Effects of Perfluorohexane Sulfonate Exposure on Immune Cell Populations in Naive Mice.

Perfluorohexane sulfonate (PFHxS) is a member of the per- and polyfluoroalkyls (PFAS) superfamily of molecules, characterized by their fluorinated carbon chains and use in a wide range of industrial applications. PFHxS and perfluorooctane sulfonate are able to accumulate in the environment and in humans with the approximated serum elimination half-life in the range of several years. More recently, some PFAS compounds have also been suggested as potential immunosuppressants. In this study, we analyze immune cell numbers in mice following 28-d repeated oral exposure to potassium PFHxS at 12, 120, 1,200, and 12,000 ng/kg/d, with resulting serum levels ranging up to ∼1,600 ng/ml, approximating ranges found in the general population and at higher levels in PFAS workers. The immunosuppressant cyclophosphamide was analyzed as a positive control. B cells, T cells, and granulocytes from the bone marrow, liver, spleen, lymph nodes, and thymus were evaluated. We found that at these exposures, there was no effect of PFHxS on major T or B cell populations, macrophages, dendritic cells, basophils, mast cells, eosinophils, neutrophils, or circulating Ab isotypes. By contrast, mice exposed to cyclophosphamide exhibited depletion of several granulocyte and T and B cell populations in the thymus, bone marrow, and spleen, as well as reductions in IgG1, IgG2b, IgG2c, IgG3, IgE, and IgM. These data indicate that exposures of up to 12,000 ng/kg of PFHxS for 28 d do not affect immune cell numbers in naive mice, which provides valuable information for assessing the risks and health influences of exposures to this compound.

Effective differentiation of double negative thymocytes requires high fidelity replication of mitochondrial DNA in an age dependent manner.

One of the most proliferative periods for T cells occurs during their development in the thymus. Increased DNA replication can result in increased DNA mutations in the nuclear genome, but also in mitochondrial genomes. A high frequency of mitochondrial DNA mutations can lead to abnormal mitochondrial function and have negative implications on human health. Furthermore, aging is accompanied by an increase in such mutations through oxidative damage and replication errors. Increased mitochondrial DNA mutations cause loss of mitochondrial protein function, and decrease energy production, substrates, and metabolites. Here we have evaluated the effect of increased mitochondrial DNA mutations on T cell development in the thymus. Using mice carrying a mutant mitochondrial DNA polymerase γ (PolG) that causes increased mitochondrial DNA mutations, we show that high fidelity replication of mitochondrial DNA is pivotal for proper T cell development. Reducing the fidelity of mitochondrial DNA replication results in a premature age-dependent reduction in the total number of CD4/CD8 double negative and double positive thymocytes. Analysis of mitochondrial density in thymocyte subpopulations suggests that this may be due to reduced proliferation in specific double negative stages. Taken together, this work suggests that T cell development is regulated by the ability of mitochondria to faithfully replicate their DNA.

Effects of PFOS and cyclophosphamide exposure on immune homeostasis in mice.

Perfluorooctane sulfonic acid (PFOS) is member of a class of molecules with fluorinated carbon chains known as polyfluoroalkyls. PFOS have been used to produce a variety of industry and comsumer uses. However, a significant concern is that it accumulates in the environment, including in animals and humans, and that it is a potential immunosuppressant. Here we analyze immune homeostasis in mice following chronic exposure to PFOS at levels up to those historically found in PFOS manufacturing workers. Mice were exposed to 0.15, 1.5, 15, or 50 µg /kg of PFOS for 28 days, after which, B cells, T cells, and granulocytes from the bone marrow, liver, spleen, lymph nodes, and thymus were evaluated. We find that at these exposures, there was no effect of PFOS on major T- or B-cell populations, macrophages, dendritic cells, basophils, mast cells, eosinophils, neutrophils, serum antibodies or select serum cytokines. By contrast, mice exposed the known immunosuppressant cyclophosphamide, which was given at 40 mg/kg for four days, exhibited depletion of several granulocyte, T- and B-cell populations of the thymus, bone marrow, and spleen, as well as circulating IgM and IgE antibodies. These data indicate that exposures of up to 50 µg /kg of PFOS for 28 days does not affect immune homeostasis in mice.

Hyaluronic acid regulates heart valve interstitial cell contraction in fibrin-based scaffolds.

Heart valve disease is associated with high morbidity and mortality worldwide resulting in hundreds of thousands of heart valve replacements each year. Tissue engineered heart valves (TEHVs) have the potential to overcome the major limitations of traditional replacement valves; however, leaflet retraction has led to the failure of TEHVs in preclinical studies. As native unmodified hyaluronic acid (HA) is known to promote healthy tissue development in native heart valves, we hypothesize that adding unmodified HA to fibrin-based scaffolds common to tissue engineering will reduce retraction by increasing cell-scaffold interactions and density of the scaffolds. Using a custom high-throughput culture system, we found that incorporating HA into millimeter-scale fibrin-based cell-populated scaffolds increases initial fiber diameter and cell-scaffold interactions, causing a cascade of mechanical, morphological, and cellular responses. These changes lead to higher levels of scaffold compaction and stiffness, increased cell alignment, and less bundling of fibrin fibers by the cells during culture. These effects significantly reduce scaffold retraction and total contractile force each by around 25%. These findings increase our understanding of how HA alters tissue remodeling and could inform the design of the next generation of tissue engineered heart valves to help reduce retraction. STATEMENT OF SIGNIFICANCE: Tissue engineered heart valves (TEHVs) have the potential to overcome the major limitations of traditional replacement valves; however, leaflet retraction induced by excessive myofibroblast activation has led to failure in preclinical studies. Developing valves are rich in hyaluronic acid (HA), which helps maintain a physiological environment for tissue remodeling without retraction. We hypothesized that adding unmodified HA to TEHVs would reduce retraction by increasing cell-scaffold interactions and density of the scaffolds. Using a high-throughput tissue culture platform, we demonstrate that HA incorporation into a fibrin-based scaffold can significantly reduce tissue retraction and total contractile force by increasing fiber bundling and altering cell-mediated matrix remodeling, therefore increasing gel density and stiffness. These finding increase our knowledge of native HA's effects within the extracellular matrix, and provide a new tool for TEHV design.