Quantitative Confocal Microscopy Research Articles

Neutrophil extracellular traps are an important component of chronic myocardial inflammation in patients with heart failure

Abstract Background and aim We have previously shown that heart failure (HF) is characterized by chronic inflammation. However, the role of neutrophils (N) and extracellular neutrophil traps (NETs) in the myocardium of patients with HF is largely unknown. The present study investigated the number of neutrophils (N) and their proportion that develop NETs in HF. Methods We used quantitative confocal microscopy and NETs markers in the left ventricular biopsies obtained from 5 controls and from patients with HF due to dilated (DCM, n=7), ischemic (ICM, n=7) and inflammatory (infCMP, n=7) cardiomyopathy. We used immunolabeling for CD45 (N) and for NETs (citrullinated histone 3 (CitH3), peptidylarginine deiminase-4 (PAD-4), neutrophil elastase (NE) and myeoloperoxidase (MPO). Results Compared to the control, the number of N was 4.3-5.7 times higher in HF. Using single labeling for NETs marker revealed that 41.1±4.6% of N in DCM, 42.9±4.1% in ICM and 47.6±2.9% in infCMP developed NETs. The use of dual labeling (MPO with CitH3, MPO with PAD-4 and CitH3 with NE) resulted in 54.5±4.7% of N-developing NETs in DCM, 59.5±4.1% in ICM and 77.6%±4.2 in infCMP. The difference between N-undergoing NETs was statistically different in infCMP than in DCM.and ICM. The proportion of N-forming NETs in control tissue was less than 5% and was significantly different from that in HF patients regardless of etiology. Conclusions This is the first study to show the presence of NETs in human hearts in situ and demonstrates that NETs are an important component of chronic inflammation in HF due to cardiomyopathies.

High-dose dexamethasone regulates microglial polarization via the GR/JAK1/STAT3 signaling pathway after traumatic brain injury.

Although microglial polarization and neuroinflammation are crucial cellular responses after traumatic brain injury, the fundamental regulatory and functional mechanisms remain insufficiently understood. As potent anti-inflammatory agents, the use of glucocorticoids in traumatic brain injury is still controversial, and their regulatory effects on microglial polarization are not yet known. In the present study, we sought to determine whether exacerbation of traumatic brain injury caused by high-dose dexamethasone is related to its regulatory effects on microglial polarization and its mechanisms of action. In vitro cultured BV2 cells and primary microglia and a controlled cortical impact mouse model were used to investigate the effects of dexamethasone on microglial polarization. Lipopolysaccharide, dexamethasone, RU486 (a glucocorticoid receptor antagonist), and ruxolitinib (a Janus kinase 1 antagonist) were administered. RNA-sequencing data obtained from a C57BL/6 mouse model of traumatic brain injury were used to identify potential targets of dexamethasone. The Morris water maze, quantitative reverse transcription-polymerase chain reaction, western blotting, immunofluorescence and confocal microscopy analysis, and TUNEL, Nissl, and Golgi staining were performed to investigate our hypothesis. High-throughput sequencing results showed that arginase 1, a marker of M2 microglia, was significantly downregulated in the dexamethasone group compared with the traumatic brain injury group at 3 days post-traumatic brain injury. Thus dexamethasone inhibited M1 and M2 microglia, with a more pronounced inhibitory effect on M2 microglia in vitro and in vivo. Glucocorticoid receptor plays an indispensable role in microglial polarization after dexamethasone treatment following traumatic brain injury. Additionally, glucocorticoid receptor activation increased the number of apoptotic cells and neuronal death, and also decreased the density of dendritic spines. A possible downstream receptor signaling mechanism is the GR/JAK1/STAT3 pathway. Overactivation of glucocorticoid receptor by high-dose dexamethasone reduced the expression of M2 microglia, which plays an anti-inflammatory role. In contrast, inhibiting the activation of glucocorticoid receptor reduced the number of apoptotic glia and neurons and decreased the loss of dendritic spines after traumatic brain injury. Dexamethasone may exert its neurotoxic effects by inhibiting M2 microglia through the GR/JAK1/STAT3 signaling pathway.

Circulating neutrophil extracellular trap-forming neutrophils in rheumatoid arthritis exacerbation are majority dual endothelin-1/signal peptide receptor+ subtype.

Neutrophil extracellular traps (NETs) are associated with rheumatoid arthritis pathogenesis and severity. Since homeostatic NET-forming neutrophils [NET+Ns] have beneficial roles in defense against pathogens, their distinction from pro-injury [NET+N] subtypes is important, especially if they are to be therapeutically targeted. Having identified circulating, pro-injury DEspR+CD11b+[NET+Ns] in patients with neutrophilic secondary tissue injury, we determined whether DEspR+[NET+Ns] are present in rheumatoid arthritis (RA) flares. Whole blood samples of patients with RA flares on maintenance therapy (n = 6) were analyzed by flow cytometry (FCM) and immunofluorescence cytology followed by semi-automated quantitative confocal microscopy (qIFC). We assessed clinical parameters, levels of neutrophils and [NET+Ns], and plasma S100A8/A9. qIFC detected circulating DEspR+CD11b+neutrophils and [NET+Ns] in RA-flare patients but not healthy controls. DEspR+[NET+Ns] were positive for citrullinated histone H3 (citH3+), extruded DNA, decondensed but recognizable polymorphic nuclei, and [NET+N] doublet interactions in mostly non-ruptured NET-forming neutrophils. Circulating DNA+/DEspR+/CD11b+/citH3+microvesicles (netMVs) were observed. FCM detected increased %DEspR+CD11b+neutrophils and DEspR+ cell-cell doublets whose levels trended with DAS28 scores, as did plasma S100A8/A9 levels. This study identifies circulating DEspR+/CD11b+neutrophils and [NET+Ns] in RA-flare patients on maintenance therapy. Detection of circulating DEspR+citH3+[NET+Ns] and netMVs indicate a systemic neutrophilic source of citH3-antigen concordant with multi-joint RA pathogenesis. Increased S100A8/A9 alarmin levels are associated with cell injury and released upon NET-formation. As a ligand for TLR4, S100A8/A9 forms a positive feedback loop for TLR4-induced DEspR+neutrophils. These data identify DEspR+neutrophils and [NET+Ns] in RA pathogenesis as a potential biomarker and/or therapeutic target.

The Immunosuppressive Drug LF15-0195 Acts Also on Glomerular Lesions, by a Change in Cytoskeleton Distribution in Podocyte

Introduction: Buffalo/Mna rats spontaneously develop nephrotic syndrome (NS) which recurs after renal transplantation. The immunosuppressive drug LF15-0195 can promote regression of the initial and post-transplantation nephropathy via induction of regulatory T cells. We investigate if this drug has an additional effect on the expression and localization of podocyte specific proteins. Methods: Buffalo/Mna kidney samples were collected before and after the occurrence of proteinuria, and after the remission of proteinuria induced by LF15-0195 treatment and compared by quantitative RT-PCR, Western blot, electron, and confocal microscopy to kidney samples of age-matched healthy rats. Cytoskeleton changes were assessed in culture by stress fibers induction by TNFα. Results: We observed, by electron microscopy, a restoration of foot process architecture in the LF15-0195-treated Buff/Mna kidneys, consistent with proteinuria remission. Nephrin, podocin, CD2AP, and α-actinin-4 mRNA levels remained low during the active disease in the Buff/Mna, in comparison with healthy rats which increase, while podocalyxin and synaptopodin transcripts were elevated before the occurrence of the disease but did not differ from healthy animals after. No difference in the mRNA and protein expression between the untreated and the LF15-0195-treated proteinuric Buff/Mna were seen for these 6 proteins. No changes were observed by confocal microscopy in the protein distribution at a cellular level, but a more homogenous distribution similar to healthy rats, was observed within the glomeruli of LF15-0195-treated rats. In addition, LF15-0195 could partially restore actin cytoskeleton of endothelial cells in TNFα-induced-cell stress experiment. Conclusion: The effect of LF15-0195 treatment appears to be mediated by 2 mechanisms: an immunomodulatory effect via regulatory T cells induction, described in our previous work and which can act on immune cell involved in the disease pathogenesis, and an effect on the restoration of podocyte cytoskeleton, independent of expression levels of the proteins involved in the slit diaphragm and podocyte function, showed in this article.

Induction of the antiviral factors APOBEC3A and RSAD2 upon CCL2 neutralization in primary human macrophages involves NF-κB, JAK/STAT, and gp130 signaling.

The CCL2/CC chemokine receptor 2 axis plays key roles in the pathogenesis of HIV-1 infection. We previously reported that exposure of monocyte-derived macrophages to CCL2 neutralizing antibody (αCCL2 Ab) restricted HIV-1 replication at postentry steps of the viral life cycle. This effect was associated with induction of transcripts coding for innate antiviral proteins, including APOBEC3A and RSAD2. This study aimed at identifying the signaling pathways involved in induction of these factors by CCL2 blocking in monocyte-derived macrophages. Through a combination of pharmacologic inhibition, quantitative reverse transcription polymerase chain reaction, Western blotting, and confocal laser-scanning microscopy, we demonstrated that CCL2 neutralization activates the canonical NF-κB and JAK/STAT pathways, as assessed by time-dependent phosphorylation of IκB, STAT1, and STAT3 and p65 nuclear translocation. Furthermore, pharmacologic inhibition of IκB kinase and JAKs strongly reduced APOBEC3A and RSAD2 transcript accumulation elicited by αCCL2 Ab treatment. Interestingly, exposure of monocyte-derived macrophages to αCCL2 Ab resulted in induction of IL-6 family cytokines, and interference with glycoprotein 130, the common signal-transducing receptor subunit shared by these cytokines, inhibited APOBEC3A and RSAD2 upregulation triggered by CCL2 neutralization. These results provide novel insights into the signal transduction pathways underlying the activation of innate responses triggered by CCL2 neutralization in macrophages. Since this response was found to be associated with protective antiviral effects, the new findings may help design innovative therapeutic approaches targeting CCL2 to strengthen host innate immunity.

Novel Environmentally Friendly RNAi Biopesticides: Targeting V-ATPase in Holotrichia parallela Larvae Using Layered Double Hydroxide Nanocomplexes.

RNA interference (RNAi)-based biopesticides offer an attractive avenue for pest control. Previous studies revealed high RNAi sensitivity in Holotrichia parallela larvae, showcasing its potential for grub control. In this study, we aimed to develop an environmentally friendly RNAi method for H. parallela larvae. The double-stranded RNA (dsRNA) of the V-ATPase-a gene (HpVAA) was loaded onto layered double hydroxide (LDH). The dsRNA/LDH nanocomplex exhibited increased environmental stability, and we investigated the absorption rate and permeability of dsRNA-nanoparticle complexes and explored the RNAi controlling effect. Silencing the HpVAA gene was found to darken the epidermis of H. parallela larvae, with growth cessation or death or mortality, disrupting the epidermis and midgut structure. Quantitative reverse transcription-polymerase chain reaction and confocal microscopy confirmed the effective absorption of the dsRNA/LDH nanocomplex by peanut plants, with distribution in roots, stems, and leaves. Nanomaterial-mediated RNAi silenced the target genes, leading to the death of pests. Therefore, these findings indicate the successful application of the nanomaterial-mediated RNAi system for underground pests, thus establishing a theoretical foundation for developing a green, safe, and efficient pest control strategy.

Neuroinflammation is associated with Alzheimer’s disease co-pathology in dementia with Lewy bodies

BackgroundNeuroinflammation and Alzheimer’s disease (AD) co-pathology may contribute to disease progression and severity in dementia with Lewy bodies (DLB). This study aims to clarify whether a different pattern of neuroinflammation, such as alteration in microglial and astroglial morphology and distribution, is present in DLB cases with and without AD co-pathology.MethodsThe morphology and load (% area of immunopositivity) of total (Iba1) and reactive microglia (CD68 and HLA-DR), reactive astrocytes (GFAP) and proteinopathies of alpha-synuclein (KM51/pser129), amyloid-beta (6 F/3D) and p-tau (AT8) were assessed in a cohort of mixed DLB + AD (n = 35), pure DLB (n = 15), pure AD (n = 16) and control (n = 11) donors in limbic and neocortical brain regions using immunostaining, quantitative image analysis and confocal microscopy. Regional and group differences were estimated using a linear mixed model analysis.ResultsMorphologically, reactive and amoeboid microglia were common in mixed DLB + AD, while homeostatic microglia with a small soma and thin processes were observed in pure DLB cases. A higher density of swollen astrocytes was observed in pure AD cases, but not in mixed DLB + AD or pure DLB cases. Mixed DLB + AD had higher CD68-loads in the amygdala and parahippocampal gyrus than pure DLB cases, but did not differ in astrocytic loads. Pure AD showed higher Iba1-loads in the CA1 and CA2, higher CD68-loads in the CA2 and subiculum, and a higher astrocytic load in the CA1-4 and subiculum than mixed DLB + AD cases. In mixed DLB + AD cases, microglial load associated strongly with amyloid-beta (Iba1, CD68 and HLA-DR), and p-tau (CD68 and HLA-DR), and minimally with alpha-synuclein load (CD68). In addition, the highest microglial activity was found in the amygdala and CA2, and astroglial load in the CA4. Confocal microscopy demonstrated co-localization of large amoeboid microglia with neuritic and classic-cored plaques of amyloid-beta and p-tau in mixed DLB + AD cases.ConclusionsIn conclusion, microglial activation in DLB was largely associated with AD co-pathology, while astrocytic response in DLB was not. In addition, microglial activity was high in limbic regions, with prevalent AD pathology. Our study provides novel insights into the molecular neuropathology of DLB, highlighting the importance of microglial activation in mixed DLB + AD.

Harnessing Microbial Effectors for Macrophage-Mediated Drug Delivery.

Macrophage-based drug delivery systems are promising, but their development is still in its infancy, with many limitations remaining to be addressed. Our aim was to design a system harnessing microbial effectors to facilitate controlled drug cargo expulsion from macrophages to enable the use of more toxic drugs without adding to the risk of off-target detrimental effects. The pore forming and actin polymerizing Listeria monocytogenes effectors listeriolysin-O (LLO) and actin assembly-inducing protein (ActA) were synthesized using a novel green fluorescent protein (GFP)-linked heterologous expression system. These effectors were coated onto polystyrene beads to generate "synthetic cargo" before loading into primary M1 macrophages. Bead uptake and release from macrophages were evaluated by using high-throughput quantitative imaging flow cytometry and confocal microscopy. In vitro results confirmed appropriate activity of synthesized effectors. Coating of these effector proteins onto polystyrene beads (simulated drug cargo) resulted in changes in cellular morphology, bead content, and intracellular bead localization, which may support an interpretation of the induced release of these beads from the cells. This forms the basis for further investigation to fully elucidate any potential release mechanisms. Bacterial effectors ActA and LLO successfully effectuated actin polarization and protrusions from cell membranes similar to those seen in cells infected with Listeria spp., illustrating the potential of using these effectors and production methods for the development of an endogenous drug delivery system capable of low-risk, targeted release of high potency drugs.

SHR and SCR coordinate root patterning and growth early in the cell cycle

Precise control of cell division is essential for proper patterning and growth during the development of multicellular organisms. Coordination of formative divisions that generate new tissue patterns with proliferative divisions that promote growth is poorly understood. SHORTROOT (SHR) and SCARECROW (SCR) are transcription factors that are required for formative divisions in the stem cell niche of Arabidopsis roots1,2. Here we show that levels of SHR and SCR early in the cell cycle determine the orientation of the division plane, resulting in either formative or proliferative cell division. We used 4D quantitative, long-term and frequent (every 15 min for up to 48 h) light sheet and confocal microscopy to probe the dynamics of SHR and SCR in tandem within single cells of living roots. Directly controlling their dynamics with an SHR induction system enabled us to challenge an existing bistable model3 of the SHR–SCR gene-regulatory network and to identify key features that are essential for rescue of formative divisions in shr mutants. SHR and SCR kinetics do not align with the expected behaviour of a bistable system, and only low transient levels, present early in the cell cycle, are required for formative divisions. These results reveal an uncharacterized mechanism by which developmental regulators directly coordinate patterning and growth.

Structural and functional characterization of siadenovirus core protein VII nuclear localization demonstrates the existence of multiple nuclear transport pathways.

Adenovirus protein VII (pVII) plays a crucial role in the nuclear localization of genomic DNA following viral infection and contains nuclear localization signal (NLS) sequences for the importin (IMP)-mediated nuclear import pathway. However, functional analysis of pVII in adenoviruses to date has failed to fully determine the underlying mechanisms responsible for nuclear import of pVII. Therefore, in the present study, we extended our analysis by examining the nuclear trafficking of adenovirus pVII from a non-human species, psittacine siadenovirus F (PsSiAdV). We identified a putative classical (c)NLS at pVII residues 120-128 (120PGGFKRRRL128). Fluorescence polarization and electrophoretic mobility shift assays demonstrated direct, high-affinity interaction with both IMPα2 and IMPα3 but not IMPβ. Structural analysis of the pVII-NLS/IMPα2 complex confirmed a classical interaction, with the major binding site of IMPα occupied by K124 of pVII-NLS. Quantitative confocal laser scanning microscopy showed that PsSiAdV pVII-NLS can confer IMPα/β-dependent nuclear localization to GFP. PsSiAdV pVII also localized in the nucleus when expressed in the absence of other viral proteins. Importantly, in contrast to what has been reported for HAdV pVII, PsSiAdV pVII does not localize to the nucleolus. In addition, our study demonstrated that inhibition of the IMPα/β nuclear import pathway did not prevent PsSiAdV pVII nuclear targeting, indicating the existence of alternative pathways for nuclear localization, similar to what has been previously shown for human adenovirus pVII. Further examination of other potential NLS signals, characterization of alternative nuclear import pathways, and investigation of pVII nuclear targeting across different adenovirus species is recommended to fully elucidate the role of varying nuclear import pathways in the nuclear localization of pVII.

In vitro inhibition of Pseudomonas aeruginosa PAO1 biofilm formation by DZ2002 through regulation of extracellular DNA and alginate production.

Pseudomonas aeruginosa (P. aeruginosa) is a common pathogen associated with biofilm infections, which can lead to persistent infections. Therefore, there is an urgent need to develop new anti-biofilm drugs. DZ2002 is a reversible inhibitor that targets S-adenosylhomocysteine hydrolase and possesses anti-inflammatory and immune-regulatory activities. However, its anti-biofilm activity has not been reported yet. Therefore, we investigated the effect of DZ2002 on P. aeruginosa PAO1 biofilm formation by crystal violet staining (CV), real-time quantitative polymerase chain reaction (RT-qPCR) and confocal laser scanning microscopy (CLSM). The results indicated that although DZ2002 didn't affect the growth of planktonic PAO1, it could significantly inhibit the formation of mature biofilms. During the inhibition of biofilm formation by DZ2002, there was a parallel decrease in the synthesis of alginate and the expression level of alginate genes, along with a weakening of swarming motility. However, these results were unrelated to the expression of lasI, lasR, rhII, rhIR. Additionally, we also found that after treatment with DZ2002, the biofilms and extracellular DNA content of PAO1 were significantly reduced. Molecular docking results further confirmed that DZ2002 had a strong binding affinity with the active site of S-adenosylhomocysteine hydrolase (SahH) of PAO1. In summary, our results indicated that DZ2002 may interact with SahH in PAO1, inhibiting the formation of mature biofilms by downregulating alginate synthesis, extracellular DNA production and swarming motility. These findings demonstrate the potential value of DZ2002 in treating biofilm infections associated with P. aeruginosa.

Targeted delivery of anti-miRNA21 sensitizes PD-L1high tumor to immunotherapy by promoting immunogenic cell death.

Rationale: Growing evidence has demonstrated that miRNA-21 (miR-21) upregulation is closely associated with tumor pathogenesis. However, the mechanisms by which miR-21 inhibition modulates the immunosuppressive tumor microenvironment (TME) and improves tumor sensitivity to immune checkpoint blockade therapies remain largely unexplored. In this study, we demonstrate the precise delivery of anti-miR-21 using a PD-L1-targeting peptide conjugate (P21) to the PD-L1high TME. Methods: Investigating miR-21 inhibition mechanisms involved conducting quantitative real-time PCR, western blot, flow cytometry, and confocal microscopy analyses. The antitumor efficacy and immune profile of P21 monotherapy, or combined with anti-PD-L1 immune checkpoint inhibitors, were assessed in mouse models bearing CT26.CL25 tumors and 4T1 breast cancer. Results Inhibition of oncogenic miR-21 in cancer cells by P21 efficiently activates tumor suppressor genes, inducing autophagy and endoplasmic reticulum stress. Subsequent cell-death-associated immune activation (immunogenic cell death) is initiated via the release of damage-associated molecular patterns. The in vivo results also illustrated that the immunogenic cell death triggered by P21 could effectively sensitize the immunosuppressive TME. That is, P21 enhances CD8+ T cell infiltration in tumor tissues by conferring immunogenicity to dying cancer cells and promoting dendritic cell maturation. Meanwhile, combining P21 with an anti-PD-L1 immune checkpoint inhibitor elicits a highly potent antitumor effect in a CT26.CL25 tumor-bearing mouse model and 4T1 metastatic tumor model. Conclusions: Collectively, we have clarified a miR-21-related immunogenic cell death mechanism through the precise delivery of anti-miR-21 to the PD-L1high TME. These findings highlight the potential of miR-21 as a target for immunotherapeutic interventions.

Type I Interferons induce endothelial destabilization in Systemic Lupus Erythematosus in a Tie2-dependent manner.

Endothelial cell (EC) dysfunction is a hallmark of Systemic Lupus Erythematosus (SLE) and Tie2 is a receptor essential for vascular stability. Inflammatory processes promote inhibition of Tie2 homeostatic activation, driving vascular dysfunction. In this work we determined whether type I Interferons (IFN) induce Tie2 signalling-mediated endothelial dysfunction in patients with SLE. Serum levels of Angiopoietin (Ang)-1, Ang-2 and soluble (s)Tie1 in patients with SLE and healthy controls were measured by ELISA. Monocytes from patients with SLE and Human Umbilical Vein EC (HUVEC) were stimulated with IFN-α, IFN-β (1000 I.U.) or SLE serum (20%). mRNA and protein expression, phosphorylation and translocation were determined by quantitative PCR, ELISA, Western Blot, flow cytometry and confocal microscopy. Viability and angiogenic capacity were determined by calcein and tube formation assays. We found that sTie1 and Ang-2 serum levels were increased and Ang-1 decreased in patients with SLE and were associated with clinical characteristics. Type I IFN significantly decreased Ang-1 and increased Ang-2 in monocytes from patients with SLE. Type I IFN increased sTie1 and Ang-2 secretion and reduced Tie2 activation in HUVEC. Functionally, type I IFN significantly reduced EC viability and impaired angiogenesis in a Tie2 signalling-dependent manner. Finally, SLE serum increased Ang-2 and sTie1 secretion and significantly decreased tube formation. Importantly, Tie1 and IFNAR1 knockdown reversed these effects in tube formation. Overall, type I IFN play an important role in the stability of EC by inhibiting Tie2 signalling, suggesting that these processes may be implicated in the cardiovascular events observed in patients with SLE.

Single-photon microscopy to study biomolecular condensates

Biomolecular condensates serve as membrane-less compartments within cells, concentrating proteins and nucleic acids to facilitate precise spatial and temporal orchestration of various biological processes. The diversity of these processes and the substantial variability in condensate characteristics present a formidable challenge for quantifying their molecular dynamics, surpassing the capabilities of conventional microscopy. Here, we show that our single-photon microscope provides a comprehensive live-cell spectroscopy and imaging framework for investigating biomolecular condensation. Leveraging a single-photon detector array, single-photon microscopy enhances the potential of quantitative confocal microscopy by providing access to fluorescence signals at the single-photon level. Our platform incorporates photon spatiotemporal tagging, which allowed us to perform time-lapse super-resolved imaging for molecular sub-diffraction environment organization with simultaneous monitoring of molecular mobility, interactions, and nano-environment properties through fluorescence lifetime fluctuation spectroscopy. This integrated correlative study reveals the dynamics and interactions of RNA-binding proteins involved in forming stress granules, a specific type of biomolecular condensates, across a wide range of spatial and temporal scales. Our versatile framework opens up avenues for exploring a broad spectrum of biomolecular processes beyond the formation of membrane-less organelles.

Effect of Camel Peptide on the Biofilm of Staphylococcus epidermidis and Staphylococcus haemolyticus Formed on Orthopedic Implants.

The increasing bacterial drug resistance and the associated challenges in the treatment of infections warrant the search for alternative therapeutic methods. Hope is placed in antimicrobial peptides, which have a broad spectrum of action and are effective against strains which are resistant to conventional antibiotics. Antimicrobial peptides are also tested for their efficacy in the treatment of infections associated with the formation of biofilm. The aim of the present study was to examine the effect of Camel peptide on S. epidermidis and S. haemolyticus adhesion to and formation of biofilm on steel cortical bone screws and also on the process of reducing mature biofilm in orthopedic implants. The tests were performed on steel implants for osteosynthesis. The MIC value and MBEC values of the peptide were determined using the microdilution method in microtiter plates. The effect of the peptide on adhesion and biofilm formation, as well as on the activity on the preformed biofilm, was evaluated using quantitative methods and confocal microscopy. The presented research results indicate that the peptide exhibits very good antimicrobial properties against the analyzed strains. Concentrations above MIC reduced biofilm in the range of 90-99%.

Lymphedema-Associated Fibroblasts Are Related to Fibrosis and Stage Progression in Patients and a Murine Microsurgical Model.

The driver of secondary lymphedema (SL) progression is chronic inflammation, which promotes fibrosis. Despite advances in preclinical research, a specific effector cell subpopulation as a biomarker for therapy response or stage progression is still missing for SL. Whole skin samples of 35 murine subjects of a microsurgically induced SL model and 12 patients with SL were collected and their fibroblasts were isolated. These lymphedema-associated fibroblasts (LAFs) were cultured in a collagen I-poly-D-lysine 3-dimensional hydrogel to mimic skin conditions. Fibroblasts from nonlymphedema skin were used as negative control and transforming growth factor β (TGF-β)-stimulated fibroblasts were used to recreate profibrotic myofibroblasts. Quantitative immunocytofluorescence confocal microscopy analysis and invasion functional assays were performed in all subpopulations and statistically compared. In contrast to normal skin fibroblasts, LAFs exhibit α-smooth muscle actin-positive stress fibers and a reduced number of tight junctions in 3-dimensional hydrogel conditions. The switch from normal E-cadherin high phenotype to an N-cadherin high -E-cadherin low morphology suggests epithelial-to-mesenchymal transition for expansion and proliferation. This pathologic behavior of LAF was confirmed by live cell imaging analysis of invasion assays. The significant activation of markers of the TGF-β receptor 2-Smad pathway and collagen synthesis (HSP-47 [heat shock protein 47]) in LAFs supports epithelial-to-mesenchymal transition phenotypic changes and previous findings relating to TGF-β1 and fibrosis with lymphedema. A characteristic SL myofibroblast subpopulation was identified and translationally related to fibrosis and TGF-β1-associated stage progression. This SL-related subpopulation was termed LAFs. A comprehensive stage-related characterization is required to validate LAFs as a reliable biomarker for SL disease progression. The authors identify a cellular effector for fibrosis and stage progression of secondary lymphedema as a possible biomarker for surgical indication and therapy response.

IFP35 aggravates Staphylococcus aureus infection by promoting Nrf2-regulated ferroptosis

IntroductionSerious Staphylococcus aureus (SA) infection is one of the most life-threatening diseases. Interferon-induced protein 35 (IFP35) is a pleiotropic factor that participates in multiple biological functions, however, its biological role in SA infection is not fully understood. Ferroptosis is a new type of regulated cell death driven by the accretion of free iron and toxic lipid peroxides and plays critical roles in tissue damage. Whether ferroptosis is involved in SA-induced immunopathology and its regulatory mechanisms remain unknown. ObjectivesWe aimed to determine the role and underlying mechanisms of IFP35 in SA-induced lung infections. MethodsSA infection models were established using wild-type (WT) and IFP35 knockout (Ifp35−/−) mice or macrophages. Histological analysis was performed to assess lung injury. Quantitative real-time PCR, western blotting, flow cytometry, and confocal microscopy were performed to detect ferroptosis. Co-IP and immunofluorescence were used to elucidate the molecular regulatory mechanisms. ResultsWe found that IFP35 levels increased in the macrophages and lung tissue of SA-infected mice. IFP35 deficiency protected against SA-induced lung damage in mice. Moreover, ferroptosis occurred and contributed to lung injury after SA infection, which was ameliorated by IFP35 deficiency. Mechanically, IFP35 facilitated the ubiquitination and degradation of nuclear factor E2-related factor 2 (Nrf2), aggravating SA-induced ferroptosis and lung injury. ConclusionsOur data demonstrate that IFP35 promotes ferroptosis by facilitating the ubiquitination and degradation of Nrf2 to exacerbate SA infection. Targeting IFP35 may be a promising approach for treating infectious diseases caused by SA.

Simultaneous Targeting of IL-1-Signaling and IL-6-Trans-Signaling Preserves Human Pulmonary Endothelial Barrier Function During a Cytokine Storm.

Systemic inflammatory diseases, such as sepsis and severe COVID-19, provoke acute respiratory distress syndrome in which the pathological hyperpermeability of the microvasculature, induced by uncontrolled inflammatory stimulation, causes pulmonary edema. Identifying the inflammatory mediators that induce human lung microvascular endothelial cell barrier dysfunction is essential to find the best anti-inflammatory treatments for critically ill acute respiratory distress syndrome patients. We have compared the responses of primary human lung microvascular endothelial cells to the main inflammatory mediators involved in cytokine storms induced by sepsis and SARS-CoV2 pulmonary infection and to sera from healthy donors and severely ill patients with sepsis. Endothelial barrier function was measured by electric cell-substrate impedance sensing, quantitative confocal microscopy, and Western blot. The human lung microvascular endothelial cell barrier was completely disrupted by IL (interleukin)-6 conjugated with soluble IL-6R (IL-6 receptor) and by IL-1β, moderately affected by TNF (tumor necrosis factor)-α and IFN (interferon)-γ and unaffected by other cytokines and chemokines, such as IL-6, IL-8, MCP (monocyte chemoattractant protein)-1 and MCP-3. The inhibition of IL-1 and IL-6R simultaneously, but not separately, significantly reduced endothelial hyperpermeability on exposing human lung microvascular endothelial cells to a cytokine storm consisting of 8 inflammatory mediators or to sera from patients with sepsis. Simultaneous inhibition of IL-1 and JAK (Janus kinase)-STAT (signal transducer and activator of transcription protein), a signaling node downstream IL-6 and IFN-γ, also prevented septic serum-induced endothelial barrier disruption. These findings strongly suggest a major role for both IL-6 trans-signaling and IL-1β signaling in the pathological increase in permeability of the human lung microvasculature and reveal combinatorial strategies that enable the gradual control of pulmonary endothelial barrier function in response to a cytokine storm.

Siglec-9 is an inhibitory receptor on human mast cells in vitro

Mast cell activation is critical for the development of allergic diseases. Ligation of sialic acid-binding immunoglobin-like lectins (Siglecs), such as Siglec-6, -7, and -8 as well as CD33, have been shown to inhibit mast cell activation. Recent studies showed that human mast cells express Siglec-9, an inhibitory receptor also expressed by neutrophils, monocytes, macrophages, and dendritic cells. We aimed to characterize Siglec-9 expression and function in human mast cells invitro. We assessed the expression of Siglec-9 and Siglec-9 ligands on human mast cell lines and human primary mast cells by real-time quantitative PCR, flow cytometry, and confocal microscopy. We used a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing approach to disrupt the SIGLEC9 gene. We evaluated Siglec-9 inhibitory activity on mast cell function by using native Siglec-9 ligands, glycophorin A(GlycA), and high-molecular-weight hyaluronic acid, a monoclonal antibody against Siglec-9, and coengagement of Siglec-9 with the high-affinity receptor for IgE (FcεRI). Human mast cells express Siglec-9 and Siglec-9 ligands. SIGLEC9 gene disruption resulted in increased expression of activation markers at baseline and increased responsiveness to IgE-dependent and IgE-independent stimulation. Pretreatment with GlycA or high-molecular-weight hyaluronic acid followed by IgE-dependent or -independent stimulation had an inhibitory effect on mast cell degranulation. Coengagement of Siglec-9 with FcεRI in human mast cells resulted in reduced degranulation, arachidonic acid production, and chemokine release. Siglec-9 and its ligands play an important role in limiting human mast cell activation invitro.

Veklury®(remdesivir) formulations inhibit initial membrane-coupled events of SARS-CoV-2 infection due to their sulfobutylether-β-cyclodextrin content.

Despite its contradictory clinical performance, remdesivir (Veklury®) has a pivotal role in COVID-19 therapy. Possible contributions of the vehicle, sulfobutylether-β-cyclodextrin (SBECD) toVeklury® effects have been overlooked. The powder and solution formulations of Veklury® are treated equivalently despite their different vehicle content. Our objective was to study Veklury® effects on initial membrane-coupled events of SARS-CoV-2 infection focusing on the cholesterol depletion-mediated role of SBECD. Using time-correlated flow cytometry and quantitative three-dimensional confocal microscopy, we studied early molecular events of SARS-CoV-2-host cell membrane interactions. Veklury® and different cholesterol-depleting cyclodextrins (CDs) reduced binding of the spike receptor-binding domain (RBD) to ACE2 and spike trimer internalization for Wuhan-Hu-1, Delta and Omicron variants. Correlations of these effects with cholesterol-dependent changes in membrane structure and decreased lipid raft-dependent ACE2-TMPRSS2 interaction establish that SBECD is not simply a vehicle but also an effector along with remdesivir due to its cholesterol-depleting potential. Veklury® solution inhibited RBD binding more efficiently due to its twice higher SBECD content. The CD-induced inhibitory effects were more prominent at lower RBD concentrations and in cells with lower endogenous ACE2 expression, indicating that the supportive CD actions can be even more pronounced during in vivo infection when viral load and ACE expression are typically low. Our findings call for the differentiation of Veklury® formulations in meta-analyses of clinical trials, potentially revealing neglected benefits of the solution formulation, and also raise the possibility of adjuvant cyclodextrin (CD) therapy, even at higher doses, in COVID-19.