were also important for this immune response. Moreover, our data indicate that the robust production of MCP-1 and granzyme B is either TLR-independent or down regulated by TLRs and occurs in the TG of TLR2/9-/- infected mice. Conclusion: Taken together, our data provide strong evidence that the responses mediated by DCs, Mo/Mϕ, NK and CD8+ T lymphocytes through IL-1β, iNOS and granzyme B production, respectively, together with the production of type I IFN early in the infection, are crucial to host defense against HSV-1. [ABSTRACT FROM AUTHOR]

Respiratory syncytial virus (RSV) infection can increase the production of IL-33 in lungs of mice. However, little is known about cellular source of IL-33, particularly the types of IL-33-producing cells in innate immune cells during RSV infection. In this study, by using BALB/c mice that were infected intranasally with RSV, it became clear that RSV infection can enhance not only the number of IL-33 + -alveolar macrophages (AMs) and dendritic cells (DCs), but also the expression of IL-33 mRNA in these cells, suggesting that innate immune cells participate in the production of IL-33. Indeed, in vitro experiments by using murine cell lines found that RSV infection results in more expression of IL-33 mRNA in AMs and DCs, further confirming that these cell types may be an important source of IL-33 during RSV infection. It should be noted that the expression of mRNA for TLR3 and TLR7 was up-regulated in pulmonary AMs during RSV infection. Blockade of TLRs by TLR3 or TLR7 antagonist significantly reduces the levels of IL-33 mRNA in AMs and DCs, suggesting that RSV-induced IL-33 production might be TLRs-dependent manner. Although the expression of TLRs mRNA in pulmonary interstitial macrophages (IMs) was enhanced after RSV infection, stimulation with agonists or inactivated RSV cannot alter the expression of IL-33 mRNA in IMs, suggesting that pulmonary IMs may not be a source of IL-33 during RSV infection. Thus, these results demonstrate that during RSV infection, respiratory macrophages and dendritic cells mediate the production of IL-33 in a TLR-dependent manner. [ABSTRACT FROM AUTHOR]

For many years, the innate immunity was of less interest than the adaptive immunity because it was perceived to have secondary importance in the functionality of the immune system. During the past decades, with the advancement of knowledge about innate immune system, interest in innate immunity has grown dramatically and thus its function has been extensively studied. Innate immunity plays fundamental roles in the initiation and induction of adaptive immune responses. It consists of several cells and receptors including natural killer (NK) cells, macrophages (MQs), dendritic cells (DCs) and pattern recognition receptors (PRRs). Two decades ago, Toll like receptors (TLRs) family was known as one of the important PRRs with unique functions especially in protection against invading pathogens. Since the female reproductive tract has access to the outside environment and has a unique interaction with different pathogens whether invading microorganisms or normal flora, allogenic sperm and semi allogenic fetus, it has an essential need for effective immune responses. It has therefore been suggested that TLRs may play important roles in the immune regulation of the female reproductive tract. In addition, it has been demonstrated that immune disturbance may be responsible for some adverse pregnancy outcomes such as preeclampsia (PE), recurrent spontaneous abortion (RSA) and intrauterine growth restriction (IUGR). Our focus in this review is to show the importance of TLRs in pregnancy with emphasis on the expression of these receptors in different tissues related to pregnancy [ABSTRACT FROM AUTHOR]

Abstract: Quantum dots (QDs) are nano-sized semiconductors. Previously, intratracheal instillation of QD705s induces persistent inflammation in mouse lungs. In our present study, QD705-COOH and QD705-PEG activated NF-κB and increased monocyte chemotactic protein-1 (MCP-1) expression in macrophages RAW264.7 via MyD88 dependent Toll-like receptor (TLR) signaling pathways. MyD88 is an adapter protein for most TLRs to activate NF-κB. Silencing expression of MyD88 or p65 with siRNA or co-treatment with a NF-κB inhibitor tremendously abolished QD705s-induced NF-κB activity and MCP-1 expression. The involved TLRs might locate either on the cell surface or inside of cells. Co-treatment with a TLR4 inhibitor completely prevented MCP-1 induction by QD705-PEG. Nevertheless, QD705-COOH readily entered cells, and co-treatment with either inhibitors of endocytosis or intracellular TLRs prevented MCP-1 induction. These findings indicate that, depending on their surface modification, OD705s activate MyD88 dependent-TLRs at the surface or inside of the cells, which is an important mechanism for nanoparticles-induced inflammatory responses. But other MyD88-independent pathways may also involve in these responses. [Copyright &y& Elsevier]

Highlights: [•] Activin A inhibited expression of TLR4, did not TLR2, on the activated macrophages. [•] Smad3 knock-down attenuated the inhibitory effect of activin A on TLR4 expression. [•] LPS promoted expression of activin type IIA receptor on macrophages. [ABSTRACT FROM AUTHOR]