Similar Cytokine Research Articles

Exploration of Cytokines and Microbiome Among Males and Females with Diarrhea-Predominant Irritable Bowel Syndrome.

Whether pathophysiological factors differ between males and females with irritable bowel syndrome-diarrhea (IBS-D) remains to be tested. To better understand potential sex differences, males with IBS-D were compared to naturally cycling females and to females with IBS-D taking hormonal contraception on plasma levels of cytokines and gut microbiome characteristics. Males and females with Rome III IBS-D completed questionnaires and kept a daily symptom diary for 28days. Blood and stool samples were collected between days 3 and 8 of the daily diary (estrogen-dominant days in naturally cycling females). Blood samples were analyzed for lipopolysaccharide (LPS)-stimulated and unstimulated cytokine levels. Stool samples were analyzed for microbiota signatures using 16S rRNA sequencing. Forty-seven participants with IBS-D (13 males, 22 naturally cycling females, 12 females with hormonal contraception use) ages 18 to 50years were studied. Males had similar unstimulated IL10, IL12P40, IL12P70, IL1β, IL8, and TNFα plasma cytokine levels compared to naturally cycling females, but higher levels compared with females using hormonal contraception. LPS-stimulated IL12P70 levels were lower in both groups of females vs. males. Alpha- and beta-diversity did not differ although differences in genus-level bacteria were found. Cytokine levels differed between males and females using hormonal contraceptives but not between males and normally cycling females. It is important to consider that naturally cycling females may have a different cytokine and microbiome profile than females using hormonal contraceptives. Whether this portends a sex difference in potential etiologic factors remains to be determined.

CCL2-mediated endothelial injury drives cardiac dysfunction in long COVID.

Evidence linking the endothelium to cardiac injury in long coronavirus disease (COVID) is well documented, but the underlying mechanisms remain unknown. Here we show that cytokines released by endothelial cells (ECs) contribute to long-COVID-associated cardiac dysfunction. Using thrombotic vascular tissues from patients with long COVID and induced pluripotent stem cell-derived ECs (iPSC-ECs), we modeled endotheliitis and observed similar dysfunction and cytokine upregulation, notably CCL2. Cardiac organoids comprising iPSC-ECs and iPSC-derived cardiomyocytes showed cardiac dysfunction after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exposure, driven by CCL2. Profiling of chromatin accessibility and gene expression at a single-cell resolution linked CCL2 to 'phenotype switching' and cardiac dysfunction, validated by high-throughput proteomics. Disease modeling of cardiac organoids and exposure of human ACE2 transgenic mice to SARS-CoV-2 spike proteins revealed that CCL2-induced oxidative stress promoted post-translational modification of cardiac proteins, leading to cardiac dysfunction. These findings suggest that EC-released cytokines contribute to cardiac dysfunction in long COVID, highlighting the importance of early vascular health monitoring in patients with long COVID.

Cytokine secretion by in vitro cultures of lung epithelial cells, differentiated macrophages and differentiated dendritic cells incubated with pneumococci and pneumococcal extracellular vesicles.

Streptococcus pneumoniae is an important human pathogen that can colonize the respiratory tract of healthy individuals. The respiratory tract mucosa is thus the first barrier for this pathogen. In this study, we have tested three models of the respiratory epithelium with immune cells: (i) monolayer of A549 human lung epithelial cells, (ii) A549 + macrophages differentiated from the human monocytic THP-1 cell line (dMφ) and (iii) A549 + dMφ + dendritic cells differentiated from THP-1 (dDC) using a two-chamber system. Pneumococcal strains Rx1 (non-encapsulated) and BHN418 (serotype 6B) were incubated with the cells and secretion of IL-6, IL-8, IL-1β, TNF-α and IL-10 was evaluated. Overall, the models using co-cultures of A549 + dMφ and A549 + dMφ + dDC elicited higher levels of pro-inflammatory cytokines and the non-encapsulated strain elicited an earlier cytokine response. BHN418 pspA (pneumococcal surface protein A) and pspC (pneumococcal surface protein C) knockouts elicited similar cytokine secretion in the co-culture models, whereas BHN18 ply (pneumolysin) knockout induced much lower levels. The results are in accordance with the activation of the inflammasome by Ply. Finally, we evaluated pneumococcal extracellular vesicles (pEVs) in the co-culture models and observed secretion of pro-inflammatory cytokines in the absence of cytotoxicity. Since pEVs are being studied as vaccine candidate against pneumococcal infections, the co-cultures of A549 + dMφ and A549 + dMφ + dDC are simple models that could be used to evaluate pEV vaccine batches.

Multi-Walled Carbon Nanotubes Accelerate Leukaemia Development in a Mouse Model.

Inflammation is associated with an increased risk of developing various cancers in both animals and humans, primarily solid tumors but also myeloproliferative neoplasms (MPNs), myelodysplastic syndromes (MDS), and acute myeloid leukemia (AML). Multi-walled carbon nanotubes (MWCNTs), a type of carbon nanotubes (CNTs) increasingly used in medical research and other fields, are leading to a rising human exposure. Our study demonstrated that exposing mice to MWCNTs accelerated the progression of spontaneous MOL4070LTR virus-induced leukemia. Additionally, similar exposures elevated pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α and induced reactive oxygen species (ROS) in a murine macrophage cell line. These effects were significantly reduced in immunodeficient mice and when mice were treated with methoxypolyethylene glycol amine (PEG)-modified MWCNTs. These findings underscore the necessity of evaluating the safety of MWCNTs, particularly for those with hematologic cancers.

Apoptotic Cell-Based Therapy for the Modification of the Inflammatory Response to Hemorrhagic Shock.

Many trauma patients die from hemorrhagic shock in the military and civilian settings. Although two-thirds of hemorrhagic shock victims die of reasons other than exsanguination, such as the consequent cytokine storm, anti-inflammatory therapies failed to be utilized. Apoptotic cell-based treatments enhance innate ability to exert systemic immunomodulation as demonstrated in several clinical applications and hence might present a novel approach in hemorrhagic shock treatment. Twenty-two rats underwent a pressure-controlled hemorrhagic shock model and followed up for 24 hours. An infusion of apoptotic cells (Allocetra-OTS, Enlivex Therapeutics Ltd, Nes Ziona, Israel) was administered to the treatment group. Hemodynamics, blood counts, biochemistry findings, and cytokine profile were compared to a saline-resuscitated control group. The treatment group's mean arterial pressure decreased from 94.8 mmHg to 28.2 mmHg, resulting in an 8.13 mg/dL increase in lactate and a 1.9 g/L decrease in hemoglobin, similar to the control group. White blood cells and platelets decreased more profoundly in the treatment group. A similar cytokine profile after 24 hours was markedly attenuated in the treatment group 2 hours after bleeding. Levels of pro-inflammatory cytokines such as interleukin (IL)-1a (28.4 pg/mL vs. 179.1 pg/mL), IL-1b (47.4 pg/mL vs. 103.9 pg/mL), IL-6 (526.2 pg/mL vs. 3492 pg/mL), interferon γ (11.4 pg/mL vs. 427.9 pg/mL), and tumor necrosis factor α (19.0 pg/mL vs. 31.7 pg/mL) were profoundly lower in the treatment group. In a pressure-control hemorrhagic shock model in rats, apoptotic cell infusion showed preliminary signs of a uniform attenuated cytokine response. Apoptotic cell-based therapies might serve as a novel immunomodulatory therapy for hemorrhagic shock.

Activin A Promotes Differentiation of a Pathogenic Multicytokine IL-9-secreting CD4+ T Cell Population.

The development of Th subsets results from cellular and cytokine cues that are present in the inflammatory environment. The developing T cell integrates multiple signals from the environment that sculpt the cytokine-producing capacity of the effector T cell. Importantly, T cells can discriminate similar cytokine signals to generate distinct outcomes, and that discrimination is critical in Th subset development. IL-9-secreting Th9 cells regulate multiple immune responses, including immunity to pathogens and tumors, allergic inflammation, and autoimmunity. In combination with IL-4, TGF-β or activin A promotes IL-9 production; yet, it is not clear if both TGF-β family members generate Th9 cells with identical phenotype and function. We observed that in contrast to TGF-β that efficiently represses Th2 cytokines in murine Th9 cultures, differentiation with activin A produced a multicytokine T cell phenotype with secretion of IL-4, IL-5, IL-13, and IL-10 in addition to IL-9. Moreover, multicytokine secreting cells are more effective at promoting allergic inflammation. These observations suggest that although TGF-β and IL-4 were identified as cytokines that stimulate optimal IL-9 production, they might not be the only cytokines that generate optimal function from IL-9-producing T cells in immunity and disease.

Vaccination with Mincle agonist UM-1098 and mycobacterial antigens induces protective Th1 and Th17 responses

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is one of the top infectious killers in the world. The only licensed vaccine against TB, Bacille Calmette-Guérin (BCG), provides variable protection against pulmonary TB, especially in adults. Hence, novel TB vaccine approaches are urgently needed. Both Th1 and Th17 responses are necessary for protection against TB, yet effective adjuvants and vaccine delivery systems for inducing robust Th1 and Th17 immunity are lacking. Herein we describe a synthetic Mincle agonist, UM-1098, and a silica nanoparticle delivery system that drives Th1/Th17 responses to Mtb antigens. Stimulation of human peripheral blood mononuclear cells (hPBMCs) with UM-1098 induced high levels of Th17 polarizing cytokines IL-6, IL-1β, IL-23 as well as IL-12p70, IL-4 and TNF-α in vitro. PBMCs from both C57BL/6 and BALB/c mice responded with a similar cytokine pattern in vitro and in vivo. Importantly, intramuscular (I.M.) vaccination with UM-1098-adjuvanted TB antigen M72 resulted in significantly higher antigen-specific IFN-γ and IL-17A levels in C57BL/6 wt mice than Mincle KO mice. Vaccination of C57BL/6 wt mice with immunodominant Mtb antigens ESAT6/Ag85B or M72 resulted in predominantly Th1 and Th17 responses and induced antigen-specific serum antibodies. Notably, in a virulent Mtb challenge model, vaccination with UM-1098 adjuvanted ESAT6/Ag85B or M72 significantly reduced lung bacterial burden when compared with unvaccinated mice and protection occurred in the absence of pulmonary inflammation. These data demonstrate that the synthetic Mincle agonist UM-1098 induces strong Th1 and Th17 immunity after vaccination with Mtb antigens and provides protection against Mtb infection in mice.

A Double-Humanized Mouse Model for Studying Host Gut Microbiome-Immune Interactions in Gulf War Illness.

Unraveling the multisymptomatic Gulf War Illness (GWI) pathology and finding an effective cure have eluded researchers for decades. The chronic symptom persistence and limitations for studying the etiologies in mouse models that differ significantly from those in humans pose challenges for drug discovery and finding effective therapeutic regimens. The GWI exposome differs significantly in the study cohorts, and the above makes it difficult to recreate a model closely resembling the GWI symptom pathology. We have used a double engraftment strategy for reconstituting a human immune system coupled with human microbiome transfer to create a humanized-mouse model for GWI. Using whole-genome shotgun sequencing and blood immune cytokine enzyme linked immunosorbent assay (ELISA), we show that our double humanized mice treated with Gulf War (GW) chemicals show significantly altered gut microbiomes, similar to those reported in a Veteran cohort of GWI. The results also showed similar cytokine profiles, such as increased levels of IL-1β, IL-6, and TNF R-1, in the double humanized model, as found previously in a human cohort. Further, a novel GWI Veteran fecal microbiota transfer was used to create a second alternative model that closely resembled the microbiome and immune-system-associated pathology of a GWI Veteran. A GWI Veteran microbiota transplant in humanized mice showed a human microbiome reconstitution and a systemic inflammatory pathology, as reflected by increases in interleukins 1β, 6, 8 (IL-1β, IL-6, IL-8), tumor necrosis factor receptor 1 (TNF R-1), and endotoxemia. In conclusion, though preliminary, we report a novel in vivo model with a human microbiome reconstitution and an engrafted human immune phenotype that may help to better understand gut-immune interactions in GWI.

Abstract 1045: Using Multiplex Imaging For Cell Phenotyping Of Early Human Atherosclerosis

Background: In the past few years, animal models and cultured cells have shown that smooth muscle cells (SMCs) and inflammatory cytokines are important new players in atherosclerotic disease. Validating the roles of these new players in human atherogenesis is crucial for the development of therapeutics targeting them. Multiplex imaging is a powerful tool for mapping cell phenotypes and microenvironment in tissue sections, enabling proof-of-concept studies using biobanked human specimens. However, this technology has seldom been applied to human atherosclerotic lesions. This study is a trial to benchmark the application of the multiplex imaging platform PhenoCycler to biobanked human coronary lesions. We hypothesize that this technology is a powerful tool to prove theories raised by previous studies: SMCs initiate foam cell formation and inflammation is associated with phenotypic switching in SMCs. Methods: We created an 8-plex imaging panel to map lesion cells previously identified as key players in atherogenesis, including foam cells from SMC and macrophage origins, CD68 + SMCs, apoptotic cells, and cells expressing inflammatory cytokines interleukin-1β and tumor necrosis factor α. They were imaged simultaneously on each section of early human lesions (n=9) to test theories related to foam cell formation, phenotypic transition, and inflammatory cytokines. Results: Foam cells of SMC origin greatly outnumbered those of macrophage origin and were enriched in the deep intima, where lipids accumulate in early atherogenesis, distinct from macrophage foam cells in the superficial intima. A higher presence of CD68 + SMCs was observed among lesion SMCs highly expressing interleukin-1β, but not TNF-α. Highly inflamed SMCs exhibited a trend of increased apoptosis compared to macrophages with similar cytokine levels. Conclusions: Our multiplex imaging results confirmed that SMCs form the majority of foam cells in early atherogenesis, and their phenotypic transition to macrophage-like is related to inflammation. Applying multiplex imaging to biobanked human lesions unlocks opportunities to prove that theories based on animal models and cultured cells apply to human atherogenesis.

Abstract LB072: Tumor-infiltrating lymphocytes (TIL) engineered with membrane-bound IL15 (cytoTIL15 cells) exhibit pharmacologically regulatable signal transduction in cis and trans

Abstract Introduction: We have previously shown the successful engineering of membrane-bound IL15 (mbIL15)-expressing tumor-infiltrating lymphocytes (TIL; cytoTIL15™ cells) from solid tumors such as melanoma, which are CD8+ enriched TIL exhibiting enhanced persistence and anti-tumor activity compared with unengineered TIL (SITC 2022, 2023; clinical candidate OBX-115: NCT05470283). The mbIL15 expressed by cytoTIL15 cells is regulated via a carbonic anhydrase 2 (CA2) drug-responsive domain, which allows expression to be induced upon delivery of the FDA-approved small molecule ligand acetazolamide (ACZ) and was designed to engage the IL2Rβ and IL2Rγ receptors in both cis and in trans. Using natural killer (NK) cells, which are potently responsive to soluble, trans-presented IL15, as an experimental model system, we examined the potential of mbIL15 on cytoTIL15 cells to potentiate activity in both cis and trans. Methods: cytoTIL15 cells and unengineered TIL were generated as described previously (SITC 2022, 2023). Cells were assessed for expression of mbIL15 and phosphorylation of downstream signaling molecules STAT5 and S6 by flow cytometry, and co-cultured with allogeneic healthy donor NK cells for 1-10 days with and without transwell inserts. Functional effects of cytoTIL15 cells were compared to those of soluble recombinant IL15. Supernatant was obtained from co-cultures for assessment of cytokine production. Results: cytoTIL15 cells demonstrated robust ACZ-dependent mbIL15 expression, and ACZ dosing regulated mbIL15 activity in cis by potentiating the phosphorylation of STAT5 (geoMFI with 0 vs. 25 µM ACZ, p=0.012) and S6 (geoMFI with 0 vs. 25 µM ACZ, p=0.016). Upon removal of ACZ, mbIL15 expression and pSTAT5 and pS6 returned to basal levels within 24 hours. mbIL15 expressed on the surface of cytoTIL15 cells could also activate cells in trans in an ACZ-dependent titratable manner. Specifically, co-culture of cytoTIL15 cells with NK cells led to an ACZ-dependent increase in pSTAT5 (geoMFI with 0 vs. 25 µM ACZ, p=0.038) in NK cells, which was not seen in co-cultures of unengineered TIL with NK cells. Additionally, NK cells showed a similar cytokine production profile when exposed to mbIL15 by cytoTIL15 cells as with soluble, trans-presented IL15. NK cell transactivation relied on direct cell contact with cytoTIL15 cells, as magnitudes of phosphorylation were decreased in co-cultures with NK and cytoTIL15 cells separated by a transwell membrane (pSTAT5 geoMFI p=0.008). Conclusions: cytoTIL15 cells demonstrate functional engagement of cytokine receptors via mbIL15 in both cis and in trans, including phosphorylation of STAT5 and S6. Taken together, these data highlight the potential for robust activity of cytoTIL15 cells through potentiation of ACZ-dependent mbIL15 activity in both cis and trans. Citation Format: Rachel Burga, Gauri Kulkarni, Zheng Ao, Dhruv K. Sethi, Jan ter Meulen, Michelle Ols. Tumor-infiltrating lymphocytes (TIL) engineered with membrane-bound IL15 (cytoTIL15 cells) exhibit pharmacologically regulatable signal transduction in cis and trans [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB072.

Amyopathic dermatomyositis may be on the spectrum of autoinflammatory disease: A clinical review.

Systemic autoinflammatory diseases (SAIDs) are distinct from autoimmune diseases. The former primarily results from abnormal innate immune response and genetic testing is crucial for disease diagnosis. Similar cutaneous involvement is a main feature for both SAID and dermatomyositis (DM), so they can be confused with each other. A literature search of PubMed and MEDLINE was conducted for relevant articles. The similarities and differences between these two types of diseases were analyzed. We found phenotypic similarities between these two types of disorders. Accumulating data supports a major role of the innate immune system and a similar cytokine profile. Molecular testing using an autoinflammatory disease gene panel may help identify SAID patients from the DM population and may offer therapeutic benefit using interleukin-1 (IL-1) inhibitors. A subset of DM, notably amyopathic dermatomyositis in the absence of autoantibodies may be on the spectrum of autoinflammatory disease.

Abstract 6335: Armoring CAR-T therapy with PD-1 blockade: a powerful strategy for enhancing anti-tumor effects in pancreatic cancer

Abstract Chimeric antigen receptor-modified T cells (CAR-T) have been widely applied in the treatment of hematological malignancies and are currently under investigation for their potential effectiveness against solid tumors. Nevertheless, the effectiveness of CAR-T in treating solid tumors remains unsatisfactory due to a range of factors, including CAR-T cell infiltration and heterogeneity. The efficacy of CAR-T therapy is substantially influenced by the tumor microenvironment. This is particularly evident when PD-L1 is activated through CAR-T, leading to the inhibition of CAR-T cell activity via the PD-1 pathway. In this study, we engineered a CAR-T therapy that targets mesothelin and concurrently expresses a membrane-bound PD-1 receptor. This design blocks the PD-1 on CAR-T cells, effectively counteracting the suppression of CAR-T function mediated by PD-L1. We observed a substantial upregulation of PD-L1 on AsPC-1 tumor cells after 4 hours of IFN-γ stimulation in vitro. CAR-T cells engineered to co-express PD-1 antibody (aMSLN-28BBZ-mPD-1) exhibited similar short-term cytotoxicity and cytokine secretion profiles to those of the traditional third-generation CAR-T (aMSLN-28BBZ). Notably, aMSLN-28BBZ-mPD-1 displayed significantly improved long-term target cell killing. Furthermore, in an in vivo model of pancreatic cancer, aMSLN-28BBZ-mPD-1 demonstrated enhanced anti-tumor activity when compared to the traditional aMSLN-28BBZ, as supported by RNAscope analysis and immunofluorescence assays of tumor tissues. These analyses revealed a marked upregulation of PD-L1 expression and enhanced CAR-T cell infiltration in the aMSLN-28BBZ-mPD-1 treatment group. In summary, our study demonstrates that co-expression of a membrane-bound PD-1 antibody overcomes PD-L1 mediated CAR-T suppression, as observed in both in vitro and in vivo. This innovative strategy underscores its therapeutic potential and value in treating solid tumors. Keywords: CAR-T, PD-1, Tumor Microenvironment, Pancreatic Cancer Citation Format: Zhixiang Zhang, Jingwei Huang, Zhifeng Zhao, Xiangyi Wang, Cnhui he, Sen Yang, Qingyang Gu. Armoring CAR-T therapy with PD-1 blockade: a powerful strategy for enhancing anti-tumor effects in pancreatic 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 6335.

Abstract 4012: SOT302: Dual expression of exogenous glutamine oxaloacetate transaminase (GOT2) and TP53-induced glycolysis and apoptosis regulator (TIGAR) enhance CAR T cell activity in preclinical solid tumor models

Abstract Background: Despite success in hematological malignancies, CAR T therapy has had limited efficacy against solid tumors. The suppressive tumor microenvironment limits CAR T activity through various mechanisms including competition for available nutrients and chronic stimulation resulting in reduced effector functions or T cell exhaustion. CAR T cells with enhanced metabolic fitness or more durable memory phenotype could potentially improve the clinical outcome in solid tumors. GOT2 plays an important role in mitochondrial function and maintenance of redox homeostasis. We have previously demonstrated enhancement of CAR T cells overexpressing GOT2. TIGAR is an enzyme known to promote antioxidative activites and reduce reactive oxygen species and has a role in protecting against apoptosis. Here we tested SOT302 CAR T cells expressing both GOT2 and TIGAR in preclinical solid tumor models. Results: Expression of GOT2 and TIGAR transgenes were confirmed by qRT-PCR and western blot. Activity of GOT2 and TIGAR were confirmed by measuring aminotransferase activity (AST) and glutathione production, respectively. Compared to control CAR T cells, SOT302 is enriched for CD8+ Tscm cells and has a higher percentage functional, non-senescent (CD28+CD57-) cells. SOT302 has similar cytokine expression and cytotoxicity in standard co-culture assays compared to controls. SOT302 cells were chronically stimulated with plate-bound antigen every 3-4 days for 4 rounds. SOT302 had greater expansion of cells and were less exhausted and had better cytolytic capacity compared to controls. SOT302 cells were then evaluated in tumor xenograft mouse models. Consistent with our previous findings, CAR T cells expressing the single GOT2 transgene were more efficacious compared to CAR T cells alone. Expression of the single TIGAR transgene had no apparent benefit over CAR alone, however SOT302 cells expressing both GOT2 and TIGAR had superior anti-tumor activity compared to CAR alone and CAR+GOT2. No overt toxicity or significant body weight loss was observed. Ex vivo analyses to measure peripheral expansion, tumor infiltration and exhaustion were also performed. Preliminary findings indicate that SOT302 has better peripheral expansion compared to CAR alone and similar expansion compared to CAR+GOT2 controls. SOT302 cells had better tumor infiltration compared to both the CAR alone and CAR+GOT2 cells. Tumor infiltrating SOT302 cells also exhibited lower levels of exhaustion compared to CAR alone or CAR+GOT2 controls. Summary: SOT302 cells express exogenous GOT2 and TIGAR transgenes and have superior anti-tumor activity in preclinical solid tumor models. These data suggest that SOT302 may be a promising candidate for patients with solid tumor cancers. Citation Format: Emily Kuiper, Samyabrata Bhaduri, Daniel Garafola, Kshitij Sharma, Jennifer Coccia, Kathleen Whiteman, Amy Jensen-Smith. SOT302: Dual expression of exogenous glutamine oxaloacetate transaminase (GOT2) and TP53-induced glycolysis and apoptosis regulator (TIGAR) enhance CAR T cell activity in preclinical solid tumor models [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 4012.

Atopic dermatitis phenotype affects expression of atopic diseases despite similar mononuclear cell cytokine response

BackgroundThe atopic march refers to the co-expression and progression of atopic diseases in childhood, often beginning with atopic dermatitis, although children may not “progress” through each atopic disease. ObjectiveWe hypothesized that future atopic disease expression is modified by atopic dermatitis phenotype, and that these differences result from underlying dysregulation of cytokine signaling. MethodsChildren (n=285) were enrolled into the Childhood Origins of ASThma birth cohort and followed prospectively. Rates of atopic dermatitis, food allergy, allergic rhinitis, and asthma were assessed longitudinally from birth to 18 years of age. Associations between atopic dermatitis phenotype and food allergy, allergic rhinitis, asthma, allergic sensitization, exhaled nitric oxide, and lung function were determined. Peripheral blood mononuclear cell responses (IL-5, IL-10, IL-13, IFN-γ) to dust mite, phytohemagglutinin, Staphylococcus aureus Cowan I, and tetanus toxoid were compared among atopic dermatitis phenotypes. ResultsAtopic dermatitis at year 1 was associated with an increased risk of food allergy (p=0.004). Both persistent and late-onset atopic dermatitis were associated with an increased risk of asthma (p=<0.001), rhinitis (p<0.001), elevated total IgE (p=<0.001), percentage of aeroallergens with detectable IgE (p<0.001), and elevated exhaled nitric oxide (p=0.002). Longitudinal analyses did not reveal consistent differences in PBMC responses among dermatitis phenotypes. ConclusionAtopic dermatitis phenotype is associated with differential expression of other atopic diseases. Our findings suggest peripheral blood cytokine dysregulation is not a mechanism underlying this process, and immune dysregulation may be mediated at mucosal surfaces or in secondary lymphoid organs.

Periodontal disease severity in patients with long COVID and non-COVID-19

Background: Previous research studies have found the persistence of various COVID-19 symptoms even after the patient tested negative on a PCR test; this incident is now known as long COVID. These long COVID symptoms are reported to appear in the oral cavity including long COVID effects on periodontal disease, as both long COVID and periodontal disease release similar proinflammatory cytokines such as Acute phase proteins, CRP, TNF-α, IL-1β, IL-2, IL-6, and IFN-g. Purpose: This study aims to show periodontal-disease severity-frequency distribution in COVID-19 survivors with long COVID and in non-COVID-19 patients. Methods: Patients’ secondary data in the Periodontics Clinic Faculty of Dentistry at Trisakti University Dental Hospital (n=40) consisted of 20 samples from COVID-19 survivors who experienced long COVID and 20 samples from the non-COVID-19 group selected according to the inclusion criteria. Afterward, the data was recapitulated and processed into a research report. Results: The distribution percentage of generalized gingivitis was highest in non-COVID-19 patients, while generalized periodontitis was highest in COVID-19 survivors with long COVID. Based on periodontitis staging and grading methods, it is not proven that long COVID increases the severity of the periodontitis. Conclusion: This research shows that the distribution of gingivitis in COVID-19 survivors with long COVID has not increased. Meanwhile, the distribution of general periodontitis increased in survivors with long COVID. However, there was no increased severity of periodontitis based on the staging and grading method of periodontitis in the COVID-19 survivors with long COVID.

The Intersection of COVID-19 and Rheumatoid Arthritis: Shared Mechanisms, Treatment Challenges, and Potential Therapeutic Approaches.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading rapidly across the world, posing a major health concern with the coronavirus disease 2019 (COVID-19). Patients with rheumatoid arthritis (RA) may be at higher risk of infection and disease progression due to impaired autoimmune systems, immunosuppressants, and comorbidities. Therefore, we review the possible immune mechanisms and pathological interactions between COVID-19 and RA, as the uncontrolled immune activation and cytokine response in COVID-19 resemble the immune inflammation of RA. We also discuss the potential mechanisms that may lead to cardiovascular complications as well as the challenges of treating RA patients with COVID-19. While several therapeutic agents are being developed to cure COVID-19, antirheumatic drugs could also be potential options due to the similar proinflammatory cytokines induced in both diseases. Additionally, we discuss the safety and effectiveness of SARS-CoV-2 vaccines and novel therapeutic approaches against RA based on the shared mechanisms between COVID-19 and RA.

Inflammatory Markers in Hemophagocytic Lymphohistiocytosis - a Single Centre Study

BACKGROUND Hemophagocytic lymphohistiocytosis (HLH) has significant clinical and biochemical overlap with other cytokine storm syndromes including Adult-Onset Still's disease (AOSD) and COVID-19 cytokine storm (CCS). Diagnosis of HLH is based largely on the HLH-2004 criteria which carry certain limitations. Many of the tests included in these criteria such as flow cytometry for NK cell cytotoxicity and soluble CD25 levels are only available in specialized centres. Moreover, they can take days to weeks to result, leading to delays in diagnosis. Biomarkers that are unique to HLH and more readily available are needed to ensure a timelier diagnosis of this life-threatening disease. C-reactive protein (CRP) is a sensitive marker of inflammation that is driven by IL-6 activity, a cytokine that is significantly elevated in AOSD and CCS but comparatively less so in HLH. Our study sought to determine whether lower levels of CRP, in conjunction with other patterns of inflammatory markers, could reliably distinguish HLH from AOSD and CCS. METHODS This was a single-centre, retrospective study examining adult ( n=45) and pediatric ( n=12) patients with HLH, and adult patients with AOSD ( n=11) and CCS ( n=13). Patients were enrolled from Vancouver General Hospital between January 1 st, 2000, to June 28 th, 2023. CRP and ferritin levels were collected for each patient if available, and values were included in analysis only if drawn prior to HLH-specific treatment. Ferritin levels were included to add to our understanding of different patterns of inflammation seen in similar cytokine storm syndromes. The Kruskal-Wallis test was used to compare CRP and ferritin values in total HLH cases compared to AOSD and CCS. Diagnostic performance of CRP was analyzed by performing a receiver operating characteristic (ROC) curve for CRP in HLH patients using the Youden index to identify the optimal cut-off point for CRP values. RESULTS In total, eighty-one patients were included in the study. Median CRP was significantly lower in HLH compared to AOSD (68.9 mg/L versus 168.0 mg/L, p &amp;lt; 0.001) and CCS (68.9 mg/L versus 121.0 mg/L, p = 0.024) (Figure 1). Median serum ferritin levels were significantly lower in CCS compared to HLH (1,386 ng/mL versus 16,722, p &amp;lt; 0.001) and AOSD (1,3860 ng/mL versus 12,480 ng/mL, p = 0.016). There was no significant difference in ferritin levels between HLH and AOSD. The ROC curve was performed for CRP values in total HLH cases which demonstrated an Area Under the Curve (AUC) of 0.799, indicating a fair diagnostic performance in differentiating HLH, with a sensitivity of 66.1% and specificity of 87.5% for CRP values less than 94.5 mg/L, as calculated by Youden's index. CONCLUSION CRP is significantly lower in HLH compared to AOSD and CCS, suggesting that mild to moderate elevations in CRP reflect the comparatively lower IL-6 activity underlying HLH. When used in conjunction with other inflammatory markers, especially marked hyperferritinemia, a comparatively lower CRP value helps capture the unique cytokine pattern underlying HLH, and is a useful, easily accessible biomarker than can aid in the diagnosis of HLH.

Modulation of NLRP3 Inflammasome in Monocytes By HT-6184 in a Single-Cell Proteomic Study

Background The NLRP3 inflammasome is associated with the activation of monocytes and plays a crucial role in the innate immune response. The NLRP3 inflammasome is a multi-protein complex expressed primarily in monocytes and other immune cells. When monocytes encounter danger signals, such as pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs), the NLRP3 inflammasome is assembled, leading to the maturation and secretion of potent pro-inflammatory cytokines, interleukin-1β (IL-1β), and interleukin-18 (IL-18). These cytokines, in turn, induce a robust inflammatory response to clear infections, promote tissue repair, and coordinate adaptive immunity. However, dysregulated NLRP3 inflammasome activation has been associated with various inflammatory disorders, such as autoimmune diseases, metabolic diseases, and neurodegenerative conditions. Purpose This study investigated the effects of a potent, allosteric NLRP3 inflammasome modulator, HT-6184, on cytokine release response from the activation of NLRP3 in human monocytes. Methods Human peripheral blood mononuclear cells (PBMCs) were utilized, and monocytes were isolated using a Miltenyi pan monocyte isolation kit. Subsequently, these isolated monocytes were exposed to different concentrations of HT-6184 (300 nM and 100 nM) before being stimulated with lipopolysaccharide (LPS) at a concentration of 10 ng/mL, a potent activator of the NLRP3 inflammasome. After 24 hours, the secreted cytokines were analyzed at the single-cell level using the Isospark, with data analysis performed using the Isospeak software. Results The results demonstrated that monocytes treated with LPS alone exhibited high levels of pro-inflammatory cytokines, including effector and stimulatory cytokines, and chemoattractive chemokines such as IL-8, MIF, MIP-1b, and TNF-alpha. In contrast, monocytes treated with HT-6184 and LPS showed reduced cytokine secretion, approaching levels observed in the control group. Notably, the HT- 6184-treated group displayed a lower incidence of polyfunctionality (2%) than the LPS-only group (11%), indicating fewer cells capable of simultaneously secreting multiple cytokines. The 3D-TSNE graph analysis revealed that the HT-6184 and LPS-treated groups clustered with the control group, signifying similar cytokine secretion profiles. In contrast, the LPS-only group exhibited a distinct pattern of cytokine secretion. Conclusions These findings indicate that HT-6184 effectively inhibits the release of pro-inflammatory cytokines upon NLRP3 activation in monocytes. Further research should explore the characteristics of HT-6184 and its ability to modulate the inflammasome in diverse in vitro and in vivo models. The modulation of the NLRP3 inflammasome by HT-6184 holds potential therapeutic implications for managing inflammatory disorders associated with dysregulated NLRP3 activation.

Neonatal immune cells have heightened responses following in-utero exposure to chorioamnionitis or COVID-19.

Chorioamnionitis alters neonatal immune responses. Gestational COVID-19 infection is associated with adverse pregnancy outcomes, but its impact on neonatal immunity is unclear. We hypothesized that gestational COVID-19 exposure would result in exaggerated neonatal immune responses, similar to chorioamnionitis-exposed neonates. Term umbilical cord blood mononuclear cells (CBMCs) were isolated from neonates exposed to chorioamnionitis, gestational COVID-19 or unexposed controls. CBMCs were cultured and stimulated with heat-killed Escherichia coli, Streptococcus agalactiae or Staphylococcus epidermidis. A multiplexed protein assay was used to measure cytokine levels in cell culture supernatants and flow cytometry was used to evaluate cellular-level cytokine expression. Both chorioamnionitis-exposed and COVID-19 exposed CBMCs demonstrated upregulation of IL-1β and IL-6 compared to unexposed CBMCs, while only COVID-19 exposure resulted in IL-8 upregulation. There were no differences between chorioamnionitis-exposed and COVID-19 exposed CBMCs when these groups were directly compared. Flow cytometry demonstrated immune cell subset specific differences in cytokine expression between the exposure groups. The fetal/neonatal response to maternal inflammation differed based on immune cell subset and etiology of inflammation, but the global neonatal cytokine responses were similar between exposure groups. This suggests that targeting perinatal inflammation rather than the specific etiology may be a possible therapeutic approach. Neonatal immune cells have similar pathogen-associated global cytokine responses, but different cell-level immune responses, following in-utero exposure to chorioamnionitis or COVID-19. This is the first study to directly compare immune responses between neonates exposed to chorioamnionitis and COVID-19. This suggests that the fetal/neonatal cellular response to perinatal inflammation differs based on the etiology and severity of maternal inflammation, but still results in a similar overall inflammatory profile regardless of the cause of perinatal inflammation.

IMMU-20. MESSENGER RNA CHALLENGE RAPIDLY PREDICTS BRAIN TUMOR RESPONSIVENESS TO CHECKPOINT INHIBITORS

Abstract BACKGROUND Pathogen recognition receptor (PRR) activation is requisite for igniting the immune response and making the tumor microenvironment ‘hot’ during oncogenesis. We theorized that these PRRs would remain present in ICI responsive tumors and could be challenged using pan-PRR mimic to prospectively predict brain cancer immunogenicity and response to immune checkpoint inhibitors (ICIs). METHODS By layering mRNA into multilamellar nanoparticles (ML RNA-NPs), multiple pathogen recognition receptors can be simultaneously activated in individualized cancer cells serving as a broad screen to test tumor immunogenicity. We challenged murine brain tumor lines known to respond (GL261 and SMA-560) or resist ICI treatment (KR158b and CT2A) with ML RNA-NPs and analyzed downstream production of proinflammatory cytokines as predictors of ICI response. RESULTS We demonstrated that ML-RNA-NPs can activate multiple PRRs simultaneously. Following ML RNA-NP challenge, ICI responsive tumors GL261 and SMA-560 (compared to more ICI resistant lines KR158b and CT2A) elicited increased production in pro-inflammatory cytokines such as IFN-β and IL-6 and in CCL4. To further develop this work into a commercially viable tool to predict ICI response, we developed 3D modeling of glial tumors for ML RNA-NP challenge and immunogenicity prediction. We enrolled canines with primary gliomas and began growing their tumoroids in 3D using liquid-like solid (LLS) technology. Using this pipeline, we successfully perfused 3D tumoroids with ML RNA-NP demonstrating ability to set up real-time patient derived explants to predict immunogenicity. We demonstrated that mRNA perfusion of 3D tumoroids elicited similar cytokine response observed with 2D culture of cell-lines. CONCLUSION Our results indicate that cytokine signatures following ML RNA-NP challenge differ significantly from ICI responsive versus non-responsive brain tumors. These findings allow for creation of a diagnostic assay to quickly assess brain tumor immunogenicity, allowing for informed treatment with ICIs.