CXCL8 Release Research Articles

HEMATOPOIETIC STEM CELL GENE THERAPY AS A NOVEL THERAPEUTIC APPROACH FOR SEVERE CROHN’S DISEASE ASSOCIATED WITH NOD2-DEFICIENCY

Abstract Pathogenic variants of the NOD2 (nucleotide-binding oligomerization domain containing protein 2) gene demonstrate the strongest genetic association to Crohn’s inflammatory bowel disease (CD). Mounting evidence links NOD2 deficiency with poor clinical outcome, particularly in pediatric and early onset CD. CD patients with pathogenic NOD2 variants, particularly carriers of more than one risk allele, frequently present an aggressive, fistulizing and fibrostenotic disease, requiring multiple surgical resections. NOD2-deficiency is implicated as a driver of CD pathogenesis through failure of gut innate immunity to resolve bacterial infections and by loss of tissue homeostasis within the intestinal microenvironment. Here we present preclinical data on OTL-104, an autologous haematopoietic stem cell gene therapy (HSC-GT) which aims to stably restore NOD2 expression in gut resident macrophages, to correct immune dysfunction linked to NOD2-deficient CD pathogenesis. We used in vitro and in vivo models of NOD2 deficiency to demonstrate the mechanism of action and the efficacy of OTL-104 autologous HSC-GT. NOD2KO human myeloid cells differentiated in vitro from CRISPR-generated NOD2KO CD34+ HSCs are unable to mount a proinflammatory cytokine response to MDP stimulation. Similarly, myeloid cells differentiated from CD34+ cells obtained from peripheral blood of genetically characterized NOD2-deficient CD patients, are also refractory to MDP stimulation and unable to generate a normal cytokine response profile (IL-8, TNFa, IL-6, CXCL1/2 and IL-10). In both NOD2 deficient CD34+ derived monocyte models, transduction with a lentiviral vector (LVV) expressing NOD2 fully restores NOD2-dependent cytokine and chemokine responses, restoring an immune profile that is comparable to monocytes derived from CD34+ cells from NOD2 wild-type healthy donors. Transplantation of lineage negative (Lin-) haematopoietic stem/progenitor cells (HSPCs) transduced with the OTL-104 LVV in Nod2KO mice was used as an in vivo model of gene therapy for CD. Compared to WT mice, Nod2KO mice fail to release systemic inflammatory mediators and recruit myeloid cells in response to MDP administration. Transplantation of transduced Lin- HSPCs restores MDP-induced systemic release of IL-6 and CXCL1 as well as innate mobilization of monocyte/macrophage cells and gut associated immune markers. Transplanted Nod2KO mice display normal hematopoiesis and stable vector copy numbers in hematopoietic cells. Key to our therapeutic approach, histopathological analysis of intestinal lamina propria from transplanted mice shows a normal biodistribution and physiological NOD2 gene expression in tissue resident cells (Figure 1). These results confirm the impact of NOD2 deficiency in primary immune activation and demonstrates the therapeutic potential of OTL-104 HSC-GT for long-term correction of NOD2-deficient CD.

OP29 Haematopoietic stem cell gene therapy as a treatment for severe Crohn’s Disease associated with pathogenic NOD2 genetic variants

Abstract Background Pathogenic variants of the NOD2 (nucleotide-binding oligomerization domain containing protein 2) gene demonstrate the strongest genetic association to Crohn’s inflammatory bowel disease (CD), with mounting evidence linking NOD2 deficiency with poor clinical outcome. CD patients with NOD2 variants, particularly carriers of more than one risk allele, frequently present an aggressive, fistulizing and fibrostenotic disease, requiring multiple surgical resections. NOD2-deficiency is implicated to drive CD pathogenesis through failure of gut innate immunity to resolve bacterial infections and by loss of tissue homeostasis within the intestinal microenvironment. Here we present preclinical data on OTL-104, an autologous haematopoietic stemcell gene therapy (HSC-GT) which aims to stably restore NOD2 expression in gut resident macrophages, to correct immune dysfunction linked to NOD2-deficient CD pathogenesis. Methods We used in vitro and in vivo models of NOD2 deficiency to demonstrate the mechanism of action and the efficacy of OTL-104 autologous HSC-GT. Results NOD2KO human myeloid cells differentiated in vitro from CRISPR-generated NOD2KO CD34+ HSCs are unable to mount a proinflammatory cytokine response to MDP stimulation. Similarly, myeloid cells differentiated from CD34+ cells obtained from peripheral blood of genetically characterized NOD2-deficient CD patients, are also refractory to MDP stimulation and unable to generate a normal cytokine response profile. In both NOD2 deficient CD34+ derived monocyte models, transduction with a lentiviral vector (LVV) expressing NOD2 fully restores NOD2-dependent cytokine and chemokine responses, restoring an immune profile that is comparable to monocytes derived from CD34+ cells from NOD2 wild-type healthy donors (Figure 1). Transplantation of lineage negative (Lin-) haematopoietic stem/progenitor cells (HSPCs) transduced with the OTL-104 LVV in NOD2KO mice was used as an in vivo model of gene therapy for CD. Compared to WT mice, NOD2KO mice fail to release systemic inflammatory mediators and recruit myeloid cells in response to MDP administration. Transplantation of transduced Lin- HSPCs restores MDP-induced systemic release of IL-6 and CXCL1 as well as innate mobilization of monocyte/macrophage cells and gut associated gene expression. Key to our therapeutic approach, histopathological analysis of intestinal lamina propria from transplanted mice shows a normal biodistribution and physiological NOD2 gene expression in tissue resident cells. Conclusion These results confirm the impact of NOD2 deficiency in primary immune activation and demonstrates the therapeutic potential of OTL-104 HSC-GT for long-term correction of NOD2-deficient CD.

The memory of airway epithelium damage in smokers and COPD patients.

Chronic obstructive pulmonary disease (COPD), a devastating and irreversible lung disease, causes structural and functional defects in the bronchial epithelium, the (ir)reversibility of which remains unexplored in vitro. This study aimed to investigate the persistence of COPD-related epithelial defects in long-term airway epithelial cultures derived from non-smokers, smokers, and COPD patients. Barrier function, polarity, cell commitment, epithelial-to-mesenchymal transition, and inflammation were evaluated and compared with native epithelium characteristics. The role of inflammation was explored using cytokines. We show that barrier dysfunction, compromised polarity, and lineage abnormalities observed in smokers and COPD persisted for up to 10 wk. Goblet cell hyperplasia was associated with recent cigarette smoke exposure. Conversely, increased IL-8/CXCL-8 release and abnormal epithelial-to-mesenchymal transition diminished over time. These ex vivo observations matched surgical samples' abnormalities. Cytokine treatment induced COPD-like changes in control cultures and reactivated epithelial-to-mesenchymal transition in COPD cells. In conclusion, these findings suggest that the airway epithelium of smokers and COPD patients retains a multidimensional memory of its original state and previous cigarette smoke-induced injuries, maintaining these abnormalities for extended periods.

Pb induces the release of CXCL10 and CCL2 chemokines via mtROS/NF-κB activation in BV-2 cells

Lead (Pb), a well-known environmental pollutant, could cause damage of microglia, the resident macrophages vitally regulating inflammation in brain. Previous studies have found that Pb exposure induces typical pro-inflammatory factors release, such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), but what effects of Pb treatment below the dose causing these factors release are unknown. Thus, cytokines assay was performed to identify the factors released from Pb-treated BV-2 cells at 2.5 μM, causing no effects on TNF-α, IL-1β, and IL-6 release and cell death. Cytokines assay identified low doses of Pb exposure mainly induce an increase in specific chemokines, including CXCL10, CCL2, and CXCL2, which were confirmed by ELISA. Subsequent assessment found Pb could damage mitochondria function and generate mitochondrial reactive oxygen species (mtROS), and Mito TEMPO, a specific inhibitor of mtROS, suppressed Pb-caused upregulation of CXCL10 and CCL2, but not CXCL2. Finally, we determined that mtROS mediated Pb-induced activation of NF-κB pathway, as Mito TEMPO treatment inhibited P-p65/p65 escalation during Pb treatment. Inhibition of NF-κB pathway by Bay11–7821 suppressed the release of CXCL10 and CCL2. Collectively, low dose of Pb induces the release of CXCL10 and CCL2 chemokines, but not TNF-α and IL-1β, via mtROS/NF-κB activation in BV-2 cells.

APOL1 Induces Pyroptosis of Fibroblasts Through NLRP3/Caspase-1/GSDMD Signaling Pathway in Ulcerative Colitis.

Pyroptosis is a form of proinfammatory gasdermin-mediated programmed cell death. Abnormal infammation in the intestine is a critical risk factor for Ulcerative colitis (UC). However, at present, it is not clear whether pyroptosis of colonic fibroblasts is involved in the pathogenesis and progression of UC. In this study, key genes associated with UC were identified by bioinformatics analysis. Datasets were downloaded from the Gene Expression Omnibus (GEO) database (GSE193677). The differentially expressed genes were analyzed, and the hub genes were screened by weighted gene co-expression network analysis (WGCNA) and differentially expressed genes. We also downloaded the dataset from GEO for single-cell RNA sequencing (GSE231993). The expression of key genes was verified by immunohistochemistry, immunofluorescence and Western blot, and the specific pathways of key genes inducing pyroptosis in cell lines were explored. The results of bioinformatics analysis showed that the expression of APOL1 and CXCL1 in UC tissues was significantly higher than that in normal tissues. The results of single-cell analysis showed that the two genes were co-localized to fibroblasts. These results were consistent with the results of immunohistochemistry and immunofluorescence colocalization in human intestinal mucosa specimens. Furthermore, APOL1 overexpression induced NLRP3-caspase1-GSDMD-mediated pyroptosis of fibroblasts, which was confirmed by Western blot. APOL1 induces pyroptosis of fibroblasts mediated by NLRP3-Caspase1-GSDMD signaling pathway and promote the release of chemokines CXCL1. Fibroblasts may play a crucial role in the pathogenesis and progression of UC.

Altered response to Toll-like receptor 4 activation in fibromyalgia: A low-dose, human experimental endotoxemia pilot study

In this pilot study, a human intravenous injection of low-dose endotoxin (lipopolysaccharide, LPS) model was used to test if fibromyalgia is associated with altered immune responses to Toll-like receptor 4 (TLR4) activation. Eight women with moderately-severe fibromyalgia and eight healthy women were administered LPS at 0.1 ng/kg in session one and 0.4 ng/kg in session two. Blood draws were collected hourly to characterize the immune response. The primary analytes of interest, leptin and fractalkine, were assayed via commercial radioimmunoassay and enzyme-linked immunosorbent assay kits, respectively. Exploratory analyses were performed on 20 secreted cytokine assays by multiplex cytokine panels, collected hourly. Exploratory analyses were also performed on testosterone, estrogen, and cortisol levels, collected hourly. Additionally, standard clinical complete blood counts with differential (CBC-D) were collected before LPS administration and at the end of the session. The fibromyalgia group demonstrated enhanced leptin and suppressed fractalkine responses to LPS administration. In the exploratory analyses, the fibromyalgia group showed a lower release of IFN-γ, CXCL10, IL-17A, and IL-12 and higher release of IL-15, TARC, MDC, and eotaxin than the healthy group. The results of this study suggest that fibromyalgia may involve an altered immune response to TLR4 activation.

Elucidating the Therapeutic Utility of Olaparib in Sulfatide-Induced Human Astrocyte Toxicity and Neuroinflammation

Metachromatic leukodystrophy (MLD) is a severe demyelinating, autosomal recessive genetic leukodystrophy, with no curative treatment. The disease is underpinned by mutations in the arylsulfatase A gene (ARSA), resulting in deficient activity of this lysosomal enzyme, and consequential accumulation of galactosylceramide-3-O-sulfate (sulfatide) in the brain. Most of the effects in the brain have been attributed to the accumulation of sulfatides in oligodendrocytes and their cell damage. In contrast, less is known regarding sulfatide toxicity in astrocytes. Poly (ADP-ribose) polymerase (PARP) inhibitors are anti-cancer therapeutics that have proven efficacy in preclinical models of many neurodegenerative and inflammatory diseases, but have never been tested for MLD. Here, we examined the toxic effect of sulfatides on human astrocytes and restoration of this cell damage by the marketed PARP-1 inhibitor, Olaparib. Cultured human astrocytes were treated with increasing concentrations of sulfatides (5–100 μM) with or without Olaparib (100 nM). Cell viability assays were used to ascertain whether sulfatide-induced toxicity was rescued by Olaparib. Immunofluorescence, calcium (Ca2+) imaging, ROS, and mitochondrial damage assays were also used to explore the effects of sulfatides and Olaparib. ELISAs were performed and chemotaxis of peripheral blood immune cells was measured to examine the effects of Olaparib on sulfatide-induced inflammation in human astrocytes. Here, we established a concentration-dependent (EC50∼20 μM at 24 h) model of sulfatide-induced astrocyte toxicity. Our data demonstrate that sulfatide-induced astrocyte toxicity involves (i) PARP-1 activation, (ii) pro-inflammatory cytokine release, and (iii) enhanced chemoattraction of peripheral blood immune cells. Moreover, these sulfatide-induced effects were attenuated by Olaparib (IC50∼100 nM). In addition, sulfatide caused impairments of ROS production, mitochondrial stress, and Ca2+ signaling in human astrocytes, that were indicative of metabolic alterations and that were also alleviated by Olaparib (100 nM) treatment. Our data support the hypothesis that sulfatides can drive astrocyte cell death and demonstrate that Olaparib can dampen many facets of sulfatide-induced toxicity, including, mitochondrial stress, inflammatory responses, and communication between human astrocytes and peripheral blood immune cells. These data are suggestive of potential therapeutic utility of PARP inhibitors in the sphere of rare demyelinating diseases, and in particular MLD.Graphical Graphical abstract. Proposed mechanism of action of Olaparib in sulfatide-treated astrocytes. Human astrocytes treated for 24 h with sulfatides increase PARP-1 expression and die. PARP-1 overexpression is modulated by Ca2+ release from the endoplasmic reticulum, thus enhancing intracellular Ca2+ concentration. PARP-1 inhibition with Olaparib reduces Ca2+ influx and cell death. Olaparib also decreases IL-6, IL-8, IL-17, and CX3CL1 release from sulfatide-stimulated astrocytes, suggesting that PARP-1 plays a role in dampening neuroinflammation in MLD. This is confirmed by the reduction of immune cell migration such as lymphocytes, NK cells, and T cells towards sulfatide-treated astrocytes. Moreover, mitochondrial stress and ROS production induced by sulfatides are rescued by PARP-1 inhibition. Future studies will focus on the signaling cascades triggered by PARP-1-mediated currents in reactive astrocytes and Olaparib as a potential therapeutic target for MLD.

Aberrant neutrophil degranulation in hospitalized patients with COVID-19 partially remains for 6 months.

Neutrophils are important players in COVID-19, contributing to tissue damage by release of inflammatory mediators, including ROS and neutrophil elastase. Longitudinal studies on the effects of COVID-19 on neutrophil phenotype and function are scarce. Here, we longitudinally investigated the phenotype and degranulation of neutrophils in COVID-19 patients (28 nonhospitalized and 35 hospitalized patients) compared with 17 healthy donors (HDs). We assessed phenotype, degranulation, CXCL8 (IL-8) release, and ROS generation within 8 days, at one or 6 month(s) after COVID-19 diagnosis. For degranulation and ROS production, we stimulated neutrophils, either with ssRNA and TNF or granulocyte-macrophage colony-stimulating factor and N-Formylmethionyl-leucyl-phenylalanine. During active COVID-19, neutrophils from hospitalized patients were more immature than from HDs and were impaired in degranulation and ROS generation, while neutrophils from nonhospitalized patients only demonstrated reduced CD66b+ granule release and ROS production. Baseline CD63 expression, indicative of primary granule release, and CXCL8 production by neutrophils from hospitalized patients were elevated for up to 6 months. These findings show that patients hospitalized due to COVID-19, but not nonhospitalized patients, demonstrated an aberrant neutrophil phenotype, degranulation, CXCL8 release, and ROS generation that partially persists up to 6 months after infection.

VPS13A knockdown impairs corticostriatal synaptic plasticity and locomotor behavior in a new mouse model of chorea-acanthocytosis

Chorea-acanthocytosis (ChAc) is an inherited neurodegenerative movement disorder caused by VPS13A gene mutations leading to the absence of protein expression. The striatum is the most affected brain region in ChAc patients. However, the study of the VPS13A function in the brain has been poorly addressed. Here we generated a VPS13A knockdown (KD) model and aimed to elucidate the contribution of VPS13A to synaptic plasticity and neuronal communication in the corticostriatal circuit. First, we infected primary cortical neurons with miR30-shRNA against VPS13A and analyzed its effects on neuronal plasticity. VPS13A-KD neurons showed a higher degree of branching than controls, accompanied by decreased BDNF and PSD-95 levels, indicative of synaptic alterations. We then injected AAV-KD bilaterally in the frontal cortex and two different regions of the striatum of mice and analyzed the effects of VPS13A-KD on animal behavior and synaptic plasticity. VPS13A-KD mice showed modification of the locomotor behavior pattern, with increased exploratory behavior and hyperlocomotion. Corticostriatal dysfunction in VPS13A-KD mice was evidenced by impaired striatal long-term depression (LTD) after stimulation of cortical afferents, which was partially recovered by BDNF administration. VPS13A-KD did not lead to neuronal loss in the cortex or the striatum but induced a decrease in the neuronal release of CX3CL1 and triggered a microglial reaction, especially in the striatum. Notably, CX3CL1 administration partially restored the impaired corticostriatal LTD in VPS13A-KD mice. Our results unveil the involvement of VPS13A in neuronal connectivity modifying BDNF and CX3CL1 release. Moreover, the involvement of VPS13A in synaptic plasticity and motor behavior provides key information to further understand not only ChAc pathophysiology but also other neurological disorders.

Role of different mechanisms in pro-inflammatory responses triggered by traffic-derived particulate matter in human bronchiolar epithelial cells

BackgroundTraffic-derived particles are important contributors to the adverse health effects of ambient particulate matter (PM). In Nordic countries, mineral particles from road pavement and diesel exhaust particles (DEP) are important constituents of traffic-derived PM. In the present study we compared the pro-inflammatory responses of mineral particles and DEP to PM from two road tunnels, and examined the mechanisms involved.MethodsThe pro-inflammatory potential of 100 µg/mL coarse (PM10-2.5), fine (PM2.5-0.18) and ultrafine PM (PM0.18) sampled in two road tunnels paved with different stone materials was assessed in human bronchial epithelial cells (HBEC3-KT), and compared to DEP and particles derived from the respective stone materials. Release of pro-inflammatory cytokines (CXCL8, IL-1α, IL-1β) was measured by ELISA, while the expression of genes related to inflammation (COX2, CXCL8, IL-1α, IL-1β, TNF-α), redox responses (HO-1) and metabolism (CYP1A1, CYP1B1, PAI-2) was determined by qPCR. The roles of the aryl hydrocarbon receptor (AhR) and reactive oxygen species (ROS) were examined by treatment with the AhR-inhibitor CH223191 and the anti-oxidant N-acetyl cysteine (NAC).ResultsRoad tunnel PM caused time-dependent increases in expression of CXCL8, COX2, IL-1α, IL-1β, TNF-α, COX2, PAI-2, CYP1A1, CYP1B1 and HO-1, with fine PM as more potent than coarse PM at early time-points. The stone particle samples and DEP induced lower cytokine release than all size-fractionated PM samples for one tunnel, and versus fine PM for the other tunnel. CH223191 partially reduced release and expression of IL-1α and CXCL8, and expression of COX2, for fine and coarse PM, depending on tunnel, response and time-point. Whereas expression of CYP1A1 was markedly reduced by CH223191, HO-1 expression was not affected. NAC reduced the release and expression of IL-1α and CXCL8, and COX2 expression, but augmented expression of CYP1A1 and HO-1.ConclusionsThe results indicate that the pro-inflammatory responses of road tunnel PM in HBEC3-KT cells are not attributed to the mineral particles or DEP alone. The pro-inflammatory responses seem to involve AhR-dependent mechanisms, suggesting a role for organic constituents. ROS-mediated mechanisms were also involved, probably through AhR-independent pathways. DEP may be a contributor to the AhR-dependent responses, although other sources may be of importance.

Impact of simulated brain interstitial fluid flow on the chemokine CXCL12 release from an alginate-based hydrogel in a new 3D in vitro model

Introduction: Extensive investigation has been undertaken regarding drug delivery systems for the management of glioblastoma multiforme (GBM). The infiltrative behavior of GBM cells within the brain tissue is primarily attributed to their heterogeneity, the movement of interstitial fluid (IFF), and the presence of chemokines. These factors contribute to the limited effectiveness of current conventional treatments. To address the dissemination of GBM cells, a proposed therapeutic approach involves utilizing a controlled release gradient of CXC-chemokine-ligand-12 (CXCL12). However, the impact of IFF on GBM cell migration within the brain underscores its critical importance as a significant parameter, which, surprisingly, has not been extensively studied in the context of localized drug delivery targeting the brain.Methods: Hydrogels are known for their inherent capacity to entrap various agents and exert precise control over their subsequent release. In the present investigation, we aimed to elucidate the release kinetics of CXCL12, whether in its free form or encapsulated within nanoparticles, from alginate-based hydrogels, both under static and dynamic conditions. To investigate the impact of convective forces mimicking the interstitial fluid flow (IFF) within the peritumoral environment of the brain, a three-dimensional in vitro model was developed. This model enabled the evaluation of CXCL12 release as a function of time and position, specifically accounting for the contribution of simulated IFF on the release behavior.Results: We first demonstrated that the release kinetic profiles under static culture conditions were independent of the initial mass loading and the predominant phenomenon occurring was diffusion. Subsequently, we investigated the release of CXCL12, which was loaded into Alginate/Chitosan-Nanoparticles (Alg/Chit-NPs) and embedded within an alginate hydrogel matrix. Mathematical modeling results also indicated the presence of electrostatic interactions between alginate and CXCL12. The Alg/Chit-NPs effectively slowed down the initial burst release, leading to a reduction in the diffusion coefficient of CXCL12. To further study the release behavior, we developed a perfusion bioreactor with a unique culture chamber designed to recapitulate the peritumoral environment and varied the fluid flow rates at 0.5 µL/min, 3 µL/min, 6.5 µL/min, and 10 µL/min. As the flow rate increased, the cumulative amount of released CXCL12 also increased for all three initial mass loadings. Beyond 3 µL/min, convection became the dominant mechanism governing CXCL12 release, whereas below this threshold, diffusion played a more prominent role.Conclusion: The indirect perfusion flow had a crucial impact on CXCL12 release and distribution inside the hydrogel in and against its direction. This system highlights the importance of considering the IFF in brain targeting delivery system and will be used in the future to study GBM cell behaviors in response to CXCL12 gradient.

Recombinant SARS-CoV-2 Spike Protein and Its Receptor Binding Domain Stimulate Release of Different Pro-Inflammatory Mediators via Activation of Distinct Receptors on Human Microglia Cells.

SARS-CoV-2 infects cells via its spike (S) protein binding to its surface receptor angiotensin converting enzyme 2 (ACE2) on target cells and results in acute symptoms involving especially the lungs known as COVID-19. However, increasing evidence indicates that SARS-CoV-2 infection produces neuroinflammation associated with neurological, neuropsychiatric, and cognitive symptoms persists well past the resolution of the infection, known as post-COVID-19 sequalae or long-COVID. The neuroimmune mechanism(s) involved in long-COVID have not been adequately characterized. In this study, we show that recombinant SARS-CoV-2 full-length S protein stimulates release of pro-inflammatory IL-1b, CXCL8, IL-6, and MMP-9 from cultured human microglia via TLR4 receptor activation. Instead, recombinant receptor-binding domain (RBD) stimulates release of TNF-α, IL-18, and S100B via ACE2 signaling. These results provide evidence that SARS-CoV-2 spike protein contributes to neuroinflammation through different mechanisms that may be involved in CNS pathologies associated with long-COVID.

Experimental traumatic occlusion drives immune changes in trigeminal ganglion

We previously demonstrated that experimental traumatic occlusion (ETO) induces a long-lasting nociceptive response. These findings were associated with altered neuronal patterns and suggestive satellite glial cell activation. This study aimed to elucidate the activation of satellite glial cells following ETO in the trigeminal ganglion. Moreover, we explored the involvement of resident and infiltrating cells in trigeminal ganglion in ETO. Finally, we investigated the overexpression of purinergic signaling and the CX3CL1/CX3CR1 axis. RT-qPCR and electrophoresis showed overexpression of GFAP in the trigeminal ganglion (TG), and immunohistochemistry corroborated these findings, demonstrating SGCs activation. ELISA reveals enhanced levels of TNF-α and IL-1β in TG after 28 d of ETO. In trigeminal ganglia, ETO groups improved the release of CX3CL1, and immunohistochemistry showed higher CX3CR1+ -immunoreactive cells in ETO groups. Immunohistochemistry and electrophoresis of the P2X7 receptor were found in ETO groups. The mRNA levels of IBA1 are upregulated in the 0.7-mm ETO group, while immunohistochemistry showed higher IBA1+ -immunoreactive cells in both ETO groups. The expression of CD68 by electrophoresis and immunohistochemistry was observed in the ETO groups. For last, ELISA revealed increased levels of IL-6, IL-12, and CCL2 in the TG of ETO groups. Furthermore, the mRNA expression revealed augmented transcription factors and cytokines associated with lymphocyte activation, such as RORγt, IL-17, Tbet, IFNγ, FOXP3, and IL-10. The findings of this study suggested that ETO activates SGCs in TG, and purinergic signaling and CX3CL1/CX3CR1 axis were upregulated. We uncovered the involvement of a distinct subtype of macrophages, named sensory neuron–associated macrophage activation (sNMAs), and detected an expanded number of infiltrated macrophages onto TG. These findings indicate that ETO induces chronic/persistent immune response.

IL-10 in combination with IL-12 and TNF-α attenuates CXCL8/CXCR1 axis in peritoneal macrophages of mice infected with Staphylococcus aureus through the TNFR1-IL-1R-NF-κB pathway

Overexpression of Staphylococcus aureus mediated CXCL8/CXCR1 axis is a major cause of sepsis and severe inflammatory diseases. This chemokine acts conjointly with various pro-inflammatory and anti-inflammatory cytokines that govern the severity of inflammation. The effects of different combinations of exogenous cytokines on CXCR1 expression in macrophages remain undetermined.Exogenous cytokine and anti-inflammatory cytokine therapy had been used to modulate CXCL8 and CXCR1 expression in peritoneal macrophages. Male Swiss albino mice were inoculated with live S. aureus (106 cells/ mouse) for the development of infection. Exogenous cytokines (TNF-α, IL-12, IFN-γ and IL-10) were administered intraperitoneally (single or combination) 24 h post S. aureus infection. The mice were sacrificed and peritoneal macrophages were isolated three days post infection. CXCL8, IL-12, IL-10 secretion, ROS generation and the bacterial phagocytic process had been evaluated. Western blot was used to study the expressions of TNFR1, IL-1R, CXCR1 and NF-κB.TNF-α, IL-12 and IFN-γ treatments aggravated CXCL8 and CXCR1 expression in the macrophages of infected mice. TNF-α + IFN-γ treatment was a major inducer of nitric oxide release and mediated maximum bacterial killing. IL-12 + TNF-α treatment was most potent in increasing ROS, CXCL8/CXCR1 expression through increased levels of TNFR1, IL-1R and NF-κB activation. IL-10 reversed the effects of exogenous cytokines but also impaired the bacterial clearance phenomenon in peritoneal lavage. Treatment with IL-12 + TNF-α + IL-10 was most effective in ameliorating oxidative stress, reduced CXCL8 release and expression levels of TNFR1, IL-1R, and NF-κB.Concludingly, IL-12 + TNF-α + IL-10 treatment mitigated CXCL8/CXCR1 expression and inflammatory signalling via downregulation of TNFR1-IL-1R-NF-κB pathway in peritoneal macrophages and inflammatory sequelae during S. aureus infection.

89 Lung Single Cell Transcriptomics to Guide the Development of AOP Anchored Cell-Based Assays in Response to Nanoparticle Exposure

Abstract Inhaled nanoparticles (NP) can cause acute and chronic inflammation. Here we seek to identify NP-specific cellular perturbation pathways and the underlying key cell types to inform adverse outcome pathway (AOP) anchored in vitro studies, by single cell transcriptomics. We exposed mice intratracheally to carbon black (CNP), tangled double-walled (DWCNT) and rigid, multi-walled carbon nanotubes (MWCNT). All lungs underwent single-cell RNA sequencing (scRNA-seq), histology and BAL analysis. At the chosen doses all NPs caused comparable airspace neutrophilia at 12h, which increased until d6 for the CNTs but remained elevated until d28 only for MWCNT, indicating the key event (KE) acute and chronic inflammation. Comparing scRNA-seq and BAL cytokine levels at 12h, differentiated the fate of inflammation NP specifically. In agreement with airspace neutrophilia, CNP uniquely triggered GM-CSF and CXCL1 release, with Csf2 and Cxcl1 mRNAs being expressed mainly in alveolar epithelial cells. DWCNT caused CCL2, -3 and -4 release involving interstitial macrophages and monocytes, associated with acute high lung monocyte and at d6 high BAL macrophage numbers. MWCNT in contrast caused ample BAL cytokine increases involving different cell types and specifically caused a Th2 cytokine response (CCL11, IL10), a crucial KE of fibrosis AOPs. MWCNT triggered cell communication analysis uncovered NP-specific networks including epithelial cells, fibroblasts, macrophages, dendritic cells and endothelial cells. Our study uncovers early NP-specific cell perturbations and identified first specific cellular response pattern to guide AOP predictive cell-based testing strategies. Further analysis of the cellular dynamics shall enhance our understanding of NP specific cell circuits and related pathologies.

Inflammatory Genes Associated with Pristine Multi-Walled Carbon Nanotubes-Induced Toxicity in Ocular Cells.

The wide application of multi-walled carbon nanotubes (MWCNTs) in various fields has raised enormous concerns regarding their safety for humans. However, studies on the toxicity of MWCNTs to the eye are rare and potential molecular mechanisms are completely lacking. This study was to evaluate the adverse effects and toxic mechanisms of MWCNTs on human ocular cells. Human retinal pigment epithelial cells (ARPE-19) were treated with pristine MWCNTs (7-11 nm) (0, 25, 50, 100 or 200 μg/mL) for 24 hours. MWCNTs uptake into ARPE-19 cells was examined using transmission electron microscopy (TEM). The cytotoxicity was evaluated by CCK-8 assay. The death cells were detected by Annexin V-FITC/PI assay. RNA profiles in MWCNT-exposed and non-exposed cells (n = 3) were analyzed using RNA-sequencing. The differentially expressed genes (DEGs) were identified through the DESeq2 method and hub of which were filtered by weighted gene co-expression, protein-protein interaction (PPI) and lncRNA-mRNA co-expression network analyses. The mRNA and protein expression levels of crucial genes were verified using quantitative polymerase chain reaction (qPCR), colorimetric analysis, ELISA and Western blotting. The toxicity and mechanisms of MWCNTs were also validated in human corneal epithelial cells (HCE-T). TEM analysis indicated the internalization of MWCNTs into ARPE-19 cells to cause cell damage. Compared with untreated ARPE-19 cells, those exposed to MWCNTs exhibited significantly decreased cell viabilities in a dose-dependent manner. The percentages of apoptotic (early, Annexin V positive; late, Annexin V and PI positive) and necrotic (PI positive) cells were significantly increased after exposure to IC50 concentration (100 μg/mL). A total of 703 genes were identified as DEGs; 254 and 56 of them were, respectively, included in darkorange2 and brown1 modules that were significantly associated with MWCNT exposure. Inflammation-related genes (including CXCL8, MMP1, CASP3, FOS, CXCL2 and IL11) were screened as hub genes by calculating the topological characteristics of genes in the PPI network. Two dysregulated long non-coding RNAs (LUCAT1 and SCAT8) were shown to regulate these inflammation-related genes in the co-expression network. The mRNA levels of all eight genes were confirmed to be upregulated, while caspase-3 activity and the release of CXCL8, MMP1, CXCL2, IL11 and FOS proteins were demonstrated to be increased in MWCNT-treated ARPE-19 cells. MWCNTs exposure also can induce cytotoxicity and increase the caspase-3 activity and the expression of LUCAT1, MMP1, CXCL2, and IL11 mRNA and protein in HCE-T cells. Our study provides promising biomarkers for monitoring MWCNT-induced eye disorders and targets for developing preventive and therapeutic strategies.

Role of altered cortisol production in the development of sarcopenia

Skeletal muscle mass and function is decreased as we age, a condition known as sarcopenia. Ageing is associated with increases in minimum and mean cortisol plasma levels and shortening of the evening cortisol quiescent period, suggesting impaired circadian function and higher basal levels with increasing age (Stamou et al, 2023). It has also been shown that the deleterious effects of bed rest on human skeletal muscle are exacerbated by hypercortisolemia (Fitts et al, 2007).The aim of this study was to examine the direct effects of increasing levels of cortisol on muscle cells. C2C12 myoblasts were fused to myotubes by serum deprivation for up to 7 days. Cellular viability was assessed using light-microscopy to identify any gross changes in cell morphology at 3, 24 and 48 hours following treatment with a range of cortisol concentrations in the physiological range (10 – 0.1uM). Indices of muscle atrophy and evidence of altered levels of inflammatory cytokines was determined by mRNA analyses and release of cytokines/chemokines by myotubes was assessed using bead based multiplex technology (Luminex immunoassay).Cortisol treatment had no significant effect on C2C12 myotube viability at any concentration or time point, or on gene expression of IL-6, RANTES (CCL5) or MCP-1 (CCL2) compared with control untreated myotubes. No increase in the release of cytokines and chemokines from C2C12 myotubes was seen following treatment with cortisol and in some instances a significant decrease in cytokine/chemokine production was seen, particularly following treatment with higher concentrations of cortisol when compared with control values (eg, 10uM cortisol, 24 hours post-treatment: IL-6: 1.6 +/- 1.6% CXCL5: 17.4 +/- 1.8%; KC (CXCL1): 11.6 +/-10.3%. These data are in contrast to previous work from our group which revealed an increase in the release of IL-6, CXCL-1 (KC), CCL2 (MCP-1) and CCL5 (RANTES) from C2C12 myotubes following treatment of myotubes 25ng/ml TNF-α (Lightfoot et al, 2015).In summary, increased levels of cortisol do not appear to have any direct deleterious effects on myotube viability or production of pro-inflammatory cytokines. However, this does not exclude any indirect effects of cortisol on muscle including the effects on other organs indirectly affecting muscle function. It may also be that cortisol has a deleterious effect when muscle is under stressful conditions and this possibility will also be examined. Fitts et al (2007). doi: 10.1152/ajpcell.00573.2006 ; Lightfoot et al. (2015) doi: 10.1016/j.redox.2015.08.007 ; Stamou et al (2023) doi.org/10.1016/j.maturitas.2022.10.006 . The authors would like to thank the EU/BBSRC and CIMA for generous funding. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 689: The codon-modified influenza virus CodaLytic™rescues immunostimulatory activity and anti-tumor efficacy in PD-1-refractory tumor models

Abstract Multiple oncolytic viruses have been shown to induce various beneficial changes in the tumor microenvironment (TME), which ultimately support induction of anti-tumor immune responses. We have previously demonstrated that CodaLytic, a codon-modified influenza virus, can kickstart the cancer immunity cycle at various steps in several mouse models with differing baseline immune contextures. Here, we are confirming the mechanisms of action of the virus in combination with immune checkpoint inhibition in murine and human preclinical models. Efficacy after intratumoral injection of 108 PFU CodaLytic in combination with αPD-1 and/or αCTLA-4 checkpoint blockade was determined in αPD-1-resistant B16-F10 melanoma and 4T1 triple-negative breast cancer mouse models. Changes in the tumor immune infiltrate after treatment were characterized using flow cytometry. Primary human tumoroids were incubated with CodaLytic +/- 10 μg/ml pembrolizumab using the 3D-Explore platform by Nilogen Oncosystems. Tumor cell killing (TCK) was detected by high-content imaging at 72h and effects on the TME were characterized by cytokine release and RNA sequencing at 24h and/or 48h. In B16-F10, addition of CodaLytic to αPD-1 blockade rescued non-significant tumor growth inhibition (TGI) with αPD-1 alone, achieving 86% TGI (p < 0.01), 40% complete regressions and 60% survival beyond 45 days (69% TGI, 20% regressions and 30% long-term survival after CodaLytic monotherapy). Mechanistically, CodaLytic-containing regimens increased tumor immune infiltration, in particular with CD8+ T cells and cross-presenting dendritic cells (DCs). 4T1 tumors did not significantly respond to αPD-1, αCTLA-4 or CodaLytic monotherapies, but achieved 86% TGI in combination with 50% long-term survival past 55 days. CTLA-4 blockade emerged as the initial driver of slowed tumor growth and αPD-1 drove long-term survival, which was further augmented by CodaLytic addition. Infiltration with CD45+ leukocytes and cross-presenting DCs correlated with tumor volumes. The benefit of CodaLytic combination treatment was also demonstrated ex vivo in human tumoroid cultures. Meaningful TCK was observed in 50% of specimens vs 17% with pembrolizumab and 33% with CodaLytic alone. In this system without immune cell recruitment from a systemic reservoir, sustained release of CXCL9 and CXCL10 and temporal decreases of MIP-1β and CCL2 associated with response. Additional transcriptional analyses are ongoing. Taken together, this and additional preclinical data confirms the emergence of CodaLytic as a potent immunostimulatory agent capable of restoring immune cell infiltration and anti-tumor efficacy when used in combination with checkpoint inhibition in PD-1 resistant models. This data supports future development of CodaLytic for immuno-virotherapy of tumor types in which checkpoint therapy is less established. Citation Format: Yiwen Zhao, Nusrat Jahan, Marcin Stawowczyk, Katie Pfeffer, Juliana Tafrova, James Rodriguez, Chen Yang, Sybil A. Tasker, Steffen Mueller, J. Robert Coleman, Johanna K. Kaufmann. The codon-modified influenza virus CodaLytic™rescues immunostimulatory activity and anti-tumor efficacy in PD-1-refractory tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 689.

SARS-CoV-2 specific T-cell humoral response assessment after COVID-19 vaccination using a rapid direct real-time PCR amplification.

The SARS-CoV-2 immune response is mediated by both humoral and cellular immunity. In this study, SARS-CoV-2 specific cellular immunity was tested by a novel direct real-time PCR (dRT-PCR) assay, targeting mRNA of CXCL10, and compared with respect to an ELISA measuring interferon gamma (IFN-γ) release. Whole blood (Li-He) and serum samples were collected from 92 healthcare workers (HCW), with three doses of homologous (Pfizer/BioNTech, n=74) or heterologous (Pfizer/BioNTech and Vaxzevria or Moderna, n=18) vaccinations. Li-He samples were incubated with SCV2 PANEL-1-T-ACTIVATION (Hyris srl, Lodi, Italy), or CoV-2 IGRA TUBE ELISA (Euroimmune, Lubeck, Germany). CXCL10 mRNA expression was analyzed by bCube/bApp (Hyris), while IFN-γ was evaluated by quant-T-Cell SARS-CoV-2 ELISA (Euroimmune). Anti-SARS-CoV-2 S-RBD IgG levels were measured in sera using a CLIA assay (Snibe, Shenzen, China). Imprecision of dRT-PCR assay was found to be satisfactory, and the two methods for measuring Tcell immunity to SARS-CoV-2 peptides agreed in 82/87 (94.2%) of results. At qualitative dRT-PCR analyses, 81 subjects (93.2%) resulted as reactive to SARS-CoV-2 peptides, 3 (3.4%) were borderline and 3 were negative (3.4%). At univariate and multivariate analyses of quantitative dRT-PCR mRNA of CXCL10 and IFN-γ release results showed no difference between HCW with previous infection, homologous/heterologous vaccination, or demographical features. Anti-SARS-CoV-2 S-RBD IgG was associated with the previous infection and thetime between the last vaccination or positivity. Direct RT-PCR appeared accurate for determining the presence or absence of immunoreactivity of SARS-CoV-2 specific Tcells, especially when rapid analyses are required, such as for organ transplantation.

Microglia/macrophages require vitamin D signaling to restrain neuroinflammation and brain injury in a murine ischemic stroke model

Vitamin D deficiency is associated with worse clinical outcomes after ischemic stroke; nevertheless, the pathophysiological mechanisms remain largely unexplored. In this study, we characterized the molecular mechanisms of how vitamin D signaling modulated stroke progression in male mouse ischemia–reperfusion stroke models. We found that vitamin D receptor (VDR) exhibited a predominant upregulation in peri-infarct microglia/macrophages following cerebral ischemia. Conditional Vdr inactivation in microglia/macrophages markedly augmented infarct volumes and neurological deficits. VDR-deficient microglia/macrophages exhibited a more primed proinflammatory phenotype with substantial secretion of TNF-α and IFN-γ. These inflammatory cytokines further enhanced CXCL10 release from endothelial cells and blood–brain barrier disruption, and ultimately infiltration of peripheral T lymphocytes. Notably, blocking TNF-α and IFN-γ significantly ameliorated stroke phenotypes in Vdr conditional knockout mice. Collectively, VDR signaling in microglia/macrophages plays a crucial role in restraining ischemia-elicited neuroinflammation and stroke progression. Our findings delineate a novel mechanism underlying the association between vitamin D deficiency and poor stroke outcomes, and underline the significance of maintaining a functional vitamin D signaling in the management of acute ischemic stroke.