Macrophages, rather than DCs, are responsible for inflammasome activity in the GM-CSF BMDC model.

Inflammasomes are one of the most important mechanisms for innate immune defense against microbial infection but are also known to drive various inflammatory disorders via processing and release of the cytokine IL-1ß. As research into the regulation and effects of inflammasomes in disease has rapidly expanded, a variety of cell types, including dendritic cells (DCs), have been suggested to be inflammasome competent. Here we describe a major fault in the widely used DC-inflammasome model of bone marrow-derived dendritic cells (BMDCs) generated with the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF). We found that among GM-CSF bone marrow-derived cell populations, monocyte-derived macrophages, rather than BMDCs, were responsible for inflammasome activation and IL-1ß secretion. Therefore, GM-CSF bone marrow-derived cells should not be used to draw conclusions about DC-dependent inflammasome biology, although they remain a useful tool for analysis of inflammasome responses in monocytes-macrophages.

Cellular Differentiation of Human Monocytes Is Regulated by Time-Dependent Interleukin-4 Signaling and the Transcriptional Regulator NCOR2.

Human in vitro generated monocyte-derived dendritic cells (moDCs) and macrophages are used clinically, e.g., to induce immunity against cancer. However, their physiological counterparts, ontogeny, transcriptional regulation, and heterogeneity remains largely unknown, hampering their clinical use. High-dimensional techniques were used to elucidate transcriptional, phenotypic, and functional differences between human in vivo and in vitro generated mononuclear phagocytes to facilitate their full potential in the clinic. We demonstrate that monocytes differentiated by macrophage colony-stimulating factor (M-CSF) or granulocyte macrophage colony-stimulating factor (GM-CSF) resembled in vivo inflammatory macrophages, while moDCs resembled in vivo inflammatory DCs. Moreover, differentiated monocytes presented with profound transcriptomic, phenotypic, and functional differences. Monocytes integrated GM-CSF and IL-4 stimulation combinatorically and temporally, resulting in a mode- and time-dependent differentiation relying on NCOR2. Finally, moDCs are phenotypically heterogeneous and therefore necessitate the use of high-dimensional phenotyping to open new possibilities for better clinical tailoring of these cellular therapies.

GM-CSF-activated human dendritic cells promote type 1 T follicular helper cell polarization in a CD40-dependent manner.

T follicular helper (Tfh) cells regulate humoral responses and present a marked phenotypic and functional diversity. Type 1 Tfh (Tfh1) cells were recently identified and associated with disease severity in infection and autoimmune diseases. The cellular and molecular requirements to induce human Tfh1 differentiation are not known. Here, using single-cell RNA sequencing (scRNAseq) and protein validation, we report that human blood CD1c+ dendritic cells (DCs) activated by GM-CSF (also known as CSF2) drive the differentiation of naive CD4+ T cells into Tfh1 cells. These Tfh1 cells displayed typical Tfh molecular features, including high levels of PD-1 (encoded by PDCD1), CXCR5 and ICOS. They co-expressed BCL6 and TBET (encoded by TBX21), and secreted large amounts of IL-21 and IFN-γ (encoded by IFNG). Mechanistically, GM-CSF triggered the emergence of two DC sub-populations defined by their expression of CD40 and ICOS ligand (ICOS-L), presenting distinct phenotypes, morphologies, transcriptomic signatures and functions. CD40High ICOS-LLow DCs efficiently induced Tfh1 differentiation in a CD40-dependent manner. In patients with mild COVID-19 or latent Mycobacterium tuberculosis infection, Tfh1 cells were positively correlated with a CD40High ICOS-LLow DC signature in scRNAseq of peripheral blood mononuclear cells or blood transcriptomics, respectively. Our study uncovered a novel CD40-dependent Tfh1 axis with potential physiopathological relevance to infection. This article has an associated First Person interview with the first author of the paper.

Treatment with Granulocyte-Macrophage Colony-Stimulating Factor Reduces Viral Titers in the Brains of West Nile Virus-Infected Mice and Improves Survival.

West Nile virus (WNV) is the leading cause of epidemic arboviral encephalitis in the United States. As there are currently no proven antiviral therapies or licensed human vaccines, understanding the neuropathogenesis of WNV is critical for rational therapeutic design. In WNV-infected mice, the depletion of microglia leads to enhanced viral replication, increased central nervous system (CNS) tissue injury, and increased mortality, suggesting that microglia play a critical role in protection against WNV neuroinvasive disease. To determine if augmenting microglial activation would provide a potential therapeutic strategy, we administered granulocyte-macrophage colony-stimulating factor (GM-CSF) to WNV-infected mice. Recombinant human GM-CSF (rHuGMCSF) (sargramostim [Leukine]) is an FDA-approved drug used to increase white blood cells following leukopenia-inducing chemotherapy or bone marrow transplantation. Daily treatment of both uninfected and WNV-infected mice with subcutaneous injections of GM-CSF resulted in microglial proliferation and activation as indicated by the enhanced expression of the microglia activation marker ionized calcium binding adaptor molecule 1 (Iba1) and several microglia-associated inflammatory cytokines, including CCL2 (C-C motif chemokine ligand 2), interleukin 6 (IL-6), and IL-10. In addition, more microglia adopted an activated morphology as demonstrated by increased sizes and more pronounced processes. GM-CSF-induced microglial activation in WNV-infected mice was associated with reduced viral titers and apoptotic activity (caspase 3) in the brains of WNV-infected mice and significantly increased survival. WNV-infected ex vivo brain slice cultures (BSCs) treated with GM-CSF also showed reduced viral titers and caspase 3 apoptotic cell death, indicating that GM-CSF specifically targets the CNS and that its actions are not dependent on peripheral immune activity. Our studies suggest that stimulation of microglial activation may be a viable therapeutic approach for the treatment of WNV neuroinvasive disease. IMPORTANCE Although rare, WNV encephalitis poses a devastating health concern, with few treatment options and frequent long-term neurological sequelae. Currently, there are no human vaccines or specific antivirals against WNV infections, so further research into potential new therapeutic agents is critical. This study presents a novel treatment option for WNV infections using GM-CSF and lays the foundation for further studies into the use of GM-CSF as a treatment for WNV encephalitis as well as a potential treatment for other viral infections.

Triptolide promotes differentiation of human monocytes into immunosuppressive MDSCs.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) negatively modulate immune activity. Prior investigations have shown much promise in using MDSCs-assisted immunotherapy for organ transplantation patients. Additionally, owing to its immunosuppressive activity, MDSCs can also be used to manage immune-associated disorders. METHODS: Granulocyte-macrophage colony-stimulating factor (GM-CSF) was employed to stimulate myeloid progenitor cell differentiation. Triptolide (PG490) was introduced toward the later phases of in vitro MDSCs induction. Lastly, real-time PCR (RT-PCR) and flow cytometry were used to assess transcript expression and cell phenotype, and a mouse skin transplantation model was established to evaluate the MDSCs-mediated immune suppression in vivo. RESULTS: Co-stimulation with PG490 and GM-CSF potently induced myeloid-derived monocytes to form MDSCs, with remarkable immune-suppressive activity. The underlying mechanism involved downregulation of T cell proliferation, activation, enhancement of inflammatory cytokine release, as well as T cell conversion to Treg cells. PG490 strongly enhanced iNOS expression in MDSCs, and iNOS inhibition successfully reversed the immune-suppression. The PG490- and GM-CSF-induced MDSCs substantially extended survival duration of murine skin grafts, thereby validating their strong immune-suppressive activity in vivo. CONCLUSIONS: Herein, we presented a new approach involving MDSCs-based immunosuppression in vitro. PG490 and GM-CSF co-treatment strongly induced immuno-suppressive activity in MDSCs both in vitro and in vivo. Our findings highlight the promise of applying MDSCs-based therapy in clinical organ transplantation treatment.

Oxidized low-density lipoproteins impair the pro-atherosclerotic effect of granulocyte-macrophage-colony-stimulating factor-producing T helper cells on macrophages.

T cells contribute to the pathogenesis of atherosclerosis. However, the presence and function of granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (ThGM) cells in atherosclerosis development is unknown. This study aims to characterize the phenotype and function of ThGM cells in experimental atherosclerosis. Atherosclerosis was induced by feeding apolipoprotein E knockout (ApoE-/-) mice with a high-fat diet. Aortic ThGM cells were detected and sorted by flow cytometry. The effect of oxidized low-density lipoprotein (oxLDL) on ThGM cells and the impact of ThGM cells on macrophages were evaluated by flow cytometry, quantitative RT-PCR, oxLDL binding/uptake assay, immunoblotting and foam cell formation assay. We found that GM-CSF+IFN-γ- ThGM cells existed in atherosclerotic aortas. Live ThGM cells were enriched in aortic CD4+CCR6-CCR8-CXCR3-CCR10+ T cells. Aortic ThGM cells triggered the expression of interleukin-1ß (IL-1ß), tumour necrosis factor (TNF), interleukin-6 (IL-6) and C-C motif chemokine ligand 2 (CCL2) in macrophages. Besides, aortic ThGM cells expressed higher CD69 than other T cells and bound to oxLDL. oxLDL suppressed the cytokine expression in ThGM cells probably via inhibiting the signal transducer and activator of transcription 5 (STAT5) signalling. Furthermore, oxLDL alleviated the effect of ThGM cells on inducing macrophages to produce pro-inflammatory cytokines and generate foam cells. The nuclear receptor subfamily 4 group A (NR4A) members NR4A1 and NR4A2 were involved in the suppressive effect of oxLDL on ThGM cells. Collectively, oxLDL suppressed the supportive effect of ThGM cells on pro-atherosclerotic macrophages.

The Cytokine GM-CSF Drives the Inflammatory Signature of CCR2+ Monocytes and Licenses Autoimmunity.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has emerged as a crucial cytokine produced by auto-reactive T helper (Th) cells that initiate tissue inflammation. Multiple cell types can sense GM-CSF, but the identity of the pathogenic GM-CSF-responsive cells is unclear. By using conditional gene targeting, we systematically deleted the GM-CSF receptor (Csf2rb) in specific subpopulations throughout the myeloid lineages. Experimental autoimmune encephalomyelitis (EAE) progressed normally when either classical dendritic cells (cDCs) or neutrophils lacked GM-CSF responsiveness. The development of tissue-invading monocyte-derived dendritic cells (moDCs) was also unperturbed upon Csf2rb deletion. Instead, deletion of Csf2rb in CCR2(+)Ly6C(hi) monocytes phenocopied the EAE resistance seen in complete Csf2rb-deficient mice. High-dimensional analysis of tissue-infiltrating moDCs revealed that GM-CSF initiates a combination of inflammatory mechanisms. These results indicate that GM-CSF signaling controls a pathogenic expression signature in CCR2(+)Ly6C(hi) monocytes and their progeny, which was essential for tissue damage.

GM-CSF suppresses antioxidant signaling and drives IL-1ß secretion through NRF2 downregulation.

GM-CSF is a potent inflammatory cytokine regulating myeloid cell differentiation, hematopoiesis, and various other functions. It is functionally associated with a number of inflammatory pathologies including rheumatoid arthritis and inflammatory bowel disease. GM-CSF has been found to promote NLRP3-dependent IL-1ß secretion, which may have a significant role in driving inflammatory pathologies. However, the molecular mechanisms remain unknown. Here, we show that GM-CSF induces IL-1ß secretion through a ROS-dependent pathway. TNF is required for reactive oxygen species (ROS) generation that strikingly does not promote NLRP3 activation, but instead drives ubiquitylation of IL-1ß, promoting its cleavage through basal NRLP3 activity. GM-CSF regulates this pathway through suppression of antioxidant responses via preventing upregulation of NRF2. Thus, the pro-inflammatory effect of GM-CSF on IL-1ß is through suppression of antioxidant responses, which leads to ubiquitylation of IL-1ß and enhanced processing. This study highlights the role of metabolic regulation of inflammatory signaling and reveals a novel mechanism for GM-CSF to promote inflammation.

Granulocyte macrophage colony stimulating factor exerts dominant effects over macrophage colony stimulating factor during macrophage differentiation in vitro to induce an inflammatory phenotype.

BACKGROUND: Macrophages (Mφ) can exist along a spectrum of phenotypes that include pro-inflammatory (M1) or anti-inflammatory (M2) immune cells. Mφ colony stimulating factor (M-CSF) and granulocyte Mφ colony stimulating factor (GM-CSF) are cytokines important in hematopoiesis, polarization and activation of Mφ. METHODS AND RESULTS: To gain a greater understanding of the relationship between GM-CSF and M-CSF, we investigated an in vitro model of differentiation to determine if GM-CSF and M-CSF can antagonize each other, in terms of Mφ phenotype and functions. We determined that Mφ cultured in mixed M-CSF: GM-CSF ratios exhibit M1-like GM-CSF-treated macrophage phenotype when the ratios of the two cytokines are 1:1 in culture. Moreover, GM-CSF is dominant over M-CSF in influencing Mφ production of proinflammatory cytokines such as IL-6, TNFα, and IL-12p40, and the anti-inflammatory cytokine IL-10. CONCLUSIONS: Our data established that GM-CSF is more dominant over M-CSF, triggering the Mφ to become pro-inflammatory cells. These findings provide insight into how GM-CSF can influence Mφ activation with implications in inflammatory diseases where the Mφ status can play a significant role in supporting the inflammatory conditions.

In vitro effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on the expression of genes related to sperm motility and energy metabolism and intracytoplasmic sperm injection outcomes in obstructive azoospermic patients.

BACKGROUND: The presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor in various testicular cells and spermatozoa suggests a potential role in enhancing spermatogonial and postmeiotic cell development. Moreover, GM-CSF activates the pivotal pathways implicated in sperm motility regulation and glucose metabolism. However, the impact of GM-CSF on testicular biopsies from patients with obstructive azoospermia (OA) remains unexplored. Therefore, this study aimed to investigate the in vitro effects of GM-CSF on the expression of genes related to glucose transporters and signaling pathways, sperm motility, and viability in testicular biopsies. METHODS AND RESULTS: Following testicular sperm extraction from 20 patients diagnosed with OA, each sample was divided into two parts: the experimental samples were incubated with medium containing 2 ng/ml GM-CSF at 37 °C for 60 min, and the control samples were incubated with medium without GM-CSF. Subsequently, the oocytes retrieved from the partner were injected with sperm from the treatment and control groups. The sperm parameters (motility and viability), the expression levels of sperm motility-related genes (PIK3R1, PIK3CA, and AKT1), and the expression levels of sperm energy metabolism-related genes (GLUT1, GLUT3, and GLUT14) were assessed. Furthermore, the fertilization and day 3 embryo development rate and embryo quality were evaluated. Compared with those in the nontreated group, the motility parameters and the mRNA expression levels of PIK3R1, AKT1, and GLUT3 in testicular sperm supplemented with GM-CSF were significantly greater (p < 0.05). However, no significant differences in the mRNA expression of PIK3CA, GLUT1, or GLUT14 were detected. According to the ICSI results, compared with the control group, the GM-CSF treatment group exhibited significantly greater fertilization rates (p = 0.027), Day 3 embryo development rate (p = 0.001), and proportions of good-quality embryos (p = 0.002). CONCLUSIONS: GM-CSF increased the expression of genes related to motility and the energy metabolism pathway and effectively promoted the motility of testis-extracted spermatozoa, consequently yielding positive clinical outcomes.

Inhibition of LXR controls the polarization of human inflammatory macrophages through upregulation of MAFB.

Monocyte-derived macrophages contribute to pathogenesis in inflammatory diseases and their effector functions greatly depend on the prevailing extracellular milieu. Whereas M-CSF primes macrophages for acquisition of an anti-inflammatory profile, GM-CSF drives the generation of T cell-stimulatory and pro-inflammatory macrophages. Liver X Receptors (LXRα and LXRß) are nuclear receptors that control cholesterol metabolism and regulate differentiation of tissue-resident macrophages. Macrophages from rheumatoid arthritis and other inflammatory pathologies exhibit an enriched LXR pathway, and recent reports have shown that LXR activation raises pro-inflammatory effects and impairs the acquisition of the anti-Inflammatory profile of M-CSF-dependent monocyte-derived macrophages (M-MØ). We now report that LXR inhibition prompts the acquisition of an anti-inflammatory gene and functional profile of macrophages generated within a pathological environment (synovial fluid from Rheumatoid Arthritis patients) as well as during the GM-CSF-dependent differentiation of human monocyte-derived macrophages (GM-MØ). Mechanistically, inhibition of LXR results in macrophages with higher expression of the v-Maf Avian Musculoaponeurotic Fibrosarcoma Oncogene Homolog B (MAFB) transcription factor, which governs the macrophage anti-inflammatory profile, as well as over-expression of MAFB-regulated genes. Indeed, gene silencing experiments on human macrophages evidenced that MAFB is required for the LXR inhibitor to enhance the anti-inflammatory nature of human macrophages. As a whole, our results demonstrate that LXR inhibition prompts the acquisition of an anti-inflammatory transcriptional and functional profile of human macrophages in a MAFB-dependent manner, and propose the use of LXR antagonists as potential therapeutic alternatives in macrophage re-programming strategies during inflammatory responses.

Radical-Generating Activity, Phagocytosis, and Mechanical Properties of Four Phenotypes of Human Macrophages.

Macrophages are the major players and orchestrators of inflammatory response. Expressed proteins and secreted cytokines have been well studied for two polar macrophage phenotypes-pro-inflammatory M1 and anti-inflammatory regenerative M2, but little is known about how the polarization modulates macrophage functions. In this study, we used biochemical and biophysical methods to compare the functional activity and mechanical properties of activated human macrophages differentiated from monocyte with GM-CSF (M0_GM) and M-CSF (M0_M) and polarized into M1 and M2 phenotypes, respectively. Unlike GM-CSF, which generates dormant cells with low activity, M-CSF confers functional activity on macrophages. M0_M and M2 macrophages had very similar functional characteristics-high reactive oxygen species (ROS) production level, and higher phagocytosis and survival compared to M1, while M1 macrophages showed the highest radical-generating activity but the lowest phagocytosis and survival among all phenotypes. All phenotypes decreased their height upon activation, but only M1 and M2 cells increased in stiffness, which can indicate a decrease in the migration ability of these cells and changes in their interactions with other cells. Our results demonstrated that while mechanical properties differ between M0 and polarized cells, all four phenotypes of monocyte-derived macrophages differ in their functional activities, namely in cytokine secretion, ROS production, and phagocytosis. Within the broad continuum of human macrophages obtained in experimental models and existing in vivo, there is a diversity of phenotypes with varying combinations of both markers and functional activities.

Mesenchymal Stem Cells from the Deciduous Tooth Pulp Lose their Ability to Suppress the Differentiation of Dendritic Cells during Long-Term Culturing.

A culture of cells expressing markers of mesenchymal stem cells (MSC) (CD73, CD90, CD44, CD29, and CD49b), but not hematopoietic cell markers, and capable of multilineage differentiation was isolated from the deciduous tooth pulp. Co-culturing with immature dendritic cells in the presence of LPS did not reveal an ability of the MSC to suppress the maturation of dendritic cells. On the contrary, co-culturing of MSC with monocytes in the presence of granulocyte-macrophage CSF and IL-4 led to complete suppression of monocyte differentiation into dendritic cells. However, long-term culturing of MSC from dental pulp showed that by the passage 11, they almost completely lose their suppressor ability. These results indicate that the immunological properties of MSC can change during culturing without changing their phenotypic markers. This should be taken into account when creating biomedical cell products.

Oncolytic virus-mediated reducing of myeloid-derived suppressor cells enhances the efficacy of PD-L1 blockade in gemcitabine-resistant pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is often refractory to treatment with gemcitabine (GEM) and immune checkpoint inhibitors including anti-programmed cell death ligand 1 (PD-L1) antibody. However, the precise relationship between GEM-resistant PDAC and development of an immunosuppressive tumor microenvironment (TME) remains unclear. In this study, we investigated the immunosuppressive TME in parental and GEM-resistant PDAC tumors and assessed the therapeutic potential of combination therapy with the telomerase-specific replication-competent oncolytic adenovirus OBP-702, which induces tumor suppressor p53 protein and PD-L1 blockade against GEM-resistant PDAC tumors. Mouse PDAC cells (PAN02) and human PDAC cells (MIA PaCa-2, BxPC-3) were used to establish GEM-resistant PDAC lines. PD-L1 expression and the immunosuppressive TME were analyzed using parental and GEM-resistant PDAC cells. A cytokine array was used to investigate the underlying mechanism of immunosuppressive TME induction by GEM-resistant PAN02 cells. The GEM-resistant PAN02 tumor model was used to evaluate the antitumor effect of combination therapy with OBP-702 and PD-L1 blockade. GEM-resistant PDAC cells exhibited higher PD-L1 expression and produced higher granulocyte-macrophage colony-stimulating factor (GM-CSF) levels compared with parental cells, inducing an immunosuppressive TME and the accumulation of myeloid-derived suppressor cells (MDSCs). OBP-702 significantly inhibited GEM-resistant PAN02 tumor growth by suppressing GM-CSF-mediated MDSC accumulation. Moreover, combination treatment with OBP-702 significantly enhanced the antitumor efficacy of PD-L1 blockade against GEM-resistant PAN02 tumors. The present results suggest that combination therapy involving OBP-702 and PD-L1 blockade is a promising antitumor strategy for treating GEM-resistant PDAC with GM-CSF-induced immunosuppressive TME formation.

Granulocyte-macrophage colony-stimulating factor-stimulated human macrophages demonstrate enhanced functions contributing to T-cell activation.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been implicated in numerous chronic inflammatory diseases, including multiple sclerosis (MS). GM-CSF impacts multiple properties and functions of myeloid cells via species-specific mechanisms. Therefore, we assessed the effect of GM-CSF on different human myeloid cell populations found in MS lesions: monocyte-derived macrophages (MDMs) and microglia. We previously reported a greater number of interleukin (IL)-15+ myeloid cells in the brain of patients with MS than in controls. Therefore, we investigated whether GM-CSF exerts its deleterious effects in MS by increasing IL-15 expression on myeloid cells. We found that GM-CSF increased the proportion of IL-15+ cells and/or IL-15 levels on nonpolarized, M1-polarized and M2-polarized MDMs from healthy donors and patients with MS. GM-CSF also increased IL-15 levels on human adult microglia. When cocultured with GM-CSF-stimulated MDMs, activated autologous CD8+ T lymphocytes secreted and expressed significantly higher levels of effector molecules (e.g. interferon-γ and GM-CSF) compared with cocultures with unstimulated MDMs. However, neutralizing IL-15 did not attenuate enhanced effector molecule expression on CD8+ T lymphocytes triggered by GM-CSF-stimulated MDMs. We showed that GM-CSF stimulation of MDMs increased their expression of CD80 and ICAM-1 and their secretion of IL-6, IL-27 and tumor necrosis factor. These molecules could participate in boosting the effector properties of CD8+ T lymphocytes independently of IL-15. By contrast, GM-CSF did not alter CD80, IL-27, tumor necrosis factor and chemokine (C-X-C motif) ligand 10 expression/secretion by human microglia. Therefore, our results underline the distinct impact of GM-CSF on human myeloid cells abundantly present in MS lesions.

GM-CSF improves endometrial receptivity in a thin endometrium rat model by upregulating HOXA10.

Endometrial receptivity is a prerequisite for the success of assisted reproduction. Patients with a consistently thin endometrium frequently fail to conceive, owing to low endometrial receptivity, and there are currently very few therapeutic options available. Our previous study demonstrated that intrauterine granulocyte-macrophage colony-stimulating factor (GM-CSF) administration resulted in a significant improvement in clinical pregnancy and implantation rates and was an effective means of increasing endometrial thickness on the day of embryo transfer in patients with thin endometrium. In order to explore the underlying process, an animal model with a thin endometrium was constructed, the homeobox A10 gene (HOXA10) was downregulated, and an inhibitor of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway (MAPK/ERK) was employed. Our findings strongly suggest a marked decrease in GM-CSF levels in the thin endometrial rat model, and the suppression of HOXA10 impeded the therapeutic efficacy of GM-CSF in this model. Moreover, we showed that GM-CSF significantly increases endometrial receptivity in the rat model and upregulates HOXA10 via the MAPK/ERK pathway. Our data provide new molecular insights into the mechanisms underlying formation of a thin endometrium and highlight a novel, potential clinical treatment strategy as well as directions for further research.

Autophagy gene Atg16L1 prevents lethal T cell alloreactivity mediated by dendritic cells.

Atg16L1 mediates the cellular degradative process of autophagy and is considered a critical regulator of inflammation based on its genetic association with inflammatory bowel disease. Here we find that Atg16L1 deficiency leads to an exacerbated graft-versus-host disease (GVHD) in a mouse model of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Atg16L1-deficient allo-HSCT recipients with GVHD displayed increased T cell proliferation due to increased dendritic cell (DC) numbers and costimulatory molecule expression. Reduced autophagy within DCs was associated with lysosomal abnormalities and decreased amounts of A20, a negative regulator of DC activation. These results broaden the function of Atg16L1 and the autophagy pathway to include a role in limiting a DC-mediated response during inflammatory disease, such as GVHD.

Colony stimulating factor 2 protects the preimplantation bovine embryo from heat shock.

Heat stress can have severe deleterious effects on embryo development and survival. The present study evaluated whether CSF2 can protect the developmental competence of the bovine embryo following exposure to a heat shock of 41°C at the zygote and morula stages. In the first experiment, putative zygotes and 2-cell embryos were assigned to receive either 10 ng/ml CSF2 or vehicle, and then cultured for 15 h at either 38.5°C or 41°C and then at 38.5°C until day 7.5. Heat shock reduced blastocyst development for embryos treated with vehicle but not for embryos cultured with CSF2. In the second experiment, day 5 embryos (morula) were treated with CSF2 or vehicle and then cultured for 15 h at either 38.5°C or 41°C and then at 38.5°C until day 7.5. Temperature treatment did not affect development to the blastocyst stage and there was no effect of CSF2 treatment or the interaction. Results indicate that CSF2 can reduce the deleterious effects of heat shock at the zygote or two-cell stage when the embryo is transcriptionally inactive.

Chitosan-Based Dressing as a Sustained Delivery System for Bioactive Cytokines.

Wounds represent a common occurrence in human life. Consequently, scientific investigations are underway to advance wound healing methodologies, with a notable focus on dressings imbued with biologically active compounds capable of orchestrating the wound microenvironment through meticulously regulated release mechanisms. Among these bioactive agents are cytokines, which, when administered to the wound milieu without appropriate protection, undergo rapid loss of their functional attributes. Within the context of this research, we present a method for fabricating dressings enriched with G-CSF (granulocyte colony-stimulating factor) or GM-CSF (granulocyte-macrophage colony-stimulating factor), showcasing both biological activity and protracted release dynamics. Based on Ligasano, a commercial polyurethane foam dressing, and chitosan crosslinked with TPP (sodium tripolyphosphate), these dressings are noncytotoxic and enable cytokine incorporation. The recovery of cytokines from dressings varied based on the dressing preparation and storage techniques (without modification, drying, freeze-drying followed by storage at 4 °C or freeze-drying followed by storage at 24 °C) and cytokine type. Generally, drying reduced cytokine levels and their bioactivity, especially with G-CSF. The recovery of G-CSF from unmodified dressings was lower compared to GM-CSF (60% vs. 80%). In summary, our freeze-drying approach enables the storage of G-CSF or GM-CSF enriched dressings at 24 °C with minimal cytokine loss, preserving their biological activity and thus enhancing future clinical availability.

Granulocyte-Macrophage-Colony-Stimulating-Factor Combined with Prostaglandin E1 Create Dendritic Cells of Leukemic Origin from AML Patients' Whole Blood and Whole Bone Marrow That Mediate Antileukemic Processes after Mixed Lymphocyte Culture.

Although several (chemotherapeutic) protocols to treat acute myeloid leukemia (AML) are available, high rates of relapses in successfully treated patients occur. Strategies to stabilize remissions are greatly needed. The combination of the (clinically approved) immune-modulatory compounds Granulocyte-Macrophage-Colony-Stimulating-Factor (GM-CSF) and Prostaglandine E1 (PGE-1) (Kit-M) converts myeloid blasts into dendritic cells of leukemic origin (DCleu). After stimulation with DCleu ex vivo, leukemia-specific antileukemic immune cells are activated. Therefore, Kit-M treatment may be an attractive immunotherapeutic tool to treat patients with myeloid leukemia. Kit-M-mediated antileukemic effects on whole bone marrow (WBM) were evaluated and compared to whole blood (WB) to evaluate the potential effects of Kit-M on both compartments. WB and WBM samples from 17 AML patients at first diagnosis, in persisting disease and at relapse after allogeneic stem cell transplantation (SCT) were treated in parallel with Kit-M to generate DC/DCleu. Untreated samples served as controls. After a mixed lymphocyte culture enriched with patients' T cells (MLC), the leukemia-specific antileukemic effects were assessed through the degranulation- (CD107a+ T cells), the intracellular IFNγ production- and the cytotoxicity fluorolysis assay. Quantification of cell subtypes was performed via flow cytometry. In both WB and WBM significantly higher frequencies of (mature) DCleu were generated without induction of blast proliferation in Kit-M-treated samples compared to control. After MLC with Kit-M-treated vs. not pretreated WB or WBM, frequencies of (leukemia-specific) immunoreactive cells (e.g., non-naive, effector-, memory-, CD3+ß7+ T cells, NK- cells) were (significantly) increased, whereas leukemia-specific regulatory T cells (Treg, CD152+ T cells) were (significantly) decreased. The cytotoxicity fluorolysis assay showed a significantly improved blast lysis in Kit-M-treated WB and WBM compared to control. A parallel comparison of WB and WBM samples revealed no significant differences in frequencies of DCleu, (leukemia-specific) immunoreactive cells and achieved antileukemic processes. Kit-M was shown to have comparable effects on WB and WBM samples regarding the generation of DCleu and activation of (antileukemic) immune cells after MLC. This was true for samples before or after SCT. In summary, a potential Kit-M in vivo treatment could lead to antileukemic effects in WB as well as WBM in vivo and to stabilization of the disease or remission in patients before or after SCT. A clinical trial is currently being planned.