Improved method to retain cytosolic reporter protein fluorescence while staining for nuclear proteins

Staining of transcription factors (TFs) together with retention of fluorescent reporter proteins is hindered by loss of fluorescence using current available methods. In this study, it is shown that current TF staining protocols do not destroy fluorescent proteins (FPs) but rather that fixation is not sufficient to retain FPs in the cytosol of the permeabilized cells. In this article, a simple and reliable protocol is elaborated, which allows efficient TF and cytokine staining while retaining FPs inside fixed cells.

Expression of the G-protein coupled receptor EBI2 in T cells is highly regulated and confers pathogenicity to myelin specific Th17 cells

Single-cell profiling reveals GPCR heterogeneity and functional patterning during neuroinflammation.

GPCR expression was intensively studied in bulk cDNA of leukocyte populations, but limited data are available with respect to expression in individual cells. Here, we show a microfluidic-based single-cell GPCR expression analysis in primary T cells, myeloid cells, and endothelial cells under naive conditions and during experimental autoimmune encephalomyelitis, the mouse model of multiple sclerosis. We found that neuroinflammation induces characteristic changes in GPCR heterogeneity and patterning, and we identify various functionally relevant subgroups with specific GPCR profiles among spinal cord-infiltrating CD4 T cells, macrophages, microglia, or endothelial cells. Using GPCRs CXCR4, S1…

The role of NG2 and OPC in induction and development of CNS inflammation

encephalitis (EAE) mouse models (J Neurosci 31:669, 2011; Brain 136:1760, 2013). Further, it has been shown that in addition to infiltrating leukocytes, astrocytes and microglia also exhibit high expression of EMMPRIN in diseased brain. EMMPRIN has been reported to interact with monocarboxylate transporter-1 (MCT-1) in astrocytes; the lattermediates lactate transport to neurons. Given that perturbation in energymetabolism can have catastrophic effects on homeostasis of the brain, it is paramount to understand EMMPRIN expression in astrocytes in normal andMS brain.We have used human fetal astrocytes (HFA) and determined their EMMPRIN levels by flow cytometry and microscopy. We found that HFA…

Alternative Splice Forms of CYLD Mediate Ubiquitination of SMAD7 to Prevent TGFB Signaling and Promote Colitis

Background & Aims The CYLD lysine 63 deubiquitinase gene (CYLD) encodes tumor suppressor protein that is mutated in familial cylindromatosus, and variants have been associated with Crohn disease (CD). Splice forms of CYLD that lack exons 7 and 8 regulate transcription factors and functions of immune cells. We examined the expression of splice forms of CYLD in colon tissues from patients with CD and their effects in mice. Methods We performed immunohistochemical analyses of colon tissues from patients with untreated CD and patients without inflammatory bowel diseases (controls). We obtained mice that expressed splice forms of CYLD (sCYLD mice) without or with SMAD7 (sCYLD/SMAD7 mice) from tr…

Dietary tryptophan is required for CNS infiltration of encephalitogenic T cells

TGF-β inhibitor Smad7 regulates dendritic cell-induced autoimmunity

TGF-β is an anti-inflammatory cytokine whose signaling is negatively controlled by Smad7. Previously, we established a role for Smad7 in the generation of autoreactive T cells; however, the function of Smad7 in dendritic cells (DCs) remains elusive. Here, we demonstrate that DC-specific Smad7 deficiency resulted in elevated expression of the transcription factors Batf3 and IRF8, leading to increased frequencies of CD8(+)CD103(+) DCs in the spleen. Furthermore, Smad7-deficient DCs expressed higher levels of indoleamine 2,3-dioxygenase (IDO), an enzyme associated with tolerance induction. Mice devoid of Smad7 specifically in DCs are resistant to the development of experimental autoimmune ence…

NF-κB inducing kinase (NIK) is an essential post-transcriptional regulator of T-cell activation affecting F-actin dynamics and TCR signaling

NF-κB inducing kinase (NIK) is the key protein of the non-canonical NF-κB pathway and is important for the development of lymph nodes and other secondary immune organs. We elucidated the specific role of NIK in T cells using T-cell specific NIK-deficient (NIKΔT) mice. Despite showing normal development of lymphoid organs, NIKΔT mice were resistant to induction of CNS autoimmunity. T cells from NIKΔT mice were deficient in late priming, failed to up-regulate T-bet and to transmigrate into the CNS. Proteomic analysis of activated NIK-/- T cells showed de-regulated expression of proteins involved in the formation of the immunological synapse: in particular, proteins involved in cytoskeleton dy…

Posttranslational modifications by ADAM10 shape myeloid antigen-presenting cell homeostasis in the splenic marginal zone

The spleen contains phenotypically and functionally distinct conventional dendritic cell (cDC) subpopulations, termed cDC1 and cDC2, which each can be divided into several smaller and less well-characterized subsets. Despite advances in understanding the complexity of cDC ontogeny by transcriptional programming, the significance of posttranslational modifications in controlling tissue-specific cDC subset immunobiology remains elusive. Here, we identified the cell-surface–expressed A-disintegrin-and-metalloproteinase 10 (ADAM10) as an essential regulator of cDC1 and cDC2 homeostasis in the splenic marginal zone (MZ). Mice with a CD11c-specific deletion of ADAM10 (ADAM10(ΔCD11c)) exhibited a …

IL-17 controls central nervous system autoimmunity through the intestinal microbiome

Interleukin-17A- (IL-17A) and IL-17F-producing CD4(+) T helper cells (T(H)17 cells) are implicated in the development of chronic inflammatory diseases, such as multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). T-H 17 cells also orchestrate leukocyte invasion of the central nervous system (CNS) and subsequent tissue damage. However, the role of IL-17A and IL-17F as effector cytokines is still confused with the encephalitogenic function of the cells that produce these cytokines, namely, T-H 17 cells, fueling a long-standing debate in the neuroimmunology field. Here, we demonstrated that mice deficient for IL-17A/F lose their susceptibility to EAE, which…

IL ‐1 signaling is critical for expansion but not generation of autoreactive GM ‐ CSF + Th17 cells

Abstract Interleukin‐1 (IL‐1) is implicated in numerous pathologies, including multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). However, the exact mechanism by which IL‐1 is involved in the generation of pathogenic T cells and in disease development remains largely unknown. We found that following EAE induction, pertussis toxin administration leads to IL‐1 receptor type 1 (IL‐1R1)‐dependent IL‐1β expression by myeloid cells in the draining lymph nodes. This myeloid‐derived IL‐1β did not vitally contribute to the generation and plasticity of Th17 cells, but rather promoted the expansion of a GM‐CSF + Th17 cell subset, thereby enhancing its encephalitog…

Introduction We and others recently showed that IL-17-producing Th17 cells are highly unstable in their phenotype and swiftly upregulate T-bet and Th1-associated cytokines in the inflamed CNS of mice with EAE [1] . This inherent plasticity was recently associated with IL-23, IFN-γ or IL-12 signalling on effector T cells [2] , [3] . Aim To understand the role of IFN-γ and IL-27 signaling for plasticity of Th17 cells in vivo. Methods We use mice lacking the IFN-γ receptor 2 chain specifically in T cells (CD4cre × IFNγR2FL/FL) as well as blocking antibodies for IFN-γ and IL-27-p28 and knockout mice for IL-27-EBI3. Further we use IL-17 reporter mice to sort Th17 cells prior adoptive transfer. W…

EBI2 – Sensor for dihydroxycholesterol gradients in neuroinflammation

Dihydroxycholesterols such as 7α,25-dihydroxysterols (7α,25-OHC) and 7α,27-OHC are generated from cholesterol by the enzymes CH25H, CYP7B1 and CYP27A1 in steady state but also in the context of inflammation. The G-protein coupled receptor (GPCR) Epstein-Barr virus-induced gene 2 (EBI2), also known as GPR183, senses these oxysterols and induces chemotactic migration of immune cells towards higher concentrations of these ligands. We recently showed that these ligands are upregulated in the CNS in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis and that EBI2 enhanced early infiltration of encephalitogenic T cells into the CNS. In this short-review we dis…

EBI2 Is Highly Expressed in Multiple Sclerosis Lesions and Promotes Early CNS Migration of Encephalitogenic CD4 T Cells

Arrival of encephalitogenic T cells at inflammatory foci represents a critical step in development of experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis. EBI2 and its ligand, 7{alpha},25-OHC, direct immune cell localization in secondary lymphoid organs. CH25H and CYP7B1 hydroxylate cholesterol to 7{alpha},25-OHC. During EAE, we found increased expression of CH25H by microglia and CYP7B1 by CNS-infiltrating immune cells elevating the ligand concentration in the CNS. Two critical pro-inflammatory cytokines, interleukin-23 (IL-23) and interleukin-1 beta (IL-1{beta}), maintained expression of EBI2 in differentiating Th17 cells. In line with this, EBI2 enhan…