Pattern Of Cytokine Production Research Articles

Changes in splenic tissue and immune response profile of Schistosoma mansoni infected mice submitted to chronic ethanol intake.

In Schistosoma mansoni infection, the spleen is one of the organs affected, causing its enlargement (splenomegaly). Intake of ethanol through alcoholic beverages can cause spleen atrophy and interfere with immune activity. To gain knowledge of this association on the spleen and on the immune response profile, male mice were used as an experimental model. These animals were divided into four groups: C. control; EC. uninfected/ethanol gavage; I. infected; and IE. infected/ethanol gavage. Groups I and IE were infected with about 100 cercariae (BH strain) of S. mansoni and in the fifth week of infection, gavage 200 μL/day/animal of 18 % ethanol was started for 28 consecutive days. At the end of the gavage (9th week of infection) all animals were euthanized. The spleen was removed and longitudinally divided in two parts. After histological processing, the sections were stained with H&E and Gomori's Reticulin for histopathological and stereological analyses, white pulp morphometry and quantification of megakaryocytes. The other fragment was macerated (in laminar flow) and the cell suspension, after adjusting the concentration (2 × 106), was plated to obtain cytokines produced by spleen cells that were measured by flow cytometry (Citometric Bead Array). Histopathological and quantitative analyzes in the spleen of the IE group showed an increase in the number of trabeculae and megakaryocytes, a decrease in reticular fibers, as well as important organizational changes in the white pulp and red pulp. Due to the decrease in the levels of cytokines measured and the result of the calculation of the ratio between the IFN-y and IL-10 cytokines (p = 0.0079) of the infected groups, we suggest that ethanol decreased the inflammatory and anti-inflammatory response generated by the infection (group IE, the production of cytokines was significantly decreased (p < 0.01). These changes demonstrate that ethanol ingestion interferes with some parameters of experimental S. mansoni infection, such as changes in splenic tissue and in the pattern of cytokine production.

SiRNA-Mediated B7H7 Knockdown in Gastric Cancer Lysate-Loaded Dendritic Cells Amplifies Expansion and Cytokine Secretion of Autologous T Cells.

Gastric cancer, ranked as the fifth most common cancer worldwide, presents multiple treatment challenges. These obstacles often arise due to cancer stem cells, which are associated with recurrence, metastasis, and drug resistance. While dendritic cell (DC)-based immunotherapy has shown promise as a therapeutic strategy, its efficacy can be limited by the tumor microenvironment and certain inhibitory immune checkpoint molecules, such as B7H7. SiRNA-medicated knockdown of B7H7 in tumor cell lysate-pulsed DCs can increase cytokine secretion and autologous T lymphocyte expansion. This study aimed to evaluate the impact of B7H7 suppression in gastric cancer cell lysate-pulsed DCs on the stimulatory potential of autologous CD3+ T lymphocytes. Peripheral blood mononuclear cells (PBMCs) were isolated and monocytes were obtained; then, they were differentiated to immature DCs (iDCs) by GM-CSF and IL-4. Tumor cell lysates from human gastric cancer cell lines were harvested, and iDCs were transformed into mature DCs (mDCs) by stimulating iDCs with tumor cell lysate and lipopolysaccharide. B7H7-siRNA was delivered into mDCs using electroporation, and gene silencing efficiency was assessed. The phenotypic characteristics of iDCs, mDCs, and B7H7-silenced mDCs were evaluated using specific surface markers, an inverted light microscope, and flow cytometry. CD3+ T cells were isolated via magnetically activated cell sorting. They were labeled with CFSE dye and co-cultured with mDCs and B7H7-silenced mDCs to evaluate their ability to induce T-cell proliferation. T-cell proliferation was assessed using flow cytometry. The concentration of TGF-β, IL-4, and IFN-γ secreted from CD3+ T cells in the co-cultured supernatant was evaluated to investigate the cytokine secretory activity of the cells. Transfection of B7H7 siRNA into mDCs was performed in optimal conditions, and the siRNA transfection effectively reduced B7H7 mRNA expression in a dose-dependent manner. SiRNA-mediated B7H7 knockdown in mDCs enhanced maturation and activation of the DCs, as demonstrated by an increased surface expression of CD11c, CD86, and CD40. Co-culture experiments revealed that B7H7-silenced mDCs had more capacity to induce T cell proliferation compared to non-transfected mDCs. The cytokine production patterns of T cells were also altered. Upon examining the levels of TGF-β, IL-4, and IFN-γ released by CD3+ T cells in the co-culture supernatant, we found that silencing B7H7 in mDCs resulted in a rise in IL-4 secretion and a reduction in TGF-β levels compared to mDCs that were not transfected. The study found that suppressing B7H7 expression in DCs significantly enhances their maturation and stimulatory activity when exposed to gastric cancer cell lysate. These B7H7-silenced DCs can substantially increase cytokine production and promote co-cultured T-cell expansion. Consequently, inhibiting B7H7 in DCs may offer a practical strategy to enhance the ability of DCs to initiate T lymphocyte responses and improve the effectiveness of DC-based cell therapy for cancer patients.

Roles of dendritic cells and macrophages during Chlamydia muridarum genital infection in stressed mice

It is well known that stress has a significant impact on public health and more evidence shows that stressors generally are greater in populations of lower socioeconomic status. Cold-induced stress inhibits immune cell activity and has been shown to cause different expressions of cytokine profiles during Chlamydia muridarum genital infection. Dendritic cells (DCs) and macrophages(MØ) are immune cells that express beta2-adrenergic receptor (β2-AR) and with key roles during chlamydia genital infection. This study is aimed to investigate the role of β2-AR by using a β2-AR knockout mouse. Wildtype (WT) and β2-AR knockout(KO) mice were stressed for five minutes daily and infected with C. muridarum intravaginally. Non-stressed infected mice of the groups were used as controls. Bone-marrow-derived DCs and MØ were tested isolated, counted differentiated, and proliferated for cytokine production. The proliferation of DCs and macrophages in the presence/absence of β2-AR agonists and antagonists was tested. Data show that non-stressed mice had a higher production of cytokines than stressed mice. Increased production of TNF-α in LPS-treated DCs and MØ of WT and β2-AR KO was observed. The effect of fenoterol and ICI118,55 antagonist showed no significant difference in cytokine production. The data indicate that β2-AR KO and WT had a similar pattern of cytokine production suggesting that deficiency in β2-AR restores the function of immune cells during genital infection. Experiments are undergoing to fully understand the mechanisms involved in modulating the function of DC and MØ during chlamydia genital infection.

Thelper1 and T helper2 cytokine production patterns in stressed beta2-adrenergic receptor-deficient mice during chlamydia genital infection.

Murine CD4+ T cells have two distinct cytokine secretion patterns to play different functions. We have demonstrated that cold-induced stress (CIS) suppresses the immune system leading to increased intensity of Chlamydia muridaum in mice. We have reported that beta2-adrenergic receptor (b2-AR) knockout (KO) mice resist chlamydia genital infection. However, the cytokine profile of  CD4+ T cells is not well explored. This study aimed to determine the cytokine production of Th1 and Th2 types in stressed and non-stressed b2-AR KO mice. We investigated the cytokine production levels of stressed and non-stressed mice during C. muridarum genital infection. Significantly increased production of cytokines was observed in plates pre-coated with anti-CD3/CD28 or in the presence of  Con A or LPS. A decrease in the production of IFN-g and IL-2, whereas an increase in the secretion of IL-10, IL-13, and IL-23 in CD4+ T cells of stressed wild-type mice was obtained; however, the secretion of IFN-g and IL-2 was restored in T cells of b2-AR KO mice. Moreover, in vitro proliferation of CD4 T cells in the presence of b2-AR antagonists, ICI 118, 551 stimulated the production of Th1 cytokines, whereas b2-AR agonist, Fenoterol, decreased the production of Th1-type cytokines. Overall, Th1 cytokine responses are reduced in stressed mice suggesting that the cytokine status was polarized toward a Th2 immune response that can be restored by removing b2-AR from immune cells.

Effects of industrially produced 2-dimensional molybdenum disulfide materials in primary human basophils

MoS2 has been increasingly used in place of graphene as a flexible and multifunctional 2D material in many biomedical applications such as cancer detection and drug delivery, which makes it crucial to evaluate downstream compatibility in human immune cells. Molybdenum is a component of stainless-steel stent implants and has previously been implicated in stent hypersensitivity. In view of this, it is important to ascertain the effect of MoS2 on allergy-relevant cells. Basophils are a less commonly used immune cell type. Unlike mast cells, basophils can be easily derived from primary human blood and can act as a sentinel for allergy. However, merely testing any one type of MoS2 in basophils could result in different biological results. We thus decided to compare 2D MoS2 from the two companies BeDimensional© (BD) and Biograph Solutions (BS), manufactured with two different but commonly exploited methods (BD, deoxycholate surfactant in a high-pressure liquid exfoliation, and BS using glycine in ball-milling exfoliation) to elucidate immunological end-points common to both MoS2 and to demonstrate the need for biological verification for end-users who may require a change of supplier. We report higher histamine production in human basophils with MoS2. No effects on either surface basophil activation markers CD63 and CD203c or reactive oxygen species (ROS) production and cell viability were observed. However, different cytokine production patterns were evidenced. IL-6 and IL-1β but not TNF and GM-CSF were increased for both MoS2. BS-MoS2 increased IL-4, while BD-MoS2 decreased IL-4 and increased IL-13. Molybdate ion itself only increased IL-1β and IL-4. Deoxycholate surfactant decreased viability at 18 h and increased ROS upon basophil activation. Therefore, these results demonstrate the safety of MoS2 in human basophils in general and highlight the importance of considering manufacturer additives and variability when selecting and investigating 2D materials such as MoS2.

High Dimensional Mapping of Bone Marrow Immune Characteristics in Immune Thrombocytopenia

Introduction Immune thrombocytopenia (ITP) is an acquired autoimmune disease that is characterized by increased antibody-mediated platelet destruction, decreased megakaryopoiesis and impaired thrombopoiesis. An abnormal bone marrow microenvironment has been proposed to play an essential role in the progression of ITP [Stem Cells Dev, 2017]. Cytometry by Time of Flight (CyTOF) is a new analytic strategy that provides single-cell proteomics for enhanced cellular analysis, and describes the components of immune cell populations and the characteristics of new biomarkers. Here, we aim to provide a high-dimensional overview and summary of the changes in cell subsets and functions in the bone marrow of patients with ITP using CyTOF. Methods A total of 40 patients diagnosed with ITP (21 men and 19 women; 23-71 years of age, median 47 years) were enrolled between July and October 2021. Based on the onset and progression of the disease, the ITP patients were classified into 3 subgroups. Group ITP-N1 included 15 newly diagnosed treatment-naïve patients ≤3 months after diagnosis, Group ITP-N2 included 10 patients with an ITP diagnosis of > 3 months who had not received any standard treatment (>3-12 months after diagnosis), and Group ITP-R included 15 patients who had received standard treatment but relapsed following remission. The healthy control (HC) group consisted of 10 donors who underwent hematopoietic stem cell transplantation. CyTOF panels were designed using 42 parameters that were measured at the single-cell level in the bone marrow of the ITP patients and HCs. The plasma levels of immune checkpoint regulators, such as PD-1, PD-L1, CTLA-4, TIM-3, LAG-3 and Galectin-9, were measured using a multianalyte flow assay kit (BioLegend). Results The platelet counts of enrolled ITP patients ranged from 1 to 96×109/L, with a median count of 25×109/L. Using CyTOF, the immune cells in the bone marrow were classified into 7 major subsets, including B cells, γδT cells, CD4+ T cells, CD8+ T cells, natural killer (NK) cells, dendritic cells (DCs), and monocytes. The proportions of DCs and monocytes were significantly different between the ITP patients and HCs. The proportion of monocytes was observed to increase significantly in the bone marrow of the ITP patients. The proportion of the pDC subset of DCs showed a trend of being reduced in the ITP patients, while the proportion of mDCs was increased significantly in the ITP patients. NK cells can be divided into 2 subsets according to the expression of CD56, and these subsets exhibit different cytokine production patterns and cytotoxic functions. The proportion of CD56low NK cells was elevated and the proportion of CD56bright NK cells decreased significantly in ITP-R patients compared to HCs; similar results were not observed in the ITP-N1 or ITP-N2 groups. NK cells in bone marrow may play an important role in the relapse of ITP. We further investigated the functional status of T cells in the ITP patients using a CyTOF panel. The results showed that the proportions of both CD4+ naïve and CD8+ naïve T cells were decreased in the bone marrow of the ITP patients. In particular, the proportion of CD8+ effector T cells was increased significantly in the ITP patients. Next, we analyzed coinhibitory and costimulatory receptor expression in each T-cell subpopulation. CTLA4 expression was observed to be reduced in CD8+ effector T cells and CD4+ Tfh cells. In addition to the changes in coinhibitory receptor expression, costimulatory molecules such as CD28 and ICOS were found to be expressed at higher levels in CD4+ central memory T cells. Combined with the soluble regulators in plasma, we found that the B7.2 levels in plasma was positively correlated with the cell surface expression of CD28 and negatively correlated with the expression of CTLA4 in T cells. Conclusions This study provides a comprehensive overview of the human bone marrow immune characteristics of patients with ITP. Using CyTOF to conduct single-cell analyses, we mapped myeloid and T-cell subset phenotypes in particular and focused on the expression of coinhibitory molecules such as CTLA4. The different proportions of bone marrow immune cells and patterns of immune checkpoint molecule expression in different stages of ITP may provide novel insights into disease etiology and offer promising therapeutic targets for patients with ITP. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Charge and receptor functional injectable hydrogels as cytokine-releasing reservoirs for wound healing

Collecting and utilizing various cytokines released by trauma and implantable materials for stimulating the body in producing a robust immune-inflammatory response would be a promising method. Therefore, an injectable “cytokine reservoir” hydrogel for cytokine management during tissue repair was developed by combining a dual network hydrogel of oxidized glucomannan aldehyde (KGM-CHO) and levodopa-grafted ε-polylysine (EPL-DOPA). The “cytokine reservoir” hydrogel actively recruited macrophages through electrostatic forces and regulated macrophages by mimicking “receptor-ligand” interactions to alter cytokine production patterns. Meanwhile, the dual network hydrogels recruited and stored cytokines by “charge” force then released them according to the degradation of hydrogels, leading to the change of cytokine release pattern. In conclusion, this study provided a unique insight in designing the cytokine management-based tissue engineering strategies for tissue repair.

Differential cytokine profiles produced by anti-epileptic drug re-exposure of peripheral blood mononuclear cells derived from severe anti-epileptic drug patients and non-allergic controls

Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) and drug reactions with eosinophilia and systemic symptoms (DRESS) are the most common severe cutaneous adverse drug reactions (SCARs). Anti-epileptic drugs are one of the most common drugs causing SCARs. Cytokine profiles of SCARs during culprit drug exposure have never been characterized. This study aimed to identify cytokine patterns between SCARs and non-SCARs in epilepsy patients and the patterns of DRESS and SJS/TEN. Epilepsy patients that showed allergic responses to anti-epileptic drugs that manifested as SJS/TEN or DRESS were recruited. Epilepsy patients with no drug allergy symptoms and healthy people were also recruited as control groups. Peripheral blood mononuclear cells (PBMCs) were isolated and co-cultured with assigned anti-epileptic drugs according to the lymphocyte transformation test (LTT). LTT and measurement of cytokine levels in supernatants were performed on day six of cell cultivation. This study identified different cytokine expression patterns between SCAR and non-SCAR in epilepsy patients. Significant levels of IL-10, IL-12, IL-17, and GM-CSF were detected in non-SCAR epilepsy. However, the levels of IL-2, IL-5, IL-13, and IFN-gamma were significantly higher in supernatants of PBMCs of DRESS cultivated with AEDs relative to those of SJS/TEN. These cytokine levels were positively correlated with the cell proliferation index. Production of IL-5 and IL-13 was a unique characteristic of DRESS PBMCs. This study was the first to demonstrate distinct differences in cytokine levels between SCAR and non-SCAR PBMCs in epilepsy, which could help explain the immune-pathomechanism of drug hypersensitivity in SCARs. Different patterns of cytokine production and cell proliferation between DRESS and SJS/TEN in AED hypersensitivity were also demonstrated. Production of IL-5 and IL-13 might be a promising marker to define drug hypersensitivity in DRESS.

Characterization of immune cells of stressed beta2-adrenergic receptor homozygous knockout mouse model during Chlamydia muridarum genital infection

Abstract The relationship of stress in chlamydia genital disease pathogenesis and its influence on the host immune response remains unknown. Beta2-adrenergic receptor(β2-AR), the major receptor of the stress hormone norepinephrine (NE), is known to impair the function of immune cells. This study was undertaken to determine whether cold-induced stress (CIS) results in differential viability percent of immune cells β2-AR KO and WT C57BL/6J treated with NE, β2-AR agonist(Fenoterol), or antagonist(ICI 118,551), and patterns of cytokine production. CIS was induced by immersing mice in cold water for five minutes daily for 21 days. Total CD4+, CD8+, differentiated bone marrow-derived monocytes were purified by negative selection and treated with chemicals for one hour before proliferation for 48 h proliferation. The vi-cell viability analyzer showed that NE results in a 30 and 40 % decrease in viability of WT C57BL/6J CD4+ T and CD8+ cells, respectively, compared to KO (p&amp;lt;0.05). Similarly, NE and Fenoterol resulted in a 20 and 27% decrease in viability of bone marrow-derived monocytes compared to the β2-AR KO mice. Cells treated with ICI 118, 551 showed the slightest reduction in viability (12%) compared to those treated with NE. ELISA results show decreased IL-4 and increased IFN-g secretion by CD4+ T cells of β2-AR KO compared to WT. The secretion of TNF-a by CD8+ cells and bone marrow cells treated with NE and β2-AR agonist or antagonist was high in all treatment groups. This study provides insights for the first time to better understand the NE-β2-AR pathway during chlamydia genital infection. Supported by WV-INBRE of NIH Grant P20GM103434 and NIH grant # 1R15AI124156-01 given to BSC.

The Age-Dependent Role of Th22, Tc22, and Tc17 Cells in the Severity of Pneumonia in COVID-19 Immunopathogenesis.

Coronavirus disease 2019 (COVID-19) has clinical manifestations ranging from mild symptoms to respiratory failure, septic shock, and multi-organ failure. Lymphocytes are divided into different subtypes based on their cytokine production pattern. In this study, we investigated the role of cytokine expressions of CD4+ T (T helper [Th]1, Th2, Th17, Th22) and CD8+ T cell subtypes (T cytotoxic [Tc]1, Tc2, Tc17, Tc22) in the pathogenesis of COVID-19. Peripheral blood mononuclear cells (PBMCs) were extracted with Ficoll by density gradient centrifugation from blood samples of 180 COVID-19 patients (children and adults) and 30 healthy controls. PBMCs were stimulated with PMA and Ionomycin and treated with Brefeldin A in the fourth hour, and a 10-colored monoclonal antibody panel was evaluated at the end of the sixth hour using flow cytometry. According to our findings, the numbers of Th22 (CD3+, CD4+, and interleukin [IL]-22+) and Tc22 (CD3+, CD8+, IL-22+) cells increased in adult patients regardless of the level of pneumonia (mild, severe, or symptom-free) as compared with healthy controls (p < 0.05). In addition, the number of Tc17 (CD3+, CD8+, and IL-17A+) cells increased in low pneumonia and severe pneumonia groups compared with the healthy controls (p < 0.05). Both IL-22 and IL-17A production decreased during a follow-up within 6 weeks of discharge. Our findings suggest that the increase in only IL-22 expressed Tc22 cells in the 0-12 age group with a general symptom-free course and higher levels of Th22 and Tc22 in uncomplicated adult cases may indicate the protective effect of IL-22. On the contrary, the association between the severity of pneumonia and the elevation of Tc17 cells in adults may reveal the damaging effect of IL-22 when it is co-expressed with IL-17.

SARS-CoV-2 variants of concern display enhanced intrinsic pathogenic properties and expanded organ tropism in mouse models.

SARS-CoV-2 variants of concern (VOCs) display enhanced transmissibility and resistance to antibody neutralization. Comparing the early 2020 isolate EU-1 to the VOCs Alpha, Beta, and Gamma in mice transgenic for human ACE2 reveals that VOCs induce a broadened scope of symptoms, expand systemic infection to the gastrointestinal tract, elicit the depletion of natural killer cells, and trigger variant-specific cytokine production patterns. Gamma infections result in accelerated disease progression associated with increased immune activation and inflammation. All four SARS-CoV-2 variants induce pDC depletion in the lungs, paralleled by reduced interferon responses. Remarkably, VOCs also use the murine ACE2 receptor for infection to replicate in the lungs of wild-type animals, which induce cellular and innate immune responses that apparently curtail the spread of overt disease. VOCs thus display distinct intrinsic pathogenic properties with broadened tissue and host range. The enhanced pathogenicity of VOCs and their potential for reverse zoonotic transmission pose challenges to clinical and pandemic management.

Quantitative contributions of TNF receptor superfamily members to CD8+ T-cell responses.

T‐cell responses to infections and cancers are regulated by co‐signalling receptors grouped into the binary categories of co‐stimulation or co‐inhibition. The co‐stimulation TNF receptor superfamily (TNFRSF) members 4‐1BB, CD27, GITR and OX40 have similar signalling mechanisms raising the question of whether they have similar impacts on T‐cell responses. Here, we screened for the quantitative impact of these TNFRSFs on primary human CD8+ T‐cell cytokine production. Although both 4‐1BB and CD27 increased production, only 4‐1BB was able to prolong the duration over which cytokine was produced, and both had only modest effects on antigen sensitivity. An operational model explained these different phenotypes using shared signalling based on the surface expression of 4‐1BB being regulated through signalling feedback. The model predicted and experiments confirmed that CD27 co‐stimulation increases 4‐1BB expression and subsequent 4‐1BB co‐stimulation. GITR and OX40 displayed only minor effects on their own but, like 4‐1BB, CD27 could enhance GITR expression and subsequent GITR co‐stimulation. Thus, different co‐stimulation receptors can have different quantitative effects allowing for synergy and fine‐tuning of T‐cell responses.

The infrapatellar fat pad produces interleukin-6-secreting T cells in response to a proteoglycan aggrecan peptide and provides dominant soluble mediators different from that present in synovial fluid.

The purpose of this study was to investigate the effects of osteoarthritis (OA) peripheral blood mononuclear cell (PBMC) -stimulating proteoglycan aggrecan peptides on T cells present in infrapatellar fat pads (IPFPs) and synovial tissues, and to correlate these findings with mediators present in synovial fluid of OA patients. We tested for interleukin-6 (IL-6) -producing T cells in IPFPs of patients with knee OA using ELISPOT. Cytokine and cytotoxic mediator production from OA PBMCs, IPFPs, synovial tissues, and synovial fluids in response to proteoglycan aggrecan peptides were quantified by cytometric bead array. Patterns of cytokine and cytotoxic mediator production were analyzed and compared. T cells from IPFPs elicited strong responses towards the p263-280 peptide by secreting IL-6. In addition, there was a trend that the p263-280 peptide stimulated higher production of cytokines/cytotoxic mediators than other proteoglycan aggrecan peptides, although this was not statistically significant. In patients with knee OA, a group of cytotoxic mediators (sFas, perforin, granzyme A, and granulysin) and IL-6 were detectable at high levels from the synovial fluid. In addition, inflammation in patients with knee OA was more pronounced in joint-surrounding tissues than levels in circulating peripheral blood. Our data suggest that T cells responding to the p263-280 peptide contribute to the secretion of various soluble mediators that are found within the synovial fluid. We also identified potential new candidates that may serve as biomarkers of knee OA.

Development of a dried pre-formulated antibody panel (7 colors/17antibodies) for the identification of human innate lymphoid cells by flow cytometry

Abstract Innate lymphoid cells (ILC) play an important role in innate immunity, lymphoid organogenesis and tissue remodeling. They form a heterogeneous population that can be categorized into 3 groups (i.e. ILC1, ILC2 and ILC3) based on their phenotype and/or distinct patterns of cytokine production. The identification and enumeration of different blood ILC subsets is important to understand immune regulation in disease models such as allergy, cancer and inflammation. Flow Cytometry is the method of choice for analysis of ILCs in human blood. However, as ILC frequencies may range below 0.1% of white blood cells, considerably elevated sample volumes and/or cell-enrichment is required. Furthermore, due to the lack of specific markers for definition of ILCs, at least 15 antibodies are required for mere identification of ILCs, including a lineage-exclusion channel to exclude most of the leukocytes. Based on current knowledge in human ILC biology, we have composed a respective set of markers to design a dry unitized 17 antibodies/7-color panel (CD294/Lineage/CD117/Nkp46/CD127/CD161/CD45) for flow cytometry. The panel in dry DURAClone format allows for the reliable identification of all ILCs subsets in whole blood or peripheral blood mononuclear cell fractions (PBMCs) in concordance with current scientific opinion. Due to the elimination of the pipetting of 17 antibodies, the method can substantially reduce variability and error related to sample preparation. Furthermore, the streamlined workflow can facilitate inter-laboratory transfer of the assays in multi-site studies.

Mechanisms That Underly T Cell Immunity in Graves' Orbitopathy.

Graves’ orbitopathy (GO), also known as thyroid-associated ophthalmopathy, is the most common ocular abnormality of Graves’ disease. It is a disfiguring, invalidating, and potentially blinding orbital disease mediated by an interlocking and complicated immune network. Self-reactive T cells directly against thyroid-stimulating hormone receptor-bearing orbital fibroblasts contribute to autoimmune inflammation and tissue remodeling in GO orbital connective tissues. To date, T helper (Th) 1 (cytotoxic leaning) and Th2 (antibody leaning) cell subsets and an emerging role of Th17 (fibrotic leaning) cells have been implicated in GO pathogenesis. The potential feedback loops between orbital native residential CD34- fibroblasts, CD34+ infiltrating fibrocytes, and effector T cells may affect the T cell subset bias and the skewed pattern of cytokine production in the orbit, thereby determining the outcomes of GO autoimmune reactions. Characterization of the T cell subsets that drive GO and the cytokines they express may significantly advance our understanding of orbital autoimmunity and the development of promising therapeutic strategies against pathological T cells.

Skewed intracellular cytokine production of iNKT cells toward Th2-related responses in alloimmunized thalassemia patients

IntroductionRed blood cell alloimmunization is a challenging issue in thalassemia patients. Several studies have investigated the role of different immune system compartment in alloimmunization, but the exact mechanism remains unclear. Considering the immunoregulatory function of iNKT cells and their subsets, in this study, we evaluated the possible role of these cells in alloimmunization status of thalassemia patients. Methods78 β-thalassemia major patients (41 alloimmunized and 37 non-alloimmunized) and 17 healthy controls were engaged in this study. Mononuclear cells were isolated from peripheral blood samples and stimulated for cytokine production. Samples were subjected to flow cytometry for enumeration of iNKT cells and characterized based on their cytokine production pattern. Finally, the results correlated with alloimmunization status, clinical and laboratory data. ResultsResults demonstrated that the number of iNKT, iNKT+IFN-ɤ+, and iNKT+IL-4+ cells in thalassemia group was significantly higher than healthy controls while no significant change was observed in the number of these cells between alloimmunized and non-alloimmunized thalassemia patients. Interestingly, the ratio of iNKT+IL-4+: iNKT+IFN-γ+ cells in alloimmunized thalassemia group represent a considerable increase in comparison to both non-alloimmunized thalassemia group and healthy controls. However, evaluating this value in non-alloimmunized group represents an approximately equal ratio of 0.94, which was almost similar to this ratio in the control group (0.99).Conclusion.Our results illustrated a noteworthy imbalance in the ratio of iNKT cell subsets in favour of IL-4 producing iNKT cells in alloimmunized thalassemia patients. Regarding the role of IL-4 in stimulating the Th2-related immune responses, this imbalance could consider as a possible mechanism in alloantibody responses of thalassemia patients.

Blood Neutrophils from Children with COVID-19 Exhibit Both Inflammatory and Anti-Inflammatory Markers

Background: Perhaps reflecting that children with COVID-19 rarely exhibit severe respiratory symptoms and often remain asymptomatic, little attention has been paid to explore the immune response in pediatric COVID-19. Here, we analyzed the phenotype and function of circulating neutrophils from children with COVID-19.Methods: One seventy-four children with COVID-19, 21 children with multisystem inflammatory syndrome (MIS-C), and 40 healthy children were studied. Neutrophil phenotype was analyzed by flow cytometry in blood samples. Cytokine production and plasma levels of IgG antibodies directed to the spike protein of SARS-CoV-2 were measured by ELISA. Cell-free DNA was quantified by fluorometry.Findings: Compared with healthy controls, neutrophils from children with COVID-19 showed a lower expression of CD11b, CD66b, and L-selectin but a higher expression of the activation markers HLA-DR, CD64 and PECAM-1 and the inhibitory receptors LAIR-1 and PD-L1. No differences in the pattern of cytokine production and NETs were observed when neutrophils from healthy children were compared with neutrophils from children with COVID-19. Interestingly, the expression of CD64 not only distinguished asymptomatic from mild and moderate COVID-19, but also positively correlated with the serum concentration of IgG antibodies directed to the spike protein of SARS-CoV-2.Interpretation: Acute lung injury is a prominent feature of severe COVID-19 in adults. A low expression of adhesion molecules together with a high expression of inhibitory receptors in neutrophils from children with COVID-19 might prevent tissue infiltration by neutrophils preserving lung function.Funding: FONCyT- ANPCyT, UBA, Argentina.Declaration of Interests: The authors have declared that no conflict of interest exists.Ethics Approval Statement: The Institutional Review Board at institutions participants reviewed and approved the sample collection and overall study.

Kinetics of Phenotypic and Functional Changes in Mouse Models of Sponge Implants: Rational Selection to Optimize Protocols for Specific Biomolecules Screening Purposes.

The sponge implant has been applied as an important in vivo model for the study of inflammatory processes as it induces the migration, proliferation, and accumulation of inflammatory cells, angiogenesis, and extracellular matrix deposition in its trabeculae. The characterization of immune events in sponge implants would be useful in identifying the immunological events that could support the selection of an appropriate experimental model (mouse strain) and time post-implant analysis in optimized protocols for novel applications of this model such as in biomolecules screening. Here, the changes in histological/morphometric, immunophenotypic and functional features of infiltrating leukocytes (LEU) were assessed in sponge implants for Swiss, BALB/c, and C57BL/6 mice. A gradual increase of fibrovascular stroma and a progressive decrease in LEU infiltration, mainly composed of polymorphonuclear cells with progressive shift toward mononuclear cells at late time-points were observed over time. Usually, Swiss mice presented a more prominent immune response with late mixed pattern (pro-inflammatory/anti-inflammatory: IL-2/IFN-γ/IL-4/IL-10/IL-17) of cytokine production. While BALB/c mice showed an early activation of the innate response with a controlled cytokine profile (low inflammatory potential), C57BL/6 mice presented a typical early pro-inflammatory (IL-6/TNF/IFN-γ) response with persistent neutrophilic involvement. A rational selection of the ideal time-point/mouse-lineage would avoid bias or tendentious results. Criteria such as low number of increased biomarkers, no recruitment of cytotoxic response, minor cytokine production, and lower biomarker connectivity (described as biomarker signature analysis and network analysis) guided the choice of the best time-point for each model (Day5/Swiss; Day7/BALB/c; Day6/C57BL/6) with wide application for screening purposes, such as identification of therapeutic biomolecules, selection of antigens/adjuvants, and follow-up of innate and adaptive immune response to vaccines candidates.

Effect of Farming on Asthma.

In this review, we discuss an immunobiology model of farm exposure towards the protective effect of asthma. Unraveling the protective effect of farming exposure could help develop novel strategies to prevent asthma. Asthma is a chronic airway inflammation that causes coughing, wheezing, chest tightness or shortness of breath. The reasons for the increase in the prevalence of asthma worldwide is still unclear but has been hypothesized to be attributable to westernization/urbanization of rural regions thus resulting in the loss of rural farming environmental. In this review we discuss the effect of the environmental factors, specifically farming, on the risk of asthma in children. Here, we will summarize the main findings of 27 studies related to 11 different cohorts. Several studies have shown preventive effect of traditional farming on the prevalence and incidence of asthma in childhood. Furthermore, consumption of unpasteurized cow's milk, exposure to farm animals as well as fodder have been shown to have a protective effect on asthma. The precise mechanism of the protective effect is still unclear. There are assumptions, that maternal/childhood exposures to farm animals result in higher microbial exposures through which the protective effect might be mediated. Also, consumption of unpasteurized milk (when consumed during pregnancy by mother or early childhood by children) can modulate cytokine production patterns which could be responsible for the observed protective effect. CONCLUSION: This review provides evidence of the protective effect of farming environment i.e., exposure to farm animals, their fodder as well as consumption of unpasteurized cow's milk suggesting that novel strategies could be developed to prevent asthma.

In vitro effects of azide-containing human CRP isoforms and oxLDL on U937-derived macrophage production of atherosclerosis-related cytokines.

Atherosclerosis is an inflammatory chronic disease of the arterial wall. Monomeric (m) and pentameric (p) C-reactive protein (CRP) and oxidized low density lipoproteins (oxLDL) seem to affect the pattern of cytokine production by macrophages, thus playing an important role in atherogenesis. Azide, the commercial preservative of CRP, may influence its action in vitro. The present study aimed to determine the effects of both isoforms of azide-containing CRP (mCRP and pCRP) with and without oxLDL on cytokine production by U937-derived macrophages. U937 monocytes were cultured and differentiated into macrophages and treated with mCRP, pCRP, oxLDL and azide individually and in combination. ELISA were performed to measure the levels of interferon-γ (IFN-γ), interleukin (IL)-4, IL-6, IL-10 and tumor necrosis factor (TNF)-α in culture supernatants collected from U937-derived macrophages following their respective treatments. Most single and combined treatments, especially in triple combination, were able to downregulate the levels of IFN-γ and IL-6 compared with control untreated cells, whilst the combination of mCRP and pCRP increased IL-4 levels. Regarding IL-10, except for an increase induced by mCRP, no significant effect was caused by any treatment compared with the control. On the other hand, the levels of TNF-α were not significantly affected by any treatment except for a decreasing trend that was observed with mCRP/oxLDL treatment compared with control. By contrast, double azide caused a significant decrease in the levels of IFN-γ and IL-6. The results of the present study indicated that mCRP, pCRP, oxLD and possibly azide, individually or in different combinations, had the tendency to upregulate the expression of IL-4 and to downregulate that of the pro-atherogenic cytokines, IFN-γ and IL-6, suggesting that the intima microenvironment serves a crucial role in atherogenesis.