IL-17+ CD8+ T cell suppression by dimethyl fumarate associates with clinical response in multiple sclerosis

IL-17-producing CD8+ (Tc17) cells are enriched in active lesions of patients with multiple sclerosis (MS), suggesting a role in the pathogenesis of autoimmunity. Here we show that amelioration of MS by dimethyl fumarate (DMF), a mechanistically elusive drug, associates with suppression of Tc17 cells. DMF treatment results in reduced frequency of Tc17, contrary to Th17 cells, and in a decreased ratio of the regulators RORC-to-TBX21, along with a shift towards cytotoxic T lymphocyte gene expression signature in CD8+ T cells from MS patients. Mechanistically, DMF potentiates the PI3K-AKT-FOXO1-T-BET pathway, thereby limiting IL-17 and RORγt expression as well as STAT5-signaling in a glutathione-dependent manner. This results in chromatin remodeling at the Il17 locus. Consequently, T-BET-deficiency in mice or inhibition of PI3K-AKT, STAT5 or reactive oxygen species prevents DMF-mediated Tc17 suppression. Overall, our data disclose a DMF-AKT-T-BET driven immune modulation and suggest putative therapy targets in MS and beyond

Mesaconate is synthesized from itaconate and exerts immunomodulatory effects in macrophages.

peer reviewedSince its discovery in inflammatory macrophages, itaconate has attracted much attention due to its antimicrobial and immunomodulatory activity1-3. However, instead of investigating itaconate itself, most studies used derivatized forms of itaconate and thus the role of non-derivatized itaconate needs to be scrutinized. Mesaconate, a metabolite structurally very close to itaconate, has never been implicated in mammalian cells. Here we show that mesaconate is synthesized in inflammatory macrophages from itaconate. We find that both, non-derivatized itaconate and mesaconate dampen the glycolytic activity to a similar extent, whereas only itaconate is able to repress tricarboxylic acid cycle activity and cellular respiration. In contrast to itaconate, mesaconate does not inhibit succinate dehydrogenase. Despite their distinct impact on metabolism, both metabolites exert similar immunomodulatory effects in pro-inflammatory macrophages, specifically a reduction of interleukin (IL)-6 and IL-12 secretion and an increase of CXCL10 production in a manner that is independent of NRF2 and ATF3. We show that a treatment with neither mesaconate nor itaconate impairs IL-1β secretion and inflammasome activation. In summary, our results identify mesaconate as an immunomodulatory metabolite in macrophages, which interferes to a lesser extent with cellular metabolism than itaconate

The TNF Family of Ligands and Receptors: Communication Modules in the Immune System and Beyond

The tumor necrosis factor (TNF) and TNF receptor (TNFR) superfamilies (TNFSF/TNFRSF) include 19 ligands and 29 receptors that play important roles in the modulation of cellular functions. The communication pathways mediated by TNFSF/TNFRSF are essential for numerous developmental, homeostatic, and stimulus-responsive processes in vivo. TNFSF/TNFRSF members regulate cellular differentiation, survival, and programmed death, but their most critical functions pertain to the immune system. Both innate and adaptive immune cells are controlled by TNFSF/TNFRSF members in a manner that is crucial for the coordination of various mechanisms driving either co-stimulation or co-inhibition of the immune response. Dysregulation of these same signaling pathways has been implicated in inflammatory and autoimmune diseases, highlighting the importance of their tight regulation. Investigation of the control of TNFSF/TNFRSF activities has led to the development of therapeutics with the potential to reduce chronic inflammation or promote anti-tumor immunity. The study of TNFSF/TNFRSF proteins has exploded over the last 30 yr, but there remains a need to better understand the fundamental mechanisms underlying the molecular pathways they mediate to design more effective anti-inflammatory and anti-cancer therapies

Recombinant p35 from bacteria can form Interleukin (IL-)12, but Not IL-35.

The Interleukin (IL)-12 family contains several heterodimeric composite cytokines which share subunits among each other. IL-12 consists of the subunits p40 (shared with IL-23) and p35. p35 is shared with the composite cytokine IL-35 which comprises of the p35/EBI3 heterodimer (EBI3 shared with IL-27). IL-35 signals via homo- or heterodimers of IL-12Rβ2, gp130 and WSX-1, which are shared with IL-12 and IL-27 receptor complexes, respectively. p35 was efficiently secreted in complex with p40 as IL-12 but not with EBI3 as IL-35 in several transfected cell lines tested which complicates the analysis of IL-35 signal transduction. p35 and p40 but not p35 and EBI3 form an inter-chain disulfide bridge. Mutation of the responsible cysteine residue (p40C197A) reduced IL-12 formation and activity only slightly. Importantly, the p40C197A mutation prevented the formation of antagonistic p40 homodimers which enabled the in vitro reconstitution of biologically active IL-12 with p35 produced in bacteria (p35bac). Reconstitution of IL-35 with p35bac and EBI3 did, however, fail to induce signal transduction in Ba/F3 cells expressing IL-12Rβ2 and gp130. In summary, we describe the in vitro reconstitution of IL-12, but fail to produce recombinant IL-35 by this novel approach

p28 and EBI3 (IL-27), p40 and p35 (IL-12) as well as p40 and p19 (IL-23) form heterodimeric cytokines and are efficiently secreted, but not EBI3 and p35 (IL-35).

<p>(<b>A</b>) HEK293 cells were transiently transfected with plasmids encoding the constructs indicated above the Western blots. Supernatant was taken 48 h post transfection. Cells were lysed, and expression and secretion of the different cytokines was assessed by Western blotting with monoclonal antibodies against the flag- or the myc-tag. Western blots shown are representative of three different experiments with similar outcomes. The asterisk denotes an unspecific band detected by the flag-antibody. (<b>B</b>) HEK293 cells were transiently transfected with plasmids containing either EBI3-Fc and p28-myc or EBI3-myc and p28-Fc. Expression and secretion of the respective proteins was analyzed by Western blotting as described in panel (A) with antibodies directed against the Fc- or the myc-tag. (<b>C</b>) Equal amounts of Ba/F3-IL-12Rβ1-IL-12Rβ2 cells were incubated with 10% of supernatants derived from transfected cells described in panel (A) and (B). Cellular proliferation was determined 48 h later as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107990#s2" target="_blank"><i>Materials and Methods</i></a>. (<b>D</b>) HEK293 cells were transiently transfected with plasmids encoding the constructs indicated above the Western blots. Supernatant was taken 48 h post transfection. Cells were lysed, and expression and secretion of the different cytokines was assessed by Western blotting with monoclonal antibodies against the flag- or the Fc-tag. Western blots shown are representative of three different experiments with similar outcomes. The asterisk denotes an unspecific band detected by the flag-antibody.</p

Recombinant p35 from bacteria forms biologically active IL-12.

<p>(<b>A</b>) Refolded p35_bac and p35_bac/C92A were subjected to size exclusion chromatography. Monomeric protein was separated from multimeric assemblies on a Superdex 75 10/300 GL equilibrated in 50 mM Tris-HCl (pH 8.0) containing 250 mM NaCl. (<b>B</b>) Purity of the bacterial produced p35_bac and p35_bac/C92A was analyzed by SDS-PAGE on a reducing gel via Coomassie brilliant blue staining. 5 µg protein were loaded per lane. (<b>C</b>) Equal amounts of Ba/F3-IL-12Rβ1-IL-12Rβ2 cells were incubated with 10 ng/ml Hyper-IL-6, 10% conditioned supernatant containing Hyper-IL-12, or 10% conditioned supernatant containing p40_C197A with increasing amounts of either monomeric or multimeric p35. (<b>D</b>) Equal amounts of Ba/F3-IL-12Rβ1-IL-12Rβ2 cells were treated as described under panel (C), but with either recombinant p35_bac or p35_bac/C92A (0–4000 ng/ml). Cellular proliferation in both experiments was determined 48 h later as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107990#s2" target="_blank"><i>Materials and Methods</i></a>. (<b>E</b>) Equal amounts of Ba/F3-gp130-IL-12Rβ1-IL-12Rβ2 cells were incubated with 50% conditioned supernatant containing p40_C197A with or without 2 or 4 µg/ml p35_bac/C92A for 15 min. Phosphorylation of STAT1 and STAT3 was determined per Western blotting. Total amounts of STAT1 and STAT3 were visualized as internal loading control. The data shown are representative of two different experiments with similar outcomes.</p

The disulfide bond p35C92-C197p40 is dispensable for the biological activity of IL-12.

<p>(<b>A</b>) Schematic representation of IL-12 comprising p35 (gray) and p40 (orange) according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107990#pone.0107990-Yoon1" target="_blank">[28]</a>. The inter-molecular disulfide bond p35C92-C197p40 is highlighted with a red circle. (<b>B</b>) 20 µl conditioned supernatant of HEK293 cells transiently transfected with either p40 wildtype or p40C197A were separated by SDS-PAGE under non-reducing conditions and proteins visualized by Western blotting with a flag-specific antibody. (<b>C</b>) HEK293 cells were transiently transfected with plasmids encoding the constructs indicated above the Western blots. Supernatant was harvested 48 h post transfection. Cells were lysed, and expression and secretion of the different cytokines was assessed by Western blotting with monoclonal antibodies against the flag-tag. Unspecific bands detected by the flag-antibody are denoted with asterisks. (<b>D</b>) Ba/F3-IL-12Rβ1-IL-12Rβ2 cells were incubated with 10% of supernatants derived from HEK293 cells transiently transfected with the constructs given below the diagram. Cellular proliferation was determined 48 h later as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107990#s2" target="_blank"><i>Materials and Methods</i></a>. (<b>E</b>) Equal amounts of Ba/F3-IL-12Rβ1-IL-12Rβ2 cells were stimulated with supernatants from HEK293 cells transiently transfected with the constructs indicated above the Western blots for 15 min. Phosphorylation of STAT3 was determined per Western blotting. Total amounts of STAT3 were visualized as internal loading control. The Western blots shown are representative of three different experiments with similar outcomes, and the proliferation assay was measured in triplicates and is representative of two performed experiments.</p

IL-12 induces the expression of Interferon-γ in murine T cells.

<p>(<b>A</b>) Murine CD4+ T cells were enriched by positive selection from single cell suspended splenocytes and lymphnode cells. 1×10⁵ cells per well were cultured on plates coated with 0.5 µg/ml anti-CD3 and 2 µg/ml soluble anti-CD28. 10% of the indicated cell culture supernatant was added and supernatants were harvested after three days. Secretion of IFN-γ was determined via ELISA. (<b>B</b>) The experiment was performed as described under panel (A). Where indicated, 2 µg/ml p35_bac/C92A were added. The experiments were performed with at least three individual mice, and data from one representative animal are shown.</p

Schematic overview of the IL-12 family of cytokines.

<p>(<b>A</b>) Interleukin-12 (consisting of the p35 and the p40 subunits) signals via a heterodimer of the two β-receptors IL-12Rβ1 and IL-12Rβ2. Interleukin-23 (consisting of the p19 and the p40 subunits) signals via a heterodimer of the two β-receptors IL-12Rβ1 and the unique IL-23R. Interleukin-27 (consisting of its subunits p28 and EBI3) engages signaling via gp130 and the unique β-receptor WSX-1. (<b>B</b>) Interleukin-35 (consisting of its subunits p35 and EBI3) is able to signal via four different combinations of β-receptors, resulting in the phosphorylation and thus activation of different STAT proteins. Binding of IL-35 to an IL-12Rβ2 homodimer induces the activation of STAT4 homodimers <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107990#pone.0107990-Collison1" target="_blank">[10]</a>, binding to a gp130 homodimer activates STAT1 homodimers <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107990#pone.0107990-Collison1" target="_blank">[10]</a>, binding to an IL-12Rβ2/gp130 heterodimer induces STAT1/STAT4 heterodimerization <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107990#pone.0107990-Collison1" target="_blank">[10]</a>, and binding of IL-35 to a IL-12Rβ2/WSX-1 heterodimer induces the activation of STAT1 and STAT3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107990#pone.0107990-Wang1" target="_blank">[12]</a>.</p