Synergistic production of TNF\u3b1 and IFN\u3b1 by human pDCs incubated with IFN\u3bb3 and IL-3

In this study, we investigated whether IFN\u3bb3 and IL-3 reciprocally influence their capacity to activate various functions of human plasmacytoid dendritic cells (pDCs). In fact, we preliminarily observed that IFN\u3bb3 upregulates the expression of the IL-3R\u3b1 (CD123), while IL-3 augments the expression of IFN\u3bbR1 in pDCs. As a result, we found that combination of IFN\u3bb3 and IL-3 induces a strong potentiation in the production of TNF\u3b1, IFN\u3b1, as well as in the expression of Interferon-Stimulated Gene (ISG) mRNAs by pDCs, as compared to either IFN\u3bb3 or IL-3 alone. In such regard, we found that endogenous IFN\u3b1 autocrinally promotes the expression of ISG mRNAs in IL-3-, but not in IFN\u3bb3 plus IL-3-, treated pDCs. Moreover, we uncovered that the production of IFN\u3b1 by IFN\u3bb3 plus IL-3-treated pDCs is mostly dependent on endogenously produced TNF\u3b1. Altogether, our data demonstrate that IFN\u3bb3 and IL-3 collaborate to promote, at maximal levels, discrete functional responses of human pDCs

RelB activation in anti-inflammatory decidual endothelial cells: a master plan to avoid pregnancy failure?

It is known that excessive inflammation at fetal-maternal interface is a key contributor in a compromised pregnancy. Female genital tract is constantly in contact with microorganisms and several strategies must be adopted to avoid pregnancy failure. Decidual endothelial cells (DECs) lining decidual microvascular vessels are the first cells that interact with pro-inflammatory stimuli released into the environment by microorganisms derived from gestational tissues or systemic circulation. Here, we show that DECs are hypo-responsive to LPS stimulation in terms of IL-6, CXCL8 and CCL2 production. Our results demonstrate that DECs express low levels of TLR4 and are characterized by a strong constitutive activation of the non-canonical NF-\u3baB pathway and a low responsiveness of the canonical pathway to LPS. In conclusion, DECs show a unique hypo-responsive phenotype to the pro-inflammatory stimulus LPS in order to control the inflammatory response at feto-maternal interface

Induction of OCT2 contributes to regulate the gene expression program in human neutrophils activated via TLR8

The transcription factors (TFs) that regulate inducible genes in activated neutrophils are not yet completely characterized. Herein, we show that the genomic distribution of the histone modification H3K27Ac, as well as PU.1 and C/EBP beta, two myeloid-lineage-determining TFs (LDTFs), significantly changes in human neutrophils treated with R848, a ligand of Toll-like receptor 8 (TLR8). Interestingly, differentially acetylated and LDTF-marked regions reveal an over-representation of OCT-binding motifs that are selectively bound by OCT2/POU2F2. Analysis of OCT2 genomic distribution in primary neutrophils and of OCT2-depletion in HL-60-differentiated neutrophils proves the requirement for OCT2 in contributing to promote, along with nuclear factor kappa B (NF-kappa B) and activator protein 1 (AP-1), the TLR8-induced gene expression program in neutrophils. Altogether, our data demonstrate that neutrophils, upon activation via TLR8, profoundly reprogram their chromatin status, ultimately displaying cell-specific, prolonged transcriptome changes. Data also show an unexpected role for OCT2 in amplifying the transcriptional response to TLR8-mediated activation

the neutrophil activating protein of helicobacter pylori crosses endothelia to promote neutrophil adhesion in vivo

Helicobacter pylori induces an acute inflammatory response followed by a chronic infection of the human gastric mucosa characterized by infiltration of neutrophils/polymorphonuclear cells (PMNs) and mononuclear cells. The H. pylori neutrophil-activating protein (HP-NAP) activates PMNs, monocytes, and mast cells, and promotes PMN adherence to the endothelium in vitro. By using intravital microscopy analysis of rat mesenteric venules exposed to HP-NAP, we demonstrated, for the first time in vivo, that HP-NAP efficiently crosses the endothelium and promotes a rapid PMN adhesion. This HP-NAP-induced adhesion depends on the acquisition of a high affinity state of β2 integrin on the plasma membrane of PMNs, and this conformational change requires a functional p38 MAPK. We also show that HP-NAP stimulates human PMNs to synthesize and release a number of chemokines, including CXCL8, CCL3, and CCL4. Collectively, these data strongly support a central role for HP-NAP in the inflammation process in vivo: indeed, HP-NAP not only recruits leukocytes from the vascular lumen, but also stimulates them to produce messengers that may contribute to the maintenance of the flogosis associated with the H. pylori infection

Human neutrophils activated by TLR8 agonists, with or without IFN\u3b3, synthesize and release EBI3, but not IL-12, IL-27, IL-35, or IL-39

The IL-12 family of cytokines plays crucial functions in innate and adaptive immunity. These cytokines include heterodimers sharing distinct \u3b1 (IL-12A, IL-23A, and IL-27A) with two \u3b2 (IL-12B and Epstein-Barr virus induced gene 3 [EBI3]) chains, respectively, IL-12 (IL-12B plus IL-12A) and IL-23 (IL-12B plus IL-23A) sharing IL-12B, IL-27 (EBI3 plus IL-27A), IL-35 (EBI3 plus IL-12A), and IL-39 (EBI3 plus IL-23A) sharing EBI3. In this context, we have recently reported that highly pure neutrophils incubated with TLR8 agonists produce functional IL-23. Previously, we showed that neutrophils incubated with LPS plus IFN\u3b3 for 20 h produce IL-12. Herein, we investigated whether highly pure, TLR8-activated, neutrophils produce EBI3, and in turn IL-27, IL-35, and IL-39, the IL-12 members containing it. We report that neutrophils incubated with TLR8 ligands, TNF\u3b1 and, to a lesser extent, LPS, produce and release remarkable amounts of EBI3, but not IL-27A, consequently excluding the possibility for an IL-27 production. We also report a series of unsuccessful experiments performed to investigate whether neutrophil-derived EBI3 associates with IL-23A to form IL-39. Furthermore, we show that neutrophils incubated with IFN\u3b3 in combination with either TLR8 or TLR4 ligands express/produce neither IL-12, nor IL-35, due to the inability of IFN\u3b3, contrary to previous findings, to activate IL12A transcription. Even IL-27 was undetectable in supernatants harvested from IFN\u3b3 plus R848-treated neutrophils, although they were found to accumulate IL27A transcripts. Finally, by immunohistochemistry experiments, EBI3-positive neutrophils were found in discrete pathologies only, including diverticulitis, cholecystitis, Gorham disease, and Bartonella Henselae infection, implying a specific role of neutrophil-derived EBI3 in vivo

Transient Decrease of Circulating and Tissular Dendritic Cells in Patients With Mycobacterial Disease and With Partial Dominant IFN\u3b3R1 Deficiency

Interferon-\u3b3 receptor 1 (IFN\u3b3R1) deficiency is one of the inborn errors of IFN-\u3b3 immunity underlying Mendelian Susceptibility to Mycobacterial Disease (MSMD). This molecular circuit plays a crucial role in regulating the interaction between dendritic cells (DCs) and T lymphocytes, thus affecting DCs activation, maturation, and priming of T cells involved in the immune response against intracellular pathogens. We studied a girl who developed at the age of 2.5 years a Mycobacterium avium infection characterized by disseminated necrotizing granulomatous lymphadenitis, and we compared her findings with other patients with the same genetic condition. The patient carried a heterozygous 818del4 mutation in the IFNGR1 gene responsible of autosomal dominant (AD) partial IFN\u3b3R1 deficiency. During the acute infection blood cells immunophenotyping showed a marked reduction in DCs counts, including both myeloid (mDCs) and plasmacytoid (pDCs) subsets, that reversed after successful prolonged antimicrobial therapy. Histology of her abdomen lymph node revealed a profound depletion of tissue pDCs, as compared to other age-matched granulomatous lymphadenitis of mycobacterial origin. Circulating DCs depletion was also observed in another patient with AD partial IFN\u3b3R1 deficiency during mycobacterial infection. To conclude, AD partial IFN\u3b3R1 deficiency can be associated with a transient decrease in both circulating and tissular DCs during acute mycobacterial infection, suggesting that DCs counts monitoring might constitute a useful marker of treatment response

SARS-CoV-2-Associated ssRNAs Activate Human Neutrophils in a TLR8-Dependent Fashion

COVID-19 disease is characterized by a dysregulation of the innate arm of the immune system. However, the mechanisms whereby innate immune cells, including neutrophils, become activated in patients are not completely understood. Recently, we showed that GU-rich RNA sequences from the SARS-CoV-2 genome (i.e., SCV2-RNA1 and SCV2-RNA2) activate dendritic cells. To clarify whether human neutrophils may also represent targets of SCV2-RNAs, neutrophils were treated with either SCV2-RNAs or, as a control, R848 (a TLR7/8 ligand), and were then analyzed for several functional assays and also subjected to RNA-seq experiments. Results highlight a remarkable response of neutrophils to SCV2-RNAs in terms of TNFα, IL-1ra, CXCL8 production, apoptosis delay, modulation of CD11b and CD62L expression, and release of neutrophil extracellular traps. By RNA-seq experiments, we observed that SCV2-RNA2 promotes a transcriptional reprogramming of neutrophils, characterized by the induction of thousands of proinflammatory genes, similar to that promoted by R848. Furthermore, by using CU-CPT9a, a TLR8-specific inhibitor, we found that SCV2-RNA2 stimulates neutrophils exclusively via TLR8-dependent pathways. In sum, our study proves that single-strand RNAs from the SARS-CoV-2 genome potently activate human neutrophils via TLR8, thus uncovering a potential mechanism whereby neutrophils may contribute to the pathogenesis of severe COVID-19 disease

Chromatin remodelling and autocrine TNFα are required for optimal interleukin-6 expression in activated human neutrophils

How IL-6 expression is regulated in human neutrophils has remained unclear. Here the authors show, using highly purified neutrophils, that TLR8 or TLR4 signalling activates latent enhancers and cooperates with autocrine TNFα to induce IL-6 transcription