Titanium Particles Modulate Lymphocyte and Macrophage Polarization in Peri-Implant Gingival Tissues.

Titanium dental implants are one of the modalities to replace missing teeth. The release of titanium particles from the implant's surface may modulate the immune cells, resulting in implant failure. However, little is known about the immune microenvironment that plays a role in peri-implant inflammation as a consequence of titanium particles. In this study, the peri-implant gingival tissues were collected from patients with failed implants, successful implants and no implants, and then a whole transcriptome analysis was performed. The gene set enrichment analysis confirmed that macrophage M1/M2 polarization and lymphocyte proliferation were differentially expressed between the study groups. The functional clustering and pathway analysis of the differentially expressed genes between the failed implants and successful implants versus no implants revealed that the immune response pathways were the most common in both comparisons, implying the critical role of infiltrating immune cells in the peri-implant tissues. The H&E and IHC staining confirmed the presence of titanium particles and immune cells in the tissue samples, with an increase in the infiltration of lymphocytes and macrophages in the failed implant samples. The in vitro validation showed a significant increase in the level of IL-1ß, IL-8 and IL-18 expression by macrophages. Our findings showed evidence that titanium particles modulate lymphocyte and macrophage polarization in peri-implant gingival tissues, which can help in the understanding of the imbalance in osteoblast-osteoclast activity and failure of dental implant osseointegration.

Expression of GPR68, an Acid-Sensing Orphan G Protein-Coupled Receptor, in Breast Cancer.

Breast cancer (BC) is the most diagnosed cancer and the leading cause of global cancer incidence in 2020. It is quite known that highly invasive cancers have disrupted metabolism that leads to the creation of an acidic tumor microenvironment. Among the proton-sensing G protein-coupled receptors is GPR68. In this study, we aimed to explore the expression pattern of GPR68 in tissues from BC patients as well as different BC cell lines. METHODS: In-silico tools were used to assess the expression of GPR68 in BC patients. The expression pattern was validated in fresh and paraffin-embedded sections of BC patients using qPCR and immunohistochemistry (IHC), respectively. Also, in-silico tools investigated GPR68 expression in different BC cell lines. Validation of GPR68 expression was performed using qPCR and immunofluorescence techniques in four different BC cell lines (MCF-7, MDA-MB-231, BT-549 and SkBr3). RESULTS: GPR68 expression was found to be significantly increased in BC patients using the in-silico tools and validation using qPCR and IHC. Upon classification according to the molecular subtypes, the luminal subtype showed the highest GPR68 expression followed by triple-negative and Her2-enriched cells. However, upon validation in the recruited cohort, the triple-negative molecular subtype of BC patients showed the highest GPR68 expression. Also, in-silico and validation data revealed that the triple-negative breast cancer cell line MDA-MB-231 showed the highest expression of GPR68. CONCLUSION: Therefore, this study highlights the potential utilization of GPR68 as a possible diagnostic and/or prognostic marker in BC.

Insights into the Role of Gremlin-1, a Bone Morphogenic Protein Antagonist, in Cancer Initiation and Progression.

The bone morphogenic protein (BMP) antagonist Gremlin-1 is a biologically significant regulator known for its crucial role in tissue differentiation and embryonic development. Nevertheless, it has been reported that Gremlin-1 can exhibit its function through BMP dependent and independent pathways. Gremlin-1 has also been reported to be involved in organ fibrosis, which has been correlated to the development of other diseases, such as renal inflammation and diabetic nephropathy. Based on growing evidence, Gremlin-1 has recently been implicated in the initiation and progression of different types of cancers. Further, it contributes to the stemness state of cancer cells. Herein, we explore the recent findings on the role of Gremlin-1 in various cancer types, including breast, cervical, colorectal, and gastric cancers, as well as glioblastomas. Additionally, we highlighted the impact of Gremlin-1 on cellular processes and signaling pathways involved in carcinogenesis. Therefore, it was suggested that Gremlin-1 might be a promising prognostic biomarker and therapeutic target in cancers.

Complement System: An Immunotherapy Target in Colorectal Cancer.

Colorectal cancer (CRC) is the third most common malignant tumor and the second most fatal cancer worldwide. Several parts of the immune system contribute to fighting cancer including the innate complement system. The complement system is composed of several players, namely component molecules, regulators and receptors. In this review, we discuss the complement system activation in cancer specifically CRC and highlight the possible interactions between the complement system and the various TME components. Additionally, the role of the complement system in tumor immunity of CRC is reviewed. Hence, such work could provide a framework for researchers to further understand the role of the complement system in CRC and explore the potential therapies targeting complement activation in solid tumors such as CRC.

Natural Killer Cell Dysfunction in Obese Patients with Breast Cancer: A Review of a Triad and Its Implications.

Natural killer cells (NK cells) are a crucial constituent of the innate immune system as they mediate immunity against viruses, bacteria, parasites, and most importantly, tumor cells. The exact mechanism of how the innate immune system and specifically NK cells interact with cancer cells is complex and is yet to be understood. Several factors that constitute the tumor microenvironment (TME) such as hypoxia and TGF-ß are believed to play a role in the complex physiological reaction of NK cells to tumor cells. On the other hand, several risk factors are implicated in the development and progression of breast cancer, most importantly: obesity. Cytokines released from adipose tissue such as adipokines, leptin, and resistin, among others, are also believed to facilitate tumor progression. In this study, we aimed to build a triad of breast cancer, obesity, and NK cell dysfunction to elucidate a link between these pillars on a cellular level. Directing efforts towards solidifying the link between these factors will help in designing a targeted immunotherapy with a low side-effect profile that can revolutionize breast cancer treatment and improve survival in obese patients.

Molecular Examination of Differentially Expressed Genes in the Brains of Experimental Autoimmune Encephalomyelitis Mice Post Herceptin Treatment.

OBJECTIVE: Herceptin (trastuzumab) is an approved drug for treating HER2+ breast cancer patients, but its use for other diseases is not established. We sought to investigate the effects of Herceptin on ameliorating experimental autoimmune encephalomyelitis (EAE) and to examine its effects on the expression of various genes. METHODS: We used in-silico analysis of publicly available data, qRT-PCR, and immunohistochemistry (IHC) to determine the expression of HER2+ cells in the brains of EAE mice. IHC was also utilized to determine the anti-inflammatory effects of Herceptin. The ability of Herceptin to alleviate the EAE clinical score was measured in these mice. Bioinformatics analysis of publicly available data and qRT-PCR were performed to investigate the differentially expressed genes that were either up-regulated or down-regulated during the high clinical score (HCS) of the disease. RESULTS: We observed that HER2/Erbb2, the receptor for Herceptin is upregulated in the brains of EAE mice when the brains were examined at the HCS stage. Further, we demonstrated that Herceptin ameliorates the EAE disease, increasing re-myelination, reducing brain inflammation, CD3+ T cell accumulation, and HER2+ cells in the brains of these mice. Molecular analysis demonstrated the expression of different genes that were either up-regulated or down-regulated during the HCS of the disease. Our combined bioinformatics and qRT-PCR analyses show increased mRNA expression of Atp6v0d2, C3, C3ar1, Ccl3, Ccl6, Cd74, Clec7a, Cybb, H2-Aa, Hspb1, Lilr4b, Lilrb4a, Mpeg1, Ms4a4a, Ms4a6c, Saa3, Serpina3n and Timp1, at HCS. Except for the mRNA levels of Cd74 and Clec7a which were increased at HCS when Herceptin was used in both prophylactic and therapeutic regimens, the levels of other described mRNAs were reduced. CONCLUSION: These novel findings show that Herceptin ameliorates the clinical score in EAE mice and are the first to investigate in detail the differential gene expression post-treatment with the drug.

Derangement of cell cycle markers in peripheral blood mononuclear cells of asthmatic patients as a reliable biomarker for asthma control.

In asthma, most of the identified biomarkers pertain to the Th2 phenotype and no known biomarkers have been verified for severe asthmatics. Therefore, identifying biomarkers using the integrative phenotype-genotype approach in severe asthma is needed. The study aims to identify novel biomarkers as genes or pathways representing the core drivers in asthma development, progression to the severe form, resistance to therapy, and tissue remodeling regardless of the sample cells or tissues examined. Comprehensive reanalysis of publicly available transcriptomic data that later was validated in vitro, and locally recruited patients were used to decipher the molecular basis of asthma. Our in-silicoanalysis revealed a total of 10 genes (GPRC5A, SFN, ABCA1, KRT8, TOP2A, SERPINE1, ANLN, MKI67, NEK2, and RRM2) related to cell cycle and proliferation to be deranged in the severe asthmatic bronchial epithelium and fibroblasts compared to their healthy counterparts. In vitro, RT qPCR results showed that (SERPINE1 and RRM2) were upregulated in severe asthmatic bronchial epithelium and fibroblasts, (SFN, ABCA1, TOP2A, SERPINE1, MKI67, and NEK2) were upregulated in asthmatic bronchial epithelium while (GPRC5A and KRT8) were upregulated only in asthmatic bronchial fibroblasts. Furthermore, MKI76, RRM2, and TOP2A were upregulated in Th2 high epithelium while GPRC5A, SFN, ABCA1 were upregulated in the blood of asthmatic patients. SFN, ABCA1 were higher, while MKI67 was lower in severe asthmatic with wheeze compared to nonasthmatics with wheezes. SERPINE1 and GPRC5A were downregulated in the blood of eosinophilic asthmatics, while RRM2 was upregulated in an acute attack of asthma. Validation of the gene expression in PBMC of locally recruited asthma patients showed that SERPINE1, GPRC5A, SFN, ABCA1, MKI67, and RRM2 were downregulated in severe uncontrolled asthma. We have identified a set of biologically crucial genes to the homeostasis of the lung and in asthma development and progression. This study can help us further understand the complex interplay between the transcriptomic data and the external factors which may deviate our understanding of asthma heterogeneity.

Chemokines and chemokine receptors during COVID-19 infection.

Chemokines are crucial inflammatory mediators needed during an immune response to clear pathogens. However, their excessive release is the main cause of hyperinflammation. In the recent COVID-19 outbreak, chemokines may be the direct cause of acute respiratory disease syndrome, a major complication leading to death in about 40% of severe cases. Several clinical investigations revealed that chemokines are directly involved in the different stages of SARS-CoV-2 infection. Here, we review the role of chemokines and their receptors in COVID-19 pathogenesis to better understand the disease immunopathology which may aid in developing possible therapeutic targets for the infection.

Understanding the Role of Innate Immune Cells and Identifying Genes in Breast Cancer Microenvironment.

The innate immune system is the first line of defense against invading pathogens and has a major role in clearing transformed cells, besides its essential role in activating the adaptive immune system. Macrophages, dendritic cells, NK cells, and granulocytes are part of the innate immune system that accumulate in the tumor microenvironment such as breast cancer. These cells induce inflammation in situ by secreting cytokines and chemokines that promote tumor growth and progression, in addition to orchestrating the activities of other immune cells. In breast cancer microenvironment, innate immune cells are skewed towards immunosuppression that may lead to tumor evasion. However, the mechanisms by which immune cells could interact with breast cancer cells are complex and not fully understood. Therefore, the importance of the mammary tumor microenvironment in the development, growth, and progression of cancer is widely recognized. With the advances of using bioinformatics and analyzing data from gene banks, several genes involved in NK cells of breast cancer individuals have been identified. In this review, we discuss the activities of certain genes involved in the cross-talk among NK cells and breast cancer. Consequently, altering tumor immune microenvironment can make breast tumors more responsive to immunotherapy.

Differentially Expressed Genes of Natural Killer Cells Can Distinguish Rheumatoid Arthritis Patients from Healthy Controls.

Rheumatoid arthritis (RA) is one of the most prevalent autoimmune diseases, while its molecular triggers are not fully understood. A few studies have shown that natural killer (NK) cells may play either a pathogenic or a protective role in RA. In this study, we sought to explore NK cell markers that could be plausibly used in evaluating the differences among healthy controls and RA patients. Publicly available transcriptome datasets from RA patients and healthy volunteers were analyzed, in order to identify differentially expressed genes (DEGs) between 1. different immune cells as compared to NK cells, and 2. NK cells of RA patients and healthy controls. The identified DEGs were validated using 16 healthy controls and 17 RA patients. Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll density gradient method, while NK cells were isolated using RosetteSep technique. RNA was extracted and gene expression was assessed using RT-qPCR. All selected genes were differentially expressed in NK cells compared to PBMCs. CD56, CXCL16, PECAM-1, ITGB7, BTK, TLR10, and IL-1ß were significantly upregulated, while CCL2, CCR4, RELA and IBTK were downregulated in the NK cells of RA patients when compared to healthy controls. Therefore, these NK specific genes might be used as promising biomarkers for RA diagnosis.

Confounding Patient Factors Affecting the Proper Interpretation of the Periostin Level as a Biomarker in Asthma Development.

INTRODUCTION: The proper use of serum periostin (POSTN) as a biomarker for asthma is hindered by inconsistent performance in different clinical settings. OBJECTIVE: To explore patient's factors that may affect POSTN expression locally and systematically and its utility as a biomarker for asthma development. MATERIALS AND METHODS: Here we used bioinformatics analysis of publicly available transcriptomics data to confirm that POSTN is an asthma specific gene involved in core signaling pathways enriched in the bronchial epithelium during asthma. We then explored a large number of datasets to identify possible confounders that may affect the POSTN gene expression and consequently, its interpretation as a reliable biomarker for asthma. Plasma and saliva levels of POSTN were determined in locally recruited asthmatic patients (mild, moderate and severe) compared to healthy controls to confirm the bioinformatics findings. RESULTS: Our bioinformatics results confirmed that POSTN was consistently upregulated in the bronchial epithelium in asthma, chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) bronchial epithelium. In asthma, its mRNA expression was affected by gender, sample anatomical site and type, steroid therapy, and smoking. In our cohort, plasma POSTN was upregulated in severe and non-severe asthmatic patients. Saliva POSTN was significantly higher in non-severe asthmatic patients compared to healthy and severe asthmatic patients (specifically those who are not on Xolair (omalizumab)). Patients' BMI, inhaled steroid use and Xolair treatment affected POSTN plasma levels. CONCLUSION: Up to our knowledge, this is the first study examining the level of POSTN in the saliva of asthmatic patients. Both plasma and saliva POSTN levels can aid in early diagnosis of asthma. Saliva POSTN level was more sensitive than plasma POSTN in differentiating between severe and non-severe asthmatics. Patients' characteristics like BMI, the use of inhaled steroids, or Xolair treatment should be carefully reviewed before any meaningful interpretation of POSTN level in clinical practice.

Gasdermin D Hypermethylation Inhibits Pyroptosis And LPS-Induced IL-1ß Release From NK92 Cells.

INTRODUCTION: Although natural killer (NK) are major cells used to treat cancer patients, recent clinical trials showed that NK92 cells can be also used for the same purpose due to their high anti-tumor activity. Here, we examined whether these cells might be inflammatory due to the release of interleukin-1ß (IL-1ß), and whether the anti-inflammatory molecules dimethyl fumarate (DMF), or monomethyl fumarate (MMF) impair this activity. METHODS: NK92 cells were examined for the synthesis and release of IL-1ß utilizing RT-PCR and ELISA assay, respectively. The expression of hydroxy-carboxylic acid receptors (HCA)1, HCA2 and HCA3 was detected by immunoblotting, flow cytometry, immunofluorescence and RT-PCR assays. The activation of caspase-1 and Gasdermin D (GSDMD) was evaluated by immunoblot assay. Pyroptosis was demonstrated by immunofluorescence imaging. Expression of DNA methyltransferases (DNMTs) mRNA was determined by whole transcriptome and immunoblot analyses. RESULTS: LPS-induced the release of IL-1ß from NK92 cells, whereas DMF or MMF inhibited this induction. The effect of these drugs was due to inhibiting the conversion of procaspase-1 into active caspase-1. NK92 cells highly expressed GSDMD, a pyroptotic-mediated molecule. However, LPS induced the distribution of GSDMD into the cell membranes, corroborated with the presence of pyroptotic bodies, an activity that was inhibited by DMF or MMF. These molecule also inhibited the generation of GSDMD through DNMT-mediated hypermethylation of the promoter region of GSDMD gene. These results were supported by increased expression of DNMTs mRNA as determined by whole transcriptome analysis. DISCUSSION: Our results are the first to show that NK92 cells utilize GSDMD pathway to release IL-1ß. Further, DMF and MMF which were previously shown to enhance NK cell cytotoxicity, also inhibit the inflammatory effects of these cells, making them most suitable for treating cancer patients. .

HCT-116 colorectal cancer cells secrete chemokines which induce chemoattraction and intracellular calcium mobilization in NK92 cells.

We recently reported that pretreatment of IL-2 activated human natural killer (NK) cells with the drugs dimethyl fumarate (DMF) and monomethyl fumarate (MMF) upregulated the expression of surface chemokine receptor CCR10. Ligands for CCR10, namely CCL27 and CCL28, induced the chemotaxis of these cells. Here, we performed a bioinformatics analysis to see which chemokines might be expressed by the human HCT-116 colorectal cancer cells. We observed that, in addition to CCL27 and CCL28, HCT-116 colorectal cancer cells profoundly express CXCL16 which binds CXCR6. Consequently, NK92 cells were treated with DMF and MMF for 24 h to investigate in vitro chemotaxis towards CXCL16, CCL27, and CCL28. Furthermore, supernatants collected from HCT-116 cells after 24 or 48 h incubation induced the chemotaxis of NK92 cells. Similar to their effects on human IL-2-activated NK cells, MMF and DMF enhanced the expression of CCR10 and CXCR6 in NK92 cells. Neutralizing anti-CXCL16 or anti-CCL28 inhibited the chemotactic effects of 24 and 48 supernatants, whereas anti-CCL27 only inhibited the 48 h supernatant activity, suggesting that 24 h supernatant contains CXCL16 and CCL28, whereas HCT-116 secretes all three chemokines after 48 h in vitro cultures. CXCL16, CCL27, and CCL28, as well as the supernatants collected from HCT-116, induced the mobilization of (Ca)2+ in NK92 cells. Cross-desensitization experiments confirmed the results of the chemotaxis experiments. Finally, incubation of NK92 cells with HCT-116 induced the lysis of the tumor cells. In summary, these results might have important implications in directing the anti-tumor effectors NK cells towards tumor growth sites.

Repressing PU.1 by miR-29a∗ in NK cells of HCV patients, diminishes its cytolytic effect on HCV infected cell models.

OBJECTIVES: Natural killer cells are immune safeguards against HCV infection. PU.1 is a pivotal transcription factor in the development of NK cells. This study aimed at studying the regulatory effect of miRNAs on both development and function of NK cells isolated from HCV patients. METHODS: NK cells were isolated from 17 chronic HCV patients and 12 healthy controls; after which miRNA and mRNA were quantified using qRT-PCR. Manipulating miRNA expression using mimics and antagomirs, was performed followed by investigating downstream targets as well as viral abundance. RESULTS: PU.1 expression levels were upregulated in NK cells of HCV patients. In silico analysis revealed PU.1 to be a potential downstream target of miR-29a(∗), where miR-29a(∗) overexpression in NK cells caused a significant downregulation in PU.1 mRNA. Forcing miR-29a(∗) caused a downregulation of the cytotoxicity determinant NK activating receptor (NKG2D) via upregulation of miR-155. Moreover, perforin-1 mRNA was found to be downregulated upon forcing the expression of miR-29a(∗) in NK cells of HCV patients. This decrease in NK cytolytic function was accompanied by an 80% viral load increase in cocultured HCVcc cell models. CONCLUSIONS: This study showed that HCV infection might abrogate NK cytotoxic potential through altering PU.1, NKG2D receptor and perforin molecules.