Intracellular IL-10 Research Articles

MicroRNA-27a Suppresses the Toxic Action of Mepivacaine on Breast Cancer Cells via Inositol-Requiring Enzyme 1-TNF Receptor-Associated Factor 2.

To investigate the toxic effects of microRNA-27a on breast cancer cells through inositol-acquiring enzyme 1-TNF receptor-associated factor 2 inhibition by mepivacaine. The elevation of miR-27a in MCF-7 of BCC lines was measured, and groups were set up as control, mepivacaine, and elevated groups. Cells from each group were examined for inflammatory progression. Elevated miR-27a in MCF-7 cells was able to distinctly augment the cell advancement (P < 0.01) and decline cell progression (P < 0.01). Meanwhile, miR-27a reduced the content of intracellular inflammatory factors IL-1β (P < 0.01) and IL-6 (P < 0.01), elevated the content of IL-10 (P < 0.01), suppressed levels of cleaved-caspase-3 and p-signal transducer and activator of transcription-3 (STAT3) (P < 0.01), and increased Bcl-2/Bax (P < 0.01). Elevated miR-27a in MCF-7 of BCC lineage was effective in reducing the toxic effects of mepivacaine on cells and enhancing cell progression. This mechanism is thought to be related to the activation of the IRE1-TRAF2 signaling pathway in BCC. The findings may provide a theoretical basis for targeted treatment of BC in clinical practice.

Amaryllidaceae alkaloids in skin cancer management: Photoprotective effect on human keratinocytes and anti-proliferative activity in melanoma cells.

Skin cancer has high rates of mortality and therapeutic failure. In this study, to develop a multi-agent strategy for skin cancer management, the selective cytotoxicity of several alkaloid fractions and pure alkaloids isolated from Amaryllidaceae species was evaluated in melanoma cells. In addition, UVB-stimulated keratinocytes (HaCaT) were exposed to seven alkaloid fractions characterized by GC-MS, and the production of intracellular reactive oxygen species (ROS) and IL-6, were measured to evaluate their photoprotection effects. The Eucharis caucana (bulb) alkaloid fraction (20 μg/ml) had a clear effect on the viability of melanoma cells, reducing it by 45.7% without affecting healthy keratinocytes. This alkaloid fraction and tazettine (both at 2.5 μg/ml) suppressed UVB-induced ROS production by 31.6% and 29.4%, respectively. The highest anti-inflammatory potential was shown by the Zephyranthes carinata (bulb) alkaloid fraction (10 μg/ml), which reduced IL-6 production by 90.8%. According to the chemometric analysis, lycoramine and tazettine had a photoprotective effect on the UVB-exposed HaCaT cells, attenuating the production of ROS and IL-6. These results suggest that Amaryllidaceae alkaloids have photoprotective and therapeutic potential in skin cancer management, especially at low concentrations.

Methylprednisolone Promotes Mycobacterium smegmatis Survival in Macrophages through NF-κB/DUSP1 Pathway

Mycobacterium tuberculosis (M. tuberculosis) is the causative agent of tuberculosis. As an important component of host immunity, macrophages are not only the first line of defense against M. tuberculosis but also the parasitic site of M. tuberculosis in the host. Glucocorticoids can cause immunosuppression, which is considered to be one of the major risk factors for active tuberculosis, but the mechanism is unclear. To study the effect of methylprednisolone on the proliferation of mycobacteria in macrophages and try to find key molecules of this phenomenon. The macrophage line RAW264.7 infected by M. smegmatis was treated with methylprednisolone, and the intracellular bacterial CFU, Reactive Oxygen Species (ROS), cytokine secretion, autophagy, and apoptosis were measured. After the cells were treated with NF-κB inhibitor BAY 11-7082 and DUSP1 inhibitor BCI, respectively, the intracellular bacterial CFU, ROS, IL-6, and TNF-α secretion were detected. After treatment with methylprednisolone, the CFU of intracellular bacteria increased, the level of ROS decreased, and the secretion of IL-6 and TNF-α decreased in infected macrophages. After BAY 11-7082 treatment, the CFU of M. smegmatis in macrophages increased, and the level of ROS production and the secretion of IL-6 by macrophages decreased. Transcriptome high-throughput sequencing and bioinformatics analysis suggested that DUSP1 was the key molecule in the above phenomenon. Western blot analysis confirmed that the expression level of DUSP1 was increased in the infected macrophages treated with methylprednisolone and BAY 11-7082, respectively. After BCI treatment, the level of ROS produced by infected macrophages increased, and the secretion of IL-6 increased. After the treatment of BCI combined with methylprednisolone or BAY 11-7082, the level of ROS produced and the secretion of IL-6 by macrophages were increased. methylprednisolone promotes the proliferation of mycobacteria in macrophages by suppressing cellular ROS production and IL-6 secretion through down-regulating NF-κB and up-regulating DUSP1 expression. BCI, an inhibitor of DUSP1, can reduce the level of DUSP1 in the infected macrophages and inhibit the proliferation of intracellular mycobacteria by promoting cellular ROS production and IL-6 secretion. Therefore, BCI may become a new molecule for host-directed therapy of tuberculosis, as well as a new strategy for the prevention of tuberculosis when treated with glucocorticoids.

Peripheral Blood Mononuclear Cells from Patients with Type 1 Diabetes and Diabetic Retinopathy Produce Higher Levels of IL-17A, IL-10 and IL-6 and Lower Levels of IFN-γ-A Pilot Study.

Inflammation is key to the pathogenesis of diabetic retinopathy (DR). This prospective study investigated alterations in inflammatory cytokines in peripheral blood mononuclear cells (PBMCs) in 41 people with type 1 diabetes (T1D), sub-grouped into mild non-proliferative DR (mNPDR; n = 13) and active and inactive (each n = 14) PDR. Age/gender-matched healthy controls (n = 13) were included. PBMCs were isolated from blood samples. Intracellular cytokine expression by PBMCs after 16-h stimulation (either E. coli lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate plus ionomycin, D-glucose or D-mannitol) were assessed by flow cytometry. Cytokine production in plasma, non-stimulated and LPS-stimulated PBMC supernatant was also assessed. Increased BMC IL-10 secretion and reduced expression of IL-6 and IFN-γ in CD3+ cells were observed in mNPDR. Reduced IL-6 and IL-10 secretion, and higher levels of intracellular IL-6 expression, especially in CD11b+ PBMCs, was detected in aPDR; levels were positively correlated with DR duration. Patients with T1D demonstrated increased intracellular expression of IL-17A in myeloid cells and reduced IFN-γ expression in CD3+ cells. Plasma levels of IL-1R1 were increased in mNPDR compared with controls. Results suggest that elevated PBMC-released IL-10, IL-6, in particular myeloid-produced IL-17A, may be involved in early stages of DR. IL-6-producing myeloid cells may play a role in PDR development.

Intracellular IFN-γ and IL-4 levels of CD4 + and CD8 + T cells in the peripheral blood of naturally infected (Leishmania infantum) symptomatic dogs before and following a 4-week treatment with miltefosine and allopurinol: a double-blinded, controlled and cross-sectional study

BackgroundCanine leishmaniosis (CanL) is a systemic disease caused by the protozoan parasite Leishmania infantum with a wide spectrum of clinical signs, with cutaneous, ocular, renal and lymphoreactive conditions prevailing in the clinical setting. The immune system plays a pivotal role in the evolution of Leishmania infection and its response to antileishmanial treatment. Cytokines are important immune response mediators that are released by activated lymphocytes and less so by other immunocytes. In dogs with leishmaniosis, IFN-γ and IL-4 have been recognized as the main activators of cellular and humoral immunity, respectively. The objective of this study was to investigate intracellular IL-4 and IFN-γ expression by CD4 + and CD8 + lymphocytes in the peripheral blood of symptomatic dogs before and after combined antileishmanial treatment with miltefosine and allopurinol.ResultsPostantileishmanial treatment CD4 + IL-4 + and CD8 + IL-4 + cell counts were significantly decreased, although no similar changes were observed in the comparisons made between the pre- and posttreatment CD4 + IFN-γ + and CD8 + IFN-γ + counts and ratios.ConclusionThe findings indicate that IL-4 production by T cells may facilitate the symptomatic phase of CanL, whereas IFN-γ production by CD4 + and CD8 + cells may indicate its negligible role in the evolution of natural CanL and perhaps the equivocal positive influence of antileishmanial treatment.

Staining of activated ß2-integrins in combination with CD137 and CD154 for sensitive identification of functional antigen-specific CD4+ and CD8+ T cells.

Common flow cytometry-based methods used for functional assessment of antigen-specific T cells rely on de novo expression of intracellular cytokines or cell surface activation induced markers. They come with some limitations such as complex experimental setting, loss of cell viability and often high unspecific background which impairs assay sensitivity. We have previously shown that staining of activated ß2-integrins either with multimers of their ligand ICAM-1 or with a monoclonal antibody can serve as a functional marker detectable on T cells after minutes (CD8+) or few hours (CD4+) of activation. Here, we present a simple method for detection of activated ß2-integrins in combination with established cell surface activation induced markers. We observed that activated ß2-integrins were still detectable after 14 hours of stimulation, allowing their detection together with CD137 and CD154. Combinatorial gating of cells expressing activated ß2-integrins and CD137 or CD154 reduced background in unstimulated samples, increasing the signal-to-noise ratio and allowing improved assessment of low-frequency T cell responses. Extracellular staining of these markers highly correlated with production of intracellular cytokines IL-2, TNF or IFNγ in CD4+ and CD8+ T cells. As an exemplary application, SARS-CoV-2 spike-specific T cell responses were assessed in individuals after COVID-19 vaccination. This method should be useful for epitope discovery projects and for the simultaneous monitoring of low-frequency antigen-specific CD4+ and CD8+ T cell responses in various physiological situations.

Effect of parasitic infection on muscular function of dystrophin gene (Dmd) deficient mouse.

Previous studies have reported many cases of Trichinella spiralis (T. spiralis) infection in normal skeletal muscle but there is little research on T. spiralis infection in abnormal muscle tissue. To identify the effect of T. spiralis infection on muscular dystrophy, this study compared aspects of infection between normal (C57BL/10) and dystrophin-deficient Duchenne muscular dystrophy (DMD) mdx mice. Infection rate was found to be lower in mdx mice than in C57BL/10 mice at early stages of infection; however, infection and inflammation in mdx mice persisted at later stages of infection while the infection rate and inflammation in C57BL/10 mice decreased gradually. The inflammation area was proportional to the degree of infection in both groups. Muscle strength was measured by the time of latency to fall in the wire-hanging test. Hanging time was shorter in the infected group than in the uninfected group in both C57BL/10 and mdx mice. Muscle strength was also reduced in mdx mice compared with C57BL/10 mice in both the un-infected and infected groups. The muscle intracellular cytokines TGF-β and IL-6 were continuously expressed from early stage to late-stage infection. IL-10 was strongly expressed at the early stage of infection but decreased as the infection progressed. TNF-α expression remained stable from early to late-stage infection in mdx mice, while TNF-α was elevated only during early-stage infection in C57BL/10 mice. The degree of muscle damage was significantly higher in mdx mice than in C57BL/10 mice because of the high level of serum creatine kinase (CK). These results suggest that mdx mice continued in infection and inflammation until the late stages of disease, which was in contrast to the C57BL/10 mice that recovered to some extent in the late stage of infection. In addition, that dystrophin-deficient mice are not suitable for T. spiralis infection compared to normal mice, and the degree of inflammation may be worse in mdx mice.

Multiple sclerosis-disease modifying therapies affect humoral and T-cell response to mRNA COVID-19 vaccine.

The mRNA vaccines help protect from COVID-19 severity, however multiple sclerosis (MS) disease modifying therapies (DMTs) might affect the development of humoral and T-cell specific response to vaccination. The aim of the study was to evaluate humoral and specific T-cell response, as well as B-cell activation and survival factors, in people with MS (pwMS) under DMTs before (T0) and after two months (T1) from the third dose of vaccine, comparing the obtained findings to healthy donors (HD). All possible combinations of intracellular IFNγ, IL2 and TNFα T-cell production were evaluated, and T-cells were labelled "responding T-cells", those cells that produced at least one of the three cytokines of interest, and "triple positive T-cells", those cells that produced simultaneously all the three cytokines. The cross-sectional evaluation showed no significant differences in anti-S antibody titers between pwMS and HD at both time-points. In pwMS, lower percentages of responding T-cells at T0 (CD4: p=0.0165; CD8: p=0.0022) and triple positive T-cells at both time-points compared to HD were observed (at T0, CD4: p=0.0007 and CD8: p=0.0703; at T1, CD4: p=0.0422 and CD8: p=0.0535). At T0, pwMS showed higher plasma levels of APRIL, BAFF and CD40L compared to HD (p<0.0001, p<0.0001 and p<0.0001, respectively) and at T1, plasma levels of BAFF were still higher in pwMS compared to HD (p=0.0022).According to DMTs, at both T0 and T1, lower anti-S antibody titers in the depleting/sequestering-out compared to the enriching-in pwMS subgroup were found (p=0.0410 and p=0.0047, respectively) as well as lower percentages of responding CD4+ T-cells (CD4: p=0.0394 and p=0.0004, respectively). Moreover, the depleting/sequestering-out subgroup showed higher percentages of IFNγ-IL2-TNFα+ T-cells at both time-points, compared to the enriching-in subgroup in which a more heterogeneous cytokine profile was observed (at T0 CD4: p=0.0187; at T0 and T1 CD8: p =0.0007 and p =0.0077, respectively). In pwMS, humoral and T-cell response to vaccination seems to be influenced by the different DMTs. pwMS under depleting/sequestering-out treatment can mount cellular responses even in the presence of a low positive humoral response, although the cellular response seems qualitatively inferior compared to HD. An understanding of T-cell quality dynamic is needed to determine the best vaccination strategy and in general the capability of immune response in pwMS under different DMT.

The immunoregulatory role of IL-35 in patients with interstitial lung disease.

Pulmonary fibrosis involves various types of immune cells and soluble mediators, including TGF-β and IL-35, a recently identified heterodimeric cytokine that belongs to the IL-12 cytokine family. However, the effect of regulatory IL-35 may play an important role in fibrotic diseases. The aim of this paper is to explore the immunoregulatory role of IL-35 in the development of fibrosis in interstitial lung disease (ILD). To gain a better understanding of this issue, the concentrations of IL-35 and different profibrotic cytokines in fibrotic (F-ILD) and non-fibrotic (NF-ILD) patients by ELISA were compared to that of intracellular IL-35 and IL-17 on CD4+ T cells stimulated in the presence of BAL or with different ratios of recombinant IL-35 (rIL-35) and TGF-β (rTGF-β), which were evaluated by flow cytometry. We observed that BAL concentration of IL-35 was lower in F patients (p < 0.001) and was negatively correlated with concentrations of TGF-β (p < 0.001) and IL-17 (p < 0.001). In supplemented cell cultures, BAL from NF but not F patients enhanced the percentage of IL-35 + CD4+ T (p < 0.001) cells and decreased the percentage of IL-17 + CD4+ T cells (p < 0.001). The percentage of IL-35 + CD4+ T cells correlated positively with BAL concentration of IL-35 (p=0.02), but correlated negatively with BAL concentrations of IL-17 (p=0.007) and TGF-β (p=0.01). After adjusting the concentrations of recombinant cytokines to establish a TGF-β: IL-35 ratio of 1:4, an enhanced percentage of IL-35 + CD4+ T cells (p < 0.001) but a decreased percentage of IL-17 + CD4+ T cells (p < 0.001) was observed. After adding recombinant IL-35 to the BAL from F patients until a 1:4 ratio of TGF-β: IL-35 was reached, a significantly increased percentage of IL-35 + CD4+ T cells (p < 0.001) and a decreased percentage of IL-17 + CD4+ T cells (p=0.003) was found. These results suggest that IL-35 may induce an anti-fibrotic response, regulating the effect of TGF-β and the inflammatory response on CD4+ T cells. In addition, the TGF-β: IL-35 ratio in BAL has been shown to be a potential biomarker to predict the outcome of F patients with ILD.

Ozone therapy restores immune dysfunction in refractory idiopathic granulomatous mastitis as a novel potential therapeutic approach.

Immunological dysfunction has been suggested to play a major role in the pathogenesis of idiopathic granulomatous mastitis (IGM). We recently showed that ozone therapy was effective in patients with steroid-resistant IGM. This study assessed alterations in intracellular cytokine expression patterns in different T-lymphocyte subsets after ozone therapy in refractory IGM. Peripheral blood T lymphocyte subsets (CD8+ , CD4+ , CD4+ CD25+ CD127- ) were analyzed via flow-cytometry for intracellular cytokine expressions IFN-γ, TNF-α, IL-10, and TGF-β before and after completion of 4-month systemic ozone therapy.Ozone therapy significantly increased the CD4+ IFN-γ+ (p = 0.032), CD4+ TNF-α+ (p = 0.028), and the CD8+ TNF-α+ (p = 0.012) T cells. In contrast, significant decreases in CD4+ IL-10+ (p = 0.047) and CD8+ IL-10+ T cells (p = 0.022) and CD4+ CD25+ CD127-//low Treg cells secreting TGF-β (p = 0.005) were found after ozone therapy. When patients were analyzed according to the response to ozone therapy, patients with a complete remission were more likely to have increased CD3- CD16+ CD56+ natural killer cells (p = 0.0027) and decreased CD19+ B lymphocytes (p = 0.046) following ozone therapy. Our results suggest that ozone therapy stimulated a T-helper-1 response associated with IFN-γ production and downregulation of TGF-β expression in CD4+ CD25+ CD127- Treg cells. These alterations in the immune system following ozone therapy can improve wound healing and restore immune dysfunction in patients with refractory IGM.

Immune modulation as a consequence of SARS-CoV-2 infection.

Erroneous immune responses in COVID-19 could have detrimental effects, which makes investigation of immune network underlying COVID-19 pathogenesis a requisite. This study aimed to investigate COVID-19 related alterations within the frame of innate and adaptive immunity. Thirty-four patients clinically diagnosed with mild, moderate and severe COVID-19 disease were enrolled in this study. Decreased ILC1 and increased ILC2 subsets were detected in mild and moderate patients compared to healthy controls. NK cell subsets and cytotoxic capacity of NK cells were decreased in severe patients. Moreover, CD3+ T cells were reduced in severe patients and a negative correlation was found between CD3+ T cells and D-dimer levels. Likewise, moderate and severe patients showed diminished CD3+CD8+ T cells. Unlike T and NK cells, plasmablast and plasma cells were elevated in patients and IgG and IgA levels were particularly increased in severe patients. Severe patients also showed elevated serum levels of pro-inflammatory cytokines such as TNF-α, IL-6 and IL-8, reduced intracellular IFN-γ and increased intracellular IL-10 levels. Our findings emphasize that SARS-CoV-2 infection significantly alters immune responses and innate and acquired immunity are differentially modulated in line with the clinical severity of the disease. Elevation of IL-10 levels in NK cells and reduction of CD3+ and CD8+ T cells in severe patients might be considered as a protective response against the harmful effect of cytokine storm seen in COVID-19.

PPAR-α Agonist Fenofibrate Ameliorates Sjögren Syndrome-Like Dacryoadenitis by Modulating Th1/Th17 and Treg Cell Responses in NOD Mice.

PurposeTo investigate the effects and mechanisms of fenofibrate, a synthetic ligand of peroxisome proliferator-activated receptor α (PPAR-α), on autoimmune dacryoadenitis in a mouse model of Sjögren syndrome (SS) dry eye.MethodsMale nonobese diabetic (NOD) mice were fed chow with or without 0.03% fenofibrate for 8 weeks, and clinical scores were determined by assessing tear secretion, fluorescein, and hematoxylin and eosin staining. Intracellular IFN-γ, IL-17, and Foxp3 in CD4+ T cells were measured by flow cytometry. The expressions of Th1, Th17, and Treg cell-related transcription factors and cytokines were detected by real-time PCR. The levels of PPAR-α and liver X receptor β (LXR-β) were detected with real-time PCR and Western blotting.ResultsFenofibrate efficiently diminished the lymphocytic inflammation in lacrimal glands (LGs), increased tear secretion, and decreased corneal fluorescein staining in NOD mice. Meanwhile, treatment of fenofibrate evidently reduced the proportion of Th1 and Th17 cells and increased the proportion of Treg cells in vivo and vitro, together with decreased expression of T-bet, IFN-γ, RORγt, and IL-17, as well as increased expression of Foxp3 and TGF-β1 in LGs. Furthermore, fenofibrate significantly upregulated the expressions of PPAR-α and LXR-β at the protein and mRNA levels.ConclusionsFenofibrate potently attenuated LG inflammation in a model of autoimmune dry eye, and this effect might partially result from regulating Th1/Th17/Treg cell responses by activating PPAR-α/LXR-β signaling. These data suggest that fenofibrate may be a novel class of therapeutic agent for SS-associated dacryoadenitis.

Dental follicle mesenchymal stem cells ameliorated glandular dysfunction in Sjögren's syndrome murine model.

Dental mesenchymal stem cells (MSCs) are potential for use in tissue regeneration in inflammatory diseases due to their rapid proliferating, multilineage differentiation, and strong anti-inflammatory features. In the present study, immunoregulatory and glandular tissue regeneration effects of the dental follicle (DF)MSCs in Sjögren's Syndrome (SS) were investigated. Dental follicle (DF) tissues were obtained from healthy individuals during tooth extraction, tissues were digested enzymatically and DFMSCs were cultured until the third passage. DFMSCs were labeled with Quantum dot 655 for cell tracking analysis. The induction of the SS mouse model was performed by the injection of Ro60-273-289 peptide intraperitoneally. DFMSCs were injected intraperitoneally, or into submandibular, or lacrimal glands. Splenocytes were analyzed for intracellular cytokine (IFN-γ, IL-17, IL-10) secretion in T helper cells, lymphocyte proliferation, and B lymphocyte subsets. Histologic analysis was done for submandibular and lacrimal glands with hematoxylin-eosin staining for morphologic examination. The systemic injection of DFMSCs significantly reduced intracellular IFN-γ and IL-17 secreting CD4+ T cells in splenocytes (p<0.05), and decreased inflammatory cell deposits and fibrosis in the glandular tissues. DFMSCs differentiated to glandular epithelial cells in submandibular and lacrimal injections with a significant reduction in lymphocytic foci. The results showed that few amounts of DFMSCs were deposited in glandular tissues when applied intraperitoneally, while high amounts of DFMSCs were located in glandular tissues and differentiated to glandular epithelial cells when applied locally in SS murine model. DFMSCs have the potential for the regulation of Th1, Th17, and Treg balance in SS, and ameliorate glandular dysfunction. DFMSCs can be a beneficial therapeutic application for SS.

The Evolution of Blood Cell Phenotypes, Intracellular and Plasma Cytokines and Morphological Changes in Critically Ill COVID-19 Patients.

Background: Severe coronavirus disease 2019 (COVID-19) causes a strong inflammatory response. To obtain an overview of inflammatory mediators and effector cells, we studied 25 intensive-care-unit patients during the timeframe after off-label chloroquine treatment and before an introduction of immunomodulatory drugs. Material and methods: Blood samples were weekly examined with flow cytometry (FCM) for surface and intracytoplasmic markers, cytokine assays were analyzed for circulating interleukins (ILs), and blood smears were evaluated for morphological changes. Samples from healthy volunteers were used for comparison. Organ function data and 30-day mortality were obtained from medical records. Results: Compared to that of the healthy control group, the expression levels of leukocyte surface markers, i.e., the cluster of differentiation (CD) markers CD2, CD4, CD8, CD158d, CD25, CD127, and CD19, were lower (p < 0.001), while those of leukocytes expressing CD33 were increased (p < 0.05). An aberrant expression of CD158d on granulocytes was found on parts of the granulocyte population. The expression levels of intracellular tumor necrosis factor alpha (TNFα) and IL-1 receptor type 2 in leukocytes were higher (p < 0.001) as well as plasma levels of TNFα, IL-2, IL-6, IL-8, IL-10 (p < 0.001), interferon gamma (IFNγ) (p < 0.01), and granulocyte-macrophage colony-stimulating factor (GM-CSF) (p < 0.05). The expression levels of CD33+ leukocytes and circulating IL-6 were higher (p < 0.05) among patients with arterial oxygen partial pressure-to-fractional inspired oxygen (PaO2/FiO2) ratios below 13.3 kPa compared to in the remaining patients. The expression levels of TNFα, IL-2, IL-4, IL-6, IL-8, and IL-10 were higher in patients treated with continuous renal replacement therapy (CRRT) (p < 0.05), and the levels of the maximum plasma creatinine and TNFα Spearman’s rank-order correlation coefficient (rho = 0.51, p < 0.05) and IL-8 (rho = 0.44, p < 0.05) correlated. Blood smears revealed neutrophil dysplasia with pseudo-Pelger forms being most common. Conclusion: These findings suggest that patients with severe COVID-19, in addition to augmented ILs, lymphopenia, and increased granulocytes, also had effects on the bone marrow.

Hepatitis B Virus-Specific Cellular Immunity Contributes to the Outcome of Occult Hepatitis B Virus Infection.

There is little known of immunologic factors leading to the occurrence of occult HBV infection (OBI). Specific cellular immune response to hepatitis B virus (HBV) core/pol peptides was compared between blood donor populations, including 37 OBIs, 53 chronic HBV infections (CHB), 47 resolved infections, and 56 non-infected controls, respectively. The rate of CD4+/CD8+ T cell proliferation in OBI or CHB carriers was higher than in HBV resolved and non-infected individuals (P < 0.05). The intensity of IFN-γ-secretion T-cell response of OBI carriers was highest, followed by CHB and resolved infections, and non-infected individuals (P < 0.05). The frequency of intracellular IFN-γ and IL-17A CD4+/CD8+ and IL-21 CD4+ T-cell responses was significantly higher in resolved infections than in OBI or CHB carriers (P < 0.05), while the level of extracellular IL-17A of peripheral blood mononuclear cells (PBMCs) was higher in OBI and CHB carriers than in resolved infections (P < 0.01). The frequency of intracellular IL-10 CD4+ T-cell response in CHB, OBI, and resolved infections was higher than in HBV non-infected individuals (P < 0.01). Intracellular IL-10 CD8+ T cell and extracellular IL-10 T-cell responses were higher in CHB than in OBI (P = 0.012) or HBV resolved infections (P < 0.01). In conclusion, the higher level of effective T-cell response with IFN-γ, IL-17A, and IL-21 contributes to resolved infection outcome, while higher levels of suppressive T-cell response with IL-10 result in HBV chronicity. OBI is an intermediary status between HBV resolved and chronic infections, in which IL-21 effector and IL-10 suppressor T-cell responses play an important role in directing the outcome of HBV infection.

Functionality of natural killer cells in obese asthma phenotypes.

Obesity-associated asthma (OA) is a difficult to treat asthma phenotype due to its severity and poor response to inhaled steroids. Early-onset allergic (EoOA) and late-onset non-allergic (LoOA) OA are suggested subtypes of this phenotype. Natural Killer (NK) cells are key elements of innate immunity involved in cytotoxicity and immune regulation, with uncertain role in OA pathogenesis. Early-onset allergic and LoOA patients together with obese non-asthmatic (ONA) controls have been enrolled in the study. Peripheral blood samples have been collected for analysis. Percentages of total NK cells, CD3- CD56dim and CD3- CD56bright NK cell subsets, cytotoxic activity, intracellular interferon-γ, interleukin (IL)-10, IL-13, IL-17secretion and activatory receptors (NKG2D, NKp46i and NKp44) have been investigated by flow cytometry. The effect of IL-12 and IL-23stimulation on NK cells and intracellular cytokines in different groups have also been analysed and compared with unstimulated conditions. Results of ONA (n=5, age 42±8), EoOA (n=5, age 42±10) and LoOA (n=8, age 46±8) patients have analysed. Body Mass Index has been found to be negatively correlated with CD69(p=.022, r=-0.534). NKG2D receptor has been significantly low in CD56dim cellsof asthma population (p=.046). NKp44 receptor expression has increased after IL-12stimulation in EoOA and control group (p=.02). Intracellular IL-10 content has increased in LoOA and control subjects (p=.018, p=.03) but not in the EoOA group. Intracellular IL-17level has found be higher in allergic OA group. LoOA patients showed a decreased NK cytotoxicity compared with the early-onset asthma group (p=.05). Our study suggests an impaired NK receptor expression, activation and reduced cytotoxicity in OA patients together with variances between different subtypes of this phenotype. This data would be beneficial for tailoring a more personalized treatment strategy combatting steroid resistance and frequent exacerbations in this group of patients.

Alveolar macrophage-derived progranulin mediated pro-inflammatory Il-6 expression via regulating Creb1 in silicosis model

Progranulin (PGRN) is a secreted factor involved in inflammatory diseases. However, the function of PGRN in silica-induced lung inflammation has not been elucidated. In this study, we demonstrated that PGRN in serum and lung tissues was markedly increased in silicosis mouse model. And immunohistochemistry results showed that PGRN was mainly expressed in alveolar macrophages, which was further confirmed in silica-treated alvelar macrophages cell line (MH-S) in vitro. PGRN promoted pro-inflammatory cytokines transcription such as interleukin (Il)-6, tumor necrosis factor-α (Tnf-α) and Il-1β in MH-S cells, and the increasing of Il-6 was most obvious. Knockdown of PGRN blocked the silica-induced elevation of intracellular Il-6 in MH-S cells. Furthermore, we also found that PGRN could increase the phosphorylation of Cyclic AMP-responsive element-binding protein 1 (Creb1), a transcriptional regulator of Il-6. Inhibition of p-Creb1 by the phosphorylation inhibitor of Creb1 (666–15) decreased PGRN-induced intracellular Il-6 production in MH-S cells. In conclusion, PGRN was highly increased in silicosis mouse model and upregulated inflammatory cytokines expression. These findings suggested that PGRN might be a key mediator in silica-induced inflammation and provided a new clue for the diagnosis and drug therapy of silicosis.

Helios expressing regulatory T cells are correlated with decreased IL-2 producing CD8 T cells and antibody diversity in Mozambican individuals living chronically with HIV-1

BackgroundHuman immunodeficiency virus type 1 (HIV-1) causes impairment of T and B cell responses, which begins during the acute phase of infection and is not completely restored by antiretroviral treatment. Regulatory T cell (Tregs) can improve overall disease outcome by controlling chronic inflammation but may also suppress beneficial HIV-1 specific immune responses. We aimed to analyze the profile of Tregs and their correlation with the status of T cells activation, the expression of IL-2 and IFNγ and the profile of HIV-1 specific antibodies response in Mozambican people living chronically with HIV-1 (PLWH-C).ResultsIn PLWH-C, the proportion of total Tregs was positively correlated with the proportion of IL-2+CD4 T cells (r = 0.647; p = 0.032) and IL-2+IFNγ+CD8 T cells (r = 0.551; p = 0.014), while the proportions of Helios+Tregs correlated inversely with levels of IL-2+CD8 T cells (r = − 0.541; p = 0.017). Overall, PLWH-C, with (82%) or without virologic suppression (64%), were seronegative for at least HIV-1 p31, gp160 or p24, and the breadth of antibody responses was positively correlated with proportions of CD38+HLA-DR+CD8 T cells (r = 0.620; p = 0.012), viral load (r = 0.452; p = 0.040) and inversely with absolute CD4 T cells count (r = − 0.481; p = 0.027). Analysis of all individuals living HIV-1 showed that the breadth of HIV-1 antibody responses was inversely correlated with the proportion of Helios+Tregs (r = − 0.45; p = 0.02).ConclusionAmong Mozambican people living with HIV-1, seronegativity to some HIV-1 proteins is common, particularly in virologically suppressed individuals. Furthermore, lower diversity of HIV-specific antibodies is correlated to lower immune activation, lower viral replication and higher CD4 counts, in PLWH-C. Elevation in the proportion of Helios+Tregs is related to a reduction of CD8 T expressing intracellular IL-2, in PLWH-C, but may contribute to impairment of B cell function.

Histone Deacetylase Isoforms Differentially Modulate Inflammatory and Autoantibody Responses in a Mouse Model of Myasthenia Gravis.

Myasthenia gravis (MG) is an autoimmune disease characterized by chronic muscle fatigue and weakness caused by autoantibodies and complement-mediated damage at neuromuscular junctions. Histone deacetylases (HDACs) are crucial epigenetic regulators of proinflammatory gene expression; however, it is unclear whether HDACs modulate chronic inflammation or autoantibody production associated with MG pathogenesis. We examined expression profiles and serum levels of key inflammatory cytokines (IL-6 and IL-21) and acetylcholine receptor (AChR)-specific autoantibodies following pharmacological inhibition of key HDAC isoforms in a mouse model of MG. We found that HDAC inhibition significantly reduced the production of IL-6, but not IL-21, in AChR-stimulated PBMCs and splenocytes (n = 5 per group). Trichostatin (pan-HDAC inhibitor) treatment of MG-PBMCs (n = 2) also exhibited reduced production of induced IL-6. Although HDAC1 inhibition lowered IL-6 levels the most, HDAC2 inhibition depleted intracellular IL-6 and markedly reduced serum anti-AChR IgG2b in EAMG mice. The transcriptomic profiling and pathway mapping also revealed that autoimmunity-linked, major cell signaling pathways were differentially altered by HDAC1/2 inhibition. HDAC inhibition-mediated reduction in IL-6 and autoantibody levels also correlated with milder disease and preservation of muscle AChR in the treated mice. Overall, our findings revealed isoform-specific functional variance of HDACs in reducing inflammation and identified HDAC-regulated many genes underlying specific inflammatory and autoantibody pathways in EAMG. Thus, the study provides a rationale for further research to evaluate the HDACs or their gene targets as a potential adjunct treatment for MG.

Involvement of Peripheral Monocytes with IL-1β in the Pathogenesis of West Syndrome.

Neuroinflammation has been implicated in the pathogenesis of West syndrome (WS). Inflammatory cytokines, including interleukin-1β(IL-1β), have been reported to be associated with epilepsy. However, the assessment of cytokine changes in humans is not always simple or deterministic. This study aimed to elucidate the immunological mechanism of WS. We examined the intracellular cytokine profiles of peripheral blood cells collected from 13 patients with WS, using flow cytometry, and measured their serum cytokine levels. These were compared with those of 10 age-matched controls. We found that the WS group had significantly higher percentages of inter IL-1β, interleukin-1 receptor antagonist (IL-1RA)-positive monocytes, and interferon gamma (IFN-γ) in their CD8+ T cells than the control group. Interestingly, the group with sequelae revealed significantly lower levels of intracellular IFN-γ and IL-6 in their CD8+ T and CD4+ T cells, respectively, than the group without sequelae. There was no correlation between the ratios of positive cells and the serum levels of a particular cytokine in the WS patients. These cytokines in the peripheral immune cells might be involved in the neuroinflammation of WS, even in the absence of infectious or immune disease. Overall, an immunological approach using flow cytometry analysis might be useful for immunological studies of epilepsy.