Production Of Colony-stimulating Factor 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.

A3 INVESTIGATING MACROPHAGE-TISSUE CELL COMMUNICATION AXES IN THE GASTROINTESTINAL TRACT

Abstract Background The gastrointestinal tract boasts the highest concentration of macrophages (MPs) in the body, acting as vital immune sentinels that oversee tissue balance. Three distinct subsets of MPs, namely CCR2+, Tim4+, and CCR2,Tim4 double-negative (DN) MPs, populate distinct gut niches. Their functional specialization suggests unique interactions within their non-hematopoietic tissue microenvironments. Despite their crucial roles, the intricacies of these interactions remain elusive. One recently discovered interaction involves a communication axis between MPs and neurons within the muscularis layer of the gut. Here, neurons secrete Colony Stimulating Factor 1 (CSF1), a survival factor for MPs. These MPs reciprocate by producing Bone Morphogenetic Protein 2 (BMP2), assisting neurons in regulating gut motility. Aims Based on the aforementioned interaction, we hypothesize that BMP2, derived from tissue-resident MPs, might influence functions of other tissue niches, subsequently prompting survival of MP via CSF1 production. Our aims are twofold: (1) Identify the role of BMP2 in gut macrophages; (2) Identify BMP2-responsive CSF1-producing tissue cells within the gut. Methods To discern the roles of MP subsets, we developed a conditional knockout mouse model, targeting Bmp2 expression in MPs. We validated successful Bmp2 deletion(BMP2 KO) through quantitative polymerase chain reaction (qPCR) and an in-situ hybridization immunofluorescence assay (RNAscope). Subsequently, single cell RNA (scRNAseq) was performed, combining samples from both control and KO small intestines. This was done to further confirm BMP2 KO and to identify BMP2-responsive CSF1-producing cells. Flow cytometry was then used to characterize the intestinal MPs in control and KO mice. Results Tim4+ MPs emerged as the primary source of BMP2. Comprehensive analysis using qPCR, RNAscope, and scRNAseq validated the complete KO of BMP2 across all macrophage subsets. The loss of MP-produced BMP2 led to a disruption in MP development. Notably, a marked reduction in DN and Tim4+ MPs was observed following deletion of Bmp2 in MPs, emphasizing the critical role of BMP2-driven CSF1 in sustaining these subsets. CSF1 producing cells co-expressing the cognate BMP receptor in the gut, were predominantly non-hematopoietic tissue cells. Among these, distinct fibroblast populations emerged as a potent but not the sole source of CSF1. Conclusions Our work underscores the involvement of multiple tissue cell populations, revealing an intricate network of interactions that extends beyond fibroblasts. This study unveils the multifaceted nature of tissue-MP interactions and their critical roles in maintaining gut homeostasis. Disturbances of these pathways may pave the way for disease by altering tissue homeostasis. Funding Agencies NSERC

Feeding spray-dried plasma to broilers early in life improved their intestinal development, immunity and performance irrespective of mycotoxins in feed.

Fungi that produce mycotoxins can grow on certain food products, such as grains and feed, and can cause a variety of health issues if consumed by animals, including chickens. The use of spray-dried plasma (SDP) is one strategy for combating the health problems caused by mycotoxins. In the present study, Ross 308 chickens (n = 960) were divided into four treatment groups. T1 group was given a control diet (corn-soybean meal), T2 group was given a control diet +2% SDP, T3 group was given a control diet +2% SDP + mixture mycotoxins and T4 group was givena control diet + mycotoxin mixture. The presence of SDP resulted in weight gain and decreased feed efficiency, whereas mycotoxins resulted in weight loss and increased feed efficiency. SDP increased the thymus' relative weight. The presence of mycotoxins increased the heterophile/lymphocyte ratio. The presence of mycotoxins reduced the production of IL-2 and macrophage inflammatory protein-3 Alpha (MIP-3a), whereas the presence of SDP increased the production of macrophage colony-stimulating Factor (M-CSF). SDP resulted in higher IgA concentrations in the intestinal and tracheal washes than mycotoxin. Finally, adding SDP to broiler diets boosts weight gain, feed efficiency, and immune system development. Our results provide information supporting that SDP is a promising tool for improving poultry immunity and performance.

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.

Differential Immune-Modulating Activities of Cell Walls and Secreted Metabolites from Probiotic Bacillus coagulans JBI-YZ6.3 under Normal versus Inflamed Culture Conditions.

Spore-forming probiotic bacteria, including Bacillus coagulans, are resilient and produce a variety of beneficial metabolites. We evaluated the immune-modulating effects of the novel probiotic strain Bacillus coagulans JBI-YZ6.3, where the germinated spores, metabolite fraction, and cell wall fraction were tested in parallel using human peripheral blood mononuclear cell cultures under both normal and lipopolysaccharide-induced inflamed culture conditions. The expression of CD25 and CD69 activation markers was evaluated via flow cytometry. Supernatants were tested for cytokines, interferons, chemokines, and growth factors using Luminex arrays. The germinated spores were highly immunogenic; both the cell wall and metabolite fractions contributed significantly. Under normal culture conditions, increased levels of immune activation were observed as increased expressions of CD25 and CD69 relative to natural killer cells, suggesting an increased ability to attack virus-infected target cells. On monocytes, a complex effect was observed, where the expression of CD25 increased under normal conditions but decreased under inflamed conditions. This, in combination with increased interleukin-10 (IL-10) and decreased monocyte chemoattractant protein-1 (MCP-1) production under inflamed conditions, points to anti-inflammatory effects. The production of the stem cell-related growth factor granulocyte colony-stimulating Factor (G-CSF) was enhanced. Further research is warranted to characterize the composition of the postbiotic metabolite fraction and document the characteristics of immunomodulating agents secreted by this probiotic strain.

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.

Abstract P180: Hypertension-linked Immune Activation Regulates Osteoclastogenesis

Osteoporosis is an age-related disease affecting 18% of adults. Hypertension is associated with increased risk for fragility fracture and accelerated bone loss in adults. We hypothesized that experimental hypertension would induce osteoporosis and examined the mechanisms involved. We used two preclinical models in 12-week-old male C57Bl/6J mice: angiotensin (ang) II infusion (490 ng/kg/min) and deoxycorticosterone acetate (DOCA)-salt. After 6 weeks of ang II or 3 weeks of DOCA-salt, the left femur and sixth lumbar vertebral body were studied. Mechanical testing showed that ang II reduced femoral ultimate strength (170.7±4.794 vs 157.3±3.670 MPa, p=0.0325; 154.1±7.403 vs. 119.9±4.375 MPa, p=0.0026) and peak moment (39.09±1.660 vs. 32.77±0.9380 Newton millimeters (Nmm), p=0.0018; 29.15±1.291 vs 19.58±1.308 Nmm, p=0.0004) with cortical and trabecular bone loss compared to controls. μCT showed that hypertension reduced vertebral body bone volume fraction (30.1±1.41 vs 25.1±0.992 %, p=0.0068; 25.9±2.03 vs 18.5±1.29 %, p=0.0119) and trabecular thickness (62±1.6 vs 57±1.4 μm, p=0.0105; 60±2.1 vs 51 ±1.6 μm, p=0.0069). Flow cytometry of bone marrow from ang II-treated mice revealed that CD4+ T cells and γδ T cells produce increased IL-17A which is known to induce osteoclastogenesis. Colony stimulating factor-1 (CSF1) is also known to stimulate osteoclast formation. Using in situ hybridization, we observed increased CSF1 mRNA in endothelial cells (ECs) of transcortical arterioles of hypertensive mice. Subsequent studies of cultured bone marrow derived ECs showed that CSF1 mRNA expression increased with 0% and 10% compared to 5% stretch. 5% stretch treated with endothelial nitric oxide synthase inhibitor, L-NAME, increased expression comparable to 0% and 10%, indicating that nitric oxide inhibits CSF1 production. Thus, T-cell activation and nitric oxide-dependent EC dysfunction in hypertension may skew the bone marrow to favor osteoclastogenesis, leading to bone loss. Understanding this mechanism will elucidate novel pharmacological targets to mitigate hypertension-induced bone loss.

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.

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.

Method for Inclusion Bodies Production via E. coli Host System: rGCSF as Model Biotherapeutic Protein.

Escherichia coli is an industrial-relevant microbial host system, which is highly preferred for the large-scale production of recombinant biotherapeutics. Overexpression of these recombinant biotherapeutics in the E. coli system often results in the formation of insoluble protein aggregates termed as inclusion bodies (IBs). The yield and quality of IBs are affected by a spectrum of parameters like temperature, optical density, medium composition, induction time, and amount of inducer. Here, we present a protocol for the formation and processing of IBs for production of recombinant human granulocytes colony-stimulating factor (rGCSF).

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.

Administration of nitro-oleic acid mitigates radiation-induced hematopoietic injury in mice

AimsLimited number of agents that provide protection against hematopoietic acute radiation syndrome led us to the evaluation of nitro-oleic acid (NO2OA) as a potential protector/mitigator against radiation-induced hematopoietic injury in C57/BL6 mice. Materials and methodsNO2OA was administered before and after sub-lethal total body irradiation (TBI) and hematological parameters were evaluated 3 or 7 days after TBI. Key findingsOur results show that NO2OA significantly increase bone marrow cellularity including the granulocyte-macrophage colony-forming cells and erythroid progenitors on the 3rd day after TBI. In addition, NO2OA enhanced recovery of white blood cells (lymphocytes, neutrophils, and monocytes) in peripheral blood 7 days after irradiation. These effects may be in part attributed to NO2OA-induced granulocyte colony-stimulating factor production after TBI. On the other hand, radiation-induced impairment of peripheral red blood cells, hemoglobin, and platelets were not affected with NO2OA treatment up to 7 days post TBI. SignificanceIn conclusion, our data show that NO2OA significantly protects hematopoiesis after irradiation, and thus showed a high potential to act as an agent for medical radiation countermeasure.

Endocannabinoids Modulate Production of Osteoclastogenic Factors by Stem Cells of the Apical Papilla In Vitro

IntroductionMany mediators are produced during pulp inflammation and necrosis, including endocannabinoids (ECbs), which might affect the function of stem cells of the apical papilla (SCAP), cells of paramount importance for root formation, and regenerative endodontic treatment. The aim of this study was to evaluate the production of osteoclastogenesis-related mediators by SCAP modulated by ECbs and lipopolysaccharide (LPS) in vitro. MethodsSCAP were cultured and treated with ECb anandamide (AEA), 2-arachidonoylglycerol, or N-arachidonoylaminophenol. All groups were incubated in the presence of a vehicle or LPS and the antagonist of transient receptor potential cation channel subfamily V member 1, capsazepine. After 24 hours, the culture medium supernatants were collected for further quantification of tumor necrosis factor alpha, CCL2, macrophage colony-stimulating factor, osteoprotegerin, and receptor activator of nuclear factor kappa B ligand. ResultsSmall amounts of tumor necrosis factor alpha and receptor activator of nuclear factor kappa B ligand were detected in SCAP supernatants, and none of the experimental conditions altered their production. A down-regulation in constitutive CCL2 production was observed in the AEA group compared with that in the LPS group. The production of macrophage colony-stimulating factor was significantly increased in all groups treated with AEA compared with the control and LPS-treated groups. Osteoprotegerin was significantly increased by AEA alone and by 2-arachidonoylglycerol and N-arachidonoylaminophenol in the presence of LPS and capsazepine. ConclusionsAEA modulates some of the osteoclastogenic factors produced by SCAP in a bone resorption protective fashion.

Haloperidol Instigates Endometrial Carcinogenesis and Cancer Progression by the NF-κB/CSF-1 Signaling Cascade.

Simple SummaryHaloperidol, a typical antipsychotic, is widely used in schizophrenia and palliative care of cancer; however, the role and impact of chronic haloperidol treatment in endometrial cancer (EC) development are unclear. Here, we showed that haloperidol is a carcinogenic compound capable of inducing endometrial hyperplasia and promoting EC progression in rodents. Mechanistically, haloperidol stimulates the production of colony-stimulating factor 1 (CSF-1) on tumor cells by activating nuclear factor kappa B (NF-κB), and its downstream autocrine oncogenic CSF-1 receptor signaling contributes to this carcinogenesis. Furthermore, we demonstrated that the use of haloperidol is associated with increased EC-specific mortality in EC patients. Overall, these findings highlighted that physicians should be cautious about the use of haloperidol in female patients.Haloperidol is a routine drug for schizophrenia and palliative care of cancer; it also has antitumor effects in several types of cancer. However, the role of haloperidol in endometrial cancer (EC) development is still unclear. Here, we show that chronic haloperidol treatment in clinically relevant doses induced endometrial hyperplasia in normal mice and promoted tumor growth and malignancy in mice with orthotopic EC. The pharmacokinetic study indicated that haloperidol highly accumulated in the uterus of mice. In vitro studies revealed that haloperidol stimulated the cellular transformation of human endometrial epithelial cells (HECCs) and promoted the proliferation, migration, and invasion of human endometrial carcinoma cells (HECCs) by activating nuclear factor kappa B (NF-κB) and its downstream signaling target, colony-stimulating factor 1 (CSF-1). Gain of function of CSF-1 promotes the cellular transformation of HEECs and the malignant progression of HECCs. Moreover, blockade of CSF-1 inhibited haloperidol-promoted EC progression in vitro and in vivo. A population-based cohort study of EC patients further demonstrated that the use of haloperidol was associated with increased EC-specific mortality. Collectively, these findings indicate that clinical use of haloperidol could potentially be harmful to female patients with EC.

Oral administration of Euglena gracilis paramylon ameliorates chemotherapy-induced leukocytopenia and gut dysbiosis in mice

Euglena gracilis (EUG) is a food supplement rich in beta-glucans, which are stored in the form of granules called paramylon. We determined whether EUG improved chemotherapy-induced leukocytopenia and dysbiosis. Mice were orally administered EUG prior to gemcitabine treatment. Analyses of the blood cell count, leukocyte population in the spleen, granulocyte/macrophage–colony-stimulating factor (GM–CSF) production by splenocytes, and fecal microbiome were conducted. The recovery of total leukocytes, neutrophils, and monocytes was accelerated after a single gemcitabine treatment. A more rapid lymphocyte recovery rate was observed after four gemcitabine treatments. No difference was observed in the percentage of T, B, or myeloid cells or in the expression of Dectin-1 in the spleens of the gemcitabine and EUG/gemcitabine groups. The EUG/gemcitabine group showed an enhanced GM–CSF production by lipopolysaccharides-stimulated splenocytes. Next-generation sequencing revealed that gemcitabine-induced dysbiosis was alleviated. This study demonstrated that EUG-derived beta-glucans could act as a biological response modifier as well as prebiotics for ameliorating chemotherapy-induced adverse effects.

Steroid-induced fibroblast growth factors drive an epithelial-mesenchymal inflammatory axis in severe asthma.

Asthma and inflammatory airway diseases restrict airflow in the lung, compromising gas exchange and lung function. Inhaled corticosteroids (ICSs) can reduce inflammation, control symptoms, and improve lung function; however, a growing number of patients with severe asthma do not benefit from ICS. Using bronchial airway epithelial brushings from patients with severe asthma or primary human cells, we delineated a corticosteroid-driven fibroblast growth factor (FGF)-dependent inflammatory axis, with FGF-responsive fibroblasts promoting downstream granulocyte colony-stimulating factor (G-CSF) production, hyaluronan secretion, and neutrophilic inflammation. Allergen challenge studies in mice demonstrate that the ICS, fluticasone propionate, inhibited type 2-driven eosinophilia but induced a concomitant increase in FGFs, G-CSF, hyaluronan, and neutrophil infiltration. We developed a model of steroid-induced neutrophilic inflammation mediated, in part, by induction of an FGF-dependent epithelial-mesenchymal axis, which may explain why some individuals do not benefit from ICS. In further proof-of-concept experiments, we found that combination therapy with pan-FGF receptor inhibitors and corticosteroids prevented both eosinophilic and steroid-induced neutrophilic inflammation. Together, these results establish FGFs as therapeutic targets for severe asthma patients who do not benefit from ICS.

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.

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.