Absence of Leucine Zipper in the Natural FOXP3Δ2Δ7 Isoform Does Not Affect Dimerization but Abrogates Suppressive Capacity

BACKGROUND: Phenotype and function of regulatory T cells (Treg) largely depend on the presence of the transcription factor FOXP3. In contrast to mice, human Treg cells express isoforms of this protein. Besides the full length version (FOXP3fl), an isoform lacking the exon 2 (FOXP3Delta2) is co-expressed in comparable amounts. Recently, a third splice variant has been described that in addition to exon 2 also misses exon 7 (FOXP3Delta2Delta7). Exon 7 encodes for a leucine zipper motif commonly used as structural dimerization element. Mutations in exon 7 have been linked to IPEX, a severe autoimmune disease suggested to be caused by impaired dimerization of the FOXP3 protein. PRINCIPAL FINDINGS: This study shows that the lack of exon 7 does not affect (homo-) dimerization. Moreover, the interaction of FOXP3Delta2Delta7 to RUNX1, NFAT and NF-kB appeared to be unchanged in co-immunoprecipitation experiments and reporter gene assays, when compared to FOXP3fl and FOXP3Delta2. Nevertheless, retroviral transduction with FOXP3Delta2Delta7 failed to induce the typical Treg-associated phenotype. The expression of FOXP3-induced surface molecules such as CD25 and CTLA-4 were not enhanced in FOXP3Delta2Delta7 transduced CD4+ T cells, which also failed to exhibit any suppressive capacity. Notably, however, co-expression of FOXP3fl with FOXP3Delta2Delta7 resulted in a reduction of CD25 expression by a dominant negative effect. CONCLUSIONS: The leucine zipper of FOXP3 does not mediate dimerization or interaction with NFAT, NF-kB and RUNX1, but is indispensable for the characteristic phenotype and function in Treg cells. FOXP3Delta2Delta7 could play a role in regulating the function of the other FOXP3 isoforms and may be involved in cancer pathogenesis, as it is overexpressed by certain malignant cells

Polyphosphate as a Target for Interference With Inflammation and Thrombosis

Activated platelets and mast cells expose the inorganic polymer, polyphosphate (polyP) on their surfaces. PolyP initiates procoagulant and proinflammatory reactions and the polymer has been recognized as a therapeutic target for interference with blood coagulation and vascular hyperpermeability. PolyP content and chain length depend on the specific cell type and energy status, which may affect cellular functions. PolyP metabolism has mainly been studied in bacteria and yeast, but its roles in eukaryotic cells and mammalian systems have remained enigmatic. In this review, we will present an overview of polyP functions, focusing on intra- and extracellular roles of the polymer and discuss open questions that emerge from the current knowledge on polyP regulation

Intrinsic coagulation pathway-mediated thrombin generation in mouse whole blood

Calibrated Automated Thrombography (CAT) is a versatile and sensitive method for analyzing coagulation reactions culminating in thrombin generation (TG). Here, we present a CAT method for analyzing TG in murine whole blood by adapting the CAT assay used for measuring TG in human plasma. The diagnostically used artificial and physiologic factor XII (FXII) contact activators kaolin, ellagic acid and polyphosphate (polyP) stimulated TG in murine blood in a dose-dependent manner resulting in a gradual increase in endogenous thrombin potential and peak thrombin, with shortened lag times and times to peak. The activated FXII inhibitor rHA-Infestin-4 and direct oral anticoagulants (DOACs) interfered with TG triggered by kaolin, ellagic acid and polyP and TG was completely attenuated in blood of FXII- (F12−/−) and FXI-deficient (F11−/−) mice. Moreover, reconstitution of blood from F12−/− mice with human FXII restored impaired contact-stimulated TG. HEK293 cell-purified polyP also initiated FXII-driven TG in mouse whole blood and addition of the selective inhibitor PPX_112 ablated natural polyP-stimulated TG. In conclusion, the data provide a method for analysis of contact activation-mediated TG in murine whole blood. As the FXII-driven intrinsic pathway of coagulation has emerged as novel target for antithrombotic agents that are validated in mouse thrombosis and bleeding models, our novel assay could expedite therapeutic drug development

Targeting NETs using dual-active DNase1 variants

Background: Neutrophil Extracellular Traps (NETs) are key mediators of immunothrombotic mechanisms and defective clearance of NETs from the circulation underlies an array of thrombotic, inflammatory, infectious, and autoimmune diseases. Efficient NET degradation depends on the combined activity of two distinct DNases, DNase1 and DNase1-like 3 (DNase1L3) that preferentially digest double-stranded DNA (dsDNA) and chromatin, respectively. Methods: Here, we engineered a dual-active DNase with combined DNase1 and DNase1L3 activities and characterized the enzyme for its NET degrading potential in vitro. Furthermore, we produced a mouse model with transgenic expression of the dual-active DNase and analyzed body fluids of these animals for DNase1 and DNase 1L3 activities. We systematically substituted 20 amino acid stretches in DNase1 that were not conserved among DNase1 and DNase1L3 with homologous DNase1L3 sequences. Results: We found that the ability of DNase1L3 to degrade chromatin is embedded into three discrete areas of the enzyme's core body, not the C-terminal domain as suggested by the state-of-the-art. Further, combined transfer of the aforementioned areas of DNase1L3 to DNase1 generated a dual-active DNase1 enzyme with additional chromatin degrading activity. The dual-active DNase1 mutant was superior to native DNase1 and DNase1L3 in degrading dsDNA and chromatin, respectively. Transgenic expression of the dual-active DNase1 mutant in hepatocytes of mice lacking endogenous DNases revealed that the engineered enzyme was stable in the circulation, released into serum and filtered to the bile but not into the urine. Conclusion: Therefore, the dual-active DNase1 mutant is a promising tool for neutralization of DNA and NETs with potential therapeutic applications for interference with thromboinflammatory disease states

Defective NET clearance contributes to sustained FXII activation in COVID-19-associated pulmonary thrombo-inflammation

BACKGROUND: Coagulopathy and inflammation are hallmarks of Coronavirus disease 2019 (COVID-19) and are associated with increased mortality. Clinical and experimental data have revealed a role for neutrophil extracellular traps (NETs) in COVID-19 disease. The mechanisms that drive thrombo-inflammation in COVID-19 are poorly understood. METHODS: We performed proteomic analysis and immunostaining of postmortem lung tissues from COVID-19 patients and patients with other lung pathologies. We further compared coagulation factor XII (FXII) and DNase activities in plasma samples from COVID-19 patients and healthy control donors and determined NET-induced FXII activation using a chromogenic substrate assay. FINDINGS: FXII expression and activity were increased in the lung parenchyma, within the pulmonary vasculature and in fibrin-rich alveolar spaces of postmortem lung tissues from COVID-19 patients. In agreement with this, plasmaaac acafajföeFXII activation (FXIIa) was increased in samples from COVID-19 patients. Furthermore, FXIIa colocalized with NETs in COVID-19 lung tissue indicating that NETs accumulation leads to FXII contact activation in COVID-19. We further showed that an accumulation of NETs is partially due to impaired NET clearance by extracellular DNases as DNase substitution improved NET dissolution and reduced FXII activation in vitro. INTERPRETATION: Collectively, our study supports that the NET/FXII axis contributes to the pathogenic chain of procoagulant and proinflammatory responses in COVID-19. Targeting both NETs and FXIIa may offer a potential novel therapeutic strategy. FUNDING: This study was supported by the European Union (840189), the Werner Otto Medical Foundation Hamburg (8/95) and the German Research Foundation (FR4239/1-1, A11/SFB877, B08/SFB841 and P06/KFO306)

Alternative Splicing of FOXP3—Virtue and Vice

FOXP3 is the lineage-defining transcription factor of CD4+ CD25+ regulatory T cells. While many aspects of its regulation, interaction, and function are conserved among species, alternatively spliced FOXP3 isoforms are expressed only in human cells. This review summarizes current knowledge about alternative splicing of FOXP3 and the specific functions of FOXP3 isoforms in health and disease. Future perspectives in research and the therapeutic potential of manipulating alternative splicing of FOXP3 are discussed

Dimerization of FOXP3fl, FOXP3Δ2 and FOXP3Δ2Δ7 with FOXP3Δ2Δ7.

<p>(A) HEK293T cells were co-transfected with FLAG-tagged FOXP3fl (left panel) or empty vector (right panel) and either control vector, FOXP3fl, FOXP3Δ2 or FOXP3Δ2Δ7. Co-immunoprecipitation experiments (IP) were performed with α-FLAG agarose and complexes were analyzed in immunoblots (IB) with FOXP3-specific antibody eBio7979 (upper panels). Expression of non-FLAG-tagged constructs was confirmed by immunoblotting of total cell lysates (lower panels). Co-immunoprecipitation shown is representative for three independent experiments. (B) HEK293T cells were co-transfected with FLAG-tagged FOXP3Δ2Δ7 and either control vector, FOXP3Δ2 or FOXP3Δ2Δ7. IP was performed with α-FLAG agarose and complexes were analyzed in IB as described above. Co-immunoprecipitation shown is representative for three independent experiments.</p

Expression of FOXP3fl, FOXP3Δ2 and FOXP3Δ2Δ7 in human Treg cells.

<p>(A) Protein expression levels of FOXP3 isoforms in human Treg cells. CD25+CD4+ T cells were isolated from PBMC by MACS using α-CD25 Treg isolation kit. FOXP3-specific eBio7979 antibody was used after Western blotting. The number of CD25+ cells loaded per lane is indicated. (B) RT-PCR analysis of human CD4+CD25+ cells. Relative transcription levels of FOXP3 isoforms in five healthy donors were investigated.</p