Formyl peptide receptor 2 regulates dendritic cell metabolism and Th17 cell differentiation during neuroinflammation.

Formyl peptide receptor 2 (FPR2) is a receptor for formylated peptides and specific pro-resolving mediators, and is involved in various inflammatory processes. Here, we aimed to elucidate the role of FPR2 in dendritic cell (DC) function and autoimmunity-related central nervous system (CNS) inflammation by using the experimental autoimmune encephalomyelitis (EAE) model. EAE induction was accompanied by increased Fpr2 mRNA expression in the spinal cord. FPR2-deficient (Fpr2 KO) mice displayed delayed onset of EAE compared to wild-type (WT) mice, associated with reduced frequencies of Th17 cells in the inflamed spinal cord at the early stage of the disease. However, FPR2 deficiency did not affect EAE severity after the disease reached its peak. FPR2 deficiency in mature DCs resulted in decreased expression of Th17 polarizing cytokines IL6, IL23p19, IL1ß, and thereby diminished the DC-mediated activation of Th17 cell differentiation. LPS-activated FPR2-deficient DCs showed upregulated Nos2 expression and nitric oxide (NO) production, as well as reduced oxygen consumption rate and impaired mitochondrial function, including decreased mitochondrial superoxide levels, lower mitochondrial membrane potential and diminished expression of genes related to the tricarboxylic acid cycle and genes related to the electron transport chain, as compared to WT DCs. Treatment with a NO inhibitor reversed the reduced Th17 cell differentiation in the presence of FPR2-deficient DCs. Together, by regulating DC metabolism, FPR2 enhances the production of DC-derived Th17-polarizing cytokines and hence Th17 cell differentiation in the context of neuroinflammation.

Genetic deletion of JAM-C in pre-leukemic cells rewires leukemic stem cell gene expression program in AML.

The leukemic stem cell (LSC) score LSC-17 based on a stemness-related gene expression signature is an indicator of poor disease outcome in acute myeloid leukemia (AML). However, our understanding of the relationships between LSC and pre-leukemic cells is still incomplete. In particular, it is not known whether "niche-anchoring" of pre-leukemic cell affects disease evolution. To address this issue, we conditionally inactivated the adhesion molecule JAM-C expressed by haematopoietic stem cells (HSC) and LSC in an inducible iMLL-AF9-driven AML mouse model. Deletion of Jam3 (encoding JAM-C) before induction of the leukemia-initiating iMLL-AF9 fusion resulted in a shift from long term to short term-HSC expansion, without affecting disease initiation and progression. In vitro experiments showed that JAM-C controlled leukemic cell nesting irrespective of the bone marrow stromal cells used. RNA sequencing performed on leukemic HSC isolated from diseased mice revealed that genes upregulated in Jam3-deficient animals belonged to Activation Protein-1 (AP-1) and TNF-/NFB pathways. Human orthologs of dysregulated genes allowed to identify a score based on AP-1/TNF-a gene expression that was distinct and complementary from LSC-17 score. Sub-stratification of AML patients with LSC-17 and AP-1/TNF-genes signature defined four groups with median survival ranging from below one year to a median not reached after 8 years. Finally, coculture experiments showed that AP-1 activation in leukemic cells was dependent on the nature of stromal cells. Altogether, our results identify the AP-1/TNF- gene signature as a proxy of LSC anchoring in specific bone marrow niches which improves the prognosis value of the LSC-17 score. NCT02320656.

Clonal hematopoiesis driven by mutated DNMT3A promotes inflammatory bone loss.

Clonal hematopoiesis of indeterminate potential (CHIP) arises from aging-associated acquired mutations in hematopoietic progenitors, which display clonal expansion and produce phenotypically altered leukocytes. We associated CHIP-DNMT3A mutations with a higher prevalence of periodontitis and gingival inflammation among 4,946 community-dwelling adults. To model DNMT3A-driven CHIP, we used mice with the heterozygous loss-of-function mutation R878H, equivalent to the human hotspot mutation R882H. Partial transplantation with Dnmt3aR878H/+ bone marrow (BM) cells resulted in clonal expansion of mutant cells into both myeloid and lymphoid lineages and an elevated abundance of osteoclast precursors in the BM and osteoclastogenic macrophages in the periphery. DNMT3A-driven clonal hematopoiesis in recipient mice promoted naturally occurring periodontitis and aggravated experimentally induced periodontitis and arthritis, associated with enhanced osteoclastogenesis, IL-17-dependent inflammation and neutrophil responses, and impaired regulatory T cell immunosuppressive activity. DNMT3A-driven clonal hematopoiesis and, subsequently, periodontitis were suppressed by rapamycin treatment. DNMT3A-driven CHIP represents a treatable state of maladaptive hematopoiesis promoting inflammatory bone loss.

Accurate redox state indication by in situ derivatization with N-ethylmaleimide - Profiling of transsulfuration and glutathione pathway metabolites by UPLC-MS/MS.

BACKGROUND: Reduced and oxidized glutathione play an important role for the intracellular detoxification of reactive oxygen species. The iron-dependent formation of such reactive oxygen species in conjunction with the inhibition of the redox-balancing enzyme glutathione peroxidase 4 underlie an imbalance in the cellular redox state, thereby resulting in a non-apoptotic form of cell death, defined as ferroptosis, which is relevant in several pathologies. METHODS: Here we present a rapid ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) based method providing the accurate quantification of 12 glutathione pathway metabolites after in situ derivatization with N-Ethylmaleimide (NEM). The method was validated regards linearity, recovery and accuracy as well as precision. The assay includes glutathione and its oxidized form glutathione disulfide. Furthermore, the related precursors cysteine, cystine, glutamic acid, γ-glutamylcysteine and cysteinylglycine, biomarkers of protein crosslinking such as cystathionine and lanthionine, as well as metabolites of the transsulfuration pathway, methionine, homocysteine and serine are simultaneously determined. RESULTS: Twelve glutathione pathway metabolites were simultaneously analyzed in four different human cell line extracts within a total LC run time of 5.5 min. Interday coefficients of variation (1.7 % to 12.0 %), the mean observed accuracy (100.0 % ± 5.2 %), linear quantification ranges over three orders of magnitude for all analytes and sufficient metabolite stability after NEM-derivatization demonstrate method reliability. Immediate derivatization with NEM at cell harvesting prevents autooxidation of glutathione, ensures accurate results for the GSH/GSSG redox ratio and thereby allows interpretation of cellular redox state. CONCLUSION: The described UPLC-MS/MS method provides a sensitive and selective tool for a fast and simultaneous analysis of glutathione pathway metabolites, its direct precursors and related compounds. Assay performance characteristics demonstrate the suitability of the method for applications in different cell cultures. Therefore, by providing glutathione related functional metabolic readouts, the method enables investigations in mechanisms of ferroptosis and alterations in oxidative stress levels in several pathophysiologies.

Potent induction of trained immunity by Saccharomyces cerevisiae ß-glucans.

Candida albicans cell wall component ß-glucan has been extensively studied for its ability to induce epigenetic and functional reprogramming of innate immune cells, a process termed trained immunity. We show that a high-complexity blend of two individual ß-glucans from Saccharomyces cerevisiae possesses strong bioactivity, resulting in an enhanced trained innate immune response by human primary monocytes. The training required the Dectin-1/CR3, TLR4, and MMR receptors, as well as the Raf-1, Syk, and PI3K downstream signaling molecules. By activating multiple receptors and downstream signaling pathways, the components of this ß-glucan preparation are able to act synergistically, causing a robust secondary response upon an unrelated challenge. In in-vivo murine models of melanoma and bladder cell carcinoma, pre-treatment of mice with the ß-glucan preparation led to a significant reduction in tumor growth. These insights may aid in the development of future therapies based on ß-glucan structures that induce an effective trained immunity response.

CD38 promotes hematopoietic stem cell dormancy.

A subpopulation of deeply quiescent, so-called dormant hematopoietic stem cells (dHSCs) resides at the top of the hematopoietic hierarchy and serves as a reserve pool for HSCs. The state of dormancy protects the HSC pool from exhaustion throughout life; however, excessive dormancy may prevent an efficient response to hematological stresses. Despite the significance of dHSCs, the mechanisms maintaining their dormancy remain elusive. Here, we identify CD38 as a novel and broadly applicable surface marker for the enrichment of murine dHSCs. We demonstrate that cyclic adenosine diphosphate ribose (cADPR), the product of CD38 cyclase activity, regulates the expression of the transcription factor c-Fos by increasing the release of Ca2+ from the endoplasmic reticulum (ER). Subsequently, we uncover that c-Fos induces the expression of the cell cycle inhibitor p57Kip2 to drive HSC dormancy. Moreover, we found that CD38 ecto-enzymatic activity at the neighboring CD38-positive cells can promote human HSC quiescence. Together, CD38/cADPR/Ca2+/c-Fos/p57Kip2 axis maintains HSC dormancy. Pharmacological manipulations of this pathway can provide new strategies to improve the success of stem cell transplantation and blood regeneration after injury or disease.

An IL-10/DEL-1 axis supports granulopoiesis and survival from sepsis in early life.

The limited reserves of neutrophils are implicated in the susceptibility to infection in neonates, however the regulation of neutrophil kinetics in infections in early life remains poorly understood. Here we show that the developmental endothelial locus (DEL-1) is elevated in neonates and is critical for survival from neonatal polymicrobial sepsis, by supporting emergency granulopoiesis. Septic DEL-1 deficient neonate mice display low numbers of myeloid-biased multipotent and granulocyte-macrophage progenitors in the bone marrow, resulting in neutropenia, exaggerated bacteremia, and increased mortality; defects that are rescued by DEL-1 administration. A high IL-10/IL-17A ratio, observed in newborn sepsis, sustains tissue DEL-1 expression, as IL-10 upregulates while IL-17 downregulates DEL-1. Consistently, serum DEL-1 and blood neutrophils are elevated in septic adult and neonate patients with high serum IL-10/IL-17A ratio, and mortality is lower in septic patients with high serum DEL-1. Therefore, IL-10/DEL-1 axis supports emergency granulopoiesis, prevents neutropenia and promotes sepsis survival in early life.

A novel macrolide-Del-1 axis to regenerate bone in old age.

Aging is associated with increased susceptibility to chronic inflammatory bone loss disorders, such as periodontitis, in large part due to the impaired regenerative potential of aging tissues. DEL-1 exerts osteogenic activity and promotes bone regeneration. However, DEL-1 expression declines with age. Here we show that systemically administered macrolide antibiotics and a non-antibiotic erythromycin derivative, EM-523, restore DEL-1 expression in 18-month-old ("aged") mice while promoting regeneration of bone lost due to naturally occurring age-related periodontitis. These compounds failed to induce bone regeneration in age-matched DEL-1-deficient mice. Consequently, these drugs promoted DEL-1-dependent functions, including alkaline phosphatase activity and osteogenic gene expression in the periodontal tissue while inhibiting osteoclastogenesis, leading to net bone growth. Macrolide-treated aged mice exhibited increased skeletal bone mass, suggesting that this treatment may be pertinent to systemic bone loss disorders. In conclusion, we identified a macrolide-DEL-1 axis that can regenerate bone lost due to aging-related disease.