High Levels Of Inflammatory Cytokines Research Articles

P34 Evaluating the adaptation of the amputee skin owing to prosthesis use: a study using in vivo Raman spectroscopy

Abstract Introduction and aims Lower limb prosthesis has proven to be an extremely successful intervention for amputees. However, to achieve adequate function, the device places significant friction and load on the skin, which leads to substantial skin breakdown. Current rehabilitation practices require skin to be ‘trained’ to perform a load-bearing role. Despite this, there are very high injury statistics for skin on the residual limb in the literature, we hypothesize that rehabilitation practices to adapt amputee skin are limited. Methods To interrogate this, we used in vivo Raman spectroscopy to evaluate the lower limbs of 13 unilateral lower limb amputees. This enabled us to assess how key constituents of the stratum corneum (SC) such as natural moisturizing factor (NMF), water and lipid content vary with depth. We also analysed sebum content to quantify cytokines present and performed mechanical characterization of the lower limb to investigate stiffness and friction. Results We found that the Raman spectra of the amputated limb show reduced NMF, lipid content and water content at the top of the SC when compared with the intact limb (P < 0.05). In addition, we found that amputee skin contains higher levels of inflammatory cytokines compared with control skin. We also recorded that skin on the residuum is four times softer than the intact limb, while friction is much higher at 0.5 on the residuum skin, compared with 0.3 on the intact limb. Conclusions Here, we show the adaptations of amputee skin in response to rehabilitation to be minimal. Contrary to the dogma that rehabilitation trains skin to bear load, we found that skin barrier function in amputee skin is impaired, as indicated by altered water and lipid profile in addition to higher baseline inflammation.

Inflammatory Response to Wear Particles: Comparisons Between Zirconia and Titanium In Vitro.

The objective of this study was to compare the inflammatory responses from peripheral blood mononuclear cells PBMCs subjected to Titanium (Ti) and/or Zirconia (Zr) particles while growing on Ti or Zr discs. In total, 240 discs were fabricated in the size of 2 mm height and 5 mm diameter. 120 discs were made of Ti (Commercially pure (CP grade 4) and 120 discs were made of Zr (3 Mol% Yttria-Stabilized Zirconia Polycrystals (3Y TZP). The PBMCs were cultured on the two-disc materials and particles with a size up to 20 μm Ti (99, 5 % Ti) and 0, 1 -2 μm Zr (3Y TZP) were added to the cultures. The concentration levels of inflammatory cytokines in culture supernatants were measured through Bioplex assay and light microscopic analysis was performed to detect cell attachment and characterize particle shape and cell-particle interaction. The inflammatory responses from PBMCs were generally higher when cells were cultured on a Ti surface compared to a Zr surface. In addition, higher cytokine levels were generally seen when cells were cultured in the presence of Ti particles compared to Zr particles in the absence of discs. However, there were only significantly increased levels for three cytokines (MCP-1, IFN-g, and TNF-a) when particles were added to the Ti discs. Higher release of NETS from neutrophils were seen in presence of Zr particles compared to Ti particles. And a lowering of cell death was seen when adding Zr particles compared to Ti particles and unstimulated control samples. Type of growth material and presence of particle affects PBMCs in vitro. Cells seeded on Ti disc and together with Ti particles generated higher levels of inflammatory cytokines compared to the Zr counterparts.

Gender disparities in lymphocyte counts and cytokine expression in COVID-19

Background: This study seeks to assess gender differences in the severity of COVID-19 infection, which have been noted in different regions during the early stages of the pandemic. Methods: A cross-sectional study conducted at Baquba Teaching Hospital in Diyala, Iraq, from October 1st to December 31st, 2020, included 132 confirmed COVID-19 patients. These patients underwent a comprehensive set of routine laboratory tests, including complete blood count, blood biochemistry, and D-dimer assessment. Statistical analysis was carried out using SPSS-20, with significance set at p < 0.05. Results: The study included patients with a mean age of 45.61 (±11.32) years, predominantly male (63.0%), residing in urban areas (57.6%), and presenting with comorbidities (78.8%). All patients exhibited positive results on CT scans (100%) and CRP tests (100%). However, PCR testing confirmed COVID-19 infection in 87.2% of cases, with 12.8% testing negative. Among males, there was a significant increase in IL-6 and IL-10 levels (42.57 ± 7.64 pg/ml and 255.27 ± 21.03 pg/ml) compared to females (16.43 ± 4.19 pg/ml and 187.48 ± 20.35 pg/ml), with p-values <0.001 and 0.003, respectively. Conversely, there was no significant difference in IFN-ɣ levels between males (165.73 ± 16.54 pg/ml) and females (176.12 ± 17.10 pg/ml), with a p-value of 0.105. However, lymphocyte levels were significantly lower in males (4.79 ± 0.85%) compared to females (14.01 ± 1.36%), with a p-value <0.001. Conclusion: Overall, COVID-19 affects males more severely than females, with males showing weaker immune responses and higher levels of inflammatory cytokines like IL-6 and IL-10. While IFN-ɣ levels do not differ significantly between genders, males have lower lymphocyte counts compared to females.

The relationship between levels of tumor necrosis factor-alpha, interleukin-6, and C-reactive protein in the serum of elderly and acute myocardial infarction.

This study aimed to explore the relationship between the serum levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and hypersensitive C-reactive protein (hs-CRP) and the prognosis of acute myocardial infarction (AMI) patients after percutaneous coronary intervention (PCI) treatment. A total of 118 early-onset AMI patients who successfully received PCI (in the PCI group, blood samples were collected before PCI, 12, 24, 48 h after PCI, and 90 d follow-up period) and 52 AMI patients who received only cardioangiography (CAG) (in the CAG group, blood samples were collected before CAG, 12, 24, 48 h after CAG, and 90 d follow-up period). The serum levels of IL-6, hs-CRP and TNF-α were detected, and the incidence of major adverse cardiac events (MACE) in the PCI group during follow-up was observed. The basic levels of IL-6, hs-CRP, and TNF-α between the PCI group and the CAG group were not statistically different (P>0.05); there was no statistically significant difference in changes of serum IL-6, hs-CRP, and TNF-α in the CAG group before and after CAG (P>0.05); IL-6, hs-CRP, and TNF-α in the PCI group were significantly higher than those before treatment (P<0.01); in the PCI group, the levels of IL-6, hs-CRP and TNF-α between the MACE group and the MACE-free group were statistically different (P<0.05). Serum IL-6, hs-CRP and TNF-α levels in AMI patients after PCI significantly increased in the short term, and PCI may induce an inflammatory response; the high levels of inflammatory cytokines, IL-6, hs-CRP, and TNF-α, in peripheral blood may have an important reference value for MACE and short-term prognosis in early-onset AMI patients after PCI.

Inflammatory profile in patients with rheumatoid arthritis and sarcopenia.

Sarcopenia is characterized by the loss of muscle mass and strength associated with aging; however, individuals with chronic diseases are at risk at the early stages. In rheumatoid arthritis (RA), sustained chronic inflammation influences muscle deterioration. It may expedite the development of sarcopenia, which has been linked to physical disability, cardiovascular events, disease activity of RA, and premature death. We aimed to compare the inflammatory profiles of patients with RA with and without sarcopenia. This cross-sectional study involved 165 women with RA. Sarcopenia was diagnosed according to criteria established by the European Working Group on Sarcopenia in Older People. To assess the inflammatory profile, concentrations of cytokines such as EGF, IL-17, IL-1α, IL-1β, IL-6, TNFα, TNFβ, and creatine kinase (CK) were measured. The prevalence of sarcopenia was 15.8% (95% CI: 8.9-18.2). The median age of patients with sarcopenia was 59.5years (49.8-65.3), compared to 50years (43-59years) p = 0.001. The disease duration was also longer in patients with sarcopenia, 21years (15-30), compared to those without sarcopenia, 13years (7.3-20) p = 0.001. The inflammatory profile differed between patients with and without sarcopenia, revealing that the cytokines IL-1α, IL-6, and TNFβ concentrations were significantly higher (p < 0.05) in patients with sarcopenia, adjusted for BMI, age, and disease duration. Patients with RA and sarcopenia were older and exhibited longer disease duration and higher levels of inflammatory cytokines compared to those without sarcopenia. These findings suggest potential implications for clinical outcomes. Key Points • The prevalence of sarcopenia in women with rheumatoid arthritis was 15.8% (95% CI, 8.9-18.2). • Levels of IL-1α, IL-6, and TNFβ cytokines were significantly higher in women with rheumatoid arthritis and sarcopenia compared with those without sarcopenia, adjusted for BMI, age, and disease duration.

Association of high disease activity and serum IL-6 levels with the incidence of inflammatory major organ events in Behçet disease: a prospective registry study.

Little is known about the relationship between the disease activity of Behçet disease (BD) and the incidence of inflammatory major organ events. In this prospective registry study, we investigated the association between the Behçet Disease Current Activity Form (BDCAF) and incidence of inflammatory major organ events, defined as the inflammation of the ocular, central nervous, intestinal, and vascular systems in BD. We enrolled participants from Japanese multicenter prospective cohorts. The BDCAF was evaluated annually. BD-related symptoms, including inflammatory major organ events, were monitored. The association between BDCAF and inflammatory major organ events was analyzed by time-to-event analysis. An unsupervised clustering of the participants' BDCAF, therapeutic agents, and multiple serum cytokines was also performed to examine their association with inflammatory major organ events. A total of 260 patients were included. The patients had a median BDCAF score of 2 [Interquartile range, 1-3] at the enrolment and remained disease active at 1- and 2-year follow-ups, indicating residual disease activity in BD. Patients with a BDCAF score of 0 had a longer inflammatory major organ event-free survival at 52 weeks than those with a score of 1 or higher (p=2.2 x 10-4). Clustering analysis revealed that patients who did not achieve remission despite treatment with tumor necrosis factor inhibitors had high serum inflammatory cytokine levels and incidences of inflammatory major organ events. Among the elevated cytokines, IL-6 was associated with inflammatory major organ events. This study suggests that treatment strategies targeting overall disease activity and monitoring residual serum IL-6 may help prevent inflammatory major organ events in BD.

Abstract 165: PVRL2 upregulation by tumor-associated macrophages confers a regulatory phenotype and is associated with poor prognosis in high-grade serous ovarian cancer

Abstract Advanced or recurrent high-grade serous ovarian cancer (HGSOC) are associated with poor prognosis. While HGSOC is moderately immunogenic, anti-PD-1/L1 therapy has fared poorly in trials. PVRL2 (Nectin2) is both a cell-adhesion molecule as well as a ligand for PVRIG, a novel co-inhibitory receptor. While PVRL2 is constitutively expressed by epithelial/tumor cells, it can also be upregulated by macrophages. Whether macrophage-expressed PVRL2 functions exclusively as an immunoregulatory ligand in HGSOC is not known. CD45+ tumor infiltrate from 6 treatment-naïve and 3 neoadjuvant chemotherapy-treated patients with HGSOC was evaluated by CITE-seq. Multiplex immunofluorescence (mIF) of PanCK, CD68 and PVRL2 was performed on 28 FFPE sections. In vivo, we monitored survival of wild-type and PVRL2−/− mice as well as corresponding bone marrow chimeras orthotopically implanted with ID8-Vegf-Defb29 ovarian tumors. CITE-seq was run on CD45+ cells sorted from wild-type vs PVRL2−/− tumors and ascites. In tumor-bearing mice, macrophages, CD8 or NK cells were depleted and differences in survival assessed. In vitro, M1 and M2 macrophages were differentiated from steady-state bone marrow progenitors and myeloid gene profiles were analyzed. CITE-seq of patient-derived immune cells revealed that PVRL2 expression was restricted to macrophage clusters. Macrophages with low PVRL2 expression showed M1 polarity and abundant expression of inflammatory cytokines and chemokines. Overall macrophage counts and PVRL2 expression were lower in post-chemotherapy cases. In mIF, PVRL2 staining was observed on CD68-positive cells as well as tumor cells. In addition, PVRL2-expressing macrophages accumulated in tumor areas, especially in residual tumor areas after chemotherapy. PVRL2−/− mice and PVRL2 bone marrow chimeras had significantly improved survival compared to their respective control cohorts. Macrophage depletion significantly shortened the prognosis of PVRL2−/− mice compared to wild-type mice. In contrast, depletion of CD8 T cells or NK cells had no effect on survival of wild-type and PVRL2−/− mice. M1 macrophages from PVRL2−/− mice expressed higher levels of inflammatory cytokines and chemokines compared to their wild-type counterparts. Furthermore, CITE-seq showed that tumor-infiltrating macrophages in PVRL2−/− mice exhibited M1 polarization compared to those from wild-type tumors. However, the inflammatory phenotype appeared to be linked more with the tumor-infiltrating macrophages than with ascites macrophages. In conclusion, our research suggests that PVRL2 upregulation by tumor-infiltrating macrophages polarizes towards a pro-tumor phenotype and affects prognosis of HGSOC. PVRL2-deficient macrophages exert their antitumor effects via inflammatory tumor killing and not via antigen-presentation to effector T cells. Citation Format: Kosuke Murakami, Hirofumi Ando, Michele Doucet, Dacheng Ding, William Huang, Sudipto Ganguly. PVRL2 upregulation by tumor-associated macrophages confers a regulatory phenotype and is associated with poor prognosis in high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 165.

Cytokine and T cell responses in post-chikungunya viral arthritis: A cross-sectional study.

To define the relationship between chronic chikungunya post-viral arthritis disease severity, cytokine response and T cell subsets in order to identify potential targets for therapy. Participants with chikungunya arthritis were recruited from Colombia from 2019-2021. Arthritis disease severity was quantified using the Disease Activity Score-28 and an Arthritis-Flare Questionnaire adapted for chikungunya arthritis. Plasma cytokine concentrations (interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, interferon-γ and tumor necrosis factor (TNF)) were measured using a Meso Scale Diagnostics assay. Peripheral blood T cell subsets were measured using flow cytometry. Among participants with chikungunya arthritis (N = 158), IL-2 levels and frequency of regulatory T cells (Tregs) were low. Increased arthritis disease activity was associated with higher levels of inflammatory cytokines (IL-6, TNF and CRP) and immunoregulatory cytokine IL-10 (p<0.05). Increased arthritis flare activity was associated with higher Treg frequencies (p<0.05) without affecting T effector (Teff) frequencies, Treg/Teff ratios and Treg subsets. Finally, elevated levels of IL-2 were correlated with increased Treg frequency, percent Tregs out of CD4+ T cells, and Treg subsets expressing immunosuppressive markers, while also correlating with an increased percent Teff out of live lymphocytes (p<0.05). Chikungunya arthritis is characterized by increased inflammatory cytokines and deficient IL-2 and Treg responses. Greater levels of IL-2 were associated with improved Treg numbers and immunosuppressive markers. Future research may consider targeting these pathways for therapy.

Modulation of Stiffness-Dependent Macrophage Inflammatory Responses by Collagen Deposition.

Macrophages are innate immune cells that interact with complex extracellular matrix environments, which have varied stiffness, composition, and structure, and such interactions can lead to the modulation of cellular activity. Collagen is often used in the culture of immune cells, but the effects of substrate functionalization conditions are not typically considered. Here, we show that the solvent system used to attach collagen onto a hydrogel surface affects its surface distribution and organization, and this can modulate the responses of macrophages subsequently cultured on these surfaces in terms of their inflammatory activation and expression of adhesion and mechanosensitive molecules. Collagen was solubilized in either acetic acid (Col-AA) or N-(2-hydroxyethyl)piperazine-N'-ethanesulfonic acid (HEPES) (Col-HEP) solutions and conjugated onto soft and stiff polyacrylamide (PA) hydrogel surfaces. Bone marrow-derived macrophages cultured under standard conditions (pH 7.4) on the Col-HEP-derived surfaces exhibited stiffness-dependent inflammatory activation; in contrast, the macrophages cultured on Col-AA-derived surfaces expressed high levels of inflammatory cytokines and genes, irrespective of the hydrogel stiffness. Among the collagen receptors that were examined, leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) was the most highly expressed, and knockdown of the Lair-1 gene enhanced the secretion of inflammatory cytokines. We found that the collagen distribution was more homogeneous on Col-AA surfaces but formed aggregates on Col-HEP surfaces. The macrophages cultured on Col-AA PA hydrogels were more evenly spread, expressed higher levels of vinculin, and exerted higher traction forces compared to those of cells on Col-HEP. These macrophages on Col-AA also had higher nuclear-to-cytoplasmic ratios of yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), key molecules that control inflammation and sense substrate stiffness. Our results highlight that seemingly slight variations in substrate deposition for immunobiology studies can alter critical immune responses, and this is important to elucidate in the broader context of immunomodulatory biomaterial design.

In Vitro Cultivation for Glugea plecoglossi (Microsporidia) Isolated from Ayu (Plecoglossus altivelis).

Glugea plecoglossi is an obligate intracellular microsporidium, which poses a significant threat to ayu (Plecoglossus altivelis). In vitro cultivation models are invaluable tools for investigating intracellular microorganisms, including G. plecoglossil. In this study, we attempted to in vitro cultivate G. plecoglossi using primary cultures derived from ayu monocytes/macrophages (MO/MΦ), a murine-derived macrophage cell line RAW264.7, and the epithelioma papulosum cyprini (EPC) cell line. The results demonstrated that MO/MΦ infected with spores exhibited a pronounced immune response which was presented by rapidly high expression levels of inflammatory cytokines, such as PaIL-1β, PaTNF-α, PaIL-10, and PaTGF-β, and detached within 96 h post-infection (hpi). Infected RAW264.7 cells remained capable of stable passage yet exhibited cellular deformation with a decrease in intracellular spores occurring around 8 days post-infection (dpi). In contrast, EPC cells promised a substantial parasite population, and the cytokine expression levels returned to normal by 8 dpi. In addition, G. plecoglossi spores recovered from EPC cells could infect young ayu, suggesting that EPC cells might be used as an in vitro cultivation system for G. plecoglossi.

Endothelial Protein C Receptor and 3K3A-Activated Protein C Protect Mice from Allergic Contact Dermatitis in a Contact Hypersensitivity Model.

Endothelial protein C receptor (EPCR) is a receptor for the natural anti-coagulant activated protein C (aPC). It mediates the anti-inflammatory and barrier-protective functions of aPC through the cleavage of protease-activated receptor (PAR)1/2. Allergic contact dermatitis is a common skin disease characterized by inflammation and defective skin barrier. This study investigated the effect of EPCR and 3K3A-aPC on allergic contact dermatitis using a contact hypersensitivity (CHS) model. CHS was induced using 1-Fluoro-2,4-dinitrobenzene in EPCR-deficient (KO) and matched wild-type mice and mice treated with 3K3A-aPC, a mutant form of aPC with diminished anti-coagulant activity. Changes in clinical and histological features, cytokines, and immune cells were examined. EPCRKO mice displayed more severe CHS, with increased immune cell infiltration in the skin and higher levels of inflammatory cytokines and IgE than wild-type mice. EPCR, aPC, and PAR1/2 were expressed by the skin epidermis, with EPCR presenting almost exclusively in the basal layer. EPCRKO increased the epidermal expression of aPC and PAR1, whereas in CHS, their expression was reduced compared to wild-type mice. 3K3A-aPC reduced CHS severity in wild-type and EPCRKO mice by suppressing immune cell infiltration/activation and inflammatory cytokines. In summary, EPCRKO exacerbated CHS, whereas 3K3A-aPC could reduce the severity of CHS in both EPCRKO and wild-type mice.

Associations between Adiponectin and the Development of Diabetes in Rheumatoid Arthritis.

We evaluated associations between adiponectin and the risk of diabetes among patients with rheumatoid arthritis (RA), a systemic inflammatory disease associated with metabolic disturbance. This prospective cohort study included adults with RA from the Veteran's Affairs Rheumatoid Arthritis Registry. Adiponectin and inflammatory cytokines/chemokines were measured at enrollment on stored serum samples. Adiponectin levels were categorized and clinical variables were described across categories (<10 μg/mL; 10-40 μg/mL; > 40 μg/mL. Multivariable Cox proportional hazard models evaluated associations between adiponectin and incident diabetes adjusting for age, sex, race, smoking status, body mass index (BMI), disease-modifying therapy use, calendar year, and comorbidity. Testing for modification of effect in the context of elevated cytokines/chemokines was performed. Among 2595 patients included in the analysis, those with adiponectin levels >40 μg/mL (N = 379; 15%) were older and had lower BMI. There were 125 new cases of diabetes among 1,689 patients without prevalent disease at enrollment. There was an inverse association between adiponectin and incident diabetes, however, the association was positive among patients with adiponectin levels >40 μg/mL. Patients with levels >40 μg/mL were at higher risk compared to those with levels 10-40 μg/mL [HR: 1.70 (1.34,2.16) p < 0.001]. Those with adiponectin levels >40 μg/mL had significantly higher levels of inflammatory cytokines with evidence of a modified effect of adiponectin on diabetes risk in the setting of inflammation. The relationship between adiponectin and incident diabetes risk is U-shaped in RA. Patients with very high adiponectin levels have greater systemic inflammation and an altered relationship between adiponectin and diabetes risk.

Investigation of the shared gene signatures and molecular mechanisms between obstructive sleep apnea syndrome and asthma

BackgroundObstructive sleep apnea syndrome (OSAS) is highly related with asthma from the epidemiology to pathogenesis, while the underlying mechanism is still unclear. Herein, we aimed to reveal the shared gene signatures and molecular mechanisms underlying the coexistence of OSAS and asthma and verified relating pathway in mouse models. We downloaded GSE75097 of OSAS and GSE165934 of asthma from GEO database and performed differential expression analysis and functional enrichment analysis to screen differentially expressed genes (DEGs) and potential pathogenic pathway. PPI network was constructed with the STRING database. Hub genes were identified with cytoHubba and immune infiltration analysis was performed with cibersort for further verification. Potential drugs were screened with Comparative Toxicogenomics Database and miRNA-gene network was constructed. Besides, to test the pulmonary function and inflammatory cytokine, mouse models with OSAS and asthma were constructed, followed by validating the involvement of NOD1/NOD2-RIPK2-NF-κB-MCPIP-1 pathway in associated diseases. ResultsIn total, 104 DEGs were identified, in which PLAUR, RIPK2, PELI1, ZC3H12A, and TNFAIP8 are the hub genes, while NOD-like receptor signaling pathway and apoptosis signaling pathway were the potential influential pathways. Increased γδT cells and neutrophils were detected in asthma patients through immune infiltration analysis. Significant difference was detected among genders in OSAS, and acetaminophen is a potential drug in the comorbidity by screening the drugs in the Comparative Toxicogenomics Database. Mice with OSAS and asthma presented with worse pulmonary function and higher levels of inflammatory cytokines. The relative proteins, including NOD1, NOD2, RIPK2, NF-κB, and MCPIP-1, were up-regulated in mice with the OSAS and asthma. ConclusionsThis research firstly elucidates NOD1/NOD2-RIPK2-NF-κB-MCPIP-1 pathway as the shared pathway in the development of OSAS and asthma through bioinformatics and experimental methods. There is an interactive deterioration model between OSAS and asthma. This study may provide some potential biomarkers in the future research of the underlying pathogenesis and treatment of comorbidity of OSAS and asthma.

Loss of CFTR function in macrophages alters the cell transcriptional program and delays lung resolution of inflammation.

Cystic fibrosis (CF) is an autosomal recessive genetic disorder caused by mutations in the CF Transmembrane-conductance Regulator (CFTR) gene. The most severe pathologies of CF occur in the lung, manifesting as chronic bacterial infection, persistent neutrophilic inflammation, and mucopurulent airway obstruction. Despite increasing knowledge of the CF primary defect and the resulting clinical sequelae, the relationship between the CFTR loss of function and the neutrophilic inflammation remains incompletely understood. Here, we report that loss of CFTR function in macrophages causes extended lung inflammation. After intratracheal inoculation with Pseudomonas aeruginosa, mice with a macrophage-specific Cftr-knockout (Mac-CF) were able to mount an effective host defense to clear the bacterial infection. However, three days post-inoculation, Mac-CF lungs demonstrated significantly more neutrophil infiltration and higher levels of inflammatory cytokines, suggesting that Mac-CF mice had a slower resolution of inflammation. Single-cell RNA sequencing revealed that absence of CFTR in the macrophages altered the cell transcriptional program, affecting the cell inflammatory and immune responses, antioxidant system, and mitochondrial respiration. Thus, loss of CFTR function in macrophages influences cell homeostasis, leading to a dysregulated cellular response to infection that may exacerbate CF lung disease.

EXTH-18. DEVELOPMENT OF A TRANSGENIC MOUSE MODEL ENABLING COMPARATIVE ANALYSIS OF CHIMERIC ANTIGEN RECEPTOR (CAR)-EXPRESSING IMMUNE CELL POPULATIONS FOR CANCER IMMUNOTHERAPY

Abstract We reported the creation of C57BL/6J-background transgenic (Tg) mice carrying the anti-epidermal growth factor receptor (EGFR)vIII-CAR with CD3z-CD28-4-1BB downstream of a Lox-Stop-Lox cassette in the Rosa26 locus. Breeding these mice with CD4-Cre Tg allows rigorous and reproducible evaluation of CAR-T cell therapy in syngeneic models (Chuntova et al. Neuro-Onc 2022). As the extension of the approach, we crossed the EGFRvIII-CAR Tg mice with mice carrying Vav-Cre, which allowed the expression of anti-EGFRvIII CAR in all hematopoietic cells. In particular, we evaluated CAR-T, CAR-NKT, CAR-NK cells, and CAR-macrophages. CAR-NK cells displayed a robust antigen-specific cytolytic activity against EGFRvIII-expressing SB28 cells, comparable to CAR-T cells in vitro and mice bearing intracerebral EGFRvIII-expressing SB28 gliomas. Remarkably, CAR-NKT cells produced higher levels of inflammatory cytokines when co-cultured with EGFRvIII-expressing SB28 cells, such as IFN-g, Perforin, IL4, and IL17. On the other hand, despite their in vitro cytotoxic effects against SB28 cells, CAR-NK cells failed to mediate a therapeutic response in mice bearing intracerebral glioma. Future studies will be directed at improving the in vivo persistence of CAR-NK cells using engineered cytokines, such as IL-2 superkine. In addition, we extensively investigated CAR-macrophages in vitro and in vivo. Unfortunately, we did not observe significant CAR-specific anti-tumor effects. Consequently, we modified the intracellular domain of CAR from CD3z-CD28-4-1BB to CD3z alone. This alteration led to increased TNF-α production through CAR signaling. This transgenic mouse model offers rigorous and reproducible evaluations of CAR-expressing hematopoietic cells. Furthermore, the model provides a platform for developing combination therapies, such as CAR-T plus CAR-NKT or CAR-T plus CAR-NK cells, with potential implications for enhanced cancer treatment strategies.

Development of a novel human triculture model of non-alcoholic fatty liver disease and identification of berberine as ameliorating steatosis, oxidative stress and fibrosis.

Objectives: Non-alcoholic fatty liver disease (NAFLD) and its progression to non-alcoholic steatohepatitis (NASH) and hepatocarcinoma is a serious and growing problem. However, the development of new therapies is severely hindered by a lack of high-throughput assays for drug testing. Methods: We have developed a simple transwell assay comprised of HepG2 hepatocytes, hepatic LX-2 stellate cells, and differentiated THP-1 cells. The cells were incubated with an activating mixture containing the NASH-associated risk factors, glucose, insulin, free fatty acids (FFAs), and lipopolysaccharide (LPS) for 72h. We compared different combinations of culture conditions to obtain a model system that recapitulates the main features of NAFLD/NASH, i.e., increased steatosis, reactive oxygen species (ROS), secretion of pro-inflammatory cytokines/chemokines, and presence of fibrosis. To confirm the usefulness of the optimized model system, we screened for compounds that inhibit steatosis in the hepatocytes and evaluated the most effective compound in the triculture model system. Results: The activating mixture stimulated HepG2 cells in this triculture to accumulate more fat and produce higher levels of reactive oxygen species (ROS) than HepG2 cells in monocultures. As well, higher levels of inflammatory cytokines and chemokines (IL-8, IL-6, MIP-1α, etc.) were produced in this triculture compared to monocultures. In addition, in all LX-2 monocultures and cocultures, exposure to the activating mixture increased markers of fibrosis. A major strength of our triculture system is that it makes possible the simultaneous monitoring of 4 main features of NASH, i.e., steatosis, oxidative stress, inflammation and fibrosis. Screening potential modulators that may reduce steatosis in HepG2 cells revealed the protective effects of the isoalkaloid, berberine. Tested using this novel triculture assay, treatment with 5µM berberine decreased steatosis and ROS in HepG2 hepatocytes, reduced inflammatory cytokine production and inhibited collagen production from LX-2 cells. Conclusion: This simple triculture model recapitulates the main features of NAFLD/NASH and should be useful for high-throughput preclinical drug discovery. In this model, berberine showed promising results in decreasing steatosis and ROS and protection against fibrosis.

Age-dependent acquisition of pathogenicity by SARS-CoV-2 Omicron BA.5.

Pathology studies of SARS-CoV-2 Omicron variants of concern (VOC) are challenged by the lack of pathogenic animal models. While Omicron BA.1 and BA.2 replicate in K18-hACE2 transgenic mice, they cause minimal to negligible morbidity and mortality, and less is known about more recent Omicron VOC. Here, we show that in contrast to Omicron BA.1, BA.5-infected mice exhibited high levels of morbidity and mortality, correlating with higher early viral loads. Neither Omicron BA.1 nor BA.5 replicated in brains, unlike most prior VOC. Only Omicron BA.5-infected mice exhibited substantial weight loss, high pathology scores in lungs, and high levels of inflammatory cells and cytokines in bronchoalveolar lavage fluid, and 5- to 8-month-old mice exhibited 100% fatality. These results identify a rodent model for pathogenesis or antiviral countermeasure studies for circulating SARS-CoV-2 Omicron BA.5. Further, differences in morbidity and mortality between Omicron BA.1 and BA.5 provide a model for understanding viral determinants of pathogenicity.

Murine Mast Cells That Are Deficient in IFNAR-Signaling Respond to Viral Infection by Producing a Large Amount of Inflammatory Cytokines, a Low Level of Reactive Oxygen Species, and a High Rate of Cell Death.

Mat cells (MCs) are located in the skin and mucous membranes at points where the body meets the environment. When activated, MCs release inflammatory cytokines, which help the immune system to fight viruses. MCs produce, and have receptors for interferons (IFNs), which belong to a family of cytokines recognized for their antiviral properties. Previously, we reported that MCs produced proinflammatory cytokines in response to a recombinant vesicular stomatitis virus (rVSVΔm51) and that IFNAR signaling was required to down-modulate these responses. Here, we have demonstrated that UV-irradiated rVSVΔm51 did not cause any inflammatory cytokines in either in vitro cultured mouse IFNAR-intact (IFNAR+/+), or in IFNAR-knockout (IFNAR-/-) MCs. However, the non-irradiated virus was able to replicate more effectively in IFNAR-/- MCs and produced a higher level of inflammatory cytokines compared with the IFNAR+/+ MCs. Interestingly, MCs lacking IFNAR expression displayed reduced levels of reactive oxygen species (ROS) compared with IFNAR+/+ MCs. Additionally, upon the viral infection, these IFNAR-/- MCs were found to coexist with many dying cells within the cell population. Based on our findings, IFNAR-intact MCs exhibit a lower rate of rVSVΔm51 infectivity and lower levels of cytokines while demonstrating higher levels of ROS. This suggests that MCs with intact IFNAR signaling may survive viral infections by producing cell-protective ROS mechanisms and are less likely to die than IFNAR-/- cells.

Monocytes from Uninfected Neonates Born to Trypanosoma cruzi-Infected Mothers Display Upregulated Capacity to Produce TNF-α and to Control Infection in Association with Maternally Transferred Antibodies.

Activated monocytes/macrophages that produce inflammatory cytokines and nitric oxide are crucial for controlling Trypanosoma cruzi infection. We previously showed that uninfected newborns from T. cruzi infected mothers (M+B- newborns) were sensitized to produce higher levels of inflammatory cytokines than newborns from uninfected mothers (M-B- newborns), suggesting that their monocytes were more activated. Thus, we wondered whether these cells might help limit congenital infection. We investigated this possibility by studying the activation status of M+B- cord blood monocytes and their ability to control T. cruzi in vitro infection. We showed that M+B- monocytes have an upregulated capacity to produce the inflammatory cytokine TNF-α and a better ability to control T. cruzi infection than M-B- monocytes. Our study also showed that T. cruzi-specific Abs transferred from the mother play a dual role by favoring trypomastigote entry into M+B- monocytes and inhibiting intracellular amastigote multiplication. These results support the possibility that some M+B- fetuses may eliminate the parasite transmitted in utero from their mothers, thus being uninfected at birth.

Highly proliferative and hypodifferentiated CAR-T cells targeting B7–H3 enhance antitumor activity against ovarian and triple-negative breast cancers

Chimeric antigen receptor (CAR)-T cell immunotherapy is highly effective against hematological neoplasms. However, owing to tumor variability, low antigen specificity, and impermanent viability of CAR-T cells, their use in the treatment of solid tumors is limited. Here, a novel CAR-T cell targeting B7–H3 and incorporating a 4-1BB costimulatory molecule with STAT3-and STAT5-related activation motifs was constructed using lentivirus transduction. B7–H3, a tumor-associated antigen, and its scFv antibody endowed CAR-T cells with tumor-specific targeting capabilities. Moreover, the integration of the trIL2RB and YRHQ motifs stimulated STAT5 and STAT3 in an antigen-dependent manner, inducing a remarkable increase in the proliferation and survival of CAR-T cells via the activation of the JAK-STAT signaling pathway. Besides, the proportion of less-differentiated T cells increased among BB-trIL2RB-z(YRHQ) CAR-T cells. Moreover, BB-trIL2RB-z(YRHQ) effectively inhibited ovarian cancer (OC) and triple-negative breast cancer (TNBC) in vivo at low doses, without high serum levels of inflammatory cytokines and organ toxicity. Therefore, our study proposes a combination of elements for the construction of superior pluripotent CAR-T cells to provide an effective strategy for the treatment of intractable solid tumors.