Pro-inflammatory Response Research Articles

A Platform for Testing the Biocompatibility of Implants: Silicone Induces a Proinflammatory Response in a 3D Skin Equivalent.

Biocompatibility testing of materials is carried out in 2D cell cultures or animal models despite serious limitations. 3D skin equivalents are advanced in vitro models for human skin. Silicone has been shown to be noncytotoxic but capable of eliciting an immune response. Our aim was to (1) establish a 3D skin equivalent to (2) assess the proinflammatory properties of silicone. We developed a coculture of keratinocytes and fibroblasts resulting in a 3D skin equivalent with an implant using samples from a breast implant. Samples with and without the silicone implant were studied histologically and immunohistochemically in comparison to native human skin samples. Cytotoxicity was assessed via LDH-assay, and cytokine response was assessed via ELISA. Histologically, our 3D skin equivalents had a four-layered epidermal and a dermal component. The presence of tight junctions was demonstrated in immunofluorescence. The only difference in 3D skin equivalents with implants was an epidermal thinning. Implanting the silicone samples did not cause more cell death, however, an inflammatory cytokine response was triggered. We were able to establish an organotypical 3D skin equivalent with an implant, which can be utilised for studies on biocompatibility of materials. This first integration of silicone into a 3D skin equivalent confirmed previous findings on silicone being non-cell-toxic but capable of exerting a proinflammatory effect.

Exposure to Mycobacterium remodels alveolar macrophages and the early innate response to Mycobacterium tuberculosis infection.

Alveolar macrophages (AMs) play a critical role during Mycobacterium tuberculosis (Mtb) infection as the first cells in the lung to encounter bacteria. We previously showed that AMs initially respond to Mtb in vivo by mounting a cell-protective, rather than pro-inflammatory response. However, the plasticity of the initial AM response was unknown. Here, we characterize how previous exposure to Mycobacterium, either through subcutaneous vaccination with Mycobacterium bovis (scBCG) or through a contained Mtb infection (coMtb) that mimics aspects of concomitant immunity, impacts the initial response by AMs. We find that both scBCG and coMtb accelerate early innate cell activation and recruitment and generate a stronger pro-inflammatory response to Mtb in vivo by AMs. Within the lung environment, AMs from scBCG vaccinated mice mount a robust interferon-associated response, while AMs from coMtb mice produce a broader inflammatory response that is not dominated by Interferon Stimulated Genes. Using scRNAseq, we identify changes to the frequency and phenotype of airway-resident macrophages following Mycobacterium exposure, with enrichment for both interferon-associated and pro-inflammatory populations of AMs. In contrast, minimal changes were found for airway-resident T cells and dendritic cells after exposures. Ex vivo stimulation of AMs with Pam3Cys, LPS and Mtb reveal that scBCG and coMtb exposures generate stronger interferon-associated responses to LPS and Mtb that are cell-intrinsic changes. However, AM profiles that were unique to each exposure modality following Mtb infection in vivo are dependent on the lung environment and do not emerge following ex vivo stimulation. Overall, our studies reveal significant and durable remodeling of AMs following exposure to Mycobacterium, with evidence for both AM-intrinsic changes and contributions from the altered lung microenvironments. Comparisons between the scBCG and coMtb models highlight the plasticity of AMs in the airway and opportunities to target their function through vaccination or host-directed therapies.

Implication of lncRNA MSTRG.81401 in Hippocampal Pyroptosis Induced by P2X7 Receptor in Type 2 Diabetic Rats with Neuropathic Pain Combined with Depression.

Major depressive disorder (MDD) is a common complication of diabetes and is often observed alongside diabetic neuropathic pain (DNP) as a comorbidity in diabetic patients. Long non-coding RNA (lncRNA) plays an important role in various pathophysiological processes. The P2X7 receptor is responsible for triggering inflammatory responses, such as pyroptosis, linked to pain and depression. The aim of this study was to investigate the effect of lncRNA MSTRG.81401 on hippocampal pyroptosis induced by the P2X7 receptor in diabetic rats with DNP combined with MDD (DNP + MDD). Our results showed that the expression of lncRNA MSTRG.81401 was significantly elevated in the hippocampus of DNP + MDD rats compared with the control group. Following the administration of shRNA targeting lncRNA MSTRG.81401, a notable elevation in mechanical and thermal pain thresholds was observed in rats with comorbid DNP and MDD. Additionally, significant improvements in depression-like behaviors were evident in the open-field test (OFT), sucrose preference test (SPT), and forced swim test (FST). In the DNP + MDD rats, elevated levels in hippocampal P2X7 receptor mRNA and protein were observed, along with increased co-expression of P2X7 and the astrocytic marker glial fibrillary acidic protein (GFAP). Meanwhile, in DNP + MDD rats, the heightened mRNA expression of NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein (ASC), pyroptosis-related protein Gasdermin D (GSDMD), caspase-1, IL-1β, IL-18, and TNF-α was detected, in addition to increased serum levels of IL-1β, IL-18 and TNF-α. After shRNA treatment with lncRNA MSTRG.81401, the above abnormal changes in indicators for pyroptosis and inflammation were improved. Therefore, our study demonstrates that shRNA of lncRNA MSTRG.81401 can alleviate the pain and depression-like behaviors in diabetic rats associated with the comorbidity of DNP and MDD by inhibiting the hippocampal P2X7 receptor-mediated pyroptosis pathway and pro-inflammatory responses. This suggests that the P2X7R/NLRP3/caspase-1 implicated pyroptosis and inflammatory scenario may serve as a potential target for the management of comorbid DNP and MDD in diabetes.

Clinical case of treatment of true drowning in fresh water

Drowning is the third leading cause of unintentional injury death worldwide. The largest number of victims of drowning falls on the young able- bodied population.Objective. There are no recent intensive care protocols for drowning, it is relevant to formulate guidelines for the treatment of drowning. Clinical observation. A 25-year-old female patient was taken by an ambulance team to the intensive care unit with a directional diagnosis ‘drowning in fresh water’ of three degree of severity. The patient had got on ventilator with closed aspiration system. Emergency bronchoscopy was performed during which 200 ml of fluid aspirated into the lungs was removed. Respiratory support was determined by stepwise increase of the PEEP level and decrease in FiO2 level taking into account respiratory index. Given the development of a pro-inflammatory mediator response, lactatacidosis, hemolysis, hyperhydration, a high risk of infectious complications, it was decided to start extracorporeal treatment early by continuous veno-venous hemodiafiltration and de-escalation of systemic antibacterial therapy with meropenem by prolonged infusion. Against the background of the ongoing treatment, we managed to achieve target respiratory parameters and stabilize central hemodynamic parameters by the end of the first day, prevent the development of renal damage, correct water-electrolyte and metabolic disorders, and avoid severe septic complications. Vasopressor support was discontinued on the 4th day, and on the 5th day the patient was switched to independent breathing. The patient`s stay in the intensive care unit was 6 days.Conclusions. Stepwise respiratory therapy using closed suction systems and urgent bronchoscopy prevent direct damage to the alveoli and ongoing translocation of fluid into the vascular bed. Prescribing broad-spectrum antibiotics prior to the growth of clinical and laboratory signs of infectious lung lesions allows to reduce the risk of severe septic complications with pulmonary destruction parenchyma. Early onset extracorporeal treatment by CVVHDF allows to correct homeostasis disorders, eliminate hypervolemia, prevent the development of acute renal damage as a result of hemolysis.

Ibuprofen-loaded bilayer electrospun mesh modulates host response toward promoting full-thickness abdominal wall defect repair.

Pro-inflammatory response impairs the constructive repair of abdominal wall defects after mesh implantation. Electrospinning-aid functionalization has the potential to improve the highly orchestrated response by attenuating the over-activation of foreign body reactions. Herein, we combined poly(L-lactic acid-co-caprolactone) (PLLA-CL) with gelatin proportionally via electrospinning, with Ibuprofen (IBU) incorporation to fabricate a bilayer mesh for the repair improvement. The PLLA-CL/gelatin/IBU (PGI) mesh was characterized in vitro and implanted into the rat model with a full-thickness defect for a comprehensive evaluation in comparison to the PLLA-CL/gelatin (PG) and off-the-shelf small intestinal submucosa (SIS) meshes. The bilayer PGI mesh presented a sustained release of IBU over 21 days with degradation in vitro and developed less-intensive intraperitoneal adhesion along with a histologically weaker inflammatory response than the PG mesh after 28 days. It elicited an M2 macrophage-dominant foreign body reaction within the process, leading to a pro-remodeling response similar to the biological SIS mesh, which was superior to the PG mesh. The PGI mesh provided preponderant mechanical supports over the SIS mesh and the native abdominal wall with similar compliance. Collectively, the newly developed mesh advances the intraperitoneal applicability of electrospun meshes by guiding a pro-remodeling response and offers a feasible functionalization approach upon immunomodulation.

TLR5M and TLR5S Synergistically Sense Flagellin in Early Endosome in Lamprey Petromyzon marinus, Switched by the N-Glycosylation Site N239.

In mammals, TLR5 functions as a homodimer to recognize bacterial flagellin on the cytomembrane. The current investigations reveal the existence of two types of TLR5, a membrane-bound PmTLR5M, and a soluble variant PmTLR5S, in lamprey (Petromyzon marinus). Although both PmTLR5M and PmTLR5S can bind flagellin, only PmTLR5M is capable of eliciting a proinflammatory response, whereas PmTLR5S can detect the flagellin and facilitate the role of PmTLR5M in early endosomes. The trafficking chaperone UNC93B1 enhances the ligand-induced signaling via PmTLR5M or the combination of PmTLR5M and PmTLR5S. PmTLR5M recruits MyD88 as an adaptor. Furthermore, chimeric receptor studies demonstrate the indispensability of the intradomain of PmTLR5M in effective activation of the proinflammatory pathway upon flagellin stimulation, and the combination of PmTLR5S with a singular intradomain in both homodimer and heterodimer ectodomain arrangements can very significantly augment the immune response. Furthermore, the flagellin binding sites between PmTLR5M and PmTLR5S are conserved, which are essential for ligand binding and signal transduction. Moreover, investigations on N-linked glycosylation modifications reveal that the N239 site in PmTLR5M and PmTLR5S plays a switch role in both flagellin binding and immune responses. In addition, PmTLR5M exhibits the high-mannose-type and complex-type N-glycosylation modifications; however, PmTLR5S shows exclusive complex-type N-glycosylation modification. The key N239 site demonstrates complex-type N-glycosylation modification. The findings address the function and mechanism of TLR5 in ligand recognition, subcellular localization, and signaling pathway in lowest vertebrate and immune system transition species, highlight the regulatory role of N-glycosylation modification in TLRs, and augment immune evolutionary research on the TLR signaling pathway.

SARS-CoV-2 Viral Replication Persists in the Human Lung for Several Weeks after Symptom Onset.

Rationale: In the upper respiratory tract, replicating (culturable) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is recoverable for ∼4-8 days after symptom onset, but there is a paucity of data about the frequency and duration of replicating virus in the lower respiratory tract (i.e., the human lung).Objectives: We undertook lung tissue sampling (needle biopsy) shortly after death in 42 mechanically ventilated decedents during the Beta and Delta waves. An independent group of 18 ambulatory patients served as a control group.Methods: Lung biopsy cores from decedents underwent viral culture, histopathological analysis, electron microscopy, transcriptomic profiling, and immunohistochemistry.Measurements and Main Results: Thirty-eight percent (16 of 42) of mechanically ventilated decedents had culturable virus in the lung for a median of 15 days (persisting for up to 4 wk) after symptom onset. Lung viral culture positivity was not associated with comorbidities or steroid use. Delta but not Beta variant lung culture positivity was associated with accelerated death and secondary bacterial infection (P < 0.05). Nasopharyngeal culture was negative in 23.1% (6 of 26) of decedents despite lung culture positivity. This hitherto undescribed biophenotype of lung-specific persisting viral replication was associated with an enhanced transcriptomic pulmonary proinflammatory response but with concurrent viral culture positivity.Conclusions: Concurrent rather than sequential active viral replication continues to drive a heightened proinflammatory response in the human lung beyond the second week of illness and was associated with variant-specific increased mortality and morbidity. These findings have potential implications for the design of interventional strategies and clinical management of patients with severe coronavirus disease (COVID-19).

Toward Clinical Applications: Transforming Nonantibiotic Antibacterials into Effective Next-Generation Supramolecular Therapeutics.

Antibiotic resistance is a major driver of morbidity and mortality worldwide, necessitating alternatives. Due to their mechanism of action, bacteriophages, endolysins, and antimicrobial peptides (coined herein as nonantibiotic antibacterials, NAA) have risen to tackle this problem and led to paradigms in treating antibiotic-resistant bacterial infections. However, their clinical applications remain challenging and have been seriously hampered by cytotoxicity, instability, weak bioactivity, low on-target bioavailability, high pro-inflammatory responses, shorter half-life, and circulatory properties. Hence, to transit preclinical phases and beyond, it has become imperative to radically engineer these alternatives into innovative and revolutionary therapeutics to overcome recalcitrant infections. This perspective highlights the promise of these agents, their limitations, promising designs, nanotechnology, and delivery approaches that can be harnessed to transform these agents. Finally, I provide an outlook on the remaining challenges that need to be tackled for their widespread clinical administration.

Pyruvate maintains and enhances the pro-inflammatory response of microglia caused by glucose deficiency in early stroke.

Pro-inflammatory microglia mainly rely on glycolysis to maintain cytokine production during ischemia, accompanied by an increase in inducible nitric oxide synthase (iNOS) and monocarboxylate transporter 1 (MCT1). The role of energy metabolism in the pro-inflammatory response of microglia is currently unclear. In this study, we tested the response of microglia in mice after cerebral ischemia and simulated an energy environment in vitro using low glucose culture medium. The research results indicate that the expression levels of iNOS and arginase 1 (ARG1) increase in the ischemic mouse brain, but the upregulation of MCT1 expression is mainly present in iNOS positive microglia. In microglia exposed to low glucose conditions, iNOS and MCT1 levels increased, while ARG1 levels decreased. Under the same conditions, knocking down MCT1 in microglia leads to a decrease in iNOS levels, while overexpression of MCT1 leads to the opposite result. The use of NF-κB inhibitors reduced the expression levels of iNOS and MCT1 in microglia. In summary, our data indicate that pyruvate maintains and enhances the NF-κB regulated pro-inflammatory response of microglia induced by low glucose.

OUTCOME OF AZITHROMYCIN AND MONOSODIUM GLUTAMATE ON HISTOLOGIC AND BIOCHEMICAL ALTERATIONS IN RATS’ LIVER

Background: Monosodium glutamate (MSG), a commonly used flavouring, mediated hepatotoxic and pro-inflammatory responses. Azithromycin (AZT), an antibiotic with anti-inflammatory activity, may be co-consumed with MSG to present unknown outcomes on the liver, a major organ for the detoxification of xenobiotics. Aim: This study evaluated the effect of AZT and MSG on histologic and biochemical changes in rats’ liver. Methods: Thirty rats in five groups were for seven successive days orally exposed to groups 1, (distilled water 1 mL Kg−1), 2, (MSG 8000 mg/kg), 3, overdose AZT, OAZT (AZT 412.5 mg Kg−1), 4, therapeutic concentration AZT, TAZT, (AZT 82.5mg Kg−1), and 5, (TAZT 82.5 mg Kg−1 + MSG 8000 mg Kg−1). Liver function markers in therats’ serum and liver tissue, and changes in the liver histologic were assessed by acceptable protocols. The mean of numeric data were tested for significance at p value less than 0.05 by analysis of variance (ANOVA). Results: MSG treatment significantly (p&lt;0.05) increased hepatic and serum alanine aminotransferase activity (ALT), aspartate aminotransferase activity (AST), alkaline phosphatase activity (ALP), total bilirubin concentration (T-BIL), and direct bilirubin concentration (D-BIL) but decreased albumin concentration (ALB) compared to control and others. TAZT treatment caused a significant (p&lt;0.05) decrease in these effects unlike MSG and OATZ treatments while TAZT + MSG co-treatment significantly (p&lt;0.05) reversed these effects compared to MSG-treated rats. Rats’ liver sections for rats in groups 2 and 3 showed severe hepatocellular degeneration and necrosis compared to others while those in group 4 (TAZT) showed the normal hepatic histo-architecture comparable to those in group 1 (control). Those in group 5 showed marked cellular swelling and leukocytic infiltration in only the centrilobular areas, suggesting active restorative responses. Conclusion: Thus, TAZT significantly mitigated the compromised histologic and biochemical integrities of liver function due to MSG treatment in rats via probable normalization of their enzymatic and non-enzymatic indicators of liver function. This suggests that TAZT could be useful in managing histologic and biochemical malfunction of rats’ liver due to MSG assault. Peer Review History: Received: 12 October 2023; Revised: 9 November; Accepted: 13 December, Available online: 15 January 2024 Academic Editor: Dr. Marwa A. A. Fayed, University of Sadat City, Egypt, maafayed@gmail.com Received file: Reviewer's Comments: Average Peer review marks at initial stage: 6.0/10 Average Peer review marks at publication stage: 7.0/10 Reviewers: Idoko Alexander, Caritas University, Enugu, Nigeria, idokoalexander1@gmail.com Dr. Dennis Amaechi, MrsFoluBabade Mini Estate, Flat 5 by Old Soldiers Quarter, Sabongari/Bwari, Abuja- Federal Capital Territory, Nigeria. amaechitoexcel@yahoo.com

Immunohistochemical profile of gingiva biopsy samples from the edentulous alveolar ridge in the jaw

Relevance. Despite the high success rate of dental implantation in restoring anatomical occlusion and achieving desired aesthetic effects, complications, often mediated by peri-implantitis, remain an area requiring further study. This study aimed to morphologically assess gingiva biopsy samples from the edentulous alveolar ridge of the jaw, using immunohistochemical markers for immunophenotyping different subpopulations of inflammatory cells, inter alia.Material and methods. Gingiva biopsy specimens from 25 patients underwent descriptive histological and immunohistochemical analyses, employing markers such as CD3, CD20, TBX21, GATA3, Foxp3, CD68, CD80, CD163, CD1a, and CD83. The study includes clinical-anamnestic data analysis, descriptive histological and immunohistochemical observations, and a quantitative analysis of the abundance of various immunopositive cell subpopulations.Results. The data indicated that approximately one third of the gum tissues exhibited signs of a chronic inflammatory response, predominantly characterized by the primary T-cell subpopulation of T-helper 1 cells, a minimal presence of regulatory T-cells, and an almost complete absence of T-helper 2 cells. Additionally, an inflammatory cell mix was observed, comprising a significant proportion of B-cells, monocyte-macrophage cells (mainly M2 macrophage subpopulation), and mature CD83+ dendritic cells. These changes reflect a pro-inflammatory local immune response, crucial to consider in future implantation planning.Conclusion. The analysis of this pilot clinical case series enhances our understanding of the gingiva condition in the edentulous alveolar ridge before dental implantation and opens avenues for further research in this field.

SARS-CoV-2 S protein activates NLRP3 inflammasome and deregulates coagulation factors in endothelial and immune cells

BackgroundHyperinflammation, hypercoagulation and endothelial injury are major findings in acute and post-COVID-19. The SARS-CoV-2 S protein has been detected as an isolated element in human tissues reservoirs and is the main product of mRNA COVID-19 vaccines. We investigated whether the S protein alone triggers pro-inflammatory and pro-coagulant responses in primary cultures of two cell types deeply affected by SARS-CoV-2, such are monocytes and endothelial cells.MethodsIn human umbilical vein endothelial cells (HUVEC) and monocytes, the components of NF-κB and the NLRP3 inflammasome system, as well as coagulation regulators, were assessed by qRT-PCR, Western blot, flow cytometry, or indirect immunofluorescence.ResultsS protein activated NF-κB, promoted pro-inflammatory cytokines release, and triggered the priming and activation of the NLRP3 inflammasome system resulting in mature IL-1β formation in both cell types. This was paralleled by enhanced production of coagulation factors such as von Willebrand factor (vWF), factor VIII or tissue factor, that was mediated, at least in part, by IL-1β. Additionally, S protein failed to enhance ADAMTS-13 levels to counteract the pro-coagulant activity of vWF multimers. Monocytes and HUVEC barely expressed angiotensin-converting enzyme-2. Pharmacological approaches and gene silencing showed that TLR4 receptors mediated the effects of S protein in monocytes, but not in HUVEC.ConclusionS protein behaves both as a pro-inflammatory and pro-coagulant stimulus in human monocytes and endothelial cells. Interfering with the receptors or signaling pathways evoked by the S protein may help preventing immune and vascular complications driven by such an isolated viral element.BJi2XdMkuptrjYtS_YhKHeVideo

Exosomes released by environmental pollutant-stimulated Keratinocytes/PBMCs can trigger psoriatic inflammation in recipient cells via the AhR signaling pathway.

Introduction: Exosomes, pivotal in intercellular communication during skin disease pathogenesis, have garnered substantial attention. However, the impact of environmental pollutants, such as benzo[a]pyrene (BaP) and 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), on exosome release amid inflammatory skin diseases remains unexplored. This study addresses this gap by examining the influence of BaP and TCDD on exosome function, specifically focusing on immune-related pathway alterations in normal recipient keratinocytes and peripheral blood mononuclear cells (PBMCs). Methods: HaCaT cells were treated with exosomes from BaP- or TCDD-treated keratinocytes. Proinflammatory cytokines and chemokines, including TNF-α, IL-1β, IL-6, IL-8, CXCL1, and CXCL5, were assessed. The involvement of the p65NF-κB/p38MAPK/ERK signaling pathway in recipient keratinocytes was investigated. Aryl hydrocarbon receptor (AhR) silencing was employed to elucidate its role in mediating the proinflammatory response induced by exosomes from BaP- or TCDD-treated keratinocytes. Results and discussion: Treatment with exosomes from BaP- or TCDD-treated keratinocytes induced a significant increase in proinflammatory cytokines and chemokines in HaCaT cells. The upregulation implicated the p65NF-κB/p38MAPK/ERK signaling pathway. AhR silencing attenuated this response, suggesting a role for AhR in mediating this response. In PBMCs from healthy controls, exosomes from BaP-stimulated PBMCs of psoriatic patients led to increased expression of proinflammatory cytokines and modulation of Th1/Th17 cell distribution via AhR activation. These findings unveil a novel dimension in the interplay between environmental xenobiotic agents (BaP and TCDD) and exosomal functions. The study establishes their influence on psoriatic inflammatory responses, shedding light on the underlying mechanisms mediated through the AhR signaling pathway in recipient keratinocytes and PBMCs.

Real-Time Exposure to 3D-Printing Emissions Elicits Metabolic and Pro-Inflammatory Responses in Human Airway Epithelial Cells.

Three-dimensional (3D) printer usage in household and school settings has raised health concerns regarding chemical and particle emission exposures during operation. Although the composition of 3D printer emissions varies depending on printer settings and materials, little is known about the impact that emissions from different filament types may have on respiratory health and underlying cellular mechanisms. In this study, we used an in vitro exposure chamber system to deliver emissions from two popular 3D-printing filament types, acrylonitrile butadiene styrene (ABS) and polylactic acid (PLA), directly to human small airway epithelial cells (SAEC) cultured in an air-liquid interface during 3D printer operation. Using a scanning mobility particle sizer (SMPS) and an optical particle sizer (OPS), we monitored 3D printer particulate matter (PM) emissions in terms of their particle size distribution, concentrations, and calculated deposited doses. Elemental composition of ABS and PLA emissions was assessed using scanning electron microscopy coupled with energy dispersive X-ray spectroscopy (SEM-EDX). Finally, we compared the effects of emission exposure on cell viability, inflammation, and metabolism in SAEC. Our results reveal that, although ABS filaments emitted a higher total concentration of particles and PLA filaments emitted a higher concentration of smaller particles, SAEC were exposed to similar deposited doses of particles for each filament type. Conversely, ABS and PLA emissions had distinct elemental compositions, which were likely responsible for differential effects on SAEC viability, oxidative stress, release of inflammatory mediators, and changes in cellular metabolism. Specifically, while ABS- and PLA-emitted particles both reduced cellular viability and total glutathione levels in SAEC, ABS emissions had a significantly greater effect on glutathione relative to PLA emissions. Additionally, pro-inflammatory cytokines including IL-1β, MMP-9, and RANTES were significantly increased due to ABS emissions exposure. While IL-6 and IL-8 were stimulated in both exposure scenarios, VEGF was exclusively increased due to PLA emissions exposures. Notably, ABS emissions induced metabolic perturbation on amino acids and energy metabolism, as well as redox-regulated pathways including arginine, methionine, cysteine, and vitamin B3 metabolism, whereas PLA emissions exposures caused fatty acid and carnitine dysregulation. Taken together, these results advance our mechanistic understanding of 3D-printer-emissions-induced respiratory toxicity and highlight the role that filament emission properties may play in mediating different respiratory outcomes.

Porcine β-Defensin 114: Creating a Dichotomous Response to Inflammation.

The immunity-related functions of defensins seem to be dependent on environmental stimuli, the cell type, and the concentration of peptides. However, the function and mechanism of porcine β-defensin 114 (pBD114) in regulating the inflammatory response to macrophages are unclear. Therefore, the modulatory effects of porcine pBD114 on the inflammatory response were investigated by treating the mouse monocyte macrophage cell line RAW264.7 with different concentrations of pBD114 with or without lipopolysaccharide (LPS). RNA-seq analysis was performed to investigate the mechanisms underlying pBD114's regulation of inflammatory responses in macrophages. In addition, the inflammatory response-modulating effects of pBD114 were also further verified with a mouse assay. The results showed that 100 μg/mL of pBD114 significantly promoted the secretion of TNF-α and IL-10 in RAW264.7. However, the LPS-induced increase in TNFα in the RAW264.7 cell cultures was significantly decreased with 10 μg/mL of pBD114. These results suggest that pBD114 can exhibit pro-inflammatory activities under normal physiological conditions with 100 μg/mL of pBD114, and anti-inflammatory activities during an excessive inflammatory response with 10 μg/mL of pBD114. RNA-seq analysis was performed to gain further insights into the effects of pBD114 on the inflammatory response. Among the pBD114-promoting RAW264.7 pro-inflammatory responses, pBD114 significantly up-regulated 1170 genes and down-regulated 724 genes. KEGG enrichment showed that the differentially expressed genes (DEGs) were significantly enriched in the immune- and signal-transduction-related signaling pathways. Protein-Protein Interaction (PPI) and key driver analysis (KDA) analyses revealed that Bcl10 and Bcl3 were the key genes. In addition, pBD114 significantly up-regulated 12 genes and down-regulated 38 genes in the anti-inflammatory response. KEGG enrichment analysis revealed that the DEGs were mainly enriched in the "Cytokine-cytokine receptor interaction" signaling pathway, and PPI and KDA analyses showed that Stat1 and Csf2 were the key genes. The results of qRT-PCR verified those of RNA-seq. In vivo mouse tests also confirmed the pro- or anti-inflammatory activities of pBD114. Although the inflammatory response is a rapid and complex physiological reaction to noxious stimuli, this study found that pBD114 plays an essential role mainly by acting on the genes related to immunity, signal transduction, signaling molecules, and interactions. In conclusion, this study provides a certain theoretical basis for the research and application of defensins.

Chimeric proteins of Mycoplasma hyopneumoniae as vaccine and preclinical model for immunological evaluation.

Mycoplasma hyopneumoniae (M. hyopneumoniae) is a primary agent of porcine enzootic pneumonia, a disease that causes significant economic losses to pig farming worldwide. Commercial vaccines induce partial protection, evidencing the need for a new vaccine against M. hyopneumoniae. In our work, three chimeric proteins were constructed, composed of potentially immunogenic domains from M. hyopneumoniae proteins. We designed three chimeric proteins (Q1, Q2, and Q3) based on bioinformatics analysis that identified five potential proteins with immunogenic potential (MHP418, MHP372, MHP199, P97, and MHP0461). The chimeric proteins were inoculated in the murine model to evaluate the immune response. The mice vaccinated with the chimeras presented IgG and IgG1 against proteins of M. hyopneumoniae. There was induction of IgG in mice immunized with Q3 starting from 30days post-vaccination, and groups Q1 and Q2 showed induction at 45days. Mice of the group immunized with Q3 showed the production of IgA. In addition, the mice inoculated with chimeric proteins showed a proinflammatory cytokine response; Q1 demonstrated higher levels of TNF, IL-6, IL2, and IL-17. In contrast, animals immunized with Q2 showed an increase in the concentrations of TNF, IL-6, and IL-4, whereas those immunized with Q3 exhibited an increase in the concentrations of TNF, IL-6, IL-10, and IL-4. The results of the present study indicate that these three chimeric proteins can be used in future vaccine trials with swine because of the promising antigenicity.

Cortical microinfarcts potentiate recurrent ischemic injury through NLRP3-dependent trained immunity

Microinfarcts are common among the elderly and patients with microinfarcts are more vulnerable to another stroke. However, the impact of microinfarcts on recurrent stroke has yet to be fully understood. The purpose of this study was to explore the negative effects of microinfarcts on recurrent stroke. To achieve this, two-photon laser was used to induce microinfarcts, while photothrombotic stroke was induced on the opposite side. The results showed that microinfarcts led to trained immunity in microglia, which worsened the pro-inflammatory response and ischemic injury in the secondary photothrombotic stroke. Additionally, the study clarified the role of NLRP3 in microglial nuclei, indicating that it interacts with the MLL1 complex through NACHT domain and increases H3K4 methylation, which suggests that NLRP3 is critical in the formation of innate immune memory caused by microinfarcts. Furthermore, the knockout of NLRP3 in microglia alleviated the trained immunity and reduced the harmful effects of microinfarcts on recurrent stroke. This study emphasizes the detrimental effect of trained immunity on recurrent stroke and highlights the critical role of NLRP3 in mediating the formation of this memory, which may offer a potential therapeutic target for mitigating recurrent strokes.

A ·OH self-suppling and dual regulation hybrid nanozyme combined with photothermal therapy to eliminate biofilms and eradicate infections

Limited by local H2O2 concentration and GSH hindrance, chemodynamic therapy (CDT) has poor antibacterial efficiency, and the pro-inflammatory response caused by CDT is often ignored. Here, a hybrid nanozyme (Fe3O4-CaO2-PDA) was synthesized, which has dual regulatory functions of ·OH and GSH, there are two stages in clearing the infection: treatment and repair. In the treatment stage, stimulated by the photothermal generated by near-infrared light (NIR), it rapidly releases a large amount of ·OH and further enhances CDT by decreasing the local GSH concentration to achieve a rapid antimicrobial effect. In the repair stage, the PDA removes the local residual ·OH, prevents over-aggregation of inflammatory cells, and regulates pro-inflammatory responses. In vitro antibacterial experiments showed that the killing rates of the hybrid nanozyme against S. aureus and E. coli were 90% and 92% respectively. At the same time, the DCFH-DA fluorescence experiment showed that the ·OH concentration was significantly controlled. in vivo, due to the function of control ·OH, nanozymes show the ability to promote collagen regeneration and regulate inflammation. In summary, this study proposed a synergistic multiple antibacterial strategy and successfully constructed a PTT-enhanced multifunctional hybrid nanozyme for efficient CDT anti-infective treatment.

Targeting host tyrosine kinase receptor EphA2 signaling via small-molecule ALW-II-41-27 inhibits macrophage pro-inflammatory signaling responses to Pneumocystis carinii β-glucans.

Pneumocystis jirovecii, the fungus that causes Pneumocystis jirovecii pneumonia (PJP), is a leading cause of morbidity and mortality in immunocompromised individuals. We have previously shown that lung epithelial cells can bind Pneumocystis spp. β-glucans via the EphA2 receptor, resulting in activation and release of proinflammatory cytokines. Herein, we show that in vivo Pneumocystis spp. β-glucans activation of the inflammatory signaling cascade in macrophages can be pharmacodynamically inhibited with the EphA2 receptor small-molecule inhibitor ALW-II-41-27. In vitro, when ALW-II-41-27 is administrated via intraperitoneal to mice prior to the administration of highly proinflammatory Saccharomyces cerevisiae β-glucans in the lung, a significant reduction in TNF-alpha release was noted in the ALW-II-41-27 pre-treated group. Taken together, our data suggest that targeting host lung macrophage activation via EphA2 receptor-fungal β-glucans interactions with ALW-II-41-27 or other EphA2 receptor kinase targeting inhibitors might be an attractive and viable strategy to reduce detrimental lung inflammation associated with PJP.

Impaired macrophage and memory T-cell responses to Bacillus Calmette-Guerin nonpolar lipid extract.

The attenuation of BCG has led to the loss of not only immunogenic proteins but also lipid antigens. Thus, we compared the macrophage and T-cell responses to nonpolar lipid extracts harvested from BCG and Mycobacterium tuberculosis (Mtb) to better understand the role of BCG lipids in the already known diminished responses of the vaccine strain. Relative to Mtb, nonpolar lipid extract from BCG presented a reduced capacity to trigger the expression of the genes encoding TNF, IL-1b, IL-6 and IL-10 in RAW 264.7 macrophages. Immunophenotyping of PBMCs isolated from healthy individuals revealed that lipids from both BCG and Mtb were able to induce an increased frequency of CD4+ and CD8+ T cells, but only the lipid extract from Mtb enhanced the frequency of CD4-CD8-double-negative, γσ+, CD4+HLA-DR+, and γσ+HLA-DR+ T cells relative to the nonstimulated control. Interestingly, only the Mtb lipid extract was able to increase the frequency of CD4+ memory (CD45RO+) T cells, whereas the BCG lipid extract induced a diminished frequency of CD4+ central memory (CD45RO+CCR7-) T cells after 48 h of culture compared to Mtb. These findings show that the nonpolar lipids of the BCG bacilli presented diminished ability to trigger both proinflammatory and memory responses and suggest a potential use of Mtb lipids as adjuvants to increase the BCG vaccine efficacy.