GM-CSF Expression Research Articles

HSF1 and GM-CSF expression, its association with cardiac health, and assessment of organ function during heat stress in crossbred Jersey cattle

The study aimed to evaluate the differential expression of HSF1 and GM-CSF mRNA in PBMCs and correlate it with myocardial injury in crossbred Jersey heifers during heat stress. The study also assessed the effect of heat stress on cardiac electrical activity, vascular health, liver function and kidney function. The experiment was conducted in two phases: for heat stressed animals; HS in June (THI ranged from 80.0 to 89.8) and for control group i.e. not exposed to heat stress in January (THI ranged between 70.1 and 71.4). Results of the study revealed that the relative abundance of HSF1 and GM-CSF mRNA increased significantly (P < 0.05) in HS. Serum cardiac biomarkers such as CK-MB, AST and CRP were significantly elevated (P < 0.05) in HS. cTnI was detected ‘positive’ in nineteen out of twenty four cases in HS. Correlation of HSF1 and GM-CSF expression with concentration of LDH, CKMB, CRP and AST in HS was negative but non-significant (P > 0.05). Significant (P < 0.05) ECG findings in HS were increased heart rate, decreased RR interval, decreased PR interval, decreased QRS amplitude and decreased amplitude of P wave. Marked reduction (P < 0.05) in serum cholesterol and triglyceride levels was observed in HS. ALP, AST, bilirubin and urea levels in serum were significantly elevated (P < 0.05) in HS. In conclusion, cardiac enzymes in serum were significantly elevated in HS indicating myocardial injury. HSF1 and GM-CSF mRNA expression alone was inadequate in conferring cytoprotection to cardiac cells in HS. Cardiac electrical activity, vascular status, liver and kidney function were significantly altered in HS.

Polygalaxanthone III downregulates inflammation in the lipopolysaccharide-stimulated RAW264.7 macrophages: A quantibody array analysis

Polygala japonica Houtt. (PJ), a member of the Polygala L. family that is suggested to exhibit detoxification properties in traditional Chinese medicine, is often used to treat upper respiratory tract infections. The anti-inflammatory effects of four main components of PJ (POL, PS-XLIX, PS-E, and PS-F) were examined using the LPS(0.3 μg·mL−1)-stimulated RAW264.7 macrophage model. The levels of NO, ROS, and iNOS were examined to analyze the anti-inflammatory activity of POL. Additionally, the levels of extracellular inflammation-related cytokines and chemokines were measured using quantibody array. The KEGG pathway analysis was performed to examine the anti-inflammatory mechanism of POL. The levels of NO in the POL-pretreated group were significantly downregulated when compared with those in the PS-E-pretreated, PS-F-pretreated, and PS-XLIX-pretreated groups. POL significantly inhibited the changes of iNOS, ROS, and inflammatory factors caused by LPS stimulation (p < 0.001). The expression levels of IL21 and GM-CSF were examined using qPCR, while those of JAK-STAT signaling pathway-related proteins in the LPS-stimulated RAW264.7 macrophages were analyzed using western blotting. POL significantly downregulated the expression of IL-21 and GM-CSF. The anti-inflammatory mechanism of POL is mediated through the JAK-STAT pathway. Thus, this study demonstrated that POL is an anti-inflammatory component of PJ and elucidated its mechanism.

Abstract 1758: Pro-inflammatory serum marker profiles that lack upregulated IL-17 are characteristic for pancreatic cancer and require for further anti-tumor immune response and strategies

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest types of cancer with 5-year survival rates between 5-8%. Chronic inflammation of the pancreas and pancreatitis is thought to be a risk factor in PDAC. The aim of this study was to elucidate characteristic pro-inflammatory cytokines and chemokines including IL-17 profiles in human pancreatic cancer. A total of 186 individuals with confirmed PDAC (n=116), chronic pancreatitis (n=32), and healthy volunteers (n=38) were included in the study. The histological stage of the tumor was determined according to the Union for International Cancer Control (UICC) TNM staging system. Tumors were evaluated for stage and differentiation grade. Data concerning age, gender, level of wall infiltration, lymph node metastasis, and distant metastases were collected in a database. Additional clinical characteristics necessary for statistical analysis of the data from the study population were collected from the Tumor Registry. Inflammation-related cytokines and chemokines in IL-17 pathway among the patients were analyzed from serum obtained pre-operatively and compared with healthy controls. Moreover, the KEGG PATHWAY, a collection of manually drawn pathway maps on molecular interaction, reaction and relation networks was used for IL-17 pathway analysis. Non-parametric tests and correlation tests were utilized to compare the factors among three groups for statistical analysis. Pancreatitis patients showed highest expression in inflammatory cytokines and chemokine panels while those with PDAC showed different profiles. Pro-inflammatory cytokines including IL-5, IL-6, CXCL8, CCL2, TNF-alpha, and IFN-gamma revealed higher expression in PDAC patients than healthy volunteers (P&amp;lt;0.05). Anti-inflammatory cytokines like IL-13 demonstrated lower expression in PDAC patients than healthy controls (P&amp;lt;0.05), which was likewise observed for IL-17. Interestingly, PDAC patients with distant metastases showed lower pro-inflammatory expression profiles than those with localized tumors. Moreover, correlation analysis demonstrated that IL-17 was correlated with IL-13 and GM-CSF expression (P&amp;lt;0.05). In conclusion, we provide evidence that PDAC patients are characterized by dominant expression of pro-inflammatory cytokine profiles, diminished IL-17 level within their serum, and also anti-inflammatory mediators within is in part divergent from other solid cancers. The IL-17 signaling. may therefore play a different role in the inflammatory pathway in pancreatic cancer. Citation Format: Yueming Luo, Martin Gasser, Amrendra Ajay, Li-LI Hsiao, Ana Maria Waaga-Gasser. Pro-inflammatory serum marker profiles that lack upregulated IL-17 are characteristic for pancreatic cancer and require for further anti-tumor immune response and strategies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1758.

Abstract 3152: NF-κB-mediated pro-inflammatory cytokine release and neutrophil recruitment in head and neck squamous cell carcinoma cell lines

Abstract Introduction: The immune system plays a pivotal role in identification and clearance of tumor cells, but can also be counterproductive and enhance tumor progression. Presence of neutrophils in solid tumors, including the sixth most common cancer globally - head and neck squamous cell carcinomas (HNSCC), is associated with poor prognosis. IL-8 and GM-CSF, whose secretion is regulated by NF-κB, are pro-inflammatory cytokines that promote extravasation of neutrophils into tissue and play an important role in the proliferation, invasion, and metastasis of HNSCC. The onset and progression of HNSCC is also associated with genetic alterations that affect several cellular signaling pathways such as transforming growth factor-β (TGFβ) pathway. TGF-β is responsible for polarization of neutrophils towards anti-tumor (N1) versus pro-tumor (N2) phenotype. Objective: The role of neutrophils in HNSCC and the possible molecular mechanisms of pro-tumor neutrophil recruitment is being investigated. Methods and Results: IL-8 and GM-CSF released at various time points by FaDu (ATCC® HTB-43™), SCC-9 (ATCC® CRL-1629™) and Cal-27 (ATCC® CRL-2095™) was measured by ELISA. Conditioned media from FaDu and Cal-27 cells exhibited consistent expression of IL-8 (115 - 117pg/mL and 103 - 110pg/ml, respectively) at 6 - 72 hours. SCC-9 conditioned media exhibited sustained levels of IL-8 expression (103 - 110pg/ml) at 6 - 24 hours that then declined at 48 - 72 hours. GM-CSF was not significantly expressed in the FaDu conditioned media; even with increasing cell density, the mean expression remained 0.142pg/ml. SCC-9 and Cal-27 expressed GM-CSF at 6 - 72 hours (16 - 20pg/mL and ~15pg/ml, respectively). In all cell lines, IL-8 expression decreased in a dose and time dependent manner after treatment with the NF-κB inhibitor, BAY 11-7082, while effect on GM-CSF expression was negligible. Expression of phosphorylated IκBα in FaDu and SCC-9 cell lysates decreased in a time and dose dependent manner, indicating inhibition of the NF-κB pathway by BAY11-7082. Expression of phosphorylated NF-κB also decreased as compared to control in these cells. In nuclear extracts, BAY 11-7082 dose-dependent alteration in NF-κB expression was observed in FaDu and Cal-27 cells at earlier time points. Conditioned media from SCC-9 and Cal-27 cells also exhibited sustained baseline expression of TGF- β (~30pg/ml) over 6 - 72 hours. Conclusion: HNSCC cells derived from tongue and hypopharyngeal squamous cell carcinoma release pro-inflammatory cytokines. The NF-κB signaling pathway appears to be involved in the release of IL-8, a cytokine that is in turn likely to be involved in recruitment of neutrophils to the cancer site that may be polarized towards the N2 pro-tumor phenotype by TGF-β secreted by HNSCC cells. Citation Format: Hina Shakeel, Fareena Bilwani, Kiran Iqbal, Kulsoom Ghias. NF-κB-mediated pro-inflammatory cytokine release and neutrophil recruitment in head and neck squamous cell carcinoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3152.

PD-1 Inhibition Enhances Blinatumomab Response in a UCB/PDX Model of B-Cell Acute Lymphoblastic Leukemia

IL-2Ry immune deficient mice engrafted with human umbilical cord blood (UCB) develop human immune systems with mixed B, T, NK, and myeloid populations. We have previously modified NOD/SCID/Gamma (NSG) and NOD/RAG/Gamma (NRG) mice to include transgenic expression of human cytokines SCF, GM-CSF, and IL-3 (NSGS/NRGS). Relative to NSG/NRG, NSGS/NRGS mice have increased myeloid and NK fractions, more complete B cell differentiation, and faster T cell reconstitution. Additionally, both innate and adaptive immune function is also improved as measured by DTH assay and human antigen specific antibody production in response to immunization. NSGS and NRGS mice also more readily engraft leukemia samples, particularly in the absence of conditioning. We took advantage of the improved nature of hematopoietic xenografts in NRGS mice to determine whether or not humanized mice can serve as an in vivo model to test immune therapies against human leukemia.Busulfan conditioned NRGS mice were humanized by engraftment with red blood cell depleted UCB. Engraftment was monitored by the appearance of significant levels of human CD45+CD3+ T cells in the peripheral blood (PB). Once mice were reconstituted with >25% T cell graft in PB, we injected an aggressive, relapsed/refractory t(4;11) patient sample to induce fatal ALL in the humanized NRGS mice. All control humanized NRGS mice succumbed to disease between 40 and 60 days after leukemia injection with rapidly expanding B-ALL in the PB, bone marrow, and spleen. Mice treated with several courses of the bi-specific T cell engager (BiTE) molecule, blinatumomab, were partially protected from disease, and only half of these mice developed detectable B-ALL at the end of the experiment (day 90 post-injection). Strikingly, the combination of blinatumomab and the PD-1 antibody, pembrolizumab, fully protected the mice from B-ALL disease. All blinatumomab/pembrolizumab treated mice survived until the end of the experiment at which time there was no evidence of disease in PB, bone marrow, or spleen preparations. Importantly, additional experiments showed that this blinatumomab effect was dependent on the presence of the human immune system. Blinatumomab did not affect initiation (early treatment) or progression (late treatment) of B-ALL in non-humanized mice that were engrafted with the same patient sample.T cell levels were monitored during the experiment and showed a slight decline upon blinatumomab treatment. In contrast, T cell levels were significantly higher in mice that received both blinatumomab and pembrolizumab, implying augmented T cell activation in vivo. This may be a relevant finding because it is likely that some patients fail blinatumomab therapy due to sub-optimal T cell levels. The addition of pembrolizumab may offer means to combat this problem. Nevertheless, mice with less than 20% CD45+CD3+ T cells in the PB failed to respond to either blinatumomab or combined blinatumomab/pembrolizumab treatment, indicating there is a lower limit to potentiating the T cell response during BiTE therapy.These results demonstrate the potential utility of dual UCB/PDX models for examining novel combinations of immune therapies in vivo and suggest that additional studies combining BiTEs with immune checkpoint inhibitors are warranted. DisclosuresNo relevant conflicts of interest to declare.

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

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

Dual interleukin-17A/F deficiency protects against acute and chronic response to cigarette smoke exposure in mice

IL-17A and IL-17F are both involved in the pathogenesis of neutrophilic inflammation observed in COPD and severe asthma. To explore this, mice deficient in both Il17a and Il17f and wild type (WT) mice were exposed to cigarette smoke or environmental air for 5 to 28 days and changes in inflammatory cells in bronchoalveolar lavage (BAL) fluid were determined. We also measured the mRNA expression of keratinocyte derived chemokine (Kc), macrophage inflammatory protein-2 (Mip2), granulocyte–macrophage colony stimulating factor (Gmcsf) and matrix metalloproteinase-9 (Mmp9 ) in lung tissue after 8 days, and lung morphometric changes after 24 weeks of exposure to cigarette smoke compared to air-exposed control animals. Macrophage counts in BAL fluid initially peaked at day 8 and again on day 28, while neutrophil counts peaked between day 8 and 12 in WT mice. Mice dual deficient with Il17a and 1l17f showed similar kinetics with macrophages and neutrophils, but cell numbers at day 8 and mRNA expression of Kc, Gmcsf and Mmp9 were significantly reduced. Furthermore, airspaces in WT mice became larger after cigarette smoke exposure for 24 weeks, whereas this was not seen dual Il17a and 1l17f deficient mice. Combined Il17a and Il17f deficiency resulted in significant attenuation of neutrophilic inflammatory response and protection against structural lung changes after long term cigarette smoke exposure compared with WT mice. Dual IL-17A/F signalling plays an important role in pro-inflammatory responses associated with histological changes induced by cigarette smoke exposure.

Oncolytic Vaccinia Virus Gene Modification and Cytokine Expression Effects on Tumor Infection, Immune Response, and Killing.

Oncolytic vaccinia viruses have promising efficacy and safety profiles in cancer therapy. Although antitumor activity can be increased by manipulating viral genes, the relative efficacy of individual modifications has been difficult to assess without side-by-side comparisons. This study sought to compare the initial antitumor activity after intravenous administration of five vaccinia virus variants of the same Western Reserve backbone and thymidine kinase gene deletion in RIP-Tag2 transgenic mice with spontaneous pancreatic neuroendocrine tumors. Tumors had focal regions of infection at 5 days after all viruses. Natural killer (NK) cells were restricted to these sites of infection, but CD8+ T cells and tumor cell apoptosis were widespread and varied among the viruses. Antitumor activity of virus VV-A34, bearing amino acid substitution A34K151E to increase viral spreading, and virus VV-IL2v, expressing a mouse IL2 variant (mIL2v) with attenuated IL2 receptor alpha subunit binding, was similar to control virus VV-GFP. However, antitumor activity was significantly greater after virus VV-A34/IL2v, which expressed mIL2v together with A34K151E mutation and viral B18R gene deletion, and virus VV-GMCSF that expressed mouse GM-CSF. Both viruses greatly increased expression of CD8 antigens Cd8a/Cd8b1 and cytotoxicity genes granzyme A, granzyme B, Fas ligand, and perforin-1 in tumors. VV-A34/IL2v led to higher serum IL2 and greater tumor expression of death receptor ligand TRAIL, but VV-GMCSF led to higher serum GM-CSF, greater expression of leukocyte chemokines and adhesion molecules, and more neutrophil recruitment. Together, the results show that antitumor activity is similarly increased by viral expression of GM-CSF or IL2v combined with additional genetic modifications.

Haplopine Ameliorates 2,4-Dinitrochlorobenzene-Induced Atopic Dermatitis-Like Skin Lesions in Mice and TNF-α/IFN-γ-Induced Inflammation in Human Keratinocyte.

This study aimed to investigate the anti-inflammatory, antioxidant, and anti-atopic dermatitis (AD) effects of haplopine, which is one of the active components in D. dasycarpus. Haplopine (12.5 and 25 μM) inhibited the mRNA expressions of inflammatory cytokines IL-6, TSLP, GM-CSF, and G-CSF and the protein expressions of IL-6 and GM-CSF in TNF-α/INF-γ-stimulated HaCaT cells. In H2O2-induced Jukat T cells, haplopine (25 and 50 μM) suppressed the productions of proinflammatory cytokines (IL-4, IL-13, and COX-2) and increased the mRNA and protein expressions of oxidative stress defense enzymes (SOD, CAT, and HO-1) in a concentration-dependent manner. In vivo, haplopine significantly attenuated the development of AD symptoms in 2,4-dinitrochlorobenzene (DNCB)-stimulated Balb/c mice, as evidenced by reduced clinical dermatitis scores, skin thickness measurements, mast cell infiltration, and serum IgE concentrations. These findings demonstrate that haplopine should be considered a novel anti-atopic agent with the potential to treat AD.

Inhibition of Jumonji Histone Demethylases Selectively Suppresses HER2+ Breast Leptomeningeal Carcinomatosis Growth via Inhibition of GMCSF Expression.

HER2+ breast leptomeningeal carcinomatosis (HER2+ LC) occurs when tumor cells spread to cerebrospinal fluid-containing leptomeninges surrounding the brain and spinal cord, a complication with a dire prognosis. HER2+ LC remains incurable, with few treatment options. Currently, much effort is devoted toward development of therapies that target mutations. However, targeting epigenetic or transcriptional states of HER2+ LC tumors might efficiently target HER2+ LC growth via inhibition of oncogenic signaling; this approach remains promising but is less explored. To test this possibility, we established primary HER2+ LC (Lepto) cell lines from nodular HER2+ LC tissues. These lines are phenotypically CD326+CD49f-, confirming that they are derived from HER2+ LC tumors, and express surface CD44+CD24-, a cancer stem cell (CSC) phenotype. Like CSCs, Lepto lines showed greater drug resistance and more aggressive behavior compared with other HER2+ breast cancer lines in vitro and in vivo. Interestingly, the three Lepto lines overexpressed Jumonji domain-containing histone lysine demethylases KDM4A/4C. Treatment with JIB04, a selective inhibitor of Jumonji demethylases, or genetic loss of function of KDM4A/4C induced apoptosis and cell-cycle arrest and reduced Lepto cell viability, tumorsphere formation, regrowth, and invasion in vitro. JIB04 treatment of patient-derived xenograft mouse models in vivo reduced HER2+ LC tumor growth and prolonged animal survival. Mechanistically, KDM4A/4C inhibition downregulated GMCSF expression and prevented GMCSF-dependent Lepto cell proliferation. Collectively, these results establish KDM4A/4C as a viable therapeutic target in HER2+ LC and spotlight the benefits of targeting the tumorigenic transcriptional network. SIGNIFICANCE: HER2+ LC tumors overexpress KDM4A/4C and are sensitive to the Jumonji demethylase inhibitor JIB04, which reduces the viability of primary HER2+ LC cells and increases survival in mouse models.

LPS Induces GM-CSF Production by Breast Cancer MDA-MB-231 Cells via Long-Chain Acyl-CoA Synthetase 1

Abstract Granulocyte–macrophage colony-stimulating factor (GM-CSF) is a monomeric glycoprotein that has been implicated in the tumor growth and progression of different types of cancer. GM-CSF is produced by various non-immune cells including MDA-MB-231 in response to various stimuli. However, the role of lipopolysaccharide (LPS) in the regulation of GM-CSF in MDA-MB-231 breast cancer cells so far remains unclear. Herein, we asked whether LPS could induce GM-CSF production in MDA-MB-231 cells, and if so, which signaling pathway was involved. MDA-MB-231 cells were treated with LPS or tumor necrosis factor alpha (TNF-α; positive control), and GM-CSF expression levels were determined by qRT-PCR, ELISA, and confocal microscopy. Phosphorylation of the mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-kB) signaling proteins were evaluated by flow cytometry. Our results show that LPS induces GM-CSF expression at both mRNA and protein levels in MDA-MBA-231 cells. Inhibition of acyl-CoA synthetase 1 (ACSL1) activity in the cells with triacsin C significantly reduces the secretion of GM-CSF. Furthermore, the inhibition of ACSL1 activity significantly blocks the LPS-mediated phosphorylation of p38 MAPK, MEK1/2, extracellular signal-regulated kinase (ERK)1/2, c-Jun NH2-terminal kinase (JNK), and nuclear factor-κB (NF-kB) in the cells. These findings provide the first evidence that LPS induces ACSL1-dependent GM-CSF gene expression in MDA-MB-231 breast cancer cells, which requires the activation of p38 MAPK, MEK1/2, ERK1/2, JNK, and NF-kB.

Single-cell multiomics dissection of basal and antigen-specific activation states of CD19-targeted CAR T cells

BackgroundAutologous T cells engineered to express a chimeric antigen receptor (CAR) specific for CD19 molecule have transformed the therapeutic landscape in patients with highly refractory leukemia and lymphoma, and the...

Exploring the Biomaterial-Induced Secretome: Physical Bone Substitute Characteristics Influence the Cytokine Expression of Macrophages.

In addition to their chemical composition various physical properties of synthetic bone substitute materials have been shown to influence their regenerative potential and to influence the expression of cytokines produced by monocytes, the key cell-type responsible for tissue reaction to biomaterials in vivo. In the present study both the regenerative potential and the inflammatory response to five bone substitute materials all based on β-tricalcium phosphate (β-TCP), but which differed in their physical characteristics (i.e., granule size, granule shape and porosity) were analyzed for their effects on monocyte cytokine expression. To determine the effects of the physical characteristics of the different materials, the proliferation of primary human osteoblasts growing on the materials was analyzed. To determine the immunogenic effects of the different materials on human peripheral blood monocytes, cells cultured on the materials were evaluated for the expression of 14 pro- and anti-inflammatory cytokines, i.e., IL-6, IL-10, IL-1β, VEGF, RANTES, IL-12p40, I-CAM, IL-4, V-CAM, TNF-α, GM-CSF, MIP-1α, Il-8 and MCP-1 using a Bio-Plex® Multiplex System. The granular shape of bone substitutes showed a significant influence on the osteoblast proliferation. Moreover, smaller pore sizes, round granular shape and larger granule size increased the expression of GM-CSF, RANTES, IL-10 and IL-12 by monocytes, while polygonal shape and the larger pore sizes increased the expression of V-CAM. The physical characteristics of a bone biomaterial can influence the proliferation rate of osteoblasts and has an influence on the cytokine gene expression of monocytes in vitro. These results indicate that the physical structure of a biomaterial has a significant effect of how cells interact with the material. Thus, specific characteristics of a material may strongly affect the regenerative potential in vivo.

Stimulation of regulatory T cells with Lactococcus lactis expressing enterotoxigenic E. coli colonization factor antigen 1 retains salivary flow in a genetic model of Sj\xf6gren\u2019s syndrome

BackgroundSjögren’s syndrome (SjS), one of the most common autoimmune diseases, impacts millions of people annually. SjS results from autoimmune attack on exocrine (salivary and lacrimal) glands, and women are nine times more likely to be affected than men. To date, no vaccine or therapeutic exists to treat SjS, and patients must rely on lifelong therapies to alleviate symptoms.MethodsOral treatment with the adhesin from enterotoxigenic Escherichia coli colonization factor antigen I (CFA/I) fimbriae protects against several autoimmune diseases in an antigen (Ag)-independent manner. Lactococcus lactis, which was recently adapted to express CFA/I fimbriae (LL-CFA/I), effectively suppresses inflammation by the induction of infectious tolerance via Ag-specific regulatory T cells (Tregs), that produce IL-10 and TGF-β. To test the hypothesis that CFA/I fimbriae can offset the development of inflammatory T cells via Treg induction, oral treatments with LL-CFA/I were performed on the spontaneous, genetically defined model for SjS, C57BL/6.NOD-Aec1Aec2 mice to maintain salivary flow.ResultsSix-week (wk)-old C57BL/6.NOD-Aec1Aec2 mice were orally dosed with LL-CFA/I and treated every 3 wks; control groups were given L. lactis vector or PBS. LL-CFA/I-treated mice retained salivary flow up to 28 wks of age and showed significantly reduced incidence of inflammatory infiltration into the submandibular and lacrimal glands relative to PBS-treated mice. A significant increase in Foxp3+ and IL-10- and TGF-β-producing Tregs was observed. Moreover, LL-CFA/I significantly reduced the expression of proinflammatory cytokines, IL-6, IL-17, GM-CSF, and IFN-γ. Adoptive transfer of CD4+ T cells from LL-CFA/I-treated, not LL vector-treated mice, restored salivary flow in diseased SjS mice.ConclusionThese data demonstrate that oral LL-CFA/I reduce or halts SjS progression, and these studies will provide the basis for future testing in SjS patients.

GM-CSF Expression and Macrophage Polarization in Joints of Undifferentiated Arthritis Patients Evolving to Rheumatoid Arthritis or Psoriatic Arthritis.

Background and AimsGM-CSF-dependent macrophage polarization has been demonstrated in rheumatoid arthritis (RA). Our aim was to seek diagnostic/prognostic biomarkers for undifferentiated arthritis (UA) by analyzing GM-CSF expression and source, macrophage polarization and density in joints of patients with UA evolving to RA or PsA compared with established RA or PsA, respectively.MethodsSynovial tissue (ST) from patients with UA evolving to RA (UA>RA, n=8), PsA (UA>PsA, n=9), persistent UA (UA, n=16), established RA (n=12) and PsA (n=10), and healthy controls (n=6), were analyzed. Cell source and quantitative expression of GM-CSF and proteins associated with pro-inflammatory (GM-CSF-driven) and anti-inflammatory (M-CSF-driven) macrophage polarization (activin A, TNFα, MMP12, and CD209, respectively) were assessed in ST CD163+ macrophages by multicolor immunofluorescence. GM-CSF and activin A levels were also quantified in paired synovial fluid samples. CD163+ macrophage density was determined in all groups by immunofluorescence.ResultsSynovial stromal cells (FAP+ CD90+ fibroblast, CD90+ endothelial cells) and CD163+ sublining macrophages were the sources of GM-CSF. ST CD163+ macrophages from all groups expressed pro-inflammatory polarization markers (activin A, TNFα, and MMP12). Expression of the M-CSF-dependent anti-inflammatory marker CD209 identified two macrophage subsets (CD163+ CD209high and CD163+ CD209low/-). CD209+ macrophages were more abundant in ST from healthy controls and PsA patients, although both macrophage subtypes showed similar levels of pro-inflammatory markers in all groups. In paired synovial fluid samples, activin A was detected in all patients, with higher levels in UA>RA and RA, while GM-CSF was infrequently detected. ST CD163+ macrophage density was comparable between UA>RA and UA>PsA patients, but significantly higher than in persistent UA.ConclusionsGM-CSF is highly expressed by sublining CD90+ FAP+ synovial fibroblasts, CD90+ activated endothelium and CD163+ macrophages in different types of arthritis. The polarization state of ST macrophages was similar in all UA and established arthritis groups, with a predominance of pro-inflammatory GM-CSF-associated markers. CD163+ macrophage density was significantly higher in the UA phases of RA and PsA compared with persistent UA. Taken together, our findings support the idea that GM-CSF is a strong driver of macrophage polarization and a potential therapeutic target not only in RA but also in PsA and all types of UA.

Difference in Host Immune response to Methicillin-Resistant and Methicillin Sensitive Staphylococcus aureus (MRSA and MSSA) Endophthalmitis

ABSTRACT Purpose The study aimed to understand the differential immune response of methicillin susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) strains in in vitro models of endophthalmitis. Methods Retinal pigment epithelium (RPE) and microglia cells (CHME-3) were exposed to MRSA and MSSA strains and analyzed for expression of inflammatory mediators by real-time quantitative PCR and validated by ELISA or immunofluorescence assay. Heatmap and STRING analysis was used to assess the differential immune expression. Results Both microglia and RPE expressed TLR-2, TLR-1, TLR-6, and TLR −9 after challenge with MRSA and MSSA strains though the expression varied. MRSA-infected cells induced higher expression of IL-1β, IL-8, 1 L-10, IL-6, and GM-CSF, while TNF-α and IFN-ϒ were downregulated in comparison to MSSA-infected cells. We also demonstrate that MRSA infection leads to increased activation of MMP-9 and MMP-2 in RPE cells, while microglia expressed only MMP-9 in MRSA-infected cells. Conclusions MRSA strain can induce an exacerbated immune response in retinal cells. Giving clues for potential targets in immunomodulatory therapies.

Glutamine Deprivation Promotes the Generation and Mobilization of MDSCs by Enhancing Expression of G-CSF and GM-CSF.

Solid tumors are often challenged by hypoxic and nutrient-deprived tumor microenvironments (TME) as tumors progress, due to limited perfusion and rapid nutrient consumption. While cancer cells can demonstrate the ability to survive in nutrient-deprived conditions through multiple intrinsic alterations, it is poorly understood how nutrient-deprived cancer cells co-opt the TME to promote cancer cell survival and tumor progression. In the present study, we found that glutamine deprivation markedly potentiated the expression of G-CSF and GM-CSF in mouse mammary cancer cells. The IRE1α-JNK pathway, which is activated by glutamine starvation, was found to be important for the upregulation of these cytokines. G-CSF and GM-CSF are well-known facilitators of myelopoiesis and mobilization of hematopoietic progenitor cells (HPC). Consistently, as tumors progressed, we found that several myeloid HPC compartments were gradually decreased in the bone marrow but were significantly increased in the spleen. Mechanistically, the HPC-maintaining capacity of the bone marrow was significantly impaired in tumor-bearing mice, with lower expression of HPC maintaining genes (i.e., CXCL12, SCF, ANGPT1, and VCAM1), and reduced levels of mesenchymal stem cells and CXCL12-producing cells. Furthermore, the mobilized HPCs that displayed the capacity for myelopoiesis were also found to accumulate in tumor tissue. Tumor-infiltrating HPCs were highly proliferative and served as important sources of immunosuppressive myeloid-derived suppressor cells (MDSCs) in the TME. Our work has identified an important role for glutamine starvation in regulating the expression of G-CSF and GM-CSF, and in facilitating the generation of immunosuppressive MDSCs in breast cancer.

Differential Expression of Serum Exosome microRNAs and Cytokines in Influenza A and B Patients Collected in the 2016 and 2017 Influenza Seasons.

MicroRNAs (miRNAs) have remarkable stability and are key regulators of mRNA transcripts for several essential proteins required for the survival of cells and replication of the virus. Exosomes are thought to play an essential role in intercellular communications by transporting proteins and miRNAs, making them ideal in the search for biomarkers. Evidence suggests that miRNAs are involved in the regulation of influenza virus replication in many cell types. During the 2016 and 2017 influenza season, we collected blood samples from 54 patients infected with influenza and from 30 healthy volunteers to identify the potential role of circulating serum miRNAs and cytokines in influenza infection. Data comparing the exosomal miRNAs in patients with influenza B to healthy volunteers showed 76 miRNAs that were differentially expressed (p < 0.05). In contrast, 26 miRNAs were differentially expressed between patients with influenza A (p < 0.05) and the controls. Of these miRNAs, 11 were commonly expressed in both the influenza A and B patients. Interferon (IFN)-inducing protein 10 (IP-10), which is involved in IFN synthesis during influenza infection, showed the highest level of expression in both influenza A and B patients. Influenza A patients showed increased expression of IFNα, GM-CSF, interleukin (IL)-13, IL-17A, IL-1β, IL-6 and TNFα, while influenza B induced increased levels of EGF, G-CSF, IL-1α, MIP-1α, and TNF-β. In addition, hsa-miR-326, hsa-miR-15b-5p, hsa-miR-885, hsa-miR-122-5p, hsa-miR-133a-3p, and hsa-miR-150-5p showed high correlations to IL-6, IL-15, IL-17A, IL-1β, and monocyte chemoattractant protein-1 (MCP-1) with both strains of influenza. Next-generation sequencing studies of H1N1-infected human lung small airway epithelial cells also showed similar pattern of expression of miR-375-5p, miR-143-3p, 199a-3p, and miR-199a-5p compared to influenza A patients. In summary, this study provides insights into the miRNA profiling in both influenza A and B virus in circulation and a novel approach to identify the early infections through a combination of cytokines and miRNA expression.

Expression of GM-CSF Is Regulated by Fli-1 Transcription Factor, a Potential Drug Target.

Friend leukemia virus integration 1 (Fli-1) is an ETS transcription factor and a critical regulator of inflammatory mediators, including MCP-1, CCL5, IL-6, G-CSF, CXCL2, and caspase-1. GM-CSF is a regulator of granulocyte and macrophage lineage differentiation and a key player in the pathogenesis of inflammatory/autoimmune diseases. In this study, we demonstrated that Fli-1 regulates the expression of GM-CSF in both T cells and endothelial cells. The expression of GM-CSF was significantly reduced in T cells and endothelial cells when Fli-1 was reduced. We found that Fli-1 binds directly to the GM-CSF promoter using chromatin immunoprecipitation assay. Transient transfection assays indicated that Fli-1 drives transcription from the GM-CSF promoter in a dose-dependent manner, and mutation of the Fli-1 DNA binding domain resulted in a significant loss of transcriptional activation. Mutation of a known phosphorylation site within the Fli-1 protein led to a significant increase in GM-CSF promoter activation. Thus, direct binding to the promoter and phosphorylation are two important mechanisms behind Fli-1-driven activation of the GM-CSF promoter. In addition, Fli-1 regulates GM-CSF expression in an additive manner with another transcription factor Sp1. Finally, we demonstrated that a low dose of a chemotherapeutic drug, camptothecin, inhibited expression of Fli-1 and reduced GM-CSF production in human T cells. These results demonstrate novel mechanisms for regulating the expression of GM-CSF and suggest that Fli-1 is a critical druggable regulator of inflammation and immunity.

Chemoprotective Effects of Geraniin against Azoxymethane Induced Colorectal Cancer by Reduction of Inflammatory Reaction.

The leading cause of cancer-related death is colorectal cancer, and inflammatory bowel disease is a risk factor for this disease. Azoxymethane (AOM) is a potent cancer inducer widely used in rats for colon cancer. The current study was scrutinizing the chemo-protective effect of geraniin against AOM induced colorectal cancer via alteration of oxidative stress and inflammatory cytokines. The rats were divided into different groups such as Group I: normal control, Group II geraniin (20 mg/kg), Group III: received AOM, Group IV-VI: AOM + geraniin (5, 10 and 20 mg/kg), respectively. All group of rats were received treatment for 16 weeks. At the end of the experimental study, the hepatic, biochemical, phase II antioxidant, antioxidant enzymes, cytokines, apoptosis and inflammatory mediators were estimated. Geraniin treatment significantly reduced tumor weight and enhanced body weight. Geraniin administration also altered the level of antioxidant parameters-superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR); phase I enzymes - cytochrome B5, cytochrome P450; phase II enzymes - Glutathione-S-Transferase (GST), UDP-Glucuronyl transferase (UDP-GT) respectively. Obtained results also demonstrate that geraniin treatment reduced the level of pro-inflammatory cytokines such as IL-2, IL-1α, IL-10, IL-1β, IL-4, IL-6, IL-12, IL-17A, IFN-γ, tumor necrosis factor-α, G-CSF, and GM-CSF. Geraniin also reduced the expression of IL-1α, IL-1β, IL-6, IFN-γ, G-CSF, and GM-CSF. On the basis of result we can conclude that geraniin reduced the colorectal cancer via inflammatory pathway.