In glomerular cells of puromycin aminonucleoside nephrosis rats both phosphorylated and total STAT3 levels increased during proteinuria

Recent studies showed that JAK/STAT pathway plays role in glomerular damages. The fact that STAT3 could be activated also by oxidative stress make Puromycin Aminonucleoside (PAN) Nephrosis model very appropriate for examination of STAT3 expression changes in glomerular pathology. Along with a control group, three PAN groups sacrificed on different days were formed by the i.p. injection of PAN for 5 consecutive days. Throughout the experiment, 24-hour-urines were collected on specific days and proteinuria levels were monitored. At the end of the experiments, tissue specimens were stained immunohistochemically for both total and phosphorylated STAT3 and evaluated subjectively. They were also examined ultrastructurally in transmission electron microscope. The proteinuria levels did not increase significantly on 5th day but showed a dramatic increase on 10th and 15th days. On 20th and 25th days, urinary protein levels gradually decreased. Ultrastructural examinations showed glomerular damages such as significant decrease in slit pore number, a significant gradual increase in glomerular basement membrane thickness and podocyte hypertrophy on 5th and 15th days; besides significant increase in mesangial matrix. The first significant increases in phosphorylated and total STAT3 levels occurred in 5th day and 15th day groups respectively. These increases diminished in 25th day group. Regarding all the findings, it was deduced that STAT3 is one of the active factors in glomerular pathologies.     

Short‐term cytokine stimulation reveals regulatory T cells with down‐regulated Foxp3 expression in human peripheral blood

The identification of regulatory T cells (Treg cells) in human peripheral blood is an important tool in diagnosis, research, and therapeutic intervention. As compared to lymphoid tissues, the frequencies of circulating Treg cells identified as CD4⁺CD25⁺Foxp3⁺ are, however, low. We here show that many of these cells remain undetected due to transient down regulation of Foxp3, which rapidly decays in the absence of cytokine‐mediated STAT5 signals. Short‐term incubation of PBMCs or isolated CD4⁺ T cells, but not of lymph node cells, with IL‐2, ‐7, or ‐15 more than doubles the frequency of Foxp3⁺CD25⁺ among CD4⁺ T cells detectable by flow cytometry. This increase is not due to cell division but to upregulation of both proteins. At the same time, the uncovered Treg cells up‐regulate CD25 and down‐regulate CD127, making them accessible to viable cell sorting. “Latent” Treg cells have a demethylated FOXP3 TSDR sequence, are enriched in naïve, non‐cycling cells, and are functional. The confirmation of our findings in RA and SLE patients shows the feasibility of uncovering latent Treg cells for immune monitoring in clinical settings. Finally, our results suggest that unmasking of latent Treg cells contributes to the increase in circulating CD4⁺CD25⁺Foxp3⁺ cells reported in IL‐2 treated patients.     

Bcl‐3 induced by IL‐22 via STAT3 activation acts as a potentiator of psoriasis‐related gene expression in epidermal keratinocytes

IL‐22 induces STAT3 phosphorylation and mediates psoriasis‐related gene expression. However, the signaling mechanism leading from pSTAT3 to the expression of these genes remains unclear. We focused on Bcl‐3, which is induced by STAT3 activation and mediates gene expression. In cultured human epidermal keratinocytes, IL‐22 increased Bcl‐3, which was translocated to the nucleus with p50 via STAT3 activation. The increases in CXCL8, S100As and human β‐defensin 2 mRNA expression caused by IL‐22 were abolished by siRNA against Bcl‐3. Although CCL20 expression was also augmented by IL‐22, the knockdown of Bcl‐3 increased its level. Moreover, the combination of IL‐22 and IL‐17A enhanced Bcl‐3 production, IL‐22‐induced gene expression, and the expression of other psoriasis‐related genes, including those encoding IL‐17C, IL‐19, and IL‐36γ. The expression of these genes (except for CCL20) was also suppressed by the knockdown of Bcl‐3. Bcl‐3 overexpression induced CXCL8 and HBD2 expression but not S100As expression. We also compared Bcl‐3 expression between psoriatic skin lesions and normal skin. Immunostaining revealed strong signals for Bcl‐3 and p50 in the nucleus of epidermal keratinocytes from psoriatic skin. The IL‐22‐STAT3‐Bcl‐3 pathway may be important in the pathogenesis of psoriasis.     

Metabolic regulation of NLRP3

A shift in our understanding of macrophage biology has come about as a result of recent discoveries in the area of metabolic reprogramming of macrophages. The NLRP3 inflammasome drives the activation of caspase-1, leading to the production of IL-1β, IL-18, and a type of cell death termed pyroptosis. The NLRP3 inflammasome has been shown to sense metabolites such as palmitate, uric acid, and cholesterol crystals and is inhibited by ketone bodies produced during metabolic flux. The NLRP3 inflammasome has also been shown to be regulated by mitochondrial reactive oxygen species and components of glycolysis, such as Hexokinase. Here, we review these findings and discuss their importance for inflammation and furthermore discuss potential therapeutic benefits of targeting NLRP3.     

Single-cell profiling of peanut-responsive T cells in patients with peanut allergy reveals heterogeneous effector TH2 subsets

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RBL cells as models for in vitro studies of mast cells and basophils

Since their establishment in 1981, RBL-2H3 cells have been widely used as a mast cell (MC) model. Their ability to be easily grown in culture in large amounts, their responsiveness to FcεRI-mediated triggers and the fact that they can be genetically manipulated, have provided advantages over primary MCs, in particular for molecular studies relying on genetic screening. Furthermore, the ability to generate clones that stably express proteins of interest, for example, a human receptor, have marked the RBL cells as an attractive MC model for drug screening. Indeed, 3 RBL reporter cell lines (RS-ATL8, NFAT-DsRed, and NPY-mRFP) have been generated providing useful models for drug and allergen screening. Similarly, RBL cells stably expressing the human MrgprX2 receptor provide a unique paradigm for analyzing ligand interactions and signaling pathways of the unique human receptor. Finally, transient co-transfections of RBL cells allow functional genomic analyses of MC secretion by combining library screening with simultaneous expression of a reporter for exocytosis. RBL cells thus comprise powerful tools for the study of intracellular membrane trafficking and exocytosis and the detection of allergens, vaccine safety studies and diagnosis of allergic sensitization. Their recent uses as an investigative tool are reviewed here.     

Axl and MerTK receptor tyrosine kinases maintain human macrophage efferocytic capacity in the presence of viral triggers

The requirement to remove apoptotic cells is equally important in homeostasis and inflammatory disease. In particular, during viral infections large quantities of infected cells undergo apoptosis and need to be efficiently cleared by phagocytes to prevent secondary necrosis. Although specific roles of several apoptotic cell sensors, such as the TAM (Tyro3, Axl, MerTK) receptor family, have been characterized in mouse models, little is known about their regulation and involvement in apoptotic cell uptake (efferocytosis) by human macrophages under inflammatory conditions. We show that whereas pro‐inflammatory stimuli consistently downregulated MerTK expression in human monocyte‐derived macrophages (MDMs), stimuli indicative of a viral infection, interferon‐α (IFN‐α) and the TLR3 ligand poly(I:C), specifically induced Axl expression and promoted binding of the bridging molecule Gas6. Axl induction by IFN‐α and poly(I:C) was associated with higher MDM efferocytic capacity compared to cells treated with other pro‐inflammatory stimuli, such as LPS and IFN‐γ. While MerTK blocking antibody uniformly suppressed apoptotic cell uptake by MDMs, Axl blocking antibody significantly reduced efferocytosis by poly(I:C)‐stimulated MDMs, but not by resting MDMs. Our observations demonstrate that Axl induction during viral infections contributes to maintaining macrophage capacity to engulf apoptotic cells, which may have important consequences for resolution of anti‐viral immune responses.     

Mouse models for the study of fate and function of innate lymphoid cells

Natural killer (NK) cells and lymphoid tissue inducer (LTi) cells were discovered more than 40 and 20 years ago, respectively. These two cell types were initially studied for their unique functions in the elimination of infected or transformed cells, and in the development of lymphoid tissues. It took an additional 10 years to realize that NK cells and LTi cells were members of a larger family of innate lymphoid cells (ILCs), whose phenotypes and functions mirror those of T cells. Many mouse models have since been developed to identify and isolate ILCs, map their developmental pathways and characterize their functions. Because of the similarity between ILCs and T cells, this exploration remains a challenge. In spite of this, a broad range of mouse models available to researchers has lead to significant progress in untangling the unique roles of ILCs early in defense, regulation of adaptive immunity and homeostasis. Here, we review these mouse models, and discuss their strengths and limitations.