Partial recovery of peripheral blood monocyte subsets in head and neck squamous cell carcinoma patients upon radio(chemo)therapy is associated with decreased plasma CXCL11.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) represents a common and heterogeneous malignancy of the oral cavity, pharynx and larynx. Surgery and radio(chemo)therapy are the standard treatment options and also have great influence on the composition of the tumor microenvironment and immune cell functions. However, the impact of radio(chemo)therapy on the distribution and characteristics of circulating monocyte subsets in HNSCC are not fully understood. METHODS: Expression patterns of adhesion molecules and chemokine receptors CD11a (integrin-α L; LFA-1), CD11b (integrin-α M; Mac-1), CD11c (integrin-α X), CX3CR1 (CX3CL1 receptor) and checkpoint molecule PD-L1 (programmed cell death ligand-1) were investigated upon radio(chemo)therapeutic treatment using flow cytometry. Furthermore, comprehensive analysis of plasma cytokines was performed before and after treatment using ELISA measurements. RESULTS: Our data reveal a partial recovery of circulating monocytes in HNSCC patients upon radio(chemo)therapeutic treatment, with differential effects of the individual therapy regimen. PD-L1 expression on non-classical monocytes significantly correlates with the individual plasma levels of chemokine CXCL11 (C-X-C motif chemokine 11). CONCLUSIONS: Further comprehensive investigations on larger patient cohorts are required to elucidate the meaningfulness of peripheral blood monocyte subsets and chemokine CXCL11 as potential bioliquid indicators in HNSCC with regard to therapy response and the individual immunological situation.

Type 2-like polarization and elevated CXCL4 secretion of monocyte derived macrophages upon internalization of plasma-derived exosomes from head and neck cancer patients.

BACKGROUND: Exosomes are closely associated with different aspects of tumor-progression in patients with head and neck squamous cell carcinoma (HNSCC), such as angiogenesis or immune regulation. As extracellular vesicles they are involved in the intercellular communication by transferring their cargo such as proteins and nucleic acids from one cell to another. However, the influence of tumor related plasma-derived exosomes on the polarization and characteristics of monocyte derived macrophages is not fully understood. METHODS: Exosomes were isolated from plasma samples of healthy donors (HD) and HNSCC patients and further evaluated with regard to morphology, size and protein composition via transmission electron microscopy, nanoparticle tracking, western blot analysis and cytokine assays. Differentiation and characteristics of monocyte derived macrophages upon exosome internalization were analyzed using flow cytometry and fluorescence microscopy. Macrophage cytokine secretion patterns were analyzed by human cytokine antibody arrays and ELISA measurements. RESULTS: Our data revealed elevated overall plasma levels of CTLA-4, PD-L1, and TIM-3 as well as elevated exosome-associated CTLA-4, PD-L2, TIM-3, and LAG-3 levels in HNSCC patients compared to HD. Furthermore, we observed a significant type 2-like polarization and elevated CXCL4 secretion of monocyte derived macrophages upon internalization of plasma-derived exosomes from HNSCC patients, which could be visualized by fluorescence microcopy of membrane stained exosomes. CONCLUSIONS: The study provides new insights regarding exosome driven pro-tumorigenic immune regulation in the circulation of patients with head and neck cancer and could help to better understand the individual immunologic situation.

Improved levels of checkpoint molecule PD-L1 on peripheral blood monocyte subsets in obstructive sleep apnea syndrome patients upon hypoglossal nerve stimulation.

Oxidative stress in patients suffering from obstructive sleep apnea syndrome (OSAS) is associated with a low-grade systemic inflammation, immune disturbance, and increased invasion of monocytes into the endothelium. Besides continuous positive airway pressure (PAP), hypoglossal nerve stimulation (HNS) has become a promising treatment option for patients with OSAS. We aimed to analyse the influence of HNS therapy on the cellular characteristics relevant for adhesion and immune regulation of circulating CD14/CD16 monocyte subsets. Whole blood flow cytometric measurements were performed to analyse the expression levels of different adhesion molecules and checkpoint molecule PD-L1 (programmed death-ligand 1) in connection with pro-inflammatory plasma cytokine IL-8 and the clinical values of BMI (body mass index), AHI (apnea-hypopnea index), ODI (oxygen desaturation index), and ESS (Epworth sleepiness scale) upon HNS treatment. Hypoglossal nerve stimulation treatment significantly improved the expression of adhesion molecule CD162 (P-selectin receptor) on non-classical monocytes and significantly downregulated the expression of PD-L1 on all three monocyte subsets. We conclude that the holistic improvement of different parameters such as the oxygenation of the peripheral blood, a reduced systemic inflammation, and the individual sleeping situation upon HNS respiratory support, leads to an improved immunologic situation.

Mutant WDR45 Leads to Altered Ferritinophagy and Ferroptosis in ß-Propeller Protein-Associated Neurodegeneration.

Beta-propeller protein-associated neurodegeneration (BPAN) is a subtype of neurodegeneration with brain iron accumulation (NBIA) caused by loss-of-function variants in WDR45. The underlying mechanism of iron accumulation in WDR45 deficiency remains elusive. We established a primary skin fibroblast culture of a new BPAN patient with a missense variant p.(Asn61Lys) in WDR45 (NM_007075.3: c.183C>A). The female patient has generalized dystonia, anarthria, parkinsonism, spasticity, stereotypies, and a distinctive cranial MRI with generalized brain atrophy, predominantly of the cerebellum. For the functional characterization of this variant and to provide a molecular link of WDR45 and iron accumulation, we looked for disease- and variant-related changes in the patient's fibroblasts by qPCR, immunoblotting and immunofluorescence comparing to three controls and a previously reported WDR45 patient. We demonstrated molecular changes in mutant cells comprising an impaired mitochondrial network, decreased levels of lysosomal proteins and enzymes, and altered autophagy, confirming the pathogenicity of the variant. Compared to increased levels of the ferritinophagy marker Nuclear Coactivator 4 (NCOA4) in control cells upon iron treatment, patients' cells revealed unchanged NCOA4 protein levels, indicating disturbed ferritinophagy. Additionally, we observed abnormal protein levels of markers of the iron-dependent cell death ferroptosis in patients' cells. Altogether, our data suggests that WDR45 deficiency affects ferritinophagy and ferroptosis, consequentially disturbing iron recycling.

1,8-Cineol Attenuates Checkpoint Molecule PDL-1 and Adhesion Molecule CX3CR1 in Circulating Monocytes in Otitis Media Patients.

BACKGROUND: Peripheral blood monocytes can be subdivided into different subsets based on the CD14/CD16 surface characteristics. Monocytes are a major source of cytokine secretion of pro-inflammatory immune responses, whereas CD16+ monocyte subsets can also contribute to persistent inflammation in the context of chronic diseases. However, the regulation and cellular characteristics of circulating monocyte subsets in patients with chronic otitis media (COM), one of the largest public health burdens, remains largely unknown. MATERIALS AND METHODS: In this study, we analyzed individual distributions of circulating monocyte subsets and associated protein expression levels of adhesion protein and chemokine receptors CD11a (integrin-α L; LFA-1), CD11b (integrin-α M; Mac-1), and CD11c (integrin-α X), CX3CR1 (CX3CL1 receptor), as well as checkpoint molecule PD-L1 (programmed cell death ligand-1), in a gender-balanced cohort of 14 patients with chronic otitis media using flow cytometry, especially in view of the therapeutic impact of the natural plant-derived monoterpene oxide 1,8-Cineol. Furthermore, using the human monocyte cell line THP-1 as a model, we investigated the influence of anti-inflammatory 1,8-Cineol on monocytic cytokine secretion patterns using human cytokine arrays and ELISA measurements. RESULTS: The data revealed significantly elevated expression levels of all analyzed adhesion molecules in certain monocyte subsets in COM patients; CX3CR1 was especially significantly down-regulated in response to 1,8-Cineol administration. Moreover, the data revealed significantly increased monocytic PD-L1 expression levels in circulating classical and intermediate monocyte subsets from COM patients compared to healthy donors, but also a significant decrease in PD-L1 in intermediate monocytes upon 1,8-Cineol therapy compared to the pre-treatment situation. Furthermore, the increased secretion of cytokine CXCL10 by THP-1 monocytes in response to LPS was found to be strongly attenuated by 1,8-Cineol. Plasma levels of CXCL10 were also significantly increased in COM patients, but no significant differences between the pre and post 1,8-Cineol situation were observed. CONCLUSIONS: The present study revealed new insights into the bioactive anti-inflammatory effects of 1,8-Cineol in terms of monocyte adhesion and immune regulation. Our data suggest the potential role of cytokine CXCL10 in COM development and maintenance, which is also involved in the activity of its concomitant disease, rheumatoid arthritis.

Distinguishing the impact of distinct obstructive sleep apnea syndrome (OSAS) and obesity related factors on human monocyte subsets.

Obstructive sleep apnea syndrome (OSAS) and obesity go hand in hand in the majority of patients and both are associated with a systemic inflammation, immune disturbance and comorbidities such as cardiovascular disease. However, the unambiguous impact of OSAS and obesity on the individual inflammatory microenvironment and the immunological consequences of human monocytes has not been distinguished yet. Therefore, aim of this study was to investigate the impact of OSAS and obesity related factors on the inflammatory microenvironment by performing flow cytometric whole blood measurements of CD14/CD16 monocyte subsets in normal weight OSAS patients, patients with obesity but without OSAS, and patients with OSAS and obesity, compared to healthy donors. Moreover, explicitly OSAS and obesity related plasma levels of inflammatory mediators adiponectin, leptin, lipocalin and metalloproteinase-9 were determined and the influence of different OSAS and obesity related factors on cytokine secretion and expression of different adhesion molecules by THP-1 monocytes was analysed. Our data revealed a significant redistribution of circulating classical and intermediate monocytes in all three patient cohorts, but differential effects in terms of monocytic adhesion molecules CD11a, CD11b, CD11c, CX3CR1, CD29, CD49d, and plasma cytokine levels. These data were reflected by differential effects of OSAS and obesity related factors leptin, TNFα and hypoxia on THP-1 cytokine secretion patterns and expression of adhesion molecules CD11b and CD49d. In summary, our data revealed differential effects of OSAS and obesity, which underlines the need for a customized therapeutic regimen with respect to the individual weighting of these overlapping diseases.

Different Influence Pattern of Conventional and Alternative Sources of Smoking on Adhesion Molecules and Cytokine Secretion in THP-1 Monocytes.

BACKGROUND/AIM: Tobacco is a carcinogen that is closely associated with the occurrence of lung cancer and head and neck squamous cell carcinoma (HNSCC). The consumption of tobacco is also leading to alterations in different immune cell subtypes. However, the impact of different conventional and alternative smoking sources on human monocytes remains elusive. MATERIALS AND METHODS: In this study, we investigated the influence of aqueous extracts of different sources of smoking (cigarettes; heated tobacco product IQOS; e-cigarettes with and without nicotine; nicotine pouches) on different monocytic adhesion molecules, chemokine receptors and checkpoint molecule PD-L1 by flow cytometry. Cytokine expression patterns were evaluated using human cytokine arrays and the human monocyte leukemia cell line THP-1 as a model. RESULTS: Data revealed differential effects of the analyzed conventional and alternative smoking devices on monocyte adhesion molecules and cytokine secretion. The examined smoking devices can be assigned to two differential monocyte activation patterns. Monocytes stimulated with aqueous extracts of cigarettes, e-cigarette without nicotine, and heat not burn product IQOS revealed distinct alterations of surface markers and cytokines compared to the monocyte activation pattern in response to aqueous extracts of nicotine, nicotine pouches, and e-cigarette with nicotine. CONCLUSION: Our data indicate differential immunological consequences of different conventional and alternative smoking sources with and without nicotine. Further comprehensive analysis as well as in vivo investigations on peripheral blood monocyte subsets from smoking individuals using different smoking sources are required to better understand the impact on monocyte characteristics, especially with regard to the development of cancer.

Obesity-related Plasma CXCL10 Drives CX3CR1-dependent Monocytic Secretion of Macrophage Migration Inhibitory Factor.

Obesity is characterized by excessive body fat accumulation and comorbidities such as diabetes mellitus, cardiovascular disease, and obstructive sleep apnea syndrome (OSAS). Both obesity and OSAS are associated with immune disturbance, alterations of systemic inflammatory mediators, and immune cell recruitment to metabolic tissues. Chemokine CXCL10 is an important regulator of proinflammatory immune responses and is significantly increased in patients with severe obesity. This research project aims to investigate the impact of CXCL10 on human monocytes in patients with obesity. We studied the distribution of the CD14/CD16 monocyte subsets as well as their CX3CR1 expression patterns in whole-blood measurements from 92 patients with obesity and/or OSAS with regard to plasma CXCL10 values and individual clinical parameters. Furthermore, cytokine secretion by THP-1 monocytes in response to CXCL10 was analyzed. Data revealed significantly elevated plasma CXCL10 in patients with obesity with an additive effect of OSAS. CXCL10 was found to drive monocytic secretion of macrophage migration inhibitory factor via receptor protein CX3CR1, which significantly correlated with the individual body mass index. Our data show, for the first time, to our knowledge, that CX3CR1 is involved in alternative CXCL10 signaling in human monocytes in obesity-related inflammation. Obesity is a multifactorial disease, and further investigations regarding the complex interplay between obesity-related inflammatory mediators and systemic immune balances will help to better understand and improve the individual situation of our patients.

Correlation of Intra-tumoral and Peripheral PD-1/PD-L1 Immunity in Head and Neck Cancer.

BACKGROUND/AIM: Head and neck squamous cell carcinoma (HNSCC) represents a heterogeneous malignant disease of the oral cavity, pharynx, and larynx. HNSCC cells evade the host immune system through alterations in their immunogenicity, production of immunosuppressive mediators, and induction of immunomodulatory cell types. The immune status of solid HNSCC can be considered as hot, cold, or excluded for each patient individually, based on the distribution of tumor infiltrating immune cells. In this context immunotherapies based on the blockade of checkpoint molecules programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1) have significantly improved therapeutic outcomes in different cancer types. In HNSCC, intra-tumoral expression levels of PD-L1 are used for decision making in checkpoint inhibitor treatment. The significance of PD-L1 as a prognostic indicator is still controversial because both PD-1 and PD-L1 are also expressed in different types of circulating immune cells and the interaction of systemic and intra-tumoral cell-type-specific expression patterns of checkpoint molecules PD-1/PD-L1 has not yet been fully unveiled. MATERIALS AND METHODS: Using immunohistochemical (IHC) staining and flow cytometry, we correlated the expression patterns of the checkpoint molecules PD1/PD-L1 in peripheral blood CD14/CD16 monocytes and CD4/CD8 T cells with intra-tumoral conditions in patients with head and neck cancer. RESULTS/CONCLUSION: Our data demonstrate significant connections between systemic and intra-tumoral PD-1/PD-L1 immune patterns, both of which may serve as promising combined biomarkers for treatment decisions in patients with head and neck cancer.

Plasma Leptin Levels, Obstructive Sleep Apnea Syndrome, and Diabetes Are Associated with Obesity-Related Alterations of Peripheral Blood Monocyte Subsets.

Obesity is a dramatically increasing disease, accompanied with comorbidities such as cardiovascular disease and obstructive sleep apnea syndrome (OSAS). Both obesity and OSAS per se are associated with systemic inflammation. However, the multifactorial impact of obesity, OSAS, and its concomitant diseases on the immunological characteristics of circulating monocytes has not yet been fully resolved. Monocyte subsets of 82 patients with obesity were analyzed in whole blood measurements in terms of the CD14/CD16 cell surface expression patterns and different monocytic adhesion molecules using flow cytometry. Plasma levels of adipokines adiponectin and leptin of all patients were evaluated and correlated with accompanying cellular and clinical values. Whole blood measurements revealed a significant overall redistribution of CD14/CD16 monocyte subsets in patients with obesity. Monocytic adhesion molecules CD11a, CD11b, and CX3CR1 were significantly elevated. The observed alterations significantly correlated with plasma leptin levels and diabetes status as crucial amplifying factors. The additive impact of obesity, diabetes, and OSAS on the immunological balance of peripheral blood monocytes requires a coordinated regimen in terms of therapeutic treatment, respiratory support, and weight loss to improve the systemic immunity in these patients.

Differential Effects of Anti-PD-1/PD-L1 Checkpoint Inhibitors on Adhesion Molecules and Cytokine Secretion by THP-1 Monocytes.

BACKGROUND/AIM: Immune checkpoint inhibitors have improved the treatment regimen for human cancers in recent years. Particularly, inhibitors of the checkpoint molecules PD-1/PD-L1 have emerged as promising therapeutic treatments by preventing T-cell anergy and exhaustion. However, the impact of different anti-PD-1/PD-L1 checkpoint inhibitors on human monocytes remains elusive. MATERIALS AND METHODS: In this study, using the human monocyte leukemia cell line THP-1 as a model, we investigated the influence of different therapeutic anti-PD-1/PD-L1 checkpoint inhibitors on monocytic adhesion molecule expression and cytokine secretion. THP-1 monocytes were treated with the anti-PD-1 checkpoint inhibitors Nivolumab and Pembrolizumab and anti-PD-L1 checkpoint inhibitors Atezolizumab and Durvalumab. Cytokine expression patterns were evaluated using cytokine arrays and enzyme-linked immunosorbent assays (ELISA) and analysis of adhesion molecules was addressed using flow cytometry. RESULTS: Our data show an overall moderate apoptosis induction upon checkpoint inhibitor treatment and significantly reduced expression levels of adhesion molecules CD29, CD49d, and CX3CR1 in response to anti-PD-1 treatment. Cytokine screening revealed overall decreased secretion levels of insulin-like growth factor binding protein 2 (IGFBP2), CD147 (basigin) and CD31 (PECAM-1) as well as elevated levels of interleukin 5 (IL-5) and interferon gamma (IFNγ) in response to checkpoint inhibitor treatment. CONCLUSION: Our data indicate differential effects of anti-PD-1/PD-L1 checkpoint inhibitors on THP-1 monocytes, both by specific anti-PD-1/PD-L1 binding and unspecific antibody IgG isotype recognition. Further investigations on peripheral blood monocyte subsets in terms of their expansion and function upon checkpoint inhibitor therapy are required to better understand the individual immunological balances in cancer patients in long-term observational studies.

1,8-cineol inhibits the Wnt/ß-catenin signaling pathway through GSK-3 dephosphorylation in nasal polyps of chronic rhinosinusitis patients.

Chronic rhinosinusitis with nasal polyps (CRSwNP) represents a benign neoplasm of the nasal mucosa, which leads to a decreased breathing capacity and reduced olfaction. The pathogenesis and the molecular mechanisms driving nasal polyps are not very well known. GSK-3 is involved in the regulation of various biosynthetic pathways and various kinases are able to regulate the GSK-3. Therefore, we investigated the effect of the monoterpene oxide 1,8-cineol on the regulation of the Wnt/ß-catenin signaling pathway with its central regulator protein GSK-3 in vitro. We determined GSK-3 expression and phosphorylation as well as the expression of negative regulators (Akt and SGK) and downstream activation of ß-catenin in nasal polyps of patients with CRSwNP by immunohistochemistry and Western blot experiments. In this study we demonstrated for the first time, that 1,8-cineol acts as a potential inhibitor of the Wnt/ß-catenin signaling pathway, by affecting the inhibitory phosphorylation of GSK-3, which is the key regulator of the ß-catenin activity. Our data provide novel insights in the regulatory networks responsible for the progression of CRSwNP and furthermore represent a new mechanism of 1,8-cineol activity, which may lead to novel treatment approaches to this natural drug.

PD-L1 is expressed on human platelets and is affected by immune checkpoint therapy.

Cancer immunotherapy has been revolutionised by drugs that enhance the ability of the immune system to detect and fight tumors. Immune checkpoint therapies that target the programmed death-1 receptor (PD-1), or its ligand (PD-L1) have shown unprecedented rates of durable clinical responses in patients with various cancer types. However, there is still a large fraction of patients that do not respond to checkpoint inhibitors, and the challenge remains to find cellular and molecular cues that could predict which patients would benefit from these therapies. Using a series of qualitative and quantitative methods we show here that PBMCs and platelets from smokers and patients with head and neck squamous cell carcinoma (HNSCC) or lung cancer express and up-regulate PD-L1 independently of tumor stage. Furthermore, treatment with Atezolizumab, a fully humanised monoclonal antibody against PD-L1, in 4 patients with lung cancer caused a decrease in PD-L1 expression in platelets, which was restored over 20 days. Altogether, our findings reveal the expression of the main therapeutic target in current checkpoint therapies in human platelets and highlight their potential as biomarkers to predict successful therapeutic outcomes.

Viral Inhibition of Bacterial Phagocytosis by Human Macrophages: Redundant Role of CD36.

Macrophages are essential to maintaining lung homoeostasis and recent work has demonstrated that influenza-infected lung macrophages downregulate their expression of the scavenger receptor CD36. This receptor has also been shown to be involved in phagocytosis of Streptococcus pneumoniae, a primary agent associated with pneumonia secondary to viral infection. The aim of this study was to investigate the role of CD36 in the effects of viral infection on macrophage phagocytic function. Human monocyte-derived macrophages (MDM) were exposed to H3N2 X31 influenza virus, M37 respiratory syncytial virus (RSV) or UV-irradiated virus. No infection of MDM was seen upon exposure to UV-irradiated virus but incubation with live X31 or M37 resulted in significant levels of viral detection by flow cytometry or RT-PCR respectively. Infection resulted in significantly diminished uptake of S. pneumoniae by MDM and significantly decreased expression of CD36 at both the cell surface and mRNA level. Concurrently, there was a significant increase in IFNß gene expression in response to infection and we observed a significant decrease in bacterial phagocytosis (p = 0.031) and CD36 gene expression (p = 0.031) by MDM cultured for 24 h in 50IU/ml IFNß. Knockdown of CD36 by siRNA resulted in decreased phagocytosis, but this was mimicked by transfection reagent alone. When MDM were incubated with CD36 blocking antibodies no effect on phagocytic ability was observed. These data indicate that autologous IFNß production by virally-infected cells can inhibit bacterial phagocytosis, but that decreased CD36 expression by these cells does not play a major role in this functional deficiency.

Viral infection of human lung macrophages increases PDL1 expression via IFNß.

Lung macrophages are an important defence against respiratory viral infection and recent work has demonstrated that influenza-induced macrophage PDL1 expression in the murine lung leads to rapid modulation of CD8+ T cell responses via the PD1 receptor. This PD1/PDL1 pathway may downregulate acute inflammatory responses to prevent tissue damage. The aim of this study was to investigate the mechanisms of PDL1 regulation by human macrophages in response to viral infection. Ex-vivo viral infection models using influenza and RSV were established in human lung explants, isolated lung macrophages and monocyte-derived macrophages (MDM) and analysed by flow cytometry and RT-PCR. Incubation of lung explants, lung macrophages and MDM with X31 resulted in mean cellular infection rates of 18%, 18% and 29% respectively. Viral infection significantly increased cell surface expression of PDL1 on explant macrophages, lung macrophages and MDM but not explant epithelial cells. Infected MDM induced IFNγ release from autologous CD8+ T cells, an effect enhanced by PDL1 blockade. We observed increases in PDL1 mRNA and IFNß mRNA and protein release by MDM in response to influenza infection. Knockdown of IFNß by siRNA, resulted in a 37.5% reduction in IFNß gene expression in response to infection, and a significant decrease in PDL1 mRNA. Furthermore, when MDM were incubated with IFNß, this cytokine caused increased expression of PDL1 mRNA. These data indicate that human macrophage PDL1 expression modulates CD8+ cell IFNγ release in response to virus and that this expression is regulated by autologous IFNß production.