Granulocyte-Macrophage Colony Stimulating Factor in Single Blastocyst Conditioned Medium as a Biomarker for Predicting Implantation Outcome of Embryo.

Background: It is highly desirable to develop new strategies based on secretomics to more accurately selection of embryos with the highest developmental potential for transfer. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been reported to promote embryo development and pregnancy establishment. However, the predictive value of GM-CSF in single blastocyst selection remains unclear. This study is to determine the concentration of GM-CSF in human single-blastocyst conditioned medium (SBCM) and to evaluate its association with embryo quality and pregnancy outcome. Methods: The patients with ≤38 years of age receiving the first cycle of assisted reproductive therapy were included in this study. The patients who had <4 top-quality embryos formed by the fertilized two pronuclear zygotes on day 3 were excluded. A total of 126 SBCM samples (SBCMs) were included, of which blastocysts from 77 SBCMs were later transferred in subsequent frozen-thawed embryo transfer. The concentrations of GM-CSF were detected by single-molecule array (SIMOA) and analyzed for their possible association with embryo quality and pregnancy outcomes. The top-quality embryo (TQ), positive HCG (HP), clinical pregnancy (CP), and ongoing pregnancy (OP) rates were determined and compared between groups divided based on GM-CSF concentrations. Results: The detection rate of GM-CSF was found to be 50% in all SBCMs. There were significant differences in TQ rate, HP rate, CP rate and OP rate among high concentration group, medium concentration group and low concentration group. Both GM-CSF alone or GM-CSF combined with the morphological score (MS) had a greater AUC of ROC curve than that of MS alone to predict the pregnancy outcome, and GM-CSF combined with MS had the highest AUC. Conclusions: The concentration of GM-CSF in SBCM was detected at fg/ml levels, which was associated with embryo quality and pregnancy outcome. Collectively, GM-CSF may be used as a biomarker for prediction of pregnancy outcome and selection of embryos with high developmental potential for transfer in assisted reproductive technology (ART).

IFN-ß Acts on Monocytes to Ameliorate CNS Autoimmunity by Inhibiting Proinflammatory Cross-Talk Between Monocytes and Th Cells.

IFN-ß has been the treatment for multiple sclerosis (MS) for almost three decades, but understanding the mechanisms underlying its beneficial effects remains incomplete. We have shown that MS patients have increased numbers of GM-CSF+ Th cells in circulation, and that IFN-ß therapy reduces their numbers. GM-CSF expression by myelin-specific Th cells is essential for the development of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. These findings suggested that IFN-ß therapy may function via suppression of GM-CSF production by Th cells. In the current study, we elucidated a feedback loop between monocytes and Th cells that amplifies autoimmune neuroinflammation, and found that IFN-ß therapy ameliorates central nervous system (CNS) autoimmunity by inhibiting this proinflammatory loop. IFN-ß suppressed GM-CSF production in Th cells indirectly by acting on monocytes, and IFN-ß signaling in monocytes was required for EAE suppression. IFN-ß increased IL-10 expression by monocytes, and IL-10 was required for the suppressive effects of IFN-ß. IFN-ß treatment suppressed IL-1ß expression by monocytes in the CNS of mice with EAE. GM-CSF from Th cells induced IL-1ß production by monocytes, and, in a positive feedback loop, IL-1ß augmented GM-CSF production by Th cells. In addition to GM-CSF, TNF and FASL expression by Th cells was also necessary for IL-1ß production by monocyte. IFN-ß inhibited GM-CSF, TNF, and FASL expression by Th cells to suppress IL-1ß secretion by monocytes. Overall, our study describes a positive feedback loop involving several Th cell- and monocyte-derived molecules, and IFN-ß actions on monocytes disrupting this proinflammatory loop.

Bioelectronic control of a microbial community using surface-assembled electrogenetic cells to route signals.

We developed a bioelectronic communication system that is enabled by a redox signal transduction modality to exchange information between a living cell-embedded bioelectronics interface and an engineered microbial network. A naturally communicating three-member microbial network is 'plugged into' an external electronic system that interrogates and controls biological function in real time. First, electrode-generated redox molecules are programmed to activate gene expression in an engineered population of electrode-attached bacterial cells, effectively creating a living transducer electrode. These cells interpret and translate electronic signals and then transmit this information biologically by producing quorum sensing molecules that are, in turn, interpreted by a planktonic coculture. The propagated molecular communication drives expression and secretion of a therapeutic peptide from one strain and simultaneously enables direct electronic feedback from the second strain, thus enabling real-time electronic verification of biological signal propagation. Overall, we show how this multifunctional bioelectronic platform, termed a BioLAN, reliably facilitates on-demand bioelectronic communication and concurrently performs programmed tasks.

The Phosphodiesterase Inhibitor Ensifentrine Reduces Production of Proinflammatory Mediators in Well Differentiated Bronchial Epithelial Cells by Inhibiting PDE4.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel that impair airway salt and fluid secretion. Excessive release of proinflammatory cytokines and chemokines by CF bronchial epithelium during airway infection leads to chronic inflammation and a slow decline in lung function; thus, there is much interest in finding safe and effective treatments that reduce inflammation in CF. We showed previously that the cyclic nucleotide phosphodiesterase (PDE) inhibitor ensifentrine (RPL554; Verona Pharma) stimulates the channel function of CFTR mutants with abnormal gating and also those with defective trafficking that are partially rescued using a clinically approved corrector drug. PDE inhibitors also have known anti-inflammatory effects; therefore, we examined whether ensifentrine alters the production of proinflammatory cytokines in CF bronchial epithelial cells. Ensifentrine reduced the production of monocyte chemoattractant protein-1 and granulocyte monocyte colony-stimulating factor (GM-CSF) during challenge with interleukin-1ß Comparing the effect of ensifentrine with milrinone and roflumilast, selective PDE3 and PDE4 inhibitors, respectively, demonstrated that the anti-inflammatory effect of ensifentrine was mainly due to inhibition of PDE4. Beneficial modulation of GM-CSF was further enhanced when ensifentrine was combined with low concentrations of the ß 2-adrenergic agonist isoproterenol or the corticosteroid dexamethasone. The results indicate that ensifentrine may have beneficial anti-inflammatory effects in CF airways particularly when used in combination with ß 2-adrenergic agonists or corticosteroids. SIGNIFICANCE STATEMENT: Airway inflammation that is disproportionate to the burden of chronic airway infection causes much of the pathology in the cystic fibrosis (CF) lung. We show here that ensifentrine beneficially modulates the release of proinflammatory factors in well differentiated CF bronchial epithelial cells that is further enhanced when combined with ß2-adrenergic agonists or low-concentration corticosteroids. The results encourage further clinical testing of ensifentrine, alone and in combination with ß2-adrenergic agonists or low-concentration corticosteroids, as a novel anti-inflammatory therapy for CF.

Amphiregulin Aggravates Glomerulonephritis via Recruitment and Activation of Myeloid Cells.

BACKGROUND: Recent studies have identified the EGF receptor (EGFR) ligand amphiregulin (AREG) as an important mediator of inflammatory diseases. Both pro- and anti-inflammatory functions have been described, but the role of AREG in GN remains unknown. METHODS: The nephrotoxic nephritis model of GN was studied in AREG-/- mice after bone marrow transplantation, and in mice with myeloid cell-specific EGFR deficiency. Therapeutic utility of AREG neutralization was assessed. Furthermore, AREG's effects on renal cells and monocytes/macrophages (M/M) were analyzed. Finally, we evaluated AREG expression in human renal biopsies. RESULTS: Renal AREG mRNA was strongly upregulated in murine GN. Renal resident cells were the most functionally relevant source of AREG. Importantly, the observation that knockout mice showed significant amelioration of disease indicates that AREG is pathogenic in GN. AREG enhanced myeloid cell responses via inducing chemokine and colony stimulating factor 2 (CSF2) expression in kidney resident cells. Furthermore, AREG directly skewed M/M to a proinflammatory M1 phenotype and protected them from apoptosis. Consequently, anti-AREG antibody treatment dose-dependently ameliorated GN. Notably, selective abrogation of EGFR signaling in myeloid cells was sufficient to protect against nephritis. Finally, strong upregulation of AREG expression was also detected in kidneys of patients with two forms of crescentic GN. CONCLUSIONS: AREG is a proinflammatory mediator of GN via (1) enhancing renal pathogenic myeloid cell infiltration and (2) direct effects on M/M polarization, proliferation, and cytokine secretion. The AREG/EGFR axis is a potential therapeutic target for acute GN.

Role of intrauterine administration of transfected peripheral blood mononuclear cells by GM-CSF on embryo implantation and pregnancy rate in mice.

One of the effective treatments in women with recurrent implantation failure (RIF) is the use of immune cells to facilitate embryo implantation. Previous studies have shown that intrauterine transmission of peripheral blood mononuclear cells (PBMC) increased the embryo implantation rate. In this study using B6D2F1 (C57BL/6 × DBA2) mice, a fragment of the granulocyte macrophage colony-stimulating factor (Gm-csf) gene was cloned into an enhanced green fluorescent protein vector (pEGFP-N1) and then transfected into PBMC. The protein level of GM-CSF was evaluated in the transfected PBMC and untransfected PBMC by ELISA. Attachment of mouse embryos and the mRNA expression levels of leukemia inhibitory factor (Lif), vascular endothelial growth factor (Vegf), matrix metalloproteinase 9 (Mmp9), Gmcsf-receptor (Gmcsf-r) and interleukin 6 (Il6) in vitro were assessed by real-time PCR in endometrial cells. To determine the pregnancy rate and number of implantation sites in vivo, the mouse uterine horns were analyzed on Day 7.5 post coitum. A greater amount of GM-CSF was produced in PBMC transfected with recombinant vector (552 pg/mL) compared with the untransfected PBMC (57 pg/mL) and PBMC transfected with empty vector (34 pg/mL) (P < 0.05). The data showed that the embryo attachment rate and mRNA expression levels (Vegf [1.7-fold], Mmp9 [1.4-fold], Lif [1.5-fold], Gm-csf r [1.6-fold] and Il6 [1.2-fold]) in the in vitro study (P < 0.01), pregnancy rate (P < 0.01) and number of implantation sites (P < 0.01) in the in vivo investigation (P < 0.05) were increased in PBMC transfected with recombinant vector compared with the PBMC group. The study demonstrated that, in mice, endometrium immunotherapy with transfected PBMC that contained recombinant GM-CSF before embryo implantation was effective in improving embryo implantation and endometrial receptivity.

GM-CSF and CXCR4 define a T helper cell signature in multiple sclerosis.

Cytokine dysregulation is a central driver of chronic inflammatory diseases such as multiple sclerosis (MS). Here, we sought to determine the characteristic cellular and cytokine polarization profile in patients with relapsing-remitting multiple sclerosis (RRMS) by high-dimensional single-cell mass cytometry (CyTOF). Using a combination of neural network-based representation learning algorithms, we identified an expanded T helper cell subset in patients with MS, characterized by the expression of granulocyte-macrophage colony-stimulating factor and the C-X-C chemokine receptor type 4. This cellular signature, which includes expression of very late antigen 4 in peripheral blood, was also enriched in the central nervous system of patients with relapsing-remitting multiple sclerosis. In independent validation cohorts, we confirmed that this cell population is increased in patients with MS compared with other inflammatory and non-inflammatory conditions. Lastly, we also found the population to be reduced under effective disease-modifying therapy, suggesting that the identified T cell profile represents a specific therapeutic target in MS.

Tubular GM-CSF Promotes Late MCP-1/CCR2-Mediated Fibrosis and Inflammation after Ischemia/Reperfusion Injury.

BACKGROUND: After bilateral kidney ischemia/reperfusion injury (IRI), monocytes infiltrate the kidney and differentiate into proinflammatory macrophages in response to the initial kidney damage, and then transition to a form that promotes kidney repair. In the setting of unilateral IRI (U-IRI), however, we have previously shown that macrophages persist beyond the time of repair and may promote fibrosis. METHODS: Macrophage homing/survival signals were determined at 14 days after injury in mice subjected to U-IRI and in vitro using coculture of macrophages and tubular cells. Mice genetically engineered to lack Ccr2 and wild-type mice were treated ±CCR2 antagonist RS102895 and subjected to U-IRI to quantify macrophage accumulation, kidney fibrosis, and inflammation 14 and 30 days after the injury. RESULTS: Failure to resolve tubular injury after U-IRI results in sustained expression of granulocyte-macrophage colony-stimulating factor by renal tubular cells, which directly stimulates expression of monocyte chemoattractant protein-1 (Mcp-1) by macrophages. Analysis of CD45+ immune cells isolated from wild-type kidneys 14 days after U-IRI reveals high-level expression of the MCP-1 receptor Ccr2. In mice lacking Ccr2 and wild-type mice treated with RS102895, the numbers of macrophages, dendritic cells, and T cell decreased following U-IRI, as did the expression of profibrotic growth factors and proimflammatory cytokines. This results in a reduction in extracellular matrix and kidney injury markers. CONCLUSIONS: GM-CSF-induced MCP-1/CCR2 signaling plays an important role in the cross-talk between injured tubular cells and infiltrating immune cells and myofibroblasts, and promotes sustained inflammation and tubular injury with progressive interstitial fibrosis in the late stages of U-IRI.

A Novel Methanol-Free Platform for Extracellular Expression of rhGM-CSF in Pichia pastoris.

The production of the recombinant proteins under the control of AOX1 promoter is a one of the most common expression systems in the methylotrophic yeast Pichia pastoris which is induced by methanol. The application of this expression platform is restricted by the toxicity and inflammatory nature of methanol, especially in food and pharmaceutical products. Human granulocyte macrophage-colony stimulating factor (hGM-CSF) is an important pharmaceutical protein, playing a crucial role in the proliferation and differentiation of innate immune cells. In this study, a methanol-free expression platform for extracellular expression of hGM-CSF was developed. To attain this goal, a novel constructed expression vector pEP(α)101, carrying the FMD promoter regulating recombinant expression by glycerol derepression was designed. The optimized hGM-CSF gene was subcloned into pEP(α)101 and transformed into P. pastoris. The expression of rhGM-CSF in three different culture media were investigated. Based on the observed heterogeneous glycosylation pattern on SDS-PAGE and western blot, the glycoproteins were deglycosylated to remove carbohydrate units. According to the results, the novel methanol independent PFMD expression platform would be a suitable candidate for driving heterologous gene expression especially for the production of food-grade and therapeutically important recombinant proteins.

RNA-Modified T Cells Mediate Effective Delivery of Immunomodulatory Cytokines to Brain Tumors.

With the presence of the blood-brain barrier (BBB), successful immunotherapeutic drug delivery to CNS malignancies remains a challenge. Immunomodulatory agents, such as cytokines, can reprogram the intratumoral microenvironment; however, systemic cytokine delivery has limited access to the CNS. To bypass the limitations of systemically administered cytokines, we investigated if RNA-modified T cells could deliver macromolecules directly to brain tumors. The abilities of T cells to cross the BBB and mediate direct cytotoxic killing of intracranial tumors make them an attractive tool as biological carriers. Using T cell mRNA electroporation, we demonstrated that activated T cells can be modified to secrete granulocyte macrophage colony-stimulating factor (GM-CSF) protein while retaining their inherent effector functions in vitro. GM-CSF RNA-modified T cells effectively delivered GM-CSF to intracranial tumors in vivo and significantly extended overall survival in an orthotopic treatment model. Importantly, GM-CSF RNA-modified T cells demonstrated superior anti-tumor efficacy as compared to unmodified T cells alone or in combination with systemic administration of recombinant GM-CSF. Anti-tumor effects were associated with increased IFN-γ secretion locally within the tumor microenvironment and systemic antigen-specific T cell expansion. These findings demonstrate that RNA-modified T cells may serve as a versatile platform for the effective delivery of biological agents to CNS tumors.

Regulatory T Cells Restrain Pathogenic T Helper Cells during Skin Inflammation.

Psoriasis is a chronic relapsing, remitting interleukin (IL)-23/IL-17-driven skin disease mediated by the interplay of T cells and polymorphonuclear granulocytes. Although preclinical studies have provided insights into the mechanisms of disease initiation, the underpinnings of natural disease remission remain largely unknown. Here, we addressed the contribution of regulatory Foxp3+ T cells (Treg cells) in psoriasiform skin inflammation and remission using the Aldara-skin inflammation model in combination with the inducible depletion of Foxp3+ Treg cells. Loss of Treg cells exacerbated skin inflammation, but this did not involve increased γδ T cell expansion or the local production of the psoriasis-associated cytokines IL-17A, IL-17F, and IL-22, which are the main driving forces of disease development. Instead, Treg cells suppressed the infiltration of granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing CD4+ T cells into the lesioned skin, and neutralizing GM-CSF in Treg cell-deficient mice reversed hyper-inflammation, resulting in disease regression. Therefore, we identified a non-redundant role of Treg cells restraining skin inflammation and mediating skin homeostasis.

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) Cells.

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly secretes GM-CSF without producing interferon (IFN)γ or interleukin (IL)-17 and is found to play an essential role in the autoimmune neuroinflammation. A method of isolation of naive CD4+ T cells from a single-cell suspension of splenocytes and THGM cell generation from naive CD4+ T cells would be a useful technique in the study of T cell-mediated immunity and autoimmune diseases. Here we describe a method that differentiates mouse naive CD4+ T cells into THGM cells promoted by IL-7. The outcome of the differentiation was assessed by the analysis of the cytokines expression using different techniques, including intracellular cytokine staining combined with flow cytometry, a quantitative real-time polymerase chain reaction (PCR), and enzyme-linked immunosorbent assays (ELISA). Using the THGM differentiation protocol as described here, about 55% of the cells expressed GM-CSF with a minimal expression of IFNα or IL-17. The predominant expression of GM-CSF by THGM cells was further confirmed by the analysis of the expression of GM-CSF, IFNα, and IL-17 at both mRNA and protein levels. Thus, this method can be used to differentiate naive CD4+ T cells to THGM cells in vitro, which will be useful in the study of THGM cell biology.

Mucosal-Associated Invariant T Cells Are Depleted and Exhibit Altered Chemokine Receptor Expression and Elevated Granulocyte Macrophage-Colony Stimulating Factor Production During End-Stage Renal Disease.

Background: End-stage renal disease (ESRD) is associated with an increased susceptibility to infectious diseases, including infection with Mycobacterium tuberculosis (Mtb). Mucosal-associated invariant T (MAIT) cells recognize vitamin B metabolites produced by many bacterial species, including Mtb, and may play an important role in providing protective immunity against tuberculosis infection in the lung. To date, little is known about MAIT cell frequency, phenotype, or function in ESRD patients. Methods: MAIT cells, identified by surface marker expression or MR1 tetramer binding, were characterized in 20 ESRD and 20 healthy control participants by multicolor flow cytometry. Ex vivo MAIT cell phenotype and cytokine production following PMA/ionomycin, IL-12/IL-18, or Escherichia coli stimulation were determined. Monocyte phenotype and plasma C-reactive protein/inflammatory cytokine levels were quantified by flow cytometry, ELISA, and multiplex bead array. Results: Peripheral blood MAIT cells were significantly depleted among ESRD patients compared to controls by both phenotypic and tetramer analysis and exhibited a loss of CXCR3 expression coupled to increased expression of CCR6 and CXCR6. ESRD was also associated with a shift in MAIT PMA-induced cytokine production away from IFNγ production and toward granulocyte macrophage-colony stimulating factor (GM-CSF) secretion, and a loss of E. coli-stimulated tumor necrosis factor α expression. Loss of IFNγ expression was associated with a combination of age, alterations in Tbet and Eomes expression, and inflammatory plasma cytokine levels. Conclusion: The loss of peripheral blood MAIT cells and associated shifts in tissue homing receptor expression and GM-CSF production may contribute to an immune environment that is permissive to bacterial replication, particularly in the lungs.

Autoimmune Th17 Cells Induced Synovial Stromal and Innate Lymphoid Cell Secretion of the Cytokine GM-CSF to Initiate and Augment Autoimmune Arthritis.

Despite the importance of Th17 cells in autoimmune diseases, it remains unclear how they control other inflammatory cells in autoimmune tissue damage. Using a model of spontaneous autoimmune arthritis, we showed that arthritogenic Th17 cells stimulated fibroblast-like synoviocytes via interleukin-17 (IL-17) to secrete the cytokine GM-CSF and also expanded synovial-resident innate lymphoid cells (ILCs) in inflamed joints. Activated synovial ILCs, which expressed CD25, IL-33Ra, and TLR9, produced abundant GM-CSF upon stimulation by IL-2, IL-33, or CpG DNA. Loss of GM-CSF production by either ILCs or radio-resistant stromal cells prevented Th17 cell-mediated arthritis. GM-CSF production by Th17 cells augmented chronic inflammation but was dispensable for the initiation of arthritis. We showed that GM-CSF-producing ILCs were present in inflamed joints of rheumatoid arthritis patients. Thus, a cellular cascade of autoimmune Th17 cells, ILCs, and stromal cells, via IL-17 and GM-CSF, mediates chronic joint inflammation and can be a target for therapeutic intervention.

PPARγ Contributes to Immunity Induced by Cancer Cell Vaccines That Secrete GM-CSF.

Peroxisome proliferator activated receptor-γ (PPARγ) is a lipid-activated nuclear receptor that promotes immune tolerance through effects on macrophages, dendritic cells (DCs), and regulatory T cells (Tregs). Granulocyte-macrophage colony stimulating factor (GM-CSF) induces PPARγ expression in multiple myeloid cell types. GM-CSF contributes to both immune tolerance and protection, but the role of PPARγ in these pathways is poorly understood. Here, we reveal an unexpected stimulatory role for PPARγ in the generation of antitumor immunity with irradiated, GM-CSF-secreting tumor-cell vaccines (GVAX). Mice harboring a deletion of pparg in lysozyme M (LysM)-expressing myeloid cells (KO) showed a decreased ratio of CD8+ T effectors to Tregs and impaired tumor rejection with GVAX. Diminished tumor protection was associated with altered DC responses and increased production of the Treg attracting chemokines CCL17 and CLL22. Correspondingly, the systemic administration of PPARγ agonists to vaccinated mice elevated the CD8+ T effector to Treg ratio through effects on myeloid cells and intensified the antitumor activity of GVAX combined with cytotoxic T lymphocyte-associated antigen-4 antibody blockade. PPARγ agonists similarly attenuated Treg induction and decreased CCL17 and CCL22 levels in cultures of human peripheral blood mononuclear cells with GM-CSF-secreting tumor cells. Together, these results highlight a key role for myeloid cell PPARγ in GM-CSF-stimulated antitumor immunity and suggest that PPARγ agonists might be useful in cancer immunotherapy. Cancer Immunol Res; 6(6); 723-32. ©2018 AACR.

Interferon-gamma inhibits seminal plasma induction of colony-stimulating factor 2 in mouse and human reproductive tract epithelial cells.

Seminal fluid interacts with the female reproductive tract to initiate a permissive immune response that facilitates embryo implantation and pregnancy success. The immune-regulatory cytokine interferon-γ (IFNG), which can be elevated in seminal plasma, is associated with reduced fertility. Here, we investigated how IFNG influences the female immune response to seminal fluid. In human Ect1 cervical epithelial cells, IFNG added at physiologically relevant concentrations substantially impaired seminal plasma-induced synthesis of key cytokines colony-stimulating factor 2 (CSF2) and interleukin-6 (IL6). Seminal fluid-induced CSF2 synthesis was also suppressed in the uterus of mice in vivo, when IFNG was delivered transcervically 12 h after mating. Transforming growth factor B1 (TGFB1) is the major seminal fluid signaling factor which elicits CSF2 induction, and IFNG exhibited potent dose-dependent suppression of CSF2 synthesis induced by TGFB1 in murine uterine epithelial cells in vitro. Similarly, IFNG suppressed TGFB1-mediated CSF2 induction in Ect1 cells and human primary cervical epithelial cells; however, IL6 regulation by IFNG was independent of TGFB1. Quantitative PCR confirmed that CSF2 regulation by IFNG in Ect1 cells occurs at the gene transcription level, secondary to IFNG suppression of TGFBR2 encoding TGFB receptor 2. Conversely, TGFB1 suppressed IFNG receptor 1 and 2 genes IFNGR1 and IFNGR2. These data identify IFNG as a potent inhibitor of the TGFB-mediated seminal fluid interaction with relevant reproductive tract epithelia in mice and human. These findings raise the prospect that IFNG in the male partner's seminal fluid impairs immune adaptation for pregnancy following coitus in women.

GM-CSF overexpression after influenza a virus infection prevents mortality and moderates M1-like airway monocyte/macrophage polarization.

BACKGROUND: Influenza A viruses cause life-threatening pneumonia and lung injury in the lower respiratory tract. Application of high GM-CSF levels prior to infection has been shown to reduce morbidity and mortality from pathogenic influenza infection in mice, but the mechanisms of protection and treatment efficacy have not been established. METHODS: Mice were infected intranasally with influenza A virus (PR8 strain). Supra-physiologic levels of GM-CSF were induced in the airways using the double transgenic GM-CSF (DTGM) or littermate control mice starting on 3 days post-infection (dpi). Assessment of respiratory mechanical parameters was performed using the flexiVent rodent ventilator. RNA sequence analysis was performed on FACS-sorted airway macrophage subsets at 8 dpi. RESULTS: Supra-physiologic levels of GM-CSF conferred a survival benefit, arrested the deterioration of lung mechanics, and reduced the abundance of protein exudates in bronchoalveolar (BAL) fluid to near baseline levels. Transcriptome analysis, and subsequent validation ELISA assays, revealed that excess GM-CSF re-directs macrophages from an "M1-like" to a more "M2-like" activation state as revealed by alterations in the ratios of CXCL9 and CCL17 in BAL fluid, respectively. Ingenuity pathway analysis predicted that GM-CSF surplus during IAV infection elicits expression of anti-inflammatory mediators and moderates M1 macrophage pro-inflammatory signaling by Type II interferon (IFN-γ). CONCLUSIONS: Our data indicate that application of high levels of GM-CSF in the lung after influenza A virus infection alters pathogenic "M1-like" macrophage inflammation. These results indicate a possible therapeutic strategy for respiratory virus-associated pneumonia and acute lung injury.

GM-CSF producing autoreactive CD4+ T cells in type 1 diabetes.

The phenotype of autoreactive T cells in type 1 diabetes is described as Th1, Th17 and/or Th21, but is largely uncharacterized. We combined multi-parameter cytokine profiling and proliferation, and identified GM-CSF producing cells as a component of the response to beta cell autoantigens proinsulin and GAD65. Overall cytokine profiles of CD4+ T cell were not altered in type 1 diabetes. In contrast, patients with recent onset type 1 diabetes had increased frequencies of proinsulin-responsive CD4+CD45RA- T cells producing GM-CSF (p=0.002), IFNγ (p=0.004), IL-17A (p=0.008), IL-21 (p=0.011), and IL-22 (p=0.007), and GAD65-responsive CD4+CD45RA- T cells producing IL-21 (p=0.039). CD4+ T cells with a GM-CSF+IFNγ-IL-17A-IL-21-IL-22- phenotype were increased in patients for responses to both proinsulin (p=0.006) and GAD65 (p=0.037). GM-CSF producing T cells are a novel phenotype in the repertoire of T helper cells in type 1 diabetes and consolidate a Th1/Th17 pro-inflammatory pathogenesis in the disease.

[Effect of the Industrial Nanoparticles TiO 2 , SiO 2 and ZnO on Cell Viability and Gene Expression in Red Bone Marrow of Mus Musculus]. / Efecto de las nanopartículas industriales TiO 2 , SiO 2 y ZnO sobre la viabilidad celular y expresión génica en médula ósea roja de mus musculus.

OBJECTIVES: To evaluate the effect of ZnO, TiO2 and SiO2 nanoparticles on cell viability and expression of the interleukin 7, interleukin 3, and granulocyte-macrophage colony stimulating factor (GM-CSF) genes in Mus musculus. MATERIAL AND METHODS: Red bone marrow was extracted from five Balb/c mice for the analysis of cell viability using the MTT test. The mice were divided into two groups of five each: one group was inoculated intraperitoneally with 0.5, 1.0, 2.5, 5.0, and 10 mg/kg of ZnO and SiO2 nanoparticles, respectively, and the other group was inoculated with 5.0, 10.0, 15.0, 20.0, and 25 mg/kg of TiO2 nanoparticles, respectively. Thirty hours later, RNA was extracted from the red bone marrow of the mice in both groups for gene expression analysis using quantitative PCR and RT-PCR. RESULTS: ZnO and SiO2 nanoparticles reduced cell viability in a dose-dependent manner by 37% and 26%, respectively, starting at a dose of 1 mg/kg. TiO2 nanoparticles at 5 mg/kg and 10 mg/kg reduced the gene expression of interleukins 7 and 3 by 55.3% and 70.2%, respectively, and SiO2 nanoparticles caused the greatest decrease (91%) in the expression of GM-CSF. ZnO nanoparticles reduced the expression of GM-CSF starting at doses of 20 mg/kg and 25 mg/kg. CONCLUSIONS: ZnO, SiO2 and TiO2 nanoparticles affect cell viability and gene expression in the mouse bone marrow.

OBJETIVOS: Evaluar el efecto de las nanopartículas de ZnO, TiO2 y SiO2 sobre la viabilidad celular y la expresión génica de las interleuquinas 7 y 3 y del factor estimulante de colonias de granulocito - macrófago (GM-CSF) en Mus musculus. MATERIALES Y MÉTODOS: Se extrajo médula ósea roja de cinco roedores (Balb/c) para el estudio de viabilidad celular mediante la prueba de MTT. Por otro lado, grupos cinco roedores fueron inoculados vía intraperitoneal con dosis de 0,5; 1; 2,5; 5 y 10 mg/kg de nanopartículas de ZnO y SiO2 y de 5; 10; 15; 20 y 25 mg/kg de nanopartículas de TiO2, 30 h después, se obtuvo el ARN a partir de la médula ósea roja para los análisis de expresión génica empleando las técnicas de PCR y RT-PCR cuantitativa. RESULTADOS: Las nanopartículas de ZnO y SiO2 redujeron la viabilidad celular de una manera dosis-dependiente en un 37 y 26%, respectivamente, a partir de una dosis de 1 mg/kg. En cuanto al efecto sobre la expresión génica, a las dosis 5 y 10 mg/kg, las nanopartículas de TiO2 redujeron en mayor porcentaje la expresión de las interleuquinas 7 y 3 (55,3 y 70,2% respectivamente), con respecto a la expresión del GM-CSF, el mayor porcentaje de reducción lo produjo las nanopartículas de SiO2 (91%). Las nanopartículas de ZnO redujeron a partir de las dosis de 20 y 25 mg/kg. CONCLUSIONES: Las nanopartículas de ZnO, SiO2 y TiO2 alteran la viabilidad celular y la expresión génica en la médula ósea de ratón.

T Cell Production of GM-CSF Protects the Host during Experimental Tuberculosis.

Although classically associated with myelopoiesis, granulocyte-macrophage colony-stimulating factor (GM-CSF) is increasingly recognized as being important for tuberculosis (TB) resistance. GM-CSF is expressed by nonhematopoietic and hematopoietic lineages following infection with Mycobacterium tuberculosis and is necessary to restrict M. tuberculosis growth in experimental models. Until the recent study by Rothchild et al. (mBio 8:e01514-17, 2017, https://doi.org/10.1128/mBio.01514-17), it was unknown whether GM-CSF-producing T cells contribute to TB resistance. Rothchild et al. identify which conventional and nonconventional T cell subsets produce GM-CSF during experimental TB, establish their protective nature using a variety of approaches, and provide a mechanistic basis for their ability to restrict M. tuberculosis growth. This commentary discusses the significance of these findings to basic and applied TB research. As translated to human disease, these findings suggest vaccine-mediated expansion of GM-CSF-producing T cells could be an effective prophylactic or therapeutic TB strategy.