TREM-1 Signaling Research Articles

Differential Immunoregulation by Human Surfactant Protein A Variants Determines Severity of SARS-CoV-2-induced Lung Disease.

COVID-19 remains a significant threat to public health globally. Infection in some susceptible individuals causes life-threatening acute lung injury (ALI/ARDS) and/or death. Human surfactant protein A (SP-A) is a C-type lectin expressed in the lung and other mucosal tissues, and it plays a critical role in host defense against various pathogens. The human SP-A genes ( SFTPA1 and SFTPA2 ) are highly polymorphic and comprise several common genetic variants, i.e., SP-A1 (variants 6A 2 , 6A 4 ) and SP-A2 (variants 1A 0 , 1A 3 ). Here, we elucidated the differential antiviral and immunoregulatory roles of SP-A variants in response to SARS-CoV-2 infection in vivo . Six genetically-modified mouse lines, expressing both hACE2 (SARS-CoV-2 receptor) and individual SP-A variants: (hACE2/6A 2 (6A 2 ), hACE2/6A 4 (6A 4 ), hACE2/1A 0 (1A 0 ), and hACE2/1A 3 (1A 3 ), one SP-A knockout (hACE2/SP-A KO (KO) and one hACE2/mouse SP-A (K18) mice, were challenged intranasally with 10 3 PFU SARS-CoV-2 or saline (Sham). Infected KO and 1A 0 mice had more weight loss and mortality compared to other mouse lines. Relative to other infected mouse lines, a more severe ALI was observed in KO, 1A 0 , and 6A 2 mice. Reduced viral titers were generally observed in the lungs of infected SP-A mice relative to KO mice. Transcriptomic analysis revealed an upregulation in genes that play central roles in immune responses such as MyD88 , Stat3 , IL-18 , and Jak2 in the lungs of KO and 1A 0 mice. However, Mapk1 was significantly downregulated in 6A 2 versus 1A 0 mice. Analysis of biological pathways identified those involved in lung host defense and innate immunity, including pathogen-induced cytokine, NOD1/2, and Trem1 signaling pathways. Consistent with the transcriptomic data, levels of cytokines and chemokines such as G-CSF, IL-6 and IL-1β were comparatively higher in the lungs and sera of KO and 1A 0 mice with the highest mortality rate. These findings demonstrate that human SP-A variants differentially modulate SARS-CoV-2-induced lung injury and disease severity by differentially inhibiting viral infectivity and regulating immune-related gene expressions.

Aging is associated with an insufficient early inflammatory response of lung endothelial cells in SARS-CoV-2 infection.

Advanced age is associated with an increased susceptibility to Coronavirus Disease (COVID)-19 and more severe outcomes, although the underlying mechanisms are understudied. The lung endothelium is located next to infected epithelial cells and bystander inflammation may contribute to thromboinflammation and COVID-19-associated coagulopathy. Here, we investigated age-associated SARS-CoV-2 pathogenesis and endothelial inflammatory responses using humanized K18-hACE2 mice. Survival was reduced to 20% in aged mice (85-112 weeks) versus 50% in young mice (12-15 weeks) at 10 days post infection (dpi). Bulk RNA-sequencing of endothelial cells from mock and infected mice at 2dpi of both age groups (aged: 72-85 weeks; young: 15 weeks) showed substantially lower significant differentially regulated genes in infected aged mice than in young mice (712 versus 2294 genes). Viral recognition and anti-viral pathways such as RIG-I-like receptor signaling, NOD-like receptor signaling and interferon signaling were regulated in response to SARS-CoV-2. Young mice showed several fold higher interferon responses (Ifitm3, Ifit1, Isg15, Stat1) and interferon-induced chemokines (Cxcl10 and Cxcl11) than aged mice. Endothelial cells from infected young mice displayed elevated expression of chemokines (Cxcl9, Ccl2) and leukocyte adhesion markers (Icam1) underscoring that inflammation of lung endothelium during infection could facilitate leukocyte adhesion and thromboinflammation. TREM1 and acute phase response signaling were particularly prominent in endothelial cells from infected young mice. Immunohistochemistry was unable to detect viral protein in pulmonary endothelium. In conclusion, our data demonstrate that the early host response of the endothelium to SARS-CoV-2 infection declines with aging, which could be a potential contributor to disease severity.

CCR2+TREM-1+ monocytes promote natural killer T cell dysfunction contributing towards HBV disease progression.

Natural killer T (NKT) cells are amongst the most important innate immune cells against hepatitis B virus (HBV) infection. Moreover, previous studies have shown that HBV infection induced TREM-1+ expression in monocyte and secretion of inflammatory cytokines. Thus, this prompted us to elucidate the role of TREM-1+ monocytes in regulating the function of iNKT cells. Ninety patients and 20 healthy participants were enrolled in the study. The percentage and phenotype of iNKT cells and TREM-1+ monocytes were measured in the peripheral blood of healthy controls (HC), patients with chronic HBV infection (CHB), HBV-related liver cirrhosis (LC), and HBV-related acute-on-chronic liver failure (ACLF) via flow cytometry. Moreover, co-culture experiments with iNKT cells and TREM-1 overexpressing THP-1 cells were performed to determine the role of TREM-1 in the regulation of NKT cell function. We observed that the percentage of iNKT cells and CD4-iNKT cells gradually decreased, whereas the percentage of CCR2+TREM-1+ monocytes increased with the progression of the disease. In addition, activation of the TREM-1 signaling pathway induced the secretion of inflammatory cytokines leading to pyroptosis of iNKT cells and secretion of IL-17 contributing towards disease progression. Therefore, this study suggests that blocking the activation of TREM-1 in monocytes could promote the elimination of HBV by inhibiting pyroptosis of iNKT cells and restoring their function. However, further studies are required to validate these results that would help in developing new treatment strategies for patients with HBV infections.

Periodic heat waves-induced neuronal etiology in the elderly is mediated by gut-liver-brain axis: a transcriptome profiling approach

Heat stress exposure in intermittent heat waves and subsequent exposure during war theaters pose a clinical challenge that can lead to multi-organ dysfunction and long-term complications in the elderly. Using an aged mouse model and high-throughput sequencing, this study investigated the molecular dynamics of the liver-brain connection during heat stress exposure. Distinctive gene expression patterns induced by periodic heat stress emerged in both brain and liver tissues. An altered transcriptome profile showed heat stress-induced altered acute phase response pathways, causing neural, hepatic, and systemic inflammation and impaired synaptic plasticity. Results also demonstrated that proinflammatory molecules such as S100B, IL-17, IL-33, and neurological disease signaling pathways were upregulated, while protective pathways like aryl hydrocarbon receptor signaling were downregulated. In parallel, Rantes, IRF7, NOD1/2, TREM1, and hepatic injury signaling pathways were upregulated. Furthermore, current research identified Orosomucoid 2 (ORM2) in the liver as one of the mediators of the liver-brain axis due to heat exposure. In conclusion, the transcriptome profiling in elderly heat-stressed mice revealed a coordinated network of liver-brain axis pathways with increased hepatic ORM2 secretion, possibly due to gut inflammation and dysbiosis. The above secretion of ORM2 may impact the brain through a leaky blood–brain barrier, thus emphasizing intricate multi-organ crosstalk.

Abstract 2846: Investigation of mechanisms that result in health disparity for Hispanic/Latino patients with B acute lymphoblastic leukemia

Abstract Although social determinants of health have a large impact on health disparity, biologic vulnerabilities - particularly in cancers that impact at-risk populations are important. Amongst children with Acute Lymphoblastic Leukemia (ALL), the Hispanic/Latino (H/L) population is vulnerable given since the risk of developing ALL is 1.2-1.75 that of Non-Hispanic White children. After corrections for socioeconomic impacts, the mortality for H/L children is 40% higher than NHW. H/L children have a higher rate of high-risk genetic variations: 2-fold rate of heterozygous IKZF1 deletion (IKZF1-del) and a 4-fold rate of CRLF2 translocation. IKZF1 deletion and CRLF2 translocations are associated increased risk of relapse and chemotherapy resistance in B ALL. IKZF1 is an important transcriptional factor and epigenetic regulator that affects global gene transcription in hematopoietic cells. Simultaneous IKZF1-del and CRLF2 translocation represented 94% of H/L children with CRLF2 translocation. Although only 49% of all H/L patients with IKZF1-del had a CRLF2 translocation. Transcriptomics were analyzed for 42 H/L and 18 NHW patient samples using RNAseq. Gene expression from H/L patients was compared to expression in NHW patients and differentially expressed genes were analyzed. Gene set enrichment analysis (GSEA) revealed downregulation of processes responsible for genome maintenance including DNA replication, DNA templated transcription elongation, double strand break repair, and recombinational repair. Ingenuity pathway analysis (IPA) revealed upregulated pathways in stress related translation, histone deacetylases, interferon, and FLT3 signaling compared to NWH. H/L specimens resulted in decreased expression for FGF pathways, ceramide, and FAK signaling. We performed the same analysis in H/L and NHW with wildtype IKZF1 to compare these two population without high risk structural variants. GSEA analysis showed downregulated Intra S DNA Damage checkpoint signaling, DNA replication checkpoint signaling, glutamine amino acid synthesis, and aspartate amino acid synthesis. IPA showed upregulated interferon, histone deacetylases, TREM1 signaling, and Tec Kinase signaling. IPA showed downregulated Ephrin and CDK5 signaling. DNA methylation patterns were compared between 8 H/L and 8 NWH B ALL samples which showed differential methylation patterns affecting multiple cancer relevant genes.In conclusion, these data represent biologic vulnerability in H/L patients with B ALL in comparison to NWH regardless of high-risk genetic variants. H/L B-ALL shows deregulated maintenance of the genome via replication and repair pathways as well as potential vulnerabilities related to regulation of glutamate/aspartate synthesis. These findings give insight into potential mechanisms that can explain increased frequency of high-risk genetic variants in H/L B ALL. Citation Format: Joseph W. Schramm, Bing He, Yali Ding, Chingakham Singh, Daniel Bogush, Katarina Dovat, Diwakar Bastihalli Tukaramrao, Riya Bhalodia, Sinisa Dovat. Investigation of mechanisms that result in health disparity for Hispanic/Latino patients with B acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2846.

Abstract 5627: PPARG-high circulating monocytes exhibit an immunosuppressive phenotype in urothelial cancer patients treated with anti-PD1

Abstract Background: PPARG is expressed in a variety of immune cells such as monocytes, macrophages, and lymphocytes, where it plays a role in their maturation and function. PPARG has been implicated in immune mediated resistance but to date, a comprehensive analysis of PPARG expression in peripheral blood mononuclear cells (PBMCs) in urothelial cancer (UC) patients is lacking. In this study, we identified the immunophenotypes associated with PPARG expression in UC patients treated with anti-PD1 therapy. Methods: We performed single-cell RNA sequencing of PBMCs from 5 UC patients and 3 normal healthy volunteers (NHV). Real-world samples (Discovery Life Sciences) were collected from patients who received prior Pembrolizumab. Time post-treatment to collection ranged from 46 to 307 days. An average of 9,500 cells were recovered per donor reflecting an ~80% QC pass rate. Count matrices were generated using 10x Genomics Cell Ranger pipeline (v7.0.0; GRCh38 reference). Downstream data processing, analysis and visualization were done on Cellenics software. Results: We evaluated 75,725 cells and identified 9 distinct cell populations. The proportion of T-cells was significantly decreased in UC samples compared to NHVs (40% vs 60%, p=0.03). Highest PPARG expression was observed on Classical Monocytes (CMs) cell population. PPARG expression was higher on CMs in UC compared to NHV samples (1.74 vs 0.11 logcounts; padj<0.001). Unsupervised clustering of all monocytes revealed two distinct CM clusters. In cluster A, 95% of the cells originated from UC samples, while most cells in cluster B (81%) originated from NHV samples. PPARG was the top differentially expressed gene upregulated in cluster A (PPARG-high) vs cluster B (PPARG-low) (1.89 vs 0.23 logcounts; padj<0.001). PPARG-high CMs are characterized by a gene signature inclusive of GPRIN3, RGCC, MAFB and IRF2BP, which have been shown to be upregulated in M2 polarized macrophages [1]. Moreover, pathway analysis using GSEA highlighted that PPARG-high CMs are enriched for TREM1 signaling genes (padj<0.001), whose high expression has been associated with immunosuppressive phenotypes in myeloid cells [2] and shorter survival of patients across other cancers [3]. Conclusion: PPARG-high circulating CMs in UC patients exhibit a transcriptomic profile associated with immunosuppression and M2 macrophage polarization. FX-909, a first-in-class PPARG inverse agonist, is being evaluated in the clinic and the study includes an exploratory biomarker approach to assess the potential effect of PPARG inhibition on the CM immune-profile of advanced UC patients enrolled in the Ph1 study [4].

Acarbose reduces Pseudomonas aeruginosa respiratory tract infection in type 2 diabetic mice

BackgroundType 2 diabetes mellitus (T2DM) is widely prevalent worldwide, and respiratory tract infections (RTIs) have become the primary cause of death for T2DM patients who develop concurrent infections. Among these, Pseudomonas aeruginosa infection has been found to exhibit a high mortality rate and poor prognosis and is frequently observed in bacterial infections that are concurrent with COVID-19. Studies have suggested that acarbose can be used to treat T2DM and reduce inflammation. Our objective was to explore the effect of acarbose on P. aeruginosa RTI in T2DM individuals and elucidate its underlying mechanism.MethodsHigh-fat diet (HFD) induction and P. aeruginosa inhalation were used to establish a RTI model in T2DM mice. The effect and mechanism of acarbose administered by gavage on P. aeruginosa RTI were investigated in T2DM and nondiabetic mice using survival curves, pathological examination, and transcriptomics.ResultsWe found that P. aeruginosa RTI was more severe in T2DM mice than in nondiabetic individuals, which could be attributed to the activation of the NF-κB and TREM-1 signaling pathways. When acarbose alleviated P. aeruginosa RTI in T2DM mice, both HIF-1α and NF-κB signaling pathways were inhibited. Furthermore, inhibition of the calcium ion signaling pathway and NF-κB signaling pathway contributed to the attenuation of P. aeruginosa RTI by acarbose in nondiabetic mice.ConclusionsThis study confirmed the attenuating effect of acarbose on P. aeruginosa RTIs in T2DM and nondiabetic mice and investigated its mechanism, providing novel support for its clinical application in related diseases.

Characterization of TREM-1 Signaling in Human Neutrophils By Kinome Array and RNA-Seq

The Triggering Receptor Expressed on Myeloid Cells (TREM)-1 is a member of the Immunoglobulin superfamily and an activating receptor mainly expressed on myeloid cells. TREM-1 ligation leads to immediate activation of polymorphonuclear neutrophils (PMN) resulting in degranulation and release of reactive oxygen species as well as various effector molecules. Beyond its role in acute and chronic inflammatory processes, TREM-1 is also involved in cancer emergence and progression probably by alteration of the tumor associated neutrophils (TAN) and macrophages (TAM). Advanced information about the TREM-1 signaling cascade may reveal novel therapeutic approaches, as various kinases can specifically be targeted with kinase inhibitors. Therefore, we investigated the protein tyrosine kinome (PTK) and serine threonine kinome (STK) of human PMN after TREM-1 activation. To gain further insights beyond the signaling cascade, we performed RNAseq to validate the TREM-1 mediated activation and to reveal the TREM-1 mediated transcriptome. Purified PMN from healthy donors were stimulated with a monoclonal TREM-1 antibody compared to an isotype matched control for 20 minutes (kinome activity) or 60 minutes (RNAseq). For kinomics, PamChip arrays (PamGene) were used to quantify the kinome activity and upstream kinase analysis (UKA) was used to predict the kinase activity. The final data analysis and the UKA was performed by PamGene. A median final score (depending on the significance and the specificity of the kinase activity compared to the control group) > 1.2 was defined as specific kinase activity according to the manufacturer's recommendations. For transcriptomics, Novogene performed library preparation and sequencing, and data analysis was performed with CLC. Differentially expressed genes were defined as FDR adjusted p value ≤ 0.05 and fold change ≥2. Gene set enrichment analysis was performed with Enrichr using the libraries Reactome 2022, WikiPathway 2021 Human, and GO Biological Process 2023 and significance was defined as adjusted p value ≤ 0.05. We identified the activation of 31 PTKs and 38 STKs upon TREM-1 ligation. The kinase statistic revealed changes in the activation state of the kinases present in the TREM-1 treated cells compared to the untreated control group ranging from 2.4 to 5.5 (PTK) and from 1.2 to 1.6 (STK) suggesting more TREM-1 mediated activity alteration of the PTKs. Interestingly, there were no downregulated kinase activities after receptor ligation. Out of the PTK family, we identified ten Src family kinases as well as the Syk family member ZAP70 and other kinases such as BTK, already previously shown to abrogate TREM-1 mediated PMN activation. The RNAseq after TREM-1 ligation revealed 542 differentially expressed genes from which 102 genes were upregulated and 440 genes were downregulated. Gene set enrichment analysis revealed upregulation of genes related to nuclear events (kinase and transcription factor activaton, R-HSA-198725) and cell chemotaxis (GO:0060326) validating the activation of several kinases after TREM-1 ligation. We found increased expressions of genes involved in adipogenesis (WP236) and lung fibrosis (WP3624) suitably to the observed increased activity of Src family kinases that are described to be involved in lung fibrosis. Moreover, some upregulated genes related to NGF-stimulated transcription (R-HSA-9031628) or regulation of transcription in response to stress (GO:0043618) in which the MAPK are known to be involved. However, we did not observe clearly increased MAPK activity. Besides upregulation, TREM-1 ligation led to decreased expression of several genes involved in (positive) regulation of cytokine production involved in inflammatory response (GO:1900017 and GO:1900015) and signaling by interleukins (R-HSA-449147). Taken together, the results contribute to the knowledge about TREM-1 in inflammatory disorders and reveal approaches of its role in cancer. This study revealed various kinases possibly involved in early TREM-1 signaling of neutrophils that may be suitable targets to inhibit TREM-1 mediated cell activation. Moreover, we linked the TREM-1 mediated kinase activity to the resulting gene expression. Further studies are needed to validate the results in vitro and in vivo and and to identify suitable pharmacological inhibitors to modulate TREM-1 mediated activities in inflammatory conditions and cancer.

Mathematical Modelling of Neutrophil Responses to Aspergillus

Abstract Invasive aspergillosis is a dangerous infection commonly affecting immunocompromised patients. The spores of the fungal pathogen, Aspergillus, are found ubiquitously in the environment and are inhaled daily by all humans. These spores are cleared quickly and effectively healthy hosts without developing infection. On the other hand, patients with impaired immunity, can develop an invasive pneumonia with poor outcomes. Neutrophils are essential to the host defense against aspergillosis, both in direct fungal killing and as modulators of immunity against the pathogen. To better understand this role, we conducted a transcriptomic time series comparing the responses of human neutrophils to Aspergillus. Differential gene expression analysis as well as gene enrichment analysis indicated an up regulation of number of transcriptional markers and pathways including NFkB, NR4A, TNF, as well as neutrophil degranulation, cytokine-mediated signaling, chemokine signaling, and TREM1 signaling among others. Combining this data with known mechanisms and pathways from the literature, we built a mathematical model describing the intracellular pathways which determine the neutrophil’s response after detecting Aspergillus. Very few mathematical models have been built modeling intracellular mechanisms of a neutrophil and none that we know of which relates to the interaction between neutrophils and this organism. We intend to use our model to make predictions on the effects of altering different pathways within the cell. Moreover we intend to use this model in a larger systems model describing the immune system response to invasive pulmonary aspergillosis. Supported by: R01AI135128, U01EB024501, Keck Foundation Medical Research Grant 994413.

Transcriptomic Analysis from Normal Glucose Tolerance to T2D of Obese Individuals Using Bioinformatic Tools

Understanding the role of white adipose tissue (WAT) in the occurrence and progression of metabolic syndrome is of considerable interest; among the metabolic syndromes are obesity and type 2 diabetes (T2D). Insulin resistance is a key factor in the development of T2D. When the target cells become resistant to insulin, the pancreas responds by producing more insulin to try to lower blood glucose. Over time, this can lead to a state of hyperinsulinemia (high levels of insulin in the blood), which can further exacerbate insulin resistance and contribute to the development of T2D. In order to understand the difference between healthy and unhealthy obese individuals, we have used published transcriptomic profiling to compare differences between the WAT obtained from obese diabetics and subjects who are obese with normal glucose tolerance and insulin resistance. The identification of aberrantly expressed messenger RNA (mRNA) and the resulting molecular interactions and signaling networks is essential for a better understanding of the progression from normal glucose-tolerant obese individuals to obese diabetics. Computational analyses using Ingenuity Pathway Analysis (IPA) identified multiple activated signaling networks in obesity progression from insulin-resistant and normal glucose-tolerant (IR-NGT) individuals to those with T2D. The pathways affected are: Tumor Necrosis Factor (TNF), Extracellular signal-Regulated protein Kinase 1/2 ERK1/2, Interleukin 1 A (IL1A), Protein kinase C (Pkcs), Convertase C5, Vascular endothelial growth factor (Vegf), REL-associated protein (RELA), Interleukin1/1 B (IL1/1B), Triggering receptor expressed on myeloid cells (TREM1) and Nuclear factor KB1 (NFKB1) networks, while functional annotation highlighted Liver X Receptor (LXR) activation, phagosome formation, tumor microenvironment pathway, LPS/IL-1 mediated inhibition of RXR function, TREM1 signaling and IL-6 signaling. Together, by conducting a thorough bioinformatics study of protein-coding RNAs, prospective targets could be exploited to clarify the molecular pathways underlying the development of obesity-related type 2 diabetes.

Investigation of the efficacy and pharmacological mechanism of Danhong injections for treating chronic obstructive pulmonary disease: A PRISMA-compliant meta-analysis and network pharmacology analysis.

Accumulating evidence supported the clinical efficacy of Danhong injection (DHI) on chronic obstructive pulmonary disease (COPD). It is urgent to summarize the effects of DHI on various outcomes in COPD patients and to elucidate the molecular mechanisms of DHI in treating COPD. Eligible studies were retrieved from 6 databases including China national knowledge infrastructure, Wangfang, VIP, web of science, PubMed, and Embase. The heterogeneity across studies was tested using the I2 statistic and the quality of studies was assessed. The pooled evaluation of outcomes was calculated using a fix- or random-effect model according to the heterogeneity. The underlying mechanism of DHI in treating COPD was analyzed using network pharmacology. A total of 34 eligible studies with a general medium quality were included in the meta-analysis. The pooled data showed that DHI intervention significantly increased clinical efficacy as compared to routine treatment. Meanwhile, our data also revealed that the addition of DHI markedly improved hemorheological indicators, lung function index, arterial blood gas index, and as well as blood coagulation functions. However, the current meta-analysis lacked sufficient data to support the significant effect of DHI on prothrombin time and activated partial thromboplastin time. Network pharmacology found 59 candidate targets of DHI in treating COPD, and enrichment analysis found these targets were associated with lymphocyte proliferation and activation, glucocorticoid receptor signaling, TREM1 signaling, IL-12 signaling and production in macrophages, and aryl hydrocarbon receptor signaling. Multiple core targets including AKT1, TNF, and IL1B, etc. Were identified and might play an important role in the action of DHI against COPD. Taken together, this study suggested that DHI could ameliorate hemorheological indicators, lung function, arterial blood gas, and as well as coagulation functions of COPD patients and elucidate the underlying mechanism of DHI against COPD.

Ingenuity pathway analysis of α-synuclein predicts potential signaling pathways, network molecules, biological functions, and its role in neurological diseases.

Despite the knowledge that mutation, multiplication, and anomalous function of α-synuclein cause progressive transformation of α-synuclein monomers into toxic amyloid fibrils in neurodegenerative diseases, the understanding of canonical signaling, interaction network molecules, biological functions, and role of α-synuclein remains ambiguous. The evolution of artificial intelligence and Bioinformatics tools have enabled us to analyze a vast pool of data to draw meaningful conclusions about the events occurring in complex biological systems. We have taken the advantage of such a Bioinformatics tool, ingenuity pathway analysis (IPA) to decipher the signaling pathways, interactome, biological functions, and role of α-synuclein. IPA of the α-synuclein NCBI gene dataset revealed neuroinflammation, Huntington's disease, TREM1, phagosome maturation, and sirtuin signaling as the key canonical signaling pathways. IPA further revealed Parkinson's disease (PD), sumoylation, and SNARE signaling pathways specific to the toxicity of α-synuclein. A frequency distribution analysis of α-synuclein-associated genes from the NCBI dataset that appeared in the predicted canonical pathways revealed that NFKB1 was the most populated gene across the predicted pathways followed by FOS, PRKCD, TNF, GSK3B, CDC42, IL6, MTOR, PLCB1, and IL1B. Overlapping of the predicted top-five canonical signaling pathways and the α-synuclein NCBI gene dataset divulged that neuroinflammation signaling was the most overlapped pathway, while NFKB1, TNF, and CASP1 were the most shared molecules among the pathways. The major diseases associated with α-synuclein were predicted to be neurological diseases, organismal injury and abnormalities, skeletal and muscular disorders, psychological disorders, and hereditary disorders. The molecule activity predictor (MAP) analysis of the principal interaction network of α-synuclein gene SNCA revealed that SNCA directly interacts with APP, CLU, and NEDD4, whereas it indirectly communicates with CALCA and SOD1. Besides, IPA also predicted amyloid plaque forming APP, cytokines/inflammatory mediators IL1B, TNF, MIF, PTGS2, TP53, and CCL2, and kinases of MAPK family Mek, ERK, and P38 MAPK as the top upstream regulators of α-synuclein signaling cascades. Taken together, the first IPA analysis of α-synuclein predicted PD as the key toxicity pathway, neurodegeneration as the major pathological outcome, and inflammatory mediators as the critical interacting partners of α-synuclein.

N-Terminal Peptide of PGLYRP1/Tag7 Is a Novel Ligand for TREM-1 Receptor.

An investigation of innate immunity receptors sheds light on the mechanisms of inflammation and associated immune reactions. One of the key immune regulators is the TREM-1 receptor, which is involved in both inflammation and antitumor immune response. In this article, we have obtained a new ligand for the TREM-1 receptor. The peptide, named N3, is a part of the innate immune protein PGLYRP1/Tag7. It is responsible for activating the TREM-1 signaling pathway. Here, we have demonstrated that the N3 peptide acts like other TREM-1 receptor ligands: its binding results in a mild inflammation response and appearance of cytotoxic lymphocytes. We have shown that cytotoxic populations of lymphocytes in N3 peptide-treated PBMCs are similar to those treated with Tag7 or Hsp70. We also determined the part of the N3 peptide responsible for binding to TREM-1. The resulting peptide (N9) consists of nine amino acids and can be considered as a potential peptide that blocks TREM-1 signaling.

Global transcriptional response of oral squamous cell carcinoma cell lines to health-associated oral bacteria - an in vitro study

ABSTRACT Background We have recently demonstrated that health-associated oral bacteria Streptococcus mitis, Neisseria flavescens, and Haemophilus parainfluenzae induce cytotoxicity in oral squamous cell carcinoma (OSCC) cell lines and downregulate CD36, a cancer-assocaited gene. Aim To explore the effect of these three species on global transcriptome of OSCC cell lines. Methods Gene expression of cell lines CAL27, SCC4 and SCC25 cocultured with the test species was assessed with Clariom-S Human microarray. Porphyromonas gingivalis was included as a pathogenic control. Data were analyzed using Ingenuity Pathway Analysis. Results The results differed by species and cell line. Overall, the transcriptional changes by S. mitis were predominantly anti-cancer including inhibition of HOTAIR regulatory pathway, JAK/Stat signaling, cyclins/cyclin-dependent kinases, and endothelin1 signaling. H. parainfluenzae and N. flavescens resulted in a mix of pro- and anti-cancer responses including activation of acute phase response, pro-inflammatory interleukins signaling, TREM-1 signaling, and tumor microenvironment pathway; but downregulation of cell cycle by inhibition of cyclins and cyclin-dependent kinases. P. gingivalis had a predominantly pro-cancer effect limited to SCC4, including upregulation of inflammatory pathways, phospholipases and PI3K signaling. Conclusion These findings provide a new insight into the role of commensal oral bacteria in OSCC. Animal studies are required to further explore them.

Silica Induction of Diverse Inflammatory Proteome in Lungs of Lupus-Prone Mice Quelled by Dietary Docosahexaenoic Acid Supplementation.

Repeated short-term intranasal instillation of lupus-prone mice with crystalline silica (cSiO2) induces inflammatory gene expression and ectopic lymphoid neogenesis in the lung, leading to early onset of systemic autoimmunity and rapid progression to glomerulonephritis. These responses are suppressed by dietary supplementation with the ω-3 polyunsaturated fatty acid docosahexaenoic acid (DHA). Here, we tested the hypothesis that dietary DHA supplementation suppresses cSiO2-induced inflammatory proteins in bronchoalveolar alveolar lavage fluid (BALF) and plasma of lupus-prone mice. Archived tissue fluid samples were used from a prior investigation in which 6 wk-old lupus-prone female NZBWF1 mice were fed isocaloric diets containing 0 or 10 g/kg DHA for 2 wks and then intranasally instilled with 1 mg cSiO2 or vehicle once weekly for 4 wks. Cohorts were terminated at 1, 5, 9 or 13 wk post-instillation (PI). BALF and plasma from each cohort were analyzed by high density multiplex array profiling of 200 inflammatory proteins. cSiO2 time-dependently induced increases in the BALF protein signatures that were highly reflective of unresolved lung inflammation, although responses in the plasma were much less robust. Induced proteins in BALF included chemokines (e.g., MIP-2, MCP-5), enzymes (e.g., MMP-10, granzyme B), adhesion molecules (e.g., sE-selectin, sVCAM-1), co-stimulatory molecules (e.g., sCD40L, sCD48), TNF superfamily proteins (e.g., sTNFRI, sBAFF-R), growth factors (e.g., IGF-1, IGFBP-3), and signal transduction proteins (e.g., MFG-E8, FcgRIIB), many of which were blocked or delayed by DHA supplementation. The BALF inflammatory proteome correlated positively with prior measurements of gene expression, pulmonary ectopic lymphoid tissue neogenesis, and induction of autoantibodies in the lungs of the control and treatment groups. Ingenuity Pathway Analysis (IPA) revealed that IL-1β, TNF-α, and IL-6 were among the top upstream regulators of the cSiO2-induced protein response. Furthermore, DHA’s effects were associated with downregulation of cSiO2-induced pathways involving i) inhibition of ARE‐mediated mRNA decay, ii) bacterial and viral pattern recognition receptor activation, or iii) TREM1, STAT3, NF-κB, and VEGF signaling and with upregulation of PPAR, LXR/RXR and PPARα/RXRα signaling. Altogether, these preclinical findings further support the contention that dietary DHA supplementation could be applicable as an intervention against inflammation-driven autoimmune triggering by cSiO2 or potentially other environmental agents.

A novel gut microbiome therapeutic derived from dietary polyphenols attenuates neuroinflammation in vivo in a model of C9orf72‐mediated frontotemporal dementia

AbstractBackgroundFrontotemporal dementia (FTD) is a neurodegenerative disorder with various genetic etiologies, the most prevalent of which include G4C2 expansions in the c9orf72 gene. Expansions produce dipeptide repeat proteins (DPR), forming aggregates with deleterious impacts on cellular processes and correlating with neuroinflammation. Glycine‐Arginine (GR) aggregates are the only significantly correlated DPR found in clinically relevant neuroanatomical regions of the brain. Our current study investigates the contribution of innate immunity to FTD pathogenesis and the attenuation of neuropathologies with the brain bioavailable microbiome‐derived metabolite, 3‐hydroxybenzoic acid (3‐HBA).MethodMice were transfected via intracerebroventricular injection with AAV9‐GFP(GR)100 or AAV9‐GFP and treated starting on Day 28 with either PBS vehicle or 50 mg kg‐1 of 3‐HBA. At 14 weeks mice were sacrificed and prefrontal cortex (PFC), somatosensory cortex (CTX), and hippocampal (HIPP) tissue was dissected and prepared for RNA sequencing. Differentially expressed genes (DEGs) were identified using DESeq2 and pathway alterations were detected using Ingenuity Pathway Analysis. ELISA for IL‐1β was conducted on PFC, CTX, and HIPP tissue. To determine the ability of 3‐HBA to reduce secretion of IL‐1β, primary microglia pretreated with 3‐HBA were stimulated with LPS+ATP and IL‐1β concentration was analyzed by ELISA.ResultDEGs associated with FTD pathology, protein expression levels of the pro‐inflammatory cytokine IL‐1β, and subsequent amelioration via treatment with 3‐HBA were demonstrated to be a result of contributions from the innate immune system and inflammasome activation in microglia. Inflammatory pathways differentially enriched among treatment groups include TREM1 Signaling, the complement system, and NFκB signaling. GR‐100 Vehicle treated animals exhibited elevated IL‐β in cortical tissue. 3‐HBA was efficacious in vivo in rescuing IL‐1β levels in GR‐100 treated mice to levels of GFP animals. Primary microglia pretreated with 3‐HBA and stimulated with LPS+ATP significantly reduced IL‐1β release, signifying the efficacy of 3‐HBA in attenuating innate immune reactivity.ConclusionPathways and genes associated with the innate immune system show enrichment in a model of FTD and are rescued by administration of 3‐HBA. Overexpression of IL‐1β in the cortex of GR‐100 Vehicle animals is attenuated by the administration of 3‐HBA. Our data implicates a gut‐brain axis mechanism of treatment for key FTD associated neuropathologies.

Transcriptomic Profiling Reveals a Role for TREM-1 Activation in Enterovirus D68 Infection-Induced Proinflammatory Responses.

Increasing cases related to the pathogenicity of Enterovirus D68 (EV-D68) have made it a growing worldwide public health concern, especially due to increased severe respiratory illness and acute flaccid myelitis (AFM) in children. There are currently no vaccines or medicines to prevent or treat EV-D68 infections. Herein, we performed genome-wide transcriptional profiling of EV-D68-infected human rhabdomyosarcoma (RD) cells to investigate host-pathogen interplay. RNA sequencing and subsequent experiments revealed that EV-D68 infection induced a profound transcriptional dysregulation of host genes, causing significantly elevated inflammatory responses and altered antiviral immune responses. In particular, triggering receptor expressed on myeloid cells 1 (TREM-1) is involved in highly activated TREM-1 signaling processes, acting as an important mediator in EV-D68 infection, and it is related to upregulation of interleukin 8 (IL-8), IL-6, IL-12p70, IL-1β, and tumor necrosis factor alpha (TNF-α). Further results demonstrated that NF-κB p65 was essential for EV-D68-induced TREM-1 upregulation. Moreover, inhibition of the TREM1 signaling pathway by the specific inhibitor LP17 dampened activation of the p38 mitogen-activated protein kinase (MAPK) signaling cascade, suggesting that TREM-1 mainly transmits activation signals to phosphorylate p38 MAPK. Interestingly, treatment with LP17 to inhibit TREM-1 inhibited viral replication and infection. These findings imply the pathogenic mechanisms of EV-D68 and provide critical insight into therapeutic intervention in enterovirus diseases.

Abstract 13322: Development of the Preclinical Science Integration and Translation (PRESCIANT) Method and Application to the Apolipoprotein E Knockout Mouse Atherosclerosis Model

Introduction: Animal models of human diseases are used extensively to interrogate molecular mechanisms in a reductionist fashion. We tested whether aggregation and integration of preclinical data can identify new causative pathways that faithfully model human disease mechanisms. We have termed this novel technique, the Preclinical Science Integration and Translation (PRESCIANT) method. Methods and Results: Data were extracted from 716 manuscripts in the journal Arteriosclerosis, Thrombosis, and Vascular Biology from 1995-2019 using the apolipoprotein E knockout mouse (ApoE-KO) to study atherosclerosis. We identified 360 unique studies in which genes were experimentally perturbed in ApoE-KO mice to impact atherosclerotic plaque size and/or composition. Impacts of the interventions on plaque size, inflammation, and lipid content were analyzed using Ingenuity Pathway Analysis. The top upstream regulatory network (sc-58125, a COX2 inhibitor) linked 37.2% (134) of the genes implicated in atherosclerosis into a single network. Further, Ingenuity Pathway Analysis identified TREM1 signaling, LXR/RXR activation, and renin-angiotensin signaling as the top 3 pathways associated with changes in atherosclerosis parameters. Specifically, early atherogenesis genes were enriched with pathways associated with inflammatory cell migration and infiltration (including TREM1) whereas late atherosclerosis genes were associated with cell metabolism and survival (including LXR/RXR activation). These two pathways were interrogated in a clinical cohort of 88,660 patients by testing for association between genetically predicted expression of the human homologs of mouse pathway genes and a composite phenotype composed of 27 human atherosclerosis diagnoses. There was a significant enrichment (p<0.01 for both pathways) in the number of human homologs associated with atherosclerosis, including 12 (57.2%) genes in the TREM1 pathway and 15 (53.6%) in the LXR/RXR pathway. Conclusion: PRESCIANT can successfully leverage decades of animal investigations to translate results from large-volume singular preclinical studies to make novel causal inferences into human disease.

Physical Activity Moderates Inflammatory Gene Expression in Rheumatoid Arthritis

Background: While general population studies have shown associations between greater physical activity and lower inflammatory markers, effects of physical activity on systemic inflammation in immune-mediated inflammatory diseases such as rheumatoid arthritis (RA) are unknown. We examined whether physical activity associates with differential expression of inflammatory genes in RA. Methods: Data derived from an observational RA cohort. Physical activity was measured with the GT9X ActiGraph Link for 7 consecutive 24-hour periods. Clinical data and peripheral whole blood for RNA sequencing were collected. Physical activity groups were defined by the highest and lowest tertiles of moderate/vigorous activity (metabolic equivalent level ≥2.00). RNA extraction, library preparation, Illumina sequencing, and gene expression data processing were performed using established methods. Genes differentially expressed in the most versus least active groups were identified using DESeq2 with the Independent Hypothesis Weighting (IHW) multiple testing procedure and adjusting for sex, age, and race/ethnicity. Ingenuity Pathway Analysis (IPA) Canonical Pathway and Upstream Regulator Analyses were employed on differentially expressed genes with a P<0·1 ranked by log2-fold change to identify biological pathways affected by physical activity. Findings: 35 participants were included (mean age 56±12 years; 91% female; race/ethnicity 31% white, 9% African American, 9% Asian, 40% Hispanic). 767 genes were differentially expressed (padj<0.1) between high versus low activity groups. The high activity group exhibited downregulation of innate and adaptive immune signaling pathways implicated in RA pathogenesis including CD40, STAT3, TREM-1, IL-17a, IL-8, toll-like receptor and interferon signaling. Upstream cytokine activation states demonstrated lower TNF-alpha and interferon among individuals in the high vs. low activity group. Interpretation: RA patients who were more physically active had lower expression of genes involved in innate and adaptive immune signaling compared to those who were more sedentary, providing mechanistic evidence that physical activity may confer protective effects on RA disease outcomes. Funding: This work is supported by NIH/NIAMS R01 AR069616 and NIH/NHLBI K23HL138461-01A1. Additional support from the Rheumatology Research Foundation. Declaration of Interest: None to declare. Ethical Approval: The UCSF Institutional Review Board approved all procedures (UCSF IRB protocol #17-21790) and all participants provided written informed consent.

Idelalisib Impairs TREM-1 Mediated Neutrophil Inflammatory Responses

Introduction: Idelalisib is a selective PI3Kδ inhibitor approved for B-cell non-Hodgkin lymphoma (B-NHL). As adverse events recurrent and severe infectious complications have been observed and experimental data suggest an influence on innate immune defense. Since PI3K is involved in signal transduction of triggering receptor expressed on myeloid cells (TREM)-1, an activating receptor on polymorphonuclear neutrophils (PMN) and macrophages that is implicated in innate immune activation of these cells, we asked, whether PMN function might be impaired by this small-molecule inhibitor.Methods: PMN from healthy humans were isolated by dextran sedimentation and density gradient centrifugation and pretreated with idelalisib, selective PI3Kα/-β/-γ inhibitors (all 1 µg/ml) or vehicle control (DMSO). Cells were activated with anti-TREM-1 agonistic antibody, an isotype matched control antibody (both own production) or Toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS, 1 µg/ml). Effector functions were evaluated by analyzing phagocytosis, CD62L shedding, degranulation and oxidative burst by flow cytometry or oxidative burst kinetics in a fluorescence reader, respectively. Interleukin-8 (IL-8) release was detected by ELISA. Western blot analyses of phosphorylated and non-phosphorylated proteins were used for the detection of the involved signaling events, and calcium flux was detected by flow cytometry. Similarly, blood samples of patients receiving idelalisib treatment were followed up for PMN functions as described above to investigate immunomudulatory effects in vivo .Results: We observed a significantly impaired oxidative burst, degranulation (CD66b and CD11b surface expression), CD62L shedding and cytokine (IL-8) release upon PMN activation via TREM-1 ligation and partly by LPS. Furthermore, TREM-1 mediated calcium flux was diminished and TREM-1 signaling was impaired regarding phosphorylation of p38MAPK, ERK1/2 and AKT indicating that the activation of Ca2+ flux as well as MAP kinases and AKT occur down stream of PI3K. Selective PI3Kα and PI3Kβ inhibitors (but not a PI3Kγ inhibitor) showed similar activation patterns, furthermore suggesting that impaired PMN functionalities are due to an unspecific PI3K class IA inhibitory effect. To assess whether our results obtained in vitro may be of relevance in vivo, we analyzed blood samples of B-NHL patients receiving idelalisib therapy ex vivo. Here we again detected an impairment of TREM-1 mediated and partly of TLR4 mediated PMN activation. PMN functions from one patient were analyzed during and after idelalisib therapy. Interestingly, PMN functions were restored after discontinuation of treatment, indicating that the functional PMN deficiencies are reversible.Conclusion: Small-molecule inhibitors might cause a neutropenia-like susceptibility to infections in patients by leading to impaired PMN functions. These side effects are largely unrecognized in daily clinical routine so far by monitoring just for counts of white blood cells, but not their functions. DisclosuresTeschner:Astellas Pharma GmbH: Other: Travel support; Gilead Sciences GmbH: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Jazz Pharmaceuticals Germany: Other: Travel Support; MSD Sharp & Dohme GmbH: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Pfizer Deutschland GmbH: Honoraria, Membership on an entity's Board of Directors or advisory committees. Hess:Celgene: Other: Grants and personal fees; Pfizer: Other: Grants and personal fees; CTI: Other: Grants; Roche: Other: Grants and personal fees; Janssen: Other: Personal Fees. Radsak:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Gilead: Other: Travel support; Acceleron: Research Funding.