Matrix-Bound Hyaluronan Molecular Weight as a Regulator of Dendritic Cell Immune Potency.

Hyaluronic acid (HA) is a glycosaminoglycan in the extracellular matrix with immunoregulatory properties depending on its molecular weight (MW). However, the impact of matrix-bound HA on dendritic cells (DCs) remains unclear due to varying distribution of HA MW under different physiological conditions. To investigate DCs in defined biosystems, 3D collagen matrices modified with HA of specific MW with similar microstructure and HA levels are used. It is found that HA MW influences cytokine binding to matrix, suggesting modulation of cytokine availability by the different HA MWs. These studies on DC immune potency reveal that low MW HA (8-15 kDa) enhances immature DC differentiation and antigen uptake, while medium (MMW-HA; 500-750 kDa) and high MW HA (HMW-HA; 1250-1500 kDa) increase cytokine secretion in mature DCs. The effect on DC phenotype and cytokine secretion by different MWs of HA is independent of CD44. However, blocking the CD44 receptor reveals its potential role in regulating acute inflammation through increased secretion of CCL2, CXCL8, and IL-6. Additionally, MMW- and HMW-HA matrices reduce migratory capacity of DCs, dependent on CD44. Overall, these findings provide insights into MW-dependent effects of matrix-bound HA on DCs, opening avenues for the design of DC-modulating materials to enhance DC-based therapy.

Effects of an aged tissue niche on the immune potency of dendritic cells using simulated microgravity.

Microgravity accelerates the aging of various physiological systems, and it is well acknowledged that aged individuals and astronauts both have increased susceptibility to infections and poor response to vaccination. Immunologically, dendritic cells (DCs) are the key players in linking innate and adaptive immune responses. Their distinct and optimized differentiation and maturation phases play a critical role in presenting antigens and mounting effective lymphocyte responses for long-term immunity. Despite their importance, no studies to date have effectively investigated the effects of microgravity on DCs in their native microenvironment, which is primarily located within tissues. Here, we address a significantly outstanding research gap by examining the effects of simulated microgravity via a random positioning machine on both immature and mature DCs cultured in biomimetic collagen hydrogels, a surrogate for tissue matrices. Furthermore, we explored the effects of loose and dense tissues via differences in collagen concentration. Under these various environmental conditions, the DC phenotype was characterized using surface markers, cytokines, function, and transcriptomic profiles. Our data indicate that aged or loose tissue and exposure to RPM-induced simulated microgravity both independently alter the immunogenicity of immature and mature DCs. Interestingly, cells cultured in denser matrices experience fewer effects of simulated microgravity at the transcriptome level. Our findings are a step forward to better facilitate healthier future space travel and enhance our understanding of the aging immune system on Earth.

The T Cell Journey: A Tour de Force.

T cells act as the puppeteers in the adaptive immune response, and their dysfunction leads to the initiation and progression of pathological conditions. During their lifetime, T cells experience myriad forces that modulate their effector functions. These forces are imposed by interacting cells, surrounding tissues, and shear forces from fluid movement. In this review, a journey with T cells is made, from their development to their unique characteristics, including the early studies that uncovered their mechanosensitivity. Then the studies pertaining to the responses of T cell activation to changes in antigen-presenting cells' physical properties, to their immediate surrounding extracellular matrix microenvironment, and flow conditions are highlighted. In addition, it is explored how pathological conditions like the tumor microenvironment can hinder T cells and allow cancer cells to escape elimination.

3D microenvironment attenuates simulated microgravity-mediated changes in T cell transcriptome.

Human space travel and exploration are of interest to both the industrial and scientific community. However, there are many adverse effects of spaceflight on human physiology. In particular, there is a lack of understanding of the extent to which microgravity affects the immune system. T cells, key players of the adaptive immune system and long-term immunity, are present not only in blood circulation but also reside within the tissue. As of yet, studies investigating the effects of microgravity on T cells are limited to peripheral blood or traditional 2D cell culture that recapitulates circulating blood. To better mimic interstitial tissue, 3D cell culture has been well established for physiologically and pathologically relevant models. In this work, we utilize 2D cell culture and 3D collagen matrices to gain an understanding of how simulated microgravity, using a random positioning machine, affects both circulating and tissue-resident T cells. T cells were studied in both resting and activated stages. We found that 3D cell culture attenuates the effects of simulated microgravity on the T cells transcriptome and nuclear irregularities compared to 2D cell culture. Interestingly, simulated microgravity appears to have less effect on activated T cells compared to those in the resting stage. Overall, our work provides novel insights into the effects of simulated microgravity on circulating and tissue-resident T cells which could provide benefits for the health of space travellers.

Fibroblast Differentiation and Matrix Remodeling Impaired under Simulated Microgravity in 3D Cell Culture Model.

Exposure to microgravity affects astronauts' health in adverse ways. However, less is known about the extent to which fibroblast differentiation during the wound healing process is affected by the lack of gravity. One of the key steps of this process is the differentiation of fibroblasts into myofibroblasts, which contribute functionally through extracellular matrix production and remodeling. In this work, we utilized collagen-based three-dimensional (3D) matrices to mimic interstitial tissue and studied fibroblast differentiation under simulated microgravity (sµG). Our results demonstrated that alpha-smooth muscle actin (αSMA) expression and translocation of Smad2/3 into the cell nucleus were reduced upon exposure to sµG compared to the 1g control, which suggests the impairment of fibroblast differentiation under sµG. Moreover, matrix remodeling and production were decreased under sµG, which is in line with the impaired fibroblast differentiation. We further investigated changes on a transcriptomic level using RNA sequencing. The results demonstrated that sµG has less effect on fibroblast transcriptomes, while sµG triggers changes in the transcriptome of myofibroblasts. Several genes and biological pathways found through transcriptome analysis have previously been reported to impair fibroblast differentiation. Overall, our data indicated that fibroblast differentiation, as well as matrix production and remodeling, are impaired in 3D culture under sµG conditions.

May the Force Be with You (Or Not): The Immune System under Microgravity.

All terrestrial organisms have evolved and adapted to thrive under Earth's gravitational force. Due to the increase of crewed space flights in recent years, it is vital to understand how the lack of gravitational forces affects organisms. It is known that astronauts who have been exposed to microgravity suffer from an array of pathological conditions including an impaired immune system, which is one of the most negatively affected by microgravity. However, at the cellular level a gap in knowledge exists, limiting our ability to understand immune impairment in space. This review highlights the most significant work done over the past 10 years detailing the effects of microgravity on cellular aspects of the immune system.

Where the infection is isolated rather than the specific species correlates with adherence strength, whereas biofilm density remains static in clinically isolated Candida and arthroconidial yeasts.

To colonize and infect the host, arthroconidial yeasts must avoid being killed by the host's defenses. The formation of biofilms on implanted devices allows fungi to avoid host responses and to disseminate into the host. To better study the mechanisms of infection by arthroconidial yeasts, adherence and biofilm formation were assayed using patient samples collected over 10 years. In clinical samples, adherence varies within species, but the relative adherence is constant for those samples isolated from the same infection site. Herein we document, for the first time, in-vitro biofilm formation by Trichosporon dohaense, T. ovoides, T. japonicum, T. coremiiforme, Cutaneotrichosporon mucoides, Cutaneotrichosporon cutaneum, Galactomyces candidus, and Magnusiomyces capitatus on clinically relevant catheter material. Analysis of biofilm biomass assays indicated that biofilm mass changes less than 2-fold, regardless of the species. Our results support the hypothesis that most pathogenic fungi can form biofilms, and that biofilm formation is a source of systemic infections.

Engineered Microvessel for Cell Culture in Simulated Microgravity.

As the number of manned space flights increase, studies on the effects of microgravity on the human body are becoming more important. Due to the high expense and complexity of sending samples into space, simulated microgravity platforms have become a popular way to study these effects on earth. In addition, simulated microgravity has recently drawn the attention of regenerative medicine by increasing cell differentiation capability. These platforms come with many advantages as well as limitations. A main limitation for usage of these platforms is the lack of high-throughput capability due to the use of large cell culture vessels. Therefore, there is a requirement for microvessels for microgravity platforms that limit waste and increase throughput. In this work, a microvessel for commercial cell culture plates was designed. Four 3D printable (polycarbonate (PC), polylactic acid (PLA) and resin) and castable (polydimethylsiloxane (PDMS)) materials were assessed for biocompatibility with adherent and suspension cell types. PDMS was found to be the most suitable material for microvessel fabrication, long-term cell viability and proliferation. It also allows for efficient gas exchange, has no effect on cell culture media pH and does not induce hypoxic conditions. Overall, the designed microvessel can be used on simulated microgravity platforms as a method for long-term high-throughput biomedical studies.

Mouse Embryonic Fibroblast Adipogenic Potential: A Comprehensive Transcriptome Analysis.

Our understanding of adipose tissue has progressed from an inert tissue for energy storage to be one of the largest endocrine organs regulating metabolic homoeostasis through its ability to synthesize and release various adipokines that regulate a myriad of pathways. The field of adipose tissue biology is growing due to this association with various chronic metabolic diseases. An important process in the regulation of adipose tissue biology is adipogenesis, which is the formation of new adipocytes. Investigating adipogenesis in vitro is currently a focus for identifying factors that might be utilized in clinically. A powerful tool for such work is high-throughput sequencing which can rapidly identify changes at gene expression level. Various cell models exist for studying adipogenesis and has been used in high-throughput studies, yet little is known about transcriptome profile that underlies adipogenesis in mouse embryonic fibroblasts. This study utilizes RNA-sequencing and computational analysis with DESeq2, gene ontology, protein-protein networks, and robust rank analysis to understand adipogenesis in mouse embryonic fibroblasts in-depth. Our analyses confirmed the requirement of mitotic clonal expansion prior to adipogenesis in this cell model and highlight the role of Cebpa and Cebpb in regulating adipogenesis through interactions of large numbers of genes.

Elucidating the role of an immunomodulatory protein in cancer: From protein expression to functional characterization.

Several fundamental discoveries made over the last two decades, in the field of cancer biology, have increased our understanding of the complex tumor micro- and macroenvironments. This has shifted the current empirical cancer therapies to more rationalized treatments targeting immunomodulatory proteins. From the point of identification, a protein target undergoes several interrogations, which are necessary to truly define its druggability. Here, we outline some basic steps that can be followed for in vitro characterization of a potential immunomodulatory protein target. We describe procedures for recombinant protein expression and purification including key annotations on protein cloning, expression systems, purification strategies and protein characterization using structural and biochemical approaches. For functional characterization, we provide detailed protocols for using flow-cytometric techniques in cell lines or primary cells to study protein expression profiles, proliferation, apoptosis and cell-cycle changes. This multilevel approach can provide valuable, in-depth understanding of any protein target with potential immunomodulatory effects.

The effect of anti-CTLA4 treatment on peripheral and intra-tumoral T cells in patients with hepatocellular carcinoma.

BACKGROUND: Checkpoint inhibitors have recently been approved for the treatment of patients with hepatocellular carcinoma (HCC). However, biomarkers, which will help identify patients responding to therapy, are missing. We recently tested the combination of anti-CTLA4 treatment (tremelimumab) with loco-regional therapy in patients with HCC and reported a partial response rate of 26%. METHODS: Here, we report updated survival analyses and results from our immune monitoring studies on peripheral blood mononuclear cells (PBMCs) and tumors from these patients. RESULTS: Tremelimumab therapy increased CD4+-HLA-DR+, CD4+PD-1+, CD8+HLA-DR+, CD8+PD-1+, CD4+ICOS+ and CD8+ICOS+ T cells in the peripheral blood of the treated patients. Patients with higher CD4+PD1+ cell frequency at baseline were more likely to respond to tremelimumab therapy. PD-1 expression was increased on alpha fetal protein (AFP) and survivin-specific CD8 T cells upon tremelimumab treatment. An increase of tumor infiltrating CD3+ T cells were also seen in these patients. Immunosequencing of longitudinal PBMC showed that one cycle of tremelimumab significantly decreased peripheral clonality, while no additional effects were seen after loco-regional therapy. CONCLUSION: In summary, we observed a clear activation of T cell responses in HCC patients treated with tremelimumab and identified potential biomarkers which will help identify patients responding to immunotherapy with anti-CTLA4.

Tremelimumab in combination with ablation in patients with advanced hepatocellular carcinoma.

BACKGROUND & AIMS: Tremelimumab is a fully human monoclonal antibody that binds to cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) on the surface of activated T lymphocytes. Ablative therapies induce a peripheral immune response which may enhance the effect of anti-CTLA4 treatment in patients with advanced hepatocellular carcinoma (HCC). This study aimed to demonstrate whether tremelimumab could be combined safely and feasibly with ablation. METHODS: Thirty-two patients with HCC were enrolled: male:female: 28:4; median age: 62 (range 36-76). Patients were given tremelimumab at two dose levels (3.5 and 10mg/kg i.v.) every 4weeks for 6 doses, followed by 3-monthly infusions until off-treatment criteria were met. On day 36, patients underwent subtotal radiofrequency ablation or chemoablation. Staging was performed by contrast-enhanced CT or MRI scan every 8weeks. RESULTS: No dose-limiting toxicities were encountered. The most common toxicity was pruritus. Of the 19 evaluable patients, five (26.3%; 95% CI: 9.1-51.2%) achieved a confirmed partial response. Twelve of 14 patients with quantifiable HCV experienced a marked reduction in viral load. Six-week tumor biopsies showed a clear increase in CD8+ T cells in patients showing a clinical benefit only. Six and 12-month probabilities of tumor progression free survival for this refractory HCC population were 57.1% and 33.1% respectively, with median time to tumor progression of 7.4months (95% CI 4.7 to 19.4months). Median overall survival was 12.3months (95% CI 9.3 to 15.4months). CONCLUSIONS: Tremelimumab in combination with tumor ablation is a potential new treatment for patients with advanced HCC, and leads to the accumulation of intratumoral CD8+ T cells. Positive clinical activity was seen, with a possible surrogate reduction in HCV viral load. LAY SUMMARY: Studies have shown that the killing of tumors by direct methods (known as ablation) can result in the immune system being activated or switched on. The immune system could potentially also recognize and kill the cancer that is left behind. There are new drugs available known as immune checkpoint inhibitors which could enhance this effect. Here, we test one of these drugs (tremelimumab) together with ablation. CLINICAL TRIAL NUMBER: ClinicalTrials.gov: NCT01853618.

NAFLD causes selective CD4(+) T lymphocyte loss and promotes hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) is the second most common cause of cancer-related death. Non-alcoholic fatty liver disease (NAFLD) affects a large proportion of the US population and is considered to be a metabolic predisposition to liver cancer. However, the role of adaptive immune responses in NAFLD-promoted HCC is largely unknown. Here we show, in mouse models and human samples, that dysregulation of lipid metabolism in NAFLD causes a selective loss of intrahepatic CD4(+) but not CD8(+) T lymphocytes, leading to accelerated hepatocarcinogenesis. We also demonstrate that CD4(+) T lymphocytes have greater mitochondrial mass than CD8(+) T lymphocytes and generate higher levels of mitochondrially derived reactive oxygen species (ROS). Disruption of mitochondrial function by linoleic acid, a fatty acid accumulated in NAFLD, causes more oxidative damage than other free fatty acids such as palmitic acid, and mediates selective loss of intrahepatic CD4(+) T lymphocytes. In vivo blockade of ROS reversed NAFLD-induced hepatic CD4(+) T lymphocyte decrease and delayed NAFLD-promoted HCC. Our results provide an unexpected link between lipid dysregulation and impaired anti-tumour surveillance.

High prevalence of Candida dubliniensis in lower respiratory tract secretions from cystic fibrosis patients may be related to increased adherence properties.

OBJECTIVES: We identified Candida spp isolated from lower respiratory tract secretions obtained from cystic fibrosis (CF) patients, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), with the aim of determining the most prevalent causative agent. We also sought to determine their adhesive properties in order to understand their biology related to CF. METHODS: Twenty-five clinical samples were collected from a cohort of 20 CF patients. Twenty-six isolates of Candida spp were isolated and identified by MALDI-TOF MS method. Adherence assays were performed using the Fluxion BioFlux 200, a flow apparatus that allows for the visualization of adhering cells. RESULTS: MALDI-TOF MS analysis revealed C. dubliniensis to be the most prevalent species (n=18, 69%), followed by C. albicans (n=4), C. tropicalis (n=3), and C. glabrata (n=1). C. dubliniensis showed the strongest adherence under constant flow when compared to the other species of Candida. In the majority of cases, C. dubliniensis was isolated in combination with Pseudomonas aeruginosa and Staphylococcus aureus. C. dubliniensis appears to be able to survive in the CF lung and coexist with bacteria. CONCLUSIONS: The data presented here show that the presence of C. dubliniensis in the lower airways of CF patients may be related to increased adherence properties.