Proinflammatory Responses In Macrophages Research Articles

Pseudomonas aeruginosa Elicits Sustained IL-1β Upregulation in Alveolar Macrophages from Lung Transplant Recipients

<h3>Purpose</h3> Clinical infection or colonization of the lung, specifically with <i>Pseudomonas aeruginosa</i> (<i>PsA</i>), is associated with increased incidence of lung allograft injury in lung transplant recipients (LTRs) and with an increase in BAL (bronchoalveolar lavage) pro-inflammatory cytokines. However, the effect of bacterial colonization on sustained innate immune activation and the mechanisms involved are not well understood. We sought to determine the mechanisms underlying persistent pro-inflammatory alveolar macrophage response to <i>PsA</i> stimulation. <h3>Methods</h3> We stimulated THP-1 derived macrophages and BAL-derived alveolar macrophages from LTRs with bacteria representative of infectious pathogens and upper respiratory commensals for up to 96 hours. Using a high dimensional flow cytometry panel, we evaluated macrophage responses including expression of costimulatory molecules (CD80, CD86, CD40); co-inhibitor molecules (B7-H1), pro-inflammatory cytokines (TNF-α, IL-6, IL-8, IL-1β), and anti-inflammatory mediators (IL-10, IL-1RA, and TGF-β). <h3>Results</h3> We observed an upregulation of pro-inflammatory cytokines (TNF-α, IL-6, IL-8, IL-1β) following stimulation by <i>PsA</i> compared to stimulation with other pathogens (<i>Staphylococcus aureus</i>) and oral-commensal bacteria (<i>Prevotella melaninogenica, Streptococcus pneumoniae</i>) in THP-1 macrophages. IL-1 β elevations were sustained for 96 hours after <i>PsA</i> exposure. Alveolar macrophages from LTRs undergoing surveillance bronchoscopy demonstrated elevated levels of IL-6 and showed a similar IL-1β responses to that observed in THP-1 macrophages when exposed to <i>PsA</i>. <h3>Conclusion</h3> <i>PsA</i> induces a sustained increase in IL-1β production in alveolar macrophages from LTRs. Given the prior described role of IL-1β in lung allograft injury and CLAD, further investigations are ongoing to determine clinical correlates of allograft injury with <i>PsA</i> and IL-1β.

Endoplasmic Reticulum Stress of Oral Squamous Cell Carcinoma Induces Immunosuppression of Neutrophils

The endoplasmic reticulum (ER) stress of cancer cells not only determined cancer cell fate but also indirectly triggered proinflammatory or immunosuppressive responses of macrophages. In addition, ER stressed neutrophils were known to acquire immunosuppressive activity with surface expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1). Since the importance of tumor ER stress and immunosuppressive neutrophils has been emphasized in head and neck cancers, we hypothesized that the ER stress of oral squamous cell carcinoma (OSCC) could transform neutrophils into LOX-1 expressing immunosuppressive phenotype. Two human OSCC cell lines, SCC25 and OML1, were treated with either vehicle or thapsigargin (THG), an ER stress inducer. These tumor conditioned media (TCM) were collected accordingly. Then human peripheral blood neutrophils from healthy donors were cultured in these TCM. The results showed that neutrophils cultured in THG-treated TCM had higher expression of LOX-1 compared with those cultured in vehicle-treated TCM. Moreover, by interleukin-2/anti-CD3/anti-CD28 activated autologous T cell proliferation assay, neutrophils conditioned by THG-treated TCM were shown to inhibit T cell proliferation more significantly than those conditioned by vehicle-treated TCM. These novel findings indicated that the ER stress of OSCC could be transmitted to neutrophils which in turn expressed LOX-1 and obtained immunosuppressive ability. Our findings further supported the existence of “transmissible” ER stress between tumor cells and neutrophils.

Ficus septica exudate, a traditional medicine used in Papua New Guinea for treating infected cutaneous ulcers: in vitro evaluation and clinical efficacy assessment by cluster randomised trial

Infected cutaneous ulcers are major health problems for children living in rural areas of Papua New Guinea. The inaccessibility of affected populations and lack of access to basic healthcare, make a local plant-based therapy an attractive treatment option. We assessed Ficus septica exudate in biological assays relevant to wound healing. We then carried out a clinical trial to determine the exudate's efficacy in healing small cutaneous ulcers compared with Savlon antiseptic cream, and soap and water washing. Pre-clinical in vitro assessment of the exudate was carried out using assays to monitor the pro-inflammatory responses of M1 macrophages and neutrophils, antibacterial assays using known ulcer pathogens, an Ames test for mutagenicity and LC-MS chemical analysis of the exudate. An open label cluster-randomised clinical trial was performed, enrolling participants from three different clusters with skin lesions less than 1 cm in diameter. Each cluster comprising 50 participants was randomly assigned to one of three treatment arms namely topical exudate, topical Savlon antiseptic cream, and standard care (soap and water treatment), all administered daily for 2 days. The primary outcome was clinical healing/improvement measured at days 7 and 14, assessed by three dermatologists using blinded photographs. The primary analysis was assessed as non-inferiority of F. septica treatment based on the risk difference for healing/improvement. In vitro, the exudate which is rich in the alkaloid ficuseptine, was found to be non-mutagenic whilst also inhibiting pro-inflammatory responses and exhibiting antibacterial activity. When administered to participants enrolled in the clinical trial, no significant differences were observed between the healing efficacy of F. septica exudate and the two comparator treatments (Savlon antiseptic cream and soap/water treatment). At day 14, but not at day 7, the efficacy of F. septica exudate for healing/improving the ulcers was non-inferior to Savlon antiseptic cream or water/soap treatment. F. septica exudate is non-mutagenic and has both bactericidal and anti-inflammatory properties. When applied topically to small cutaneous ulcers, the exudate has a healing effect that is non-inferior to Savlon antiseptic cream and standard treatment with soap and water at day 14. Our findings, which should be confirmed in larger clinical trials, have important public health implications.

PTIP governs NAD+ metabolism by regulating CD38 expression to drive macrophage inflammation.

NAD+ metabolism is involved in many biological processes. However, the underlying mechanism of how NAD+ metabolism is regulated remains elusive. Here, we find that PTIP governs NAD+ metabolism in macrophages by regulating CD38 expression and is required for macrophage inflammation. Through integrating histone modifications with NAD+ metabolic gene expression profiling, we identify PTIP as a key factor in regulating CD38 expression, the primary NAD+-consuming enzyme in macrophages. Interestingly, we find that PTIP deletion impairs the proinflammatory response of primary murine and human macrophages, promotes their metabolic switch from glycolysis to oxidative phosphorylation, and alters NAD+ metabolism via downregulating CD38 expression. Mechanistically, an intronic enhancer of CD38 is identified. PTIP regulates CD38 expression by cooperating with acetyltransferase p300 in establishing the CD38 active enhancer with enriched H3K27ac. Overall, our findings reveal a critical role for PTIP in fine-tuning the inflammatory responses of macrophages via regulating NAD+ metabolism.

3H-1,2-dithiole-3-thione suppresses LPS-induced proinflammatory responses in macrophages: potential involvement of antioxidant induction, NF-κB, and Nrf2.

Previously, we reported that 3H-1,2-dithiole-3-thione (D3T), an Nrf2 activator, acted as a potential chemoprotectant against lipopolysaccharide (LPS)-induced mortality in mice. In view of the critical involvement of macrophages in the pathogenesis of LPS-induced endotoxemia, in the present study, we investigated the protective effects of D3T on LPS-induced proinflammatory responses in cultured murine RAW 264.7 macrophage cell line and primary peritoneal macrophages and the potential involvement of antioxidant induction, NF-κB, and Nrf2. We showed that treatment with D3T resulted in increased levels of a series of antioxidants in RAW 264.7 cells in a concentration-dependent manner. These included the reduced form of glutathione, glutathione peroxidase, glutathione reductase, glutathione S-transferase, and NADPH:quinone oxidoreductase 1. Catalase was also potently induced by D3T which, however, did not show a concentration dependency. Concurrent with the ability to induce the above cellular antioxidants, D3T pretreatment of RAW 264.7 cells also led to a concentration-dependent suppression of LPS-induced interleukin-1beta (IL-1β) production and nitric oxide release. LPS-stimulated tumor necrosis factor-alpha (TNF-α) production was also suppressed by D3T, but to a much lesser extent. Using NF-κB reporter gene-expressing RAW 264.7 cells, we further showed that D3T pretreatment also suppressed LPS-induced NF-κB activation. To investigate the potential involvement of Nrf2, a chief regulator of cellular antioxidant genes, we used peritoneal macrophages isolated from Nrf2+/+ and Nrf2-/- mice. Our results showed that D3T pretreatment suppressed LPS-induced proinflammatory responses in Nrf2+/+ macrophages, and this inhibitory effect of D3T was completely lost in Nrf2-/- macrophages. Collectively, the results of the present study demonstrated that D3T acted as a potent suppressor of LPS-induced proinflammatory responses in macrophages. Antioxidant induction, NF-κB suppression, and Nrf2 activation appeared to contribute to the anti-proinflammatory activity of D3T in macrophages.

Blood Bacteria-Free DNA in Septic Mice Enhances LPS-Induced Inflammation in Mice through Macrophage Response.

Although bacteria-free DNA in blood during systemic infection is mainly derived from bacterial death, translocation of the DNA from the gut into the blood circulation (gut translocation) is also possible. Hence, several mouse models with experiments on macrophages were conducted to explore the sources, influences, and impacts of bacteria-free DNA in sepsis. First, bacteria-free DNA and bacteriome in blood were demonstrated in cecal ligation and puncture (CLP) sepsis mice. Second, administration of bacterial lysate (a source of bacterial DNA) in dextran sulfate solution (DSS)-induced mucositis mice elevated blood bacteria-free DNA without bacteremia supported gut translocation of free DNA. The absence of blood bacteria-free DNA in DSS mice without bacterial lysate implies an impact of the abundance of bacterial DNA in intestinal contents on the translocation of free DNA. Third, higher serum cytokines in mice after injection of combined bacterial DNA with lipopolysaccharide (LPS), when compared to LPS injection alone, supported an influence of blood bacteria-free DNA on systemic inflammation. The synergistic effects of free DNA and LPS on macrophage pro-inflammatory responses, as indicated by supernatant cytokines (TNF-α, IL-6, and IL-10), pro-inflammatory genes (NFκB, iNOS, and IL-1β), and profound energy alteration (enhanced glycolysis with reduced mitochondrial functions), which was neutralized by TLR-9 inhibition (chloroquine), were demonstrated. In conclusion, the presence of bacteria-free DNA in sepsis mice is partly due to gut translocation of bacteria-free DNA into the systemic circulation, which would enhance sepsis severity. Inhibition of the responses against bacterial DNA by TLR-9 inhibition could attenuate LPS-DNA synergy in macrophages and might help improve sepsis hyper-inflammation in some situations.

Chemical modification of uridine modulates mRNA-mediated proinflammatory and antiviral response in primary human macrophages

In vitro transcribed (IVT)-mRNA has been accepted as a promising therapeutic modality. Advances in facile and rapid production technologies make IVT-mRNA an appealing alternative to protein- or virus-based medicines. Robust expression levels, lack of genotoxicity, and their manageable immunogenicity benefit its clinical applicability. We postulated that innate immune responses of therapeutically relevant human cells can be tailored or abrogated by combinations of 5′-end and internal IVT-mRNA modifications. Using primary human macrophages as targets, our data show the particular importance of uridine modifications for IVT-mRNA performance. Among five nucleotide modification schemes tested, 5-methoxy-uridine outperformed other modifications up to 4-fold increased transgene expression, triggering moderate proinflammatory and non-detectable antiviral responses. Macrophage responses against IVT-mRNAs exhibiting high immunogenicity (e.g., pseudouridine) could be minimized upon HPLC purification. Conversely, 5′-end modifications had only modest effects on mRNA expression and immune responses. Our results revealed how the uptake of chemically modified IVT-mRNA impacts human macrophages, responding with distinct patterns of innate immune responses concomitant with increased transient transgene expression. We anticipate our findings are instrumental to predictively address specific cell responses required for a wide range of therapeutic applications from eliciting controlled immunogenicity in mRNA vaccines to, e.g., completely abrogating cell activation in protein replacement therapies.

Stiffness- and Bioactive Factor-Mediated Protection of Self-Assembled Cartilage against Macrophage Challenge in a Novel Co-Culture System.

Objective:Tissue-engineered cartilage implants must withstand the potential inflammatory and joint loading environment for successful long-term repair of defects. The work’s objectives were to develop a novel, direct cartilage-macrophage co-culture system and to characterize interactions between self-assembled neocartilage and differentially stimulated macrophages.Design:In study 1, it was hypothesized that the proinflammatory response of macrophages would intensify with increasing construct stiffness; it was expected that the neocartilage would display a decrease in mechanical properties after co-culture. In study 2, it was hypothesized that bioactive factors would protect neocartilage properties during macrophage co-culture. Also, it was hypothesized that interleukin 10 (IL-10)-stimulated macrophages would improve neocartilage mechanical properties compared to lipopolysaccharide (LPS)-stimulated macrophages.Results:As hypothesized, stiffer neocartilage elicited a heightened proinflammatory macrophage response, increasing tumor necrosis factor alpha (TNF-α) secretion by 5.47 times when LPS-stimulated compared to construct-only controls. Interestingly, this response did not adversely affect construct properties for the stiffest neocartilage but did correspond to a significant decrease in aggregate modulus for soft and medium stiffness constructs. In addition, bioactive factor-treated constructs were protected from macrophage challenge compared to chondrogenic medium-treated constructs, but IL-10 did not improve neocartilage properties, although stiff constructs appeared to bolster the anti-inflammatory nature of IL-10-stimulated macrophages. However, co-culture of bioactive factor-treated constructs with LPS-treated macrophages reduced TNF-α secretion by over 4 times compared to macrophage-only controls.Conclusions:In conclusion, neocartilage stiffness can mediate macrophage behavior, but stiffness and bioactive factors prevent macrophage-induced degradation. Ultimately, this co-culture system could be utilized for additional studies to develop the burgeoning field of cartilage mechano-immunology.

BET protein inhibitor apabetalone represses Porphyromonas gingivalis LPS-induced macrophage M1 polarization via regulating miR-130a/STAT3 axis

Periodontitis is a frequent chronic inflammatory disorder destroying periodontium. Recent studies have revealed the role of bromodomain and extraterminal domain inhibitor (BETi) and microRNA (miR)-130a in regulating macrophage polarization and pro-inflammatory response. However, little is known about whether apabetalone (a novel BETi) and miR-130a are correlated with chronic inflammatory state in periodontitis by regulating macrophage polarization. Here murine RAW264.7 macrophages were applied as an in vitro inflammatory model. After treatment with Porphyromonas gingivalis-derived lipopolysaccharide (Pg LPS) and apabetalone, the expression of macrophage M1 polarization markers and inflammatory cytokines was assessed using real-time PCR, western blot, and enzyme-linked immuno sorbent assay (ELISA). MiR-130a level was assessed using real-time PCR, and the target gene was identified using dual luciferase reporter assay. We demonstrated that apabetalone repressed Pg LPS-induced macrophage M1 polarization in a dose-dependent manner, as evidenced by decreased expression of inducible nitric oxide synthase (iNOS), CD86, and pro-inflammatory cytokines, and increased expression of Arg-1 and CD206. Mechanistically, Pg LPS increased miR-130a expression in macrophages, whereas apabetalone treatment repressed the effect. Functionally, forced expression of miR-130a promoted macrophage M1 polarization, and signal transducer and activator of transcription (STAT)-3 was the direct target gene of miR-130a in the process. Taken together, apabetalone decreases Pg LPS-induced macrophage M1 polarization via regulating miR-130a-3p/STAT3 axis, and may be a promising target for the clinical management of periodontitis.

Danger signal extracellular calcium initiates differentiation of monocytes into SPP1/osteopontin-producing macrophages

The danger signal extracellular calcium is pathophysiologically increased in the synovial fluid of patients with rheumatoid arthritis (RA). Calcium activates the NLRP3-inflammasome via the calcium-sensing receptor in monocytes/macrophages primed by lipopolysaccharide, and this effect is mediated by the uptake of calciprotein particles (CPPs) formed out of calcium, phosphate, and fetuin-A. Aim of the study was to unravel the influence of calcium on monocytes when the priming signal is not present. Monocytes were isolated from the blood of healthy controls and RA patients. Macrophages were characterized using scRNA-seq, DNA microarray, and proteomics. Imaging flow cytometry was utilized to study intracellular events. Here we show that extracellular calcium and CPPs lead to the differentiation of monocytes into calcium-macrophages when the priming signal is absent. Additional growth factors are not needed, and differentiation is triggered by calcium-dependent CPP-uptake, lysosomal alkalization due to CPP overload, and TFEB- and STAT3-dependent increased transcription of the lysosomal gene network. Calcium-macrophages have a needle-like shape, are characterized by excessive, constitutive SPP1/osteopontin production and a strong pro-inflammatory cytokine response. Calcium-macrophages differentiated out of RA monocytes show a stronger manifestation of this phenotype, suggesting the differentiation process might lead to the pro-inflammatory macrophage response seen in the RA synovial membrane.

(Pro)renin Receptor Inhibition Reduces Plasma Cholesterol and Triglycerides but Does Not Attenuate Atherosclerosis in Atherosclerotic Mice.

Objective: Elevated plasma cholesterol concentrations contributes to ischemic cardiovascular diseases. Recently, we showed that inhibiting hepatic (pro)renin receptor [(P)RR] attenuated diet-induced hypercholesterolemia and hypertriglyceridemia in low-density lipoprotein receptor (LDLR) deficient mice. The purpose of this study was to determine whether inhibiting hepatic (P)RR could attenuate atherosclerosis.Approach and Results: Eight-week-old male LDLR−/− mice were injected with either saline or N-acetylgalactosamine-modified antisense oligonucleotides (G-ASOs) primarily targeting hepatic (P)RR and were fed a western-type diet (WTD) for 16 weeks. (P)RR G-ASOs markedly reduced plasma cholesterol concentrations from 2,211 ± 146 to 1,128 ± 121 mg/dL. Fast protein liquid chromatography (FPLC) analyses revealed that cholesterol in very low-density lipoprotein (VLDL) and intermediate density lipoprotein (IDL)/LDL fraction were potently reduced by (P)RR G-ASOs. Moreover, (P)RR G-ASOs reduced plasma triglyceride concentrations by more than 80%. Strikingly, despite marked reduction in plasma lipid concentrations, atherosclerosis was not reduced but rather increased in these mice. Further testing in ApoE−/− mice confirmed that (P)RR G-ASOs reduced plasma lipid concentrations but not atherosclerosis. Transcriptomic analysis of the aortas revealed that (P)RR G-ASOs induced the expression of the genes involved in immune responses and inflammation. Further investigation revealed that (P)RR G-ASOs also inhibited (P)RR in macrophages and in enhanced inflammatory responses to exogenous stimuli. Moreover, deleting the (P)RR in macrophages resulted in accelerated atherosclerosis in WTD fed ApoE−/− mice.Conclusion: (P)RR G-ASOs reduced the plasma lipids in atherosclerotic mice due to hepatic (P)RR deficiency. However, augmented pro-inflammatory responses in macrophages due to (P)RR downregulation counteracted the beneficial effects of lowered plasma lipid concentrations on atherosclerosis. Our study demonstrated that hepatic (P)RR and macrophage (P)RR played a counteracting role in atherosclerosis.

A DAMP-scavenging, IL-10-releasing hydrogel promotes neural regeneration and motor function recovery after spinal cord injury

Spinal cord injury (SCI) creates an inflammatory microenvironment characterized by damage-associated molecular patterns (DAMPs) and immune cell activation that exacerbate secondary damage and impair neurological recovery. Here we develop an immunoregulatory hydrogel scaffold for treating SCI that scavenges DAMPs and slowly releases the anti-inflammatory cytokine interleukin-10 (IL-10). We created this dual-functional scaffold by modifying a photocrosslinked gelatin hydrogel with the cationic, DAMP-binding polymer poly (amidoamine) and with IL-10, and compared the therapeutic activity of this scaffold with that of gelatin-only, gelatin + poly (amidoamine), and gelatin + IL-10 scaffolds in vitro and in vivo. In vitro, the dual-functional scaffold scavenged anionic DAMPs and exhibited sustained release of IL-10, reduced the proinflammatory responses of macrophages and microglia, and enhanced the neurogenic differentiation of neural stem cells. In a complete transection SCI mouse model, the injected dual-functional scaffold suppressed proinflammatory cytokine production, promoted the M2 macrophage/microglia phenotype, and led to neural regeneration and axon growth without scar formation to a greater extent than the single-function or control scaffolds. This DAMP-scavenging, IL-10-releasing scaffold provides a new strategy for promoting neural regeneration and motor function recovery following severe SCI.

Abstract 10573: Dietary 7-Ketocholesterol Exacerbates Myocardial Ischemia-Reperfusion Injury Through Macrophage-Mediated Inflammation in Mice

Background: Myocardial ischemia-reperfusion (IR) injury limits the beneficial effects of early reperfusion therapy for acute myocardial infarction. Emerging evidence suggests that 7-ketocholesterol (7-KC), one of the most common dietary oxysterols, has pro-inflammatory properties and correlates with cardiovascular diseases. However, the underlying mechanisms remain scant. Here we investigated the effects of 7-KC on myocardial IR injury in mice. Methods and Results: Wild type mice were fed either a control high-fat high-cholesterol diet (HFHCD) or HFHCD containing 7-KC (7KC-HFHCD) for three weeks. In a murine model of 30-min myocardial ischemia and subsequent reperfusion, dietary 7KC-HFHCD increased plasma 7-KC level (145 ± 89.7 ng/mL vs 399 ± 175 ng/mL, P&lt;0.0005, N=8) and infarct size after myocardial IR (52 ± 7.3% vs 64 ± 6.9%, P&lt;0.05, N=8-10) without affecting blood pressure and heart rate. The ratio of Ly-6C high inflammatory monocytes to total monocytes increased in 7KC-HFHCD group as assessed by flow cytometric analysis (49 ± 15% vs 61 ± 9.3%, P&lt;0.05, N=6). We then took a systems approach to explore the pro-inflammatory effects of 7-KC on macrophages and performed unbiased RNA-sequencing using murine peritoneal macrophages stimulated with 7-KC. Pathway analysis of differentially expressed transcripts revealed that 7-KC regulated the expression of transcripts related to inflammation, cholesterol biosynthesis and endoplasmic reticulum (ER) stress. We further validated in vitro that 7-KC induced ER stress, mitochondrial reactive oxygen species, and nuclear factor-kappa B activation associated with increased mRNA levels of pro-inflammatory cytokines such as MCP-1 and TNF-α in murine peritoneal macrophages. Administration of N-acetyl-L-cysteine (5mM), an antioxidant, decreased 7-KC-induced pro-inflammatory cytokines, and this decrease was not observed in Tlr4 -/- murine macrophages, or in macrophages transfected with liver X receptor siRNA. Conclusions: Dietary 7-KC exacerbated myocardial IR injury through macrophage-mediated inflammation in mice. Oxidative stress is involved in the 7-KC-induced pro-inflammatory response in macrophages. Dietary oxysterols are a promising therapeutic target for IR injury.

δPKC-Mediated DRP1 Phosphorylation Impacts Macrophage Mitochondrial Function and Inflammatory Response to Endotoxin.

Recent studies have demonstrated that alterations in mitochondrial dynamics can impact innate immune function. However, the upstream mechanisms that link mitochondrial dynamics to innate immune phenotypes have not been completely elucidated. This study asks if Protein Kinase C, subunit delta (δPKC)-mediated phosphorylation of dynamin-related protein 1 (Drp1), a key driver of mitochondrial fission, impacts macrophage pro-inflammatory response following bacterial-derived lipopolysaccharide (LPS) stimulation. Using RAW 264.7 cells, bone marrow-derived macrophages from C57BL/6J mice, as well as human monocyte-derived macrophages, we first characterized changes in δPKC-mediated phosphorylation of Drp1 following LPS stimulation. Next, using rationally designed peptides that inhibit δPKC activation (δV1-1) and δPKC-Drp1 interaction (ψDrp1), we determined whether δPKC-mediated phosphorylation of Drp1 impacts LPS-induced changes in mitochondrial morphology, mitochondrial function, and inflammatory response. Our results demonstrated that δPKC-dependent Drp1 activation is associated with increased mitochondrial fission, impaired cellular respiration, and increased mitochondrial reactive oxygen species in LPS-treated macrophages. This is reversed using a rationally designed peptide that selectively inhibits δPKC phosphorylation of Drp1 (ψDrp1). Interestingly, limiting excessive mitochondrial fission using ψDrp1 reduced LPS-triggered pro-inflammatory response, including a decrease in NF-κB nuclear localization, decreased iNOS induction, and a reduction in pro-inflammatory cytokines (IL-1β, TNFα, IL-6). These data suggest that inhibiting Drp1 phosphorylation by δPKC abates the excessive mitochondrial fragmentation and mitochondrial dysfunction that is seen following LPS treatment. Furthermore, these data suggest that limiting δPKC-dependent Drp1 activation decreases the pro-inflammatory response following LPS treatment. Altogether, δPKC-dependent Drp1 phosphorylation might be an upstream mechanistic link between alterations in mitochondrial dynamics and innate immune phenotypes, and may have therapeutic potential.

Extracellular Vesicles Secreted by TDO2-Augmented Fibroblasts Regulate Pro-inflammatory Response in Macrophages.

Extracellular vesicles (EVs) are secreted lipid bilayer vesicles that mediate cell to cell communication and are effectors of cell therapy. Previous work has shown that canonical Wnt signaling is necessary for cell and EV therapeutic potency. Tryptophan 2,3-dioxygenase (TDO2) is a target gene of canonical Wnt signaling. Augmenting TDO2 in therapeutically inert fibroblasts endows their EVs with immunomodulatory capacity including attenuating inflammatory signaling in macrophages. Transcriptomic analysis showed that macrophages treated with EVs from fibroblasts overexpressing TDO2 had blunted inflammatory response compared to control fibroblast EVs. In vivo, EVs from TDO2-overexpressing fibroblasts preserved cardiac function. Taken together, these results describe the role of a major canonical Wnt-target gene (TDO2) in driving the therapeutic potency of cells and their EVs.

Interaction of Opioids with TLR4-Mechanisms and Ramifications.

Simple SummaryRecent evidence indicates that opioids can be active at a receptor that is abundantly expressed on innate immune cells as well as cancer cells: the receptor is termed toll-like receptor 4 (TLR4). TLR4 is increasingly recognised as playing key roles in tumour biology and anticancer defences. However, the issue of whether TLR4 mediates some of the effects of opioids on tumour growth and metastasis is entirely unknown. We review existing evidence, mechanisms, and functional consequences of the action of opioids at TLR4. This opens new avenues of research on the role of opioids in cancer.The innate immune receptor toll-like receptor 4 (TLR4) is known as a sensor for the gram-negative bacterial cell wall component lipopolysaccharide (LPS). TLR4 activation leads to a strong pro-inflammatory response in macrophages; however, it is also recognised to play a key role in cancer. Recent studies of the opioid receptor (OR)-independent actions of opioids have identified that TLR4 can respond to opioids. Opioids are reported to weakly activate TLR4, but to significantly inhibit LPS-induced TLR4 activation. The action of opioids at TLR4 is suggested to be non-stereoselective, this is because OR-inactive (+)-isomers of opioids have been shown to activate or to inhibit TLR4 signalling, although there is some controversy in the literature. While some opioids can bind to the lipopolysaccharide (LPS)-binding cleft of the Myeloid Differentiation factor 2 (MD-2) co-receptor, pharmacological characterisation of the inhibition of opioids on LPS activation of TLR4 indicates a noncompetitive mechanism. In addition to a direct interaction at the receptor, opioids affect NF-κB activation downstream of both TLR4 and opioid receptors and modulate TLR4 expression, leading to a range of in vivo outcomes. Here, we review the literature reporting the activity of opioids at TLR4, its proposed mechanism(s), and the complex functional consequences of this interaction.

Standardized Extract of Asparagus officinalis Stem Attenuates SARS-CoV-2 Spike Protein-Induced IL-6 and IL-1β Production by Suppressing p44/42 MAPK and Akt Phosphorylation in Murine Primary Macrophages.

Excessive host inflammation following infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is associated with severity and mortality in coronavirus disease 2019 (COVID-19). We recently reported that the SARS-CoV-2 spike protein S1 subunit (S1) induces pro-inflammatory responses by activating toll-like receptor 4 (TLR4) signaling in macrophages. A standardized extract of Asparagus officinalis stem (EAS) is a unique functional food that elicits anti-photoaging effects by suppressing pro-inflammatory signaling in hydrogen peroxide and ultraviolet B-exposed skin fibroblasts. To elucidate its potential in preventing excessive inflammation in COVID-19, we examined the effects of EAS on pro-inflammatory responses in S1-stimulated macrophages. Murine peritoneal exudate macrophages were co-treated with EAS and S1. Concentrations and mRNA levels of pro-inflammatory cytokines were assessed using enzyme-linked immunosorbent assay and reverse transcription and real-time polymerase chain reaction, respectively. Expression and phosphorylation levels of signaling proteins were analyzed using western blotting and fluorescence immunomicroscopy. EAS significantly attenuated S1-induced secretion of interleukin (IL)-6 in a concentration-dependent manner without reducing cell viability. EAS also markedly suppressed the S1-induced transcription of IL-6 and IL-1β. However, among the TLR4 signaling proteins, EAS did not affect the degradation of inhibitor κBα, nuclear translocation of nuclear factor-κB p65 subunit, and phosphorylation of c-Jun N-terminal kinase p54 subunit after S1 exposure. In contrast, EAS significantly suppressed S1-induced phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) and Akt. Attenuation of S1-induced transcription of IL-6 and IL-1β by the MAPK kinase inhibitor U0126 was greater than that by the Akt inhibitor perifosine, and the effects were potentiated by simultaneous treatment with both inhibitors. These results suggest that EAS attenuates S1-induced IL-6 and IL-1β production by suppressing p44/42 MAPK and Akt signaling in macrophages. Therefore, EAS may be beneficial in regulating excessive inflammation in patients with COVID-19.

Impact of tribocorrosion and titanium particles release on dental implant complications - A narrative review.

Titanium particles as a product of degradation have been detected in periimplant oral tissues and it has been assumed that implants were the source of these particles. Periimplantitis sites had higher concentrations of particles in comparison to healthy implant sites. Several factors have been identified in the degradation of dental implant surface, such as mechanical wear, contact with chemical agents, and the effects of biofilm adhesion. Titanium particles silently prompt the immune-system activation and generate a pro-inflammatory response in macrophages, T lymphocytes and monocytes. During the activation, inflammatory cytokines are released including, granulocyte–macrophage colony-stimulating factor (GM-CSF), prostaglandin, and TNF-α, IL-1β, IL-6. The nanoparticles depict unique features such as high level of biological reactivity and potentially harmful compared to microparticles since they have a relatively greater surface area to volume ratio. Allergic response to titanium as a cause of implant failure has not been well documented. Evidence demonstrating biological complication due to titanium particles release includes peri-implant tissue inflammation that lead terminally to implant loss. There is a biological probability for a relation between the presence of titanium particles and ions, biological complication, and corrosion, but there is no justifiable evidence for unidirectional series of causative actions.

BHLHE40 promotes macrophage pro-inflammatory gene expression and functions.

Macrophages are the principal innate immune cells that populate all major organs and provide the first line of cellular defense against infections and/or injuries. The immediate and early-responding macrophages must mount a robust pro-inflammatory response to protect the host by eliminating deleterious agents. The effective pro-inflammatory macrophage response requires the activation of complex transcriptional programs that modulate the dynamic regulation of inflammatory and metabolic gene expression. Therefore, transcription factors that govern pro-inflammatory and metabolic gene expression play an essential role in shaping the macrophage inflammatory response. Herein, we identify the basic helix-loop-helix family member e40 (BHLHE40), as a critical transcription factor that promotes broad pro-inflammatory and glycolytic gene expression by elevating HIF1α levels in macrophages. Our in vivo studies revealed that myeloid-BHLHE40 deficiency significantly attenuates macrophage and neutrophil recruitment to the site of inflammation. Our integrated transcriptomics and gene set enrichment analysis (GSEA) studies show that BHLHE40 deficiency broadly curtails inflammatory signaling pathways, hypoxia response, and glycolytic gene expression in macrophages. Utilizing complementary gain- and loss-of-function studies, our analyses uncovered that BHLHE40 promotes LPS-induced HIF1α mRNA and protein expression in macrophages. More importantly, forced overexpression of oxygen stable form of HIF1α completely reversed attenuated pro-inflammatory and glycolytic gene expression in BHLHE40-deficient macrophages. Collectively, these results demonstrate that BHLHE40 promotes macrophage pro-inflammatory gene expression and functions by elevating HIF1α expression in macrophages.

Krueppel-Like Factor 4 Expression in Phagocytes Regulates Early Inflammatory Response and Disease Severity in Pneumococcal Pneumonia.

The transcription factor Krueppel-like factor (KLF) 4 fosters the pro-inflammatory immune response in macrophages and polymorphonuclear neutrophils (PMNs) when stimulated with Streptococcus pneumoniae, the main causative pathogen of community-acquired pneumonia (CAP). Here, we investigated the impact of KLF4 expression in myeloid cells such as macrophages and PMNs on inflammatory response and disease severity in a pneumococcal pneumonia mouse model and in patients admitted to hospital with CAP. We found that mice with a myeloid-specific knockout of KLF4 mount an insufficient early immune response with reduced levels of pro-inflammatory cytokines and increased levels of the anti-inflammatory cytokine interleukin (IL) 10 in bronchoalveolar lavage fluid and plasma and an impaired bacterial clearance from the lungs 24 hours after infection with S. pneumoniae. This results in higher rates of bacteremia, increased lung tissue damage, more severe symptoms of infection and reduced survival. Higher KLF4 gene expression levels in the peripheral blood of patients with CAP at hospital admission correlate with a favourable clinical presentation (lower sequential organ failure assessment (SOFA) score), lower serum levels of IL-10 at admission, shorter hospital stay and lower mortality or requirement of intensive care unit treatment within 28 days after admission. Thus, KLF4 in myeloid cells such as macrophages and PMNs is an important regulator of the early pro-inflammatory immune response and, therefore, a potentially interesting target for therapeutic interventions in pneumococcal pneumonia.