Granulocyte-macrophage Colony-stimulating Factor Production Research Articles

A comprehensive study of glucose and oxygen gradients in a scaled-down model of recombinant HuGM-CSF production in thermoinduced Escherichia coli fed-batch cultures

The effect of gradients of elevated glucose and low dissolved oxygen in the addition zone of fed-batch E. coli thermoinduced recombinant high cell density cultures can be evaluated through two-compartment scale-down models. Here, glucose was fed in the inlet of a plug flow bioreactor (PFB) connected to a stirred tank bioreactor (STB). E. coli cells diminished growth from 48.2 ± 2.2 g/L in the stage of RP production if compared to control (STB) with STB-PFB experiments, when residence time inside the PFB was 25 s (34.1 ± 3.5 g/L) and 40 s (25.6 ± 5.1 g/L), respectively. The recombinant granulocyte-macrophage colony-stimulating factor (rHuGM-CSF) production decreased from 34 ± 7% of RP in inclusion bodies (IB) in control cultures to 21 ± 8%, and 7 ± 4% during the thermoinduction production phase when increasing residence time inside the PFB to 25 s and 40 s, respectively. This, along with the accumulation of acetic and formic acid (up to 4 g/L), indicates metabolic redirection of central carbon routes through metabolic flow and mixed acid fermentation. Special care must be taken when producing a recombinant protein in heat-induced E. coli, because the yield and productivity of the protein decreases as the size of the bioreactors increases, especially if they are carried at high cell density.

Enhancing productivity of Chinese hamster ovary (CHO) cells: synergistic strategies combining low-temperature culture and mTORC1 signaling engineering

Introduction: The growing demand for recombinant proteins in medicine has prompted biopharmaceutical companies to seek ways to maximize the manufacturing process. Despite its known negative impact on cell growth, temperature shift (TS) has emerged as a cost-effective strategy to enhance protein quantity and quality in Chinese Hamster Ovary cells (CHO). As cells adapt their growth and protein synthesis rate to the environment through influencing mTOR complex 1 (mTORC1), here we evaluated the potential of mTORC1 signaling engineering to improve the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) protein in stable CHO cells at low temperature.Methods: First, the expression of genes that negatively control mTORC1 functions in response to environmental fluctuations, including TSC1, AMPK, MAPKAPK5, and MARK4 genes, was assessed via real-time qPCR in CHO-K1 after a temperature shift from 37°C to 30°C. Then, plasmids harboring the shRNAs targeting these genes were constructed into the PB513B-1 plasmid with expression driven by either the constitutive CMV promoter or the cold-inducible HSP90 promoter. Finally, the impact of transient gene downregulation was evaluated on GM-CSF and mTOR proteins productivity in GM-CSF-producing CHO-K1 cells using ELISA and Western-blot assays, respectively. The growth rate of the transfected cells at the two temperatures was evaluated using flow cytometry.Results: Hypothermic conditions promote the upregulation of mTORC1 inhibitor genes, especially TSC1 and MAPKAPK5, while downregulating S6K, a key effector of the mTORC1 signaling pathway, in CHO-K1 cells. Transcription and protein levels of mTOR increased upon transfection, “pB513-b CMV-P/4shRNAs/GFP” plasmid, “pB513-bHSP90-P/4sh-RNAs/GFP” and pB513B-1 plasmid as mock group in GM-CSF-producing CHO-K1 cells (approximately 60%), along with a high transcript level of S6K. Cell growth-related characteristics were improved, albeit with distinct effects at different temperatures. Notably, these changes were more efficient at 30°C when utilizing the HSP90 promoter, resulting in a three-fold increase in GM-CSF production after 3 days.Conclusion: This study highlights the importance of temperature regulation and mTORC1 modulation in CHO cellular processes, particularly in recombinant protein production. Understanding these mechanisms paves the way for developing innovative strategies to enhance cell growth, protein synthesis, and overall bioprocess performance, particularly in manufacturing human therapeutic proteins.

Blockade of Proteinase-Activated Receptor 2 (PAR2) Attenuates Neuroinflammation in Experimental Autoimmune Encephalomyelitis.

Proteinase-activated receptor-2 (PAR2), which modulates inflammatory responses, is elevated in the central nervous system in multiple sclerosis (MS) and in its murine model, experimental autoimmune encephalomyelitis (EAE). In PAR2-null mice, disease severity of EAE is markedly diminished. We therefore tested whether inhibiting PAR2 activation in vivo might be a viable strategy for the treatment of MS. Using the EAE model, we show that a PAR2 antagonist, the pepducin palmitoyl-RSSAMDENSEKKRKSAIK-amide (P2pal-18S), attenuates EAE progression by affecting immune cell function. P2pal-18S treatment markedly diminishes disease severity and reduces demyelination, as well as the infiltration of T-cells and macrophages into the central nervous system. Moreover, P2pal-18S decreases granulocyte-macrophage colony-stimulating factor (GM-CSF) production and T-cell activation in cultured splenocytes and prevents macrophage polarization in vitro. We conclude that PAR2 plays a key role in regulating neuroinflammation in EAE and that PAR2 antagonists represent promising therapeutic agents for treating MS and other neuroinflammatory diseases. SIGNIFICANCE STATEMENT: Proteinase-activated receptor-2 modulates inflammatory responses and is increased in multiple sclerosis lesions. We show that the proteinase-activated receptor-2 antagonist palmitoyl-RSSAMDENSEKKRKSAIK-amide reduces disease in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis by inhibiting T-cell and macrophage activation and infiltration into the central nervous system, making it a potential treatment for multiple sclerosis.

Gene Ontology Analysis Highlights Biological Processes Influencing Responsiveness to Biological Therapy in Psoriasis.

Psoriasis is a chronic, immune-mediated and inflammatory skin disease. Although various biological drugs are available for psoriasis treatment, some patients have poor responses or do not respond to treatment. The aim of the present study was to highlight the molecular mechanism of responsiveness to current biological drugs for psoriasis treatment. To this end, we reviewed previously published articles that reported genes associated with treatment response to biological drugs in psoriasis, and gene ontology analysis was subsequently performed using the Cytoscape platform. Herein, we revealed a statistically significant association between NF-kappaB signaling (p value = 3.37 × 10-9), regulation of granulocyte macrophage colony-stimulating factor production (p value = 6.20 × 10-6), glial cell proliferation (p value = 2.41 × 10-5) and treatment response in psoriatic patients. To the best of our knowledge, we are the first to directly associate glial cells with treatment response. Taken together, our study revealed gene ontology (GO) terms, some of which were previously shown to be implicated in the molecular pathway of psoriasis, as novel GO terms involved in responsiveness in psoriatic disease patients.

Decidual lymphatic endothelial cell-derived granulocyte-macrophage colony-stimulating factor induces M1 macrophage polarization via the NF-κB pathway in severe pre-eclampsia.

Direct interactions between macrophages and lymphatic vessels have been shown previously.In pre-eclampsia (PE), macrophages are dominantly polarized into a proinflammatory M1 phenotype and lymphangiogenesis is defective in the decidua.Here, we investigated whether decidual lymphatic endothelial cells (dLECs) affect macrophage polarization in PE. THP-1 macrophages were cocultured with dLECs or cultured in the conditioned medium (CM) of dLECs. Macrophage polarization was measured using flow cytometry. Granulocyte-macrophage colony-stimulating factor (GM-CSF) expression in dLECs was measured using qRT-PCR and ELISA. The activation of nuclear translocation of nuclear factor-κ (NF-κB), an upstream signaling molecule of GM-CSF, was assessed by immunocytochemical localization of p65. Through GM-CSF knockdown and NF-κB inhibition in dLEC, we evaluated whether the GM-CSF/NF-κB pathway of PE dLEC affects decidual macrophage polarization. The ratio of inflammatory M1 macrophages with HLA-DR+ /CD80+ markers significantly increased following coculturing with PE dLECs or culturing in PE dLEC CM, indicating that the PE dLEC-derived soluble factor acts in a paracrine manner. GM-CSF expression was significantly upregulated in PE dLECs. Recombinant human GM-CSF induced macrophage polarization toward an M1-like phenotype, whereas its knockdown in PE dLECs suppressed it, suggesting PE dLECs induce M1 macrophage polarization by secreting GM-CSF. The NF-κB p65 significantly increased in PE dLECs compared to the control, and pretreatment with an NF-κB inhibitor significantly suppressed GM-CSF production from PE dLECs. In PE, dLECs expressing high levels of GM-CSF via the NF-κB-dependent pathway play a role in inducing decidual M1 macrophage polarization.

Functional Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) Delivered by Canine Histiocytic Sarcoma Cells Persistently Infected with Engineered Attenuated Canine Distemper Virus.

The immune response plays a key role in the treatment of malignant tumors. One important molecule promoting humoral and cellular immunity is granulocyte-macrophage colony-stimulating factor (GM-CSF). Numerous successful trials have led to the approval of this immune-stimulating molecule for cancer therapy. However, besides immune stimulation, GM-CSF may also accelerate tumor cell proliferation, rendering this molecule a double-edged sword in cancer treatment. Therefore, detailed knowledge about the in vitro function of GM-CSF produced by infected tumor cells is urgently needed prior to investigations in an in vivo model. The aim of the present study was to functionally characterize a persistent infection of canine histiocytic sarcoma cells (DH82 cells) with the canine distemper virus strain Onderstepoort genetically engineered to express canine GM-CSF (CDV-Ondneon-GM-CSF). The investigations aimed (1) to prove the overall functionality of the virally induced production of GM-CSF and (2) to determine the effect of GM-CSF on the proliferation and motility of canine HS cells. Infected cells consistently produced high amounts of active, pH-stable GM-CSF, as demonstrated by increased proliferation of HeLa cells. By contrast, DH82 cells lacked increased proliferation and motility. The significantly increased secretion of GM-CSF by persistently CDV-Ondneon-GM-CSF-infected DH82 cells, the pH stability of this protein, and the lack of detrimental effects on DH82 cells renders this virus strain an interesting candidate for future studies aiming to enhance the oncolytic properties of CDV for the treatment of canine histiocytic sarcomas.

Pre-conditioning of gingival epithelial cells with sub-apoptotic concentrations of curcumin prevents pro-inflammatory cytokine release.

Plaque-induced gingival inflammation (gingivitis) is ubiquitous in humans. The epithelial barrier reacts to the presence of oral bacteria and induces inflammatory cascades. The objective of this study was to investigate the mechanism by which the small molecule micronutrient curcumin could decrease inflammatory response in vitro to oral bacterium heat-killed Fusobacterium nucleatum as curcumin could be a useful compound for combatting gingivitis already consumed by humans. H400 oral epithelial cell line was pre-conditioned with curcumin and the production of cytokines was measured by enzyme-linked immunosorbent assay (ELISA) and translocation of transcription factors was used to monitor inflammatory responses. Haem oxygenase (HO-1) expression and molecules that HO-1 releases were evaluated for their potential to reduce the quantity of cytokine production. Immunofluorescence microscopy and Western blotting were used to evaluate changes in transcription factor and enzyme location. Pre-conditioning of H400 cells with a sub-apoptotic concentration of curcumin (20 μM) attenuated secretion of Granulocyte-Macrophage - Colony-Stimulating Factor (GM-CSF) and reduced NFkB nuclear translocation. This pre-conditioning caused an increase in nuclear Nrf2; an initial drop (at 8h) followed by an adaptive increase (at 24 h) in glutathione; and an increase in haem oxygenase (HO-1) expression. Inhibition of HO-1 by SnPPIX prevented the curcumin-induced attenuation of GM-CSF production. HO-1 catalyses the breakdown of haem to carbon monoxide, free iron and biliverdin: the HO-1/CO anti-inflammatory pathway. Elevations in carbon monoxide, achieved using carbon monoxide releasing molecule-2 (CORM2) treatment alone abrogated F. nucleatum-induced cytokine production. Biliverdin is converted to bilirubin by biliverdin reductase (BVR). This pleiotropic protein was found to increase in cell membrane expression upon curcumin treatment. Curcumin decreased inflammatory cytokine production induced by Fusobacterium nucleatum in H400 oral epithelial cells. The mechanism of action appears to be driven by the increase of haem oxygenase and the production of carbon monoxide.

Differentially Expressed Genes and Signalling Pathways Regulated by High Selenium Involved in Antioxidant and Immune Functions of Goats Based on Transcriptome Sequencing.

The objective of this study is to observe the effect of high selenium on the antioxidant and immune functions of growing goats based on transcriptome sequencing. Eighteen goats were randomly divided into three groups: (1) the control (CON) group was fed a basal diet, and (2) the treatment 1 group (LS) and treatment 2 group (HS) were fed a basal diet with 2.4 and 4.8 mg/kg selenium-yeast (SY), respectively. The results indicate that HS treatment significantly (p < 0.05) increased the apparent digestibility of either extract and significantly increased (p < 0.05) total antioxidant capacity, whereas it significantly (p < 0.05) decreased plasma aspartate aminotransferase and malondialdehyde relative to the control group. The LS treatment had significantly (p < 0.05) increased glutathione S-transferase and catalase compared to CON. A total of 532 differentially expressed genes (DEGs) between the CON and HS were obtained using transcriptome sequencing. Kyoto Encyclopedia of Genes and Genomes analysis identified upregulated (p < 0.05) DEGs mainly related to vascular smooth muscle contraction, alpha-linolenic acid metabolism, biosynthesis of unsaturated fatty acids, the VEGF signalling pathway, and proteoglycans in cancer; downregulated (p < 0.05) DEGs mainly related to the NOD-like receptor signalling pathway, influenza A, cytokine-cytokine receptor interaction, haematopoietic cell lineage, and African trypanosomiasis. Ontology analyses of the top genes show that the identified DEGs are mainly involved in the regulation of granulocyte macrophage colony-stimulating factor production for biological processes, the external side of the plasma membrane for cellular components, and carbohydrate derivative binding for molecular functions. Seven genes are considered potential candidate genes for regulating antioxidant activity, including selenoprotein W, 1, glutathione peroxidase 1, glutathione S-transferase A1, tumour necrosis factor, tumour necrosis factor superfamily member 10, tumour necrosis factor superfamily member 8, and tumour necrosis factor superfamily member 13b. The experimental observations indicate that dietary supplementation with 4.8 mg/kg SY can enhance antioxidant and immune functions by improving muscle immunity, reducing the concentrations of inflammatory molecules, and modulating antioxidant and inflammatory signalling pathways in growing goats.

Корреляции между клинико-лабораторными показателями и маркерами ангиогенеза и фиброза в операционном материале при миомэктомии

Introduction. The search for the relationships between clinical features of patients with uterine fibroids (uterine myoma, leiomyoma) and the characteristics of their myomatous nodules is relevant for identifying prognostic markers. Aim. In patients of reproductive age with uterine fibroids, to identify the presence of correlations between clinical laboratory parameters and markers of inflammation and fibrosis in the surgical specimens. Materials and methods. Myomectomy was performed in 226 female patients with myomatous no¬dules. Examination of patients included clinical investigations (ultrasound, magnetic resonance imaging), extragenital abnormalities were assessed. Samples of the dominant myomatous nodules and myometrium were examined using enzyme-linked immunosorbent assay (ELISA) for the content of baseline and stimulated by polyclonal activators (PA) vascular endothelial growth factor (VEGF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in the incubation medium. The expression of progesterone receptors (PgR) and VEGF using immunohistochemistry (IHC) was assessed. Results. The combination of uterine fibroids with endometriosis in reproductive-aged women influenced a number of histological and molecular characteristics of the dominant myomatous nodule: positively – on the grade of fibrosis (p &lt; 0.0001), negatively – on the PA-induced production of GM-CSF and VEGF (p = 0.0054 and p = 0.0016, respectively). Patients with adenomyosis had more myomatous nodules (p = 0.0192) compared with patients without any form of endometriosis, while the largest nodule diameter and the degree of fibrosis were in women without adenomyosis (p = 0.0397 and p = 0.0344, respectively). PA-induced VEGF production in the nodule is greater in adenomyosis (p = 0.0326), while PgR expression in the perifocal myometrium is less (p = 0.045). In patients with uterine myoma, the body mass index had a weak positive correlation with VEGF expression in the perifocal myometrium. The number of myomatous nodules had weak positive correlation with the stimulation index of PA (SIPA) which reflects their influence of the VEGF production in the dominant nodule in vitro. The degree of fibrosis of the dominant nodule was characterized by moderately positive correlation with the expression of VEGF in the perifocal myometrium according to IHC. Conclusion. The results of the study reflect the systemic nature of myomatous transformation with multifocal myometrium tumor growth and the potential for the formation of new myomatous nodules, especially in women with elevated body mass index.

Red Ginseng Extract and γ-Aminobutyric Acid Synergistically Enhance Immunity Against Cancer Cells and Antitumor Metastasis Activity in Mice.

The effects of combined administration of red ginseng (RG) extracts and gamma-aminobutyric acid (GABA) on immunostimulatory activity and tumor metastasis inhibition were investigated in mice. For the immunostimulatory activity, splenocyte proliferation, natural killer (NK) cell activity, including the production of granzyme B (GrB) and interferon gamma (IFN-γ), and serum level of cytokine such as IFN-γ, interleukin (IL)-17, and IL-21 were assessed. Peyer's patch cells obtained from mice administered with RG+GABA were cultured, and the cytokine level in the culture supernatant and bone marrow (BM) cell proliferation activity were examined. The proliferative activity of splenocytes was significantly higher in the RG-GABA treatment group than in RG or GABA alone (P < .05). In the experimental tumor metastasis model, oral administration of RG+GABA showed a higher antitumor metastatic effect compared to that of RG or GABA alone. Oral administration of RG+GABA significantly augmented NK cell-mediated cytotoxicity against YAC-1 tumor cells. In addition, the production of GrB and IFN-γ was stimulated in the culture supernatant of NK cells and YAC-1 cells. Serum concentrations of IFN-γ, IL-17, and IL-21 in mice with RG+GABA were significantly higher compared to the corresponding blood levels in mice administered with RG or GABA alone. The RG+GABA group showed significant BM cell proliferation and increased production of IL-6 and granulocyte-macrophage colony-stimulating factor compared to that in the monotherapy groups. Therefore, RG may have a synergistic effect with GABA for enhancing the host defense system such as BM proliferation and NK cell activity in a tumor metastasis model.

Blocking DCIR mitigates colitis and prevents colorectal tumors by enhancing the GM-CSF-STAT5 pathway.

Dendritic cell immunoreceptor (DCIR; Clec4a2), a member of the C-type lectin receptor family, plays important roles in homeostasis of the immune and bone systems. However, the intestinal role of this molecule is unclear. Here, we show that dextran sodium sulfate (DSS)-induced colitis and azoxymethane-DSS-induced intestinal tumors are reduced in Clec4a2-/- mice independently of intestinal microbiota. STAT5 phosphorylation and expression of Csf2 and tight junction genes are enhanced, while Il17a and Cxcl2 are suppressed in the Clec4a2-/- mouse colon, which exhibits reduced infiltration of neutrophils and myeloid-derived suppressor cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF) administration ameliorates DSS colitis associated with reduced Il17a and enhanced tight junction gene expression, whereas anti-GM-CSF exacerbates symptoms. Furthermore, anti-NA2, a ligand for DCIR, ameliorates colitis and prevents colorectal tumors. These observations indicate that blocking DCIR signaling ameliorates colitis and suppresses colonic tumors, suggesting DCIR as a possible target for the treatment of these diseases.

Persistent Infection of a Canine Histiocytic Sarcoma Cell Line with Attenuated Canine Distemper Virus Expressing Vasostatin or Granulocyte-Macrophage Colony-Stimulating Factor.

Canine histiocytic sarcoma (HS) represents a neoplasia with poor prognosis. Due to the high metastatic rate of HS, there is urgency to improve treatment options and to prevent tumor metastases. Canine distemper virus (CDV) is a single-stranded negative-sense RNA (ssRNA (-)) virus with potentially oncolytic properties. Moreover, vasostatin and granulocyte-macrophage colony-stimulating factor (GM-CSF) are attractive molecules in cancer therapy research because of their anti-angiogenetic properties and potential modulation of the tumor microenvironment. In the present study, an in vitro characterization of two genetically engineered viruses based on the CDV strain Onderstepoort (CDV-Ond), CDV-Ondneon-vasostatin and CDV-Ondneon-GM-CSF was performed. Canine histiocytic sarcoma cells (DH82 cells) were persistently infected with CDV-Ond, CDV-Ondneon, CDV-Ondneon-vasostatin and CDV-Ondneon-GM-CSF and characterized on a molecular and protein level regarding their vasostatin and GM-CSF production. Interestingly, DH82 cells persistently infected with CDV-Ondneon-vasostatin showed a significantly increased number of vasostatin mRNA transcripts. Similarly, DH82 cells persistently infected with CDV-Ondneon-GM-CSF displayed an increased number of GM-CSF mRNA transcripts mirrored on the protein level as confirmed by immunofluorescence and Western blot. In summary, modified CDV-Ond strains expressed GM-CSF and vasostatin, rendering them promising candidates for the improvement of oncolytic virotherapies, which should be further detailed in future in vivo studies.

AMFR drives allergic asthma development by promoting alveolar macrophage–derived GM-CSF production

Alveolar macrophages (AMs) are specialized tissue-resident macrophages that orchestrate the immune response in allergic inflammation and asthma. However, what signals direct AMs to cross talk with other immune cells remains unclear. Here, we report that autocrine motility factor receptor (AMFR), an endoplasmic reticulum-resident E3 ubiquitin ligase, is upregulated in AMs of asthma and is critical for this condition. AMFR deficiency significantly decreased allergy-induced T helper 2 (Th2) and eosinophilic inflammation, with less granulocyte-macrophage colony-stimulating factor (GM-CSF) production in AMs. Mechanistically, following thymic stromal lymphopoietin (TSLP) stimulation, AMFR associated directly with cytokine-inducible SH2-containing protein (CIS), induced the ubiquitination of Lys48-linked polyubiquitination of CIS, and consequently blocked the inhibitory effect of CIS on signal transducer and activator of transcription 5 (STAT5) phosphorylation and the downstream pathway activation in AMs. In conclusion, our results demonstrate that AMFR serves a crucial role in promoting inflammation in asthma through regulating AM function, and may emerge as a new potential drug target for asthma therapy.

Setd2 determines distinct properties of intestinal ILC3 subsets to regulate intestinal immunity.

Subsets of group 3 innate lymphoid cells (ILC3s) are heterogeneous in development and function and play differential roles in intestinal immunity. Histone modifications are involved in the fate commitment of immune cells, including ILC3s. Here, we report that deletion of Setd2, histone H3K36 methyltransferase, in ILC3s results in increased generation of NKp46+ILC3s with enhanced cytotoxic signatures and tumor-suppressive capacity. Meanwhile, Rag1-/-RorcCreSetd2flox/flox mice have fewer CCR6+ILC3s and less defective solitary intestinal lymphoid tissue formation, accompanied by reduced granulocyte-macrophage colony-stimulating factor (GM-CSF) production by NKp46-ILC3s and decreased CD11b+CD103+ dendritic cell accumulation. The deficiency of Setd2-/-NKp46-ILC3s may contribute to disturbed RORγt+Treg homeostasis and intestinal inflammation in Rag1-/-RorcCreSetd2flox/flox mice upon Tcell reconstitution. Setd2 regulates genome accessibility imprinting gene mRNA expression, with a more profound effect on NKp46+ILC3s than NKp46-ILC3s. Therefore, Setd2 determines distinct chromatin status and transcriptomic programs of ILC3 subsets to affect their function and intestinal immunity.

JAK1 signaling in dendritic cells promotes peripheral tolerance in autoimmunity through PD-L1-mediated regulatory Tcell induction.

Dendritic cells (DCs) induce peripheral Tcell tolerance, but cell-intrinsic signaling cascades governing their stable tolerogenesis remain poorly defined. Janus Kinase 1 (JAK1) transduces cytokine-receptor signaling, and JAK inhibitors (Jakinibs), including JAK1-specific filgotinib, break inflammatory cycles in autoimmunity. Here, we report in heterogeneous DC populations of multiple secondary lymphoid organs that JAK1 promotes peripheral Tcell tolerance during experimental autoimmune encephalomyelitis (EAE). Mice harboring DC-specific JAK1 deletion exhibit elevated peripheral CD4+ Tcell expansion, less regulatory Tcells (Tregs), and worse EAE outcomes, whereas adoptive DC transfer ameliorates EAE pathogenesis by inducing peripheral Tregs, programmed cell death ligand 1 (PD-L1) dependently. This tolerogenic program is substantially reduced upon the transfer of JAK1-deficient DCs. DC-intrinsic IFN-γ-JAK1-STAT1 signaling induces PD-L1, which is required for DCs to convert CD4+ Tcells into Tregs invitro and attenuated upon JAK1 deficiency and filgotinib treatment. Thus, DC-intrinsic JAK1 promotes peripheral tolerance, suggesting potential unwarranted DC-mediated effects of Jakinibs in autoimmune diseases.

Изучение маркеров ангиогенеза и воспаления в доминантных миоматозных узлах и миометрии

Th e aim of the research. To study markers of angiogenesis and infl ammation in dominant fi broids and myometrium. Material and methods. Myomectomy in 226 patients of reproductive age with uterine myoma was carried out. Th e resected samples of dominant myomatous nodules and perifocal myometrium of 35 females including 12 treated using selective progesterone receptor (PgR) modulator were examined for the content of granulocyte-macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF) and 17-hydroxyprogesterone in the incubation medium using enzyme-linked immunosorbent assay (ELISA). Spontaneous and stimulated by polyclonal activators (PA) levels of the markers were studied. Additionally, the expression of PgR and VEGF was analysed through immunohistochemistry (IHC). Nonparametric statistics methods were used. Th e Wilcoxon test was used for intragroup comparison, the Mann-Whitney test was used for intergroup comparison. Th e correlations between biomarkers were studied using the Spearman correlation coeffi cient. Results. In the surgical specimens of leiomyoma and myometrium treated without hormone therapy, statistically signifi cant diff erences were revealed for PA-stimulated GM-CSF production incubated in vitro (2-fold lower in the nodule than in the myometrium), as well as for VEGF expression in tissue samples (2-fold higher in the nodule than in the myometrium). PgR expression in the myometrium directly correlated with the infl ammatory infi ltration of the nodule. Th e use of the selective PgR modulator contributed to the downregulation of GM-CSF production and VEGF expression in the nodule with no eff ect on the myometrium, as well as to a decrease in 17-OH-progesterone in the myometrium without changes in the nodules, and to a negative correlation between PgR expression and VEGF production in leiomyomas. Conclusion. For the dominant fi broid, application of the selective PgR modulator contributed to a downregulation of VEGF expression and to the decrease of in-vitro production of GM-CSF without infl uence on the perifocal myometrium, which demonstrates its proangiogenic and proinfl ammatory potential in patients with uterine myoma.

GM-CSF production by non-classical monocytes controls antagonistic LPS-driven functions in allergic inflammation.

SUMMARYLipopolysaccharide (LPS) can either promote or prevent T helper 2 (Th2) cell allergic responses. However, the underlying mechanism remains unknown. We show here that LPS activity switches from pro-pathogenic to protective depending on the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) by non-classical monocytes. In the absence of GM-CSF, LPS can favor pathogenic Th2 cell responses by supporting the trafficking of lung-migratory dendritic cells (mDC2s) into the lung-draining lymph node. However, when non-classical monocytes produce GM-CSF, LPS and GM-CSF synergize to differentiate monocyte-derived DCs from classical Ly6Chi monocytes that instruct mDC2s for Th2 cell suppression. Importantly, only allergens with cysteine protease activity trigger GM-CSF production by non-classical monocytes. Hence, the therapeutic effect of LPS is restricted to allergens with this enzymatic activity. Treatment with GM-CSF, however, restores the protective effects of LPS. Thus, GM-CSF produced by non-classical monocytes acts as a rheostat that fine-tunes the pathogenic and therapeutic functions of LPS.

A case of improvement of clozapine-induced low leukocyte counts by adenine, cepharanthin and ninjin-yoei-to in a patient with treatment-resistant schizophrenia

BackgroundAlthough clozapine is the optimal drug for patients with treatment-resistant schizophrenia, the drug has harmful adverse effects such as leukopenia. Adenine and cepharanthine are known to be effective for radiation- or drug-induced leukopenia. Furthermore, ninjin-yoei-to, a Chinese herbal medicine, augments the production of granulocyte-macrophage colony-stimulating factor. Thus, these drugs may be useful for clozapine-induced leukopenia.Case presentationA 21 years-old woman with schizophrenia was hospitalized for initiation of clozapine treatment. Despite concomitant use of adenine, cepharanthine, and lithium carbonate having activities of increasing leukocytes, a decrease in leukocyte counts occurred after the initiation of clozapine. Additional administration of ninjin-yoei-to increased leukocyte counts, which prevented the development of leukopenia.ConclusionsThis is the first case that concomitant use of adenine, cepharanthin, and ninjin-yoei-to exhibited the effectiveness of reversing the decrease in leukocytes caused by clozapine. Monitoring leukocyte counts and preventing leukopenia are essential for successful treatment with clozapine for refractory schizophrenia. These medicines may be a potential option for preventing clozapine-induced leukopenia.

Deficiency of IL-27 Signaling Exacerbates Experimental Autoimmune Uveitis with Elevated Uveitogenic Th1 and Th17 Responses.

Human uveitis is an autoimmune disease of the central nervous system that is characterized by ocular inflammation with the involvement of uveitogenic Th1 and Th17 responses. In experimental autoimmune uveitis (EAU), the animal model for human uveitis, both responses are proven to be critical in disease development. Therefore, targeting both Th1 and Th17 cells has therapeutic implication for disease resolution. IL-27 is a multifunctional cytokine that can either promote or inhibit T cell responses and is implicated in both autoimmune and infectious diseases. The aim of this study is to characterize the role of IL-27/IL-27R signaling in regulating uveitogenic Th1/Th17 responses in EAU. By immunizing IL-27Rα−/− mice and their wild-type (WT) littermates for EAU, we demonstrated that IL-27 signaling deficiency exacerbated EAU with severe ocular inflammation and impairment of visual function. Furthermore, there was a significant increase in the eye-infiltrating Th1 and Th17 cells in IL-27Rα−/− EAU mice compared to WT. Their retinal antigen-specific Th1 and Th17 responses were also significantly increased, as represented by the elevation of their signature cytokines, IFN-γ and IL-17A, respectively. We also observed the upregulation of another pathogenic cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF), from effector T cells in IL-27Rα−/− EAU mice. Mechanistic studies confirmed that IL-27 inhibited GM-CSF production from Th17 cells. In addition, the induction of IL-10 producing type 1 regulatory T (Tr1) cells was impaired in IL-27Rα−/− EAU mice. These results identified that IL-27 signaling plays a suppressive role in EAU by regulating multiple CD4+ cell subsets, including the effector Th1 and Th17 cells and the regulatory Tr1 cells. Our findings provide new insights for therapeutic potential in controlling uveitis by enhancing IL-27 signaling.

POS0112 CD8+ T-CELL INFILTRATION IS ASSOCIATED WITH LESIONAL GM-CSF OVEREXPRESSION IN GCA

Background:Giant cell arteritis (GCA) is a large-vessel vasculitis characterized by granulomatous T cell- and macrophage infiltration. Granulocyte-macrophage colony stimulating factor (GM-CSF) is present in GCA-affected vessels where it may be involved in skewing of tissue destructive macrophages (1). Furthermore, a recent phase 2 clinical trial demonstrated that GM-CSF receptor blockade was superior to placebo at 26 weeks in GCA. However, the cellular source of GM-CSF in GCA is still unclear.Objectives:As T cells have been identified as a major source of GM-CSF in other immune-mediated diseases such as multiple sclerosis, we investigated whether T cells are GM-CSF producers in GCA patients, both systemically and locally.Methods:GM-CSF production capacity by PMA-stimulated PBMCs from newly diagnosed, untreated GCA patients and HCs (N=10 each) was assessed using flow cytometry. Temporal artery biopsies (TABs, N=5) were immunohistochemically stained for CD3, CD8 and GM-CSF. Colocalization of these markers was assessed with immunofluorescence.Results:Proportions of CD4+ (median GCA=3.1%; HCs=2.76%) and CD8+ (median GCA=5.8%; HCs=5.8%) T cells produced GM-CSF after in vitro stimulation, but no significant differences were found between the groups. Immunofluorescence staining confirmed that CD3+ T cells (both CD8+ and CD8-) were positive for GM-CSF in TABs. Interestingly, GM-CSF positivity in TABs correlated strongly with the extent of CD8+ T-cell infiltration (r=0.74, p&lt;0.01), but not with CD3+ T-cell infiltration (r=0.38, p=0.16).Conclusion:Our data imply that T cells are an important source of GM-CSF in GCA lesions. The correlation of the extent of CD8+ T-cell infiltration with GM-CSF positivity suggests that CD8+ T cells are the major source of local GM-CSF overexpression in tissue.