Chemokine Receptor Expression Research Articles

Transcriptomic analysis of reproductive organs of pregnant mice post toxoplasma gondii infection reveals the potential factors that contribute to poor prognosis.

Toxoplasma gondii is an obligate intracellular parasite of phylum Apicomplexa that poses a huge threat to pregnant hosts, and induces tragic outcomes for pregnant hosts, fetuses and newborns. However, the molecular mechanism underlying the tragic consequences caused by T. gondii remains to be revealed. In the present study, we applied RNA-seq to study the transcriptomic landscape of the whole reproductive organ of pregnant mice post T. gondii infection, aiming to reveal the key altered biological characters of reproductive organs of pregnant mice that could contribute to the tragic outcomes caused by T. gondii infection. The results of the present study showed that the transcriptome of reproductive organs of pregnant mice was significantly altered by T. gondii infection. A total of 2,598 differentially expressed genes (DEGs) were identified, including 1,449 upregulated genes and 1,149 downregulated genes. Enrichment analysis of the DEGs showed that the significantly altered features of reproductive organs of pregnant mice were excessive inflammatory responses, downregulated metabolism processes, and congenital diseases. The chemotaxis of immune cells in the reproductive organs of infected pregnant mice could also be reshaped by 19 differentially expressed chemokines and 6 differentially expressed chemokine receptors that could contribute to the damages of reproductive organ in pregnant mice. Overall, the findings of present study may help to understand the pathogenic mechanism of the acute T. gondii infection in reproductive organs of pregnant mice, and it could also help to improve toxoplasmosis therapeutics for pregnant individuals.

P-Glycoprotein Exacerbates Brain Injury Following Experimental Cerebral Ischemia by Promoting Proinflammatory Microglia Activation.

Microglia are activated following cerebral ischemic insult. P-glycoprotein (P-gp) is an efflux transporter on microvascular endothelial cells and upregulated after cerebral ischemia. This study evaluated the effects and possible mechanisms of P-gp on microglial polarization/activation in mice after ischemic stroke. P-gp-specific siRNA and adeno-associated virus (p-AAV) were used to silence and overexpress P-gp, respectively. Middle cerebral artery occlusion/reperfusion (MCAO/R) and oxygen-glucose deprivation/reoxygenation (OGD/R) were performed in mice and cerebral microvascular endothelial cells (bEnd.3) in vitro, respectively. OGD/R-injured bEnd.3 cells were cocultured with mouse microglial cells (BV2) in Transwell. Influences on acute ischemic stroke outcome, the expression of inflammatory cytokines, and chemokines and chemokines receptors, microglial polarization, glucocorticoid receptor (GR) nuclear translocation, and GR-mediated mRNA decay (GMD) activation were evaluated via reverse transcription real-time polymerase chain reaction, western blot, or immunofluorescence. Silencing P-gp markedly alleviated experimental ischemia injury as indicated by reduced cerebral infarct size, improved neurological deficits, and reduced the expression of interleukin-6 (IL-6) and IL-12 expression. Silencing P-gp also mitigated proinflammatory microglial polarization and the expression of C-C motif chemokine ligand 2 (CCL2) and its receptor CCR2 expression, whereas promoted anti-inflammatory microglia polarization. Additionally, P-gp silencing promoted GR nuclear translocation and the expression of GMD relative proteins in endothelial cells. Conversely, overexpressing P-gp via p-AAV transfection offset all these effects. Furthermore, silencing endothelial GR counteracted all effects mediated by silencing or overexpressing P-gp. Elevated P-gp expression aggravated inflammatory response and brain damage after ischemic stroke by augmenting proinflammatory microglial polarization in association with increased endothelial CCL2 release due to GMD inhibition by P-gp.

Aqueous Macrophages Contribute to Conserved CCL2 and CXCL10 Gradients in Uveitis

Uveitis is a heterogenous group of inflammatory eye disease for which current cytokine-targeted immune therapies are effective for only a subset of patients. We hypothesized that despite pathophysiologic nuances that differentiate individual disease states, all forms of eye inflammation might share common mechanisms for immune cell recruitment. Identifying these mechanisms is critical for developing novel, broadly acting therapeutic strategies. Experimental study. Biospecimens from patients with active or inactive uveitis and healthy controls. Protein concentration and single cell gene expression were assessed in aqueous fluid biopsies and plasma samples from deidentified patients with uveitis or healthy controls. The concentration of 31 inflammatory proteins was measured in all aqueous samples, as well as plasma samples from patients with active uveitis. Chemokine and cytokine ligand and receptor expression were assessed in individual cell types from aqueous biopsies obtained from patients with active uveitis. We identified 6 chemokines that were both elevated in active uveitis compared with controls and enriched in aqueous compared with plasma during active uveitis (C-C motif chemokine ligand [CCL]2, C-X-C motif chemokine ligand [CXCL]10, CXCL9, CXCL8, CCL3, and CCL14), forming potential gradients for migration of immune cells from the blood to the eye. Of these, CCL2 and CXCL10 were consistently enriched in the aqueous of all patients in our cohort, as well as in a larger cohort of patients from a previously published study. These data suggest that CCL2 and CXCL10 are key mediators in immune cell migration to the eye during uveitis. Next, single cell RNA sequencing suggested that macrophages contribute to aqueous enrichment of CCL2 and CXCL10 during human uveitis. Finally, using chemokine ligand and receptor expression mapping, we identified a broad signaling network for macrophage-derived CCL2 and CXCL10 in human uveitis. These data suggest that ocular macrophages may play a central role, via CCL2 and CXCL10 production, in recruiting inflammatory cells to the eye in patients with uveitis. Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Prostaglandin E₂ impacts multiple stages of the natural killer cell antitumor immune response.

Tumor immune escape is a major factor contributing to cancer progression and unresponsiveness to cancer therapies. Tumors can produce prostaglandin E2 (PGE2 ), an inflammatory mediator that directly acts on Natural killer (NK) cells to inhibit antitumor immunity. However, precisely how PGE2 influences NK cell tumor-restraining functions remains unclear. Here, we report that following PGE₂ treatment, human NK cells exhibited altered expression of specific activating receptors and a reduced ability to degranulate and kill cancer targets. Transcriptional analysis uncovered that PGE₂ also differentially modulated the expression of chemokine receptors by NK cells, inhibiting CXCR3 but increasing CXCR4. Consistent with this, PGE₂-treated NK cells exhibited decreased migration to CXCL10 but increased ability to migrate toward CXCL12. Using live cell imaging, we showed that in the presence of PGE2 , NK cells were slower and less likely to kill cancer target cells following conjugation. Imaging the sequential stages of NK cell killing revealed that PGE₂ impaired NK cell polarization, but not the re-organization of synaptic actin or the release of perforin itself. Together, these findings demonstrate that PGE₂ affects multiple but select NK cell functions. Understanding how cancer cells subvert NK cells is necessary to more effectively harness the cancer-inhibitory function of NK cells in treatments.

Mechanistic insights into the anti-restenotic effects of HSP27 and HO1 modulated by reconstituted HDL on neointimal hyperplasia

High-density lipoprotein (HDL) therapy has demonstrated beneficial effects in acute stroke and acute myocardial infarction models by reducing infarct size. In this study, we investigated the inhibitory effects of reconstituted HDL (rHDL) on neointimal hyperplasia and elucidated its underlying mechanism using a balloon injury rat model. Our finding revealed a significant 37% reduction in the intima to media ratio in the arteries treated with 80 mg/kg rHDL compared to those subjected to injury alone (p < 0.05), indicating a specific inhibition of neointimal hyperplasia. In vivo analysis further supported the positive effects of rHDL by demonstrating a reduction in smooth muscle cell (SMC) proliferation and an increase in endothelial cell (EC) proliferation. Additionally, rHDL treatment led to decreased infiltration of leukocytes and downregulated the expression of matrix metallopeptidase 9 (MMP9) in the neointimal area. Notably, rHDL administration resulted in decreased expression of VCAM1 and HIF1α, alongside increased expression of heme oxygenase 1 (HO1) and heat shock protein 27 (HSP27). Overexpression of HSP27 and HO1 effectively inhibited SMC proliferation. Moreover, rHDL-mediated suppression of injury-induced HIF1α coincided with upregulation of HSP27. Interestingly, HSP27 and HO1 had varying effects on the expression of chemokine receptors and rHDL did not exert significant effect on chemokine receptor expression in THP1 cells. These findings underscore the distinct roles of HSP27 and HO1 as potential regulatory factors in the progression of restenosis. Collectively, our study demonstrates that rHDL exerts a potent anti-neointimal hyperplasia effect by reducing leukocytes infiltration and SMC proliferation while promoting EC proliferation.

Role of non-canonical T cells in homeostasis and pathology

In addition to the subsets of T lymphocytes and innate lymphocytes (innate lymphoid cells), the well-known players in adaptive immunity, there is an intermediate group of lymphocytes (innate-like cells) that already possess the T cell receptor, but with a restricted repertoire. This group includes γδT cells, subsets of type I and II NKT cells carrying both T cell receptor and NK-cell receptors, and mucosal-associated invariant T (MAIT) cells. The development of innate-like cells occurs in the thymus, but their positive and negative selection takes place without the participation of thymic epithelial cells. A distinctive feature is that innate-like cells acquire an effector phenotype already in the thymus, and therefore do not require complex activation reactions during antigen recognition. Upon exit from the thymus, noncanonical T cells express chemokine receptors, allowing them to migrate into barrier tissues at an early age. A characteristic feature of the T cell receptor innate-like cells is the recognition of non-peptide antigens presented in non-polymorphic histocompatibility molecules (MHC-Ib). This type of molecule includes the CD1 a/b/c/d/e molecule and the MR1 molecule. These molecules present lipid, glycolipid antigens and metabolites of B vitamins, synthesized by various representatives of the microbiota. The presence of functionally different subpopulations of innate-like cells with an activated phenotype allows them to quickly respond to the antigen by producing cytokines typical of Th1, Th2, Th17. They also exhibit cytotoxic and immunoregulatory activity. These cells are actively involved in regulation of barrier tissue homeostasis and interaction with microbiota. They synthesize growth factors for epithelial cells, fibroblasts, and vascular endothelium, which are required for regeneration of damaged tissues. They also participate in anti-infectious defense, directing the development of the immune response. Moreover, they have been found to be involved in many autoimmune diseases. The special functions of innate-like cells make them a promising target for therapeutic interventions. It has been shown that antibiotics, salicylates and some other well-known drugs exert certain effects on the innate-like cells. Different dietary options also affect the activity of these cells.

CD49d Shapes B Cell Receptor Responsiveness and Tissue Distribution in an Aggressive Chronic Lymphocytic Leukemia Murine Model

Background: High expression of the very late antigen-4 (VLA-4) integrin subunit CD49d predicts disease progression in chronic lymphocytic leukemia (CLL). We previously demonstrated that B cell receptor (BCR) signals induce activation of VLA-4 (Tissino et al., J Exp Med. 2018) and characterized Eµ-TCL1-transgenic (TCL1-tg) mice as an adequate model for the human high-risk CD49d-high CLL patient subgroup, with VLA-4 inhibition hampering the disease (Szenes et al., Leukemia 2020). Here, we address the consequences of genetic B cell-specific ablation of CD49d on disease development, engraftment capacity and treatment susceptibility in a novel CLL mouse model. Methods: We crossed ITGA4fl/fl mice, kindly provided by T. Papayannopoulou, University of Washington, with TCL1-tg mice and CD19 Cre mice, subsequently named CD49d∆B TCL1-tg. Tumor development was continuously monitored by regular blood checks and mice were sacrificed at 10 months. Tumor infiltration was quantified in peritoneal cavity (PC), spleen and bone marrow by flow cytometry using anti-CD5/CD19 antibodies. Integrin and chemokine receptor expression was profiled. In vitro responsiveness to BCR stimulation of CD49d-deficient and proficient leukemic cells was assessed using phospho-specific flow cytometry. Furthermore, purified leukemic cells derived from PC or spleen were subjected to RNA-seq analyses. CD49d-deficient or proficient leukemic cells were transplanted into wild-type C57BL/6J recipients in short and long term experiments. Ongoing: CD49d-deficient or proficient leukemic cells were transplanted competitively into C57BL/6J recipients and are receiving continuous Bruton's tyrosine kinase (BTK)-inhibitor (acalabrutinib) treatment upon tumor onset. Results: CD49d∆B TCL1-tg mice showed a B cell-specific integrin loss in all organs. Blood tumor burden was measured monthly. At months seven and eight, leukemic cells of CD49d∆B TCL1-tg mice were reduced by approx. 25% compared to TCL1-tg littermates (Fig. 1A). This trend was reversed at month nine, with approx. 45% increased lymphocytosis in CD49d∆B TCL1-tg mice. CD49d expression in leukemic cells of TCL1-tg mice was unaffected by disease progression. At sacrifice, CD49d∆B TCL1-tg mice showed an 80% ( p=&amp;lt;0.0001) reduction of tumor burden in the bone marrow (Fig. 1B), but comparable leukemic cell numbers in PC and spleen. Leukemic splenocytes derived from CD49d∆B TCL1-tg mice displayed 38% ( p=0.032) higher IgM and 33% higher CD44 expression but 43% ( p=0.015) reduced expression of CXCR4. BCR signaling was modulated by the integrin loss and BCR stimulation with anti-IgM induced 43% ( p=0.022) higher PLCγ2 and 45% ( p=0.022) higher SYK phosphorylation in leukemic cells derived from CD49d∆B TCL1-tg mice compared to littermates, but comparable BTK phosphorylation. RNA-seq analysis revealed strong differences in leukemic cell transcriptomes between PC and spleen, indicating tissue dependent effects on leukemic cells. Particularly, TNF-α signaling via NF-κB, MTORC1, MAPK signaling and JAK-STAT signaling pathways were upregulated in leukemic cells derived from spleen compared to PC irrespective of CD49d expression, while chemokine signaling pathways were downregulated in PC. When comparing CD49d deficient and proficient leukemic cells, the gene set MYC targets v1 was enriched in deficient cells. When injecting CD49d-deficient leukemic splenocytes intravenously, more cells were recovered after 5 days from blood ( p=0.048) and spleen compared to CD49d-proficient cells, but did not engraft in bone marrow or peritoneum. When injected intraperitonally, CD49d-deficient leukemic cells were found in peritoneum in contrast to TCL1-tg littermates ( p=&amp;lt;0.0001). Conclusion: The TCL1 mouse model resembles a human high-risk CLL (e.g. IGHV unmutated, CD49d-expressing), and thus allows to investigate the impact of CD49d expression in this CLL subset. Loss of CD49d expression resulted in an altered redistribution of leukemic cells in tissue sites with a significant reduction of leukemic cells in the bone marrow. Lack of integrin signals resulted in increased antigen-induced BCR signaling and IgM expression, as well as a B cell receptor activation phenotype associated with MYC gene set enrichment, indicating a feedback loop from integrins to the BCR. Currently, the susceptibility of CD49d∆B TCL1-tg mice to BTK inhibitors is under investigation.

Crosstalk between NFAT and NFκB Signaling Regulates NK Cell Immunosurveillance

One of the major transcriptional regulators in lymphoid cells is NFAT (Nuclear Factor of Activated T Cells), controlling lymphocyte development and activity. The role of NFAT signaling is well defined in T cells, the cytotoxic lymphocytes of the adaptive immunity. However, surprisingly little is known regarding the relevance of this transcription factor family in NK cells as effector cells of the innate immunity. Available data indicate that NFAT activity is dispensable for development of NK cells, whereas effects of the immunosuppressive drugs cyclosporin A and tacrolimus, that inhibit calcineurin and consecutively NFAT, implicate an involvement of the NFAT family in NK cell function. We here employed different genetic mouse models and functional analyses to unravel the role of NFAT1 (NFATc2) and NFAT2 (NFATc1) in NK cell reactivity. In vitro knockout (KO) of NFAT1 (NFATc2) or NFAT2 (NFATc1) enhanced NK cell degranulation and resulted in increased production of granzyme B and perforin upon stimulation of activating receptors like NK1.1 or Nkp46 or upon co-culture with different leukemia and solid tumor cells. In line, cytotoxicity assays revealed increased lysis of YAC-1 and B16F10 tumor cells by both NFAT1- and NFAT2-deficient NK cells as compared to wildtype (WT) controls. The inhibitory effect of NFAT transcription factors on NK cell effector function could also be confirmed in vivo by employing WT and NFAT KO animals in syngeneic B16F10 melanoma, RMA-S lymphoma and LL/2-luc lung carcinoma models, which revealed a significantly reduced tumor burden in NFAT1 and NFAT2 KO mice. Furthermore, we detected higher numbers of tumor infiltrating NK cells in NFAT KO mice, which suggested improved NK cell migration and homing to the tumor side. Comparative analyses with single NFAT as well as NFAT1+NFAT2 double KO and WT animals further confirmed the inhibitory effect of NFAT1 and NFAT2 and pointed to additive effects of NFAT1 and NFAT2 in NK cell tumor immunosurveillance. Proteomics analysis of NK cells from the different NFAT KO strains revealed altered expression of molecules which are regulating chemokine receptor expression as well as a decreased expression of NFκB inhibitory molecules, like NFKBIB (NFKB inhibitor beta) Commd6 (COMM domain containing 6). NFκB signaling was shown to regulate granzyme B and perforin synthesis and expression. Thus, we suggest that NFAT1 and NFAT2 inhibit NFκB signaling in NK cells, and NFAT1 and NFAT2 KO reinforce NFκB induced granzyme B and perforin expression and thereby increase NK cell cytotoxicity. Taken together, our results identify NFAT as a negative regulator of NK cell function. In addition, we provide the first evidence for a direct functional involvement of NFAT1 and NFAT2 in NK cell antitumor reactivity by regulation tumor infiltration and cytotoxicity of NK cell.

High-dimensional phenotyping of the peripheral immune response in community-acquired pneumonia.

Community-acquired pneumonia (CAP) represents a major health burden worldwide. Dysregulation of the immune response plays an important role in adverse outcomes in patients with CAP. We analyzed peripheral blood mononuclear cells by 36-color spectral flow cytometry in adult patients hospitalized for CAP (n=40), matched control subjects (n=31), and patients hospitalized for COVID-19 (n=35). We identified 86 immune cell metaclusters, 19 of which (22.1%) were differentially abundant in patients with CAP versus matched controls. The most notable differences involved classical monocyte metaclusters, which were more abundant in CAP and displayed phenotypic alterations reminiscent of immunosuppression, increased susceptibility to apoptosis, and enhanced expression of chemokine receptors. Expression profiles on classical monocytes, driven by CCR7 and CXCR5, divided patients with CAP into two clusters with a distinct inflammatory response and disease course. The peripheral immune response in patients with CAP was highly similar to that in patients with COVID-19, but increased CCR7 expression on classical monocytes was only present in CAP. CAP is associated with profound cellular changes in blood that mainly relate to classical monocytes and largely overlap with the immune response detected in COVID-19.

Differential expression of interleukin-35 receptor distinguishes different subsets of granulocyte-macrophage-colony-stimulating factor-producing T helper cells in a mouse endometriosis model

The immune system contributes to the pathophysiology of endometriosis. The role of ThGM cells, which produce granulocyte macrophage-colony-stimulating factor (GM-CSF), in the pathogenesis of endometriosis remains unknown. To analyze the features of ThGM cells in endometriosis, a mouse endometriosis model was established. ThGM cells in the spleen, peritoneal fluid (PF), and endometriotic lesions (EL) were measured by flow cytometry, based on the expression of surface markers and intracellular proteins. Live ThGM cells were sorted according to chemokine receptor expression profiles and their effects on other CD4+ T cell subsets were determined by co-culture assays. An adoptive transfer assay was performed to characterize the effect of ThGM cells on endometriosis. We found that ThGM cells were present in endometriotic PF and EL. Live EL ThGM cells were enriched in CD4+CXCR3-CCR8-CCR4+CCR10+ T cells. EL ThGM cells differentially express interleukin-35 receptor (IL-35R), consisting of an IL-35R+ subset and an IL-35R- subset. The IL-35R+ subset expressed less GM-CSF, interleukin-2 (IL-2), and tumor necrosis factor-alpha (TNF-α) and proliferated slower than the IL-35R- subset. Meanwhile, the IL-35R+ subset was weaker than the IL-35R- subset in promoting the functions of Th1 and Th17 cells. ThGM cell transfer did not influence EL development but significantly alleviated pro-inflammatory cytokines in PF and ELs. Interleukin-35 (IL-35), the ligand of IL-35R, suppressed ThGM cell function and proliferation in an IL-35R-dependent manner. In summary, ThGM cells in the PF and ELs might exacerbate endometriotic inflammation. IL-35 might suppress the function of ThGM cells via IL-35R.

Sting-Mediated Interferon Signaling Exerts Potent Antileukemic Effects in High-Risk Myeloid and Monocytic Malignancies

Clinical outcomes of patients (pts) with high-risk (HR) myeloid neoplasms remain dismal despite available therapies. Interferons (IFN) regulate hematopoietic stem-progenitor cell (HSPC) differentiation, proliferation and self-renewal capabilities and preferentially impair clonal HSPC fitness. To evaluate if myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML) HSPCs activate IFN responses we performed RNA sequencing in bone marrow (BM) CD34 + cells obtained from pts with MDS (n=58), CMML (n=27) and age-matched healthy donors (HDs, n=17). This analysis revealed significant deregulation of IFN pathway genes in HSPCs from pts with CMML and to a lesser extent in those of pts with MDS. Activation of the stimulator of interferon genes (STING) can inhibit stem-cell function and induce myeloid and monocytic cell death via IRF3-dependent and independent mechanisms. Therefore, we hypothesized that STING pathway activation might have therapeutic potential in high risk (HR) myeloid and monocytic (MyMo) neoplasms. In vitro treatment of monocytic MOLM13 and THP1 cell lines with the novel synthetic STING agonist dazostingag resulted in induction of IRF3 phosphorylation, caspase 3 cleavage and apoptosis. Dazostingag was also able to induce enhanced cell death in multiple myeloid cell line cultures. Given the ability of dazostingag to induce apoptosis of monocytic cell lines, we sought to evaluate the activity of monocyte-directed STING activation using TAK-500, an IgG1 anti-CCR2 antibody linked to dazostingag as a STING agonist payload. To do so, we first analyzed the expression of the monocyte-specific cysteine-cysteine chemokine receptor 2 (CCR2) in primary pts samples and cell lines. Both CCR2 RNA and protein expression levels were higher in CMML compared to HDs or MDS. In accordance with this finding, monocytic cell lines (MOLM13, THP1) had significantly higher CCR2 expression compared to non-monocytic cell lines (SKM1, HL60, U937, KG1). Treatment of MOLM13 and THP1 cell lines with TAK-500 induced strong IRF3 phosphorylation, caspase 3 cleavage and apoptosis. In vitro treatment of BM mononuclear cells (MNCs) from pts with MDS (n=10), CMML (n=11) or AML (n=8) in co-culture with mesenchymal stromal cells with TAK-500 (0.5 mM) or dazostingag (1 mM) resulted in increased cell death of MNCs from pts with CMML and AML. TAK-500 (0.5 mM) significantly depleted CCR2 + BM MNCs from all disease groups but did not affect CCR2 + cells from HDs. Next, to evaluate whether STING activation impairs repopulation potential of neoplastic HSPCs we performed colony-formation studies in CD34 + BM cells from pts with CMML (n=8), MDS (n=3) and AML (n=4). Treatment with TAK-500 (0.5mM) and dazostingag (1mM) resulted in significantly impaired myeloid colony-formation in all disease groups without significantly impairing erythroid colony-formation in MDS or CMML samples. Taken together, these data suggests that STING activation might induce aberrant myelomonocytic cell death and deplete aberrant HSPCs in HR-MyMo neoplasms. To evaluate the in vivo efficacy of TAK-500 and dazostingag, we established xenograft models by inoculating NOG-IL15 transgenic mice with MOLM13 cells. Treatment with TAK-500 and dazostingag resulted in reduction of tumor burden evaluated by bioluminescence with no observed toxicities. Administration of weekly doses of TAK-500 induced enhanced and sustained tumor burden suppression. To determine the in vivo effect of STING activation in an immune competent model of CMML, we then evaluated the effects of TAK-500 and dazostingag in Vav-KDM6B/Tet2 f/f/Vav-Cre mice, a previously reported (Wei et al Leukemia 2022) animal model with CMML phenotype. Treatment with TAK-500 led to enhanced apoptosis of Ccr2 + BM cells in these mice. Moreover, the number of colonies formed by lineage negative/Sca1 positive/cKit positive BM progenitor (LSK) cells isolated from TAK-500 treated mice was significantly lower than in vehicle treated control mice, suggesting a negative regulation of repopulating capacity of Tet2 deficient/ KDM6B overexpressing BM HSPCs by TAK-500. Evaluation of the efficacy of TAK-500 and dazostingag in patient-derived xenograft models of primary CMML samples is planned. Taken together, our data suggests that CCR2-directed STING activation might have therapeutic potential in HR-MyMo neoplasms by depleting CCR2 + cells and impairing clonal HSPC repopulation potential.

T regulatory lymphocytes specific for SARS-CoV-2 display increased functional plasticity

The study of phenotypic and functional characteristics of immune cells involved in host response to SARS-CoV-2 is relevant for understanding COVID-19 pathogenesis and individual differences in disease progression. We have analyzed chemokine receptor expression in SARS-CoV-2-specific CD4+ T lymphocytes from vaccinated donors, and have found an increase of CCR9+ and CCR6+ cells. CCR9+ specific CD4+ cells are enriched in T regulatory (Treg) lymphocytes. These cells specifically show heterogeneous regulatory activity, associated with different profiles of CCR9/CCR6 expression, individual differences in IL-10 and IL-17 production, and variable FoxP3 and Notch4 expression. A higher heterogeneity in FoxP3 is selectively observed in convalescent individuals within vaccinated population. Accordingly, SARS-CoV-2-specific CD4+ lymphocytes from COVID-19 patients are also enriched in CCR9+ and CCR6+ cells. CCR6+ specific Treg lymphocytes are mainly increased in critically ill individuals, indicating a preferential role for these cells in lung injury pathogenesis. We provide experimental evidence for a SARS-CoV-2-specific Treg population with increased plasticity, which may contribute to the differential pathogenic response against SARS-CoV-2 among individuals, and underlie the development of autoimmune conditions following SARS-CoV-2 infection.

Regenerative potential of mesenchymal stromal cells in wound healing: unveiling the influence of normoxic and hypoxic environments.

The innate and adaptive immune systems rely on the skin for various purposes, serving as the primary defense against harmful environmental elements. However, skin lesions may lead to undesirable consequences such as scarring, accelerated skin aging, functional impairment, and psychological effects over time. The rising popularity of mesenchymal stromal cells (MSCs) for skin wound treatment is due to their potential as a promising therapeutic option. MSCs offer advantages in terms of differentiation capacity, accessibility, low immunogenicity, and their central role in natural wound-healing processes. To accelerate the healing process, MSCs promote cell migration, angiogenesis, epithelialization, and granulation tissue development. Oxygen plays a critical role in the formation and expansion of mammalian cells. The term "normoxia" refers to the usual oxygen levels, defined at 20.21 percent oxygen (160 mm of mercury), while "hypoxia" denotes oxygen levels of 2.91 percent or less. Notably, the ambient O2 content (20%) in the lab significantly differs from the 2%-9% O2 concentration in their natural habitat. Oxygen regulation of hypoxia-inducible factor-1 (HIF-1) mediated expression of multiple genes plays a crucial role in sustaining stem cell destiny concerning proliferation and differentiation. This study aims to elucidate the impact of normoxia and hypoxia on MSC biology and draw comparisons between the two. The findings suggest that expanding MSC-based regenerative treatments in a hypoxic environment can enhance their growth kinetics, genetic stability, and expression of chemokine receptors, ultimately increasing their effectiveness.

Regulatory Mechanism of Mangiferin Combined with Bortezomib on Malignant Biological Behavior of Burkitt Lymphoma and Its Effect on Expression of CXC Chemokine Receptors

To analyze the effects of mangiferin combined with bortezomib on the proliferation, invasion, apoptosis and autophagy of human Burkitt lymphoma Raji cells, as well as the expression of CXC chemokine receptors (CXCRs) family, and explore the molecular mechanism between them to provide scientific basis for basic research and clinical work of Burkitt lymphoma. Raji cells were intervened with different concentrations of mangiferin and bortezomib alone or in combination, then cell proliferation was detected by CCK-8 assay, cell invasion ability was detected by Transwell chamber method, cell apoptosis was detected by Annexin V/PI double-staining flow cytometry, apoptosis, autophagy and Akt/mTOR pathway protein expression were detected by Western blot, and the expression changes of CXCR family was detected by real-time quantitative PCR (RT-qPCR). Different concentrations of mangiferin intervened Raji cells for different time could inhibit cell viability in a concentration- and time-dependent manner (r =-0.682, r =-0.836). When Raji cells were intervened by combination of mangiferin and bortezomib, compared with single drug group, the proliferation and invasion abilities were significantly decreased, while the apoptosis level was significantly increased (P <0.01). Mangiferin combined with bortezomib could significantly up-regulate the expression of pro-apoptotic protein Bax and down-regulate the expression of anti-apoptotic protein Bcl-2 after intervention in Raji cells. Caspase-3 was also hydrolyzed and activated, and then induced the apoptosis of Raji cells. Mangiferin combined with bortezomib could up-regulate the expression of LC3Ⅱ protein in Raji cells, and the ratio of LC3Ⅱ/LC3Ⅰ in cells was significantly up-regulated compared with single drug or control group (P <0.01). Mangiferin combined with bortezomib could significantly inhibit the phosphorylation levels of Akt and mTOR, inhibit the proliferation and invasion of Raji cells by inhibiting Akt/mTOR pathway, and induce cell autophagy and apoptosis. Mangiferin and bortezomib could down-regulate the expressions of CXCR4 and CXCR7 mRNA after single-agent intervention in Raji cells, and the down-regulations of CXCR4 and CXCR7 mRNA expression were more significant when the two drugs were combined (P <0.01). Mangiferin alone or combined with bortezomib had no significant effect on CXCR5 mRNA expression in Raji cells (P >0.05), while the combination of the two drugs could down-regulate the expression of CXCR3 (P <0.05). Mangiferin combined with bortezomib can synergistically inhibit the proliferation and invasion of Raji cells, and induce autophagy and apoptosis. The mechanism may be related to the inhibition of Akt/mTOR signaling pathway, down-regulation of anti-apoptotic protein Bcl-2 and up-regulation of pro-apoptotic protein Bax, and the inhibition of the expression of CXCR family.

Risk factors for a differentiation syndrome in patients with acute promyelocytic leukemia

Differentiation syndrome (DS) is a severe complication of acute promyelocytic leukemia and its treatment, which is one of the causes of high early mortality. the similarity of clinical manifestations of DS and other complications that may develop during acute promyelocytic leukemia therapy makes it difficult to diagnose ds. at the same time, untimely initiation of DS therapy with glucocorticosteroids can lead to the patient’s death. The only generally accepted risk factor for ds is initial leukocytosis. Specific markers confirming ds have not yet been found. A number of studies show that in patients with diagnosed DS, the expression of CD56, CD54, CD2, CD15, CD13, markers of immature granulocytes, β2-integrins was more often found on blast cells. exposure to tretinoin increased the expression of chemokine receptors, chemokines, and cytokines by blast cells and vascular endothelium. The influence exerted by atypical promyelocytes, due to their biological characteristics, on the coagulation system suggests an association between hemostasis state and ds development. However, the value of the above markers as predictors or signs of DS still needs to be tested, especially when it comes to non-chemotherapeutic treatment of acute promyelocytic leukemia with arsenic trioxide.

Impact of azithromycin on the migration of peripheral blood T lymphocytes from patients with chronic obstructive pulmonary disease to RANTES and IP-10

The inflammatory process specific for chronic obstructive pulmonary disease (COPD) is accompanied by T lymphocyte migration from peripheral blood to the respiratory tract. Suppression of T cell chemotaxis by drugs may attenuate the inflammatory response in patients with COPD.The aim of this study was to determine the ability of azithromycin in combination with glucocorticoids to affect the migration of blood T cells in patients with COPD.The percentage of T lymphocytes expressing chemokine receptors CCR5, CCR6, CCR7, CXCR3, CXCR4, CXCR6 was analyzed by flow cytometry in the peripheral blood of 54 smokers with COPD, 21 healthy smokers, and 20 healthy non-smokers, as well as in bronchoalveolar lavage (BAL) of 7 smokers with COPD and 7 healthy smokers. Additionally, we determined the effect of azithromycin (10 μg/ml) and budesonide (10 nM) on the migration of peripheral blood T helper cells and cytotoxic T lymphocytes from patients with COPD (n = 8) to chemokines RANTES (10 nM) and IP-10 (10 nM).The percentage of T lymphocytes expressing chemokine receptors CXCR3 and CCR5 increased in the peripheral blood of COPD smokers compared with healthy smokers and healthy non-smokers, as well as in the BAL of COPD smokers compared with healthy smokers. The proportion of T cells expressing chemokine receptors CXCR4, CXCR6, CCR6, and CCR7 did not differ in the peripheral blood and the BAL between COPD patients and healthy controls. Budesonide only inhibited the migration of cytotoxic T lymphocytes to RANTES. Azithromycin, alone and combined with budesonide, inhibited the migration of T helper cells and cytotoxic T lymphocytes to both RANTES and IP-10. Moreover, the inhibitory effect of azithromycin, in combination with budesonide and without it, on the T cell migration was significantly greater than the effect of budesonide alone.Our results suggest a role for CXCR3 and CCR5 in T cell recruitment into the lungs of COPD patients and demonstrate the ability of azithromycin to inhibit T lymphocyte migration.

Combination therapy of irreversible electroporation and cytokine-induced killer cells for treating mice bearing panc02 pancreatic-cancer xenografts

The current study aimed to investigate the antitumor effects and potent mechanism of cytokine-induced killer (CIK) cells combined with irreversible electroporation (IRE) via Panc02 cell-bearing mouse model in vivo. CIK cells were isolated from the spleens of Panc02 pancreatic-cancer (PC) subcutaneous-xenograft model and the proportion of different lymphocytes was also determined. The antitumor effect of the combination of IRE and CIK cells in a PC subcutaneous-xenograft model was also investigated. The proportion of cells that were positive for CD3+CD8+ and the proportion of CD3+CD56+ cells were both significantly increased after 21 days of in vitro culture. Combined treatment of IRE and CIK cell significantly inhibited tumor growth and increased the survival rate of Panc02 cell–bearing mice. Furthermore, infiltration of lymphocytes into tumor tissue was significantly increased by this combination therapy compared with the untreated group or monotherapy group. In addition, IRE significantly enhanced the expression of chemokine receptors elicited by CIK cells. In conclusion, IRE combined with CIK cells showed superior antitumor efficacy in a PC xenograft model, which we attributed to the promotion of lymphocytic infiltration, as well as to upregulation of chemokine receptor expression and the regulators of CIK cell proliferation.

Aberrant expression of suppressor of cytokine signaling (SOCS) molecules contributes to the development of allergic diseases.

Suppressor of cytokine signalling (SOCS) proteins bind to certain cytokine receptors, Janus kinases and signalling molecules to regulate signalling pathways, thus controlling immune and inflammatory responses. Dysregulated expression of various types of SOCS molecules was indicated in multiple types of allergic diseases. SOCS1, SOCS2, SOCS3, SOCS5, and cytokine-inducible SH2 domain protein (CISH) can differentially exert anti-allergic impacts through different mechanisms, such as suppressing Th2 cell development and activation, reducing eosinophilia, decreasing IgE production, repressing production of pro-allergic chemokines, promoting Treg cell differentiation and activation, suppressing Th17 cell differentiation and activation, increasing anti-allergic Th1 responses, inhibiting M2 macrophage polarization, modulating survival and development of mast cells, reducing pro-allergic activity of keratinocytes, and suppressing pulmonary fibrosis. Although some anti-allergic effects were attributed to SOCS3, it can perform pro-allergic impacts through several pathways, such as promoting Th2 cell development and activation, supporting eosinophilia, boosting pro-allergic activity of eosinophils, increasing IgE production, enhancing the expression of the pro-allergic chemokine receptor, reducing Treg cell differentiation, increasing pro-allergic Th9 responses, as well as supporting mucus secretion and collagen deposition. In this review, we discuss the contrasting roles of SOCS proteins in contexts of allergic disorders to provide new insights regarding the pathophysiology of these diseases and possibly explore SOCS proteins as potential therapeutic targets for alleviating allergies.

Pancancer network analysis reveals key master regulators for cancer invasiveness

BackgroundTumor invasiveness reflects numerous biological changes, including tumorigenesis, progression, and metastasis. To decipher the role of transcriptional regulators (TR) involved in tumor invasiveness, we performed a systematic network-based pan-cancer assessment of master regulators of cancer invasiveness.Materials and methodsWe stratified patients in The Cancer Genome Atlas (TCGA) into invasiveness high (INV-H) and low (INV-L) groups using consensus clustering based on an established robust 24-gene signature to determine the prognostic association of invasiveness with overall survival (OS) across 32 different cancers. We devise a network-based protocol to identify TRs as master regulators (MRs) unique to INV-H and INV-L phenotypes. We validated the activity of MRs coherently associated with INV-H phenotype and worse OS across cancers in TCGA on a series of additional datasets in the Prediction of Clinical Outcomes from the Genomic Profiles (PRECOG) repository.ResultsBased on the 24-gene signature, we defined the invasiveness score for each patient sample and stratified patients into INV-H and INV-L clusters. We observed that invasiveness was associated with worse survival outcomes in almost all cancers and had a significant association with OS in ten out of 32 cancers. Our network-based framework identified common invasiveness-associated MRs specific to INV-H and INV-L groups across the ten prognostic cancers, including COL1A1, which is also part of the 24-gene signature, thus acting as a positive control. Downstream pathway analysis of MRs specific to INV-H phenotype resulted in the identification of several enriched pathways, including Epithelial into Mesenchymal Transition, TGF-β signaling pathway, regulation of Toll-like receptors, cytokines, and inflammatory response, and selective expression of chemokine receptors during T-cell polarization. Most of these pathways have connotations of inflammatory immune response and feasibility for metastasis.ConclusionOur pan-cancer study provides a comprehensive master regulator analysis of tumor invasiveness and can suggest more precise therapeutic strategies by targeting the identified MRs and downstream enriched pathways for patients across multiple cancers.Graphical

Identification of cow milk epitopes to characterize and quantify disease-specific T cells in allergic children

Cow milk (CM) allergy is the most prevalent food allergy in young children in the United States and Great Britain. Current diagnostic tests are either unreliable (IgE test and skin prick test) or resource-intensive with risks (food challenges). We sought to determine whether allergen-specific T cells in CM-allergic (CMA) patients have a distinct quality and/or quantity that could potentially be used as a diagnostic marker. Using PBMCs from 147 food-allergic pediatric subjects, we mapped T-cell responses to a set of reactive epitopes in CM that we compiled in a peptide pool. This pool induced cytokine responses in invitro cultured cells distinguishing subjects with CMA from subjects without CMA. We further used the pool to isolate and characterize antigen-specific CD4 memory T cells using flow cytometry and single-cell RNA/TCR sequencing assays. We detected significant changes in the transcriptional program and clonality of CM antigen-specific (CM+) T cells elicited by the pool in subjects with CMA versus subjects without CMA exvivo. CM+ T cells from subjects with CMA had increased percentages of FOXP3+ cells over FOXP3- cells. FOXP3+ cells are often equated with regulatory T cells that have suppressive activity, but CM+ FOXP3+ cells from subjects with CMA showed significant expression of interferon-responsive genes and dysregulated chemokine receptor expression compared with subjects without CMA, suggesting that these are not conventional regulatory T cells. The CM+ FOXP3+ cells were also more clonally expanded than the FOXP3- population. We were further able to use surface markers (CD25, CD127, and CCR7) in combination with our peptide pool stimulation to quantify these CM+ FOXP3+ cells by a simple flow-cytometry assay. We show increased percentages of CM+ CD127-CD25+ cells from subjects with CMA in an independent cohort, which could be used for diagnostic purposes. Looking specifically for TH2 cells normally associated with allergic diseases, we found a small population of clonally expanded CM+ cells that were significantly increased in subjects with CMA and that had high expression of TH2 cytokines and pathogenic TH2/T follicular helper markers. Overall, these findings suggest that there are several differences in the phenotypes of CM+ T cells with CM allergy and that the increase in CM+ FOXP3+ cells is a potential diagnostic marker of an allergic state. Such markers have promising applications in monitoring natural disease outgrowth and/or the efficacy of immunotherapy that will need to be validated in future studies.