Tetramerization of STAT5 promotes autoimmune-mediated neuroinflammation

Signal tranducer and activator of transcription 5 (STAT5) plays a critical role in mediating cellular responses following cytokine stimulation. STAT proteins critically signal via the formation of dimers, but additionally, STAT tetramers serve key biological roles, and we previously reported their importance in T and natural killer (NK) cell biology. However, the role of STAT5 tetramerization in autoimmune-mediated neuroinflammation has not been investigated. Using the STAT5 tetramer-deficient Stat5a-Stat5b N-domain double knockin (DKI) mouse strain, we report here that STAT5 tetramers promote the pathogenesis of experimental autoimmune encephalomyelitis (EAE). The mild EAE phenotype observed in DKI mice correlates with the impaired extravasation of pathogenic T-helper 17 (Th17) cells and interactions between Th17 cells and monocyte-derived cells (MDCs) in the meninges. We further demonstrate that granulocyte-macrophage colony-stimulating factor (GM-CSF)–mediated STAT5 tetramerization regulates the production of CCL17 by MDCs. Importantly, CCL17 can partially restore the pathogenicity of DKI Th17 cells, and this is dependent on the activity of the integrin VLA-4. Thus, our study reveals a GM-CSF-STAT5 tetramer-CCL17 pathway in MDCs that promotes autoimmune neuroinflammation.

Simultaneous assessment of eight phosphorylated STAT residues in T-cells by flow cytometry.

Signal transducer and activator of transcription (STAT) proteins are a family of transcription factors controlling functions in immune responses and other cell types. Given their importance, we developed a flow cytometry panel to assess eight phosphorylated STAT residues in human T-cells, including six tyrosine residues across six STAT proteins (STAT1, STAT2, STAT3, STAT4, STAT5a, STAT6) and additional serine residues on STAT1 and STAT3. We applied this protocol to test the in vitro induction of pSTATs in response to CD3/CD28 activation and a panel of recombinant cytokines. We also assessed the pSTAT expression profiles of naive CD4+ T-cells polarized to Th1, Th2, Th17 or iTregs. pSTAT1(S727), pSTAT2(Y689) and pSTAT3(S727) were constitutively expressed in most T-cells, even in the absence of stimulation. For pSTAT1(S727) and pSTAT3(S727), we observed two positive states, high and low. Conversely, expression of pSTAT1(Y701), pSTAT3(Y705), pSTAT4(Y693) and pSTAT6(Y641) were absent in resting T-cells and only expressed with CD3/CD28 activation or with specific cytokines. Variable frequencies of pSTAT5a(Y694) expression were observed in resting T-cells, which increased with activation or specific cytokine stimulation (e.g. IL-2). IFN-beta stimulation enhanced frequencies of expressing cells for all pSTATs. Correlations among several pSTATs, particularly pSTAT1(S727)high and pSTAT3(S727)high were observed. While polarization resulted in increases in canonically associated pSTATs, other non-canonical pSTAT changes were also observed. Collectively, we developed, optimized, and tested a sensitive and rapid approach for simultaneously assessing phosphorylation of six STAT proteins. Using this approach, we made several novel observations of T-cell pSTAT induction in response to stimuli.

An HLA-I signature favouring KIR-educated Natural Killer cells mediates immune control of HIV in children and contrasts with the HLA-B-restricted CD8+ T-cell-mediated immune control in adults

Natural Killer (NK) cells contribute to HIV control in adults, but HLA-B-mediated T-cell activity has a more substantial impact on disease outcome. However, the HLA-B molecules influencing immune control in adults have less impact on paediatric infection. To investigate the contribution NK cells make to immune control, we studied >300 children living with HIV followed over two decades in South Africa. In children, HLA-B alleles associated with adult protection or disease-susceptibility did not have significant effects, whereas Bw4 (p = 0.003) and low HLA-A expression (p = 0.002) alleles were strongly associated with immunological and viral control. In a comparator adult cohort, Bw4 and HLA-A expression contributions to HIV disease outcome were dwarfed by those of protective and disease-susceptible HLA-B molecules. We next investigated the immunophenotype and effector functions of NK cells in a subset of these children using flow cytometry. Slow progression and better plasma viraemic control were also associated with high frequencies of less terminally differentiated NKG2A+NKp46+CD56dim NK cells strongly responsive to cytokine stimulation and linked with the immunogenetic signature identified. Future studies are indicated to determine whether this signature associated with immune control in early life directly facilitates functional cure in children.

STAT1 Gain-of-Function Variants Drive Altered T Cell Prevalence, Metabolism, and Heightened IL-6 Sensitivity

Patients with Signal Transducer and Activator of Transcription 1 (STAT1) gain-of-function (GOF) pathogenic variants exhibit susceptibility to infections, autoimmunity, and cancer due to enhanced or prolonged STAT1 phosphorylation following cytokine stimulation. While interferons (IFNs) are canonical STAT1 activators, other cytokines that may also contribute to pathology in STAT1 GOF patients have been less well defined. Here we analyzed the immune profiles and cytokine responses of two patients with heterozygous GOF mutations in the STAT1 coiled-coil domain. A systems immunology approach revealed major changes in the T cell compartment and minor changes in the B cells, NK cells, and myeloid cells. Both patients with STAT1 GOF differed from healthy individuals in the abundance and phenotype of effector memory, Th17, and Treg populations. STAT1 GOF T cells displayed a pattern of increased activation and had elevated markers of glycolysis and lipid oxidation. Hypersensitivity of T cells to IL-6 was observed with intense, sustained STAT1 phosphorylation in memory T cell populations that exceeded that induced by IFNs. Together, these results show a role for STAT1 in T cell metabolism and suggest that IL-6 may play a critical role to promote T cell memory formation and activation in patients with STAT1 GOF.

The Effect of Influenza Vaccines on Maturation of Dendritic Cells Generated from Bone Marrow

Background: Possibility to control immune system by regulating the activity of Dendritic Cells (DC) with the help of vaccines or other immunobiological drugs opens great prospects for infectious, oncological and autoimmune control. The aim of this study was to evaluate in vitro the effect of adjuvant subunit and non-adjuvant split influenza vaccines on maturation of DCs from human bone marrow. Methods: From bone marrow cells of healthy volunteers, DCs were obtained using rGM-CSF and IL-4. On the 8th day of cultivation, 10μl of vaccines against influenza were introduced into the culture of Immature DCs (i-DCs): a non-adjuvant split vaccine (Vaxigripp, Sanofi Pasteur) and an immunoadjuvant subunit vaccine (Grippol plus, Petrovax), as well as immunomodulator Polyoxidonium. Results: Insertion of influenza vaccines into i-DC culture induced the acquisition by DCs typical morphological signs of maturation. DCs became large with eccentrically located of irregular shape nucleus, densified cytoplasm, numerous processes. By immunophenotypic examination decrease in monocyte/macrophage pool, cells with expression of CD34 immaturity marker, increase in expressing CD11c/CD86 costimulatory molecules and CD83 terminal differentiation molecules were observed. Although Polyoxidonium caused a decrease in number of CD11c/CD14 cells (18, 5%), but compared to vaccines, its activity was lower (p<0, 05). Grippol plus more actively induced differentiation of TLR2 and TLR8 expressing cells, whereas Vaxigripp-expression of TLR4 and TLR8 on DCs. Conclusion: The possibility of using in vitro model of DCs obtained from human bone marrow cells by cytokine stimulation for examination of the ability of influenza vaccines to induce DC maturation processes has been demonstrated.

Loss of Endothelial Barrier Function in the Inflammatory Setting: Indication for a Cytokine-Mediated Post-Transcriptional Mechanism by Virtue of Upregulation of miRNAs miR-29a-3p, miR-29b-3p, and miR-155-5p

Dysfunction of the endothelial barrier plays a central role in the pathogenesis of both acute and chronic inflammatory processes such as sepsis or atherosclerosis. Due to attenuation of endothelial cell contacts, there is an increased transfer of blood proteins and fluid into the surrounding tissue, which relates to edema formation and distribution disorders. However, the mechanisms underlying these responses are not fully understood. In this study, we used human endothelial cells to mimic the loss of barrier function in an inflammatory milieu. We found that a weakened endothelial barrier after cytokine stimulation was accompanied by a significantly changed transcriptome. Apparent was a depletion of mRNAs encoding cell adhesion molecules. Furthermore, we found that cytokine treatment of endothelial cells induced upregulation of miR-29a-3p, miR-29b-3p, and miR-155-5p. miRNAs are known to negatively affect stability and translational efficiency of target mRNAs. Remarkably, miR-29a-3p, miR-29b-3p, and miR-155-5p have already been described to target the mRNAs of central tight and adherent junction proteins including F11 receptor, claudin 1, β-catenin, p120-catenin, and eplin. This taken together points to the existence of a posttranscriptional mechanism for expression inhibition of central adhesion proteins, which is triggered by inflammatory cytokines and mediated by miR-29a-3p, miR-29b-3p, and miR-155-5p.

Antifungical and immunomodulatory activities from Caesalpinia pulcherrima extracts

Caesalpinia pulcherrima is a species widely used in folk medicine for various diseases such as fever, infections and mouth ulcers. In addition, scientific studies have reported medicinal properties such as antitumor, antiviral, antibacterial and antifungal. Therefore, the objective of this work was to evaluate if organic extracts obtained from leaves of C. pulcherrima have medicinal properties. Three organic extracts were obtained (hexane, ethyl acetate and ethanol) from the leaves collected from the Soxhlet equipment. The characterization was made by GC-MS and UPLC-MS and biological properties as antioxidant (AAT, DPPH, ABTS and lipid peroxidation radicals), antimicrobial, cytotoxicity and immunostimulant (using splenocytes of mice Balb/c) were investigated. Results showed several classes of secondary metabolites, but the ethyl acetate showed more phenols and flavonoids than the other extracts. Extracts showed good results in antioxidant tests, especially the ethyl acetate, but did not show antibacterial activity. The fungal evaluation showed high antifungal properties, especially the hexanic and the ethyl acetate against Candida tropicalis, Aspergillus terreu, Candida krusei and Cryptococcus neoformans strains. Cytotoxicity evaluation showed that under 25 μg/mL are the safety doses that can be used for in vitro tests, besides that, extracts were able to induce cytokine stimulation. These results show that the ethyl acetate extract was the best formulation found in this study to be used against fungal infections, for antioxidant proposes and for promote immunostimulation.

Transcriptome Signature of Vγ9Vδ2 T Cells Treated With Phosphoantigens and Notch Inhibitor Reveals Interplay Between TCR and Notch Signaling Pathways

Gamma delta (γδ) T cells, especially the Vγ9Vδ2 subtype, have been implicated in cancer therapy and thus have earned the spotlight in the past decade. Although one of the most important properties of γδ T cells is their activation by phosphoantigens, which are intermediates of the Mevalonate and Rohmer pathway of isoprenoid biosynthesis, such as IPP and HDMAPP, respectively, the global effects of such treatments on Vγ9Vδ2 T cells remain elusive. Here, we used the high-throughput transcriptomics approach to elucidate the transcriptional changes in human Vγ9Vδ2 T cells upon HDMAPP, IPP, and anti-CD3 treatments in combination with interleukin 2 (IL2) cytokine stimulation. These activation treatments exhibited a dramatic surge in transcription with distinctly enriched pathways. We further assessed the transcriptional dynamics upon inhibition of Notch signaling coupled with activation treatments. We observed that the metabolic processes are most affected upon Notch inhibition via GSI-X. The key effector genes involved in gamma–delta cytotoxic function were downregulated upon Notch blockade even in combination with activation treatment, suggesting a transcriptional crosstalk between T-cell receptor (TCR) signaling and Notch signaling in Vγ9Vδ2 T cells. Collectively, we demonstrate the effect of the activation of TCR signaling by phosphoantigens or anti-CD3 on the transcriptional status of Vγ9Vδ2 T cells along with IL2 stimulation. We further show that the blockade of Notch signaling antagonistically affects this activation.

(20S) Ginsenoside Rh2 Inhibits STAT3/VEGF Signaling by Targeting Annexin A2

Signal transducers and activators of transcription 3 (STAT3) acts as a transcriptional signal transducer, converting cytokine stimulation into specific gene expression. In tumor cells, aberrant activation of the tyrosine kinase pathway leads to excessive and continuous activation of STAT3, which provides further signals for tumor cell growth and surrounding angiogenesis. In this process, the tumor-associated protein Annexin A2 interacts with STAT3 and promotes Tyr705 phosphorylation and STAT3 transcriptional activation. In this study, we found that (20S) ginsenoside Rh2 (G-Rh2), a natural compound inhibitor of Annexin A2, inhibited STAT3 activity in HepG2 cells. (20S) G-Rh2 interfered with the interaction between Annexin A2 and STAT3, and inhibited Tyr705 phosphorylation and subsequent transcriptional activity. The inhibitory activity of STAT3 leaded to the negative regulation of the four VEGFs, which significantly reduced the enhanced growth and migration ability of HUVECs in co-culture system. In addition, (20S)G-Rh2 failed to inhibit STAT3 activity in cells overexpressing (20S)G-Rh2 binding-deficient Annexin A2-K301A mutant, further proving Annexin A2-mediated inhibition of STAT3 by (20S)G-Rh2. These results indicate that (20S)G-Rh2 is a potent inhibitor of STAT3, predicting the potential activity of (20S)G-Rh2 in targeted therapy applications.

HIF1α is required for NK cell metabolic adaptation during virus infection

Natural killer (NK) cells are essential for early protection against virus infection, and must metabolically adapt to the energy demands of activation. Here, we found upregulation of the metabolic adaptor hypoxia inducible factor-1α (HIF-1α) is a feature of mouse NK cells during murine cytomegalovirus (MCMV) infection in vivo. HIF-1 α -deficient NK cells failed to control viral load, causing increased morbidity. No defects were found in effector functions of HIF-1α KO NK cells however, their numbers were significantly reduced. Loss of HIF-1 α did not affect NK cell proliferation during in vivo infection and in vitro cytokine stimulation. Instead, we found HIF-1α -deficient NK cells showed increased expression of the pro-apoptotic protein Bim and glucose metabolism was impaired during cytokine stimulation in vitro. Similarly, during MCMV infection HIF-1α -deficient NK cells upregulated Bim and had increased caspase activity. Thus, NK cells require HIF-1α-dependent metabolic functions to repress Bim expression and sustain cell numbers for an optimal virus response.