LPS Induces Opposing Memory-like Inflammatory Responses in Mouse Bone Marrow Neutrophils

A growing body of evidence suggests that innate immune cells can respond in a memory-like (adaptive) fashion, which is referred to as trained immunity. Only few in vivo studies have shown training effects in neutrophils; however, no in vitro setup has been established to study the induction of trained immunity or tolerance in neutrophils by microbial agents. In light of their short lifespan (up to 48 h), we suggest to use the term trained sensitivity for neutrophils in an in vitro setting. Here, we firstly describe a feasible two-hit model, using different doses of lipopolysaccharide (LPS) in bone marrow neutrophils. We found that low doses (10 pg/mL) induce pro-inflammatory activation (trained sensitivity), whereas priming with high doses (100 ng/mL) leads to suppression of pro-inflammatory mediators such as TNF-α or IL-6 (tolerance) (p < 0.05). On a functional level, trained neutrophils displayed increased phagocytic activity and LFA-1 expression as well as migrational capacity and CD11a expression, whereas tolerant neutrophils show contrasting effects in vitro. Mechanistically, TLR4/MyD88/PI3Ks regulate the activation of p65, which controls memory-like responses in mouse bone marrow neutrophils (p < 0.05). Our results open a new window for further in vitro studies on memory-like inflammatory responses of short-lived innate immune cells such as neutrophils.     

Phosphorus flow in production of soy protein concentrate and isolate from defatted soybean flour

Implications of excess phosphorus (P) in waste streams obtained from soy-based protein preparation processes on the environment and their potential utilization as P-source are two significant understudied areas. Soybean-based protein ingredients for food products retain comparatively enhanced functional properties and are cheaper than other plant-based proteins. Soybean protein can be extracted and utilized as a food ingredient primarily by preparing soy protein concentrates (SPC) and soy protein isolates (SPI) from soybean meal/defatted soy flour (DSF). In a typical soybean processing facility, along with the soy products and soy-protein preparations, the recovery of phosphorus as a coproduct will enhance the economic feasibility of the overall process as the recovered P can be used as fertilizer. In this study, the SPC and SPI were prepared from the DSF following widely used conventional protocols and P flow in these processes was tracked. In SPC production, ~59% of the total P was retained with SPC and ~34% was in the aqueous waste streams. For SPI process ~24% of total P was retained with SPI and ~59% went in the waste solid residue (~40%) and aqueous streams (~19%). About 80%–89% P removal from the waste aqueous streams was achieved by Ca-phytate precipitation. This work demonstrated that in the process of SPC and SPI preparation the phosphorus from the waste aqueous streams can be precipitated out to avoid subsequent eutrophication and the waste solid residue with ~40% P can be reused as a P-fertilizer as other applications of this residue are unspecified.     

PCSK9 Induces Tissue Factor Expression by Activation of TLR4/NFkB Signaling

Proprotein convertase subtilisin kexin 9 (PCSK9) increases LDL cholesterol (C) concentration by accelerating the hepatic degradation of the LDL receptor (R) thus promoting atherogenesis. The molecule, however, also exerts proinflammatory effects independent of circulating LDL-C by enhancing local cytokine production and activation of NFkB, a process that might involve Toll-like receptor 4 (TLR4), a crucial component of the innate immunity system. Tissue factor (TF), a glycoprotein which plays an essential role in coagulation and inflammation, is rapidly induced by circulating monocytes stimulated by proinflammatory agents through NFkB-dependent mechanisms. The aims of our study were (1) to assess whether PCSK9 may induce monocytic TF expression and (2) to evaluate whether the TLR4/NFkB signaling pathway may contribute to that effect. Experiments were carried out in peripheral blood mononuclear cells (PBMCs), THP-1 cells, and HEK293 cells transfected with plasmids encoding the human TLR4 complex. PCSK9 increased procoagulant activity (PCA), mRNA and TF protein expression in both PBMCs and THP-1 cultures. Pre-treatment with inhibitors of TLR4/NFkB signaling such as LPS-RS, CLI-095, and BAY 11-7082, downregulated PCSK9-induced TF expression. A similar effect was obtained by incubating cell cultures with anti-PCSK9 human monoclonal antibody. In TLR4-HEK293 cells, PCSK9 activated the TLR4/NFkB signaling pathway to an extent comparable to LPS, the specific agonist of TLR4s and quantitative confocal microscopy documented the colocalization of PCSK9 and TLR4s. In conclusion, PCSK9 induces TF expression through activation of TLR4/NFkB signaling.     

Exposome and Immunity Training: How Pathogen Exposure Order Influences Innate Immune Cell Lineage Commitment and Function

Immune memory is a defining characteristic of adaptive immunity, but recent work has shown that the activation of innate immunity can also improve responsiveness in subsequent exposures. This has been coined “trained immunity” and diverges with the perception that the innate immune system is primitive, non-specific, and reacts to novel and recurrent antigen exposures similarly. The “exposome” is the cumulative exposures (diet, exercise, environmental exposure, vaccination, genetics, etc.) an individual has experienced and provides a mechanism for the establishment of immune training or immunotolerance. It is becoming increasingly clear that trained immunity constitutes a delicate balance between the dose, duration, and order of exposures. Upon innate stimuli, trained immunity or tolerance is shaped by epigenetic and metabolic changes that alter hematopoietic stem cell lineage commitment and responses to infection. Due to the immunomodulatory role of the exposome, understanding innate immune training is critical for understanding why some individuals exhibit protective phenotypes while closely related individuals may experience immunotolerant effects (e.g., the order of exposure can result in completely divergent immune responses). Research on the exposome and trained immunity may be leveraged to identify key factors for improving vaccination development, altering inflammatory disease development, and introducing potential new prophylactic treatments, especially for diseases such as COVID-19, which is currently a major health issue for the world. Furthermore, continued exposome research may prevent many deleterious effects caused by immunotolerance that frequently result in host morbidity or mortality.     

Comparison of Cellular Uptake and Inflammatory Response via Toll-Like Receptor 4 to Lipopolysaccharide and Titanium Dioxide Nanoparticles

The innate immune response is the earliest cellular response to infectious agents and mediates the interactions between microbes and cells. Toll-like receptors (TLRs) play an important role in these interactions. We have already shown that TLRs are involved with the uptake of titanium dioxide nanoparticles (TiO₂ NPs) and promote inflammatory responses. In this paper, we compared role of cellular uptake and inflammatory response via TLR 4 to lipopolysaccharide (LPS) and TiO₂ NPs. In the case of LPS, LPS binds to LPS binding protein (LBP) and CD 14, and then this complex binds to TLR 4. In the case of TiO₂ NPs, the necessity of LBP and CD 14 to induce the inflammatory response and for uptake by cells was investigated using over-expression, antibody blocking, and siRNA knockdown experiments. Our results suggested that for cellular uptake of TiO₂ NPs, TLR 4 did not form a complex with LBP and CD 14. In the TiO₂ NP-mediated inflammatory response, TLR 4 acted as the signaling receptor without protein complex of LPS, LBP and CD 14. The results suggested that character of TiO₂ NPs might be similar to the complex of LPS, LBP and CD 14. These results are important for development of safer nanomaterials.     

Interaction of TLR4 and TLR8 in the Innate Immune Response against Mycobacterium Tuberculosis

The interaction and crosstalk of Toll-like receptors (TLRs) is an established pathway in which the innate immune system recognises and fights pathogens. In a single nucleotide polymorphisms (SNP) analysis of an Indian cohort, we found evidence for both TLR4-399T and TRL8-1A conveying increased susceptibility towards tuberculosis (TB) in an interdependent manner, even though there is no established TLR4 ligand present in Mycobacterium tuberculosis (Mtb), which is the causative pathogen of TB. Docking studies revealed that TLR4 and TLR8 can build a heterodimer, allowing interaction with TLR8 ligands. The conformational change of TLR4-399T might impair this interaction. With immunoprecipitation and mass spectrometry, we precipitated TLR4 with TLR8-targeted antibodies, indicating heterodimerisation. Confocal microscopy confirmed a high co-localisation frequency of TLR4 and TLR8 that further increased upon TLR8 stimulation. The heterodimerisation of TLR4 and TLR8 led to an induction of IL12p40, NF-κB, and IRF3. TLR4-399T in interaction with TLR8 induced an increased NF-κB response as compared to TLR4-399C, which was potentially caused by an alteration of subsequent immunological pathways involving type I IFNs. In summary, we present evidence that the heterodimerisation of TLR4 and TLR8 at the endosome is involved in Mtb recognition via TLR8 ligands, such as microbial RNA, which induces a Th1 response. These findings may lead to novel targets for therapeutic interventions and vaccine development regarding TB.     

Toll-like Receptor Response to Hepatitis C Virus Infection: A Recent Overview

Hepatitis C virus (HCV) infection remains a major global health burden, causing chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Toll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors that detect pathogen-associated molecular patterns and activate downstream signaling to induce proinflammatory cytokine and chemokine production. An increasing number of studies have suggested the importance of TLR responses in the outcome of HCV infection. However, the exact role of innate immune responses, including TLR response, in controlling chronic HCV infection remains to be established. A proper understanding of the TLR response in HCV infection is essential for devising new therapeutic approaches against HCV infection. In this review, we discuss the progress made in our understanding of the host innate immune response to HCV infection, with a particular focus on the TLR response. In addition, we discuss the mechanisms adopted by HCV to avoid immune surveillance mediated by TLRs.     

The Promoter of the Immune-Modulating Gene TIR-Containing Protein C of the Uropathogenic Escherichia coli Strain CFT073 Reacts to the Pathogen’s Environment

The TIR-containing protein C (TcpC) of the uropathogenic Escherichia coli strain CFT073 modulates innate immunity by interfering with the Toll-like receptor and NALP3 inflammasome signaling cascade. During a urinary tract infection the pathogen encounters epithelial and innate immune cells and replicates by several orders of magnitude. We therefore analyzed whether these cell types and also the density of the pathogen would induce the recently defined promoter of the CFT073 tcpC gene to, in time, dampen innate immune responses. Using reporter constructs we found that the uroepithelial cell line T24/83 and the monocytic cell line THP-1 induced the tcpC promoter. Differentiation of monocytic THP-1 cells to macrophages increased their potential to switch on the promoter. Cell-associated CFT073 displayed the highest promoter activity. Since potassium represents the most abundant intracellular ion and is secreted to induce the NLRP3 inflammasome, we tested its ability to activate the tcpC promoter. Potassium induced the promoter with high efficiency. Sodium, which is enriched in the renal cortex generating an antibacterial hypersalinity, also induced the tcpC promoter. Finally, the bacterial density modulated the tcpC promoter activity. In the search for promoter-regulating proteins, we found that the DNA-binding protein H-NS dampens the promoter activity. Taken together, different cell types and salts, present in the kidney, are able to induce the tcpC promoter and might explain the mechanism of TcpC induction during a kidney infection with uropathogenic E. coli strains.     

Modified Low Density Lipoprotein and Lipoprotein-Containing Circulating Immune Complexes as Diagnostic and Prognostic Biomarkers of Atherosclerosis and Type 1 Diabetes Macrovascular Disease

In atherosclerosis; blood low-density lipoproteins (LDL) are subjected to multiple enzymatic and non-enzymatic modifications that increase their atherogenicity and induce immunogenicity. Modified LDL are capable of inducing vascular inflammation through activation of innate immunity; thus, contributing to the progression of atherogenesis. The immunogenicity of modified LDL results in induction of self-antibodies specific to a certain type of modified LDL. The antibodies react with modified LDL forming circulating immune complexes. Circulating immune complexes exhibit prominent immunomodulatory properties that influence atherosclerotic inflammation. Compared to freely circulating modified LDL; modified LDL associated with the immune complexes have a more robust atherogenic and proinflammatory potential. Various lipid components of the immune complexes may serve not only as diagnostic but also as essential predictive markers of cardiovascular events in atherosclerosis. Accumulating evidence indicates that LDL-containing immune complexes can also serve as biomarker for macrovascular disease in type 1 diabetes.     

Helicobacter pylori Neutrophil-Activating Protein Directly Interacts with and Activates Toll-like Receptor 2 to Induce the Secretion of Interleukin-8 from Neutrophils and ATRA-Induced Differentiated HL-60 Cells

Helicobacter pylori neutrophil-activating protein (HP-NAP)-induced production of reactive oxygen species (ROS) by neutrophils and monocytes is regulated by pertussis toxin (PTX)-sensitive G proteins, whereas HP-NAP-induced cytokine secretion by monocytes is mediated by Toll-like receptor 2 (TLR2). However, it is unclear whether TLR2 participates in HP-NAP-induced cytokine secretion by neutrophils. Here, all-trans retinoic acid (ATRA)-induced differentiated HL-60 cells were first employed as a neutrophil model to investigate the molecular mechanisms underlying neutrophil responses to HP-NAP. HP-NAP-induced ROS production in ATRA-induced differentiated HL-60 cells is mediated by the PTX-sensitive heterotrimeric G protein-dependent activation of extracellular signal-regulated kinase 1/2 and p38-mitogen-activated protein kinase, which is consistent with the findings reported for human neutrophils. Next, whether TLR2 participated in HP-NAP-induced secretion of interleukin-8 (IL-8) was investigated in neutrophils and ATRA-induced differentiated HL-60 cells. In both cells, TLR2 participated in HP-NAP-induced IL-8 secretion but not HP-NAP-induced ROS production. Interestingly, PTX-sensitive G proteins also contributed to the HP-NAP-induced secretion of IL-8 from neutrophils and the differentiated HL-60 cells. Our ELISA-based binding assay further revealed the competitive binding of Pam₃CSK₄, a TLR2 agonist, and HP-NAP to TLR2, which suggests the presence of specific and direct interactions between HP-NAP and TLR2. Thus, HP-NAP directly interacts with and activates TLR2 to induce IL-8 secretion in neutrophils and ATRA-induced differentiated HL-60 cells.     

Injury-Induced Innate Immune Response During Segment Regeneration of the Earthworm,Eisenia andrei

Regeneration of body parts and their interaction with the immune response is a poorly understood aspect of earthworm biology. Consequently, we aimed to study the mechanisms of innate immunity during regeneration in Eisenia andrei earthworms. In the course of anterior and posterior regeneration, we documented the kinetical aspects of segment restoration by histochemistry. Cell proliferation peaked at two weeks and remitted by four weeks in regenerating earthworms. Apoptotic cells were present throughout the cell renewal period. Distinct immune cell (e.g., coelomocyte) subsets were accumulated in the newly-formed blastema in the close proximity of the apoptotic area. Regenerating earthworms have decreased pattern recognition receptors (PRRs) (e.g., TLR, except for scavenger receptor) and antimicrobial peptides (AMPs) (e.g., lysenin) mRNA patterns compared to intact earthworms. In contrast, at the protein level, mirroring regulation of lysenins became evident. Experimental coelomocyte depletion caused significantly impaired cell divisions and blastema formation during anterior and posterior regeneration. These obtained novel data allow us to gain insight into the intricate interactions of regeneration and invertebrate innate immunity.     

Toll-Like Receptors in the Pathogenesis of Essential Hypertension. A Forthcoming Immune-Driven Theory in Full Effect

Essential hypertension (EH) is a highly heterogenous disease with a complex etiology. Recent evidence highlights the significant contribution of subclinical inflammation, triggered and sustained by excessive innate immune system activation in the pathogenesis of the disease. Toll-like receptors (TLRs) have been implied as novel effectors in this inflammatory environment since they can significantly stimulate the production of pro-inflammatory cytokines, the migration and proliferation of smooth muscle cells and the generation of reactive oxygen species (ROS), facilitating a low-intensity inflammatory background that is evident from the very early stages of hypertension. Furthermore, the net result of their activation is oxidative stress, endothelial dysfunction, vascular remodeling, and finally, vascular target organ damage, which forms the pathogenetic basis of EH. Importantly, evidence of augmented TLR expression and activation in hypertension has been documented not only in immune but also in several non-immune cells located in the central nervous system, the kidneys, and the vasculature which form the pathogenetic core systems operating in hypertensive disease. In this review, we will try to highlight the contribution of innate immunity in the pathogenesis of hypertension by clarifying the deleterious role of TLR signaling in promoting inflammation and facilitating hypertensive vascular damage.     

Escherichia coli Maltose-Binding Protein Induces M1 Polarity of RAW264.7 Macrophage Cells via a TLR2- and TLR4-Dependent Manner

Maltose-binding protein (MBP) is a critical player of the maltose/maltodextrin transport system in Escherichia coli. Our previous studies have revealed that MBP nonspecifically induces T helper type 1 (Th1) cell activation and activates peritoneal macrophages obtained from mouse. In the present study, we reported a direct stimulatory effect of MBP on RAW264.7 cells, a murine macrophage cell line. When stimulated with MBP, the production of nitric oxide (NO), IL-1β, IL-6 and IL-12p70, and the expressions of CD80, MHC class II and inducible nitric oxide synthase (iNOS) were all increased in RAW264.7 cells, indicating the activation and polarization of RAW264.7 cells into M1 macrophages induced by MBP. Further study showed that MBP stimulation upregulated the expression of TLR2 and TLR4 on RAW264.7 cells, which was accompanied by subsequent phosphorylation of IκB-α and p38 MAPK. Pretreatment with anti-TLR2 or anti-TLR4 antibodies largely inhibited the phosphorylation of IκB-α and p38 MAPK, and greatly reduced MBP-induced NO and IL-12p70 production, suggesting that the MBP-induced macrophage activation and polarization were mediated by TLR2 and TLR4 signaling pathways. The observed results were independent of lipopolysaccharide contamination. Our study provides a new insight into a mechanism by which MBP enhances immune responses and warrants the potential application of MBP as an immune adjuvant in immune therapies.     

IgG and IgA with Potential Microbial-Binding Activity Are Expressed by Normal Human Skin Epidermal Cells

The innate immune system of the skin is thought to depend largely on a multi-layered mechanical barrier supplemented by epidermis-derived antimicrobial peptides. To date, there are no reports of antimicrobial antibody secretion by the epidermis. In this study, we report the expression of functional immunoglobulin G (IgG) and immunoglobulin A (IgA), previously thought to be only produced by B cells, in normal human epidermal cells and the human keratinocyte line HaCaT. While B cells express a fully diverse Ig, epidermal cell-expressed IgG or IgA showed one or two conservative V_HDJ_H rearrangements in each individual. These unique VDJ rearrangements in epidermal cells were found neither in the B cell-derived Ig VDJ databases published by others nor in our positive controls. IgG and IgA from epidermal cells of the same individual had different VDJ rearrangement patterns. IgG was found primarily in prickle cells, and IgA was mainly detected in basal cells. Both epidermal cell-derived IgG and IgA showed potential antibody activity by binding pathogens like Staphylococcus aureus, the most common pathogenic skin bacteria, but the microbial-binding profile was different. Our data indicates that normal human epidermal cells spontaneously express IgG and IgA, and we speculate that these Igs participate in skin innate immunity.     

Determination of IgG1 and IgG3 SARS-CoV-2 Spike Protein and Nucleocapsid Binding—Who Is Binding Who and Why?

The involvement of immunoglobulin (Ig) G3 in the humoral immune response to SARS-CoV-2 infection has been implicated in the pathogenesis of acute respiratory distress syndrome (ARDS) in COVID-19. The exact molecular mechanism is unknown, but it is thought to involve this IgG subtype’s differential ability to fix, complement and stimulate cytokine release. We examined the binding of convalescent patient antibodies to immobilized nucleocapsids and spike proteins by matrix-assisted laser desorption/ionization–time of flight (MALDI-ToF) mass spectrometry. IgG3 was a major immunoglobulin found in all samples. Differential analysis of the spectral signatures found for the nucleocapsid versus the spike protein demonstrated that the predominant humoral immune response to the nucleocapsid was IgG3, whilst for the spike protein it was IgG1. However, the spike protein displayed a strong affinity for IgG3 itself, as it would bind from control plasma samples, as well as from those previously infected with SARS-CoV-2, similar to the way protein G binds IgG1. Furthermore, detailed spectral analysis indicated that a mass shift consistent with hyper-glycosylation or glycation was a characteristic of the IgG3 captured by the spike protein.     

Lysin Motif (LysM) Proteins: Interlinking Manipulation of Plant Immunity and Fungi

The proteins with lysin motif (LysM) are carbohydrate-binding protein modules that play a critical role in the host-pathogen interactions. The plant LysM proteins mostly function as pattern recognition receptors (PRRs) that sense chitin to induce the plant’s immunity. In contrast, fungal LysM blocks chitin sensing or signaling to inhibit chitin-induced host immunity. In this review, we provide historical perspectives on plant and fungal LysMs to demonstrate how these proteins are involved in the regulation of plant’s immune response by microbes. Plants employ LysM proteins to recognize fungal chitins that are then degraded by plant chitinases to induce immunity. In contrast, fungal pathogens recruit LysM proteins to protect their cell wall from hydrolysis by plant chitinase to prevent activation of chitin-induced immunity. Uncovering this coevolutionary arms race in which LysM plays a pivotal role in manipulating facilitates a greater understanding of the mechanisms governing plant-fungus interactions.