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The introduction of immune checkpoint inhibitors has demonstrated significant improvements in survival for subsets of cancer patients. However, they carry significant and sometimes life-threatening toxicities. Prompt prediction and monitoring of immune toxicities have the potential to maximise the benefits of immune checkpoint therapy. Herein, we develop a digital nanopillar SERS platform that achieves real-time single cytokine counting and enables dynamic tracking of immune toxicities in cancer patients receiving immune checkpoint inhibitor treatment - broader applications are anticipated in other disease indications. By analysing four prospective cytokine biomarkers that initiate inflammatory responses, the digital nanopillar SERS assay achieves both highly specific and highly sensitive cytokine detection down to attomolar level. Significantly, we report the capability of the assay to longitudinally monitor 10 melanoma patients during immune inhibitor blockade treatment. Here, we show that elevated cytokine concentrations predict for higher risk of developing severe immune toxicities in our pilot cohort of patients. There is a clinical need to monitor immune-related toxicities of immune checkpoint blockade therapy. Here, the authors develop a digital SERS platform for multiplexed single cytokine counting to track immune-toxicities and demonstrate the ability to use pre-screening to identify patients at higher risk.

As an indispensable inflammatory mediator during sepsis, granulocyte colony-stimulating factor (G-CSF) facilitates neutrophil production by activating G-CSFR. However, little is known about the role of intracellular downstream signalling pathways in the induction of inflammation. To explore the functions of molecules in regulating G-CSFR signalling, RNA sequencing and integrated proteomic and phosphoproteomic analyses were conducted to predict the differentially expressed molecules in modulating the inflammatory response after G-CSFR expression was either up- or downregulated, in addition to the confirmation of their biological function by diverse experimental methods. In the integrated bioinformatic analysis, 3190 differentially expressed genes (DEGs) and 1559 differentially expressed proteins (DEPs) were identified in multiple-group comparisons (p < 0.05, FC >  ± 1.5) using enrichment analyses, as well as those classic pathways such as the TNF, NFkappaB, IL-17, and TLR signalling pathways. Among them, 201 proteins, especillay intercellular cell adhesion molecule-1 (ICAM1) and PKCa, were identified as potential molecules involved in inflammation according to the protein–protein interaction (PPI) analysis, and the leukocyte transendothelial migration (TEM) pathway was attributed to the intervention of G-CSFR. Compared with the control and TNF-a treatment, the G-CSFR (G-CSFROE)-overexpressing led to an obvious increase in the number of leukocytes with the TEM phenotype. Mechanically, the expression of ICAM1 and PKCa was significantly up- and downregulated by G-CSFROE, which directly led to increased TEM; moreover, PKCa expression was negatively regulated by ICAM1 expression, leading to aberrant leukocyte TEM. Altogether, the ICAM1‒PKCa axis was found a meaningful target in the leukocyte TEM induced by G-CSFR upregulation.

Sciatic nerve crush injury triggers sterile inflammation within the distal nerve and axotomized dorsal root ganglia (DRGs). Granulocytes and pro-inflammatory Ly6C high monocytes infiltrate the nerve first and rapidly give way to Ly6C negative inflammation-resolving macrophages. In axotomized DRGs, few hematogenous leukocytes are detected and resident macrophages acquire a ramified morphology. Single-cell RNA-sequencing of injured sciatic nerve identifies five macrophage subpopulations, repair Schwann cells, and mesenchymal precursor cells. Macrophages at the nerve crush site are molecularly distinct from macrophages associated with Wallerian degeneration. In the injured nerve, macrophages ‘eat’ apoptotic leukocytes, a process called efferocytosis, and thereby promote an anti-inflammatory milieu. Myeloid cells in the injured nerve, but not axotomized DRGs, strongly express receptors for the cytokine GM-CSF. In GM-CSF-deficient ( Csf2 -/- ) mice, inflammation resolution is delayed and conditioning-lesion-induced regeneration of DRG neuron central axons is abolished. Thus, carefully orchestrated inflammation resolution in the nerve is required for conditioning-lesion-induced neurorepair.

Granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF) and/or their receptors are increasingly detected in solid human tumors, although little is known about their function in tumor growth and invasion. We analyzed RNA and protein expression of both factors and their receptors in 22 human gliomas (WHO grade II, III, and IV) and derived cell cultures. G-CSF, GM-CSF, and/or their receptors were expressed in all tumors and derived cell cultures, but coexpression of both factors and receptors was almost exclusively found in grade IV glioblastomas and thus correlated with advanced tumor stage. The functional significance of G-CSF and GM-CSF as regulators for glioma cells was demonstrated by 1. stimulation of proliferation and migration in tumor cells expressing one or both receptors by the corresponding factor; 2) inhibition of growth and migration of glioma cells expressing G-CSF, GM-CSF, and their receptors by neutralizing antibodies to both factors. These results indicate a significant role for both factors in the autocrine regulation of growth and migration in late-stage malignant gliomas and suggest a shift from paracrine to autocrine regulation with tumor progression. The implication of G-CSF and GM-CSF in glioblastoma growth regulation could make these factors further prognostic indicators and raises questions concerning their use in cancer therapy.

A crystal structure of the signaling complex between human granulocyte colony-stimulating factor (GCSF) and a ligand binding region of GCSF receptor (GCSF-R), has been determined to 2.8 Å resolution. The GCSF:GCSF-R complex formed a 2:2 stoichiometry by means of a cross-over interaction between the Ig-like domains of GCSF-R and GCSF. The conformation of the complex is quite different from that between human GCSF and the cytokine receptor homologous domain of mouse GCSF-R, but similar to that of the IL-6/gp130 signaling complex. The Ig-like domain cross-over structure necessary for GCSF-R activation is consistent with previously reported thermodynamic and mutational analyses.

Background Microglia are resident macrophage-like cells in the central nervous system (CNS) and cause innate immune responses via the LPS receptors, Toll-like receptor (TLR) 4 and CD14, in a variety of neuroinflammatory disorders including bacterial infection, Alzheimer’s disease, and amyotrophic lateral sclerosis. Granulocyte macrophage-colony stimulating factor (GM-CSF) activates microglia and induces inflammatory responses via binding to GM-CSF receptor complex composed of two different subunit GM-CSF receptor α (GM-CSFRα) and common β chain (βc). GM-CSF has been shown to be associated with neuroinflammatory responses in multiple sclerosis and Alzheimer’s disease. However, the mechanisms how GM-CSF promotes neuroinflammation still remain unclear. Methods Microglia were stimulated with 20 ng/ml GM-CSF and the levels of TLR4 and CD14 expression were evaluated by RT-PCR and flowcytometry. LPS binding was analyzed by flowcytometry. GM-CSF receptor complex was analyzed by immunocytechemistry. The levels of IL-1β, IL-6 and TNF-α in culture supernatant of GM-CSF-stimulated microglia and NF-κB nuclear translocation were determined by ELISA. Production of nitric oxide (NO) was measured by the Griess method. The levels of p-ERK1/2, ERK1/2, p-p38 and p38 were assessed by Western blotting. Statistically significant differences between experimental groups were determined by one-way ANOVA followed by Tukey test for multiple comparisons. Results GM-CSF receptor complex was expressed in microglia. GM-CSF enhanced TLR4 and CD14 expressions in microglia and subsequent LPS-binding to the cell surface. In addition, GM-CSF priming increased LPS-induced NF-κB nuclear translocation and production of IL-1β, IL-6, TNF-α and NO by microglia. GM-CSF upregulated the levels of p-ERK1/2 and p-p38, suggesting that induction of TLR4 and CD14 expression by GM-CSF was mediated through ERK1/2 and p38, respectively. Conclusions These results suggest that GM-CSF upregulates TLR4 and CD14 expression in microglia through ERK1/2 and p38, respectively, and thus promotes the LPS receptor-mediated inflammation in the CNS.

Peripheral T cell tolerance is promoted by the regulatory cytokine TGF-β and Foxp3-expressing Treg cells. However, whether TGF-β and Treg cells are part of the same regulatory module, or exist largely as distinct pathways to repress self-reactive T cells remains incompletely understood. Using a transgenic model of autoimmune diabetes, here we show that ablation of TGF-β receptor II (TβRII) in T cells, but not Foxp3 deficiency, resulted in early-onset diabetes with complete penetrance. The rampant autoimmune disease was associated with enhanced T cell priming and elevated T cell expression of the inflammatory cytokine GM-CSF, concomitant with pancreatic infiltration of inflammatory monocytes that triggered immunopathology. Ablation of the GM-CSF receptor alleviated the monocyte response and inhibited disease development. These findings reveal that TGF-β promotes T cell tolerance primarily via Foxp3-independent mechanisms and prevents autoimmunity in this model by repressing the cross talk between adaptive and innate immune systems.

Granulocyte–macrophage colony-stimulating factor (GM-CSF) is often used to treat leucopenia. Other haematopoietins may increase the number of circulating leucocytes with higher efficiency, but GM-CSF has additional effects that may be far more relevant than its haematopoietic activity. GM-CSF induces differentiation, proliferation and activation of macrophages and dendritic cells which are necessary for the subsequent T helper cell type 1 and cytotoxic T lymphocyte activation. GM-CSF haematopoietic and non-haematopoietic functions have pro-inflammatory and immune regulatory potential to treat a variety of autoimmune diseases and tumours. On the other hand, GM-CSF deficiency leads to various immune dysfunctions and the current utilization of GM-CSF as haematopoietic factor might be an accurate but very incomplete indication for a cytokine with vast clinical potential.

Colony-stimulating factor 2 (CSF2) functions in the reproductive tract to modulate the function of the preimplantation embryo. The β subunit of the CSF2 receptor (CSF2RB) is not expressed in the embryo, and signal transduction is therefore different than for myeloid cells where the receptor is composed of α (CSF2RA) and β subunits. Here, we produced embryos in which exons 5 and 6 of CSF2RA were disrupted using the CRISPR/Cas 9 system to test whether CSF2RA signaling was essential for actions of CSF2 in the bovine embryo. Wild-type and CSF2RA knockout embryos were treated with 10 ng/mL CSF2 or vehicle at day 5 of development. Blastocysts were harvested at day 8 to determine transcript abundance of 90 genes by real-time polymerase chain reaction (PCR). Responses in female blastocysts were examined separately from male blastocysts because actions of CSF2 are sex-dependent. For wild-type embryos, CSF2 altered expression of 10 genes in females and 20 in males. Only three genes were affected by CSF2 in a similar manner for both sexes. Disruption of CSF2RA prevented the effect of CSF2 on expression for 9 of 10 CSF2-regulated genes in females and 19 of 20 genes in males. The results confirm the importance of CSF2RA for regulation of gene expression by CSF2 in the blastocyst. Summary sentence Disruption of CSF2RA blocked most CSF2-induced changes in gene expression in the blastocyst, indicating that, despite the absence of the β subunit of the receptor, the preimplantation embryo uses CSF2RA for signal transduction.

Background Exercise training is known as a practical way to increase cardioprotection against stress, and it seems that stem cell recruitment is one of its mechanisms. The purpose of the present study was to investigate the effect of preconditioning with High-intensity interval training (HIIT) on tissue levels of G-CSF, its receptor and C-Kit following acute myocardial infarction in male rats. Methods Twenty Male Wistar rats were randomly divided into 4 groups of control, MI, HIIT, and HIIT+MI. Training groups performed 2 weeks of high intensity interval training in 4 sections. The first section consisted training in 3 days and 2 sessions in each day (4 × 2 min with 35–40 m/min and 3 × 2 min with 25–30 m/min between high intervals. The second part included 2 days of training (4 × 2 min with 40 to 45 m/min and 3 × 2 min with 28 to 32 m /min). The third part was performed in 3 days with one more repetition. The fourth section consisted 2 days of training and with one more repetition compared to section 3. For induction of myocardial infarction, subcutaneous injection of isoprenaline was used. CK, total CK, LDH, and troponin T were measured in serum and G-CSF, G-CSFR and C-Kit proteins were measured by the Western Blot method in the heart tissue. Results The results of this study showed that enzymes of CK, total CK, LDH, troponin T had a significant increase in both MI and HIIT+MI groups compared to the other two groups ( P  < 0.001) and these indices in the MI group were significantly higher than the HIIT+MI group. Also, the results demonstrated that G-CSF, G-CSFR and C-Kit protein expression in the heart tissue significantly increased after MI. As well as, 2 weeks of HIIT training significantly increased G-CSF and C-kit in the training group compared to the control group, but the training caused that these proteins does not increase in HIIT+MI group as much as MI group. Conclusions Along with other protective pathways, high intensity interval training can increase cardioprotection and decrease heart injuries through the increase in G-CSF, G-CSFR and C-kit level.